WO2012016145A2 - Mitochondrial apoptosis-induced inflammation - Google Patents

Mitochondrial apoptosis-induced inflammation Download PDF

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WO2012016145A2
WO2012016145A2 PCT/US2011/045893 US2011045893W WO2012016145A2 WO 2012016145 A2 WO2012016145 A2 WO 2012016145A2 US 2011045893 W US2011045893 W US 2011045893W WO 2012016145 A2 WO2012016145 A2 WO 2012016145A2
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disease
nlrp3
secretion
inflammatory disease
apoptosis
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PCT/US2011/045893
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French (fr)
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WO2012016145A3 (en
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Kenichi Shimada
Moshe Arditi
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Cedars-Sinai Medical Center
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
    • A61K31/708—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid having oxo groups directly attached to the purine ring system, e.g. guanosine, guanylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]

Definitions

  • This invention relates to the treatment and diagnosis of inflammation, inflammatory diseases and inflammatory disease conditions.
  • Chlamydophila pneumoniae ⁇ CP Chlamydophila pneumoniae ⁇ CP is a widely prevalent (Blasi et al, 2009) intracellular Gram-negative pathogen that causes upper respiratory infections and contributes to the development of chronic inflammatory conditions such as asthma (Sutherland and Martin, 2007), atherosclerosis (Watson and Alp, 2008), arthritis (Gerard et al, 2009), and chronic obstructive pulmonary lung disease (COPD) (Papaetis et al, 2009).
  • TLR/MyD88 Naiki et al, 2005
  • NOD/RIP2 Shiada et al, 2009
  • Toll-like receptor (TLR) 2 and TLR4 both use MyD88 to sense CP (Joyee and Yang, 2008), yet TLR2 _/" and TLR4 _/" mice clear CP and recover from infection (Rodriguez et al., 2006).
  • This finding suggests redundant roles for TLR2 and TLR4 and also implicates the IL-1 receptor— a MyD88-dependent cytokine receptor— in host defense against CP.
  • CP infection elicits robust IL- ⁇ secretion in a number of experimental models (Kaukoranta-Tolvanen et al., 1996; Netea et al, 2004; Netea et al., 2000) and aberrant IL-1R modulation plays a role in the pathogenesis of CP infection in COPD patients (Rupp et al, 2003).
  • Secretion of IL- ⁇ a potent pyrogen that elicits a strong pro-inflammatory response (Dinarello, 2009), is tightly controlled by a diverse class of cytosolic complexes known as inflammasomes (Latz, 2010).
  • NLR NOD-like Receptor family member NLRP3 forms cytosolic oligomers with apoptosis-associated speck like protein (ASC) in dendritic cells (Ghiringhelli et al., 2009) and macrophages (Franchi et al, 2009), triggering autocatalytic activation of caspase-1 (Martinon et al, 2009). Caspase-1, in turn, cleaves pro-IL- ⁇ , producing mature IL-1 ⁇ .
  • ASC apoptosis-associated speck like protein
  • NLRP3 undergoes bipartite activation (Latz, 2010).
  • the first signal often NF- ⁇ activation, induces pro-IL- ⁇ and NLRP3 expression.
  • the second signal any one of a variety of unrelated entities— particulate matter (Dostert et al., 2008), crystals (Duewell et al, 2010), aggregated ⁇ -amyloid (Halle et al., 2008), extracellular ATP (Hogquist et al., 1991; Mariathasan et al., 2006) and microbial toxins (Meixenberger et al., 2010)— activates NLRP3. Exactly how the NLRP3 inflammasome responds to such wide range of danger signals was heretofore unclear.
  • ROS reactive oxygen species
  • Various embodiments of the present invention provide a method, comprising: providing a composition comprising a mitochondrial apoptosis inhibitor; and administering the composition to a subject in need of treatment for inflammation to treat the inflammation.
  • the mitochondrial apoptosis inhibitor can be selected from the group consisting of Bcl-2, Mcl-l, Bcl-xL, Bcl-w, Bfl-l, nfh, XIAP, Boo/Diva, Nrl3, BH4 domain, Inositol 1,4,5-trisphosphate receptor (IP3R) peptide, GSK-3 , cyclosporine A, viral mitochondrial inhibitor of apoptosis (vMIA) peptide/protein, MitoQ and combinations thereof.
  • the treatment for inflammation can treat an inflammatory disease, an inflammatory disease condition, an autoimmune disease, or combinations thereof.
  • the inflammatory disease, inflammatory disease condition, or autoimmune disease can be where IL-1 beta plays a role.
  • the inflammatory disease, inflammatory disease condition, or autoimmune disease where IL-1 beta can play a role can be selected from the group consisting of type 2 diabetes, rheumatoid arthritis, psoriasis, Alzheimer's disease, silicosis and asbestosis, gout, pseudogout, familial cold autoinflammatory syndrome (FCAS), Muckel-Wells syndrome (MWS), neonatal-onset multisystem inflammatory disease (NOMID), and combinations thereof.
  • the inflammatory disease or inflammatory disease condition can be selected from the group consisting of arthritis, Crohn's disease, inflammatory bowel disease, Alzheimer's disease, diabetes, gout, atherosclerosis, asbestosis/silicosis induced lung fibrosis and combinations thereof.
  • the autoimmune disease can be selected from the group consisting of Hashimoto's thyroiditis, Pernicious anemia, Addison's disease, type I diabetes, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, Sjogren syndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis, reactive arthritis, Grave's disease, celiac disease, and combinations thereof.
  • compositions comprising an oxidative nucleotide
  • administering the composition to a subject in need of treatment for inflammation to treat the inflammation.
  • the oxidative nucleotide can be selected from the group consisting of (i) 8-Hydroxy-2'-deoxyguranosine, (ii) 8-hydroxy Guanosine, (iii) 8-Oxo-2'- deoxyadenosine, (iv) 5-formyl-2'-deoxycytidine, (v) 5-formyl-2'-deoxyuridine, (vi) 5- hydroxymethyl-2'-deoxyuridine, (vii) 5-Hydroxymethyl-2'-deoxycytidine, (viii) 5-hydroxy- 2'-deoxyuridine, (ix) 5-hydroxy-2'-deoxycytidine, and (x) combinations thereof.
  • the treatment for inflammation can treat an inflammatory disease, an inflammatory disease condition, an autoimmune disease, or combinations thereof.
  • the inflammatory disease, inflammatory disease condition, or autoimmune disease can be where IL-1 beta plays a role.
  • the inflammatory disease, inflammatory disease condition, or autoimmune disease where IL-1 beta can play a role can be selected from the group consisting of type 2 diabetes, rheumatoid arthritis, psoriasis, Alzheimer's disease, silicosis and asbestosis, gout, pseudogout, familial cold autoinflammatory syndrome (FCAS), Muckel-Wells syndrome (MWS), neonatal-onset multisystem inflammatory disease (NOMID), and combinations thereof.
  • the inflammatory disease or inflammatory disease condition can be selected from the group consisting of arthritis, Crohn's disease, inflammatory bowel disease, Alzheimer's disease, diabetes, gout, atherosclerosis, asbestosis/silicosis induced lung fibrosis and combinations thereof.
  • the autoimmune disease can be selected from the group consisting of Hashimoto's thyroiditis, Pernicious anemia, Addison's disease, type I diabetes, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, Sjogren syndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis, reactive arthritis, Grave's disease, celiac disease, and combinations thereof.
  • FIG. 1 shows that Caspl plays a critical protective role during CP lung infection in accordance with various embodiments of the present invention.
  • Caspl deficiency confers higher mortality.
  • Caspl "7" mice or WT were infected intratracheally with 2xl0 6 , 1.5xl0 6 or lxl 0 6 inclusion forming units (IFU) of CP. The Kaplan-Meier survival curve is shown. Statistical significance was determined by Fisher's exact test.
