WO2023023538A2 - Treating tissue inflammation - Google Patents
Treating tissue inflammation Download PDFInfo
- Publication number
- WO2023023538A2 WO2023023538A2 PCT/US2022/075051 US2022075051W WO2023023538A2 WO 2023023538 A2 WO2023023538 A2 WO 2023023538A2 US 2022075051 W US2022075051 W US 2022075051W WO 2023023538 A2 WO2023023538 A2 WO 2023023538A2
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- WO
- WIPO (PCT)
- Prior art keywords
- mammal
- aspartate
- cells
- tnf
- tissue inflammation
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Definitions
- tissue inflammation e.g., autoimmune tissue inflammation
- one or more inhibitors of production of a tumor necrosis factor-a (TNF-a) polypeptide can be administered to a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation) to treat the mammal.
- TNF-a tumor necrosis factor-a
- TNF-a is a prime therapeutic target in rheumatoid arthritis (RA) and specific blockade of the cytokine is now considered standard therapy (Rubbert-Roth et al. , Autoimmun. Rev., 17:24-28 (2016)).
- RA rheumatoid arthritis
- specific blockade of the cytokine is now considered standard therapy (Rubbert-Roth et al. , Autoimmun. Rev., 17:24-28 (2016)).
- RA rheumatoid arthritis
- Low rates of disease remission, the development of adverse effects, and the generation of antibodies against biologic TNF-a inhibitors (Rubbert-Roth et al. , Autoimmun. Rev., 17:24- 28 (2018)) all curb the efficacy of anti-TNF therapy.
- tissue inflammation e.g., autoimmune tissue inflammation
- one or more inhibitors of TNF-a polypeptide production can be administered to a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation) to treat the mammal.
- tissue inflammation e.g., autoimmune tissue inflammation
- intact mitochondria can be administered to a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation) to treat the mammal (e.g., to reduce TNF-a polypeptide production by, for example, T cells and treat the mammal).
- one or more inhibitors of unfolded protein response (UPR) signaling can be administered to a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation) to treat the mammal (e.g., to reduce TNF-a polypeptide production by, for example, T cells and treat the mammal).
- tissue inflammation e.g., autoimmune tissue inflammation
- one or more inhibitors for co-translational translocation can be administered to a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation) to treat the mammal (e.g., to reduce TNF-a polypeptide production by, for example, T cells and treat the mammal).
- tissue inflammation e.g., autoimmune tissue inflammation
- one or more inhibitors for co-translational translocation can be administered to a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation) to treat the mammal (e
- one or more mitochondrial metabolites can be administered to a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation) to treat the mammal (e.g., to reduce TNF-a polypeptide production by, for example, T cells and treat the mammal).
- tissue inflammation e.g., autoimmune tissue inflammation
- one or more inhibitors of aspartate transport and/or aspartate metabolism can be administered to a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation) to treat the mammal (e.g., to reduce TNF-a polypeptide production by, for example, T cells and treat the mammal).
- the interventions described herein that block TNF-a polypeptide production can be used to suppress tissue inflammation.
- Having the ability to reduce TNF-a production by, for example, T cells as described herein provides a unique and unrealized strategy for treating tissue inflammation.
- blocking production of a TNF-a polypeptide by, for example, T cells instead of inhibiting the produced TNF-a polypeptide provides an opportunity to treat tissue inflammation with a reduced risk of disease remission, the development of adverse effects, and the generation of antibodies against biologic TNF-a inhibitors.
- one aspect of this document features methods for restoring aspartate homeostasis in a mammal having tissue inflammation.
- the methods can include, or consist essentially of, administering, to a mammal having tissue inflammation, a composition comprising cells, where a population of intact mitochondria was introduced into the cells, and where the production of a TNF-a polypeptide within the mammal is reduced following the administering step.
- the composition can include T cells containing the intact mitochondria.
- the T cells can be obtained from the mammal.
- the mammal can be a human.
- the tissue inflammation can be associated with an autoimmune disease.
- the autoimmune disease can be polymyositis, dermatomyositis, rheumatoid arthritis (RA), scleroderma, sjogren's syndrome, systemic lupus erythematosus, vasculitis, mixed connective tissue disease (MCTD), autoimmune hepatitis, psoriasis, or ankylosing spondylitis.
- the autoimmune disease can be RA.
- the tissue inflammation can be associated with irritable bowel syndrome (IBS).
- the method can include administering to the mammal an anti-TNF-a therapy.
- the anti-TNF-a therapy can be adalimumab, adalimumab-adbm, adalimumab-adaz, adalimumab-atto, certolizumab pegol, etanercept, etanercept-szzs, infliximab, infliximab- abda, or infliximab-dyyb.
- this document features methods for restoring aspartate homeostasis in a mammal having tissue inflammation.
- the methods can include, or consist essentially of, administering, to a mammal having tissue inflammation, an inhibitor of UPR signaling, where the method is effective to reduce production of a TNF-a polypeptide within the mammal.
- the inhibitor of UPR signaling can target an UPR signaling pathway component selected from the group consisting of binding immunoglobulin protein (BiP) polypeptides, IREla polypeptides, PERK polypeptides, ATF6 polypeptides, eIF2a polypeptides, CHOP polypeptides, XBP-1 polypeptides, and XBP-1S polypeptides.
- BiP binding immunoglobulin protein
- the inhibitor of UPR signaling can be eeyarestatin I, mycolactone, exotoxin A, NSC 630668-R/l, MAL3-39, MAL3-101, E6 berbamine, ophiobolin A, equisetin, CJ-21058, Rose Bengal, erythrosin B, P97-A4, P87-A4, 17D9, P91-E9, 16F6, bisthiouracil, SCA-21, HUN-7293, cotransin, CAM741, apratoxin A, decatransin, valinomycin, CAD A, kinase inhibiting RNase attenuator 6 (KIRA6), 3-hydroxy-2-naphthoic acid (3HNA), MKC-3946, 4-Phenylbutyric acid (4- PBA), taurine-conjugated ursodeoxycholic acid (TUDCA), olmesartan, N-acetylcysteine (NA
- the mammal can be a human.
- the tissue inflammation can be associated with an autoimmune disease.
- the autoimmune disease can be polymyositis, dermatomyositis, rheumatoid arthritis (RA), scleroderma, sjogren's syndrome, systemic lupus erythematosus, vasculitis, mixed connective tissue disease (MCTD), autoimmune hepatitis, psoriasis, or ankylosing spondylitis.
- the autoimmune disease can be RA.
- the tissue inflammation can be associated with irritable bowel syndrome (IBS).
- the method can include administering to the mammal an anti-TNF-a therapy.
- the anti-TNF-a therapy can be adalimumab, adalimumab-adbm, adalimumab-adaz, adalimumab- atto, certolizumab pegol, etanercept, etanercept-szzs, infliximab, infliximab-abda, or infliximab-dyyb.
- this document features methods for restoring aspartate homeostasis in a mammal having tissue inflammation.
- the methods can include, or consist essentially of, administering, to a mammal having tissue inflammation, an inhibitor of co-translational translocation, where the method is effective to reduce production of a TNF-a polypeptide within the mammal.
- the inhibitor of co-translational translocation can target a co- translational translocation pathway component that is selected from the group consisting of Sec61 polypeptides, Hsp70 polypeptides, ATPase polypeptides, calmodulin polypeptides, transmembrane domains of newly produced polypeptides, signal peptides, and signal peptidase complex polypeptides.
- the mammal can be a human.
- the tissue inflammation can be associated with an autoimmune disease.
- the autoimmune disease can be polymyositis, dermatomyositis, rheumatoid arthritis (RA), scleroderma, sjogren's syndrome, systemic lupus erythematosus, vasculitis, mixed connective tissue disease (MCTD), autoimmune hepatitis, psoriasis, or ankylosing spondylitis.
- the autoimmune disease can be RA.
- the tissue inflammation can be associated with irritable bowel syndrome (IBS).
- the method can include administering to the mammal an anti-TNF-a therapy.
- the anti-TNF-a therapy can be adalimumab, adalimumab-adbm, adalimumab-adaz, adalimumab-atto, certolizumab pegol, etanercept, etanercept-szzs, infliximab, infliximab-abda, or infliximab- dyyb.
- this document features methods for restoring aspartate homeostasis in a mammal having tissue inflammation.
- the methods can include, or consist essentially of, administering, to a mammal having tissue inflammation, a composition comprising a mitochondrial metabolite, where the method is effective to reduce production of a TNF-a polypeptide within the mammal.
- the mitochondrial metabolite can be L-aspartate, D- aspartate, NAD, alpha-ketobutyrate, malate, oxaloacetate, or nicotinamide riboside.
- the composition can include L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate.
- the mammal can be a human.
- the tissue inflammation can be associated with an autoimmune disease.
- the autoimmune disease can be polymyositis, dermatomyositis, rheumatoid arthritis (RA), scleroderma, sjogren's syndrome, systemic lupus erythematosus, vasculitis, mixed connective tissue disease (MCTD), autoimmune hepatitis, psoriasis, or ankylosing spondylitis.
- the autoimmune disease can be RA.
- the tissue inflammation can be associated with irritable bowel syndrome (IBS).
- the method can include administering to the mammal an anti-TNF-a therapy.
- the anti-TNF-a therapy can be adalimumab, adalimumab-adbm, adalimumab-adaz, adalimumab-atto, certolizumab pegol, etanercept, etanercept-szzs, infliximab, infliximab-abda, or infliximab-dyyb.
- this document features methods for restoring aspartate homeostasis in a mammal having tissue inflammation.
- the methods can include, or consist essentially of, administering, to a mammal having tissue inflammation, an inhibitor of aspartate transport or aspartate metabolism, where the method is effective to reduce production of a TNF-a polypeptide within the mammal.
- the inhibitor of aspartate transport or aspartate metabolism can be DL-TBOA, L-(-)-threo-3 -hydroxyaspartic acid, L-trans-2,4-PDC, TFB-TBOA, or GOT1 inhibitor 2c.
- the mammal can be a human.
- the tissue inflammation can be associated with an autoimmune disease.
- the autoimmune disease can be polymyositis, dermatomyositis, rheumatoid arthritis (RA), scleroderma, sjogren's syndrome, systemic lupus erythematosus, vasculitis, mixed connective tissue disease (MCTD), autoimmune hepatitis, psoriasis, or ankylosing spondylitis.
- the autoimmune disease can be RA.
- the tissue inflammation can be associated with irritable bowel syndrome (IBS).
- the method can include administering to the mammal an anti-TNF-a therapy.
- the anti-TNF-a therapy can be adalimumab, adalimumab-adbm, adalimumab-adaz, adalimumab-atto, certolizumab pegol, etanercept, etanercept-szzs, infliximab, infliximab-abda, or infliximab-dyyb.
- this document features methods for reducing tissue inflammation in a mammal.
- the methods can include, or consist essentially of, administering, to a mammal, a composition comprising cells, where a population of intact mitochondria was introduced into the cells, and where the production of a TNF-a polypeptide within the mammal is reduced following the administering step.
- the composition can include T cells containing the intact mitochondria.
- the T cells can be obtained from the mammal.
- the mammal can be a human.
- the tissue inflammation can be associated with an autoimmune disease.
- the autoimmune disease can be polymyositis, dermatomyositis, RA, scleroderma, sjogren's syndrome, systemic lupus erythematosus, vasculitis, MCTD, autoimmune hepatitis, psoriasis, or ankylosing spondylitis.
- the autoimmune disease can be RA.
- the tissue inflammation can be associated with IBS.
- the method also can include administering to the mammal an anti- TNF-a therapy.
- the anti-TNF-a therapy can be adalimumab, adalimumab-adbm, adalimumab-adaz, adalimumab-atto, certolizumab pegol, etanercept, etanercept-szzs, infliximab, infliximab-abda, or infliximab-dyyb.
- this document features methods for reducing tissue inflammation in a mammal.
- the methods can include, or consist essentially of, administering, to a mammal, an inhibitor of UPR signaling, where the method is effective to reduce production of a TNF-a polypeptide within the mammal.
- the inhibitor of UPR signaling can target an UPR signaling pathway component selected from the group consisting of BiP polypeptides, IRE la polypeptides, PERK polypeptides, ATF6 polypeptides, eIF2a polypeptides, CHOP polypeptides, XBP-1 polypeptides, and XBP-1S polypeptides.
- the inhibitor of UPR signaling can be eeyarestatin I, mycolactone, exotoxin A, NSC 630668-R/l, MAL3-39, MAL3-101, E6 berbamine, ophiobolin A, equisetin, CJ-21058, Rose Bengal, erythrosin B, P97-A4, P87-A4, 17D9, P91-E9, 16F6, bisthiouracil, SCA-21, HUN-7293, cotransin, CAM741, apratoxin A, decatransin, valinomycin, CAD A, kinase inhibiting RNase attenuator 6 (KIRA6), 3-hydroxy-2-naphthoic acid (3HNA), MKC-3946, 4-Phenylbutyric acid (4- PBA), taurine-conjugated ursodeoxycholic acid (TUDCA), olmesartan, N-acetylcysteine (NA
- the mammal can be a human.
- the tissue inflammation can be associated with an autoimmune disease.
- the autoimmune disease can be polymyositis, dermatomyositis, RA, scleroderma, sjogren's syndrome, systemic lupus erythematosus, vasculitis, MCTD, autoimmune hepatitis, psoriasis, or ankylosing spondylitis.
- the autoimmune disease can be RA.
- the tissue inflammation can be associated with IBS.
- the method also can include administering to the mammal an anti-TNF-a therapy.
- the anti- TNF-a therapy can be adalimumab, adalimumab-adbm, adalimumab-adaz, adalimumab-atto, certolizumab pegol, etanercept, etanercept-szzs, infliximab, infliximab-abda, or infliximab- dyyb.
- this document features methods for reducing tissue inflammation in a mammal.
- the methods can include, or consist essentially of, administering, to a mammal, an inhibitor of co-translational translocation, where the method is effective to reduce production of a TNF-a polypeptide within said mammal.
- the inhibitor of co-translational translocation can target a co-translational translocation pathway component that is selected from the group consisting of Sec61 polypeptides, Hsp70 polypeptides, ATPase polypeptides, calmodulin polypeptides, transmembrane domains of newly produced polypeptides, signal peptides, and signal peptidase complex polypeptides.
- the mammal can be a human.
- the tissue inflammation can be associated with an autoimmune disease.
- the autoimmune disease can be polymyositis, dermatomyositis, RA, scleroderma, sjogren's syndrome, systemic lupus erythematosus, vasculitis, MCTD, autoimmune hepatitis, psoriasis, or ankylosing spondylitis.
- the autoimmune disease can be RA.
- the tissue inflammation can be associated with IBS.
- the method also can include administering to the mammal an anti-TNF-a therapy.
- the anti- TNF-a therapy can be adalimumab, adalimumab-adbm, adalimumab-adaz, adalimumab-atto, certolizumab pegol, etanercept, etanercept-szzs, infliximab, infliximab-abda, or infliximab- dyyb.
- this document features methods for reducing tissue inflammation in a mammal.
- the methods can include, or consist essentially of, administering, to a mammal, a composition comprising a mitochondrial metabolite, where the method is effective to reduce production of a TNF-a polypeptide within the mammal.
- the mitochondrial metabolite can be L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, oxaloacetate, or nicotinamide riboside.
- the composition can include L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate.
- the mammal can be a human.
- the tissue inflammation can be associated with an autoimmune disease.
- the autoimmune disease can be polymyositis, dermatomyositis, RA, scleroderma, sjogren's syndrome, systemic lupus erythematosus, vasculitis, MCTD, autoimmune hepatitis, psoriasis, or ankylosing spondylitis.
- the autoimmune disease can be RA.
- the tissue inflammation can be associated with IBS.
- the method also can include administering to the mammal an anti-TNF-a therapy.
- the anti- TNF-a therapy can be adalimumab, adalimumab-adbm, adalimumab-adaz, adalimumab-atto, certolizumab pegol, etanercept, etanercept-szzs, infliximab, infliximab-abda, or infliximab- dyyb.
- this document features methods for reducing tissue inflammation in a mammal.
- the methods can include, or consist essentially of, administering, to a mammal, an inhibitor of aspartate transport or aspartate metabolism, where the method is effective to reduce production of a TNF-a polypeptide within the mammal.
- the inhibitor of aspartate transport or aspartate metabolism can be DL-TBOA, L-(-)-threo-3-hydroxyaspartic acid, L- trans-2,4-PDC, TFB-TBOA, or GOT I inhibitor 2c.
- the mammal can be a human.
- the tissue inflammation can be associated with an autoimmune disease.
- the autoimmune disease can be polymyositis, dermatomyositis, RA, scleroderma, sjogren's syndrome, systemic lupus erythematosus, vasculitis, MCTD, autoimmune hepatitis, psoriasis, or ankylosing spondylitis.
- the autoimmune disease can be RA.
- the tissue inflammation can be associated with IBS.
- the method also can include administering to the mammal an anti-TNF-a therapy.
- the anti- TNF-a therapy can be adalimumab, adalimumab-adbm, adalimumab-adaz, adalimumab-atto, certolizumab pegol, etanercept, etanercept-szzs, infliximab, infliximab-abda, or infliximab- dyyb.
- this document features methods for treating a mammal having tissue inflammation.
- the methods can include, or consist essentially of, administering, to a mammal, a composition comprising cells, where a population of intact mitochondria was introduced into the cells, and where the production of a TNF-a polypeptide within the mammal is reduced following the administering step.
- the composition can include T cells containing the intact mitochondria.
- the T cells are obtained from the mammal.
- the mammal can be a human.
- the tissue inflammation can be associated with an autoimmune disease.
- the autoimmune disease can be polymyositis, dermatomyositis, RA, scleroderma, sjogren's syndrome, systemic lupus erythematosus, vasculitis, MCTD, autoimmune hepatitis, psoriasis, or ankylosing spondylitis.
- the autoimmune disease can be RA.
- the tissue inflammation can be associated with IBS.
- the method also can include administering to the mammal an anti-TNF-a therapy.
- the anti-TNF-a therapy can be adalimumab, adalimumab- adbm, adalimumab-adaz, adalimumab-atto, certolizumab pegol, etanercept, etanercept-szzs, infliximab, infliximab-abda, or infliximab-dyyb.
- this document features methods for treating a mammal having tissue inflammation.
- the methods can include, or consist essentially of, administering, to a mammal, an inhibitor of UPR signaling, where the method is effective to reduce production of a TNF-a polypeptide within the mammal.
- the inhibitor of UPR signaling can target an UPR signaling pathway component selected from the group consisting of BiP polypeptides, IREla polypeptides, PERK polypeptides, ATF6 polypeptides, eIF2a polypeptides, CHOP polypeptides, XBP-1 polypeptides, and XBP-1S polypeptides.
- the inhibitor of UPR signaling can be eeyarestatin I, mycolactone, exotoxin A, NSC 630668-R/l, MAL3-39, MAL3-101, E6 berbamine, ophiobolin A, equisetin, CJ-21058, Rose Bengal, erythrosin B, P97-A4, P87-A4, 17D9, P91-E9, 16F6, bisthiouracil, SCA-21, HUN-7293, cotransin, CAM741, apratoxin A, decatransin, valinomycin, CAD A, kinase inhibiting RNase attenuator 6 (KIRA6), 3-hydroxy-2-naphthoic acid (3HNA), MKC-3946, 4-Phenylbutyric acid (4- PBA), taurine-conjugated ursodeoxycholic acid (TUDCA), olmesartan, N-acetylcysteine (NA
- the mammal can be a human.
- the tissue inflammation can be associated with an autoimmune disease.
- the autoimmune disease can be polymyositis, dermatomyositis, RA, scleroderma, sjogren's syndrome, systemic lupus erythematosus, vasculitis, MCTD, autoimmune hepatitis, psoriasis, or ankylosing spondylitis.
- the autoimmune disease can be RA.
- the tissue inflammation can be associated with IBS.
- the method also can include administering to the mammal an anti-TNF-a therapy.
- the anti- TNF-a therapy can be adalimumab, adalimumab-adbm, adalimumab-adaz, adalimumab-atto, certolizumab pegol, etanercept, etanercept-szzs, infliximab, infliximab-abda, or infliximab- dyyb.
- this document features methods for treating a mammal having tissue inflammation.
- the methods can include, or consist essentially of, administering, to a mammal, an inhibitor of co-translational translocation, where the method is effective to reduce production of a TNF-a polypeptide within the mammal.
- the inhibitor of co- translational translocation can target a co-translational translocation pathway component that is selected from the group consisting of Sec61 polypeptides, Hsp70 polypeptides, ATPase polypeptides, calmodulin polypeptides, transmembrane domains of newly produced polypeptides, signal peptides, and signal peptidase complex polypeptides.
- the mammal can be a human.
- the tissue inflammation can be associated with an autoimmune disease.
- the autoimmune disease can be polymyositis, dermatomyositis, RA, scleroderma, sjogren's syndrome, systemic lupus erythematosus, vasculitis, MCTD, autoimmune hepatitis, psoriasis, or ankylosing spondylitis.
- the autoimmune disease can be RA.
- the tissue inflammation can be associated with IBS.
- the method also can include administering to the mammal an anti- TNF-a therapy.
- the anti-TNF-a therapy can be adalimumab, adalimumab-adbm, adalimumab-adaz, adalimumab-atto, certolizumab pegol, etanercept, etanercept-szzs, infliximab, infliximab-abda, or infliximab-dyyb.
- this document features methods for treating a mammal having tissue inflammation.
- the methods can include, or consist essentially of, administering, to a mammal, a composition comprising a mitochondrial metabolite, where the method is effective to reduce production of a TNF-a polypeptide within the mammal.
- the mitochondrial metabolite can be L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, oxaloacetate, or nicotinamide riboside.
- the composition can include L-aspartate, D- aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate.
- the mammal can be a human.
- the tissue inflammation can be associated with an autoimmune disease.
- the autoimmune disease can be polymyositis, dermatomyositis, RA, scleroderma, sjogren's syndrome, systemic lupus erythematosus, vasculitis, MCTD, autoimmune hepatitis, psoriasis, or ankylosing spondylitis.
- the autoimmune disease can be RA.
- the tissue inflammation can be associated with IBS.
- the method also can include administering to the mammal an anti- TNF-a therapy.
- the anti-TNF-a therapy can be adalimumab, adalimumab-adbm, adalimumab-adaz, adalimumab-atto, certolizumab pegol, etanercept, etanercept-szzs, infliximab, infliximab-abda, or infliximab-dyyb.
- this document features methods for treating a mammal having tissue inflammation.
- the methods can include, or consist essentially of, administering, to a mammal, an inhibitor of aspartate transport or aspartate metabolism, where the method is effective to reduce production of a TNF-a polypeptide within the mammal.
- the inhibitor of aspartate transport or aspartate metabolism can be DL-TBOA, L-(-)-threo-3-hydroxyaspartic acid, L-trans-2,4-PDC, TFB-TBOA, or GOT1 inhibitor 2c.
- the mammal can be a human.
- the tissue inflammation can be associated with an autoimmune disease.
- the autoimmune disease can be polymyositis, dermatomyositis, RA, scleroderma, sjogren's syndrome, systemic lupus erythematosus, vasculitis, MCTD, autoimmune hepatitis, psoriasis, or ankylosing spondylitis.
- the autoimmune disease can be RA.
- the tissue inflammation can be associated with IBS.
- the method also can include administering to the mammal an anti- TNF-a therapy.
- the anti-TNF-a therapy can be adalimumab, adalimumab-adbm, adalimumab-adaz, adalimumab-atto, certolizumab pegol, etanercept, etanercept-szzs, infliximab, infliximab-abda, or infliximab-dyyb.
- this document features methods for treating a mammal having tissue inflammation.
- the methods can include, or consist essentially of, administering, to a mammal, a) a composition comprising aspartate, and b) adalimumab; where the method is effective to reduce production of a TNF-a polypeptide within the mammal.
- the composition comprising aspartate and the adalimumab can be administered together.
- the composition comprising aspartate and said adalimumab are administered separately.
- the mammal can be a human.
- composition comprising aspartate also can include a mitochondrial metabolite selected from the group consisting of NAD, alpha-ketobutyrate, malate, oxaloacetate, and nicotinamide riboside.
- the composition comprising aspartate can include L-aspartate, D- aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate.
- this document features methods for treating a mammal having tissue inflammation.
- the methods can include, or consist essentially of, administering, to said mammal, a) a composition comprising aspartate, and b) etanercept; where the method is effective to reduce production of a TNF-a polypeptide within the mammal.
- the composition comprising aspartate and the etanercept can be administered together.
- the composition comprising aspartate and the etanercept can be administered separately.
- the mammal can be a human.
- composition comprising aspartate also can include a mitochondrial metabolite selected from the group consisting of NAD, alpha-ketobutyrate, malate, oxaloacetate, and nicotinamide riboside.
- the composition comprising aspartate can include L-aspartate, D- aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate.
- this document features methods for treating a mammal having tissue inflammation .
- the methods can include, or consist essentially of, administering, to a mammal, a) a composition comprising aspartate, and b) infliximab; where the method is effective to reduce production of a TNF-a polypeptide within the mammal.
- the composition comprising aspartate and the infliximab can be administered together.
- the composition comprising aspartate and the infliximab can be administered separately.
- the mammal can be a human.
- composition comprising aspartate also can include a mitochondrial metabolite selected from the group consisting of NAD, alpha-ketobutyrate, malate, oxaloacetate, and nicotinamide riboside.
- the composition comprising aspartate can include L-aspartate, D- aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate.
- Figure 1 A) Mitochondrial oxygen consumption rates (OCR) measured by Seahorse Analyzer. n 6 each.
- Figure IE Confocal microscopy imaging of the ER enzyme protein disulfide isomerase (PDI) and mitochondrial membrane potential (MitoTracker Red).
- PDI protein disulfide isomerase
- MitoTracker Red mitochondrial membrane potential
- Scale bar 10 pm.
- Figure IF Correlation of ER size (ER Tracker intensity) and mitochondrial membrane potential in cells gated based on cell size.
- Figures 1G - II Expanded ER size in RA T cells.
- Figures 1H and II Confocal microscopy imaging of the ER chaperon protein calnexin in RA and healthy T cells. Scale bar, 10 pm. Single cell calnexin intensity quantification (50 T cells from 5 controls and 5 patients).
- Figures 1J - IN ER stress response and ER expansion in T cells with low mitochondrial activity.
- Figure IL Representative transmission electron microscope image of healthy and RA T cells. Arrows indicate ER. Scale bar, 1 pm.
- Figures IQ - IS Mitochondrial transfer corrects ER size.
- Figure IQ Experimental scheme.
- FIGs 2A - 20 Mitochondria-derived aspartate controls ER size.
- Naive CD4 + CD45RA + T cells from patients with RA, and age-matched healthy individuals were stimulated for 72 hours.
- Figure 2A Mitochondrial intermediates determine ER size.
- Figure 2B Scheme for the malate/aspartate shuttle.
- Figure 2C and 2D RA T cells are aspartate/oxaloacetate deficient.
- Intracellular aspartate ( Figure 2C) and oxaloacetate (Figure 2D) concentrations in healthy and RA T cells each dot indicates one sample from a healthy control or a RA patient.
- Figures 2E and 2F Aspartate inhibits ER stress signals and Phosphatidylcholine synthesis.
- RA T cells were treated with aspartate for 3 days.
- ER stress gene expression ( Figure 2E) and Phosphatidylcholine content (Figure 2F) were measured.
- Figures 2G and 2H) Aspartate concentrations depend on intact mitochondrial function.
- Figures 21 - 2K Glutamic-Oxaloacetic Transaminase 2 (GOT2) regulates ER size. GOT2 was knocked down by si-RNA in healthy T cells.
- Synovitis was induced in chimeric mice engrafted with human synovial tissue and immunoreconstituted with RA PBMC. Chimeras were treated with vehicle or aspartate i.p. 5 mg/kg.
- Figure 2L H&E staining of explanted synovial tissue.
- Figure 2M Tissue transcriptomic analysis (qPCR) of synovial explants. Shown are data for T cell receptor (TRB) transcripts and the lineage-determining transcription factors TBX21 and RORC.
- Figure 2N Co-immunofluorescence staining for IFN-y-producing CD3 + T cells in the synovial tissue. Representative images; scale bar, 10 pm.
- Figure 20 Tissue transcriptomic analysis of key inflammatory cytokines.
- Figures 3 A - 30 Aspartate is required for NAD regeneration and ADP-ribosylation of BiP.
- Figures 3D and 3F Aspartate and intact mitochondria regenerate NAD + .
- Figure 3D) NAD+/NADH ratios in RA T cells treated with or without aspartate, n 4.
- Figures 3G and 3H Representative histograms. ER size measurements from 4 experiments.
- Figure 3K) ADP-ribosylation of BiP in healthy and RA T cells, n 3.
- Figure 3L) ADP-ribosylation of BiP in healthy CD4 T cells treated with or without Rotenone (10 nM) for 24 hours. n 3.
- FIGS 4A - 4R T cells rich in rough ER are TNF-a-super producers. Naive CD4 + CD45RA + T cells from RA patients and age-matched controls were stimulated for 72 hours.
- Figures 4A - 4E Enrichment of rough ER in RA T cells.
- Figures 4 A and 4B Immunoblot analysis of the ER chaperon protein calnexin and the ribosomal proteins L-17 and S-7 in CD4 + T cells from 5 healthy individuals and 6 RA patients.
- Figures 4C and 4D Rough ER was isolated from healthy and RA T cells 2 hours after restimulation.
- Figure 4G T cell stimulation induces enrichment of ER-bound mRNA for secretory proteins.
- FIG 4J Flow cytometric measurement of intracellular TNF-a in CD4 + T cells from RA patients and healthy individuals before and after PMA/ION stimulation.
- Figure 4K TNF-a secreted into the extracellular space by unstimulated and stimulated RA and control CD4 + T cells.
- Figures 4L - 4P Mitochondrial function and aspartate control TNF-a production.
- Figure 4N GOT2 knockdown in healthy T cells, combined with or without aspartate rescue.
- Figure 4R Scheme showing the aspartate-NAD-BiP pathway controlling TNF-a secretion. All data are mean ⁇ SEM. Unpaired Mann- Whitney-Wilcoxon rank test ( Figures 4A - 4K). Paired t test ( Figures 4M - 4Q). *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001.
- FIGS 5 A - 5N TNF-a-producing CD4 T cells function as key arthritogenic effector cells.
- Figure 5 A and 5E Cellular composition of leucocyte-rich and leukocyte-poor tissues collected from patients with rheumatoid synovitis.
- Figures 5B - 5D and 5F - 5H TNF-a is a product of tissue T cells. Flow cytometric analysis of intracellular TNF-a in T cells, B cells and macrophages after stimulation with LPS/PMA/ION/BFA for 4 hours.
- Figures 5B and 5F Histogram of TNF-a staining.
- Figures 5C and 5G Frequencies of TNF- a-producing cell populations.
- FIGS 5D and 5H MFI of TNF-a staining in different cell populations.
- Figures 51 - 5K Spontaneous TNF-a production in T cells and macrophages residing in the synovium. Freshly harvested synovial tissue from RA patients was incubated with or without the secretion inhibitor BFA for 4 hours, before cells were dissociated from the tissue and intracellular TNF-a was detected by flow cytometry. TNF-a + CD45 + CD68 + macrophages ( Figure 51) and TNF-a + CD45 + CD3 + T cells (Figure 5J) in synovial tissue before and after BFA treatment.
- Figure 5K Fold change in the frequency of TNF-a + macrophage and TNF-a + T cells after BFA treatment.
- TNF-a- producing CD4 + T cells are an absolute requirement for rheumatoid synovitis.
- Rheumatoid synovitis was induced in human synovial tissues engrafted into NSG mice.
- CD4 + T cells from RA patients were transfected with control or TNFA siRNA and adoptively transferred into the chimeric mice. Synovial grafts were explanted two weeks later. 8 tissues in each group.
- Figure 5L H&E staining of explanted synovial tissues.
- Figure 5M Immunofluorescence staining of CD3 + T cells in synovial infiltrates. Scale bar; 10 pm.
- Figure 5N Synovial tissue transcriptome for TRB, TBTT'T. RORG and other key inflammatory markers. All data are mean ⁇ SEM. Paired t test ( Figure 5K). Unpaired Mann-Whitney-Wilcoxon rank test ( Figure 5N). *P ⁇ 0.05, **P ⁇ 0.01, ***p ⁇ 0.001.
- FIG. 7 Gating strategy to analyze ER and mitochondrial function on the single cell level.
- CD4 + CD45RA + T cells were stimulated for 72 hours.
- ER biomass was determined with ER tracker and mitochondrial function was assessed with the mitochondrial membrane potential.
- Gate #1 small cellular size.
- Gate #2 medium cellular size.
- Gate #3 large cellular size.
- FIG. 1 ER size in T cells from patients with psoriatic arthritis (PsA).
- CD4 + CD45RA + T cells were isolated from patients with PsA and age-matched healthy controls and stimulated for 72 hours.
- Flow cytometric quantification of ER size (ER Tracker MFI) in n 4 control-patient pairs. Data are mean ⁇ SEM. Unpaired Mann- Whitney- Wilcoxon rank test.
- Figures 9 A - 9E XBP1S overexpression induces ER expansion. Healthy CD4 + T cells were stimulated and transfected with control or XBP1S overexpression plasmid before the ER size was determined.
- Figure 9C Confocal microscopy imaging of the ER protein calnexin.
- FIGs 11 A - 11C Mitochondria transfer efficiency in Jurkat T cells.
- Figure 11 A Experimental scheme. In Jurkat T cells, mitochondria were labeled with MitoTrackerRed and isolated. Mitochondria from donor cells were transferred into recipient cells.
- Figure 1 IB Flow cytometric analysis of MitoTrackerRed intensity after mitochondria transfer. Ratio indicates donor cell number/recipient cell number.
- Figure 11C Confocal imaging of exogenous mitochondria transferred into Jurkat T cells.
- FIG. 12 Expression of the Glutamic-Oxaloacetic Transaminases (GOT) 1 and 2 in healthy and RA T cells.
- GOT Glutamic-Oxaloacetic Transaminases
- Peripheral blood CD4 + CD45RA + T cells from RA patients and age- matched healthy individuals were isolated and stimulated for 72 hours.
- mRNA levels of GOT1 and GOT2 were determined by qPCR.
- n 4 in each group. All data are mean ⁇ SEM. Unpaired Mann- Whitney-Wilcoxon rank test.
- FIGs 14A - 14B Asparagine does not affect ER size and TNF-a production.
- Naive CD4 + T cells from RA patients were activated for 72 hours in the presence of Aspartate (1 mM) or Asparagine (1 mM).
- Figure 14 A) ER size was determined flow cytometrically with ER tracker (n 6).
- FIGS 15 A - 15B Pyruvate and a-ketobutyrate (a-KB) inhibit ER expansion and TNF-a production in RA T cells.
- FIG. 16A Naive CD4 + T cells were purified from peripheral blood mononuclear cells and stimulated with anti-CD3/CD28 for 72 hours. The rough ER was isolated by calcium precipitation and the isolate was immunoblotted for the ER protein calnexin, the ribosomal protein LI 7 and the cytoplasmic protein a-actin.
- Figure 16B Healthy CD4 + T cells were activated with PMA/Ionomycin for 2 hours before isolation of the rough ER and immunoblotting of the ER protein calnexin, the ribosomal protein S7 and the cytosolic protein P-actin.
- FIGs 17A - 17B TNF-a production is unaffected by Tunicamycin.
- Naive CD4 + T cells were purified from peripheral blood mononuclear cells and stimulated with anti- CD3/CD28 beads for 72 hours in the presence of the ER stress inducer Tunicamycin.
- Figures 18A - 18B Scheme for in vivo experiments.
- Figure 18A NSG mice were engrafted with human synovial tissue and reconstituted with RA PBMC. Before the transfer, CD4 T cells were sorted and transfected with control or TNFA siRNA.