  • Caspl "7" mice show increased lymphocytic infiltration in the lung during late-stage infection.
  • C Caspl deficiency results in delayed cytokine production and reduced bacterial clearance. Cytokine (IL-6, IFNy, IL-12p40 and IL-1 ⁇ ) levels in both BALF and lung homogenates were determined using ELISA.
  • D Bacterial burden in infected WT and Caspl "7" lung homogenates was also quantitated. Data shown are representative of three independent experiments.
  • FIG. 2 shows that IL-1 signaling is crucial for host survival and bacterial clearance during CP lung infection in accordance with various embodiments of the present invention.
  • a and B Blocked IL-1 signaling leads to higher mortality and reduced bacterial clearance.
  • WT mice were daily given an IL-IRA (500 ⁇ g) or vehicle control and then infected with CP (1.5xl0 6 IFU/mouse). The Kaplan-Meier survival curve is shown. Bacterial burden in lung homogenates was determined for IL-IRA and vehicle control treated mice 5 days post infection with lxl 0 6 IFU.
  • C and D Caspl "7" mice given recombinant rIL- ⁇ exhibit reduced mortality and enhanced bacterial clearance.
  • Caspl "7" mice were treated with rIL- ⁇ (8 ng) daily for 3 days or vehicle control and then infected with CP (1.5xl0 6 IFU/mouse). The Kaplan-Meier survival curve is shown. Bacterial burden in lung homogenates of rIL- ⁇ and vehicle control treated mice 5 days after infection with lxl 0 6 IFU. Note on statistical significance: one-way ANOVA with Tukey's post-hoc test - * p ⁇ 0.05, ** p ⁇ 0.01, *** pO.001.
  • FIG. 3 depicts macrophage TLR2/MyD88 signaling and the NLRP3/ASC/Caspase-l inflammasome are required for IL- ⁇ secretion in response to CP in accordance with various embodiments of the present invention.
  • TLR2/MyD88 signaling is necessary for IL- ⁇ secretion in response to live CP.
  • IL- ⁇ concentration in the culture supernatant was determined using ELISA after a 24 h treatment with various stimuli— UVCP (MOI 2.5, 5 or 10), live CP (MOI 2.5, 5 or 10), LPS or PGN— in WT, MyD88 _/" , TRIF _/ ⁇ , and RIP2 _/" BMDM and in WT, TLR2 _/" , TLR4 _/ ⁇ , and TLR9 _/ ⁇ BMDM.
  • Data shown are representative of at least three independent experiments.
  • C, D, E and F Live CP, not UVCP, stimulates maximal IL- 1 ⁇ secretion from macrophages 24 hours after infection.
  • IL- ⁇ and TNFa concentrations were measured in the culture supernatants of WT and Caspl "7" BMDM treated for 24 h with the aforementioned stimuli. A proportion of cells were also treated with 5 mM ATP for the final 2 h of culture. Data shown are representative of at least three independent experiments.
  • G and H The NLRP3/ASC inflammasome is required for macrophage IL- ⁇ secretion in response to live CP.
  • IL-1 ⁇ and TNFa concentrations in the culture supernatants of WT, AS /_ , and NLRP3 " " BMDM were measured 24 hours after treatment with UVCP (MOI 2.5, 5, 10), live CP (MOI 2.5, 5, 10), LPS or PGN. Data shown are representative of at least three independent experiments.
  • Figure 4 shows that phagocytosis and bacterial de novo protein synthesis are necessary to activate the NLRP3 inflammasome in CP infected macrophages in accordance with various embodiments of the present invention.
  • A Bacterial internalization by macrophages is necessary for IL- ⁇ secretion in response to CP. IL- ⁇ (black bars) and TNFa (gray bars) secretion by CP infected (MOI 10, 24 h) BMDM in the presence or absence of cytochalasin D was quantified using ELISA. Data shown are representative of two or more independent experiments.
  • CP activation of the NLRP3 inflammasome in macrophages is cathepsin independent.
  • IL- ⁇ concentration in culture supematants of CP infected (MOI 10, 24 h) BMDM was determined in the presence of increasing amounts of Ac-LLM.
  • LPS-primed (1 ⁇ g/ml, 8 h) BMDM treated with alum (ALM, 130 ⁇ g/ml, final 2 h of culture) were given increasing amounts of Ac-LLM.
  • Data shown are representative of two or more independent experiments. (** p ⁇ 0.01, *** p ⁇ 0.001; Student's t test).
  • C and D Antioxidant (N-acetylcysteine, NAC) treatment does not specifically inhibit IL- ⁇ secretion.
  • ELISA was used to determine IL- ⁇ and TNFa concentration in the culture supematants of CP (MOI 10, 24 h) infected BMDM and LPS- primed (8 h), ATP treated (5 mM, final 2 h culture) BMDM in the presence of increasing doses of NAC.
  • CP activation of the NLRP3 inflammasome is reactive oxygen species (ROS) independent.
  • ROS reactive oxygen species
  • ELISA was used to determine IL- ⁇ and TNFa concentration in the culture supematants of CP (MOI 10, 24 h) infected BMDM in the presence of increasing doses of chloramphenicol. Data shown are representative of two or more independent experiments.
  • FIG. 5 shows that mitochondrial dysfunction is linked to NLRP3 inflammasome activation in accordance with various embodiments of the present invention.
  • A Mitochondrial protein synthesis is important in the NLRP3 inflammasome activation cascade.
  • ELISA was used to determine IL- ⁇ and TNFa concentration in the culture supematants LPS-primed (1 ⁇ g/ml, 8 h), ATP treated (5 mM, final 2 h culture) BMDM in the presence of increasing doses of chloramphenicol. Also shown is an LDH release assay of the same BMDM. Data shown are representative of two or more independent experiments.
  • mPTP mitochondrial permeability transition pore
  • IL- ⁇ and TNFa secretion by CP and LPS+ATP treated BMDM were measured using ELISA in the presence of increasing doses of cyclosporin A (CsA). Data shown are representative of two or more independent experiments.
  • CsA cyclosporin A
  • Loss of mitochondrial membrane potential ( ⁇ ) correlates with NLRP3 inflammasome activation.
  • BMDM were treated with UVCP or infected with live CP (MOI 2.5, 5, or 10; 24 h) and then examined for TMRM incorporation.
  • LPS-primed BMDM (1 ⁇ g/ml, 8 h) were treated with ATP (5mM) or staurosporine (STS, 5 ⁇ ) and examined for TMRM incorporation. The kinetics of ⁇ loss were monitored over time using a similar TMRM incorporation assay.
  • BMDM were treated with live CP (MOI 10), ATP (5 mM), STS (5 ⁇ ), alum (ALM, 130 ⁇ g/ml), or nigericin (NIG, 10 ⁇ ). Data shown are representative of two or more independent experiments (** p ⁇ 0.01, *** p ⁇ 0.001; Student's t test).
  • Extracellular ATP a potent NLRP3 activator, causes rapid, irreversible loss of ⁇ .
  • BMDM were stimulated with LPS (1 ⁇ g/ml, 3 h) and then loaded with 200 nM TMRM in Ringer's solution for 30 min. Following probe loading, 5 mM ATP was added and the live cell kinetics of ⁇ were analyzed every 20 seconds thereafter. The kinetic profile shown depicts the mean value +/- the SEM at each time point. Representative image of mitochondrial TMRM fluorescence and associated 3 dimensional intensity map are shown. Data are representative of two or more independent experiments.
  • Figure 6 depicts apoptotic stimuli activate the NLRP3 inflammasome in macrophages in accordance with various embodiments of the present invention.
  • NLRP3 activators cause cell death.
  • BMDM were treated with UVCP (MOI 10), live Cpn (MOI 10), ATP (5 mM), STS (5 ⁇ ) or alum (ALM, 130 ⁇ g/ml) and LDH release was determined at the indicated time points. Data shown are representative of three or more independent experiments.