- Figure 18B NSG mice were engrafted with human synovial tissue and reconstituted with RA PBMC. Before PBMC reconstitution, CD4 T cells were sorted and transferred with mitochondria isolated from healthy T cells.
- FIGs 19A - 19C Mitochondria transfer into CD4 + T cells protects synovial tissue from inflammation. Mitochondria were isolated from healthy T cells and transferred into RA CD4 + T cells prior to their adoptive transfer into synovium-NSG chimeras. Explanted synovial grafts were analyzed by immunohistochemical staining and tissue transcriptomics (RT-PCR). 8 tissues in each group.
- Figure 19A H&E staining of synovial tissue sections.
- Figure 19B Immunofluorescence staining for CD3 + T cells in synovial infiltrates. Representative images. Scale bar; 10 pm.
- Figure 19C Gene expression profiling (RT-PCR) of TRB, TBET, RORG and other key inflammatory markers. All data are mean ⁇ SEM. Unpaired Mann- Whitney-Wilcoxon rank test. *P ⁇ 0.05, **P ⁇ 0.01, ***p ⁇ 0.001.
- Figures 20A - 20B Knockdown efficiency of TNFA and GOT2.
- Figure 21 Chemical structure of exemplary inhibitors of co-translational translocation including natural and synthetic inhibitors of co-translational translocation.
- tissue inflammation e.g., autoimmune tissue inflammation
- one or more inhibitors of TNF-a polypeptide production can be administered to a mammal (e.g, a human) having tissue inflammation (e.g, autoimmune tissue inflammation) to treat the mammal.
- tissue inflammation e.g., autoimmune tissue inflammation
- intact mitochondria can be administered to a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation) to treat the mammal (e.g., to reduce TNF-a polypeptide production by, for example, T cells and treat the mammal).
- one or more inhibitors of unfolded protein response (UPR) signaling can be administered to a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation) to treat the mammal (e.g., to reduce TNF-a polypeptide production by, for example, T cells and treat the mammal).
- tissue inflammation e.g., autoimmune tissue inflammation
- one or more inhibitors for co- translational translocation can be administered to a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation) to treat the mammal (e.g., to reduce TNF-a polypeptide production by, for example, T cells and treat the mammal).
- tissue inflammation e.g., autoimmune tissue inflammation
- one or more inhibitors for co- translational translocation can be administered to a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation) to treat the mammal (e.g
- one or more mitochondrial metabolites can be administered to a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation) to treat the mammal (e.g., to reduce TNF-a polypeptide production by, for example, T cells and treat the mammal).
- tissue inflammation e.g., autoimmune tissue inflammation
- one or more inhibitors of aspartate transport and/or aspartate metabolism can be administered to a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation) to treat the mammal (e.g., to reduce TNF-a polypeptide production by, for example, T cells and treat the mammal).
- one or more (e.g., one, two, three, four, or more) inhibitors of TNF-a polypeptide production can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having tissue inflammation such as autoimmune tissue inflammation) to reduce tissue inflammation present within a mammal.
- a mammal e.g., a human
- tissue inflammation e.g., autoimmune tissue inflammation
- the materials and methods described herein can be used to reduce tissue inflammation present within a mammal having tissue inflammation (e.g., autoimmune tissue inflammation) by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.
- one or more (e.g., one, two, three, four, or more) inhibitors of TNF-a polypeptide production can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having tissue inflammation such as autoimmune tissue inflammation) to reduce one or more symptoms of tissue inflammation (e.g., autoimmune tissue inflammation).
- tissue inflammation e.g., autoimmune tissue inflammation
- symptoms of tissue inflammation include, without limitation, tender joints, warm joints, swollen joints, joint stiffness, fatigue, fever, and loss of appetite.
- tissue inflammation e.g., autoimmune tissue inflammation
- tissue inflammation e.g., autoimmune tissue inflammation
- a mammal having tissue inflammation e.g., autoimmune tissue inflammation
- tissue inflammation e.g., autoimmune tissue inflammation
- one or more (e.g., one, two, three, four, or more) inhibitors of TNF-a polypeptide production can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having tissue inflammation such as autoimmune tissue inflammation) to reduce a level of TNF-a polypeptides within the mammal.
- a mammal e.g., a human
- tissue inflammation e.g., autoimmune tissue inflammation
- the materials and methods described herein can be used to reduce a level of TNF-a polypeptides within a mammal having tissue inflammation (e.g., autoimmune tissue inflammation) by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.
- one or more (e.g., one, two, three, four, or more) inhibitors of TNF-a polypeptide production can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having tissue inflammation such as autoimmune tissue inflammation) to restore aspartate homeostasis within the mammal.
- a mammal e.g., a human
- tissue inflammation such as autoimmune tissue inflammation
- the materials and methods described herein can be used to restore a level of aspartate in cells within a mammal having tissue inflammation (e.g., autoimmune tissue inflammation) to from about 3 nmol/10 6 cells to about 10 nmol/10 6 (e.g., from about 3 nmol/10 6 cells to about 9 nmol/10 6 , from about 3 nmol/10 6 cells to about 8 nmol/10 6 , from about 3 nmol/10 6 cells to about 7 nmol/10 6 , from about 3 nmol/10 6 cells to about 6 nmol/10 6 , from about 3 nmol/10 6 cells to about 5 nmol/10 6 , from about 3 nmol/10 6 cells to about 4 nmol/10 6 , from about 4 nmol/10 6 cells to about 10 nmol/10 6 , from about 5 nmol/10 6 cells to about 10 nmol/10 6 , from about 6 nmol/10 6 cells to about 10 nmol/10 6 , from about 7 nmol/10 6 cells to about 10 nmol/10 6
- one or more (e.g., one, two, three, four, or more) inhibitors of TNF-a polypeptide production can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having tissue inflammation such as autoimmune tissue inflammation) to restore NAD + /NADH balance within the mammal.
- a mammal e.g., a human
- tissue inflammation such as autoimmune tissue inflammation
- the materials and methods described herein can be used to restore NAD + /NADH balance within a mammal having tissue inflammation (e.g., autoimmune tissue inflammation) to from about 4 NAD + /NADH to about 8 NAD + /NADH (e.g., from about 4 NAD + /NADH to about 7 NAD + /NADH, from about 4 NAD + /NADH to about 6 NAD + /NADH, from about 4 NAD + /NADH to about 5 NAD + /NADH, from about 5 NAD + /NADH to about 8 NAD + /NADH, from about 6 NAD + /NADH to about 8 NAD + /NADH, from about 7 NAD + /NADH to about 8 NAD + /NADH, from about 5 NAD7N ADH to about 6 NAD7N ADH, or from about 6 NAD7NADH to about 7 NAD7NADH).
- tissue inflammation e.g., autoimmune tissue inflammation
- one or more (e.g., one, two, three, four, or more) inhibitors of TNF-a polypeptide production can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having tissue inflammation such as autoimmune tissue inflammation) to restore phosphatidylcholine levels in cells within the mammal.
- a mammal e.g., a human
- tissue inflammation such as autoimmune tissue inflammation
- the materials and methods described herein can be used to restore phosphatidylcholine levels in cells within a mammal having tissue inflammation (e.g., autoimmune tissue inflammation) to from about 5 nmol/10 6 cells to about 10 nmol/10 6 cells (e.g., from about 5 nmol/10 6 cells to about 9 nmol/10 6 cells, from about 5 nmol/10 6 cells to about 8 nmol/10 6 cells, from about 5 nmol/10 6 cells to about 7 nmol/10 6 cells, from about 5 nmol/10 6 cells to about 6 nmol/10 6 cells, from about 6 nmol/10 6 cells to about 10 nmol/10 6 cells, from about 7 nmol/10 6 cells to about 10 nmol/10 6 cells, from about 8 nmol/10 6 cells to about 10 nmol/10 6 cells, from about 9 nmol/10 6 cells to about 10 nmol/10 6 cells, from about 6 nmol/10 6 cells to about 9 nmol/10 6 cells, from about 7 nmol/10 6 cells to about 8
- tissue inflammation e.g., autoimmune tissue inflammation
- mammals that can have tissue inflammation include, without limitation, humans, non-human primates (e.g., monkeys), dogs, cats, horses, cows, pigs, sheep, mice, and rats.
- a human having tissue inflammation e.g., autoimmune tissue inflammation
- tissue inflammation e.g., autoimmune tissue inflammation
- tissue inflammation can be associated with a disease.
- tissue inflammation can be associated with an autoimmune disease (e.g., autoimmune tissue inflammation).
- tissue inflammation can be induced by one or more medications.
- tissue inflammation examples include, without limitation, polymyositis, dermatomyositis, RA, scleroderma, sjogren’s syndrome, systemic lupus erythematosus, vasculitis, mixed connective tissue disease (MCTD), autoimmune hepatitis, irritable bowel syndrome (IBS), psoriasis, cancer immunotherapy induced inflammation, uveitis, and ankylosing spondylitis.
- tissue inflammation e.g., autoimmune tissue inflammation
- the tissue inflammation can be present in any appropriate tissue within the mammal.
- tissues that can be inflamed in association with a disease include, without limitation, synovial tissue, connective tissue, gastrointestinal tract (bowel) tissue, lung tissue, nervous system tissue, skin tissue, eye tissue, ear tissue, and blood vessels.
- the methods described herein can include identifying a mammal (e.g., a human) as having tissue inflammation (e.g., autoimmune tissue inflammation). Any appropriate method can be used to identify a mammal as having tissue inflammation (e.g., autoimmune tissue inflammation).
- tissue inflammation e.g., autoimmune tissue inflammation
- physical examinations e.g., to check joints for swelling, redness, and/or warmth or to check reflexes and/or muscle strength
- blood tests e.g., to determine erythrocyte sedimentation rates (ESR) and/or to detect C-reactive protein (CRP) levels, rheumatoid factors, and/or anti-cyclic citrullinated peptide (anti-CCP) antibodies
- imaging tests e.g., X-rays, magnetic resonance imaging (MRI), and ultrasound tests
- laboratory tests e.g., to check for autoantibodies and/or genetic markers, and/or for metabolomics
- mammals e.g., humans
- tissue inflammation e.g., autoimmune tissue inflammation
- a mammal e.g., a human having tissue inflammation (e.g., autoimmune tissue inflammation) can be administered or instructed to self-administer any or more (e.g., one, two, three, four, or more) inhibitors of TNF-a polypeptide production (e.g., inhibitors of TNF-a polypeptide production by T cells).
- the mammal can be administered or instructed to self-administer any one or more agents that can inhibit TNF- a polypeptide expression.
- nucleic acid molecules designed to induce RNA interference of TNF-a polypeptide expression e.g., siRNA molecules and shRNA molecules
- the mammal when treating a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation), the mammal can be administered or instructed to self-administer mitochondria (e.g., intact mitochondria).
- mitochondria e.g., intact mitochondria
- intact mitochondria e.g., a composition including intact mitochondria
- tissue inflammation e.g., autoimmune tissue inflammation
- treat the mammal e.g., to reduce TNF-a polypeptide production by, for example, T cells and treat the mammal.
- Mitochondria e.g., intact mitochondria
- a mammal e.g., a human
- tissue inflammation e.g., autoimmune tissue inflammation
- mitochondria can be obtained from any type of cell.
- cells that mitochondria can be obtained from include, without limitation, T cells, cord blood cells, myeloid cells, stromal cells, and cell lines.
- Mitochondria e.g., intact mitochondria
- mitochondria can be obtained from any appropriate source.
- mitochondria can be obtained from the mammal to be treated as described herein.
- mitochondria can be obtained from a donor mammal.
- mitochondria can be obtained as described in Example 1.
- mitochondria can be administered to a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation) by administration directly into the inflamed tissue.
- tissue inflammation e.g., autoimmune tissue inflammation
- Examples of tissues that can be inflamed and can be loaded with mitochondria include, without limitation, joint tissue, muscle, nervous tissue, skin tissue, connective tissue, and bone marrow.
- mitochondria can be administered to a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation) using cells loaded with the exogenously added intact mitochondria.
- cells containing (e.g., cells engineered to contain) mitochondria that are exogenous to those cells can be administered to a mammal (e.g., a human) having tissue inflammation to treat the mammal.
- a cell can be engineered to contain (e.g., can be loaded with) mitochondria that are exogenous to that cell.
- Any appropriate cell can be loaded with mitochondria. Examples of cells that can be loaded with mitochondria include, without limitation, T cells, monocytes, macrophages, B cells, endothelial cells, stromal cells, and cells within bone marrow. Any appropriate method can be used to load a cell with mitochondria.
- a cell can be loaded with mitochondria as described in Example 1.
- T cells can be obtained from a mammal (e.g., a human) to be treated as described herein, the T cells can be loaded with intact mitochondria obtained from, e.g., healthy cells from that mammal or another mammal of the same species, and the mitochondria-loaded T cells can be administered to the mammal.
- cells other than T cells can be obtained from a mammal (e.g., a human) to be treated and mitochondria can be obtained from those cells, T cells can be obtained from the mammal, the T cells can be loaded with the obtained mitochondria, and the mitochondria-loaded T cells can be administered to the mammal.
- the mammal when treating a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation), the mammal can be administered or instructed to self-administer one or more inhibitors of UPR signaling.
- one or more inhibitors of UPR signaling e.g., a composition including one or more inhibitors of UPR signaling
- tissue inflammation e.g., autoimmune tissue inflammation
- the mammal e.g., to reduce TNF-a polypeptide production by, for example, T cells and treat the mammal.
- An inhibitor of UPR signaling can be any type of molecule (e.g., small molecules, polypeptides such as antibodies, and nucleic acids such as DNA, RNA, or DNA/RNA hybrids).
- an inhibitor of UPR signaling can target any appropriate component within the UPR signaling pathway.
- components within the UPR signaling pathway that can be targeted by an inhibitor of UPR signaling include, without limitation, BiP polypeptides, IRE la polypeptides, PERK polypeptides, ATF6 polypeptides, eIF2a polypeptides, CHOP polypeptides, XBP-1 polypeptides, and XBP-1S polypeptides.
- an inhibitor of UPR signaling can target any appropriate component within the co-translational translocation pathway.
- components within the co-translational translocation pathway that can be targeted by an inhibitor of UPR signaling include, without limitation, Sec61 polypeptides, Hsp70 polypeptides, ATPase polypeptides, calmodulin polypeptides, transmembrane domains of newly produced polypeptides, signal peptides, and signal peptidase complex polypeptides.
- an inhibitor of UPR signaling can be an inhibitor of ER stress.
- inhibitors of UPR signaling include, without limitation, eeyarestatin I (3-(4-Chlorophenyl)-4- [ [(4-chlorophenyl)amino]carbonyl] hydroxyamino] -5 , 5 -dimethyl-2-oxo- 1 -imidazolidineacetic acid 2-[3-(5-nitro-2-furanyl)-2-propen-l-ylidene]hydrazide), mycolactone((6S,7S,9E,12R)- 12-[(2S,4E,6R,7R,9R)-7,9-Dihydroxy-4,6-dimethyldec-4-en-2-yl]-7,9-dimethyl-2-oxo-l- oxacy clododec-9-en-6-yl (2E,4E,6E, 8E, 10E, 12S,13S,15S)-12,13,15 -trihydroxy-4, 6, 10- trimethylhexa
- the mammal when treating a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation), the mammal can be administered or instructed to self-administer any one or more metabolites (e.g., mitochondrial metabolites).
- one or more mitochondrial metabolites e.g., a composition including one or more mitochondrial metabolites
- a mammal e.g., a human
- tissue inflammation e.g., autoimmune tissue inflammation
- treat the mammal e.g., to reduce TNF-a polypeptide production by, for example, T cells and treat the mammal.
- Any appropriate metabolite or combination of metabolites can be administered to a mammal (e.g., a human) as described herein.
- a mammal e.g., a human
- metabolites that can be administered to a mammal (e.g., a human) as described herein include, without limitation, aspartate (e.g., L- aspartate and D-aspartate), NAD, alpha-ketobutyrate, malate, oxaloacetate, and nicotinamide riboside.
- L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate can be administered to a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation) to treat the mammal.
- tissue inflammation e.g., autoimmune tissue inflammation
- Any appropriate amount of a metabolite can be administered to a mammal (e.g., a human) as described herein.
- a composition including from about 10 mg to about 3000 mg (e.g., from about 10 mg to about 2500 mg, from about 10 mg to about 2000 mg, from about 10 mg to about 1500 mg, from about 10 mg to about 1000 mg, from about 10 mg to about 800 mg, from about 10 mg to about 500 mg, from about 10 mg to about 200 mg, from about 10 mg to about 100 mg, from about 100 mg to about 3000 mg, from about 300 mg to about 3000 mg, from about 500 mg to about 3000 mg, from about 700 mg to about 3000 mg, from about 1000 mg to about 3000 mg, from about 1500 mg to about 3000 mg, from about 2000 mg to about 3000 mg, from about 2500 mg to about 3000 mg, from about 100 mg to about 2500 mg, from about 500 mg to about 2000 mg, from about 1000 mg to about 1500 mg, from about 500 mg to about 1500 mg, or from about 1500 mg to about 1500 mg) of a metabolite can be administered to a mammal (e.g., a human) having tissue inflammation (e.g.
- a composition including from about 10 mg to about 3000 mg of (e.g., e.g., from about 10 mg to about 2500 mg, from about 10 mg to about 2000 mg, from about 10 mg to about 1500 mg, from about 10 mg to about 1000 mg, from about 10 mg to about 800 mg, from about 10 mg to about 500 mg, from about 10 mg to about 200 mg, from about 10 mg to about 100 mg, from about 100 mg to about 3000 mg, from about 300 mg to about 3000 mg, from about 500 mg to about 3000 mg, from about 700 mg to about 3000 mg, from about 1000 mg to about 3000 mg, from about 1500 mg to about 3000 mg, from about 2000 mg to about 3000 mg, from about 2500 mg to about 3000 mg, from about 100 mg to about 2500 mg, from about 500 mg to about 2000 mg, from about 1000 mg to about 1500 mg, from about 500 mg to about 1500 mg, or from about 1500 mg to about 1500 mg) D-aspartate can be administered to a mammal (e.g., a human)
- a composition including from about 10 mg to about 1000 mg (e.g., from about 10 mg to about 800 mg, from about 10 mg to about 500 mg, from about 10 mg to about 300 mg, from about 10 mg to about 100 mg, from about 10 mg to about 50 mg, from about 50 mg to about 1000 mg, from about 100 mg to about 1000 mg, from about 500 mg to about 1000 mg, from about 700 mg to about 1000 mg, from about 100 mg to about 800 mg, from about 300 mg to about 500 mg, from about 100 mg to about 300 mg, or from about 500 mg to about 700 mg) of NAD can be administered to a mammal (e.g., a human) having tissue inflammation to treat the mammal.
- a mammal e.g., a human
- a mammal e.g., a human
- the mammal when treating a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation), the mammal can be administered or instructed to self-administer any one or more inhibitors of aspartate transport and/or aspartate metabolism.
- tissue inflammation e.g., autoimmune tissue inflammation
- one or more inhibitors of aspartate transport and/or aspartate metabolism can be administered to a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation) to treat the mammal (e.g., to reduce TNF-a polypeptide production by, for example, T cells and treat the mammal).
- An inhibitor of aspartate transport and/or aspartate metabolism can be any type of molecule (e.g., small molecules, polypeptides such as antibodies, and nucleic acids such as DNA, RNA, or DNA/RNA hybrids).
- an inhibitor of aspartate transport and/or aspartate metabolism can inhibit a GOT1 polypeptide.
- an inhibitor of aspartate transport and/or aspartate metabolism can agonize a GOT2 polypeptide.
- an inhibitor of aspartate transport and/or aspartate metabolism can inhibit an EAAT1 polypeptide.
- inhibitors of aspartate transport and/or aspartate metabolism include, without limitation, DL-TBOA (dl-threo-P- benzyloxyaspartate), L-(-)-threo-3-hydroxyaspartic acid, L-trans-2,4-PDC (L-trans- Pyrrolidine-2,4-dicarboxylic acid), TFB-TBOA ((3S)-3-[[3-[[4- (Trifluoromethyl)benzoyl]amino]phenyl]methoxy]-L-aspartic acid), and GOT1 inhibitor 2c ( 1 -piperazinecarboxamide, n-(4-chlorophenyl)-4-(l h-indol-4-yl)-) .
- one or more (e.g., one, two, three, four, or more) inhibitors of TNF-a polypeptide production can be formulated into a composition (e.g., a pharmaceutically acceptable composition) for administration to a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation).
- a composition e.g., a pharmaceutically acceptable composition
- tissue inflammation e.g., autoimmune tissue inflammation
- one or more inhibitors of TNF-a polypeptide production can be formulated together with one or more pharmaceutically acceptable carriers (additives), excipients, and/or diluents.
- Examples of pharmaceutically acceptable carriers, excipients, and diluents that can be used in a composition described herein include, without limitation, sucrose, lactose, starch (e.g., starch glycolate), cellulose, cellulose derivatives (e.g., modified celluloses such as microcrystalline cellulose, and cellulose ethers like hydroxypropyl cellulose (HPC) and cellulose ether hydroxypropyl methylcellulose (HPMC)), xylitol, sorbitol, mannitol, gelatin, polymers (e.g., polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), crosslinked polyvinylpyrrolidone (crospovidone), carboxymethyl cellulose, polyethylene-polyoxypropylene-block polymers, and crosslinked sodium carboxymethyl cellulose (croscarmellose sodium)), titanium oxide, azo dyes, silica gel, fumed silica, talc, magnesium carbonate, vegetable stearin
- compositions containing one or more inhibitors of TNF-a polypeptide production e.g., inhibitors of TNF-a polypeptide production by T cells
- a mammal e.g., a human
- tissue inflammation e.g., autoimmune tissue inflammation
- the composition can be designed for oral or parenteral (including, without limitation, a subcutaneous, intravenous, intramuscular, intradermal, transdermal, intrathecal, or intraperitoneal (i.p.)) administration to the mammal.
- compositions suitable for oral administration include, without limitation, liquids, tablets, capsules, pills, powders, gels, and granules.
- compositions suitable for parenteral administration include, without limitation, aqueous and non-aqueous sterile injection solutions that can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient.
- a composition e.g., a pharmaceutically acceptable composition
- tissue inflammation e.g., autoimmune tissue inflammation
- the composition can deliver the one or more inhibitors of TNF-a polypeptide production in any manner.
- a composition including inhibitors of TNF-a polypeptide production can be an immediate release composition (e.g., an immediate release oral dosage form).
- a composition including one or more inhibitors of TNF-a polypeptide production can be a controlled (e.g., delayed and/or sustained) release compositions (e.g., a controlled release oral dosage form).
- a controlled release composition including one or more inhibitors of TNF-a polypeptide production can be designed to release the one or more inhibitors of TNF-a polypeptide production over 1, 2, 4, 7, or 10 days.
- a composition containing one or more inhibitors of TNF-a polypeptide production can be administered to a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation) in any appropriate amount (e.g., any appropriate dose).
- tissue inflammation e.g., autoimmune tissue inflammation
- an effective dose of a metabolite can be from about 0.15 milligrams per kilogram body weight (mg/kg) to about 45 mg/kg (e.g., from about 0.15 mg/kg to about 35 mg/kg, from about 0.15 mg/kg to about 25 mg/kg, from about 0.15 mg/kg to about 15 mg/kg, from about 0.15 mg/kg to about 10 mg/kg, from about 0.15 mg/kg to about 5 mg/kg, from about 0.15 mg/kg to about 2 mg/kg, from about 1 mg/kg to about 45 mg/kg, from about 5 mg/kg to about 45 mg/kg, from about 10 mg/kg to about 45 mg/kg, from about 15 mg/kg to about 45 mg/kg, from about 25 mg/kg to about 45 mg/kg, from about 35 mg/kg to about 45 mg/kg, from about 1 mg/kg to about 35 mg/kg, from about 5 mg/kg to about 25 mg/kg, from about 10 mg/kg to about 15 mg/kg, from about 5 mg/kg to about 45 mg/
- an effective dose of L-aspartate can be from about 0.15 mg/kg to about 45 mg/kg (e.g., from about 0.15 mg/kg to about 35 mg/kg, from about 0.15 mg/kg to about 25 mg/kg, from about 0.15 mg/kg to about 15 mg/kg, from about 0.15 mg/kg to about 10 mg/kg, from about 0.15 mg/kg to about 5 mg/kg, from about 0.15 mg/kg to about 2 mg/kg, from about 1 mg/kg to about 45 mg/kg, from about 5 mg/kg to about 45 mg/kg, from about 10 mg/kg to about 45 mg/kg, from about 15 mg/kg to about 45 mg/kg, from about 25 mg/kg to about 45 mg/kg, from about 35 mg/kg to about 45 mg/kg, from about 1 mg/kg to about 35 mg/kg, from about 5 mg/kg to about 25 mg/kg, from about 10 mg/kg to about 15 mg/kg, from about 5 mg/kg to about 15 mg/kg, or from about 15 mg/kg (e
- an effective dose of D-aspartate can be from about 0.15 mg/kg to about 45 mg/kg (e.g., e.g., from about 0.15 mg/kg to about 35 mg/kg, from about 0.15 mg/kg to about 25 mg/kg, from about 0.15 mg/kg to about 15 mg/kg, from about 0.15 mg/kg to about 10 mg/kg, from about 0.15 mg/kg to about 5 mg/kg, from about 0.15 mg/kg to about 2 mg/kg, from about 1 mg/kg to about 45 mg/kg, from about 5 mg/kg to about 45 mg/kg, from about 10 mg/kg to about 45 mg/kg, from about 15 mg/kg to about 45 mg/kg, from about 25 mg/kg to about 45 mg/kg, from about 35 mg/kg to about 45 mg/kg, from about 1 mg/kg to about 35 mg/kg, from about 5 mg/kg to about 25 mg/kg, from about 10 mg/kg to about 15 mg/kg, from about 5 mg/kg to about 15 mg/kg, or
- an effective dose of NAD can be from about 0.75 mg/kg to about 15 mg/kg (e.g., from about 0.75 mg/kg to about 12 mg/kg, from about 0.75 mg/kg to about 10 mg/kg, from about 0.75 mg/kg to about 7 mg/kg, from about 0.75 mg/kg to about 5 mg/kg, from about 0.75 mg/kg to about 2 mg/kg, from about 3 mg/kg to about 15 mg/kg, from about 5 mg/kg to about 15 mg/kg, from about 8 mg/kg to about 15 mg/kg, from about 10 mg/kg to about 15 mg/kg, from about 12 mg/kg to about 15 mg/kg, from about 1 mg/kg to about 12 mg/kg, from about 5 mg/kg to about 10 mg/kg, from about 1 mg/kg to about 5 mg/kg, from about 3 mg/kg to about 7 mg/kg, or from about 8 mg/kg to about 12 mg/kg).
- an effective dose of alpha-ketobutyrate can be from about 0.15 mg/kg to about 30 mg/kg (e.g., from about 0.15 mg/kg to about 25 mg/kg, from about 0.15 mg/kg to about 20 mg/kg, from about 0.15 mg/kg to about 15 mg/kg, from about 0.15 mg/kg to about 10 mg/kg, from about 0.15 mg/kg to about 5 mg/kg, from about 0.15 mg/kg to about 2 mg/kg, from about 1 mg/kg to about 30 mg/kg, from about 5 mg/kg to about 30 mg/kg, from about 10 mg/kg to about 30 mg/kg, from about 15 mg/kg to about 30 mg/kg, from about 20 mg/kg to about 30 mg/kg, from about 25 mg/kg to about 30 mg/kg, from about 1 mg/kg to about 25 mg/kg, from about 5 mg/kg to about 20 mg/kg, from about 10 mg/kg to about 15 mg/kg, from about 1 mg/kg to about 10 mg/kg, or from about 10 mg/kg
- an effective dose of malate can be from about 0.15 mg/kg to about 45 mg/kg (e.g., e.g., from about 0.15 mg/kg to about 35 mg/kg, from about 0.15 mg/kg to about 25 mg/kg, from about 0.15 mg/kg to about 15 mg/kg, from about 0.15 mg/kg to about 10 mg/kg, from about 0.15 mg/kg to about 5 mg/kg, from about 0.15 mg/kg to about 2 mg/kg, from about 1 mg/kg to about 45 mg/kg, from about 5 mg/kg to about 45 mg/kg, from about 10 mg/kg to about 45 mg/kg, from about 15 mg/kg to about 45 mg/kg, from about 25 mg/kg to about 45 mg/kg, from about 35 mg/kg to about 45 mg/kg, from about 1 mg/kg to about 35 mg/kg, from about 5 mg/kg to about 25 mg/kg, from about 10 mg/kg to about 15 mg/kg, from about 5 mg/kg to about 15 mg/kg, or from about 15 mg/kg
- an effective dose of oxaloacetate can be from about 0.15 mg/kg to about 45 mg/kg (e.g., e.g., from about 0.15 mg/kg to about 35 mg/kg, from about 0.15 mg/kg to about 25 mg/kg, from about 0.15 mg/kg to about 15 mg/kg, from about 0.15 mg/kg to about 10 mg/kg, from about 0.15 mg/kg to about 5 mg/kg, from about 0.15 mg/kg to about 2 mg/kg, from about 1 mg/kg to about 45 mg/kg, from about 5 mg/kg to about 45 mg/kg, from about 10 mg/kg to about 45 mg/kg, from about 15 mg/kg to about 45 mg/kg, from about 25 mg/kg to about 45 mg/kg, from about 35 mg/kg to about 45 mg/kg, from about 1 mg/kg to about 35 mg/kg, from about 5 mg/kg to about 25 mg/kg, from about 10 mg/kg to about 15 mg/kg, from about 5 mg/kg to about 15 mg/kg, from about
- an effective dose of the total metabolite(s) can be from about 0.15 mg/kg to about 45 mg/kg (e.g., e.g., from about 0.15 mg/kg to about 35 mg/kg, from about 0.15 mg/kg to about 25 mg/kg, from about 0.15 mg/kg to about 15 mg/kg, from about 0.15 mg/kg to about 10 mg/kg, from about 0.15 mg/kg to about 5 mg/kg, from about 0.15 mg/kg to about 2 mg/kg, from about 1 mg/kg to about 45 mg/kg, from about 5 mg/kg to about 45 mg/kg, from about 10 mg/kg to about 45 mg/kg, from about 15 mg/kg to about 45 mg/kg, from about 25 mg/kg to about 45 mg/kg, from about 35 mg/kg to about 45 mg/kg, from about 1 mg/kg to about 35 mg/kg, from about 5 mg/kg to about 25 mg/kg, from about 10 mg/kg to about 15 mg/kg, from about 5 mg/kg to about 15 mg/kg,
- An effective amount of a composition containing one or more inhibitors of TNF-a polypeptide production can be any amount that can treat a mammal having tissue inflammation (e.g., autoimmune tissue inflammation) as described herein without producing significant toxicity to the mammal.
- the effective amount can remain constant or can be adjusted as a sliding scale or variable dose depending on the mammal’s response to treatment.
- Various factors can influence the actual effective amount used for a particular application. For example, the frequency of administration, duration of treatment, use of multiple treatment agents, route of administration, and/or severity of the cancer in the mammal being treated may require an increase or decrease in the actual effective amount administered.
- a composition containing one or more inhibitors of TNF-a polypeptide production can be administered to a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation) in any appropriate frequency.
- the frequency of administration can be any frequency that can treat a mammal having tissue inflammation (e.g., autoimmune tissue inflammation) without producing significant toxicity to the mammal.
- the frequency of administration can be from about once a day to about once a week, from about once a week to about once a month, or from about twice a month to about once a month.
- the frequency of administration can remain constant or can be variable during the duration of treatment.
- various factors can influence the actual frequency of administration used for a particular application. For example, the effective amount, duration of treatment, use of multiple treatment agents, and/or route of administration may require an increase or decrease in administration frequency.
- a composition containing one or more inhibitors of TNF-a polypeptide production can be administered to a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation) for any appropriate duration.
- tissue inflammation e.g., autoimmune tissue inflammation
- An effective duration for administering or using a composition described herein can be any duration that can treat a mammal having tissue inflammation (e.g., autoimmune tissue inflammation) without producing significant toxicity to the mammal.
- the effective duration can vary from several weeks to several months, from several months to several years, or from several years to a lifetime. Multiple factors can influence the actual effective duration used for a particular treatment.
- an effective duration can vary with the frequency of administration, effective amount, use of multiple treatment agents, and/or route of administration.
- methods for treating a mammal can include administering to the mammal one or more inhibitors of TNF-a polypeptide production (e.g., inhibitors of TNF-a polypeptide production by T cells) as the sole active ingredient to treat the mammal.
- tissue inflammation e.g., autoimmune tissue inflammation
- a composition containing one or more inhibitors of TNF-a polypeptide production can include the one or more inhibitors of TNF-a polypeptide production as the sole active ingredient in the composition that is effective to treat a mammal having tissue inflammation (e.g., autoimmune tissue inflammation).
- a composition containing one or more metabolites can include the one or metabolites as the sole active ingredient in the composition that is effective to treat a mammal having tissue inflammation (e.g., autoimmune tissue inflammation).
- tissue inflammation e.g., autoimmune tissue inflammation
- methods for treating a mammal e.g., a human having tissue inflammation (e.g., autoimmune tissue inflammation) as described herein (e.g., by administering one or more inhibitors of TNF-a polypeptide production) also can include administering to the mammal one or more (e.g., one, two, three, four, five or more) additional agents/therapies used to treat tissue inflammation (e.g., autoimmune tissue inflammation).
- an agent that can be used to treat tissue inflammation can be an anti-TNF-a therapy.
- anti-TNF-a therapies that can be used to treat tissue inflammation (e.g., autoimmune tissue inflammation) and can be used together with one or more inhibitors of TNF-a polypeptide production described herein include, without limitation, adalimumab (e.g., HUMIRA®), adalimumab-adbm (e.g., Cyltezo®), adalimumab-adaz (e.g., HYRIMOZTM), adalimumab-atto (e.g., AMJEVITATM), certolizumab pegol (e.g., CIMZIA®), etanercept (e.g., Enbrel®), etanercept-szzs (e.g., ERELZITM), infliximab (e.g., REMICADE®), infliximab-abda (e.g., KEVZARA®), inflixima
- an agent that can be used to treat tissue inflammation can be a diseasemodifying antirheumatic drug (DMARD; e.g., biologic DMARDs and synthetic DMARDs).
- DMARDs that can be used to treat tissue inflammation (e.g., autoimmune tissue inflammation) and can be used together with one or more inhibitors of TNF-a polypeptide production described herein include, without limitation, methotrexate (e.g., Trexall® and OTREXUP®), leflunomide (e.g., Arava®), hydroxychloroquine (e.g., Plaquenil®), sulfasalazine (e.g., Azulfidine®), abatacept (e.g., ORENCIA®), adalimumab (e.g., HUMIRA®), adalimumab-adbm (e.g., Cyltezo®), adalimumab-adaz (e.g., met
- an agent that can be used to treat tissue inflammation can be a nonsteroidal anti-inflammatory drug (NSAID).
- NSAID nonsteroidal anti-inflammatory drug
- NSAIDs that can be used to treat tissue inflammation (e.g., autoimmune tissue inflammation) and can be used together with one or more inhibitors of TNF-a polypeptide production (e.g., inhibitors of TNF-a polypeptide production by T cells) described herein include, without limitation, ibuprofen (e.g., Advil® and MOTRIN® IB) and naproxen sodium (e.g., ALEVE®).
- tissue inflammation e.g., autoimmune tissue inflammation
- inhibitors of TNF-a polypeptide production by T cells include, without limitation, ibuprofen (e.g., Advil® and MOTRIN® IB) and naproxen sodium (e.g., ALEVE®).
- ibuprofen e.g., Advil® and MOTRIN® IB
- naproxen sodium e.g., ALEVE®
- an agent that can be used to treat tissue inflammation can be a steroid (e.g., a corticosteroid).