  • BMDM were treated with UVCP (MOI 10), live CP (MOI 10), ATP (5 mM) or STS (5 ⁇ ) for the indicated times.
  • Immunoblotting was used to analyze mouse caspase-1, pro-IL- ⁇ , and IL- ⁇ in culture supernatants and lysates of BMDM treated with (left to right) UVCP (MOI 10, 8 h), live CP (MOI 10, 8 h), LPS (1 8 h), LPS + ATP (5 mM, final 2 h of culture), ATP alone, LPS + STS (5 ⁇ , final 2 h of culture), or STS alone.
  • Data shown are representative of three or more independent experiments.
  • Staurosporine induces IL- ⁇ secretion from primed macrophages without affecting TNFa secretion.
  • IL- ⁇ and TNFa secretion by WT and Caspl ' BMDM was measured using ELISA.
  • BMDM were primed with UVCP (MOI 10, 8 h), live Cpn (MOI 10, 8 h) or LPS (1 ⁇ / ⁇ 1, 8 h). Cells were then treated with STS (2.5 ⁇ or 5 ⁇ ) for the final 2 h of culture. Data shown are representative of three or more independent experiments.
  • (E) Staurosporine-induced IL- ⁇ ⁇ secretion by macrophages depends on the NLRP3/ASC inflammasome. WT, ASC /_ or NLRP3 " " BMDM were given UVCP (MOI 10, 8 h), live CP (MOI 10, 8 h) or LPS (1 8 h) and then treated with STS
  • BMDM were treated with UVCP (MOI 10, 8 h), live CP (MOI 10, 8 h), LPS (1 ⁇ g/ml, 8 h), UVCP + ATP (5 mM, final 2 h of culture), live CP + ATP, LPS + ATP, UVCP + STS (5 ⁇ , final 2 h of culture), live CP + STS, or LPS + STS and then exposed to various concentrations of AntA (0, 2.5, 5, 10 ⁇ ). IL- ⁇ and TNFa secretion were then measured using ELISA. Data shown are representative of three or more independent experiments. (H) Apoptotic cells release IL- ⁇ in a Caspl -dependent manner.
  • BMDM were treated with live CP (MOI 10, 8 h), LPS (1 ⁇ g/ml, 5 h), LPS + ATP (5 mM, final 2 h of culture), or ATP alone. Then, cells were fixed in paraformaldehyde and stained with an antibody specific for intracellular IL- ⁇ and with DAPI. Data shown are representative of three or more independent experiments.
  • Figure 7 shows that Bcl2 overexpression reduced IL- ⁇ ⁇ secretion, mitochondrial collapse and apoptosis, but not TNFa production in accordance with various embodiments of the present invention.
  • a and B IL- ⁇ and TNFa in supernatant was measured in Bcl2- overexpressed macrophages.
  • C ⁇ was determined by TMRM incorporation assay.
  • D Apoptotic cell was determined by DAPI staining.
  • FIG. 8 depicts Alveolar macrophages are reservoir for C. pneumoniae in Caspl-/- mice in accordance with various embodiments of the present invention.
  • Caspl -/-phagocytes Compared to WT, Caspl -/-phagocytes contain more Chlamydophila and macrophages are the principal C. pneumoniae harboring cell type. Single-cell suspensions from C. pneumoniae infected lungs of WT and Caspl -/-were prepared 12 days following infection. Cells were then stained for characteristic leukocyte markers and stained intracellular C. pneumoniae with a FITC conjugated anti-Chlamydia monoclonal antibody (mAb), and analyzed by flow cytometry to determine which cell types contain C. pneumoniae.
  • mAb FITC conjugated anti-Chlamydia monoclonal antibody
  • CD45+ cells Representative flow cytometry histograms of CD45+ cells, F4/80+ cells (CDl lc+ gated), and Ly6G+ (CDl lc+ gated) cells are shown. Also shown are the proportions of total lung leukocytes that contain C. pneumoniae in WT and Caspl-/-mice and the absolute numbers of leukocytes in C. pneumoniae infected lungs from WT and Caspl-/-mice. Data for all experiments shown represent at least two independent experiments.
  • FIG. 9 shows that TLR2/MyD88 is indispensable for C. pneumoniae-induced TNF- ⁇ production by macrophages in accordance with various embodiments of the present invention.
  • A WT, MyD88-/-, TRIF-/-, and RIP2-/-BMDM were treated with live C. pneumoniae (MOI 2.5, 5, 10), LPS (1 ⁇ g/ml), PGN, poly I:C, muramyldipeptide (MDP), and CpG DNA and assessed for TNFa production by ELISA.
  • B WT, TLR2-/-, TLR4-/-, and TLR9-/-BMDM were treated with live C. pneumoniae (MOI 2.5, 5, 10), LPS (1 ⁇ g/ml), PGN, and CpG DNA and assessed for TNFaproduction by ELISA.
  • FIG. 10 shows that Caspl deficiency does not affect macrophage phagocytic activity or Chlamydophilal infectivity in accordance with various embodiments of the present invention.
  • Caspl -/-macrophages are as effective as WT macrophages in internalizing C. pneumoniae.
  • BMDMs were infected with labeled C. pneumoniae (solid line histogram, MOI 2.5, 5, 10, and 20) or vehicle control (gray-filled histogram). The mean fluorescence intensity (MFI) and percentage of labeled C. pneumoniae internalized cells are indicated.
  • MFI mean fluorescence intensity
  • B WT and Caspl -/-BMDMs were infected with C. pneumoniae (MOI 10). Cell lysates were harvested at indicated time points and viable bacteria were quantified by infecting HEp2 cells.
  • FIG. 11 shows C. pneumoniae-induced ⁇ loss in macrophages is NLRP3 inflammasome independent.
  • A WT, ASC-/-, NLRP3-/-and Caspl -/-BMDM were treated with UVCP (MOI 2.5, 5, 10), live C. pneumoniae (MOI 2.5, 5, 10), or STS (5 ⁇ ) and then examined for TMRM incorporation.
  • Statistical significance was determined by Student's t test in comparison to non-treated cells (* p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001).
  • the LDH release was determined in C. pneumoniae-mfected BMDM at 24hs in WT and Caspl-/-BMDM (B). Data shown are representative of three or more independent experiments.
  • Figure 12 also shows that mitochondrial dysfunction is linked to NLRP3 inflammasome activation in accordance to various embodiments of the present application,
  • (a-c) ELISA was used to determine IL- ⁇ and TNFa concentrations in BMDM culture supernatants treated with (a) various NLRP3 inflammasome pathway-activating stimuli indicated or
  • LPS (1 ⁇ g/ml) plus ATP (5 mM, final 2h culture
  • Cyclosporin A (CsA) inhibits LPS+ATP-induced IL- ⁇ ⁇ in WT BMDM and cyclophilin D (CypD) deficient BMDM in a dose-dependent manner
  • CsA Cyclosporin A
  • Loss of mitochondrial membrane potential ( ⁇ ⁇ ⁇ ) correlates with NLRP3 inflammasome activation.
  • ATP mM
  • STS 5 ⁇
  • Extracellular ATP causes rapid, irreversible loss of A X V M by TMRM microscopy.
  • a representative image of mitochondrial TMRM fluorescence and associated 3D intensity map are shown in (f).
  • the kinetic profile shown in (g) depicts the mean value +/- the SEM at each time point.
  • High concentrations of extracellular K + (h) block IL- ⁇ secretion and (i) prevent ⁇ ⁇ ⁇ reduction as measured by TMRM incorporation.
  • BMDM were transferred to culture media containing either 5 mM or 100 mM K + .
  • BMDM were then treated with ATP (5 mM), STS (5 ⁇ ) or NIG (10 ⁇ ) and cultured for an additional 2 h before culture supernatants were collected and IL- 1 ⁇ concentration was measured.
  • ATP 5 mM
  • STS 5 ⁇
  • NIG 10 ⁇
  • CP infected BMDM were cultured for an additional 20 h before supernatants were collected for IL- ⁇ ⁇ measurement
  • OCR Oxygen consumption rate
  • the base line OCR was significantly reduced 2 h after ATP (5 mM), STS (5 ⁇ ) or NIG (10 ⁇ ) treatment, (k) The change of OCR was monitored after ⁇ ⁇ oligomycin, 1 ⁇ FCCP, and ⁇ ⁇ Rotenone sequential additions.
  • the kinetics of OCR was monitored in macrophages in response to NLRP3 activator. Data shown are representative of two or more independent experiments. Note on statistical significance: * p ⁇ 0.05, ** p ⁇ 0.01 , ***p ⁇ 0.001.
  • Figure 13 also shows that Bcl-2 inversely regulates mitochondrial dysfunction and NLRP3 inflammasome activation in accordance to various embodiments of the present application, (a) BCI-2-UQOX or empty vector (EV, neor) was stably expressed in MCL macrophages, and Bcl2 expression was determined by immunoblot.
  • Figure 14 shows that Salmonella type III secretion protein and invasion factor SipB induces mitochondrial depolarization and caspase-1 dependent IL- ⁇ secretion in accordance to various embodiments of the present application.
  • (a) Three h LPS-primed Nlrp3-/-BMDM were exposed to S. typhimurium (MOI 5) and nonflagellated mutant, and IL- ⁇ in the culture supernatant was quantified by ELISA. Significant differences are indicated; *** p ⁇ 0.001, by one-way ANOVA and Tukey's HSD post-hoc test.
  • Figure 15 shows that oxidized mitochondrial DNA binds to NLRP3 and activates the inflammasome in accordance to various embodiments of the present application.
  • Flag- NLRP3 and empty vector (EV) stably-expressing 293 cells were exposed to BrdU-labeled mitochondrial DNA (mtDNA) with lipofectamine. Cell lysates were collected 3 h later and immunoprecipitated.
  • mtDNA was detected by BrdU dot-blot, and NLRP3 Ab was used for immunoblotting as a loading control,
  • BMDM were preloaded with BrdU (10 ⁇ ), and cells were treated with LPS (1 ⁇ g/ml, 3 h), followed by 3-MA (2.5 mM, 1 h), ATP (5 mM, 1 h) or NIG (10 ⁇ , 1 h).
  • Cell lysates were collected and immunoprecipitated with anti- NLRP3 Ab, then detected by dot-blots probed with anti-BrdU or anti-80H-dG Abs.
  • IL- ⁇ and TNFa levels were quantified in supematants by ELISA.
  • LPS-primed Aim2 ⁇ f ⁇ BMDM were treated with exogenous oxDNA (2 ⁇ / ⁇ 1) and IL- ⁇ and TNFa levels were determined in culture supematants by ELISA.
  • BMDM were stimulated with LPS (1 ⁇ / ⁇ 1) in the presence of deoxyguanosine (dG) and 8-OH-dG, then treated with (h) ATP, NIG or (i) poly(dA:dT) (2 ⁇ / ⁇ 1), and IL- ⁇ release was quantified by ELISA.
  • BMDM were primed with LPS (1 ⁇ , 3 h) in the presence of dG or 8-OH-dG (200 ⁇ ), pretreated with 3-MA (2.5 mM for lh), and then treated with ATP (5 mM, 1 h).
  • Immunoprecipitation with anti-NLRP3 Ab was performed, and BrdU or 8-OH-dG dot-blotting was performed with immunoblotting for NLRP3 as a loading control.
  • Data shown are representative of three or more independent experiments. Note on statistical significance: * p ⁇ 0.05.
  • FIG 16 shows that macrophage NLRP3, ASC, CASP1 deficiency does not alter TNFa secretion in accordance to various embodiments of the present application.
  • BMDM were exposed to UV-killed CP or live CP (MOI 10, 24hrs), or treated with LPS (1 ⁇ g/ml, 5 h), LPS + ATP (5 mM, final 2 h of culture), ATP.
  • IL- ⁇ and TNFa secretion were then measured using ELISA. Data shown are representative of three independent experiments.
  • FIG. 17 shows that Bcl2 overexpression suppresses apoptosis in accordance to various embodiments of the present application.
  • BMDM were stimulated with LPS (1 ⁇ g/ml, 3 h) and then treated with ATP (5 mM, 4h) and NIG (10 ⁇ , 4h).
  • Cells were labeled with annexin V and 7AAD. Apoptotic cell was analyzed by flow cytometry. Data shown are three independent experiments.
  • FIG. 18 shows that pyroptosis and necrosis do not play a role in NLRP3 inflammasome activation in accordance to various embodiments of the present application
  • CASP1-/- BMDM were infected with CP (MOI 10), treated with ATP (5 mM), STS (5 ⁇ ), NIG (10 ⁇ ) or Alum (130 ⁇ g/ml) for 4 h. and examined for TMRM incorporation. The change of ⁇ were examined using a TMRM incorporation assay
  • the LDH release was determined in CP-infected BMDM at 24hs in WT and CASP1-/- BMDM.
  • Figure 19 shows Bcl2 overexpression suppressed IL- ⁇ secretion not TNFa secretion in accordance to various embodiments of the present application.
  • the stably expressing Bcl2- neor, Empty vector (EV, neor), and YFP-neor macrophages were stimulated as indicated.
  • IL- 1 ⁇ and TNFa level in the supernatant were determined.
  • Data shown are three independent experiments. * p ⁇ 0.05, Student's t test.
  • Figure 20 shows that mitochondrial DNA contributes NLRP3 inflammasome.
  • pOMCL was generated with 50 ⁇ g/ml of EtBr for more than 20 passages. Mitochondrial DNA contents were determined by qPCR of mitochondria specific gene mtCOXl compared to nuclear DNA (GAPDH).
  • GPDH nuclear DNA
  • b-c Metabolic and non-metabolic mitochondrial labeling were compared in pOMCL.
  • pOMCL is attenuated IL- ⁇ secretion in response to NLRP3 stimuli.
  • LPS-primed BMDM were treated with ATP (5 mM) and NIG (10 ⁇ ) for 15 and 30 min. Cytosolic fraction was collected from homogenates. Mitochondrial DNA was detected by qPCR of mtCOXl . Data shown are representative of three or more independent experiments. Note on statistical significance: *** p ⁇ 0.001 (Student's t test used unless otherwise noted).
  • Figure 21 shows that mitochondrial DNA is colocalized with NLRP3.
  • NLRP3 stably expressed 293 cells were exposed to BrdU-labeled mitochondrial DNA with lipofectamin in accordance to various embodiments of the present application. 3hrs after exposure, Cell were fixed with 2% PF A/PBS, permeabilized, treated with 10 U/ml for 30 min, and then stained with anti-BldU mAb. The co-localization of YFP-NLRP3 and mtDNA were analyzed by confocal microscopy, (b) The representative 3 dimensional analysis for co- localization of YFP-NLRP3 and mtDNA.
  • the present invention provides for methods to treat inflammation and inflammatory diseases and disease conditions; particularly, inflammatory diseases and disease conditions wherein the role of IL- ⁇ ⁇ has been established.
  • inflammatory diseases and disease conditions that can be treated by the present invention include but are not limited to autoimmune diseases, arthritis, Crohn's disease, inflammatory bowel disease, Alzheimer's disease, diabetes, gout, atherosclerosis, and asbestosis/silicosis induced lung fibrosis.
  • autoimmune diseases include but are not limited to Hashimoto's thyroiditis, Pernicious anemia, Addison's disease, type I diabetes, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, Sjogren syndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis, reactive arthritis, Grave's disease, and celiac disease - sprue (gluten sensitive enteropathy).
  • IL- ⁇ is a highly inflammatory cytokine and inhibitors, such as mitochondrial apoptosis inhibitors, can be used to block the IL- ⁇ induced inflammatory pathway in multiple inflammatory diseases where the role of IL- ⁇ has been established.