- a steroid e.g., a corticosteroid
- steroids that can be used to treat tissue inflammation (e.g., autoimmune tissue inflammation) and can be used together with one or more inhibitors of TNF-a polypeptide production (e.g., inhibitors of TNF-a polypeptide production by T cells) described herein include, without limitation, prednisone, and prednisolone.
- one or more inhibitors of TNF-a polypeptide production e.g., inhibitors of TNF-a polypeptide production by T cells
- additional agents used to treat tissue inflammation e.g., autoimmune tissue inflammation
- the one or more additional agents can be administered at the same time (e.g., in a single composition containing both one or more inhibitors of TNF-a polypeptide production and the one or more additional agents) or independently.
- one or more inhibitors of TNF-a polypeptide production described herein can be administered first, and the one or more additional agents administered second, or vice versa.
- tissue inflammation e.g., autoimmune tissue inflammation
- one or more inhibitors of TNF-a polypeptide production e.g., inhibitors of TNF-a polypeptide production by T cells
- the one or more additional therapies can be performed at the same time or independently of the administration of one or more inhibitors of TNF-a polypeptide production described herein.
- one or more inhibitors of TNF-a polypeptide production can be administered before, during, or after the one or more additional therapies are performed.
- a mammal e.g., a human having tissue inflammation (e.g., autoimmune tissue inflammation) can be administered one or more (e.g., one, two, three, four, five or more) metabolites (e.g., mitochondrial metabolites such as L-aspartate, D- aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate) and can be administered one or more (e.g., one, two, three, four, five or more) anti-TNF-a therapies to treat the mammal.
- tissue inflammation e.g., a mammal having tissue inflammation can be administered aspartate (e.g., a composition including D-aspartate and/or L-aspartate) and can be administered one or more anti-TNF-a therapies.
- a mammal having tissue inflammation can be administered (e.g., by subcutaneous injection) adalimumab (e.g., from about 40 mg to about 80 mg adalimumab) and can be administered aspartate (e.g., a composition including from about 10 mg to about 3000 mg of aspartate (e.g., D-aspartate and/or L-aspartate)).
- a mammal having tissue inflammation can be administered a composition that includes both adalimumab and aspartate.
- a mammal having tissue inflammation can be administered adalimumab and can be separately administered aspartate.
- adalimumab and the aspartate can be administered within 1 to 15 minutes of each other.
- the administration of adalimumab and aspartate can be repeated (e.g., can be repeated about every week or about every 2 weeks).
- a mammal having tissue inflammation can be administered (e.g., by subcutaneous injection) etanercept (e.g., about 50 mg etanercept) and can be administered aspartate (e.g., a composition including from about 10 mg to about 3000 mg of aspartate (e.g., D-aspartate and/or L-aspartate)).
- etanercept e.g., about 50 mg etanercept
- aspartate e.g., a composition including from about 10 mg to about 3000 mg of aspartate (e.g., D-aspartate and/or L-aspartate)
- a mammal having tissue inflammation can be administered a composition that includes both etanercept and aspartate.
- a mammal having tissue inflammation can be administered etanercept and can be separately administered aspartate.
- the etanercept and the aspartate can be administered within 1 to 15 minutes of each other. In some cases, the administration of etanercept and aspartate can be repeated (e.g., can be repeated about every week).
- a mammal having tissue inflammation can be administered (e.g., by subcutaneous injection) infliximab (e.g., from about 5 mg/kg to about 10 mg/kg infliximab) and can be administered aspartate (e.g., a composition including from about 10 mg to about 3000 mg of aspartate (e.g., D-aspartate and/or L-aspartate)).
- infliximab e.g., from about 5 mg/kg to about 10 mg/kg infliximab
- aspartate e.g., a composition including from about 10 mg to about 3000 mg of aspartate (e.g., D-aspartate and/or L-aspartate)
- a mammal having tissue inflammation can be administered a composition that includes both infliximab and aspartate.
- a mammal having tissue inflammation can be administered infliximab and can be separately administered aspartate.
- the infliximab and the aspartate can be administered within 1 to 15 minutes of each other. In some cases, the administration of infliximab and aspartate can be repeated (e.g., can be repeated about every 8 weeks).
- a mammal having tissue inflammation can be administered two or more of mitochondrial metabolites (e.g., a composition including L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate) and can be administered one or more anti-TNF-a therapies.
- mitochondrial metabolites e.g., a composition including L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate
- a mammal having tissue inflammation can be administered (e.g., by subcutaneous injection) adalimumab (e.g., from about 40 mg to about 80 mg adalimumab) and can be administered two or more mitochondrial metabolites (e.g., L- aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate such as a composition including from about 10 mg to about 3000 mg of total metabolites of L- aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate).
- mitochondrial metabolites e.g., L- aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate
- oxaloacetate e.g., L- aspartate, D-aspartate, NAD, alpha-ketobutyrate,
- a mammal having tissue inflammation can be administered a composition that includes adalimumab, L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate.
- a mammal having tissue inflammation can be administered adalimumab and can be separately administered two or more mitochondrial metabolites (e.g., L-aspartate, D- aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate).
- adalimumab and the two or more mitochondrial metabolites can be administered within 1 to 15 minutes of each other.
- the administration of adalimumab, L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate can be repeated (e.g., can be repeated about every week or about every 2 weeks).
- a mammal having tissue inflammation can be administered (e.g., by subcutaneous injection) etanercept (e.g., about 50 mg etanercept) and can be administered two or more mitochondrial metabolites (e.g., L-aspartate, D-aspartate, NAD, alpha- ketobutyrate, malate, and oxaloacetate such as a composition including from about 10 mg to about 3000 mg of total metabolites of L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate).
- mitochondrial metabolites e.g., L-aspartate, D-aspartate, NAD, alpha- ketobutyrate, malate, and oxaloacetate
- a mammal having tissue inflammation can be administered a composition that includes etanercept, L-aspartate, D-aspartate, NAD, alpha- ketobutyrate, malate, and oxaloacetate.
- a mammal having tissue inflammation can be administered etanercept and can be separately administered two or more mitochondrial metabolites (e.g., L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate).
- etanercept When a mammal is administered etanercept and is separately administered two or more mitochondrial metabolites (e.g., L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate), the etanercept and the two or more mitochondrial metabolites can be administered within 1 to 15 minutes of each other. In some cases, the administration of etanercept, L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate can be repeated (e.g., can be repeated about every week).
- two or more mitochondrial metabolites e.g., L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate
- a mammal having tissue inflammation can be administered (e.g., by subcutaneous injection) infliximab (e.g., about 5 mg/kg to about 10 mg/kg infliximab) and can be administered two or more mitochondrial metabolites (e.g., L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate such as a composition including from about 10 mg to about 3000 mg of total metabolites of L-aspartate, D-aspartate, NAD, alpha- ketobutyrate, malate, and oxaloacetate).
- mitochondrial metabolites e.g., L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate
- oxaloacetate such as a composition including from about 10 mg to about 3000 mg of total metabolites of L-aspartate, D-
- a mammal having tissue inflammation can be administered a composition that includes infliximab, L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate.
- a mammal having tissue inflammation can be administered infliximab and can be separately administered two or more mitochondrial metabolites (e.g., L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate).
- infliximab and the two or more mitochondrial metabolites can be administered within 1 to 15 minutes of each other.
- the administration of infliximab, L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate can be repeated (e.g., can be repeated about every 8 weeks).
- Example 1 Mitochondrial aspartate regulates TNF-a biogenesis and autoimmune tissue inflammation
- This Example identifies a deficiency of mitochondrial aspartate production as a key abnormality in such autoimmune T cells.
- Shortage of mitochondrial aspartate disrupted the regeneration of the metabolic cofactor nicotinamide adenine dinucleotide (NAD), causing ADP-deribosylation of the endoplasmic reticulum (ER) sensor GRP78/BiP.
- NAD nicotinamide adenine dinucleotide
- ER endoplasmic reticulum
- ribosome-rich ER membranes expanded, promoting co-translational translocation and enhanced biogenesis of the transmembrane cytokine TNF-a.
- ER nch T cells were the predominant TNF-a producers in the arthritic joint.
- T cell-specific transfer of intact mitochondria as well as supplementation of exogenous aspartate rescued the mitochondria- instructed expansion of ER membranes and suppressed TNF-a release and rheumatoid tissue inflammation.
- Naive CD4 + T cells from RA patients have a mitochondrial defect, manifesting with low oxygen consumption and low mitochondrial membrane potential (MMP) ( Figures 1 A-1C), while mitochondrial mass is similar between healthy and RA T cells ( Figure 6).
- MMP mitochondrial membrane potential
- Figures 1 A-1C mitochondrial mass is similar between healthy and RA T cells
- Figure 6 mitochondrial mass was quantified.
- Flow cytometry for ER-positioned ATP-sensitive K + channels (marked by ER Tracker) and confocal imaging for the ER-resident enzyme protein disulfide isomerase (PDI) revealed an inverse relationship between the mitochondrial membrane potential and the biomass of the ER ( Figures ID and IE).
- ER Tracker signals in activated CD4 + T cells from patients with psoriatic arthritis were indistinguishable from age-matched controls ( Figure 8), indicating that the expansion of ER size is an RA specific phenotype, and not simply a consequence of systemic inflammation.
- RA T cells synthesized significantly more phosphatidylcholine, one of the major phospholipids needed for biomembrane generation (Figure IK). Transmission electron microscopy confirmed highly abundant and elongated ER structures in activated CD4 + T cells from RA patients ( Figure IL).
- amino acid aspartate as a sensitive biomarker of mitochondrial respiration and as a signal transducer between mitochondria and the ER membrane system.
- Aspartate is dispatched from mitochondria as an electron acceptor, facilitating the cytoplasmic regeneration of NAD + from NADH, and then malate reenters the mitochondria as an electron carrier (Figure 2B).
- NAD + and NADH concentrations are affected by aspartate shortage, intracellular NAD + /NADH were measured in healthy and patient-derived CD4 + T cells. Healthy T cells reached NAD + levels of 550 pmol/10 A6 cells and NADH concentrations of 70 pmol/10 A6 cells. The NAD + /NADH ratio in healthy T cells exceeded 7. With reduced NAD + and elevated NADH, RA T cells achieved only an NAD + /NADH ratio of 3 ( Figures 3B and 3C).
- RA T cells were treated with aspartate or received a transfer of exogenous mitochondria. Both interventions significantly improved NAD + regeneration ( Figures 3D and 3E). Inhibiting mitochondrial respiration with the complex I inhibitor Rotenone essentially prevented conversion of NADH into NAD + ( Figure 3F), implicating aspartate and mitochondrial intactness in maintaining the NAD + /NADH balance.
- RA T cells were activated with or without NAD + supplement. Like aspartate, exogenous NAD + lowered the ER biomass ( Figures 3G and 3H) and reduced the ER stress signature ( Figure 31).
- Bip senses and binds to unfolded proteins, releasing PERK, ATF6 and IRE and triggering their respective ER stress pathways (Figure 3 J).
- Bip is ideally positioned to communicate variations in mitochondrial function to the endomembrane system, with posttranslational protein modifications providing fast access to this communication pathway.
- NAD + availability in RA T cells whether NAD- dependent ADP-ribosylation is relevant for Bip function was tested. Compared to healthy T cells, only 30% of Bip molecules in RA T cells were ADP-ribosylated (Figure 3K).
- the BipADP-R 10 phenotype could be reproduced by blocking the electron transport chain with rotenone (Figure 3L) or Piercidin A ( Figure 13). In contrast, surplus exogenous NAD + restored ADP-ribosylation of Bip in RA T cells ( Figure 3M).
- Ribosome-rich rough ER was quantified in healthy and patient-derived T cells ( Figures 4A - 4D). ER biomass was expanded in RA T cells, as indicated by the increased load of the ER chaperone calnexin ( Figures 4A and 4B) while global ribosomal protein levels were indistinguishable in healthy and RA T cells.
- the ribosome-bonded rough ER responsible for co-translational translocation
- mRNAs contained in the ER-bonded ribosomes were analyzed. Considering that the rough ER sheets serve as the main site of synthesis for secreted and membrane-integrated proteins, we concentrated on T cell effector cytokines. In naive CD4 + T cells, transcripts for the lineage-determining effector cytokines IFN-y, IL-4 and IL- 17 were barely detectable, but IL2, and even more so, TNFA mRNA were abundant ( Figure 4E). Considering the critical role of TNF-a in the RA disease process, the transcription and translation of this cytokine was focused on.
- mRNA for the 4 cytokines IFN- y, IL-2, IL- 17 and TNF-a were all highly enriched amongst the ER-bound fraction, with IL2 and TNFA mRNA being >100fold more abundant at the ER membrane ( Figure 4G).
- RA CD4 + T cells recruited a higher proportion of TNFA mRNA to the organelle’s surface ( Figure 4H), where selected mRNAs become subject to co- translational translocation. This redistribution of TNFA mRNA resulted in highly efficient TNF-a biogenesis ( Figures 4L4K).
- TNF-a-producing CD4+ T cells function as key arthritogenic effector cells.
- Rheumatoid synovial lesions consist of a mixture of cell types, most prominently, T cells, B cells, macrophages, and synovial fibroblasts.
- TNF-a production was analyzed. Tissue-residing T cells contained abundant amounts of TNFA mRNA, while macrophages and B cells were low positive, and fibroblasts were negative. TNF-a production was allocated to the different cellular subsets by determining intracellular TNF-a by flow cytometry.
- TNF-a production is a feature of tissue-embedded cells and not just detectable after LPS/PMA/ionomycin stimulation of disaggregated cells.
- a method to analyze cytokine production in freshly harvested synovial tissues from RA patients was established. Cytokine secretion from the cells was blocked by treating intact tissue slices with BFA for 4 hours. Subsequently, cells were isolated from the tissue and intracellular TNF-a was detected by flow cytometry ( Figures 51 - 5K). In fresh synovial tissues, about 40% of synovial CD68 + macrophages produced TNF-a, independent of BFA treatment, indicating intracellular retention of TNF-a in tissue-residing macrophages.
- tissue-residing T cells appeared to immediately release TNF-a into the tissue microenvironment.
- the frequencies of TNF-a + T cells increased 10-20-fold upon BFA-induced secretion blockade (Figure 5K).
- T cell-derived TNF-a is relevant for synovial inflammation
- a humanized mouse model was used in which human synovial tissue is engrafted into NSG mice and the chimeric host is immuno-reconstituted with peripheral blood mononuclear cells (PBMC) from RA patients.
- PBMC peripheral blood mononuclear cells
- FACS-sorted CD4 + T cells were transfected with control siRNA or siRNA targeting TNFA ( Figure 18 A).
- the knockdown lowered TNFA transcripts to about 50% of controls ( Figure 20A). mimicking a physiologic situation. Histological evaluation of explanted synovial tissues demonstrated that suppressing T cell-derived TNF-a was strongly anti-inflammatory.
- Control chimeras injected with control-si-RNA transfected CD4 + T cells, developed robust synovitis.
- Tissues harvested from the control mice were densely infiltrated with CD3 + T cells ( Figures 5L and 5M).
- Tissue transcriptomic analysis revealed abundance of TCR, TBX21 and RORC transcripts ( Figure 5N).
- CD4 + T cells with intact TNF-a production infiltrated into the synovial tissue space and triggered induction of IFNG, IL 17, IL21, TNFA, IL6 and IL1B ( Figure 5N).
- PBMCs Peripheral blood mononuclear cells
- CD4 + CD45RA + naive T cells were isolated from PBMCs with the EasySep Human Naive CD4 + T Cell Enrichment Kit (STEMCELL Technologies). Purity of cell populations was consistently > 95%.
- Anti-CD3/anti-CD28- coated Dynabeads were used to activate naive CD4 + T cells at a ratio of 2 cells: 1 bead for 72 hours.
- Cell Activation Cocktail (PMA/Ionomycin) (Biolegend) was used to stimulate cytokine production in T cells with or without Brefeldin A (eBioscience) for 2 hours.
- the pCMV5-Flag-XBPls plasmid was purchased from Addgene.
- Human TNFA siRNA, GOT2 siRNA and control siRNA were obtained from Thermo Fisher Scientific.
- the mitochondrial respiration inhibitors Antimycin A and Rotenone were purchased from Agilent Technologies.
- NAD + was obtained from Cayman Chemical.
- a-Ketoglutaric acid, succinic acid, L-aspartic acid were from Sigma-Aldrich. Malic acid was from Santa Cruz Biotechnology.
- CD4 + CD45RA + naive T cells were activated for 72 hours and stimulated with PMA/ION/BFA for 2 hours. 4% PFA was used for fixation and 0.1% saponin to permeabilize the cells. Cells were stained with PE AntiHuman TNF-a (1 TOO, BD, Cat# 554513) antibody for 1 hour. Flow cytometry was performed on an LSR II flow cytometer (BD Biosciences). Data were analyzed with FlowJo software (Tree Star Inc).
- ER tracker green (Thermo Fisher Scientific, Cat# E34251) was used to stain the ER- localized ATP-sensitive K + channels following the manufacture’s instruction. Flow cytometry was performed to detect ER tracker signal on an LSR II flow cytometer (BD Biosciences). Intensity of ER tracker staining represents the size of the ER membranes. Data were analyzed with FlowJo software (Tree Star Inc).
- the sample was rinsed 2 times in distilled water and dehydrated in an ethanolic series culminating in two changes of 100% acetone.
- the cell pellet was then placed in a mixture of Spurr resin and acetone (1 : 1) for 30 minutes, followed by 2 hours in 100% resin with 2 changes.
- the cell pellet was placed into 100% Spurr resin in an embedding mold and polymerized at 65°C for 12 hours or longer.
- Ultrathin (70-90 nm) sections were cut on an ultramicrotome with a diamond knife, stained with lead citrate and examined with a JEOL 1400 transmission electron microscope.
- Endoplasmic Reticulum Isolation Kit (Sigma- Aldrich, Catalog Number: ER0100) was used to isolate rough ER.
- MitoTracker Red (Thermo Fisher Scientific, Cat# M7512) is a fluorescent dye that stains mitochondria in live cells and its accumulation is dependent upon mitochondrial membrane potential (MMP). Staining intensity was analyzed with an LSR II flow cytometer (BD Biosciences) and data were analyzed with FlowJo software (Tree Star Inc).
- RNA or Rough ER bound RNA were extracted with Trizol (Thermo Fisher Scientific) and Direct-zol RNA MiniPrep Kit (ZYMO Research).
- cDNA was synthesized using Maxima First Strand cDNA Synthesis Kits for qPCR with reverse transcription (RT- qPCR) (Thermo Fisher Scientific). Quantitative PCR analyses were performed using SYBR Green qPCR Master Mix (Bimake) and gene expression was normalized to P-actin. Primers were listed in Table 2. Attorney Docket No. 07039-2075W01 / 2021-418
- naive CD4 + T cells from healthy donor were isolated and activated with anti-CD3/CD28 beads.
- naive CD4 + T cells from RA patient were purified and activated.
- 4 million healthy CD4+ T cells were harvested, and mitochondria were isolated using Mitochondria Isolation Kit for Cultured Cells (Thermo Fisher Scientific, Catalog number: 89874).
- 0.2 million activated RA CD4 + T cells were collected and spun at 300xg for 5 minutes. Resuspend cells were mixed with the mitochondria suspension and carefully resuspended. The cell/mitochondria mixture and the control cell/PBS mixture were centrifuged at l,500xg for 5 minutes. After washing, the cells were stimulated with anti-CD3/CD28 beads for one more day.
- mice from the Jackson Laboratory were kept in a pathogen-free facility. Wedges of human synovial tissue were subcutaneously implanted into 8-12-week-old mice. After 7 days, the mice were infused with 10 million PBMC collected from RA patients with active disease.
- CD4 + T cells in PBMC were FACS sorted and transfected with siRNA targeting TNFA or control siRNA before the cells were injected into the mice. Alternatively, healthy mitochondria were transferred into RA CD4 + T cells prior to the immune reconstitution.
- CD4 + T cells were FACS sorted and mitochondria from healthy T cells were transferred into RA CD4 + T cells as described above.
- synovial tissues were explanted from the chimeric mice, OCT-embedded (4583; Sakura Finetek USA) or shock-frozen for further experiments (tissue staining or RNA extraction).
- IP Lysis Buffer Thermo Fisher Scientific, Catalog#: 87788
- BiP protein in whole cell lysates was pulled down by incubation with agarose conjugated anti-BiP antibody (Santa Cruz Biotechnology, Catalog#: sc-13539) for 4 hours at 4°C.
- Agarose was washed 5 times with IP lysis buffer and boiled for 5 minutes in loading buffer. Eluted protein was separated by SDS-page and ADP-ribosylated BiP was detected using antibody against ADP- ribose (Cell Signaling Technology, Catalog#: 83732).
- IP Lysis Buffer Thermo Fisher Scientific, Catalog#: 87788
- protease and phosphatase inhibitors were lysed with IP Lysis Buffer (Thermo Fisher Scientific, Catalog#: 87788) containing protease and phosphatase inhibitors.
- Whole cell lysates were incubated with 2pg anti-BiP antibody (Santa Cruz Biotechnology, Catalog#: sc-13539) and Protein A/G PLUS- Agarose (Santa Cruz Biotechnology, Catalog#: sc-2003) for 4 hours at 4°C.
- Normal rat IgG (Santa Cruz Biotechnology, Catalog#: sc-2026) was used as IgG control.
- the immunocomplexes were washed with IP lysis buffer 5 times, then eluted with loading buffer and separated by SDS-PAGE. Immunoblotting for IREl-a was performed following standard procedures for Western blotting. Quantification and Statistical Analysis
- T cells are isolated from a human identified as having tissue inflammation, loaded with intact mitochondria, and the mitochondria-loaded T cells are administered back to the human.
- the administered mitochondria-loaded T cells can reduce production of a TNF-a polypeptide within the inflamed tissue to reduce tissue inflammation present within the human.
- Healthy cells that are not T cells are isolated from a human identified as having tissue inflammation, and healthy mitochondria are obtained from the healthy cells.
- T cells are isolated from a human identified as having tissue inflammation, loaded with intact, healthy mitochondria isolated from the healthy cells that are not T cells, and the mitochondria-loaded T cells are administered back to the human.
- the administered mitochondria-loaded T cells can reduce production of a TNF-a polypeptide within the inflamed tissue to reduce tissue inflammation present within the human.
- Example 4 Treating tissue inflammation
- T cells are isolated from a human identified as having tissue inflammation, loaded with intact, healthy mitochondria isolated from cord blood cells, and the mitochondria-loaded T cells are administered back to the human.
- the administered mitochondria-loaded T cells can reduce production of a TNF-a polypeptide within the inflamed tissue to reduce tissue inflammation present within the human.
- a human identified as having tissue inflammation is administered (e.g., by subcutaneous injection) 40 mg to 80 mg adalimumab (e.g., HUMIRA®) together with 10 mg to 3000 mg of aspartate (e.g., D-aspartate and/or L-aspartate).
- adalimumab e.g., HUMIRA®
- aspartate e.g., D-aspartate and/or L-aspartate
- the administration is repeated every 2 weeks. In some cases, the administration is repeated every week.
- the administered combination of adalimumab and aspartate can reduce tissue inflammation present within the human.
- a human identified as having tissue inflammation is administered (e.g., by subcutaneous injection) 50 mg etanercept (e.g., Enbrel®) together with 10 mg to 3000 mg of aspartate (e.g., D-aspartate and/or L-aspartate). In some cases, the administration is repeated every week.
- etanercept e.g., Enbrel®
- aspartate e.g., D-aspartate and/or L-aspartate
- the administered combination of etanercept and aspartate can reduce tissue inflammation present within the human.
- a human identified as having tissue inflammation is administered (e.g., by subcutaneous injection) 5 mg/kg to 10 mg/kg infliximab (e.g., REMICADE®) together with 10 mg to 3000 mg of aspartate (e.g., D-aspartate and/or L-aspartate). In some cases, the administration is repeated every 8 weeks.
- infliximab e.g., REMICADE®
- aspartate e.g., D-aspartate and/or L-aspartate
- the administration is repeated every 8 weeks.
- the administered combination of infliximab and aspartate can reduce tissue inflammation present within the human.
- a human identified as having tissue inflammation is administered (e.g., by subcutaneous injection) 40 mg to 80 mg adalimumab (e.g., HUMIRA®) and is administered, separately but within 1 to 15 minutes, 10 mg to 3000 mg of aspartate (e.g., D-aspartate and/or L-aspartate).
- aspartate e.g., D-aspartate and/or L-aspartate.
- the administration is repeated every 2 weeks. In some cases, the administration is repeated every week.
- the administered combination of adalimumab and aspartate can reduce tissue inflammation present within the human.
- a human identified as having tissue inflammation is administered (e.g., by subcutaneous injection) 50 mg etanercept (e.g., Enbrel®) and is administered, separately but within 1 to 15 minutes, 10 mg to 3000 mg of aspartate (e.g., D-aspartate and/or L-aspartate). In some cases, the administration is repeated every week.
- etanercept e.g., Enbrel®
- aspartate e.g., D-aspartate and/or L-aspartate
- the administered combination of etanercept and aspartate can reduce tissue inflammation present within the human.
- a human identified as having tissue inflammation is administered (e.g., by subcutaneous injection) 5 mg/kg to 10 mg/kg infliximab (e.g., REMICADE®) and is administered, separately but within 1 to 15 minutes, 10 mg to 3000 mg of aspartate (e.g., D- aspartate and/or L-aspartate). In some cases, the administration is repeated every 8 weeks.
- infliximab e.g., REMICADE®
- aspartate e.g., D- aspartate and/or L-aspartate.
- the administration is repeated every 8 weeks.
- the administered combination of infliximab and aspartate can reduce tissue inflammation present within the human.
- a human identified as having tissue inflammation is administered (e.g., by subcutaneous injection) 40 mg to 80 mg adalimumab (e.g., HUMIRA®) together with 10 mg to 3000 mg of total metabolites of L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate.
- adalimumab e.g., HUMIRA®
- the administration is repeated every 2 weeks. In some cases, the administration is repeated every week.
- the administered combination of adalimumab and metabolites can reduce tissue inflammation present within the human.
- a human identified as having tissue inflammation is administered (e.g., by subcutaneous injection) 50 mg etanercept (e.g., Enbrel®) together with 10 mg to 3000 mg of total metabolites of L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate. In some cases, the administration is repeated every week.
- etanercept e.g., Enbrel®
- the administration is repeated every week.
- the administered combination of etanercept and metabolites can reduce tissue inflammation present within the human.
- a human identified as having tissue inflammation is administered (e.g., by subcutaneous injection) 5 mg/kg to 10 mg/kg infliximab (e.g., REMICADE®) together with 10 mg to 3000 mg of total metabolites of L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate. In some cases, the administration is repeated every 8 weeks.
- infliximab e.g., REMICADE®
- the administered combination of infliximab and metabolites can reduce tissue inflammation present within the human.
- a human identified as having tissue inflammation is administered (e.g., by subcutaneous injection) 40 mg to 80 mg adalimumab (e.g., HUMIRA®) and is administered, separately but within 1 to 15 minutes, 10 mg to 3000 mg of total metabolites of L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate.
- adalimumab e.g., HUMIRA®
- the administration is repeated every 2 weeks. In some cases, the administration is repeated every week.
- Example 15 Treating tissue inflammation
- a human identified as having tissue inflammation is administered (e.g., by subcutaneous injection) 50 mg etanercept (e.g., Enbrel®) and is administered, separately but within 1 to 15 minutes, 10 mg to 3000 mg of total metabolites of L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate. In some cases, the administration is repeated every week.
- etanercept e.g., Enbrel®
- the administered combination of etanercept and metabolites can reduce tissue inflammation present within the human.
- a human identified as having tissue inflammation is administered (e.g., by subcutaneous injection) 5 mg/kg to 10 mg/kg infliximab (e.g., REMICADE®) and is administered, separately but within 1 to 15 minutes, 10 mg to 3000 mg of total metabolites of L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate. In some cases, the administration is repeated every 8 weeks.
- infliximab e.g., REMICADE®
- the administered combination of infliximab and metabolites can reduce tissue inflammation present within the human.
- Example 17 Exemplary Embodiments
- Embodiment 1 A method for restoring aspartate homeostasis in a mammal having tissue inflammation, wherein said method comprises administering, to said mammal, a composition comprising cells, wherein a population of intact mitochondria was introduced into said cells, and wherein the production of a tumor necrosis factor-a (TNF-a) polypeptide within said mammal is reduced following said administering step.
- a composition comprising cells, wherein a population of intact mitochondria was introduced into said cells, and wherein the production of a tumor necrosis factor-a (TNF-a) polypeptide within said mammal is reduced following said administering step.
- TNF-a tumor necrosis factor-a
- Embodiment 2 The method of embodiment 1, wherein said composition comprises T cells containing said intact mitochondria.
- Embodiment 3 The method of embodiment 1 or embodiment 2, where said T cells are obtained from said mammal.
- Embodiment 4 A method for restoring aspartate homeostasis in a mammal having tissue inflammation, wherein said method comprises administering, to said mammal, an inhibitor of UPR signaling, wherein said method is effective to reduce production of a TNF-a polypeptide within said mammal.
- Embodiment 5 The method of embodiment 4, wherein said inhibitor of UPR signaling can target an UPR signaling pathway component selected from the group consisting of binding immunoglobulin protein (BiP) polypeptides, IRE la polypeptides, PERK polypeptides, ATF6 polypeptides, eIF2a polypeptides, CHOP polypeptides, XBP-1 polypeptides, and XBP-1S polypeptides.
- BiP binding immunoglobulin protein
- Embodiment 6 The method of embodiment 4, wherein said inhibitor of UPR signaling is selected from the group consisting of eeyarestatin I, mycolactone, exotoxin A, NSC 630668-R/l, MAL3-39, MAL3-101, E6 berbamine, ophiobolin A, equisetin, CJ-21058, Rose Bengal, erythrosin B, P97-A4, P87-A4, 17D9, P91-E9, 16F6, bisthiouracil, SCA-21, HUN- 7293, cotransin, CAM741, apratoxin A, decatransin, valinomycin, CADA, kinase inhibiting RNase attenuator 6 (KIRA6), 3-hydroxy-2-naphthoic acid (3HNA), MKC-3946, 4- Phenylbutyric acid (4-PBA), taurine-conjugated ursodeoxycholic acid (TUDCA), o
- Embodiment 7 A method for restoring aspartate homeostasis in a mammal having tissue inflammation, wherein said method comprises administering, to said mammal, an inhibitor of co-translational translocation, wherein said method is effective to reduce production of a TNF-a polypeptide within said mammal.
- Embodiment 8 The method of embodiment 7, wherein said inhibitor of co- translational translocation can target a co-translational translocation pathway component that is selected from the group consisting of Sec61 polypeptides, Hsp70 polypeptides, ATPase polypeptides, calmodulin polypeptides, transmembrane domains of newly produced polypeptides, signal peptides, and signal peptidase complex polypeptides.
- a co-translational translocation pathway component that is selected from the group consisting of Sec61 polypeptides, Hsp70 polypeptides, ATPase polypeptides, calmodulin polypeptides, transmembrane domains of newly produced polypeptides, signal peptides, and signal peptidase complex polypeptides.
- Embodiment 9 A method for restoring aspartate homeostasis in a mammal having tissue inflammation, wherein said method comprises administering, to said mammal, a composition comprising a mitochondrial metabolite, wherein said method is effective to reduce production of a TNF-a polypeptide within said mammal.
- Embodiment 10 The method of embodiment 9, wherein said mitochondrial metabolite is selected from the group consisting of L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, oxaloacetate, and nicotinamide riboside.
- Embodiment 11 The method of embodiment 9 or embodiment 10, wherein said composition comprises L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate.
- Embodiment 12 A method for restoring aspartate homeostasis in a mammal having tissue inflammation, wherein said method comprises administering, to said mammal, an inhibitor of aspartate transport or aspartate metabolism, wherein said method is effective to reduce production of a TNF-a polypeptide within said mammal.
- Embodiment 13 The method of embodiment 12, wherein said inhibitor of aspartate transport or aspartate metabolism is selected from the group consisting of DL-TBOA, L-(-)- threo-3-hydroxyaspartic acid, L-trans-2,4-PDC, TFB-TBOA, and GOT1 inhibitor 2c.
- Embodiment 14 The method of any one of embodiments 1-13, wherein said mammal is a human.
- Embodiment 15 The method of any one of embodiments 1-14, wherein said tissue inflammation is associated with an autoimmune disease.
- Embodiment 16 The method of embodiment 15, wherein said autoimmune disease is selected from the group consisting of polymyositis, dermatomyositis, rheumatoid arthritis (RA), scleroderma, sjogren's syndrome, systemic lupus erythematosus, vasculitis, mixed connective tissue disease (MCTD), autoimmune hepatitis, psoriasis, and ankylosing spondylitis.
- RA rheumatoid arthritis
- scleroderma sjogren's syndrome
- systemic lupus erythematosus vasculitis
- mixed connective tissue disease MCTD
- autoimmune hepatitis psoriasis
- ankylosing spondylitis ankylosing spondylitis.
- Embodiment 17 The method of embodiment 15, wherein said autoimmune disease is RA.
- Embodiment 18 The method of any one of embodiments 1-14, wherein said tissue inflammation is associated with irritable bowel syndrome (IBS).
- IBS irritable bowel syndrome
- Embodiment 19 The method of any one of embodiments 1-18, said method comprising administering to said mammal an anti-TNF-a therapy.
- Embodiment 20 The method of embodiment 19, wherein said anti-TNF-a therapy is selected from the group consisting of adalimumab, adalimumab-adbm, adalimumab-adaz, adalimumab-atto, certolizumab pegol, etanercept, etanercept-szzs, infliximab, infliximab- abda, and infliximab-dyyb.
- said anti-TNF-a therapy is selected from the group consisting of adalimumab, adalimumab-adbm, adalimumab-adaz, adalimumab-atto, certolizumab pegol, etanercept, etanercept-szzs, infliximab, infliximab- abda, and infliximab-d
- Embodiment 21 A method for reducing tissue inflammation in a mammal, wherein said method comprises administering, to said mammal, a composition comprising cells, wherein a population of intact mitochondria was introduced into said cells, and wherein the production of a TNF-a polypeptide within said mammal is reduced following said administering step.
- Embodiment 22 The method of embodiment 21, wherein said composition comprises T cells containing said intact mitochondria.
- Embodiment 23 The method of embodiment 21 or embodiment 22, where said T cells are obtained from said mammal.
- Embodiment 24 A method for reducing tissue inflammation in a mammal, wherein said method comprises administering, to said mammal, an inhibitor of UPR signaling, wherein said method is effective to reduce production of a TNF-a polypeptide within said mammal.
- Embodiment 25 The method of embodiment 24, wherein said inhibitor of UPR signaling can target an UPR signaling pathway component selected from the group consisting of BiP polypeptides, IREla polypeptides, PERK polypeptides, ATF6 polypeptides, eIF2a polypeptides, CHOP polypeptides, XBP-1 polypeptides, and XBP-1S polypeptides.
- an UPR signaling pathway component selected from the group consisting of BiP polypeptides, IREla polypeptides, PERK polypeptides, ATF6 polypeptides, eIF2a polypeptides, CHOP polypeptides, XBP-1 polypeptides, and XBP-1S polypeptides.
- Embodiment 26 The method of embodiment 24, wherein said inhibitor of UPR signaling is selected from the group consisting of eeyarestatin I, mycolactone, exotoxin A, NSC 630668-R/l, MAL3-39, MAL3-101, E6 berbamine, ophiobolin A, equisetin, CJ-21058, Rose Bengal, erythrosin B, P97-A4, P87-A4, 17D9, P91-E9, 16F6, bisthiouracil, SCA-21, HUN-7293, cotransin, CAM741, apratoxin A, decatransin, valinomycin, CAD A, kinase inhibiting RNase attenuator 6 (KIRA6), 3-hydroxy-2-naphthoic acid (3HNA), MKC-3946, 4- Phenylbutyric acid (4-PBA), taurine-conjugated ursodeoxycholic acid (TUDCA),
- Embodiment 27 A method for reducing tissue inflammation in a mammal, wherein said method comprises administering, to said mammal, an inhibitor of co-translational translocation, wherein said method is effective to reduce production of a TNF-a polypeptide within said mammal.