  • mitochondrial apoptosis inhibitors include but are not limited to Bcl-2, Mcl-1, Bcl-xL, Bcl-w, Bfl-1, nfh, XIAP, Boo/Diva, Nrl3, BH4 domain, Inositol 1,4,5- trisphosphate receptor (IP3R) peptide, ⁇ 8 ⁇ -3 ⁇ , cyclosporine A, viral mitochondrial inhibitor of apoptosis (vMIA) peptide/protein, and MitoQ.
  • Bcl-2 Mcl-1, Bcl-xL, Bcl-w, Bfl-1, nfh, XIAP, Boo/Diva, Nrl3, BH4 domain, Inositol 1,4,5- trisphosphate receptor (IP3R) peptide, ⁇ 8 ⁇ -3 ⁇ , cyclosporine A, viral mitochondrial inhibitor of apoptosis (vMIA) peptide/protein, and MitoQ.
  • IP3R Inosito
  • apoptosis is thought to be a 'silent death' that fails to provoke inflammation.
  • the inventors report that in the presence of signal 1, the NLRP3 inflammasome is activated by mitochondrial apoptotic signaling that licenses production of the pro-inflammatory cytokine, interleukin- ⁇ ⁇ (IL- ⁇ ).
  • IL- ⁇ interleukin- ⁇ ⁇
  • NLRP3 secondary signal activators such as ATP induce mitochondrial dysfunction and apoptosis, which results in the release of oxidized mitochondrial DNA (mtDNA).
  • mtDNA oxidized mitochondrial DNA
  • the anti-apoptotic protein Bcl-2 inversely regulates mitochondrial dysfunction and NLRP3 inflammasome activation.
  • Mitochondrial DNA directly induces NLRP3 inflammasome activation, because macrophages lacking mtDNA have severely attenuated IL- ⁇ production, yet still undergo apoptosis.
  • Both binding of oxidized mtDNA to the NLRP3 inflammasome and IL- ⁇ ⁇ secretion can be competitively inhibited by the oxidized nucleoside, 8-OH-dG.
  • the inventors' data reveal that oxidized mtDNA released during programmed cell death causes activation of the NLRP3 inflammasome.
  • caspase-1 dependent IL- ⁇ secretion is critical for host defense during CP lung infection and that CP infection alone triggers caspase-1 activation via the NLRP3 inflammasome.
  • CP infected macrophages secreted less IL- ⁇ in the presence of inhibitors of apoptotic signals. Closer examination of this phenomenon revealed that mitochondrial damage which specifically resulted in apoptotic signals induced NLRP3 -dependent IL- ⁇ secretion in primed macrophages.
  • Caspl -dependent IL- ⁇ secretion is required for host defense against CP lung infection.
  • Caspl "7" mice displayed delayed pulmonary bacterial clearance leading to increased mortality compared to WT mice. Macrophages play a key role in this process, responding to CP via the NLRP3 inflammasome.
  • Caspl "7" mice showed delayed IFN- ⁇ production and defective iNOS activation in Caspl "7” AM, consistent with reports showing the critical role of IFN- ⁇ and iNOS in clearing CP infection (Rottenberg et al, 1999).
  • the inventors discovered evidence that the mitochondria play a role within the
  • NLRP3 activation cascade Closer examination of this phenomenon led the inventors to discover a novel mode of NLRP3 activation whereby the mitochondria sense cellular danger and disseminate apoptotic signals that trigger the NLRP3 inflammasome.
  • NLRP3 contains a leucine rich repeat (LRR) domain, a central NACHT domain and a pyrin domain (PYD) (Martinon et al, 2007). Hoffman and colleagues showed that BMDM from mice lacking the LRR domain of NLRP3 exhibit attenuated but not ablated IL- ⁇ secretion (Hoffman et al., 2010). As such, it is thought that, in a mechanism distinct from pattern recognition receptors (PRR), NLRP3 does not sense each of these diverse ligands directly with its LRR.
  • PRR pattern recognition receptors
  • ROS reactive oxygen species
  • K+ potassium cation
  • lysosomal leakage activate the NLRP3 inflammasome
  • CP infectious elementary body
  • RB vegetative reticulate body
  • Chlamydophila are known to inhibit actively the process of phagolysosomal fusion.
  • the mitochondrial K+ cycle inexorably linked to intracellular K+ concentration, is absolutely central to maintaining mitochondrial volume, controlling metabolic ROS (Garlid and Paucek, 2003) and induction of apoptosis (Park and Kim, 2002). Perhaps the mitochondria have been overlooked as the control station for NLRP3 activation because all three physiological perturbations not only lead to but also result from mitochondrial apoptotic cascades.
  • NLRP3 triggers such as alum, ATP, nigericin and live CP cause mitochondrial dysfunction and cell death in macrophages.
  • antimycin A an anti-apoptotic
  • cyclosporin A an inhibitor of mitochondrial pore formation
  • NALP1 interacts with Caspase-9 and the pro-apoptotic protein Apaf-1 (Chu et al, 2001), the anti-apoptotic protein Bcl-2 suppresses NALP1 activation (Bruey et al., 2007) and Bcl-xL and Bcl-2 gene transfection attenuates muramyl dipeptide induced caspase-1 activity (Faustin et al, 2009). It is also interesting that many inflammasome associated proteins have homologs which act as apoptotic factors in C. elegans (Miura et al, 1993).
  • both the apoptosome and the NLRP3 inflammasome share common upstream activating factors derived from the mitochondria. Yet, the apoptosome and the inflammasome are functionally distinct. Mice deficient in Apaf-1, Caspase-9 or Caspase- 3 exhibit infertility, abnormal brain development, and lethality (Honarpour et al., 2000; Kuida et al, 1998; Kuida et al, 1996) whereas no such phenotype has been found for Caspase-1, NLRP3 or ASC deficient mice.
  • IL- ⁇ a potent pyrogen that elicits a strong pro-inflammatory response l
  • NLR NOD-like Receptor family member
  • NLRP3 forms cytosolic oligomers with apoptosis-associated speck like protein (ASC) in dendritic cells 3 and macrophages 4 , triggering autocatalytic activation of caspase-1 5 .
  • ASC apoptosis-associated speck like protein
  • Caspase-1 cleaves pro-IL- ⁇ , producing mature IL- ⁇ .
  • NLRP3 undergoes bipartite activation 2 .
  • the first signal often NF- ⁇ activation, induces pro-IL- ⁇ and NLRP3 expression.
  • the second signal any one of a variety of unrelated entities— particulate matter 6 , crystals 7 , aggregated ⁇ -amyloid 8 , extracellular ATP 9 ' 10 , or microbial toxins 11 activates the NLRP3 inflammasome.
  • Mitochondria are extraordinarly complex regulators of cytosolic homeostasis. These organelles sense and respond to changes in intracellular K + 17 and ROS 18 , and have an important relationship with the lysosome 19 . Perturbation of intracellular K + , ROS or lysosomal stability can result in mt dysfunction and apoptosis 20 ⁇ 22 . Therefore, these cellular organelles are well positioned to regulate the NLRP3 activation pathway.
  • CP Chlamydia pneumoniae
  • mt damage which specifically triggers apoptotic signals, induces NLRP3 -dependent IL- ⁇ secretion in primed macrophages.
  • Evidence is provided herein for a novel innate immune defense program by which primed macrophages, in response to danger signals and cytosolic stress, activate an mt-dependent apoptotic cascade, which is necessary and sufficient for caspase-1 activation by the NLRP3 inflammasome in the presence of signal 1.
  • This apoptotic cascade activates the NLRP3 inflammasome through cytosolic release of oxidized mtDNA, which binds to and activates the NLRP3 inflammasome.
  • NLRP3 inflammasome has become a critical nexus mediating IL- ⁇ and IL-18 responses to pathogens and innate immune stimuli. Because inflammasome stimuli are diverse and often unrelated, discerning the mechanism of NLRP3 activation has proven difficult and remains elusive. Recent evidence has begun to indicate mt as key players in NLRP3 inflammasome signaling 15 ' 16 . During the analysis of C. pneumoniae infection, the inventors uncovered evidence that mt play an important role in activating the NLRP3 inflammasome. Herein, the inventors provide a mechanistic explanation for NLRP3 activation.
  • mt as key players in NLRP3 inflammasome induction unifies the three physiological perturbations under the single umbrella of apoptosis.
  • mt rapidly respond by releasing cytochrome c and inducing apoptosis 39 .
  • cathepsins and ROS released during lysosomal rupture can also profoundly impact mt membrane integrity, causing membrane permeabilization and subsequent initiation of apoptosis 40 ' 41 .
  • the mt K + cycle inexorably linked to intracellular K + concentration, is obligatorily central to maintaining mt volume, controlling metabolic ROS 17 and inducing
  • NLRP3 triggers such as alum, ATP, NIG and live CP cause mt dysfunction and cell death in macrophages. It is also shown herein that STS, a pro-apoptotic compound, is sufficient to act as a second signal for NLRP3 activation. Moreover, LPS- primed macrophages treated with NLRP3 activators secrete less IL- ⁇ ⁇ , but the same amount of TNFa in the presence of cyclosporin A.
  • Apoptosis is critical for NLRP3 inflammasome induction, because overexpression of anti-apoptotic Bcl2 attenuates IL- ⁇ secretion by LPS- primed macrophages and Bcl2 knock-down results in the converse.
  • the inventors also find that S. typhimurium type III secretion mutants that cannot induce apoptosis are unable to induce IL- ⁇ secretion, and Bcl2 overexpression inhibits wild-type S. typhimurium infection- induced IL- ⁇ secretion.
  • apoptosis is an indispensable step in NLRP3 inflammasome activation. While apoptosis is often portrayed as 'silent' cell death, the inventors' data suggest that in the presence of proinflammatory signal 1, the apoptotic machinery activates the NLRP3 inflammasome. Thus, intrinsic apoptosis has the capability of activating the NLRP3 inflammasome, but remains a silent death unless in the context of signal 1.
  • NLRP3 inflammasome complexes sense such a wide range of cytosolic danger signals, including ATP, K + efflux, alum, uric acid crystals, ⁇ - amyloid, and various microbial infections 43 ' 44 .
  • ATP ATP
  • K + efflux alum
  • uric acid crystals alum
  • ⁇ - amyloid a microbial infections 43 ' 44
  • NLRP3 does not seem to sense each of these diverse ligands directly with its leucine-rich repeat (LRR) domain. Instead, it was believed that three broad physiological changes— ROS generation, K + efflux, or lysosomal leakage— activate the NLRP3 inflammasome 45 .
  • LRR leucine-rich repeat
  • Mitochondrial ROS are now appreciated to play a central role in NLRP3 inflammasome activation 15 ' 16 .
  • ROS are generated and oxidize mtDNA 36 .
  • the inventors' data indicate that it is this oxidized form of mtDNA that binds to and activates the NLRP3 inflammasome and that this interaction can be competitively inhibited by oxidized dG. Therefore the inventors' data are in agreement with these previous studies regarding the importance of mtROS, and now provide the likely mechanism for NLRP3 inflammasome activation.
  • transfected oxidized mtDNA can activate both NLRP3 and AIM2
  • AIM2 is preferentially activated by normal DNA, while NLRP3 , by oxidized DNA.
  • NLRP3 inflammasome activation it was not known at what point in the signaling cascade the two events diverge. According to the inventors' model, both the apoptosome and the NLRP3 inflammasome share common upstream activating factors derived from mt. However, it is a byproduct of apoptosis, oxidized mtDNA released into the cytosol, which seems to be the activating factor for the NLRP3 inflammasome.
  • mice deficient in Apaf-1, caspase-9 or caspase-3 exhibit infertility, abnormal brain development, and lethality 53 55 whereas no such phenotypes have been found for caspase-1, NLRP3- or ASC deficient mice.
  • the two pathways share a common origin, their downstream effects are different.
  • the inventors show that mt dysfunction leading to apoptosis occurs with, and is necessary and sufficient for, NLRP3 activation in the presence of signal 1.
  • a key implication of the present study is that inhibition of apoptosis by intracellular microbes serves a dual role: attenuation of IL- ⁇ secretion and maintenance of a viable host cell for intracellular growth.
  • the inventors' results suggest that evolution has developed an innate immune strategy that relies on mt to determine the right time to sacrifice a jeopardized host cell for the sake of initiating a strong inflammatory cascade via IL- ⁇ . Therefore, apoptosis is not always 'silent'; rather, it can be a powerful voice to instruct nearby cells of imminent danger in the presence of NF-KB-activating signal 1.
  • oxidative mitochondrial DNA mtDNA
  • mtDNA oxidative mitochondrial DNA
  • oxidative nucleotides can competitively inhibit the oxidized mtDNA from binding and activating NLRP3, therefore inducing IL-lbeta.
  • Embodiments of the present invention are based, at least in part, on these findings.
  • various embodiments of the present invention provide a method of treating inflammation in a subject in need thereof.
  • the method can comprise providing a composition comprising a mitochondrial apoptosis inhibitor and administering the composition to the subject to treat the inflammation.
  • the mitochondrial apoptosis inhibitor can be selected from the group consisting of Bcl-2, Mcl-1, Bcl-xL, Bcl-w, Bfl-1, nfh, XIAP, Boo/Diva, Nrl3, BH4 domain, Inositol 1,4,5-trisphosphate receptor (IP3R) peptide, GSK-3 , cyclosporine A, viral mitochondrial inhibitor of apoptosis (vMIA) peptide/protein, MitoQ and combinations thereof.
  • the method can also comprise providing a composition comprising an oxidative nucleotide and administering the composition to the subject to treat the inflammation.
  • the inflammation is where IL-1 beta plays a role.
  • the oxidative nucleotide is selected from the group consisting of
  • inventions of the present invention provide a method of treating an inflammatory disease, an inflammatory disease condition, or an autoimmune disease in a subject in need thereof.
  • the method can comprise providing a composition comprising a mitochondrial apoptosis inhibitor and administering the composition to the subject to treat the inflammatory disease, the inflammatory disease condition or the autoimmune disease.
  • the method can also comprise providing a composition comprising an oxidative nucleotide and administering the composition to the subject to treat the inflammatory disease, the inflammatory disease condition or the autoimmune disease.
  • inflammatory disease, the inflammatory disease condition or the autoimmune disease is one where IL-1 beta plays a role.
  • inflammatory disease the inflammatory disease condition or the autoimmune disease where IL-1 beta plays a role is selected from the group consisting of type 2 diabetes, rheumatoid arthritis, psoriasis, Alzheimer's disease, silicosis and asbestosis, gout/pseudogout, familial cold autoinflammatory syndrome (FCAS), Muckel- Wells syndrome (MWS), neonatal-onset multisystem inflammatory disease (NOMID), an combinations thereof.
  • FCAS familial cold autoinflammatory syndrome
  • MWS Muckel- Wells syndrome
  • NOMID neonatal-onset multisystem inflammatory disease
  • the inflammatory disease or the inflammatory disease condition can be selected from the group consisting of an autoimmune disease, arthritis, Crohn's disease, inflammatory bowel disease, Alzheimer's disease, diabetes, gout, atherosclerosis, asbestosis/silicosis induced lung fibrosis and combinations thereof.
  • the autoimmune disease can be selected from the group consisting of Hashimoto's thyroiditis, Pernicious anemia, Addison's disease, type I diabetes, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, Sjogren syndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis, reactive arthritis, Grave's disease, celiac disease - sprue (gluten sensitive enteropathy) and combinations thereof.