- Embodiment 28 The method of embodiment 27, wherein said inhibitor of co- translational translocation can target a co-translational translocation pathway component that is selected from the group consisting of Sec61 polypeptides, Hsp70 polypeptides, ATPase polypeptides, calmodulin polypeptides, transmembrane domains of newly produced polypeptides, signal peptides, and signal peptidase complex polypeptides.
- a co-translational translocation pathway component that is selected from the group consisting of Sec61 polypeptides, Hsp70 polypeptides, ATPase polypeptides, calmodulin polypeptides, transmembrane domains of newly produced polypeptides, signal peptides, and signal peptidase complex polypeptides.
- Embodiment 29 A method for reducing tissue inflammation in a mammal, wherein said method comprises administering, to said mammal, a composition comprising a mitochondrial metabolite, wherein said method is effective to reduce production of a TNF-a polypeptide within said mammal.
- Embodiment 30 The method of embodiment 29, wherein said mitochondrial metabolite is selected from the group consisting of L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, oxaloacetate, and nicotinamide riboside.
- Embodiment 31 The method of embodiment 29 or embodiment 30, wherein said composition comprises L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate.
- Embodiment 32 A method for reducing tissue inflammation in a mammal, wherein said method comprises administering, to said mammal, an inhibitor of aspartate transport or aspartate metabolism, wherein said method is effective to reduce production of a TNF-a polypeptide within said mammal.
- Embodiment 34 The method of embodiment 32, wherein said inhibitor of aspartate transport or aspartate metabolism is selected from the group consisting of DL-TBOA, L-(-)- threo-3-hydroxyaspartic acid, L-trans-2,4-PDC, TFB-TBOA, and GOT1 inhibitor 2c.
- Embodiment 35 The method of any one of embodiments 21-34, wherein said mammal is a human.
- Embodiment 36 The method of any one of embodiments 21-35, wherein said tissue inflammation is associated with an autoimmune disease.
- Embodiment 37 The method of embodiment 36, wherein said autoimmune disease is selected from the group consisting of polymyositis, dermatomyositis, rheumatoid arthritis (RA), scleroderma, sjogren's syndrome, systemic lupus erythematosus, vasculitis, mixed connective tissue disease (MCTD), autoimmune hepatitis, psoriasis, and ankylosing spondylitis.
- RA rheumatoid arthritis
- scleroderma scleroderma
- sjogren's syndrome systemic lupus erythematosus
- vasculitis vasculitis
- mixed connective tissue disease (MCTD) mixed connective tissue disease
- autoimmune hepatitis psoriasis
- ankylosing spondylitis ankylosing spondylitis.
- Embodiment 38 The method of embodiment 36, wherein said autoimmune disease is RA.
- Embodiment 39 The method of any one of embodiments 21-35, wherein said tissue inflammation is associated with IBS.
- Embodiment 40 The method of any one of embodiments 21-39, said method comprising administering to said mammal an anti-TNF-a therapy.
- Embodiment 41 The method of embodiment 40, wherein said anti-TNF-a therapy is selected from the group consisting of adalimumab, adalimumab-adbm, adalimumab-adaz, adalimumab-atto, certolizumab pegol, etanercept, etanercept-szzs, infliximab, infliximab- abda, and infliximab-dyyb.
- Embodiment 42 Embodiment 42.
- a method for treating a mammal having tissue inflammation comprises administering, to said mammal, a composition comprising cells, wherein a population of intact mitochondria was introduced into said cells, and wherein the production of a TNF-a polypeptide within said mammal is reduced following said administering step.
- Embodiment 43 The method of embodiment 42, wherein said composition comprises T cells containing said intact mitochondria.
- Embodiment 44 The method of embodiment 42 or embodiment 43, where said T cells are obtained from said mammal.
- Embodiment 45 A method for treating a mammal having tissue inflammation, wherein said method comprises administering, to said mammal, an inhibitor of UPR signaling, wherein said method is effective to reduce production of a TNF-a polypeptide within said mammal.
- Embodiment 46 The method of embodiment 45, wherein said inhibitor of UPR signaling can target an UPR signaling pathway component selected from the group consisting of BiP polypeptides, IREla polypeptides, PERK polypeptides, ATF6 polypeptides, eIF2a polypeptides, CHOP polypeptides, XBP-1 polypeptides, and XBP-1S polypeptides.
- an UPR signaling pathway component selected from the group consisting of BiP polypeptides, IREla polypeptides, PERK polypeptides, ATF6 polypeptides, eIF2a polypeptides, CHOP polypeptides, XBP-1 polypeptides, and XBP-1S polypeptides.
- Embodiment 47 The method of embodiment 46, wherein said inhibitor of UPR signaling is selected from the group consisting of eeyarestatin I, mycolactone, exotoxin A, NSC 630668-R/l, MAL3-39, MAL3-101, E6 berbamine, ophiobolin A, equisetin, CJ-21058, Rose Bengal, erythrosin B, P97-A4, P87-A4, 17D9, P91-E9, 16F6, bisthiouracil, SCA-21, HUN-7293, cotransin, CAM741, apratoxin A, decatransin, valinomycin, CAD A, kinase inhibiting RNase attenuator 6 (KIRA6), 3-hydroxy-2-naphthoic acid (3HNA), MKC-3946, 4- Phenylbutyric acid (4-PBA), taurine-conjugated ursodeoxycholic acid (TUDCA),
- Embodiment 48 A method for treating a mammal having tissue inflammation, wherein said method comprises administering, to said mammal, an inhibitor of co-translational translocation, wherein said method is effective to reduce production of a TNF-a polypeptide within said mammal.
- Embodiment 49 The method of embodiment 48, wherein said inhibitor of co- translational translocation can target a co-translational translocation pathway component that is selected from the group consisting of Sec61 polypeptides, Hsp70 polypeptides, ATPase polypeptides, calmodulin polypeptides, transmembrane domains of newly produced polypeptides, signal peptides, and signal peptidase complex polypeptides.
- a co-translational translocation pathway component that is selected from the group consisting of Sec61 polypeptides, Hsp70 polypeptides, ATPase polypeptides, calmodulin polypeptides, transmembrane domains of newly produced polypeptides, signal peptides, and signal peptidase complex polypeptides.
- Embodiment 50 A method for treating a mammal having tissue inflammation, wherein said method comprises administering, to said mammal, a composition comprising a mitochondrial metabolite, wherein said method is effective to reduce production of a TNF-a polypeptide within said mammal.
- Embodiment 51 The method of embodiment 50, wherein said mitochondrial metabolite is selected from the group consisting of L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, oxaloacetate, and nicotinamide riboside.
- Embodiment 52 The method of embodiment 50 or embodiment 51, wherein said composition comprises L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate.
- Embodiment 53 A method for treating a mammal having tissue inflammation, wherein said method comprises administering, to said mammal, an inhibitor of aspartate transport or aspartate metabolism, wherein said method is effective to reduce production of a TNF-a polypeptide within said mammal.
- Embodiment 54 The method of embodiment 53, wherein said inhibitor of aspartate transport or aspartate metabolism is selected from the group consisting of DL-TBOA, L-(-)- threo-3-hydroxyaspartic acid, L-trans-2,4-PDC, TFB-TBOA, and GOT1 inhibitor 2c.
- Embodiment 55 The method of any one of embodiments 42-54, wherein said mammal is a human.
- Embodiment 56 The method of any one of embodiments 42-55, wherein said tissue inflammation is associated with an autoimmune disease.
- Embodiment 57 The method of embodiment 56, wherein said autoimmune disease is selected from the group consisting of polymyositis, dermatomyositis, rheumatoid arthritis (RA), scleroderma, sjogren's syndrome, systemic lupus erythematosus, vasculitis, mixed connective tissue disease (MCTD), autoimmune hepatitis, psoriasis, and ankylosing spondylitis.
- RA rheumatoid arthritis
- scleroderma scleroderma
- sjogren's syndrome systemic lupus erythematosus
- vasculitis vasculitis
- mixed connective tissue disease (MCTD) mixed connective tissue disease
- autoimmune hepatitis psoriasis
- ankylosing spondylitis ankylosing spondylitis.
- Embodiment 58 The method of embodiment 57, wherein said autoimmune disease is RA.
- Embodiment 59 The method of any one of embodiments 42-55, wherein said tissue inflammation is associated with IBS.
- Embodiment 60 The method of any one of embodiments 42-46, said method comprising administering to said mammal an anti-TNF-a therapy.
- Embodiment 61 The method of embodiment 60, wherein said anti-TNF-a therapy is selected from the group consisting of adalimumab, adalimumab-adbm, adalimumab-adaz, adalimumab-atto, certolizumab pegol, etanercept, etanercept-szzs, infliximab, infliximab- abda, and infliximab-dyyb.
- Embodiment 62 A method for treating a mammal having tissue inflammation, wherein said method comprises administering, to said mammal, a) a composition comprising aspartate, and b) adalimumab; wherein said method is effective to reduce production of a TNF-a polypeptide within said mammal.
- Embodiment 63 The method of embodiment 62, wherein said composition comprising aspartate and said adalimumab are administered together.
- Embodiment 64 The method of embodiment 62, wherein said composition comprising aspartate and said adalimumab are administered separately.
- Embodiment 65 A method for treating a mammal having tissue inflammation, wherein said method comprises administering, to said mammal, a) a composition comprising aspartate, and b) etanercept; wherein said method is effective to reduce production of a TNF- a polypeptide within said mammal.
- Embodiment 66 The method of embodiment 65, wherein said composition comprising aspartate and said etanercept are administered together.
- Embodiment 67 The method of embodiment 65, wherein said composition comprising aspartate and said etanercept are administered separately.
- Embodiment 68 A method for treating a mammal having tissue inflammation, wherein said method comprises administering, to said mammal, a) a composition comprising aspartate, and b) infliximab; wherein said method is effective to reduce production of a TNF- a polypeptide within said mammal.
- Embodiment 69 The method of embodiment 68, wherein said composition comprising aspartate and said infliximab are administered together.
- Embodiment 70 The method of embodiment 68, wherein said composition comprising aspartate and said infliximab are administered separately.
- Embodiment 7E The method of any one of embodiments 62-70, wherein said mammal is a human
- Embodiment 72 The method of any one of embodiments 62-71, wherein said composition comprising aspartate further comprises a mitochondrial metabolite selected from the group consisting of NAD, alpha-ketobutyrate, malate, oxaloacetate, and nicotinamide riboside.
- a mitochondrial metabolite selected from the group consisting of NAD, alpha-ketobutyrate, malate, oxaloacetate, and nicotinamide riboside.
- Embodiment 73 The method of any one of embodiments 62-72, wherein said composition comprising aspartate comprises L-aspartate, D-aspartate, NAD, alpha- ketobutyrate, malate, and oxaloacetate.
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Abstract
This document provides methods and materials involved in treating mammals (e.g., humans) having tissue inflammation (e.g., autoimmune tissue inflammation). For example, one or more inhibitors of production of a tumor necrosis factor-α (TNF-α) polypeptide by, for example, T cells can be administered to a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation) to treat the mammal.
Description
TREATING TISSUE INFLAMMATION
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Patent Application Serial No. 63/234,636, filed on August 18, 2021. The disclosure of the prior application is considered part of (and is incorporated by reference in) the disclosure of this application.
SEQUENCE LISTING
This application contains a Sequence Listing that has been submitted electronically as an XML file named “07039-2075 WO 1. XML.” The XML file, created on August 1, 2022, is 41,000 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.
TECHNICAL FIELD
This document relates to methods and materials involved in treating mammals (e.g., humans) having tissue inflammation (e.g., autoimmune tissue inflammation). For example, one or more inhibitors of production of a tumor necrosis factor-a (TNF-a) polypeptide can be administered to a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation) to treat the mammal.
BACKGROUND INFORMATION
Direct targeting of TNF-a with biologic therapies has revolutionized the management of multiple autoimmune diseases (Rubbert-Roth et al., Autoimmun. Rev., 17:24-28 (2018); and Kalliolias et al., Nat. Rev. Rheumatol., 12:49-62 (2016)). TNF-a is a prime therapeutic target in rheumatoid arthritis (RA) and specific blockade of the cytokine is now considered standard therapy (Rubbert-Roth et al. , Autoimmun. Rev., 17:24-28 (2018)). However, low rates of disease remission, the development of adverse effects, and the generation of antibodies against biologic TNF-a inhibitors (Rubbert-Roth et al. , Autoimmun. Rev., 17:24- 28 (2018)) all curb the efficacy of anti-TNF therapy.
SUMMARY
This document provides methods and materials for treating mammals (e.g., humans) having tissue inflammation (e.g., autoimmune tissue inflammation). In some cases, one or more inhibitors of TNF-a polypeptide production can be administered to a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation) to treat the mammal. For example, intact mitochondria can be administered to a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation) to treat the mammal (e.g., to reduce TNF-a polypeptide production by, for example, T cells and treat the mammal). In another example, one or more inhibitors of unfolded protein response (UPR) signaling can be administered to a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation) to treat the mammal (e.g., to reduce TNF-a polypeptide production by, for example, T cells and treat the mammal). For example, one or more inhibitors for co-translational translocation can be administered to a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation) to treat the mammal (e.g., to reduce TNF-a polypeptide production by, for example, T cells and treat the mammal). In yet another example, one or more mitochondrial metabolites can be administered to a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation) to treat the mammal (e.g., to reduce TNF-a polypeptide production by, for example, T cells and treat the mammal). In some cases, one or more inhibitors of aspartate transport and/or aspartate metabolism can be administered to a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation) to treat the mammal (e.g., to reduce TNF-a polypeptide production by, for example, T cells and treat the mammal).
As demonstrated herein, the interventions described herein that block TNF-a polypeptide production can be used to suppress tissue inflammation. Having the ability to reduce TNF-a production by, for example, T cells as described herein provides a unique and unrealized strategy for treating tissue inflammation. For example, blocking production of a TNF-a polypeptide by, for example, T cells instead of inhibiting the produced TNF-a polypeptide provides an opportunity to treat tissue inflammation with a reduced risk of disease remission, the development of adverse effects, and the generation of antibodies against biologic TNF-a inhibitors.
In general, one aspect of this document features methods for restoring aspartate homeostasis in a mammal having tissue inflammation. The methods can include, or consist essentially of, administering, to a mammal having tissue inflammation, a composition comprising cells, where a population of intact mitochondria was introduced into the cells, and where the production of a TNF-a polypeptide within the mammal is reduced following the administering step. The composition can include T cells containing the intact mitochondria. The T cells can be obtained from the mammal. The mammal can be a human. The tissue inflammation can be associated with an autoimmune disease. The autoimmune disease can be polymyositis, dermatomyositis, rheumatoid arthritis (RA), scleroderma, sjogren's syndrome, systemic lupus erythematosus, vasculitis, mixed connective tissue disease (MCTD), autoimmune hepatitis, psoriasis, or ankylosing spondylitis. The autoimmune disease can be RA. The tissue inflammation can be associated with irritable bowel syndrome (IBS). The method can include administering to the mammal an anti-TNF-a therapy. The anti-TNF-a therapy can be adalimumab, adalimumab-adbm, adalimumab-adaz, adalimumab-atto, certolizumab pegol, etanercept, etanercept-szzs, infliximab, infliximab- abda, or infliximab-dyyb.
In another aspect, this document features methods for restoring aspartate homeostasis in a mammal having tissue inflammation. The methods can include, or consist essentially of, administering, to a mammal having tissue inflammation, an inhibitor of UPR signaling, where the method is effective to reduce production of a TNF-a polypeptide within the mammal. The inhibitor of UPR signaling can target an UPR signaling pathway component selected from the group consisting of binding immunoglobulin protein (BiP) polypeptides, IREla polypeptides, PERK polypeptides, ATF6 polypeptides, eIF2a polypeptides, CHOP polypeptides, XBP-1 polypeptides, and XBP-1S polypeptides. The inhibitor of UPR signaling can be eeyarestatin I, mycolactone, exotoxin A, NSC 630668-R/l, MAL3-39, MAL3-101, E6 berbamine, ophiobolin A, equisetin, CJ-21058, Rose Bengal, erythrosin B, P97-A4, P87-A4, 17D9, P91-E9, 16F6, bisthiouracil, SCA-21, HUN-7293, cotransin, CAM741, apratoxin A, decatransin, valinomycin, CAD A, kinase inhibiting RNase attenuator 6 (KIRA6), 3-hydroxy-2-naphthoic acid (3HNA), MKC-3946, 4-Phenylbutyric acid (4- PBA), taurine-conjugated ursodeoxycholic acid (TUDCA), olmesartan, N-acetylcysteine
(NAC), oleanolic acid (OA), ursolic acid, telmisartan, quercetin, 4p8C, STF-083010, B-109, GSK2606414, GSK2656157, AMG PERK44, melatonin, ceapin, IRSIB, AID 2732, salubrinal, ISRIB, guanabenz, sephinl, salicylaldimines, APY29, sunitinib, toyocamycin, 3- ethoxy-5,6-dibromosalicylal- dehyde, apigenin, FIRE peptide, baicalein, kaempferol, compound 147, compound 263, or 16F16. The mammal can be a human. The tissue inflammation can be associated with an autoimmune disease. The autoimmune disease can be polymyositis, dermatomyositis, rheumatoid arthritis (RA), scleroderma, sjogren's syndrome, systemic lupus erythematosus, vasculitis, mixed connective tissue disease (MCTD), autoimmune hepatitis, psoriasis, or ankylosing spondylitis. The autoimmune disease can be RA. The tissue inflammation can be associated with irritable bowel syndrome (IBS). The method can include administering to the mammal an anti-TNF-a therapy. The anti-TNF-a therapy can be adalimumab, adalimumab-adbm, adalimumab-adaz, adalimumab- atto, certolizumab pegol, etanercept, etanercept-szzs, infliximab, infliximab-abda, or infliximab-dyyb.
In another aspect, this document features methods for restoring aspartate homeostasis in a mammal having tissue inflammation. The methods can include, or consist essentially of, administering, to a mammal having tissue inflammation, an inhibitor of co-translational translocation, where the method is effective to reduce production of a TNF-a polypeptide within the mammal. The inhibitor of co-translational translocation can target a co- translational translocation pathway component that is selected from the group consisting of Sec61 polypeptides, Hsp70 polypeptides, ATPase polypeptides, calmodulin polypeptides, transmembrane domains of newly produced polypeptides, signal peptides, and signal peptidase complex polypeptides. The mammal can be a human. The tissue inflammation can be associated with an autoimmune disease. The autoimmune disease can be polymyositis, dermatomyositis, rheumatoid arthritis (RA), scleroderma, sjogren's syndrome, systemic lupus erythematosus, vasculitis, mixed connective tissue disease (MCTD), autoimmune hepatitis, psoriasis, or ankylosing spondylitis. The autoimmune disease can be RA. The tissue inflammation can be associated with irritable bowel syndrome (IBS). The method can include administering to the mammal an anti-TNF-a therapy. The anti-TNF-a therapy can be adalimumab, adalimumab-adbm, adalimumab-adaz, adalimumab-atto,
certolizumab pegol, etanercept, etanercept-szzs, infliximab, infliximab-abda, or infliximab- dyyb.
In another aspect, this document features methods for restoring aspartate homeostasis in a mammal having tissue inflammation. The methods can include, or consist essentially of, administering, to a mammal having tissue inflammation, a composition comprising a mitochondrial metabolite, where the method is effective to reduce production of a TNF-a polypeptide within the mammal. The mitochondrial metabolite can be L-aspartate, D- aspartate, NAD, alpha-ketobutyrate, malate, oxaloacetate, or nicotinamide riboside. The composition can include L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate. The mammal can be a human. The tissue inflammation can be associated with an autoimmune disease. The autoimmune disease can be polymyositis, dermatomyositis, rheumatoid arthritis (RA), scleroderma, sjogren's syndrome, systemic lupus erythematosus, vasculitis, mixed connective tissue disease (MCTD), autoimmune hepatitis, psoriasis, or ankylosing spondylitis. The autoimmune disease can be RA. The tissue inflammation can be associated with irritable bowel syndrome (IBS). The method can include administering to the mammal an anti-TNF-a therapy. The anti-TNF-a therapy can be adalimumab, adalimumab-adbm, adalimumab-adaz, adalimumab-atto, certolizumab pegol, etanercept, etanercept-szzs, infliximab, infliximab-abda, or infliximab-dyyb.
In another aspect, this document features methods for restoring aspartate homeostasis in a mammal having tissue inflammation. The methods can include, or consist essentially of, administering, to a mammal having tissue inflammation, an inhibitor of aspartate transport or aspartate metabolism, where the method is effective to reduce production of a TNF-a polypeptide within the mammal. The inhibitor of aspartate transport or aspartate metabolism can be DL-TBOA, L-(-)-threo-3 -hydroxyaspartic acid, L-trans-2,4-PDC, TFB-TBOA, or GOT1 inhibitor 2c. The mammal can be a human. The tissue inflammation can be associated with an autoimmune disease. The autoimmune disease can be polymyositis, dermatomyositis, rheumatoid arthritis (RA), scleroderma, sjogren's syndrome, systemic lupus erythematosus, vasculitis, mixed connective tissue disease (MCTD), autoimmune hepatitis, psoriasis, or ankylosing spondylitis. The autoimmune disease can be RA. The tissue inflammation can be associated with irritable bowel syndrome (IBS). The method can
include administering to the mammal an anti-TNF-a therapy. The anti-TNF-a therapy can be adalimumab, adalimumab-adbm, adalimumab-adaz, adalimumab-atto, certolizumab pegol, etanercept, etanercept-szzs, infliximab, infliximab-abda, or infliximab-dyyb.
In another aspect, this document features methods for reducing tissue inflammation in a mammal. The methods can include, or consist essentially of, administering, to a mammal, a composition comprising cells, where a population of intact mitochondria was introduced into the cells, and where the production of a TNF-a polypeptide within the mammal is reduced following the administering step. The composition can include T cells containing the intact mitochondria. The T cells can be obtained from the mammal. The mammal can be a human. The tissue inflammation can be associated with an autoimmune disease. The autoimmune disease can be polymyositis, dermatomyositis, RA, scleroderma, sjogren's syndrome, systemic lupus erythematosus, vasculitis, MCTD, autoimmune hepatitis, psoriasis, or ankylosing spondylitis. The autoimmune disease can be RA. The tissue inflammation can be associated with IBS. The method also can include administering to the mammal an anti- TNF-a therapy. The anti-TNF-a therapy can be adalimumab, adalimumab-adbm, adalimumab-adaz, adalimumab-atto, certolizumab pegol, etanercept, etanercept-szzs, infliximab, infliximab-abda, or infliximab-dyyb.
In another aspect, this document features methods for reducing tissue inflammation in a mammal. The methods can include, or consist essentially of, administering, to a mammal, an inhibitor of UPR signaling, where the method is effective to reduce production of a TNF-a polypeptide within the mammal. The inhibitor of UPR signaling can target an UPR signaling pathway component selected from the group consisting of BiP polypeptides, IRE la polypeptides, PERK polypeptides, ATF6 polypeptides, eIF2a polypeptides, CHOP polypeptides, XBP-1 polypeptides, and XBP-1S polypeptides. The inhibitor of UPR signaling can be eeyarestatin I, mycolactone, exotoxin A, NSC 630668-R/l, MAL3-39, MAL3-101, E6 berbamine, ophiobolin A, equisetin, CJ-21058, Rose Bengal, erythrosin B, P97-A4, P87-A4, 17D9, P91-E9, 16F6, bisthiouracil, SCA-21, HUN-7293, cotransin, CAM741, apratoxin A, decatransin, valinomycin, CAD A, kinase inhibiting RNase attenuator 6 (KIRA6), 3-hydroxy-2-naphthoic acid (3HNA), MKC-3946, 4-Phenylbutyric acid (4- PBA), taurine-conjugated ursodeoxycholic acid (TUDCA), olmesartan, N-acetylcysteine
(NAC), oleanolic acid (OA), ursolic acid, telmisartan, quercetin, 4p8C, STF-083010, B-109, GSK2606414, GSK2656157, AMG PERK44, melatonin, ceapin, IRSIB, AID 2732, salubrinal, ISRIB, guanabenz, sephinl, salicylaldimines, APY29, sunitinib, toyocamycin, 3- ethoxy-5,6-dibromosalicylal- dehyde, apigenin, FIRE peptide, baicalein, kaempferol, compound 147, compound 263, or 16F16. The mammal can be a human. The tissue inflammation can be associated with an autoimmune disease. The autoimmune disease can be polymyositis, dermatomyositis, RA, scleroderma, sjogren's syndrome, systemic lupus erythematosus, vasculitis, MCTD, autoimmune hepatitis, psoriasis, or ankylosing spondylitis. The autoimmune disease can be RA. The tissue inflammation can be associated with IBS. The method also can include administering to the mammal an anti-TNF-a therapy. The anti- TNF-a therapy can be adalimumab, adalimumab-adbm, adalimumab-adaz, adalimumab-atto, certolizumab pegol, etanercept, etanercept-szzs, infliximab, infliximab-abda, or infliximab- dyyb.
In another aspect, this document features methods for reducing tissue inflammation in a mammal. The methods can include, or consist essentially of, administering, to a mammal, an inhibitor of co-translational translocation, where the method is effective to reduce production of a TNF-a polypeptide within said mammal. The inhibitor of co-translational translocation can target a co-translational translocation pathway component that is selected from the group consisting of Sec61 polypeptides, Hsp70 polypeptides, ATPase polypeptides, calmodulin polypeptides, transmembrane domains of newly produced polypeptides, signal peptides, and signal peptidase complex polypeptides. The mammal can be a human. The tissue inflammation can be associated with an autoimmune disease. The autoimmune disease can be polymyositis, dermatomyositis, RA, scleroderma, sjogren's syndrome, systemic lupus erythematosus, vasculitis, MCTD, autoimmune hepatitis, psoriasis, or ankylosing spondylitis. The autoimmune disease can be RA. The tissue inflammation can be associated with IBS. The method also can include administering to the mammal an anti-TNF-a therapy. The anti- TNF-a therapy can be adalimumab, adalimumab-adbm, adalimumab-adaz, adalimumab-atto, certolizumab pegol, etanercept, etanercept-szzs, infliximab, infliximab-abda, or infliximab- dyyb.
In another aspect, this document features methods for reducing tissue inflammation in a mammal. The methods can include, or consist essentially of, administering, to a mammal, a composition comprising a mitochondrial metabolite, where the method is effective to reduce production of a TNF-a polypeptide within the mammal. The mitochondrial metabolite can be L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, oxaloacetate, or nicotinamide riboside. The composition can include L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate. The mammal can be a human. The tissue inflammation can be associated with an autoimmune disease. The autoimmune disease can be polymyositis, dermatomyositis, RA, scleroderma, sjogren's syndrome, systemic lupus erythematosus, vasculitis, MCTD, autoimmune hepatitis, psoriasis, or ankylosing spondylitis. The autoimmune disease can be RA. The tissue inflammation can be associated with IBS. The method also can include administering to the mammal an anti-TNF-a therapy. The anti- TNF-a therapy can be adalimumab, adalimumab-adbm, adalimumab-adaz, adalimumab-atto, certolizumab pegol, etanercept, etanercept-szzs, infliximab, infliximab-abda, or infliximab- dyyb.
In another aspect, this document features methods for reducing tissue inflammation in a mammal. The methods can include, or consist essentially of, administering, to a mammal, an inhibitor of aspartate transport or aspartate metabolism, where the method is effective to reduce production of a TNF-a polypeptide within the mammal. The inhibitor of aspartate transport or aspartate metabolism can be DL-TBOA, L-(-)-threo-3-hydroxyaspartic acid, L- trans-2,4-PDC, TFB-TBOA, or GOT I inhibitor 2c. The mammal can be a human. The tissue inflammation can be associated with an autoimmune disease. The autoimmune disease can be polymyositis, dermatomyositis, RA, scleroderma, sjogren's syndrome, systemic lupus erythematosus, vasculitis, MCTD, autoimmune hepatitis, psoriasis, or ankylosing spondylitis. The autoimmune disease can be RA. The tissue inflammation can be associated with IBS. The method also can include administering to the mammal an anti-TNF-a therapy. The anti- TNF-a therapy can be adalimumab, adalimumab-adbm, adalimumab-adaz, adalimumab-atto, certolizumab pegol, etanercept, etanercept-szzs, infliximab, infliximab-abda, or infliximab- dyyb.
In another aspect, this document features methods for treating a mammal having tissue inflammation. The methods can include, or consist essentially of, administering, to a mammal, a composition comprising cells, where a population of intact mitochondria was introduced into the cells, and where the production of a TNF-a polypeptide within the mammal is reduced following the administering step. The composition can include T cells containing the intact mitochondria. The T cells are obtained from the mammal. The mammal can be a human. The tissue inflammation can be associated with an autoimmune disease. The autoimmune disease can be polymyositis, dermatomyositis, RA, scleroderma, sjogren's syndrome, systemic lupus erythematosus, vasculitis, MCTD, autoimmune hepatitis, psoriasis, or ankylosing spondylitis. The autoimmune disease can be RA. The tissue inflammation can be associated with IBS. The method also can include administering to the mammal an anti-TNF-a therapy. The anti-TNF-a therapy can be adalimumab, adalimumab- adbm, adalimumab-adaz, adalimumab-atto, certolizumab pegol, etanercept, etanercept-szzs, infliximab, infliximab-abda, or infliximab-dyyb.
In another aspect, this document features methods for treating a mammal having tissue inflammation. The methods can include, or consist essentially of, administering, to a mammal, an inhibitor of UPR signaling, where the method is effective to reduce production of a TNF-a polypeptide within the mammal. The inhibitor of UPR signaling can target an UPR signaling pathway component selected from the group consisting of BiP polypeptides, IREla polypeptides, PERK polypeptides, ATF6 polypeptides, eIF2a polypeptides, CHOP polypeptides, XBP-1 polypeptides, and XBP-1S polypeptides. The inhibitor of UPR signaling can be eeyarestatin I, mycolactone, exotoxin A, NSC 630668-R/l, MAL3-39, MAL3-101, E6 berbamine, ophiobolin A, equisetin, CJ-21058, Rose Bengal, erythrosin B, P97-A4, P87-A4, 17D9, P91-E9, 16F6, bisthiouracil, SCA-21, HUN-7293, cotransin, CAM741, apratoxin A, decatransin, valinomycin, CAD A, kinase inhibiting RNase attenuator 6 (KIRA6), 3-hydroxy-2-naphthoic acid (3HNA), MKC-3946, 4-Phenylbutyric acid (4- PBA), taurine-conjugated ursodeoxycholic acid (TUDCA), olmesartan, N-acetylcysteine (NAC), oleanolic acid (OA), ursolic acid, telmisartan, quercetin, 4p8C, STF-083010, B-109, GSK2606414, GSK2656157, AMG PERK44, melatonin, ceapin, IRSIB, AID 2732, salubrinal, ISRIB, guanabenz, sephinl, salicylaldimines, APY29, sunitinib, toyocamycin, 3-
ethoxy-5, 6-dibromosalicylal- dehyde, apigenin, FIRE peptide, baicalein, kaempferol, compound 147, compound 263, or 16F16. The mammal can be a human. The tissue inflammation can be associated with an autoimmune disease. The autoimmune disease can be polymyositis, dermatomyositis, RA, scleroderma, sjogren's syndrome, systemic lupus erythematosus, vasculitis, MCTD, autoimmune hepatitis, psoriasis, or ankylosing spondylitis. The autoimmune disease can be RA. The tissue inflammation can be associated with IBS. The method also can include administering to the mammal an anti-TNF-a therapy. The anti- TNF-a therapy can be adalimumab, adalimumab-adbm, adalimumab-adaz, adalimumab-atto, certolizumab pegol, etanercept, etanercept-szzs, infliximab, infliximab-abda, or infliximab- dyyb.
In another aspect, this document features methods for treating a mammal having tissue inflammation. The methods can include, or consist essentially of, administering, to a mammal, an inhibitor of co-translational translocation, where the method is effective to reduce production of a TNF-a polypeptide within the mammal. The inhibitor of co- translational translocation can target a co-translational translocation pathway component that is selected from the group consisting of Sec61 polypeptides, Hsp70 polypeptides, ATPase polypeptides, calmodulin polypeptides, transmembrane domains of newly produced polypeptides, signal peptides, and signal peptidase complex polypeptides. The mammal can be a human. The tissue inflammation can be associated with an autoimmune disease. The autoimmune disease can be polymyositis, dermatomyositis, RA, scleroderma, sjogren's syndrome, systemic lupus erythematosus, vasculitis, MCTD, autoimmune hepatitis, psoriasis, or ankylosing spondylitis. The autoimmune disease can be RA. The tissue inflammation can be associated with IBS. The method also can include administering to the mammal an anti- TNF-a therapy. The anti-TNF-a therapy can be adalimumab, adalimumab-adbm, adalimumab-adaz, adalimumab-atto, certolizumab pegol, etanercept, etanercept-szzs, infliximab, infliximab-abda, or infliximab-dyyb.
In another aspect, this document features methods for treating a mammal having tissue inflammation. The methods can include, or consist essentially of, administering, to a mammal, a composition comprising a mitochondrial metabolite, where the method is effective to reduce production of a TNF-a polypeptide within the mammal. The
mitochondrial metabolite can be L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, oxaloacetate, or nicotinamide riboside. The composition can include L-aspartate, D- aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate. The mammal can be a human. The tissue inflammation can be associated with an autoimmune disease. The autoimmune disease can be polymyositis, dermatomyositis, RA, scleroderma, sjogren's syndrome, systemic lupus erythematosus, vasculitis, MCTD, autoimmune hepatitis, psoriasis, or ankylosing spondylitis. The autoimmune disease can be RA. The tissue inflammation can be associated with IBS. The method also can include administering to the mammal an anti- TNF-a therapy. The anti-TNF-a therapy can be adalimumab, adalimumab-adbm, adalimumab-adaz, adalimumab-atto, certolizumab pegol, etanercept, etanercept-szzs, infliximab, infliximab-abda, or infliximab-dyyb.
In another aspect, this document features methods for treating a mammal having tissue inflammation. The methods can include, or consist essentially of, administering, to a mammal, an inhibitor of aspartate transport or aspartate metabolism, where the method is effective to reduce production of a TNF-a polypeptide within the mammal. The inhibitor of aspartate transport or aspartate metabolism can be DL-TBOA, L-(-)-threo-3-hydroxyaspartic acid, L-trans-2,4-PDC, TFB-TBOA, or GOT1 inhibitor 2c. The mammal can be a human. The tissue inflammation can be associated with an autoimmune disease. The autoimmune disease can be polymyositis, dermatomyositis, RA, scleroderma, sjogren's syndrome, systemic lupus erythematosus, vasculitis, MCTD, autoimmune hepatitis, psoriasis, or ankylosing spondylitis. The autoimmune disease can be RA. The tissue inflammation can be associated with IBS. The method also can include administering to the mammal an anti- TNF-a therapy. The anti-TNF-a therapy can be adalimumab, adalimumab-adbm, adalimumab-adaz, adalimumab-atto, certolizumab pegol, etanercept, etanercept-szzs, infliximab, infliximab-abda, or infliximab-dyyb.
In another aspect, this document features methods for treating a mammal having tissue inflammation. The methods can include, or consist essentially of, administering, to a mammal, a) a composition comprising aspartate, and b) adalimumab; where the method is effective to reduce production of a TNF-a polypeptide within the mammal. The composition comprising aspartate and the adalimumab can be administered together. The composition
comprising aspartate and said adalimumab are administered separately. The mammal can be a human. The composition comprising aspartate also can include a mitochondrial metabolite selected from the group consisting of NAD, alpha-ketobutyrate, malate, oxaloacetate, and nicotinamide riboside. The composition comprising aspartate can include L-aspartate, D- aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate.
In another aspect, this document features methods for treating a mammal having tissue inflammation. The methods can include, or consist essentially of, administering, to said mammal, a) a composition comprising aspartate, and b) etanercept; where the method is effective to reduce production of a TNF-a polypeptide within the mammal. The composition comprising aspartate and the etanercept can be administered together. The composition comprising aspartate and the etanercept can be administered separately. The mammal can be a human. The composition comprising aspartate also can include a mitochondrial metabolite selected from the group consisting of NAD, alpha-ketobutyrate, malate, oxaloacetate, and nicotinamide riboside. The composition comprising aspartate can include L-aspartate, D- aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate.
In another aspect, this document features methods for treating a mammal having tissue inflammation . The methods can include, or consist essentially of, administering, to a mammal, a) a composition comprising aspartate, and b) infliximab; where the method is effective to reduce production of a TNF-a polypeptide within the mammal. The composition comprising aspartate and the infliximab can be administered together. The composition comprising aspartate and the infliximab can be administered separately. The mammal can be a human. The composition comprising aspartate also can include a mitochondrial metabolite selected from the group consisting of NAD, alpha-ketobutyrate, malate, oxaloacetate, and nicotinamide riboside. The composition comprising aspartate can include L-aspartate, D- aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
DESCRIPTION OF THE DRAWINGS
Figures 1A - IS. Mitochondrial insufficiency promotes endoplasmic reticulum (ER) expansion. Naive CD4+CD45RA+ T cells from patients with rheumatoid arthritis (RA) and age-matched healthy individuals were stimulated for 72 hours. Figures 1 A - 1C) Reduced mitochondrial fitness in RA T cells. Figure 1 A) Mitochondrial oxygen consumption rates (OCR) measured by Seahorse Analyzer. n=6 each. Figures IB and 1C) Mitochondrial membrane potential measured by flow cytometry (MitoTracker Red). n=7 each. Figure ID) Correlation of ER size (ER Tracker MFI) and mitochondrial membrane potential in healthy T cells (n=10). Figure IE) Confocal microscopy imaging of the ER enzyme protein disulfide isomerase (PDI) and mitochondrial membrane potential (MitoTracker Red). PDI (ER) arrow marks a cell with abundant ER, MitoTracker Red arrow marks a cell with high mitochondrial activity. Scale bar, 10 pm. Figure IF) Correlation of ER size (ER Tracker intensity) and mitochondrial membrane potential in cells gated based on cell size. Figures 1G - II) Expanded ER size in RA T cells. Figure 1G) Flow cytometric quantification of ER size (ER Tracker MFI); n = 5 each. Figures 1H and II) Confocal microscopy imaging of the ER chaperon protein calnexin in RA and healthy T cells. Scale bar, 10 pm. Single cell calnexin intensity quantification (50 T cells from 5 controls and 5 patients). Figures 1J - IN) ER stress response and ER expansion in T cells with low mitochondrial activity. Figure 1 J) ER
stress gene expression (qPCR) in healthy and RA T cells, n=6 each. Figure IK) Intracellular phosphatidylcholine concentrations in healthy and RA T cells, n = 6 healthy and 8 RA. Figure IL) Representative transmission electron microscope image of healthy and RA T cells. Arrows indicate ER. Scale bar, 1 pm. Figures IM and IN) Separation of two CD4+ T cell subpopulations based on mitochondrial membrane potential and qPCR analysis of ER stress genes in Mito MP111 and Mito MP10 T cells, n=4. Figures 10 and IP) Mitochondrial stress expands ER size. Healthy T cells were treated with or w/o the complex I inhibitor Rotenone (10 nM). Histogram of ER Tracker staining and collective MFI, n=4. Figures IQ - IS) Mitochondrial transfer corrects ER size. Figure IQ) Experimental scheme. CD4+CD45RA+ T cells from RA patients were stimulated with anti-CD3/CD28 for 48 hours, mitochondria isolated from healthy or RA CD4 T cells were transferred into RA T cells (donor cell number/recipient cell number = 10: 1). Mitochondrial membrane potential (Figure 1R) and ER size (Figure IS) in RA T cells after mitochondria transfer, n = 6. All data are mean ± SEM. Unpaired Mann- Whitney-Wilcoxon rank test (Figures 1 A - 1C and 1G - 1 J). Paired t test (Figures IN and IP). One-way ANOVA and post-ANOVA pair-wise two-group comparisons conducted with Tukey’s method (Figures 1R and IS). *P < 0.05, **P < 0.01, ***P < 0.001.
Figures 2A - 20. Mitochondria-derived aspartate controls ER size. Naive CD4+CD45RA+ T cells from patients with RA, and age-matched healthy individuals were stimulated for 72 hours. Figure 2A) Mitochondrial intermediates determine ER size. RA T cells were supplemented with the indicated mitochondrial intermediates (all 1 mM) and ER size was quantified flow cytometrically, n=6. Figure 2B) Scheme for the malate/aspartate shuttle. Figure 2C and 2D) RA T cells are aspartate/oxaloacetate deficient. Intracellular aspartate (Figure 2C) and oxaloacetate (Figure 2D) concentrations in healthy and RA T cells, each dot indicates one sample from a healthy control or a RA patient. Figures 2E and 2F) Aspartate inhibits ER stress signals and Phosphatidylcholine synthesis. RA T cells were treated with aspartate for 3 days. ER stress gene expression (Figure 2E) and Phosphatidylcholine content (Figure 2F) were measured. Figures 2G and 2H) Aspartate concentrations depend on intact mitochondrial function. Intracellular aspartate concentrations were measured after treatment of healthy T cells with the complex I inhibitor
Rotenone (lOnM; n=3) or after the transfer of healthy mitochondria into RA T cells (n=3). Figures 21 - 2K) Glutamic-Oxaloacetic Transaminase 2 (GOT2) regulates ER size. GOT2 was knocked down by si-RNA in healthy T cells. Figure 21) Intracellular aspartate levels, n=5; Figure 2J) ER size quantified by flow cytometric analysis (ER Tracker MFI), n=4; Figure 2K) ER stress genes quantified by qPCR, n=4. Figures 2L - 20) Aspartate is antiinflammatory. Synovitis was induced in chimeric mice engrafted with human synovial tissue and immunoreconstituted with RA PBMC. Chimeras were treated with vehicle or aspartate i.p. 5 mg/kg. Figure 2L) H&E staining of explanted synovial tissue. Figure 2M) Tissue transcriptomic analysis (qPCR) of synovial explants. Shown are data for T cell receptor (TRB) transcripts and the lineage-determining transcription factors TBX21 and RORC. Figure 2N) Co-immunofluorescence staining for IFN-y-producing CD3+ T cells in the synovial tissue. Representative images; scale bar, 10 pm. Figure 20) Tissue transcriptomic analysis of key inflammatory cytokines. 8 tissues in each study arm. All data are mean±SEM. One-way ANOVA and post-ANOVA pair-wise two-group comparisons conducted with Tukey’s method (Figures 2A and 2J). Paired t test (Figures 2E - 21 and 2K). Unpaired Mann- Whitney-Wilcoxon rank test (Figures 2C, 2D, 2M, and 20). *P < 0.05, **P < 0.01, ***P < 0.001.
Figures 3 A - 30. Aspartate is required for NAD regeneration and ADP-ribosylation of BiP. Naive CD4+CD45RA+ T cells from patients with RA, and age-matched healthy individuals were stimulated for 72 hours. Figures 3 A - 3C) NAD+ deficiency in RA T cells. Quantification of intracellular NAD+, NADH and the NAD/NADH ratio in healthy and RA T cells, n=6. Figures 3D and 3F) Aspartate and intact mitochondria regenerate NAD+. Figure 3D) NAD+/NADH ratios in RA T cells treated with or without aspartate, n=4. Figure 3E) NAD+/NADH ratios in RA T cells with or without mitochondrial transfer, n=3. Figure 3F) NAD+/NADH ratios in healthy T cells treated with or without the complex I inhibitor Rotenone (10 nM), n=3. Figures 3G - 31) NAD+ controls ER size and ER stress. RA T cells were treated with or withouto NAD. ER size was determined flow cytometrically (ER Tracker MFI). Figures 3G and 3H) Representative histograms. ER size measurements from 4 experiments. Figure 31) ER stress gene expression profiling (qPCR) in RA T cells treated with or without NAD+, n=4. Figures 3 J - 30) NAD-dependent ribosylation of BiP prevents
ER expansion and stabilizes Ire- la binding. Figure 3 J) Scheme of IREl-a activity controlled by BiP in the ER lumen. Figure 3K) ADP-ribosylation of BiP in healthy and RA T cells, n=3. Figure 3L) ADP-ribosylation of BiP in healthy CD4 T cells treated with or without Rotenone (10 nM) for 24 hours. n=3. Figure 3M) ADP-ribosylation of BiP in RA CD4 T cells treated with or without NAD (10 pM) for 24 hours. n=3. Figure 3N) BiP- IRE-la binding in healthy CD4+ T cells treated with 0, 10, 50 nM rotenone for 24 hours. n=3. Figure 30) BiP- IRE-la binding in activated RA CD4+ T cells treated with 0, 10, 20 pM NAD for 24 hours. n=3. All data are mean ± SEM. Unpaired Mann- Whitney -Wilcoxon rank test (Figures 3 A - 3C and 3K). Paired t test (Figures 3D - 31). One-way ANOVA and post-ANOVA pair-wise two-group comparisons conducted with Tukey’s method (Figures 3N and 30) . *P < 0.05, **P < 0.01, ***P < 0.001.
Figures 4A - 4R. T cells rich in rough ER are TNF-a-super producers. Naive CD4+CD45RA+ T cells from RA patients and age-matched controls were stimulated for 72 hours. Figures 4A - 4E) Enrichment of rough ER in RA T cells. Figures 4 A and 4B) Immunoblot analysis of the ER chaperon protein calnexin and the ribosomal proteins L-17 and S-7 in CD4+ T cells from 5 healthy individuals and 6 RA patients. Figures 4C and 4D) Rough ER was isolated from healthy and RA T cells 2 hours after restimulation. Figure 4C) Calnexin, ribosomal S7 and P-actin were quantified by immunoblotting. Figure 4D) Quantification of blot intensity for rough ER in each group, n=5 healthy and 4 RA. Figure 4E) TNF-a is the predominant cytokine expressed by activated naive CD4 T cells. Transcripts for T cell effector cytokines in activated naive CD4 T cells quantified by qPCR, n=3. Figure 4F) Healthy and RA T cells express similar level of TNFA mRNA. TNFA mRNA concentrations in healthy and RA T cells before (n=8 healthy and 8 RA) and after (n=5 healthy and 7 RA) PMA/ION stimulation (qPCR). Figure 4G) T cell stimulation induces enrichment of ER-bound mRNA for secretory proteins. Fold change of ER-bound mRNA for secretory proteins and intracellular proteins after PMA/ION stimulation, n=3. Figure 4H) Enrichment for ER-bound TNFA mRNA in RA T cells. Rough ER was isolated from healthy and RA T cells 2 hours after stimulation. mRNA associated with rough ER was quantified by qPCR. n=6. Figures 41 - 4K) ERnch RA T cells are TNF-a-superproducers. Figure 41) Representative confocal image of the ER chaperone protein Calnexin and TNF-a
in healthy and RA T cells. Figure 4J) Flow cytometric measurement of intracellular TNF-a in CD4+ T cells from RA patients and healthy individuals before and after PMA/ION stimulation. Figure 4K) TNF-a secreted into the extracellular space by unstimulated and stimulated RA and control CD4+ T cells. Figures 4L - 4P) Mitochondrial function and aspartate control TNF-a production. Figures 4L and 4M) Electron transfer was inhibited in healthy T cells with Rotenone (10 nM) or Piercidin A (10 pM). TNF-a was measured by flow cytometry, n=4 in each series. Figure 4N) GOT2 knockdown in healthy T cells, combined with or without aspartate rescue. TNF-a was measured by flow cytometry, n=4. Figure 40) TNF-a production in RA T cells treated with or without aspartate, n=3. Figure 4P) TNF-a production in RA T cells reconstituted with or without healthy mitochondria, n=3. Figure 4Q) TNF-a production in RA T cells treated with or w/o NAD+, n=4. Figure 4R) Scheme showing the aspartate-NAD-BiP pathway controlling TNF-a secretion. All data are mean ± SEM. Unpaired Mann- Whitney-Wilcoxon rank test (Figures 4A - 4K). Paired t test (Figures 4M - 4Q). *P < 0.05, **P < 0.01, ***P < 0.001.
Figures 5 A - 5N. TNF-a-producing CD4 T cells function as key arthritogenic effector cells. Figure 5 A and 5E) Cellular composition of leucocyte-rich and leukocyte-poor tissues collected from patients with rheumatoid synovitis. Figures 5B - 5D and 5F - 5H) TNF-a is a product of tissue T cells. Flow cytometric analysis of intracellular TNF-a in T cells, B cells and macrophages after stimulation with LPS/PMA/ION/BFA for 4 hours. Figures 5B and 5F) Histogram of TNF-a staining. Figures 5C and 5G) Frequencies of TNF- a-producing cell populations. Figures 5D and 5H) MFI of TNF-a staining in different cell populations. Figures 51 - 5K) Spontaneous TNF-a production in T cells and macrophages residing in the synovium. Freshly harvested synovial tissue from RA patients was incubated with or without the secretion inhibitor BFA for 4 hours, before cells were dissociated from the tissue and intracellular TNF-a was detected by flow cytometry. TNF-a+ CD45+ CD68+ macrophages (Figure 51) and TNF-a+ CD45+ CD3+ T cells (Figure 5J) in synovial tissue before and after BFA treatment. Figure 5K) Fold change in the frequency of TNF-a+ macrophage and TNF-a+ T cells after BFA treatment. n=3 tissues. Figures 5L - 5N) TNF-a- producing CD4+ T cells are an absolute requirement for rheumatoid synovitis. Rheumatoid synovitis was induced in human synovial tissues engrafted into NSG mice. CD4+T cells
from RA patients were transfected with control or TNFA siRNA and adoptively transferred into the chimeric mice. Synovial grafts were explanted two weeks later. 8 tissues in each group. Figure 5L) H&E staining of explanted synovial tissues. Figure 5M) Immunofluorescence staining of CD3+ T cells in synovial infiltrates. Scale bar; 10 pm. Figure 5N) Synovial tissue transcriptome for TRB, TBTT'T. RORG and other key inflammatory markers. All data are mean ± SEM. Paired t test (Figure 5K). Unpaired Mann-Whitney-Wilcoxon rank test (Figure 5N). *P < 0.05, **P < 0.01, ***p < 0.001.
Figure 6. Mitochondria mass in healthy and RA T cells. CD4+CD45RA+ T cells were stimulated for 72 hours. Flow cytometric quantification of mitochondria mass (MitoTrack Green MFI); n = 6 healthy and 6 RA. Data are mean ± SEM. Unpaired Mann- Whitney-Wilcoxon rank test.
Figure 7. Gating strategy to analyze ER and mitochondrial function on the single cell level. CD4+CD45RA+ T cells were stimulated for 72 hours. ER biomass was determined with ER tracker and mitochondrial function was assessed with the mitochondrial membrane potential. Gate #1: small cellular size. Gate #2: medium cellular size. Gate #3: large cellular size.
Figure 8. ER size in T cells from patients with psoriatic arthritis (PsA). CD4+CD45RA+ T cells were isolated from patients with PsA and age-matched healthy controls and stimulated for 72 hours. Flow cytometric quantification of ER size (ER Tracker MFI) in n=4 control-patient pairs. Data are mean ± SEM. Unpaired Mann- Whitney- Wilcoxon rank test.
Figures 9 A - 9E. XBP1S overexpression induces ER expansion. Healthy CD4+ T cells were stimulated and transfected with control or XBP1S overexpression plasmid before the ER size was determined. Figures 9A and 9B) Flow cytometry for ER Tracker staining; n=4. Figure 9C) Confocal microscopy imaging of the ER protein calnexin. Figures 9D and 9E) Flow cytometry for calnexin expression; n=3. All data are mean±SEM. Paired t test. *P < 0.05, ***P < 0.001.
Figure 10. Inhibitors of mitochondrial respiration promote ER expansion. Healthy naive CD4+ T cells were stimulated for 72 hours in the presence of the mitochondrial respiration inhibitors Piericidin A (10 pM), Antimycin A (10 nM) or Oligomycin (1 nM).
ER size was determined by flow cytometry measuring ER tracker (n=4). Data are mean±SEM. One-way ANOVA and post-ANOVA pair-wise two-group comparisons conducted with Tukey’s method. **P < 0.01.
Figures 11 A - 11C. Mitochondria transfer efficiency in Jurkat T cells. Figure 11 A) Experimental scheme. In Jurkat T cells, mitochondria were labeled with MitoTrackerRed and isolated. Mitochondria from donor cells were transferred into recipient cells. Figure 1 IB) Flow cytometric analysis of MitoTrackerRed intensity after mitochondria transfer. Ratio indicates donor cell number/recipient cell number. Figure 11C) Confocal imaging of exogenous mitochondria transferred into Jurkat T cells.
Figure 12. Expression of the Glutamic-Oxaloacetic Transaminases (GOT) 1 and 2 in healthy and RA T cells. Peripheral blood CD4+CD45RA+ T cells from RA patients and age- matched healthy individuals were isolated and stimulated for 72 hours. mRNA levels of GOT1 and GOT2 were determined by qPCR. n = 4 in each group. All data are mean ± SEM. Unpaired Mann- Whitney-Wilcoxon rank test.
Figure 13. Mitochondrial complex I inhibitor Piercidin A inhibits ADP-ribosylation of Bip. ADP-ribosylation of BiP in healthy CD4 T cells treated with or without Piercidin A (10 pM) for 24 hours, n = 2.
Figures 14A - 14B. Asparagine does not affect ER size and TNF-a production. Naive CD4+ T cells from RA patients were activated for 72 hours in the presence of Aspartate (1 mM) or Asparagine (1 mM). Figure 14 A) ER size was determined flow cytometrically with ER tracker (n=6). Figure 14B) Intracellular TNF-a was quantified by flow cytometry (n=3). Data are mean ± SEM. One-way ANOVA and post-ANOVA pair- wise two-group comparisons conducted with Tukey’s method. *P < 0.05, **P < 0.01.
Figures 15 A - 15B. Pyruvate and a-ketobutyrate (a-KB) inhibit ER expansion and TNF-a production in RA T cells. Naive CD4+ T cells from RA patients were stimulated for 72 hours in the presence of pyruvate (1 mM) or a-KB (1 mM), and ER size was determined with ER tracker (Figure 15 A, n=6). Intracellular TNF-a was measured by flow cytometry (Figure 15B, n=4). Data are mean ± SEM. One-way ANOVA and post-ANOVA pair- wise two-group comparisons conducted with Tukey’s method. *P < 0.01, **P < 0.01.
Figures 16A - 16B. Isolation of Rough ER. Figure 16A) Naive CD4+ T cells were purified from peripheral blood mononuclear cells and stimulated with anti-CD3/CD28 for 72 hours. The rough ER was isolated by calcium precipitation and the isolate was immunoblotted for the ER protein calnexin, the ribosomal protein LI 7 and the cytoplasmic protein a-actin. Figure 16B) Healthy CD4+ T cells were activated with PMA/Ionomycin for 2 hours before isolation of the rough ER and immunoblotting of the ER protein calnexin, the ribosomal protein S7 and the cytosolic protein P-actin.
Figures 17A - 17B. TNF-a production is unaffected by Tunicamycin. Naive CD4+ T cells were purified from peripheral blood mononuclear cells and stimulated with anti- CD3/CD28 beads for 72 hours in the presence of the ER stress inducer Tunicamycin. Figure 17A) Fold change of ER size compared to the control group, n=6. Figure 17B) Intracellular TNF-a concentrations measured by flow cytometry after PMA/ION stimulation for 2 hours in the presence of the secretion inhibitor BFA, n=4. All data are mean ± SEM, one-way ANOVA and post-ANOVA pair-wise two-group comparisons conducted with Tukey’s method, ***P < 0.001.
Figures 18A - 18B. Scheme for in vivo experiments. Figure 18A) NSG mice were engrafted with human synovial tissue and reconstituted with RA PBMC. Before the transfer, CD4 T cells were sorted and transfected with control or TNFA siRNA. Figure 18B) NSG mice were engrafted with human synovial tissue and reconstituted with RA PBMC. Before PBMC reconstitution, CD4 T cells were sorted and transferred with mitochondria isolated from healthy T cells.
Figures 19A - 19C. Mitochondria transfer into CD4+ T cells protects synovial tissue from inflammation. Mitochondria were isolated from healthy T cells and transferred into RA CD4+ T cells prior to their adoptive transfer into synovium-NSG chimeras. Explanted synovial grafts were analyzed by immunohistochemical staining and tissue transcriptomics (RT-PCR). 8 tissues in each group. Figure 19A) H&E staining of synovial tissue sections. Figure 19B) Immunofluorescence staining for CD3+ T cells in synovial infiltrates. Representative images. Scale bar; 10 pm. Figure 19C) Gene expression profiling (RT-PCR) of TRB, TBET, RORG and other key inflammatory markers. All data are mean ± SEM. Unpaired Mann- Whitney-Wilcoxon rank test. *P < 0.05, **P < 0.01, ***p < 0.001.
Figures 20A - 20B. Knockdown efficiency of TNFA and GOT2. Figure 20 A) TNFA mRNA transcripts measured by qPCR after knocking down TNFA with siRNA in healthy T cells, n=2. Figure 20B) GOT2 mRNA transcripts measured by qPCR after knocking down GOT2 with siRNA in healthy T cells, n=3. Paired t test. *P < 0.05, **P < 0.01.
Figure 21. Chemical structure of exemplary inhibitors of co-translational translocation including natural and synthetic inhibitors of co-translational translocation.
DETAILED DESCRIPTION
This document provides methods and materials for treating mammals (e.g., humans) having tissue inflammation (e.g., autoimmune tissue inflammation). In some cases, one or more inhibitors of TNF-a polypeptide production (e.g., inhibitors of TNF-a polypeptide production by T cells) can be administered to a mammal (e.g, a human) having tissue inflammation (e.g, autoimmune tissue inflammation) to treat the mammal. For example, intact mitochondria can be administered to a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation) to treat the mammal (e.g., to reduce TNF-a polypeptide production by, for example, T cells and treat the mammal). In some cases, one or more inhibitors of unfolded protein response (UPR) signaling can be administered to a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation) to treat the mammal (e.g., to reduce TNF-a polypeptide production by, for example, T cells and treat the mammal). For example, one or more inhibitors for co- translational translocation can be administered to a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation) to treat the mammal (e.g., to reduce TNF-a polypeptide production by, for example, T cells and treat the mammal). In some cases, one or more mitochondrial metabolites can be administered to a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation) to treat the mammal (e.g., to reduce TNF-a polypeptide production by, for example, T cells and treat the mammal). In some cases, one or more inhibitors of aspartate transport and/or aspartate metabolism can be administered to a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation) to treat the mammal (e.g., to reduce TNF-a polypeptide production by, for example, T cells and treat the mammal).
In some cases, one or more (e.g., one, two, three, four, or more) inhibitors of TNF-a polypeptide production (e.g., inhibitors of TNF-a polypeptide production by T cells) can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having tissue inflammation such as autoimmune tissue inflammation) to reduce tissue inflammation present within a mammal. For example, the materials and methods described herein can be used to reduce tissue inflammation present within a mammal having tissue inflammation (e.g., autoimmune tissue inflammation) by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.
In some cases, one or more (e.g., one, two, three, four, or more) inhibitors of TNF-a polypeptide production (e.g., inhibitors of TNF-a polypeptide production by T cells) can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having tissue inflammation such as autoimmune tissue inflammation) to reduce one or more symptoms of tissue inflammation (e.g., autoimmune tissue inflammation). Examples of symptoms of tissue inflammation (e.g., autoimmune tissue inflammation) include, without limitation, tender joints, warm joints, swollen joints, joint stiffness, fatigue, fever, and loss of appetite. For example, the materials and methods described herein can be used to reduce one or more symptoms of tissue inflammation (e.g., autoimmune tissue inflammation) in a mammal having tissue inflammation (e.g., autoimmune tissue inflammation) by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.
In some cases, one or more (e.g., one, two, three, four, or more) inhibitors of TNF-a polypeptide production (e.g., inhibitors of TNF-a polypeptide production by T cells) can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having tissue inflammation such as autoimmune tissue inflammation) to reduce a level of TNF-a polypeptides within the mammal. For example, the materials and methods described herein can be used to reduce a level of TNF-a polypeptides within a mammal having tissue inflammation (e.g., autoimmune tissue inflammation) by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.
In some cases, one or more (e.g., one, two, three, four, or more) inhibitors of TNF-a polypeptide production (e.g., inhibitors of TNF-a polypeptide production by T cells) can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having tissue
inflammation such as autoimmune tissue inflammation) to restore aspartate homeostasis within the mammal. For example, the materials and methods described herein can be used to restore a level of aspartate in cells within a mammal having tissue inflammation (e.g., autoimmune tissue inflammation) to from about 3 nmol/106 cells to about 10 nmol/106 (e.g., from about 3 nmol/106 cells to about 9 nmol/106, from about 3 nmol/106 cells to about 8 nmol/106, from about 3 nmol/106 cells to about 7 nmol/106, from about 3 nmol/106 cells to about 6 nmol/106, from about 3 nmol/106 cells to about 5 nmol/106, from about 3 nmol/106 cells to about 4 nmol/106, from about 4 nmol/106 cells to about 10 nmol/106, from about 5 nmol/106 cells to about 10 nmol/106, from about 6 nmol/106 cells to about 10 nmol/106, from about 7 nmol/106 cells to about 10 nmol/106, from about 8 nmol/106 cells to about 10 nmol/106, from about 9 nmol/106 cells to about 10 nmol/106, from about 4 nmol/106 cells to about 9 nmol/106, from about 5 nmol/106 cells to about 8 nmol/106, from about 6 nmol/106 cells to about 7 nmol/106, from about 4 nmol/106 cells to about 6 nmol/106, from about 5 nmol/106 cells to about 7 nmol/106, from about 6 nmol/106 cells to about 8 nmol/106, or from about 7 nmol/106 cells to about 9 nmol/106).
In some cases, one or more (e.g., one, two, three, four, or more) inhibitors of TNF-a polypeptide production (e.g., inhibitors of TNF-a polypeptide production by T cells) can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having tissue inflammation such as autoimmune tissue inflammation) to restore NAD+/NADH balance within the mammal. For example, the materials and methods described herein can be used to restore NAD+/NADH balance within a mammal having tissue inflammation (e.g., autoimmune tissue inflammation) to from about 4 NAD+/NADH to about 8 NAD+/NADH (e.g., from about 4 NAD+/NADH to about 7 NAD+/NADH, from about 4 NAD+/NADH to about 6 NAD+/NADH, from about 4 NAD+/NADH to about 5 NAD+/NADH, from about 5 NAD+/NADH to about 8 NAD+/NADH, from about 6 NAD+/NADH to about 8 NAD+/NADH, from about 7 NAD+/NADH to about 8 NAD+/NADH, from about 5 NAD7N ADH to about 6 NAD7N ADH, or from about 6 NAD7NADH to about 7 NAD7NADH).
In some cases, one or more (e.g., one, two, three, four, or more) inhibitors of TNF-a polypeptide production (e.g., inhibitors of TNF-a polypeptide production by T cells) can be
administered to a mammal (e.g., a human) in need thereof (e.g., a human having tissue inflammation such as autoimmune tissue inflammation) to restore phosphatidylcholine levels in cells within the mammal. For example, the materials and methods described herein can be used to restore phosphatidylcholine levels in cells within a mammal having tissue inflammation (e.g., autoimmune tissue inflammation) to from about 5 nmol/106 cells to about 10 nmol/106 cells (e.g., from about 5 nmol/106 cells to about 9 nmol/106 cells, from about 5 nmol/106 cells to about 8 nmol/106 cells, from about 5 nmol/106 cells to about 7 nmol/106 cells, from about 5 nmol/106 cells to about 6 nmol/106 cells, from about 6 nmol/106 cells to about 10 nmol/106 cells, from about 7 nmol/106 cells to about 10 nmol/106 cells, from about 8 nmol/106 cells to about 10 nmol/106 cells, from about 9 nmol/106 cells to about 10 nmol/106 cells, from about 6 nmol/106 cells to about 9 nmol/106 cells, from about 7 nmol/106 cells to about 8 nmol/106 cells, from about 6 nmol/106 cells to about 8 nmol/106 cells, or from about 7 nmol/106 cells to about 9 nmol/106 cells).
Any appropriate mammal having tissue inflammation (e.g., autoimmune tissue inflammation) can be treated as described herein. Examples of mammals that can have tissue inflammation (e.g., autoimmune tissue inflammation) and can be treated as described herein include, without limitation, humans, non-human primates (e.g., monkeys), dogs, cats, horses, cows, pigs, sheep, mice, and rats. In some cases, a human having tissue inflammation (e.g., autoimmune tissue inflammation) can be treated as described herein.
When treating a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation) as described herein, the tissue inflammation can be associated with a disease. In some cases, tissue inflammation can be associated with an autoimmune disease (e.g., autoimmune tissue inflammation). In some cases, tissue inflammation can be induced by one or more medications. Examples of diseases that can include tissue inflammation that can be treated as described herein include, without limitation, polymyositis, dermatomyositis, RA, scleroderma, sjogren’s syndrome, systemic lupus erythematosus, vasculitis, mixed connective tissue disease (MCTD), autoimmune hepatitis, irritable bowel syndrome (IBS), psoriasis, cancer immunotherapy induced inflammation, uveitis, and ankylosing spondylitis.
When treating a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation) as described herein, the tissue inflammation can be present in any appropriate tissue within the mammal. Examples of tissues that can be inflamed in association with a disease (e.g., an autoimmune disease) include, without limitation, synovial tissue, connective tissue, gastrointestinal tract (bowel) tissue, lung tissue, nervous system tissue, skin tissue, eye tissue, ear tissue, and blood vessels.
In some cases, the methods described herein can include identifying a mammal (e.g., a human) as having tissue inflammation (e.g., autoimmune tissue inflammation). Any appropriate method can be used to identify a mammal as having tissue inflammation (e.g., autoimmune tissue inflammation). For example, physical examinations (e.g., to check joints for swelling, redness, and/or warmth or to check reflexes and/or muscle strength), blood tests (e.g., to determine erythrocyte sedimentation rates (ESR) and/or to detect C-reactive protein (CRP) levels, rheumatoid factors, and/or anti-cyclic citrullinated peptide (anti-CCP) antibodies), imaging tests (e.g., X-rays, magnetic resonance imaging (MRI), and ultrasound tests), and/or laboratory tests (e.g., to check for autoantibodies and/or genetic markers, and/or for metabolomics) can be used to identify mammals (e.g., humans) having tissue inflammation (e.g., autoimmune tissue inflammation).
A mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation) can be administered or instructed to self-administer any or more (e.g., one, two, three, four, or more) inhibitors of TNF-a polypeptide production (e.g., inhibitors of TNF-a polypeptide production by T cells). In some cases when treating a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation), the mammal can be administered or instructed to self-administer any one or more agents that can inhibit TNF- a polypeptide expression. For example, nucleic acid molecules designed to induce RNA interference of TNF-a polypeptide expression (e.g., siRNA molecules and shRNA molecules) can be used to inhibit TNF-a polypeptide expression.
In some cases when treating a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation), the mammal can be administered or instructed to self-administer mitochondria (e.g., intact mitochondria). For example, intact mitochondria (e.g., a composition including intact mitochondria) can be administered to a mammal (e.g., a
human) having tissue inflammation (e.g., autoimmune tissue inflammation) to treat the mammal (e.g., to reduce TNF-a polypeptide production by, for example, T cells and treat the mammal). Mitochondria (e.g., intact mitochondria) to be administered to a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation) can be obtained from any type of cell. Examples of cells that mitochondria can be obtained from include, without limitation, T cells, cord blood cells, myeloid cells, stromal cells, and cell lines. Mitochondria (e.g., intact mitochondria) to be administered to a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation) can be obtained from any appropriate source. In some cases, mitochondria can be obtained from the mammal to be treated as described herein. In some cases, mitochondria can be obtained from a donor mammal. Any appropriate method can be used to obtain mitochondria (e.g., to isolate mitochondria from a cell). In some cases, mitochondria can be obtained as described in Example 1. In some cases, mitochondria can be administered to a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation) by administration directly into the inflamed tissue. Examples of tissues that can be inflamed and can be loaded with mitochondria include, without limitation, joint tissue, muscle, nervous tissue, skin tissue, connective tissue, and bone marrow. In some cases, mitochondria can be administered to a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation) using cells loaded with the exogenously added intact mitochondria. For example, cells containing (e.g., cells engineered to contain) mitochondria that are exogenous to those cells can be administered to a mammal (e.g., a human) having tissue inflammation to treat the mammal. In some cases, a cell can be engineered to contain (e.g., can be loaded with) mitochondria that are exogenous to that cell. Any appropriate cell can be loaded with mitochondria. Examples of cells that can be loaded with mitochondria include, without limitation, T cells, monocytes, macrophages, B cells, endothelial cells, stromal cells, and cells within bone marrow. Any appropriate method can be used to load a cell with mitochondria. In some cases, a cell can be loaded with mitochondria as described in Example 1. In some cases, T cells can be obtained from a mammal (e.g., a human) to be treated as described herein, the T cells can be loaded with intact mitochondria obtained from, e.g., healthy cells from that mammal or another mammal of the same species, and the
mitochondria-loaded T cells can be administered to the mammal. In some cases, cells other than T cells can be obtained from a mammal (e.g., a human) to be treated and mitochondria can be obtained from those cells, T cells can be obtained from the mammal, the T cells can be loaded with the obtained mitochondria, and the mitochondria-loaded T cells can be administered to the mammal.