  • the mitochondrial apoptosis inhibitor can be selected from the group consisting of Bcl-2, Mcl-1, Bcl-xL, Bcl-w, Bfl-1, nfh, XIAP, Boo/Diva, Nrl3, BH4 domain, Inositol 1,4,5-trisphosphate receptor (IP3R) peptide, GSK-3 , cyclosporine A, viral mitochondrial inhibitor of apoptosis (vMIA) peptide/protein, MitoQ and combinations thereof.
  • Bcl-2 Mcl-1, Bcl-xL, Bcl-w, Bfl-1, nfh, XIAP, Boo/Diva, Nrl3, BH4 domain, Inositol 1,4,5-trisphosphate receptor (IP3R) peptide, GSK-3 , cyclosporine A, viral mitochondrial inhibitor of apoptosis (vMIA) peptide/protein, MitoQ and combinations thereof.
  • the oxidative nucleotide is selected from the group consisting of (i) 8-Hydroxy-2'-deoxyguranosine (CAS 88847-89-6; also known as 8-OH-dG, 8- OHdG, 8-Oxo-7,8-dihydro-2'-deoxyguanosine, 8-Oxo-7,8-dihydrodeoxyguanosine, 8-Oxo- dG, 8-hydroxydeoxyguanosine, oh8dG),
  • Hy droxythymidine 5 -hy droxymethy 1-2 ' -D ; alpha-hy droxythymidine; 5 - hydroxymethyldeoxyuridine;5 -hydroxymethyl-2 ' -deoxyuridine;2 ' -deoxy-5 - (hy droxymethy l)uridine;2 ' -deoxy-5 -(hydroxymethyl)-uridin;2 ' -Deoxy-5 - hydroxymethyluridine, 98%; 1 -[(2R,4S,5R)-4-hydroxy-5-methylol-tetrahydrofuran-2-yl]-5- methylol-pyrimidine-2,4-quinone; 1 -[(2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]- 5-(hydroxymethyl)pyrimidine-2,4-dione),
  • the present invention provides pharmaceutical compositions including a pharmaceutically acceptable excipient along with a therapeutically effective amount of the mitochondrial apoptosis inhibitor or the oxidative nucleotide.
  • “Pharmaceutically acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients may be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous.
  • compositions according to the invention may be formulated for delivery via any route of administration.
  • Route of administration may refer to any administration pathway known in the art, including but not limited to aerosol, nasal, oral, transmucosal, transdermal or parenteral.
  • Transdermal administration may be accomplished using a topical cream or ointment or by means of a transdermal patch.
  • Parenteral refers to a route of administration that is generally associated with injection, including intraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal.
  • the compositions may be in the form of solutions or suspensions for infusion or for injection, or as lyophilized powders.
  • the pharmaceutical compositions can be in the form of tablets, gel capsules, sugar-coated tablets, syrups, suspensions, solutions, powders, granules, emulsions, microspheres or nanospheres or lipid vesicles or polymer vesicles allowing controlled release.
  • the compositions may be in the form of solutions or suspensions for infusion or for injection.
  • the pharmaceutical compositions based on compounds according to the invention may be formulated for treating the skin and mucous membranes and are in the form of ointments, creams, milks, salves, powders, impregnated pads, solutions, gels, sprays, lotions or suspensions.
  • compositions can also be in the form of microspheres or nanospheres or lipid vesicles or polymer vesicles or polymer patches and hydrogels allowing controlled release.
  • topical-route compositions can be either in anhydrous form or in aqueous form depending on the clinical indication. Via the ocular route, they may be in the form of eye drops.
  • compositions according to the invention can also contain any pharmaceutically acceptable carrier.
  • “Pharmaceutically acceptable carrier” as used herein refers to a pharmaceutically acceptable material, composition, or vehicle that is involved in carrying or transporting a compound of interest from one tissue, organ, or portion of the body to another tissue, organ, or portion of the body.
  • the carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or a combination thereof.
  • Each component of the carrier must be “pharmaceutically acceptable” in that it must be compatible with the other ingredients of the formulation. It must also be suitable for use in contact with any tissues or organs with which it may come in contact, meaning that it must not carry a risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that excessively outweighs its therapeutic benefits.
  • compositions according to the invention can also be encapsulated, tableted or prepared in an emulsion or syrup for oral administration.
  • Pharmaceutically acceptable solid or liquid carriers may be added to enhance or stabilize the composition, or to facilitate preparation of the composition.
  • Liquid carriers include syrup, peanut oil, olive oil, glycerin, saline, alcohols and water.
  • Solid carriers include starch, lactose, calcium sulfate, dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, acacia, agar or gelatin.
  • the carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax.
  • the pharmaceutical preparations are made following the conventional techniques of pharmacy involving milling, mixing, granulation, and compressing, when necessary, for tablet forms; or milling, mixing and filling for hard gelatin capsule forms.
  • a liquid carrier When a liquid carrier is used, the preparation will be in the form of a syrup, elixir, emulsion or an aqueous or non-aqueous suspension.
  • Such a liquid formulation may be administered directly p.o. or filled into a soft gelatin capsule.
  • the pharmaceutical compositions according to the invention may be delivered in a therapeutically effective amount.
  • the precise therapeutically effective amount is that amount of the composition that will yield the most effective results in terms of efficacy of treatment in a given subject. This amount will vary depending upon a variety of factors, including but not limited to the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication), the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and the route of administration.
  • Typical dosages of an effective the mitochondrial apoptosis inhibitor can be in the ranges recommended by the manufacturer where known therapeutic compounds are used, and also as indicated to the skilled artisan by the in vitro responses or responses in animal models. Such dosages typically can be reduced by up to about one order of magnitude in concentration or amount without losing the relevant biological activity.
  • the actual dosage will depend upon the judgment of the physician, the condition of the patient, and the effectiveness of the therapeutic method based, for example, on the in vitro responsiveness of the relevant primary cultured cells or histocultured tissue sample, such as biopsied malignant tumors, or the responses observed in the appropriate animal models, as previously described.
  • the present invention is also directed to a kit to treat inflammation, inflammatory disease and/or inflammatory disease condition.
  • the kit is useful for practicing the inventive method of treating inflammation, inflammatory disease and inflammatory disease condition.
  • the kit is an assemblage of materials or components, including at least one of the inventive compositions.
  • the kit contains a composition including a mitochondrial apoptosis inhibitor as described above.
  • the kit contains a composition including an oxidative nucleotide as described above.
  • kits are configured for the purpose of treating inflammation; other embodiments are configured for the purpose of treating inflammatory diseases; other embodiments are configured for the purpose of treating inflammatory disease conditions; and other embodiments are configured for the purpose of treating autoimmune diseases.
  • the kit is configured particularly for the purpose of treating mammalian subjects.
  • the kit is configured particularly for the purpose of treating human subjects.
  • the kit is configured for veterinary applications, treating subjects such as, but not limited to, farm animals, domestic animals, and laboratory animals. Instructions for use may be included in the kit.
  • kits for use typically include a tangible expression describing the technique to be employed in using the components of the kit to effect a desired outcome, such as to treat inflammation, inflammatory disease and/or inflammatory disease condition.
  • the kit also contains other useful components, such as, diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring tools, bandaging materials or other useful paraphernalia as will be readily recognized by those of skill in the art.
  • the materials or components assembled in the kit can be provided to the practitioner stored in any convenient and suitable ways that preserve their operability and utility.
  • the components can be in dissolved, dehydrated, or lyophilized form; they can be provided at room, refrigerated or frozen temperatures.
  • the components are typically contained in suitable packaging material(s).
  • packaging material refers to one or more physical structures used to house the contents of the kit, such as inventive compositions and the like.
  • the packaging material is constructed by well-known methods, preferably to provide a sterile, contaminant- free environment.