In some cases when treating a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation), the mammal can be administered or instructed to self-administer one or more inhibitors of UPR signaling. For example, one or more inhibitors of UPR signaling (e.g., a composition including one or more inhibitors of UPR signaling) can be administered to a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation) to treat the mammal (e.g., to reduce TNF-a polypeptide production by, for example, T cells and treat the mammal). An inhibitor of UPR signaling can be any type of molecule (e.g., small molecules, polypeptides such as antibodies, and nucleic acids such as DNA, RNA, or DNA/RNA hybrids). In some cases, an inhibitor of UPR signaling can target any appropriate component within the UPR signaling pathway. Examples of components within the UPR signaling pathway that can be targeted by an inhibitor of UPR signaling include, without limitation, BiP polypeptides, IRE la polypeptides, PERK polypeptides, ATF6 polypeptides, eIF2a polypeptides, CHOP polypeptides, XBP-1 polypeptides, and XBP-1S polypeptides. In some cases, an inhibitor of UPR signaling can target any appropriate component within the co-translational translocation pathway. Examples of components within the co-translational translocation pathway that can be targeted by an inhibitor of UPR signaling include, without limitation, Sec61 polypeptides, Hsp70 polypeptides, ATPase polypeptides, calmodulin polypeptides, transmembrane domains of newly produced polypeptides, signal peptides, and signal peptidase complex polypeptides. In some cases, an inhibitor of UPR signaling can be an inhibitor of ER stress. Examples of inhibitors of UPR signaling include, without limitation, eeyarestatin I (3-(4-Chlorophenyl)-4- [ [(4-chlorophenyl)amino]carbonyl] hydroxyamino] -5 , 5 -dimethyl-2-oxo- 1 -imidazolidineacetic acid 2-[3-(5-nitro-2-furanyl)-2-propen-l-ylidene]hydrazide), mycolactone((6S,7S,9E,12R)- 12-[(2S,4E,6R,7R,9R)-7,9-Dihydroxy-4,6-dimethyldec-4-en-2-yl]-7,9-dimethyl-2-oxo-l- oxacy clododec-9-en-6-yl (2E,4E,6E, 8E, 10E, 12S,13S,15S)-12,13,15 -trihydroxy-4, 6, 10-
trimethylhexadeca-2,4,6,8, 10-pentaenoate), exotoxin A, NSC 630668-R/l (6-[5- (ethoxycarbonylcarbamoyl)-2, 4-dioxopyrimidin- 1 -yl] hexyl N- [6- [6-[ 5 - (ethoxycarbonylcarbamoyl)-2,4-dioxopyrimidin-l- yl]hexoxycarbonylamino]hexyl]carbamate), MAL3-39, MAL3-101 (4-[l,l'-Biphenyl]-4-yl- l-[6-[[2-(butylamino)-l-[3-(methoxycarbonyl)-4-(2-methoxy-2-oxoethoxy)phenyl]-2- oxoethyl]?hexylamino]-6-oxohexyl]-l,2,3,4-tetrahydro-6-methyl-2-oxo-5- pyrimidinecarboxylic acid phenylmethyl ester), E6 berbamine ([(1 S,14R)-20, 21,25- trimethoxy- 15,30-dimethyl-7, 23 -dioxa- 15,30- diazaheptacyclo[22.6.2.23,6.18,12.114,18.027,31.022,33]hexatriaconta- 3(36),4,6(35),8,10,12(34),18,20,22(33),24,26,31-dodecaen-9-yl] 4-nitrobenzoate), ophiobolin A ((rR,2S,3S,3'S,4'R,5R,7'S,8'E,H'R)-4'-hydroxy-r,3,4'-trimethyl-5-(2-methylprop-l-enyl)- 6'-oxospiro[oxolane-2,12'-tricyclo[9.3.0.03,7]tetradec-8-ene]-8'-carbaldehyde), equisetin ((3E,5S)-3-[[(lS,2R,4aS,6R,8aR)-l,6-dimethyl-2-[(E)-prop-l-enyl]-4a,5,6,7,8,8a-hexahydro- 2H-naphthalen-l-yl]-hydroxymethylidene]-5-(hydroxymethyl)-l-methylpyrrolidine-2,4- dione), CJ-21058 ((5R,Z)-3-(Hydroxy((lR,2S,6S,8aS)-l,3,6-trimethyl-2-((E)-prop-l-en-l- yl)-l,2,4a,5,6,7,8,8a-octahydro-naphthalen-l-yl)methylene)-5-(hydroxymethyl)-l- methylpyrrolidine-2, 4-dione), Rose Bengal (2-[2,7-bis(131I)(iodanyl)-4,5-diiodo-3-oxido-6- oxoxanthen-9-yl]-3,4,5,6-tetrachlorobenzoate), erythrosin B (3',6'-dihydroxy-2',4',5',7'- tetraiodospiro[2-benzofiiran-3,9'-xanthene]-l-one), P97-A4, P87-A4, 17D9, P91-E9, 16F6, bisthiouracil, SCA-21 (pyrimidine, 4,6-bis[[3-[3,5-bis(trifluoromethyl)phenyl]-lh-l,2,4- triazol-5 -yl]thio]-2-(methylthio)- / 4, 6-bis [ [3 - [3 , 5 -bis(trifluoromethyl)phenyl] - 1 h- 1 ,2, 4- triazol-5-yl]thio]-2-(methylthio)pyrimidine), HUN-7293 (3-[14-[(l-methoxyindol-3- yl)methyl]-7, 13,19,20-tetramethyl-5, 17-bis(2-methylhexyl)-8,ll-bis(2-methylpropyl)- 3 ,6,9, 12, 15, 18,21 -heptaoxo- 1 -oxa-4,7, 10, 13 , 16, 19-hexazacyclohenicos-2-yl]propanenitrile), cotransin ((3S,6S,9S,12S,15S, 18S,21R)-9-benzyl-3, 4, 10,16, 21-pentamethyl-6, 12, 15,18- tetrakis(2-methylpropyl)- 1 -oxa-4,7, 10, 13 , 16, 19-hexazacyclohenicosane-2, 5, 8, 11,14,17,20- heptone), CAM741 (propyl 3-[(2R,5S,8S,l lS,14S,17S,20S)-14-[(l-methoxyindol-3- yl)methyl]-7, 13,19,20-tetramethyl-5, 17-bis[(2R)-2-methylhexyl]-8,ll-bis(2-methylpropyl)- 3 ,6,9, 12, 15, 18,21 -heptaoxo- 1 -oxa-4,7, 10, 13 , 16, 19-hexazacyclohenicos-2-yl]propanoate), apratoxin A ((2S,3S,5S,7S,10S,16S,19S,22S,25E,27S)-16-[(2S)-butan-2-yl]-7-tert-butyl-3-
hydroxy-22-[(4-methoxyphenyl)methyl]-2,5,17,19,20,25-hexamethyl-8-oxa-29-thia- 14,17,20,23 , 30-pentazatricyclo[25.2.1.010,14]triaconta- 1 (30),25-diene-9, 15,18,21 ,24- pentone), decatransin, valinomycin ((3S,6S,9R,12R,15S,18S,21R,24R,27S,30S,33R,36R)- 6,18,30-trimethyl-3 ,9 ,12, 15 ,21 ,24,27 ,33 ,36-nona(propan-2-yl)-l ,7 ,13 ,19,25 ,31-hexaoxa- 4,10,16,22,28,34-hexazacyclohexatriacontane-2,5,8,ll,14,17,20,23,26,29,32,35-dodecone), CAD A ( 1 , 5 , 9 -tri azacyclododecane, 3 -methylene- 1 , 5 -bi s[(4-methylphenyl) sulfonyl] -9- (phenylmethyl)- / 3-methylene- l,5-bis[(4-methylphenyl)sulfonyl]-9-(phenylmethyl)- 1,5,9- triazacyclododecane), kinase inhibiting RNase attenuator 6 (KIRA6) (urea, n-[4-[8-amino-3- (1,1 -dimethylethyl)imidazo[ 1 , 5-a]pyrazin- 1 -y 1] - 1 -naphthalenyl]-n'-[3 - (trifluoromethyl)phenyl]- / n-[4- [8-amino-3 -(1,1 -dimethylethyl)imidazo[ 1 , 5-a]pyrazin- 1 -y 1] - l-naphthalenyl]-n'-[3-(trifluoromethyl)phenyl]urea), 3HNA (3-hydroxy-2-naphthoic acid), MKC-3946 (1-naphthalenecarboxaldehyde, 2-hydroxy-6-[5-[(4-methyl-l- piperazinyl)carbonyl]-2-thienyl]- / 2-hydroxy-6-[5-[(4-methyl-l-piperazinyl)carbonyl]-2- thienyl]-l-naphthalenecarboxaldehyde), 4-PB A (4-Phenylbutyric acid), taurine-conjugated ursodeoxycholic acid (TUDCA) (2-{(4R)-4-[(lR,3aS,3bR,4S,5aS,7R,9aS,9bS,l laR)-4,7- Dihydroxy-9a, 11 a-dimethylhexadecahydro-lH-cyclopenta[a]phenanthren- 1 - yl]pentanamido} ethane- 1 -sulfonic acid), olmesartan (5-(2-hydroxypropan-2-yl)-2-propyl-3- [[4-[2-(2H-tetrazol-5-yl)phenyl]phenyl]methyl]imidazole-4-carboxylic acid), NAC (N- acetylcysteine), oleanolic acid (OA), ursolic acid, telmisartan (2-[4-[[4-methyl-6-(l- methylbenzimidazol-2-yl)-2-propylbenzimidazol-l-yl]methyl]phenyl]benzoic acid), quercetin (2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxychromen-4-one), 4p8C (7-Hydroxy-4- methyl-2-oxo-2H-l -benzop yran-8-carboxaldehyde), STF-083010 (2 -thiophenesulfonamide, n-[(2-hydroxy-l-naphthalenyl)methylene]- / n-[(2-hydroxy-l-naphthalenyl)methylene]-2- thiophenesulfonamide), B-109 (N-[(4,6-dimethyl-2-oxo-3,4-dihydro-lH-pyridin-3- yl)methyl]-l,6-di(propan-2-yl)pyrazolo[3,4-b]pyridine-4-carboxamide), GSK2606414 (ethanone, l-[5-(4-amino-7-methyl-7h-pyrrolo[2,3-d]pyrimidin-5-yl)-2,3-dihydro-lh-indol- 1 -yl] -2- [3 -(trifluoro methyl)phenyl]- / 1 -[5 -(4-amino-7-methyl-7h-pyrrolo [2, 3 -d]pyrimidin-5 - yl)-2, 3 -dihydro- 1 h-indol- 1 - y 1 ] -2 - [3 -(trifluoro methyl)phenyl] ethenone), GSK2656157 (ethanone, l-[5-(4-amino-7-methyl-7h-pyrrolo[2,3-d]pyrimidin-5-yl)-4-fluoro-2,3-dihydro- 1 h-indol- l-yl]-2-(6-methyl-2-pyridinyl)- / l-[5-(4-amino-7-methyl-7h-pyrrolo[2,3-
d]pyrimidin-5-yl)-4-fluoro-2,3-dihydro-lh-indol-l-yl]-2-(6-methyl-2-pyridinyl)ethenone), AMG PERK44 (4- [2- Amino-4-methyl-3 -(2-methyl-6-quinolinyl)benzoyl]- 1 ,2-dihydro- 1 - methyl-2,5-diphenyl-3H-pyrazol-3-one hydrochloride), melatonin (N-[2-(5-methoxy-lH- indol-3-yl)ethyl]acetamide), ceapin (N-[l-[[2,4-bis(trifluoromethyl)phenyl]methyl]pyrazol-4- yl]-5-(fiiran-2-yl)-l,2-oxazole-3-carboxamide), ISRIB (acetamide, n,n'-trans-l,4- cyclohexanediylbis[2-(4-chlorophenoxy)- / n,n'-trans-l,4-cyclohexanediylbis[2-(4- chlorophenoxy)acetamide]), salubrinal (salubrinal / 2-propenamide, 3-phenyl-n-[2,2,2- trichloro-l-[[(8-quinolinylamino)thioxomethyl]amino]ethyl]-, (2e)- / (2e)-3 -phenyl-n- [2,2,2- trichloro-l-[[(8-quinolinylamino)thioxomethyl]amino]ethyl]-2-propenamide), guanabenz (2- [(E)-(2,6-dichlorophenyl)methylideneamino]guanidine), sephinl (2-[(E)-(2- chlorophenyl)methylideneamino]guanidine), 2-(Iminomethyl)phenol, APY29 (2-N-(3H- benzimidazol-5-yl)-4-N-(5-cyclopropyl-lH-pyrazol-3-yl)pyrimidine-2,4-diamine), sunitinib (N-[2-(diethylamino)ethyl]-5-[(Z)-(5-fluoro-2-oxo-lH-indol-3-ylidene)methyl]-2,4- dimethyl- 1 H-pyrrole-3 -carboxamide), toyocamy cin (4-amino-7- [(2R, 3R,4 S, 5R)-3 ,4- dihydroxy-5-(hydroxymethyl)oxolan-2-yl]pyrrolo[2,3-d]pyrimidine-5-carbonitrile), 3- ethoxy-5,6-dibromosalicylal- dehyde, apigenin (5,7-dihydroxy-2-(4- hydroxyphenyl)chromen-4-one), FIRE peptide, baicalein (5,6,7-trihydroxy-2- phenylchromen-4-one), kaempferol (3,5,7-trihydroxy-2-(4-hydroxyphenyl)chromen-4-one), compound 147 (benzenesulfonamide, 4-(3-thienyl)-n-[3-(trifhioromethyl)phenyl]-3-[[[[3- (trifluoromethyl)phenyl] amino] carbonyl] amino] - / 4-(3 -thienyl)-n- [3 - (trifluoromethy l)p heny 1] -3 - [ [ [ [3 - (trifluoromethyl)phenyl]amino]carbonyl]amino]benzenesulfonamide), compound 263 (benzaldehyde, 2-hydroxy-, 2-(4-nitrophenyl)hydrazone, [c(e)]- / [c(e)]-2- hydroxybenzaldehyde 2-(4-nitrophenyl)hydrazone), and 16F16 (lh-pyrido[3,4-b]indole-l- carboxylic acid, 2-(2-chloroacetyl)-2,3,4,9-tetrahydro-l-methyl-, methyl ester / methyl 2-(2- chloroacetyl)-2,3,4,9-tetrahydro-l-methyl-lh-pyrido[3,4-b]indole-l-carboxylate). . In some cases, inhibitors of UPR signaling can be as shown in Figure 21.
In some cases when treating a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation), the mammal can be administered or instructed to self-administer any one or more metabolites (e.g., mitochondrial metabolites). For example,
one or more mitochondrial metabolites (e.g., a composition including one or more mitochondrial metabolites) can be administered to a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation) to treat the mammal (e.g., to reduce TNF-a polypeptide production by, for example, T cells and treat the mammal). Any appropriate metabolite or combination of metabolites can be administered to a mammal (e.g., a human) as described herein. Examples of metabolites that can be administered to a mammal (e.g., a human) as described herein include, without limitation, aspartate (e.g., L- aspartate and D-aspartate), NAD, alpha-ketobutyrate, malate, oxaloacetate, and nicotinamide riboside. For example, L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate can be administered to a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation) to treat the mammal. Any appropriate amount of a metabolite can be administered to a mammal (e.g., a human) as described herein. In some cases, a composition including from about 10 mg to about 3000 mg (e.g., from about 10 mg to about 2500 mg, from about 10 mg to about 2000 mg, from about 10 mg to about 1500 mg, from about 10 mg to about 1000 mg, from about 10 mg to about 800 mg, from about 10 mg to about 500 mg, from about 10 mg to about 200 mg, from about 10 mg to about 100 mg, from about 100 mg to about 3000 mg, from about 300 mg to about 3000 mg, from about 500 mg to about 3000 mg, from about 700 mg to about 3000 mg, from about 1000 mg to about 3000 mg, from about 1500 mg to about 3000 mg, from about 2000 mg to about 3000 mg, from about 2500 mg to about 3000 mg, from about 100 mg to about 2500 mg, from about 500 mg to about 2000 mg, from about 1000 mg to about 1500 mg, from about 500 mg to about 1500 mg, or from about 1500 mg to about 1500 mg) of a metabolite can be administered to a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation). For example, a composition including from about 10 mg to about 3000 mg (e.g., e.g., from about 10 mg to about 2500 mg, from about 10 mg to about 2000 mg, from about 10 mg to about 1500 mg, from about 10 mg to about 1000 mg, from about 10 mg to about 800 mg, from about 10 mg to about 500 mg, from about 10 mg to about 200 mg, from about 10 mg to about 100 mg, from about 100 mg to about 3000 mg, from about 300 mg to about 3000 mg, from about 500 mg to about 3000 mg, from about 700 mg to about 3000 mg, from about 1000 mg to about 3000 mg, from about 1500 mg to about 3000 mg,
from about 2000 mg to about 3000 mg, from about 2500 mg to about 3000 mg, from about 100 mg to about 2500 mg, from about 500 mg to about 2000 mg, from about 1000 mg to about 1500 mg, from about 500 mg to about 1500 mg, or from about 1500 mg to about 1500 mg) of L-aspartate can be administered to a mammal (e.g., a human) having tissue inflammation to treat the mammal. For example, a composition including from about 10 mg to about 3000 mg of (e.g., e.g., from about 10 mg to about 2500 mg, from about 10 mg to about 2000 mg, from about 10 mg to about 1500 mg, from about 10 mg to about 1000 mg, from about 10 mg to about 800 mg, from about 10 mg to about 500 mg, from about 10 mg to about 200 mg, from about 10 mg to about 100 mg, from about 100 mg to about 3000 mg, from about 300 mg to about 3000 mg, from about 500 mg to about 3000 mg, from about 700 mg to about 3000 mg, from about 1000 mg to about 3000 mg, from about 1500 mg to about 3000 mg, from about 2000 mg to about 3000 mg, from about 2500 mg to about 3000 mg, from about 100 mg to about 2500 mg, from about 500 mg to about 2000 mg, from about 1000 mg to about 1500 mg, from about 500 mg to about 1500 mg, or from about 1500 mg to about 1500 mg) D-aspartate can be administered to a mammal (e.g., a human) having tissue inflammation to treat the mammal. For example, a composition including from about 10 mg to about 1000 mg (e.g., from about 10 mg to about 800 mg, from about 10 mg to about 500 mg, from about 10 mg to about 300 mg, from about 10 mg to about 100 mg, from about 10 mg to about 50 mg, from about 50 mg to about 1000 mg, from about 100 mg to about 1000 mg, from about 500 mg to about 1000 mg, from about 700 mg to about 1000 mg, from about 100 mg to about 800 mg, from about 300 mg to about 500 mg, from about 100 mg to about 300 mg, or from about 500 mg to about 700 mg) of NAD can be administered to a mammal (e.g., a human) having tissue inflammation to treat the mammal. For example, a composition including from about 10 mg to about 2000 mg (e.g., from about 10 mg to about 1500 mg, from about 10 mg to about 1000 mg, from about 10 mg to about 500 mg, from about 10 mg to about 100 mg, from about 100 mg to about 2000 mg, from about 500 mg to about 2000 mg, from about 700 mg to about 2000 mg, from about 1000 mg to about 2000 mg, from about 1500 mg to about 2000 mg, from about 100 mg to about 1500 mg, from about 500 mg to about 1000 mg, from about 100 mg to about 500 mg, from about 500 mg to about 800 mg, from about 800 mg to about 1000 mg, from about 1000 mg to about 1200 mg, from about
1200 mg to about 1500 mg, or from about 1500 mg to about 1800 mg) of alpha-ketobutyrate can be administered to a mammal (e.g., a human) having tissue inflammation to treat the mammal. For example, a composition including from about 10 mg to about 3000 mg (e.g., e.g., from about 10 mg to about 2500 mg, from about 10 mg to about 2000 mg, from about 10 mg to about 1500 mg, from about 10 mg to about 1000 mg, from about 10 mg to about 800 mg, from about 10 mg to about 500 mg, from about 10 mg to about 200 mg, from about 10 mg to about 100 mg, from about 100 mg to about 3000 mg, from about 300 mg to about 3000 mg, from about 500 mg to about 3000 mg, from about 700 mg to about 3000 mg, from about 1000 mg to about 3000 mg, from about 1500 mg to about 3000 mg, from about 2000 mg to about 3000 mg, from about 2500 mg to about 3000 mg, from about 100 mg to about 2500 mg, from about 500 mg to about 2000 mg, from about 1000 mg to about 1500 mg, from about 500 mg to about 1500 mg, or from about 1500 mg to about 1500 mg) of malate can be administered to a mammal (e.g., a human) having tissue inflammation to treat the mammal. For example, a composition including from about 10 mg to about 3000 mg (e.g., from about 10 mg to about 2500 mg, from about 10 mg to about 2000 mg, from about 10 mg to about 1500 mg, from about 10 mg to about 1000 mg, from about 10 mg to about 800 mg, from about 10 mg to about 500 mg, from about 10 mg to about 200 mg, from about 10 mg to about 100 mg, from about 100 mg to about 3000 mg, from about 300 mg to about 3000 mg, from about 500 mg to about 3000 mg, from about 700 mg to about 3000 mg, from about 1000 mg to about 3000 mg, from about 1500 mg to about 3000 mg, from about 2000 mg to about 3000 mg, from about 2500 mg to about 3000 mg, from about 100 mg to about 2500 mg, from about 500 mg to about 2000 mg, from about 1000 mg to about 1500 mg, from about 500 mg to about 1500 mg, or from about 1500 mg to about 1500 mg) of oxaloacetate can be administered to a mammal (e.g., a human) having tissue inflammation to treat the mammal.
In some cases when treating a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation), the mammal can be administered or instructed to self-administer any one or more inhibitors of aspartate transport and/or aspartate metabolism. For example, one or more inhibitors of aspartate transport and/or aspartate metabolism (e.g., a composition including inhibitors of aspartate transport and/or aspartate metabolism) can be administered to a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune
tissue inflammation) to treat the mammal (e.g., to reduce TNF-a polypeptide production by, for example, T cells and treat the mammal). An inhibitor of aspartate transport and/or aspartate metabolism can be any type of molecule (e.g., small molecules, polypeptides such as antibodies, and nucleic acids such as DNA, RNA, or DNA/RNA hybrids). In some cases, an inhibitor of aspartate transport and/or aspartate metabolism can inhibit a GOT1 polypeptide. In some cases, an inhibitor of aspartate transport and/or aspartate metabolism can agonize a GOT2 polypeptide. In some cases, an inhibitor of aspartate transport and/or aspartate metabolism can inhibit an EAAT1 polypeptide. Examples of inhibitors of aspartate transport and/or aspartate metabolism include, without limitation, DL-TBOA (dl-threo-P- benzyloxyaspartate), L-(-)-threo-3-hydroxyaspartic acid, L-trans-2,4-PDC (L-trans- Pyrrolidine-2,4-dicarboxylic acid), TFB-TBOA ((3S)-3-[[3-[[4- (Trifluoromethyl)benzoyl]amino]phenyl]methoxy]-L-aspartic acid), and GOT1 inhibitor 2c ( 1 -piperazinecarboxamide, n-(4-chlorophenyl)-4-(l h-indol-4-yl)-) .
In some cases, one or more (e.g., one, two, three, four, or more) inhibitors of TNF-a polypeptide production (e.g., inhibitors of TNF-a polypeptide production by T cells) can be formulated into a composition (e.g., a pharmaceutically acceptable composition) for administration to a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation). For example, one or more inhibitors of TNF-a polypeptide production can be formulated together with one or more pharmaceutically acceptable carriers (additives), excipients, and/or diluents. Examples of pharmaceutically acceptable carriers, excipients, and diluents that can be used in a composition described herein include, without limitation, sucrose, lactose, starch (e.g., starch glycolate), cellulose, cellulose derivatives (e.g., modified celluloses such as microcrystalline cellulose, and cellulose ethers like hydroxypropyl cellulose (HPC) and cellulose ether hydroxypropyl methylcellulose (HPMC)), xylitol, sorbitol, mannitol, gelatin, polymers (e.g., polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), crosslinked polyvinylpyrrolidone (crospovidone), carboxymethyl cellulose, polyethylene-polyoxypropylene-block polymers, and crosslinked sodium carboxymethyl cellulose (croscarmellose sodium)), titanium oxide, azo dyes, silica gel, fumed silica, talc, magnesium carbonate, vegetable stearin, magnesium stearate, aluminum stearate, stearic acid, antioxidants (e.g., vitamin A, vitamin E, vitamin C, retinyl palmitate, and selenium),
citric acid, sodium citrate, parabens (e.g., methyl paraben and propyl paraben), petrolatum, dimethyl sulfoxide, mineral oil, serum proteins (e.g., human serum albumin), glycine, sorbic acid, potassium sorbate, water, salts or electrolytes (e.g., saline, protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, and zinc salts), colloidal silica, magnesium trisilicate, polyacrylates, waxes, wool fat, lecithin, and corn oil.
In some cases, when a composition containing one or more inhibitors of TNF-a polypeptide production (e.g., inhibitors of TNF-a polypeptide production by T cells) is administered to a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation), the composition can be designed for oral or parenteral (including, without limitation, a subcutaneous, intravenous, intramuscular, intradermal, transdermal, intrathecal, or intraperitoneal (i.p.)) administration to the mammal. Compositions suitable for oral administration include, without limitation, liquids, tablets, capsules, pills, powders, gels, and granules. Compositions suitable for parenteral administration include, without limitation, aqueous and non-aqueous sterile injection solutions that can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient.
When one or more (e.g., one, two, three, four, or more) inhibitors of TNF-a polypeptide production (e.g., inhibitors of TNF-a polypeptide production by T cells) are formulated into a composition (e.g., a pharmaceutically acceptable composition) for administration to a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation), the composition can deliver the one or more inhibitors of TNF-a polypeptide production in any manner. In some cases, a composition including inhibitors of TNF-a polypeptide production can be an immediate release composition (e.g., an immediate release oral dosage form). In some cases, a composition including one or more inhibitors of TNF-a polypeptide production can be a controlled (e.g., delayed and/or sustained) release compositions (e.g., a controlled release oral dosage form). For example, a controlled release composition including one or more inhibitors of TNF-a polypeptide production can be designed to release the one or more inhibitors of TNF-a polypeptide production over 1, 2, 4, 7, or 10 days.
A composition containing one or more inhibitors of TNF-a polypeptide production (e.g., inhibitors of TNF-a polypeptide production by T cells) can be administered to a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation) in any appropriate amount (e.g., any appropriate dose). In some cases, an effective dose of a metabolite can be from about 0.15 milligrams per kilogram body weight (mg/kg) to about 45 mg/kg (e.g., from about 0.15 mg/kg to about 35 mg/kg, from about 0.15 mg/kg to about 25 mg/kg, from about 0.15 mg/kg to about 15 mg/kg, from about 0.15 mg/kg to about 10 mg/kg, from about 0.15 mg/kg to about 5 mg/kg, from about 0.15 mg/kg to about 2 mg/kg, from about 1 mg/kg to about 45 mg/kg, from about 5 mg/kg to about 45 mg/kg, from about 10 mg/kg to about 45 mg/kg, from about 15 mg/kg to about 45 mg/kg, from about 25 mg/kg to about 45 mg/kg, from about 35 mg/kg to about 45 mg/kg, from about 1 mg/kg to about 35 mg/kg, from about 5 mg/kg to about 25 mg/kg, from about 10 mg/kg to about 15 mg/kg, from about 5 mg/kg to about 15 mg/kg, or from about 15 mg/kg to about 35 mg/kg). For example, an effective dose of L-aspartate can be from about 0.15 mg/kg to about 45 mg/kg (e.g., from about 0.15 mg/kg to about 35 mg/kg, from about 0.15 mg/kg to about 25 mg/kg, from about 0.15 mg/kg to about 15 mg/kg, from about 0.15 mg/kg to about 10 mg/kg, from about 0.15 mg/kg to about 5 mg/kg, from about 0.15 mg/kg to about 2 mg/kg, from about 1 mg/kg to about 45 mg/kg, from about 5 mg/kg to about 45 mg/kg, from about 10 mg/kg to about 45 mg/kg, from about 15 mg/kg to about 45 mg/kg, from about 25 mg/kg to about 45 mg/kg, from about 35 mg/kg to about 45 mg/kg, from about 1 mg/kg to about 35 mg/kg, from about 5 mg/kg to about 25 mg/kg, from about 10 mg/kg to about 15 mg/kg, from about 5 mg/kg to about 15 mg/kg, or from about 15 mg/kg to about 35 mg/kg). For example, an effective dose of D-aspartate can be from about 0.15 mg/kg to about 45 mg/kg (e.g., e.g., from about 0.15 mg/kg to about 35 mg/kg, from about 0.15 mg/kg to about 25 mg/kg, from about 0.15 mg/kg to about 15 mg/kg, from about 0.15 mg/kg to about 10 mg/kg, from about 0.15 mg/kg to about 5 mg/kg, from about 0.15 mg/kg to about 2 mg/kg, from about 1 mg/kg to about 45 mg/kg, from about 5 mg/kg to about 45 mg/kg, from about 10 mg/kg to about 45 mg/kg, from about 15 mg/kg to about 45 mg/kg, from about 25 mg/kg to about 45 mg/kg, from about 35 mg/kg to about 45 mg/kg, from about 1 mg/kg to about 35 mg/kg, from about 5 mg/kg to about 25 mg/kg, from about 10 mg/kg to about 15 mg/kg, from about 5 mg/kg to
about 15 mg/kg, or from about 15 mg/kg to about 35 mg/kg). For example, an effective dose of NAD can be from about 0.75 mg/kg to about 15 mg/kg (e.g., from about 0.75 mg/kg to about 12 mg/kg, from about 0.75 mg/kg to about 10 mg/kg, from about 0.75 mg/kg to about 7 mg/kg, from about 0.75 mg/kg to about 5 mg/kg, from about 0.75 mg/kg to about 2 mg/kg, from about 3 mg/kg to about 15 mg/kg, from about 5 mg/kg to about 15 mg/kg, from about 8 mg/kg to about 15 mg/kg, from about 10 mg/kg to about 15 mg/kg, from about 12 mg/kg to about 15 mg/kg, from about 1 mg/kg to about 12 mg/kg, from about 5 mg/kg to about 10 mg/kg, from about 1 mg/kg to about 5 mg/kg, from about 3 mg/kg to about 7 mg/kg, or from about 8 mg/kg to about 12 mg/kg). For example, an effective dose of alpha-ketobutyrate can be from about 0.15 mg/kg to about 30 mg/kg (e.g., from about 0.15 mg/kg to about 25 mg/kg, from about 0.15 mg/kg to about 20 mg/kg, from about 0.15 mg/kg to about 15 mg/kg, from about 0.15 mg/kg to about 10 mg/kg, from about 0.15 mg/kg to about 5 mg/kg, from about 0.15 mg/kg to about 2 mg/kg, from about 1 mg/kg to about 30 mg/kg, from about 5 mg/kg to about 30 mg/kg, from about 10 mg/kg to about 30 mg/kg, from about 15 mg/kg to about 30 mg/kg, from about 20 mg/kg to about 30 mg/kg, from about 25 mg/kg to about 30 mg/kg, from about 1 mg/kg to about 25 mg/kg, from about 5 mg/kg to about 20 mg/kg, from about 10 mg/kg to about 15 mg/kg, from about 1 mg/kg to about 10 mg/kg, or from about 10 mg/kg to about 20 mg/kg). For example, an effective dose of malate can be from about 0.15 mg/kg to about 45 mg/kg (e.g., e.g., from about 0.15 mg/kg to about 35 mg/kg, from about 0.15 mg/kg to about 25 mg/kg, from about 0.15 mg/kg to about 15 mg/kg, from about 0.15 mg/kg to about 10 mg/kg, from about 0.15 mg/kg to about 5 mg/kg, from about 0.15 mg/kg to about 2 mg/kg, from about 1 mg/kg to about 45 mg/kg, from about 5 mg/kg to about 45 mg/kg, from about 10 mg/kg to about 45 mg/kg, from about 15 mg/kg to about 45 mg/kg, from about 25 mg/kg to about 45 mg/kg, from about 35 mg/kg to about 45 mg/kg, from about 1 mg/kg to about 35 mg/kg, from about 5 mg/kg to about 25 mg/kg, from about 10 mg/kg to about 15 mg/kg, from about 5 mg/kg to about 15 mg/kg, or from about 15 mg/kg to about 35 mg/kg). For example, an effective dose of oxaloacetate can be from about 0.15 mg/kg to about 45 mg/kg (e.g., e.g., from about 0.15 mg/kg to about 35 mg/kg, from about 0.15 mg/kg to about 25 mg/kg, from about 0.15 mg/kg to about 15 mg/kg, from about 0.15 mg/kg to about 10 mg/kg, from about 0.15 mg/kg to about 5 mg/kg, from about 0.15 mg/kg to about 2
mg/kg, from about 1 mg/kg to about 45 mg/kg, from about 5 mg/kg to about 45 mg/kg, from about 10 mg/kg to about 45 mg/kg, from about 15 mg/kg to about 45 mg/kg, from about 25 mg/kg to about 45 mg/kg, from about 35 mg/kg to about 45 mg/kg, from about 1 mg/kg to about 35 mg/kg, from about 5 mg/kg to about 25 mg/kg, from about 10 mg/kg to about 15 mg/kg, from about 5 mg/kg to about 15 mg/kg, or from about 15 mg/kg to about 35 mg/kg). In some cases, an effective dose of the total metabolite(s) can be from about 0.15 mg/kg to about 45 mg/kg (e.g., e.g., from about 0.15 mg/kg to about 35 mg/kg, from about 0.15 mg/kg to about 25 mg/kg, from about 0.15 mg/kg to about 15 mg/kg, from about 0.15 mg/kg to about 10 mg/kg, from about 0.15 mg/kg to about 5 mg/kg, from about 0.15 mg/kg to about 2 mg/kg, from about 1 mg/kg to about 45 mg/kg, from about 5 mg/kg to about 45 mg/kg, from about 10 mg/kg to about 45 mg/kg, from about 15 mg/kg to about 45 mg/kg, from about 25 mg/kg to about 45 mg/kg, from about 35 mg/kg to about 45 mg/kg, from about 1 mg/kg to about 35 mg/kg, from about 5 mg/kg to about 25 mg/kg, from about 10 mg/kg to about 15 mg/kg, from about 5 mg/kg to about 15 mg/kg, or from about 15 mg/kg to about 35 mg/kg). An effective amount of a composition containing one or more inhibitors of TNF-a polypeptide production can be any amount that can treat a mammal having tissue inflammation (e.g., autoimmune tissue inflammation) as described herein without producing significant toxicity to the mammal. The effective amount can remain constant or can be adjusted as a sliding scale or variable dose depending on the mammal’s response to treatment. Various factors can influence the actual effective amount used for a particular application. For example, the frequency of administration, duration of treatment, use of multiple treatment agents, route of administration, and/or severity of the cancer in the mammal being treated may require an increase or decrease in the actual effective amount administered.
A composition containing one or more inhibitors of TNF-a polypeptide production (e.g., inhibitors of TNF-a polypeptide production by T cells) can be administered to a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation) in any appropriate frequency. The frequency of administration can be any frequency that can treat a mammal having tissue inflammation (e.g., autoimmune tissue inflammation) without producing significant toxicity to the mammal. For example, the frequency of administration
can be from about once a day to about once a week, from about once a week to about once a month, or from about twice a month to about once a month. The frequency of administration can remain constant or can be variable during the duration of treatment. As with the effective amount, various factors can influence the actual frequency of administration used for a particular application. For example, the effective amount, duration of treatment, use of multiple treatment agents, and/or route of administration may require an increase or decrease in administration frequency.
A composition containing one or more inhibitors of TNF-a polypeptide production (e.g., inhibitors of TNF-a polypeptide production by T cells) can be administered to a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation) for any appropriate duration. An effective duration for administering or using a composition described herein can be any duration that can treat a mammal having tissue inflammation (e.g., autoimmune tissue inflammation) without producing significant toxicity to the mammal. For example, the effective duration can vary from several weeks to several months, from several months to several years, or from several years to a lifetime. Multiple factors can influence the actual effective duration used for a particular treatment. For example, an effective duration can vary with the frequency of administration, effective amount, use of multiple treatment agents, and/or route of administration.
In some cases, methods for treating a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation) can include administering to the mammal one or more inhibitors of TNF-a polypeptide production (e.g., inhibitors of TNF-a polypeptide production by T cells) as the sole active ingredient to treat the mammal. For example, a composition containing one or more inhibitors of TNF-a polypeptide production can include the one or more inhibitors of TNF-a polypeptide production as the sole active ingredient in the composition that is effective to treat a mammal having tissue inflammation (e.g., autoimmune tissue inflammation). For example, a composition containing one or more metabolites (e.g., mitochondrial metabolites such as L-aspartate, D-aspartate, NAD, alphaketobutyrate, malate, and oxaloacetate) can include the one or metabolites as the sole active ingredient in the composition that is effective to treat a mammal having tissue inflammation (e.g., autoimmune tissue inflammation).
In some cases, methods for treating a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation) as described herein (e.g., by administering one or more inhibitors of TNF-a polypeptide production) also can include administering to the mammal one or more (e.g., one, two, three, four, five or more) additional agents/therapies used to treat tissue inflammation (e.g., autoimmune tissue inflammation). In some cases, an agent that can be used to treat tissue inflammation can be an anti-TNF-a therapy. Examples of anti-TNF-a therapies that can be used to treat tissue inflammation (e.g., autoimmune tissue inflammation) and can be used together with one or more inhibitors of TNF-a polypeptide production described herein include, without limitation, adalimumab (e.g., HUMIRA®), adalimumab-adbm (e.g., Cyltezo®), adalimumab-adaz (e.g., HYRIMOZ™), adalimumab-atto (e.g., AMJEVITA™), certolizumab pegol (e.g., CIMZIA®), etanercept (e.g., Enbrel®), etanercept-szzs (e.g., ERELZI™), infliximab (e.g., REMICADE®), infliximab-abda (e.g., KEVZARA®), infliximab-dyyb (e.g., INFLECTRA®), and bio-similars of anti-TNF-a therapies. In cases where an agent that can be used to treat tissue inflammation is an anti-TNF-a therapy, the anti-TNF-a therapy can be administered every 2, 4, 6, or 8 weeks.
In some cases, an agent that can be used to treat tissue inflammation can be a diseasemodifying antirheumatic drug (DMARD; e.g., biologic DMARDs and synthetic DMARDs). Examples of DMARDs that can be used to treat tissue inflammation (e.g., autoimmune tissue inflammation) and can be used together with one or more inhibitors of TNF-a polypeptide production described herein include, without limitation, methotrexate (e.g., Trexall® and OTREXUP®), leflunomide (e.g., Arava®), hydroxychloroquine (e.g., Plaquenil®), sulfasalazine (e.g., Azulfidine®), abatacept (e.g., ORENCIA®), adalimumab (e.g., HUMIRA®), adalimumab-adbm (e.g., Cyltezo®), adalimumab-adaz (e.g., HYRIMOZ™), adalimumab-atto (e.g., AMJEVITA™), anakinra (e.g., KINERET®), certolizumab pegol (e.g., CIMZIA®), etanercept (e.g., Enbrel®), etanercept-szzs (e.g., ERELZI™), golimumab (e.g., SIMPONI®), infliximab (e.g., REMICADE®), infliximab-abda (e.g., KEVZARA®), infliximab-dyyb (e.g. , INFLECTRA®), rituximab (e.g. , RITUXAN®), sarilumab (e.g. , KEVZARA®), tocilizumab (e.g. , Actemra®), baricitinib (e.g. , Olumiant®), tofacitinib (e.g. , Xeljanz®), upadacitinib (e.g., RINVOQ™), azathioprin, thiopurine, and mycophenolate.
In some cases, an agent that can be used to treat tissue inflammation can be a nonsteroidal anti-inflammatory drug (NSAID). Examples of NSAIDs that can be used to treat tissue inflammation (e.g., autoimmune tissue inflammation) and can be used together with one or more inhibitors of TNF-a polypeptide production (e.g., inhibitors of TNF-a polypeptide production by T cells) described herein include, without limitation, ibuprofen (e.g., Advil® and MOTRIN® IB) and naproxen sodium (e.g., ALEVE®).
In some cases, an agent that can be used to treat tissue inflammation can be a steroid (e.g., a corticosteroid). Examples of steroids that can be used to treat tissue inflammation (e.g., autoimmune tissue inflammation) and can be used together with one or more inhibitors of TNF-a polypeptide production (e.g., inhibitors of TNF-a polypeptide production by T cells) described herein include, without limitation, prednisone, and prednisolone.
In cases where one or more inhibitors of TNF-a polypeptide production (e.g., inhibitors of TNF-a polypeptide production by T cells) are used in combination with additional agents used to treat tissue inflammation (e.g., autoimmune tissue inflammation), the one or more additional agents can be administered at the same time (e.g., in a single composition containing both one or more inhibitors of TNF-a polypeptide production and the one or more additional agents) or independently. For example, one or more inhibitors of TNF-a polypeptide production described herein can be administered first, and the one or more additional agents administered second, or vice versa.
Examples of therapies that can be used to treat tissue inflammation (e.g., autoimmune tissue inflammation) include, without limitation, surgery (e.g., synovectomy, tendon repair, joint fusion, and total joint replacement), gentle exercise, applying heat or cold, and relaxation (e.g., guided imagery, deep breathing, and muscle relaxation).
In cases where one or more inhibitors of TNF-a polypeptide production (e.g., inhibitors of TNF-a polypeptide production by T cells) described herein are used in combination with one or more additional therapies used to treat tissue inflammation (e.g., autoimmune tissue inflammation), the one or more additional therapies can be performed at the same time or independently of the administration of one or more inhibitors of TNF-a polypeptide production described herein. For example, one or more inhibitors of TNF-a
polypeptide production can be administered before, during, or after the one or more additional therapies are performed.
In some cases, a mammal (e.g., a human) having tissue inflammation (e.g., autoimmune tissue inflammation) can be administered one or more (e.g., one, two, three, four, five or more) metabolites (e.g., mitochondrial metabolites such as L-aspartate, D- aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate) and can be administered one or more (e.g., one, two, three, four, five or more) anti-TNF-a therapies to treat the mammal. For example, a mammal having tissue inflammation can be administered aspartate (e.g., a composition including D-aspartate and/or L-aspartate) and can be administered one or more anti-TNF-a therapies.
In some cases, a mammal having tissue inflammation can be administered (e.g., by subcutaneous injection) adalimumab (e.g., from about 40 mg to about 80 mg adalimumab) and can be administered aspartate (e.g., a composition including from about 10 mg to about 3000 mg of aspartate (e.g., D-aspartate and/or L-aspartate)). For example, a mammal having tissue inflammation can be administered a composition that includes both adalimumab and aspartate. For example, a mammal having tissue inflammation can be administered adalimumab and can be separately administered aspartate. When a mammal is administered adalimumab and is separately administered aspartate, the adalimumab and the aspartate can be administered within 1 to 15 minutes of each other. In some cases, the administration of adalimumab and aspartate can be repeated (e.g., can be repeated about every week or about every 2 weeks).
In some cases, a mammal having tissue inflammation can be administered (e.g., by subcutaneous injection) etanercept (e.g., about 50 mg etanercept) and can be administered aspartate (e.g., a composition including from about 10 mg to about 3000 mg of aspartate (e.g., D-aspartate and/or L-aspartate)). For example, a mammal having tissue inflammation can be administered a composition that includes both etanercept and aspartate. For example, a mammal having tissue inflammation can be administered etanercept and can be separately administered aspartate. When a mammal is administered etanercept and is separately administered aspartate, the etanercept and the aspartate can be administered within 1 to 15
minutes of each other. In some cases, the administration of etanercept and aspartate can be repeated (e.g., can be repeated about every week).
In some cases, a mammal having tissue inflammation can be administered (e.g., by subcutaneous injection) infliximab (e.g., from about 5 mg/kg to about 10 mg/kg infliximab) and can be administered aspartate (e.g., a composition including from about 10 mg to about 3000 mg of aspartate (e.g., D-aspartate and/or L-aspartate)). For example, a mammal having tissue inflammation can be administered a composition that includes both infliximab and aspartate. For example, a mammal having tissue inflammation can be administered infliximab and can be separately administered aspartate. When a mammal is administered infliximab and is separately administered aspartate, the infliximab and the aspartate can be administered within 1 to 15 minutes of each other. In some cases, the administration of infliximab and aspartate can be repeated (e.g., can be repeated about every 8 weeks).
In some cases, a mammal having tissue inflammation can be administered two or more of mitochondrial metabolites (e.g., a composition including L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate) and can be administered one or more anti-TNF-a therapies. For example, a mammal having tissue inflammation can be administered (e.g., by subcutaneous injection) adalimumab (e.g., from about 40 mg to about 80 mg adalimumab) and can be administered two or more mitochondrial metabolites (e.g., L- aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate such as a composition including from about 10 mg to about 3000 mg of total metabolites of L- aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate). In some cases, a mammal having tissue inflammation can be administered a composition that includes adalimumab, L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate. In some cases, a mammal having tissue inflammation can be administered adalimumab and can be separately administered two or more mitochondrial metabolites (e.g., L-aspartate, D- aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate). When a mammal is administered adalimumab and is separately administered two or more mitochondrial metabolites (e.g., L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate), the adalimumab and the two or more mitochondrial metabolites can be administered within 1 to 15 minutes of each other. In some cases, the administration of
adalimumab, L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate can be repeated (e.g., can be repeated about every week or about every 2 weeks).
In some cases, a mammal having tissue inflammation can be administered (e.g., by subcutaneous injection) etanercept (e.g., about 50 mg etanercept) and can be administered two or more mitochondrial metabolites (e.g., L-aspartate, D-aspartate, NAD, alpha- ketobutyrate, malate, and oxaloacetate such as a composition including from about 10 mg to about 3000 mg of total metabolites of L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate). For example, a mammal having tissue inflammation can be administered a composition that includes etanercept, L-aspartate, D-aspartate, NAD, alpha- ketobutyrate, malate, and oxaloacetate. For example, a mammal having tissue inflammation can be administered etanercept and can be separately administered two or more mitochondrial metabolites (e.g., L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate). When a mammal is administered etanercept and is separately administered two or more mitochondrial metabolites (e.g., L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate), the etanercept and the two or more mitochondrial metabolites can be administered within 1 to 15 minutes of each other. In some cases, the administration of etanercept, L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate can be repeated (e.g., can be repeated about every week).
In some cases, a mammal having tissue inflammation can be administered (e.g., by subcutaneous injection) infliximab (e.g., about 5 mg/kg to about 10 mg/kg infliximab) and can be administered two or more mitochondrial metabolites (e.g., L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate such as a composition including from about 10 mg to about 3000 mg of total metabolites of L-aspartate, D-aspartate, NAD, alpha- ketobutyrate, malate, and oxaloacetate). For example, a mammal having tissue inflammation can be administered a composition that includes infliximab, L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate. For example, a mammal having tissue inflammation can be administered infliximab and can be separately administered two or more mitochondrial metabolites (e.g., L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate). When a mammal is administered infliximab and is separately administered two or more mitochondrial metabolites (e.g., L-aspartate, D-aspartate, NAD,
alpha-ketobutyrate, malate, and oxaloacetate), the infliximab and the two or more mitochondrial metabolites can be administered within 1 to 15 minutes of each other. In some cases, the administration of infliximab, L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate can be repeated (e.g., can be repeated about every 8 weeks).
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
EXAMPLES
Example 1: Mitochondrial aspartate regulates TNF-a biogenesis and autoimmune tissue inflammation
This Example identifies a deficiency of mitochondrial aspartate production as a key abnormality in such autoimmune T cells. Shortage of mitochondrial aspartate disrupted the regeneration of the metabolic cofactor nicotinamide adenine dinucleotide (NAD), causing ADP-deribosylation of the endoplasmic reticulum (ER) sensor GRP78/BiP. As a result, ribosome-rich ER membranes expanded, promoting co-translational translocation and enhanced biogenesis of the transmembrane cytokine TNF-a. ERnch T cells were the predominant TNF-a producers in the arthritic joint. T cell-specific transfer of intact mitochondria as well as supplementation of exogenous aspartate rescued the mitochondria- instructed expansion of ER membranes and suppressed TNF-a release and rheumatoid tissue inflammation. These results connect autoimmune tissue inflammation to dysfunctional inter- organellar communication and define mitochondrial intactness in T cells as a key component of tissue tolerance.
Results
Naive CD4+ T cells from RA patients have a mitochondrial defect, manifesting with low oxygen consumption and low mitochondrial membrane potential (MMP) (Figures 1 A-1C), while mitochondrial mass is similar between healthy and RA T cells (Figure 6). To understand how the lack of mitochondrial fitness impacts other organelles, endoplasmic reticulum (ER) mass was quantified. Flow cytometry for ER-positioned ATP-sensitive K+ channels (marked by ER Tracker) and confocal imaging for the ER-resident enzyme protein
disulfide isomerase (PDI) revealed an inverse relationship between the mitochondrial membrane potential and the biomass of the ER (Figures ID and IE). Cell size-restricted gating (Figure 7) confirmed the negative correlation between MMP and ER-Tracker intensity in all subpopulations (Figure IF). Compared to healthy T cells, CD4+ T cells from RA patients had a higher ER Tracker signal (Figure 1G) and expressed more of the ER chaperone Calnexin (Figures 1H and II). To assess whether expansion of ER structures is a feature specific for RA T cells, patients with psoriatic arthritis (PsA) were recruited. ER Tracker signals in activated CD4+ T cells from patients with psoriatic arthritis were indistinguishable from age-matched controls (Figure 8), indicating that the expansion of ER size is an RA specific phenotype, and not simply a consequence of systemic inflammation. RA T cells synthesized significantly more phosphatidylcholine, one of the major phospholipids needed for biomembrane generation (Figure IK). Transmission electron microscopy confirmed highly abundant and elongated ER structures in activated CD4+ T cells from RA patients (Figure IL).
Whether ER stress signals contribute to the ERnch phenotype of RA T cells was explored. When activated, naive CD4+ T cells from RA patients disproportionally upregulated the ER stress gene signature (Figure 1 J), suggesting persistent ER stress signaling. That ER stress promotes expansion of the ER membrane system was verified by overexpressing the active form of XBP1 (Xbp-ls) in healthy T cells and quantifying ER biomass (Figure 9). To define whether mitochondrial activity regulates ER stress signaling, T cells were sorted into MMP10 and MMP111 subpopulations (Figure IM). ER stress gene expression was a feature of T cells with low mitochondrial activity (Figure IN).
To examine whether mitochondria-derived signals determine ER size and function, ER-associated membranes were quantified after inhibiting the electron transport chain complex I with Rotenone (10 nM). Impaired electron transport prompted an increase in ER mass (Figures 10 and IP). Other mitochondrial respiration inhibitors (Piericidin A, Antimycin A, Oligomycin) had similar effects (Figure 10). To repair defective mitochondria in RA T cells, exogenous mitochondria were harvested and transferred into RA recipient cells (Figure IQ). To establish and validate the mitochondrial transfer, a donor/recipient cell ratio of 10: 1 was used, which yielded excellent post-transfer improvement in MMP (Figure
11). Delivery of exogenous mitochondria from healthy T cells into RA T cells restored the MMP to normal levels (Figure 1R) and suppressed ER biomass by >30% (Figure IS). Mitochondria harvested from RA T cells failed to restore the ER size.
These data identified mitochondrial metabolism as a key regulator of ER biogenesis and classified CD4+ T cells from RA patients as MMP10 ERnch.
To address which mitochondrial signals restrain ER expansion and clarify how the ER senses impaired mitochondrial fitness, key mitochondrial intermediates were screened. Exogenous a-KG left ER mass unaffected (Figure 2A), while succinate, malate, and more so, oxaloacetate and aspartate rapidly suppressed formation of ER membranes in RA T cells (Figure 2A). The amino acid aspartate, part of the malate-aspartate shuttle, was most effective in communicating with the ER. Aspartate is synthesized in the mitochondrial matrix out of malate-oxaloacetate by the transaminase GOT2 and then transported into the cytosol. Under conditions of NADH availability, cytosolic aspartate is converted back into malate to reenter the mitochondria in the malate-aspartate shuttle (Figure 2B). Activation of healthy T cells resulted in accumulation of aspartate (4 nmol/l 0 6 cells), while RA T cells remained aspartate low, generating less than 2.5 nmol/lCk6 cells (Figure 2C). Similarly, RA T cells lacked behind in producing adequate concentrations of oxaloacetate, the precursor of aspartate in the TCA cycle (Figure 2D). The aspartate10 phenotype in RA T cells was not due to insufficient glutamic-oxaloacetic transaminase; GOT1 and GOT2 transcript concentrations were indistinguishable in healthy and RA T cells (Figure 12). Exogenous aspartate was rapidly taken up into RA T cells to correct the ER stress gene signature (Figure 2E) and inhibit the inappropriate biosynthesis of phosphatidylcholine, a membrane building block required for ER biomass generation (Figure 2F). Maintenance of cellular aspartate concentrations required intactness of mitochondrial metabolism. Inhibiting mitochondrial respiration through the complex I inhibitor Rotenone dramatically decreased aspartate levels in healthy T cells (Figure 2G), while the transfer of healthy mitochondria into RA T cells restored aspartate generation (Figure 2H). Knockdown of GOT2, the gate keeper enzyme in aspartate synthesis, successfully mimicked the aspartate10 phenotype of RA T cells (Figure 21) and healthy T cells responded to GOT2 loss-of- function with increased formation of ER
membranes and upregulation of the ER stress gene signature (Figure 2J and 2K). Exogenous aspartate rescued the ER expansion induced by GOT2 knockdown (Figure 2J).
These data identified the amino acid aspartate as a sensitive biomarker of mitochondrial respiration and as a signal transducer between mitochondria and the ER membrane system.
To test whether the aspartate10 phenotype in RA T cells is disease-relevant and if supplementation of aspartate can protect tissue from inflammatory damage, synovitis was induced in human synovium-engrafted NSG mice and the chimeras were treated with aspartate. RA PBMC induced robust synovitis, which was successfully suppressed by aspartate treatment (Figure 2L). Transcriptomic analysis of the tissue lesions revealed a marked beneficial effect of aspartate supplementation lowering T cell receptor (TRB) expression and curtailing TBX21 and RORG mRNA accrual (Figure 2M). Immunohistochemical analysis confirmed that the frequency of tissue residing CD3+ IFNy+ T cells was highly dependent on aspartate availability, declining by 65% with aspartate supplementation (Figure 2N). mRNA levels for the inflammatory mediators IFN-y, IL- 17, IL-6, TNF-a, IL-ip all decreased (Figure 20) after aspartate treatment, indicating successful containment of tissue inflammation. Conversely, the anti-inflammatory genes TGFB and IL 10 were not affected.
Together, these data identified the mitochondrial intermediate aspartate as a regulator of ER mass and classified the amino acid as an anti-inflammatory substrate.
Aspartate is dispatched from mitochondria as an electron acceptor, facilitating the cytoplasmic regeneration of NAD+ from NADH, and then malate reenters the mitochondria as an electron carrier (Figure 2B). To test whether NAD+ and NADH concentrations are affected by aspartate shortage, intracellular NAD+/NADH were measured in healthy and patient-derived CD4+ T cells. Healthy T cells reached NAD+ levels of 550 pmol/10A6 cells and NADH concentrations of 70 pmol/10A6 cells. The NAD+/NADH ratio in healthy T cells exceeded 7. With reduced NAD+ and elevated NADH, RA T cells achieved only an NAD+/NADH ratio of 3 (Figures 3B and 3C). To restore the NAD+/NADH balance, RA T cells were treated with aspartate or received a transfer of exogenous mitochondria. Both interventions significantly improved NAD+ regeneration (Figures 3D and 3E). Inhibiting
mitochondrial respiration with the complex I inhibitor Rotenone essentially prevented conversion of NADH into NAD+ (Figure 3F), implicating aspartate and mitochondrial intactness in maintaining the NAD+/NADH balance. To examine whether NAD+ functions similarly to aspartate in regulating ER mass/function, RA T cells were activated with or without NAD+ supplement. Like aspartate, exogenous NAD+ lowered the ER biomass (Figures 3G and 3H) and reduced the ER stress signature (Figure 31).
Collectively, these data delineated a mechanistic connection between insufficient mitochondrial aspartate production, failed regeneration of the electron acceptor NAD+ and expansion of the ER membrane system.
Linder ER stress conditions, Bip senses and binds to unfolded proteins, releasing PERK, ATF6 and IRE and triggering their respective ER stress pathways (Figure 3 J). Bip is ideally positioned to communicate variations in mitochondrial function to the endomembrane system, with posttranslational protein modifications providing fast access to this communication pathway. Given the low NAD+ availability in RA T cells, whether NAD- dependent ADP-ribosylation is relevant for Bip function was tested. Compared to healthy T cells, only 30% of Bip molecules in RA T cells were ADP-ribosylated (Figure 3K). The BipADP-R10 phenotype could be reproduced by blocking the electron transport chain with rotenone (Figure 3L) or Piercidin A (Figure 13). In contrast, surplus exogenous NAD+ restored ADP-ribosylation of Bip in RA T cells (Figure 3M).
To link ADP-ribosylation to Bip function, the binding between Bip and its target protein Ire- la was analyzed by immunoprecipitation. Increasing doses of Rotenone disrupted the binding of Bip to Ire- la (Figure 3N), whereas NAD+ supplementation doubled Bip-Ire-la complex formation (Figure 30).
Together, these data define NAD+-dependent ADP-ribosylation of Bip as an on-off switch of ER stress signals, placing mitochondrial fitness upstream of ER size and function (Figure 4R), and specifying aspartate as a mitochondria-to-ER messenger.
To explore the functional consequence of ER expansion in RA T cells, cytokine product! on/secretion, which relies heavily on the endomembrane system, was focused on. Ribosome-rich rough ER was quantified in healthy and patient-derived T cells (Figures 4A - 4D). ER biomass was expanded in RA T cells, as indicated by the increased load of the ER
chaperone calnexin (Figures 4A and 4B) while global ribosomal protein levels were indistinguishable in healthy and RA T cells. To connect the unrestrained expansion of the ER in RA T cells to their functional behavior, the ribosome-bonded rough ER, responsible for co-translational translocation, was focused on. Immunoblotting showed high purity of rough ER when isolated out of the T cell cytosol (Figure 16A). T cells responded to activation with a dramatic increase in the ribosome-occupied rough ER membrane system (Figure 16B). Comparative analysis of healthy and RA T cells demonstrated that the diagnosis of RA was associated with a >2 fold enlargement of ribosome-occupied ER membrane sheets (Figures 4C and 4D).
To understand the functional implications of the enlarged rough ER in RA T cells, mRNAs contained in the ER-bonded ribosomes were analyzed. Considering that the rough ER sheets serve as the main site of synthesis for secreted and membrane-integrated proteins, we concentrated on T cell effector cytokines. In naive CD4+ T cells, transcripts for the lineage-determining effector cytokines IFN-y, IL-4 and IL- 17 were barely detectable, but IL2, and even more so, TNFA mRNA were abundant (Figure 4E). Considering the critical role of TNF-a in the RA disease process, the transcription and translation of this cytokine was focused on. The overall pool of TNFA mRNA in resting and stimulated T cells from healthy individuals and patients were indistinguishable (Figure 4F). To explore whether the expansion of ER membrane sheets in RA T cells impacts TNF-a transcription, ER-bound mRNAs were measured upon PMA/Ionomycin activation. Non-secretory cytoplasmic proteins, including the glycolytic enzyme GAPDH and the cytoskeletal protein ACTIN, were underrepresented amongst ER-bound mRNAs (Figure 4G). mRNA for the 4 cytokines IFN- y, IL-2, IL- 17 and TNF-a were all highly enriched amongst the ER-bound fraction, with IL2 and TNFA mRNA being >100fold more abundant at the ER membrane (Figure 4G). With their expanded ER morphology, RA CD4+ T cells recruited a higher proportion of TNFA mRNA to the organelle’s surface (Figure 4H), where selected mRNAs become subject to co- translational translocation. This redistribution of TNFA mRNA resulted in highly efficient TNF-a biogenesis (Figures 4L4K). In patient-derived T cells, confocal imaging revealed a strong signal for TNF-a co-localizing with calnexin but also embedded into the plasma membrane (Figure 41). Intracellular staining for TNF-a protein yielded higher concentrations
of TNF-a in resting and stimulated T cells (Figure 4J). Quantification of secreted TNF-a confirmed doubling of cytokine release during the resting and the stimulated state in patient- derived cells (Figure 4K). Whether the unfolded protein response (UPR), a stress response program triggered by the accumulation of unfolded proteins in the ER lumen, is sufficient to enhance TNF-a biogenesis and can mimic the ERnch phenotype of RA T cells was tested. Treatment of healthy CD4 T cells with tunicamycin failed to upregulate TNF-a synthesis (Figure 17), suggesting that the mitochondria-induced ER expansion in RA T cells represents a state of pseudo-ER stress.
The mechanistic link between ER expansion and enhanced recruitment of TNFA mRNA to the ER surface resulting in a TNF-a superproducer phenotype raised the question whether correcting the growth of the endomembrane system could repair the uncontrolled cytokine release. Disrupting mitochondrial function with the respiration inhibitors Rotenone and Piercidin A or by knocking down GOT2 mRNA transformed healthy T cells into TNF-a111 producers (Figures 4L - 4N). GOT2 lose-of-fimction could be rescued by supplementation of aspartate (Figure 4N). Three interventions repaired the excessive TNF-a secretion of RA T cells: the supplementation of exogenous aspartate, NAD+ and the transfer of intact mitochondria into the patients’ T cells (Figures 40 - 4Q). Interestingly, asparagine, the amino acid structurally similar to aspartate, failed to affect ER size and TNF-a production (Figure 14), indicating a unique role of aspartate in the NAD+/NADH metabolism. Finally, the metabolic intermediates pyruvate and a-ketobutyrate, which can regenerate NAD+ through a distinct mechanism were tested. Both metabolites inhibited ER expansion (Figure 15 A) in RA T cells and suppressed TNF-a production (Figure 15B).
Collectively, these data indicate that the enlargement of ribosome-occupied ER sheets in RA T cells have profound implications for TNF-a biogenesis by targeting the process of co-translational translocation, imparting a competitive advantage to TNFA mRNA (Figure 4R).
TNF-a-producing CD4+ T cells function as key arthritogenic effector cells.
Rheumatoid synovial lesions consist of a mixture of cell types, most prominently, T cells, B cells, macrophages, and synovial fibroblasts. To implicate some or all cell
populations in TNF-a production, recently published single cell RNAseq data (Zhang et al., Nat. Immunol., 20:928-942 (2019)) was analyzed. Tissue-residing T cells contained abundant amounts of TNFA mRNA, while macrophages and B cells were low positive, and fibroblasts were negative. TNF-a production was allocated to the different cellular subsets by determining intracellular TNF-a by flow cytometry. Synovial tissues from RA patients were disaggregated and stimulated with LPS/PMA/ionomycin in the presence of the Golgi blocker Brefeldin A (BFA). Intracellular TNF-a was measured cytometrically in T cells (CD45+CD3+), B cells (CD45+CD19+) and macrophages (CD45+CD68+). In leukocyte-rich synovial tissues (Figure 5 A), 80% of T cells and B cells were TNF-a+, while only 40% of macrophage produced TNF-a (Figures 5B and 5C). TNF-a was highly abundant in T cells (Figure 5D), multifold higher than in B cells and macrophages. In leukocyte-poor RA tissues, B cells were barely detected (Figure 5E), but most T cells contained high amounts of TNF-a (Figures 5F - 5H). These data identified synovial T cells as a dominant cellular source of TNF-a, in both leukocyte-poor and leukocyte-rich tissue lesions.
To validate that TNF-a production is a feature of tissue-embedded cells and not just detectable after LPS/PMA/ionomycin stimulation of disaggregated cells, a method to analyze cytokine production in freshly harvested synovial tissues from RA patients was established. Cytokine secretion from the cells was blocked by treating intact tissue slices with BFA for 4 hours. Subsequently, cells were isolated from the tissue and intracellular TNF-a was detected by flow cytometry (Figures 51 - 5K). In fresh synovial tissues, about 40% of synovial CD68+ macrophages produced TNF-a, independent of BFA treatment, indicating intracellular retention of TNF-a in tissue-residing macrophages. In contrast, tissue-residing T cells appeared to immediately release TNF-a into the tissue microenvironment. The frequencies of TNF-a+ T cells increased 10-20-fold upon BFA-induced secretion blockade (Figure 5K). These data confirmed ongoing TNF-a generation and secretion by synovia- embedded T cells.
To investigate whether T cell-derived TNF-a is relevant for synovial inflammation, a humanized mouse model was used in which human synovial tissue is engrafted into NSG mice and the chimeric host is immuno-reconstituted with peripheral blood mononuclear cells (PBMC) from RA patients. Prior to the PBMC transfer, FACS-sorted CD4+ T cells were
transfected with control siRNA or siRNA targeting TNFA (Figure 18 A). The knockdown lowered TNFA transcripts to about 50% of controls (Figure 20A). mimicking a physiologic situation. Histological evaluation of explanted synovial tissues demonstrated that suppressing T cell-derived TNF-a was strongly anti-inflammatory. Control chimeras, injected with control-si-RNA transfected CD4+ T cells, developed robust synovitis. Tissues harvested from the control mice were densely infiltrated with CD3+ T cells (Figures 5L and 5M). Tissue transcriptomic analysis revealed abundance of TCR, TBX21 and RORC transcripts (Figure 5N). CD4+ T cells with intact TNF-a production infiltrated into the synovial tissue space and triggered induction of IFNG, IL 17, IL21, TNFA, IL6 and IL1B (Figure 5N). Synovial explants harvested from mice reconstituted with CD4+TNF-a10 T cells had few tissue-infiltrating cells (Figure 51), density of tissue-residing CD3+ T cell was low (Figure 5M), and all inflammatory genes were expressed at low abundance (Figure 5N). In a parallel approach, whether reconstitution of intact mitochondria affected synovitis was tested (Figure 18B). Analysis of the synovial explants documented obvious anti-inflammatory potency of mitochondria transfer. Synovial grafts harvested from mice reconstituted with T cells transferred with intact mitochondria had few tissue-infiltrating cells (Figure 19A) and low-density CD3+ T cell infiltrates (Figure 19B). Transcripts for key inflammatory genes were consistently low (Figure 19C). Collectively, these data established that TNF-a- producing T cells preferentially home to the synovial tissue environment and are indispensable for the induction and maintenance of synovitis.
Materials and Methods
Patients and samples
Patients enrolled into the study fulfilled the diagnostic criteria for RA and tested positive for rheumatoid factor and/or anti-CCP antibodies. All patients (n=107) recruited had active disease and clinical characteristics are presented in Table 1. The following criteria excluded individuals from enrollment: current or previous diagnosis of cancer, uncontrolled medical disease, chronic inflammatory syndrome. Age-matched healthy donors without a personal history of cancer or autoimmune disease served as controls.
Table 1. Demographic and clinical characteristics of RA patient cohort.
Cell preparation and culture Peripheral blood mononuclear cells (PBMCs) were isolated by gradient centrifugation with Lymphocyte Separation Medium (Lonza). CD4+CD45RA+ naive T cells were isolated from PBMCs with the EasySep Human Naive CD4+ T Cell Enrichment Kit (STEMCELL Technologies). Purity of cell populations was consistently > 95%. Anti-CD3/anti-CD28- coated Dynabeads (Gibco) were used to activate naive CD4+ T cells at a ratio of 2 cells: 1 bead for 72 hours. Cell Activation Cocktail (PMA/Ionomycin) (Biolegend) was used to stimulate cytokine production in T cells with or without Brefeldin A (eBioscience) for 2 hours.
Reagents
The pCMV5-Flag-XBPls plasmid was purchased from Addgene. Human TNFA siRNA, GOT2 siRNA and control siRNA were obtained from Thermo Fisher Scientific. The mitochondrial respiration inhibitors Antimycin A and Rotenone were purchased from Agilent
Technologies. NAD+ was obtained from Cayman Chemical. a-Ketoglutaric acid, succinic acid, L-aspartic acid were from Sigma-Aldrich. Malic acid was from Santa Cruz Biotechnology.
Intracellular TNF-a measurement
To measure intracellular TNF-a production, CD4+CD45RA+ naive T cells were activated for 72 hours and stimulated with PMA/ION/BFA for 2 hours. 4% PFA was used for fixation and 0.1% saponin to permeabilize the cells. Cells were stained with PE AntiHuman TNF-a (1 TOO, BD, Cat# 554513) antibody for 1 hour. Flow cytometry was performed on an LSR II flow cytometer (BD Biosciences). Data were analyzed with FlowJo software (Tree Star Inc).
Labelling of ER membranes
ER tracker green (Thermo Fisher Scientific, Cat# E34251) was used to stain the ER- localized ATP-sensitive K+ channels following the manufacture’s instruction. Flow cytometry was performed to detect ER tracker signal on an LSR II flow cytometer (BD Biosciences). Intensity of ER tracker staining represents the size of the ER membranes. Data were analyzed with FlowJo software (Tree Star Inc).
Transmission electron microscopy
Cells were resuspended in McDowell's and Trump's fixative for 1 hour at room temperature and then pelleted with a microcentrifuge. Cells were washed with 0. IM phosphate buffer for 5 minutes and recentrifuged and the buffer was aspirated (twice). Liquid agar was added to the cell pellet and the cells were resuspended followed immediately by centrifugation. Once the sample had cooled the agar was removed and the sample pellet removed with a razor blade and placed into 0. IM phosphate buffer. Following 2 rinses in 0. IM phosphate buffer (pH 7.2), the sample was placed in 1% osmium tetroxide in the same buffer for 1 hour at room temperature. The sample was rinsed 2 times in distilled water and dehydrated in an ethanolic series culminating in two changes of 100% acetone. The cell pellet was then placed in a mixture of Spurr resin and acetone (1 : 1) for 30 minutes, followed by 2 hours in 100% resin with 2 changes. The cell pellet was placed into 100% Spurr resin in
an embedding mold and polymerized at 65°C for 12 hours or longer. Ultrathin (70-90 nm) sections were cut on an ultramicrotome with a diamond knife, stained with lead citrate and examined with a JEOL 1400 transmission electron microscope.
Rough ER isolation
Endoplasmic Reticulum Isolation Kit (Sigma- Aldrich, Catalog Number: ER0100) was used to isolate rough ER.
Measurement of mitochondrial membrane potential
MitoTracker Red (Thermo Fisher Scientific, Cat# M7512) is a fluorescent dye that stains mitochondria in live cells and its accumulation is dependent upon mitochondrial membrane potential (MMP). Staining intensity was analyzed with an LSR II flow cytometer (BD Biosciences) and data were analyzed with FlowJo software (Tree Star Inc).
Immunoblotting
Cellular proteins or rough ER proteins were extracted with RIPA buffer (Sigma- Aldrich). Protein expression levels were examined by western blotting. Monoclonal antibodies specific for ribosomal protein LI 7, ribosomal protein S7 and Bip were purchased from Santa Cruz Biotechnology. Antibodies specific for calnexin and Ire la were from Cell Signaling Technology. P-actin expression detected with anti-P-actin antibody (Cell Signaling Technology, 8H10D10) served as the internal control.
Real time PCR
Total RNA or Rough ER bound RNA were extracted with Trizol (Thermo Fisher Scientific) and Direct-zol RNA MiniPrep Kit (ZYMO Research). cDNA was synthesized using Maxima First Strand cDNA Synthesis Kits for qPCR with reverse transcription (RT- qPCR) (Thermo Fisher Scientific). Quantitative PCR analyses were performed using SYBR Green qPCR Master Mix (Bimake) and gene expression was normalized to P-actin. Primers were listed in Table 2.
Attorney Docket No. 07039-2075W01 / 2021-418
Mitochondria transfer
On day 0, naive CD4+ T cells from healthy donor were isolated and activated with anti-CD3/CD28 beads. On day 3, naive CD4+ T cells from RA patient were purified and activated. On day 5, 4 million healthy CD4+ T cells were harvested, and mitochondria were isolated using Mitochondria Isolation Kit for Cultured Cells (Thermo Fisher Scientific, Catalog number: 89874). In parallel, 0.2 million activated RA CD4+ T cells were collected and spun at 300xg for 5 minutes. Resuspend cells were mixed with the mitochondria suspension and carefully resuspended. The cell/mitochondria mixture and the control cell/PBS mixture were centrifuged at l,500xg for 5 minutes. After washing, the cells were stimulated with anti-CD3/CD28 beads for one more day.
Human synovial tissue- -NSG mouse chimeras
NSG mice from the Jackson Laboratory were kept in a pathogen-free facility. Wedges of human synovial tissue were subcutaneously implanted into 8-12-week-old mice. After 7 days, the mice were infused with 10 million PBMC collected from RA patients with active disease. In some experiments, CD4+ T cells in PBMC were FACS sorted and transfected with siRNA targeting TNFA or control siRNA before the cells were injected into the mice. Alternatively, healthy mitochondria were transferred into RA CD4+ T cells prior to the immune reconstitution. For these experiments, CD4+ T cells were FACS sorted and mitochondria from healthy T cells were transferred into RA CD4+ T cells as described above. On day 14, synovial tissues were explanted from the chimeric mice, OCT-embedded (4583; Sakura Finetek USA) or shock-frozen for further experiments (tissue staining or RNA extraction).
Immunohistochemistry
Frozen sections of synovial tissues were stained with mouse anti-human CD3 (1 : 100; DAKO, Clone F7.2.38) and rabbit anti-human IFN-y (1 : 100, Abeam, ab25101). Alexa Fluor 594 anti-mouse IgG (1 :200, Thermo Fisher Scientific, A- 11032) and Alexa Fluor 488 antirabbit IgG (1 :200, Thermo Fisher Scientific, A-11034) were used as secondary antibodies.
Images of CD3/IFN-y staining were obtained using a LSM710 confocal microscope (Carl Zeiss) with a Plan-Neofluar x 40/1.3-NA oil objective lens.
Immunofluorescence
To visualize intracellular proteins, cells were collected and fixed with 4% PFA for 10 min and permeabilized using 0.5% saporin. The following primary antibodies were used: Calnexin rabbit mAb (1 :100, Cell signaling technology), protein disulfide isomerase (PDI) mouse mAb (1 TOO, Thermo Fisher Scientific). The following secondary antibodies were used: Alexa Fluor 594 goat anti-mouse IgG (1 :200, Thermo Fisher Scientific, A-l 1032), Alexa Fluor 488 goat anti-rabbit IgG (1 :200, Thermo Fisher Scientific, A-l 1008). Nuclei were stained with DAPI. The LSM710 system (Carl Zeiss) with a Plan Apochromat 63x/1.40-NA oil DICIII objective lens (Carl Zeiss) was used to acquire images.
BiP ADP-ribosylation
5x10A6 activated naive CD4+ T cells were lysed with IP Lysis Buffer (Thermo Fisher Scientific, Catalog#: 87788) containing protease and phosphatase inhibitors. BiP protein in whole cell lysates was pulled down by incubation with agarose conjugated anti-BiP antibody (Santa Cruz Biotechnology, Catalog#: sc-13539) for 4 hours at 4°C. Agarose was washed 5 times with IP lysis buffer and boiled for 5 minutes in loading buffer. Eluted protein was separated by SDS-page and ADP-ribosylated BiP was detected using antibody against ADP- ribose (Cell Signaling Technology, Catalog#: 83732).
BiP/ IRE 1 -a binding
Cells were lysed with IP Lysis Buffer (Thermo Fisher Scientific, Catalog#: 87788) containing protease and phosphatase inhibitors. Whole cell lysates were incubated with 2pg anti-BiP antibody (Santa Cruz Biotechnology, Catalog#: sc-13539) and Protein A/G PLUS- Agarose (Santa Cruz Biotechnology, Catalog#: sc-2003) for 4 hours at 4°C. Normal rat IgG (Santa Cruz Biotechnology, Catalog#: sc-2026) was used as IgG control. The immunocomplexes were washed with IP lysis buffer 5 times, then eluted with loading buffer and separated by SDS-PAGE. Immunoblotting for IREl-a was performed following standard procedures for Western blotting.
Quantification and Statistical Analysis
Statistical analyses were performed using GraphPad Prism software (GraphPad Software). To compare data within two groups, the paired Wilcoxon test or the Mann- Whitney test were used when the sample size per group was >5. Parametric t test was only used if the sample size per group was <=5. To adjust for multiple testing, Hochberg’s stepdown method was used to control for a family-wise-error rate at the 0.05 levels. One-way ANOVA was used and pair-wise comparison using Tukey’s method was applied for comparisons between 3 or more groups. All data points were included in the analysis and no outliers were detected using Grubbs’ test. All data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 and statistical parameters are presented in each figure legend.
Example 2: Treating tissue inflammation
T cells are isolated from a human identified as having tissue inflammation, loaded with intact mitochondria, and the mitochondria-loaded T cells are administered back to the human. The administered mitochondria-loaded T cells can reduce production of a TNF-a polypeptide within the inflamed tissue to reduce tissue inflammation present within the human.
Example 3: Treating tissue inflammation
Healthy cells that are not T cells are isolated from a human identified as having tissue inflammation, and healthy mitochondria are obtained from the healthy cells.
T cells are isolated from a human identified as having tissue inflammation, loaded with intact, healthy mitochondria isolated from the healthy cells that are not T cells, and the mitochondria-loaded T cells are administered back to the human.
The administered mitochondria-loaded T cells can reduce production of a TNF-a polypeptide within the inflamed tissue to reduce tissue inflammation present within the human.
Example 4: Treating tissue inflammation
T cells are isolated from a human identified as having tissue inflammation, loaded with intact, healthy mitochondria isolated from cord blood cells, and the mitochondria-loaded T cells are administered back to the human.
The administered mitochondria-loaded T cells can reduce production of a TNF-a polypeptide within the inflamed tissue to reduce tissue inflammation present within the human.
Example 5: Treating tissue inflammation
A human identified as having tissue inflammation is administered (e.g., by subcutaneous injection) 40 mg to 80 mg adalimumab (e.g., HUMIRA®) together with 10 mg to 3000 mg of aspartate (e.g., D-aspartate and/or L-aspartate). In some cases, the administration is repeated every 2 weeks. In some cases, the administration is repeated every week.
The administered combination of adalimumab and aspartate can reduce tissue inflammation present within the human.
Example 6: Treating tissue inflammation
A human identified as having tissue inflammation is administered (e.g., by subcutaneous injection) 50 mg etanercept (e.g., Enbrel®) together with 10 mg to 3000 mg of aspartate (e.g., D-aspartate and/or L-aspartate). In some cases, the administration is repeated every week.
The administered combination of etanercept and aspartate can reduce tissue inflammation present within the human.
Example 7: Treating tissue inflammation
A human identified as having tissue inflammation is administered (e.g., by subcutaneous injection) 5 mg/kg to 10 mg/kg infliximab (e.g., REMICADE®) together with 10 mg to 3000 mg of aspartate (e.g., D-aspartate and/or L-aspartate). In some cases, the administration is repeated every 8 weeks.
The administered combination of infliximab and aspartate can reduce tissue inflammation present within the human.
Example 8: Treating tissue inflammation
A human identified as having tissue inflammation is administered (e.g., by subcutaneous injection) 40 mg to 80 mg adalimumab (e.g., HUMIRA®) and is administered, separately but within 1 to 15 minutes, 10 mg to 3000 mg of aspartate (e.g., D-aspartate and/or L-aspartate). In some cases, the administration is repeated every 2 weeks. In some cases, the administration is repeated every week.
The administered combination of adalimumab and aspartate can reduce tissue inflammation present within the human.
Example 9: Treating tissue inflammation
A human identified as having tissue inflammation is administered (e.g., by subcutaneous injection) 50 mg etanercept (e.g., Enbrel®) and is administered, separately but within 1 to 15 minutes, 10 mg to 3000 mg of aspartate (e.g., D-aspartate and/or L-aspartate). In some cases, the administration is repeated every week.
The administered combination of etanercept and aspartate can reduce tissue inflammation present within the human.
Example 10: Treating tissue inflammation
A human identified as having tissue inflammation is administered (e.g., by subcutaneous injection) 5 mg/kg to 10 mg/kg infliximab (e.g., REMICADE®) and is administered, separately but within 1 to 15 minutes, 10 mg to 3000 mg of aspartate (e.g., D- aspartate and/or L-aspartate). In some cases, the administration is repeated every 8 weeks.
The administered combination of infliximab and aspartate can reduce tissue inflammation present within the human.
Example 11: Treating tissue inflammation
A human identified as having tissue inflammation is administered (e.g., by subcutaneous injection) 40 mg to 80 mg adalimumab (e.g., HUMIRA®) together with 10 mg
to 3000 mg of total metabolites of L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate. In some cases, the administration is repeated every 2 weeks. In some cases, the administration is repeated every week.
The administered combination of adalimumab and metabolites can reduce tissue inflammation present within the human.
Example 12: Treating tissue inflammation
A human identified as having tissue inflammation is administered (e.g., by subcutaneous injection) 50 mg etanercept (e.g., Enbrel®) together with 10 mg to 3000 mg of total metabolites of L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate. In some cases, the administration is repeated every week.
The administered combination of etanercept and metabolites can reduce tissue inflammation present within the human.
Example 13: Treating tissue inflammation
A human identified as having tissue inflammation is administered (e.g., by subcutaneous injection) 5 mg/kg to 10 mg/kg infliximab (e.g., REMICADE®) together with 10 mg to 3000 mg of total metabolites of L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate. In some cases, the administration is repeated every 8 weeks.
The administered combination of infliximab and metabolites can reduce tissue inflammation present within the human.
Example 14: Treating tissue inflammation
A human identified as having tissue inflammation is administered (e.g., by subcutaneous injection) 40 mg to 80 mg adalimumab (e.g., HUMIRA®) and is administered, separately but within 1 to 15 minutes, 10 mg to 3000 mg of total metabolites of L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate. In some cases, the administration is repeated every 2 weeks. In some cases, the administration is repeated every week.
The administered combination of adalimumab and metabolites can reduce tissue inflammation present within the human.
Example 15: Treating tissue inflammation
A human identified as having tissue inflammation is administered (e.g., by subcutaneous injection) 50 mg etanercept (e.g., Enbrel®) and is administered, separately but within 1 to 15 minutes, 10 mg to 3000 mg of total metabolites of L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate. In some cases, the administration is repeated every week.
The administered combination of etanercept and metabolites can reduce tissue inflammation present within the human.
Example 16: Treating tissue inflammation
A human identified as having tissue inflammation is administered (e.g., by subcutaneous injection) 5 mg/kg to 10 mg/kg infliximab (e.g., REMICADE®) and is administered, separately but within 1 to 15 minutes, 10 mg to 3000 mg of total metabolites of L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate. In some cases, the administration is repeated every 8 weeks.
The administered combination of infliximab and metabolites can reduce tissue inflammation present within the human.
Example 17: Exemplary Embodiments
Embodiment 1. A method for restoring aspartate homeostasis in a mammal having tissue inflammation, wherein said method comprises administering, to said mammal, a composition comprising cells, wherein a population of intact mitochondria was introduced into said cells, and wherein the production of a tumor necrosis factor-a (TNF-a) polypeptide within said mammal is reduced following said administering step.
Embodiment 2. The method of embodiment 1, wherein said composition comprises T cells containing said intact mitochondria.
Embodiment 3. The method of embodiment 1 or embodiment 2, where said T cells are obtained from said mammal.
Embodiment 4. A method for restoring aspartate homeostasis in a mammal having tissue inflammation, wherein said method comprises administering, to said mammal, an inhibitor of UPR signaling, wherein said method is effective to reduce production of a TNF-a polypeptide within said mammal.
Embodiment 5. The method of embodiment 4, wherein said inhibitor of UPR signaling can target an UPR signaling pathway component selected from the group consisting of binding immunoglobulin protein (BiP) polypeptides, IRE la polypeptides, PERK polypeptides, ATF6 polypeptides, eIF2a polypeptides, CHOP polypeptides, XBP-1 polypeptides, and XBP-1S polypeptides.
Embodiment 6. The method of embodiment 4, wherein said inhibitor of UPR signaling is selected from the group consisting of eeyarestatin I, mycolactone, exotoxin A, NSC 630668-R/l, MAL3-39, MAL3-101, E6 berbamine, ophiobolin A, equisetin, CJ-21058, Rose Bengal, erythrosin B, P97-A4, P87-A4, 17D9, P91-E9, 16F6, bisthiouracil, SCA-21, HUN- 7293, cotransin, CAM741, apratoxin A, decatransin, valinomycin, CADA, kinase inhibiting RNase attenuator 6 (KIRA6), 3-hydroxy-2-naphthoic acid (3HNA), MKC-3946, 4- Phenylbutyric acid (4-PBA), taurine-conjugated ursodeoxycholic acid (TUDCA), olmesartan, N-acetylcysteine (NAC), oleanolic acid (OA), ursolic acid, telmisartan, quercetin, 4p8C, STF-083010, B-109, GSK2606414, GSK2656157, AMG PERK44, melatonin, ceapin, IRSIB, AID 2732, salubrinal, ISRIB, guanabenz, sephinl, salicylaldimines, APY29, sunitinib, toyocamycin, 3-ethoxy-5,6-dibromosalicylal- dehyde, apigenin, FIRE peptide, baicalein, kaempferol, compound 147, compound 263, and 16F16.
Embodiment 7. A method for restoring aspartate homeostasis in a mammal having tissue inflammation, wherein said method comprises administering, to said mammal, an
inhibitor of co-translational translocation, wherein said method is effective to reduce production of a TNF-a polypeptide within said mammal.
Embodiment 8. The method of embodiment 7, wherein said inhibitor of co- translational translocation can target a co-translational translocation pathway component that is selected from the group consisting of Sec61 polypeptides, Hsp70 polypeptides, ATPase polypeptides, calmodulin polypeptides, transmembrane domains of newly produced polypeptides, signal peptides, and signal peptidase complex polypeptides.
Embodiment 9. A method for restoring aspartate homeostasis in a mammal having tissue inflammation, wherein said method comprises administering, to said mammal, a composition comprising a mitochondrial metabolite, wherein said method is effective to reduce production of a TNF-a polypeptide within said mammal.
Embodiment 10. The method of embodiment 9, wherein said mitochondrial metabolite is selected from the group consisting of L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, oxaloacetate, and nicotinamide riboside.
Embodiment 11. The method of embodiment 9 or embodiment 10, wherein said composition comprises L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate.
Embodiment 12. A method for restoring aspartate homeostasis in a mammal having tissue inflammation, wherein said method comprises administering, to said mammal, an inhibitor of aspartate transport or aspartate metabolism, wherein said method is effective to reduce production of a TNF-a polypeptide within said mammal.
Embodiment 13. The method of embodiment 12, wherein said inhibitor of aspartate transport or aspartate metabolism is selected from the group consisting of DL-TBOA, L-(-)- threo-3-hydroxyaspartic acid, L-trans-2,4-PDC, TFB-TBOA, and GOT1 inhibitor 2c.
Embodiment 14. The method of any one of embodiments 1-13, wherein said mammal is a human.
Embodiment 15. The method of any one of embodiments 1-14, wherein said tissue inflammation is associated with an autoimmune disease.
Embodiment 16. The method of embodiment 15, wherein said autoimmune disease is selected from the group consisting of polymyositis, dermatomyositis, rheumatoid arthritis (RA), scleroderma, sjogren's syndrome, systemic lupus erythematosus, vasculitis, mixed connective tissue disease (MCTD), autoimmune hepatitis, psoriasis, and ankylosing spondylitis.
Embodiment 17. The method of embodiment 15, wherein said autoimmune disease is RA.
Embodiment 18. The method of any one of embodiments 1-14, wherein said tissue inflammation is associated with irritable bowel syndrome (IBS).
Embodiment 19. The method of any one of embodiments 1-18, said method comprising administering to said mammal an anti-TNF-a therapy.
Embodiment 20. The method of embodiment 19, wherein said anti-TNF-a therapy is selected from the group consisting of adalimumab, adalimumab-adbm, adalimumab-adaz, adalimumab-atto, certolizumab pegol, etanercept, etanercept-szzs, infliximab, infliximab- abda, and infliximab-dyyb.
Embodiment 21. A method for reducing tissue inflammation in a mammal, wherein said method comprises administering, to said mammal, a composition comprising cells, wherein a
population of intact mitochondria was introduced into said cells, and wherein the production of a TNF-a polypeptide within said mammal is reduced following said administering step.
Embodiment 22. The method of embodiment 21, wherein said composition comprises T cells containing said intact mitochondria.
Embodiment 23. The method of embodiment 21 or embodiment 22, where said T cells are obtained from said mammal.
Embodiment 24. A method for reducing tissue inflammation in a mammal, wherein said method comprises administering, to said mammal, an inhibitor of UPR signaling, wherein said method is effective to reduce production of a TNF-a polypeptide within said mammal.
Embodiment 25. The method of embodiment 24, wherein said inhibitor of UPR signaling can target an UPR signaling pathway component selected from the group consisting of BiP polypeptides, IREla polypeptides, PERK polypeptides, ATF6 polypeptides, eIF2a polypeptides, CHOP polypeptides, XBP-1 polypeptides, and XBP-1S polypeptides.
Embodiment 26. The method of embodiment 24, wherein said inhibitor of UPR signaling is selected from the group consisting of eeyarestatin I, mycolactone, exotoxin A, NSC 630668-R/l, MAL3-39, MAL3-101, E6 berbamine, ophiobolin A, equisetin, CJ-21058, Rose Bengal, erythrosin B, P97-A4, P87-A4, 17D9, P91-E9, 16F6, bisthiouracil, SCA-21, HUN-7293, cotransin, CAM741, apratoxin A, decatransin, valinomycin, CAD A, kinase inhibiting RNase attenuator 6 (KIRA6), 3-hydroxy-2-naphthoic acid (3HNA), MKC-3946, 4- Phenylbutyric acid (4-PBA), taurine-conjugated ursodeoxycholic acid (TUDCA), olmesartan, N-acetylcysteine (NAC), oleanolic acid (OA), ursolic acid, telmisartan, quercetin, 4p8C, STF-083010, B-109, GSK2606414, GSK2656157, AMG PERK44, melatonin, ceapin, IRSIB, AID 2732, salubrinal, ISRIB, guanabenz, sephinl, salicylaldimines, APY29, sunitinib, toyocamycin, 3-ethoxy-5,6-dibromosalicylal- dehyde, apigenin, FIRE peptide, baicalein, kaempferol, compound 147, compound 263, and 16F16.
Embodiment 27. A method for reducing tissue inflammation in a mammal, wherein said method comprises administering, to said mammal, an inhibitor of co-translational translocation, wherein said method is effective to reduce production of a TNF-a polypeptide within said mammal.
Embodiment 28. The method of embodiment 27, wherein said inhibitor of co- translational translocation can target a co-translational translocation pathway component that is selected from the group consisting of Sec61 polypeptides, Hsp70 polypeptides, ATPase polypeptides, calmodulin polypeptides, transmembrane domains of newly produced polypeptides, signal peptides, and signal peptidase complex polypeptides.
Embodiment 29. A method for reducing tissue inflammation in a mammal, wherein said method comprises administering, to said mammal, a composition comprising a mitochondrial metabolite, wherein said method is effective to reduce production of a TNF-a polypeptide within said mammal.
Embodiment 30. The method of embodiment 29, wherein said mitochondrial metabolite is selected from the group consisting of L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, oxaloacetate, and nicotinamide riboside.
Embodiment 31. The method of embodiment 29 or embodiment 30, wherein said composition comprises L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate.
Embodiment 32. A method for reducing tissue inflammation in a mammal, wherein said method comprises administering, to said mammal, an inhibitor of aspartate transport or aspartate metabolism, wherein said method is effective to reduce production of a TNF-a polypeptide within said mammal.
Embodiment 34. The method of embodiment 32, wherein said inhibitor of aspartate transport or aspartate metabolism is selected from the group consisting of DL-TBOA, L-(-)- threo-3-hydroxyaspartic acid, L-trans-2,4-PDC, TFB-TBOA, and GOT1 inhibitor 2c.
Embodiment 35. The method of any one of embodiments 21-34, wherein said mammal is a human.
Embodiment 36. The method of any one of embodiments 21-35, wherein said tissue inflammation is associated with an autoimmune disease.
Embodiment 37. The method of embodiment 36, wherein said autoimmune disease is selected from the group consisting of polymyositis, dermatomyositis, rheumatoid arthritis (RA), scleroderma, sjogren's syndrome, systemic lupus erythematosus, vasculitis, mixed connective tissue disease (MCTD), autoimmune hepatitis, psoriasis, and ankylosing spondylitis.
Embodiment 38. The method of embodiment 36, wherein said autoimmune disease is RA.
Embodiment 39. The method of any one of embodiments 21-35, wherein said tissue inflammation is associated with IBS.
Embodiment 40. The method of any one of embodiments 21-39, said method comprising administering to said mammal an anti-TNF-a therapy.
Embodiment 41. The method of embodiment 40, wherein said anti-TNF-a therapy is selected from the group consisting of adalimumab, adalimumab-adbm, adalimumab-adaz, adalimumab-atto, certolizumab pegol, etanercept, etanercept-szzs, infliximab, infliximab- abda, and infliximab-dyyb.
Embodiment 42. A method for treating a mammal having tissue inflammation, wherein said method comprises administering, to said mammal, a composition comprising cells, wherein a population of intact mitochondria was introduced into said cells, and wherein the production of a TNF-a polypeptide within said mammal is reduced following said administering step.
Embodiment 43. The method of embodiment 42, wherein said composition comprises T cells containing said intact mitochondria.
Embodiment 44. The method of embodiment 42 or embodiment 43, where said T cells are obtained from said mammal.
Embodiment 45. A method for treating a mammal having tissue inflammation, wherein said method comprises administering, to said mammal, an inhibitor of UPR signaling, wherein said method is effective to reduce production of a TNF-a polypeptide within said mammal.
Embodiment 46. The method of embodiment 45, wherein said inhibitor of UPR signaling can target an UPR signaling pathway component selected from the group consisting of BiP polypeptides, IREla polypeptides, PERK polypeptides, ATF6 polypeptides, eIF2a polypeptides, CHOP polypeptides, XBP-1 polypeptides, and XBP-1S polypeptides.
Embodiment 47. The method of embodiment 46, wherein said inhibitor of UPR signaling is selected from the group consisting of eeyarestatin I, mycolactone, exotoxin A, NSC 630668-R/l, MAL3-39, MAL3-101, E6 berbamine, ophiobolin A, equisetin, CJ-21058, Rose Bengal, erythrosin B, P97-A4, P87-A4, 17D9, P91-E9, 16F6, bisthiouracil, SCA-21, HUN-7293, cotransin, CAM741, apratoxin A, decatransin, valinomycin, CAD A, kinase inhibiting RNase attenuator 6 (KIRA6), 3-hydroxy-2-naphthoic acid (3HNA), MKC-3946, 4- Phenylbutyric acid (4-PBA), taurine-conjugated ursodeoxycholic acid (TUDCA), olmesartan, N-acetylcysteine (NAC), oleanolic acid (OA), ursolic acid, telmisartan, quercetin, 4p8C,
STF-083010, B-109, GSK2606414, GSK2656157, AMG PERK44, melatonin, ceapin, IRSIB, AID 2732, salubrinal, ISRIB, guanabenz, sephinl, salicylaldimines, APY29, sunitinib, toyocamycin, 3-ethoxy-5,6-dibromosalicylal- dehyde, apigenin, FIRE peptide, baicalein, kaempferol, compound 147, compound 263, and 16F16.
Embodiment 48. A method for treating a mammal having tissue inflammation, wherein said method comprises administering, to said mammal, an inhibitor of co-translational translocation, wherein said method is effective to reduce production of a TNF-a polypeptide within said mammal.
Embodiment 49. The method of embodiment 48, wherein said inhibitor of co- translational translocation can target a co-translational translocation pathway component that is selected from the group consisting of Sec61 polypeptides, Hsp70 polypeptides, ATPase polypeptides, calmodulin polypeptides, transmembrane domains of newly produced polypeptides, signal peptides, and signal peptidase complex polypeptides.
Embodiment 50. A method for treating a mammal having tissue inflammation, wherein said method comprises administering, to said mammal, a composition comprising a mitochondrial metabolite, wherein said method is effective to reduce production of a TNF-a polypeptide within said mammal.
Embodiment 51. The method of embodiment 50, wherein said mitochondrial metabolite is selected from the group consisting of L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, oxaloacetate, and nicotinamide riboside.
Embodiment 52. The method of embodiment 50 or embodiment 51, wherein said composition comprises L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate.
Embodiment 53. A method for treating a mammal having tissue inflammation, wherein said method comprises administering, to said mammal, an inhibitor of aspartate transport or aspartate metabolism, wherein said method is effective to reduce production of a TNF-a polypeptide within said mammal.
Embodiment 54. The method of embodiment 53, wherein said inhibitor of aspartate transport or aspartate metabolism is selected from the group consisting of DL-TBOA, L-(-)- threo-3-hydroxyaspartic acid, L-trans-2,4-PDC, TFB-TBOA, and GOT1 inhibitor 2c.
Embodiment 55. The method of any one of embodiments 42-54, wherein said mammal is a human.
Embodiment 56. The method of any one of embodiments 42-55, wherein said tissue inflammation is associated with an autoimmune disease.
Embodiment 57. The method of embodiment 56, wherein said autoimmune disease is selected from the group consisting of polymyositis, dermatomyositis, rheumatoid arthritis (RA), scleroderma, sjogren's syndrome, systemic lupus erythematosus, vasculitis, mixed connective tissue disease (MCTD), autoimmune hepatitis, psoriasis, and ankylosing spondylitis.
Embodiment 58. The method of embodiment 57, wherein said autoimmune disease is RA.
Embodiment 59. The method of any one of embodiments 42-55, wherein said tissue inflammation is associated with IBS.
Embodiment 60. The method of any one of embodiments 42-46, said method comprising administering to said mammal an anti-TNF-a therapy.
Embodiment 61. The method of embodiment 60, wherein said anti-TNF-a therapy is selected from the group consisting of adalimumab, adalimumab-adbm, adalimumab-adaz, adalimumab-atto, certolizumab pegol, etanercept, etanercept-szzs, infliximab, infliximab- abda, and infliximab-dyyb.
Embodiment 62. A method for treating a mammal having tissue inflammation, wherein said method comprises administering, to said mammal, a) a composition comprising aspartate, and b) adalimumab; wherein said method is effective to reduce production of a TNF-a polypeptide within said mammal.
Embodiment 63. The method of embodiment 62, wherein said composition comprising aspartate and said adalimumab are administered together.
Embodiment 64. The method of embodiment 62, wherein said composition comprising aspartate and said adalimumab are administered separately.
Embodiment 65. A method for treating a mammal having tissue inflammation, wherein said method comprises administering, to said mammal, a) a composition comprising aspartate, and b) etanercept; wherein said method is effective to reduce production of a TNF- a polypeptide within said mammal.
Embodiment 66. The method of embodiment 65, wherein said composition comprising aspartate and said etanercept are administered together.
Embodiment 67. The method of embodiment 65, wherein said composition comprising aspartate and said etanercept are administered separately.
Embodiment 68. A method for treating a mammal having tissue inflammation, wherein said method comprises administering, to said mammal, a) a composition comprising
aspartate, and b) infliximab; wherein said method is effective to reduce production of a TNF- a polypeptide within said mammal.
Embodiment 69. The method of embodiment 68, wherein said composition comprising aspartate and said infliximab are administered together.
Embodiment 70. The method of embodiment 68, wherein said composition comprising aspartate and said infliximab are administered separately.
Embodiment 7E The method of any one of embodiments 62-70, wherein said mammal is a human
Embodiment 72. The method of any one of embodiments 62-71, wherein said composition comprising aspartate further comprises a mitochondrial metabolite selected from the group consisting of NAD, alpha-ketobutyrate, malate, oxaloacetate, and nicotinamide riboside.
Embodiment 73. The method of any one of embodiments 62-72, wherein said composition comprising aspartate comprises L-aspartate, D-aspartate, NAD, alpha- ketobutyrate, malate, and oxaloacetate.
OTHER EMBODIMENTS
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
Claims
1. A method for restoring aspartate homeostasis in a mammal having tissue inflammation, wherein said method comprises administering, to said mammal, a composition comprising cells, wherein a population of intact mitochondria was introduced into said cells, and wherein the production of a tumor necrosis factor-a (TNF-a) polypeptide within said mammal is reduced following said administering step.
2. A method for restoring aspartate homeostasis in a mammal having tissue inflammation, wherein said method comprises administering, to said mammal, an inhibitor of UPR signaling, wherein said method is effective to reduce production of a TNF-a polypeptide within said mammal.
3. A method for restoring aspartate homeostasis in a mammal having tissue inflammation, wherein said method comprises administering, to said mammal, an inhibitor of co-translational translocation, wherein said method is effective to reduce production of a TNF-a polypeptide within said mammal.
4. A method for restoring aspartate homeostasis in a mammal having tissue inflammation, wherein said method comprises administering, to said mammal, a composition comprising a mitochondrial metabolite, wherein said method is effective to reduce production of a TNF-a polypeptide within said mammal.
5. A method for restoring aspartate homeostasis in a mammal having tissue inflammation, wherein said method comprises administering, to said mammal, an inhibitor of aspartate transport or aspartate metabolism, wherein said method is effective to reduce production of a TNF-a polypeptide within said mammal.
6. A method for reducing tissue inflammation in a mammal, wherein said method comprises administering, to said mammal, a composition comprising cells, wherein a
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population of intact mitochondria was introduced into said cells, and wherein the production of a TNF-a polypeptide within said mammal is reduced following said administering step.
7. A method for reducing tissue inflammation in a mammal, wherein said method comprises administering, to said mammal, an inhibitor of UPR signaling, wherein said method is effective to reduce production of a TNF-a polypeptide within said mammal.
8. A method for reducing tissue inflammation in a mammal, wherein said method comprises administering, to said mammal, an inhibitor of co-translational translocation, wherein said method is effective to reduce production of a TNF-a polypeptide within said mammal.
9. A method for reducing tissue inflammation in a mammal, wherein said method comprises administering, to said mammal, a composition comprising a mitochondrial metabolite, wherein said method is effective to reduce production of a TNF-a polypeptide within said mammal.
10. A method for reducing tissue inflammation in a mammal, wherein said method comprises administering, to said mammal, an inhibitor of aspartate transport or aspartate metabolism, wherein said method is effective to reduce production of a TNF-a polypeptide within said mammal.
11. A method for treating a mammal having tissue inflammation, wherein said method comprises administering, to said mammal, a composition comprising cells, wherein a population of intact mitochondria was introduced into said cells, and wherein the production of a TNF-a polypeptide within said mammal is reduced following said administering step.
12. The method of any one of claims 1, 6, and 11, wherein said composition comprises T cells containing said intact mitochondria.
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13. The method of claim 12, where said T cells are obtained from said mammal.
14. A method for treating a mammal having tissue inflammation, wherein said method comprises administering, to said mammal, an inhibitor of UPR signaling, wherein said method is effective to reduce production of a TNF-a polypeptide within said mammal.
15. The method of any one of claims 2, 7, and 14, wherein said inhibitor of UPR signaling can target an UPR signaling pathway component selected from the group consisting of BiP polypeptides, IREla polypeptides, PERK polypeptides, ATF6 polypeptides, eIF2a polypeptides, CHOP polypeptides, XBP-1 polypeptides, and XBP-1S polypeptides.
16. The method of claim 15, wherein said inhibitor of UPR signaling is selected from the group consisting of eeyarestatin I, mycolactone, exotoxin A, NSC 630668-R/l, MAL3-39, MAL3-101, E6 berbamine, ophiobolin A, equisetin, CJ-21058, Rose Bengal, erythrosin B, P97-A4, P87-A4, 17D9, P91-E9, 16F6, bisthiouracil, SCA-21, HUN-7293, cotransin, CAM741, apratoxin A, decatransin, valinomycin, CAD A, kinase inhibiting RNase attenuator 6 (KIRA6), 3-hydroxy-2-naphthoic acid (3HNA), MKC-3946, 4-Phenylbutyric acid (4- PBA), taurine-conjugated ursodeoxycholic acid (TUDCA), olmesartan, N-acetylcysteine (NAC), oleanolic acid (OA), ursolic acid, telmisartan, quercetin, 4p8C, STF-083010, B-109, GSK2606414, GSK2656157, AMG PERK44, melatonin, ceapin, IRSIB, AID 2732, salubrinal, ISRIB, guanabenz, sephinl, salicylaldimines, APY29, sunitinib, toyocamycin, 3- ethoxy-5,6-dibromosalicylal- dehyde, apigenin, FIRE peptide, baicalein, kaempferol, compound 147, compound 263, and 16F16.
17. A method for treating a mammal having tissue inflammation, wherein said method comprises administering, to said mammal, an inhibitor of co-translational translocation, wherein said method is effective to reduce production of a TNF-a polypeptide within said mammal.
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18. The method of any one of claims 3, 8, and 17, wherein said inhibitor of co- translational translocation can target a co-translational translocation pathway component that is selected from the group consisting of Sec61 polypeptides, Hsp70 polypeptides, ATPase polypeptides, calmodulin polypeptides, transmembrane domains of newly produced polypeptides, signal peptides, and signal peptidase complex polypeptides.
19. A method for treating a mammal having tissue inflammation, wherein said method comprises administering, to said mammal, a composition comprising a mitochondrial metabolite, wherein said method is effective to reduce production of a TNF-a polypeptide within said mammal.
20. The method of any one of claims 4, 9, and 19, wherein said mitochondrial metabolite is selected from the group consisting of L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, oxaloacetate, and nicotinamide riboside.
21. The method of claim 20, wherein said composition comprises L-aspartate, D- aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate.
22. A method for treating a mammal having tissue inflammation, wherein said method comprises administering, to said mammal, an inhibitor of aspartate transport or aspartate metabolism, wherein said method is effective to reduce production of a TNF-a polypeptide within said mammal.
23. The method of any one of claims 5, 6, and 22, wherein said inhibitor of aspartate transport or aspartate metabolism is selected from the group consisting of DL-TBOA, L-(-)- threo-3-hydroxyaspartic acid, L-trans-2,4-PDC, TFB-TBOA, and GOT1 inhibitor 2c.
24. The method of any one of claims 5, 6, and 22, wherein said mammal is a human.
79
25. The method of any one of claims 5, 6, and 22, wherein said tissue inflammation is associated with an autoimmune disease.
26. The method of claim 25, wherein said autoimmune disease is selected from the group consisting of polymyositis, dermatomyositis, rheumatoid arthritis (RA), scleroderma, sjogren's syndrome, systemic lupus erythematosus, vasculitis, mixed connective tissue disease (MCTD), autoimmune hepatitis, psoriasis, and ankylosing spondylitis.
27. The method of claim 26, wherein said autoimmune disease is RA.
28. The method of any one of claims 5, 6, and 22, wherein said tissue inflammation is associated with IBS.
29. The method of any one of claims 5, 6, and 22, said method comprising administering to said mammal an anti-TNF-a therapy.
30. The method of claim 29, wherein said anti-TNF-a therapy is selected from the group consisting of adalimumab, adalimumab-adbm, adalimumab-adaz, adalimumab-atto, certolizumab pegol, etanercept, etanercept-szzs, infliximab, infliximab-abda, and infliximab- dyyb.
31. A method for treating a mammal having tissue inflammation, wherein said method comprises administering, to said mammal, a) a composition comprising aspartate, and b) adalimumab; wherein said method is effective to reduce production of a TNF-a polypeptide within said mammal.
32. The method of claim 31, wherein said composition comprising aspartate and said adalimumab are administered together.
80
33. The method of claim 31, wherein said composition comprising aspartate and said adalimumab are administered separately.
34. A method for treating a mammal having tissue inflammation, wherein said method comprises administering, to said mammal, a) a composition comprising aspartate, and b) etanercept; wherein said method is effective to reduce production of a TNF-a polypeptide within said mammal.
35. The method of claim 34, wherein said composition comprising aspartate and said etanercept are administered together.
36. The method of claim 34, wherein said composition comprising aspartate and said etanercept are administered separately.
37. A method for treating a mammal having tissue inflammation, wherein said method comprises administering, to said mammal, a) a composition comprising aspartate, and b) infliximab; wherein said method is effective to reduce production of a TNF-a polypeptide within said mammal.
38. The method of claim 37, wherein said composition comprising aspartate and said infliximab are administered together.
39. The method of claim 37, wherein said composition comprising aspartate and said infliximab are administered separately.
40. The method of any one of claims 31-39, wherein said mammal is a human
41. The method of any one of claims 31-40, wherein said composition comprising aspartate further comprises a mitochondrial metabolite selected from the group consisting of NAD, alpha-ketobutyrate, malate, oxaloacetate, and nicotinamide riboside.
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42. The method of any one of claims 31-41, wherein said composition comprising aspartate comprises L-aspartate, D-aspartate, NAD, alpha-ketobutyrate, malate, and oxaloacetate.
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