  • the packaging materials employed in the kit are those customarily utilized in treating inflammation, inflammatory disease and/or inflammatory disease condition.
  • a package refers to a suitable solid matrix or material such as glass, plastic, paper, foil, and the like, capable of holding the individual kit components.
  • a package can be a glass vial used to contain suitable quantities of an inventive composition containing a mitochondrial apoptosis inhibitor.
  • the packaging material generally has an external label which indicates the contents and/or purpose of the kit and/or its components.
  • Example 1 is provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.
  • Example 1 is provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.
  • Example 1 is provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.
  • Example 1 is provided to
  • mice were kindly provided by Dr. Richard Flavell (Yale Univ, New Haven, CT).
  • NLRP3 ⁇ ⁇ mice and ASC ⁇ ⁇ mice were kindly provided by Dr. Katherine Fitzgerald (University of Massachusetts Medical School, Worcester, MA).
  • C57BL/6 mice, TRIF ⁇ ⁇ mice and Ppif ( ypD) were obtained from Jackson Labs.
  • mice MyD88 _/" , RIP2 _/” , TLR2 _/” , TLR4 _/” , and TLR9 _/" (Naiki et al, 2005; Shimada et al., 2009) mice were maintained according to Cedars-Sinai Medical Center Institutional Animal Care and Use Committee guidelines. All mice were used at 8-12 weeks of age.
  • CM-1 C. pneumoniae
  • ATCC ATCC
  • Manassa VA
  • BALF and lung homogenates from C. pneumoniae (CM-1, ATCC, Manassa, VA) infected mice were propagated in HEp2 cells and counted as previously described (Shimada et al, 2009).
  • Lungs were fixed in formalin buffer, paraffin-embedded, and hematoxylin and eosin
  • Isolated single cells were stained with anti-F4/80 mAb (clone BM8), anti-CDl lc mAb (clone HL3).
  • mAb anti-F4/80 mAb
  • anti-CDl lc mAb anti-CDl lc mAb
  • HL3 high-density cells
  • iNOS staining cells were permeabilized using Cytofix/Cytoperm kit (BD Biosciences) and stained with -conjugated anti-mouse iNOS mAb (clone 6/iNOS/NOS Type II, BD Bioscienses).
  • Flow cytometric analysis was performed by CyAnTM Flow cytometer (Beckman Coulter) and the data was analyzed by Summit (Dako, Carpinteria, CA, USA).
  • TMRM mitochondrial membrane potential
  • LDH release was assessed in cell-free medium at indicated times following the manufacturer's instructions (Cytotoxicity Detection Kit, Roche Diagnostics). The data are expressed as percentage of maximum LDH release for the particular treatment at each time point.
  • BMDM were stimulated for indicated time, supernatants were collected and proteins were precipitated by methanol-chloroform extraction, and cell lysates were collected. Immunoblot analysis was done with described antibodies; anti mouse caspase-1 plO (sc-514; Santa Cruz Biotechnology), anti-mouse IL- ⁇ (AF-401-NA; R&D Systems), anti-GAPDH (6C5; Santa Cruz Biotechnology).
  • IL-1 receptor antagonist (IL-IRA) (Kineret, Amgen), N-Acetyl-L- leucyl-L-leucyl-L-methional (Tocris bioscience, Ellisville, MO), adenosine 5 "-triphosphate, chloramphenicol, cytochalasin D, staurosporine, cyclosporine A, antimycin A and N-Acetyl- L-cysteine, nigericin, Alum, 2'-deoxyguanocine (Sigma, St. Louis, MO), LPS from E.
  • IL-IRA Recombinant IL-1 receptor antagonist
  • coli InvivoGen, San Diego, CA
  • 8-Hydroxy-2'-deoxyguanosine, 7-Bromoindirubin-3'- monoxime Enzo life sciences, Madison Meeting, PA
  • 8-OHdG quantitation kit Cell Biolabs, Inc., San Diego, CA
  • NLRP3 cDNA was kindly provided by Dr. Eicke Latz (University of Massachusetts Medical School, Worcester, MA).
  • Salmonella typhimurium IR715, ⁇ , AhilA, AorgA, AspiB, AprgH, AmsbB, AfljB/fliC, AsspB
  • Dr. Andreas Baumler University of California in Davis, Davis, CA
  • Chlamydia pneumoniae CM-1, ATCC, Manassa, VA was propagated in Hep2 cells.
  • cytokine concentrations in the BALF, lung homogenates or culture supernatant were determined using by OptiEIA Mouse IL-6 ELISA Set (BD Biosciences, San Jose, CA, USA) and Mouse ⁇ ELISA, Mouse IL-12p40 ELISA, Mouse IL- ⁇ ELISA and Mouse TNFa ELISA (eBioscience, San Diego, CA). The assays were performed as described in manufacturers' protocols.
  • lymphocytes were isolated by digesting the lung tissue at 37°C for lh with HANKS' containing 100 ⁇ g/ml Blenzyme (Roche Diagnostics, Indianapolis, IN, USA) and 50 units/ml DNase I (Roche Diagnostics) and filtering through a 70 ⁇ cell strainer (BD Biosciences). Erythrocytes were depleted by lysis buffer before staining.
  • Isolated single cells were stained with following specific mAbs: CD 16/32 (clone 93), Ly6G (clone 1A8), CDl lb (clone Ml/70), F4/80 (clone BM8), CDl lc (clone HL3), CD45 (clone 30-F11), CD4 (clone RM4-5), CD8 (clone 53-6.7), NK1.1 (clone PK136) and B220 (clone RA3-6B2) were purchased from eBioscience as direct conjugates to FITC, PE or PECy5.
  • pulmonary macrophages F4/80+ and CD1 lc+
  • DC F4/80- and CD1 lc+
  • Neutrophils Ly6G+ and CD1 lb+
  • T cells CD3+
  • NK cells NK1.1+
  • B cells B220+ and CD19+
  • Chlamydophila staining cells were permeabilized using Cytofix/Cytoperm kit (BD Biosciences) and stained with FITC-conjugated anti-Chlamydia LPS mAb (Accurate Chemical and Scientific Corporation, Westbury, NY, USA).
  • Flow cytometric analysis was performed by CyAnTM Flow cytometer (Beckman Coulter) and the data was analyzed by Summit (Dako, Carpinteria, CA, USA).
  • BMDM bone marrow-derived macrophages
  • RPMI 1640 media cell culture medium
  • BMDM were harvested at day 7 and infected with C. pneumoniae by centrifugation at 500x g for 30 min.
  • Caspase-1 and IL- ⁇ are critical in host innate immune responses against pulmonary CP infection
  • Caspase-1 Caspl
  • Caspl "7" mice were infected intratracheally with CP (2xl0 6 , 1.5xl0 6 or lxlO 6 IFU). Caspl deficiency resulted in greater mortality at 1.5xl0 6 IFU, compared to WT mice ( Figure 1A).
  • the bronchoalveolar lavage fluid (BALF) and lung homogenates from Caspl "7” mice exhibited significantly higher leukocyte recruitment, particularly macrophages and lymphocytes, at days 5 and 12 post infection compared to WT mice (Figure IB). Histological examination of Caspl "7" lungs 12 days after infection mice revealed higher inflammation and tissue damage scores compared to WT lungs ( Figure IF).
  • the inventors next sought to determine the predominant cell type infected by CP in the lungs. Since CP is an obligate intracellular pathogen, the cell types infected by CP in Caspl "7" mice compared to wild-type mice ( Figure 8) were analyzed by flow cytometry. CP was found predominantly in alveolar macrophages (AM) and, to a lesser degree, in neutrophils and dendritic cells (DC) on day 12 ( Figure 8). There were relatively more infected Caspl "7" AM than their wild-type counterparts, revealing that AM are likely a reservoir of bacterial replication after pulmonary infection in Caspl "7" mice.
  • AM alveolar macrophages
  • DC neutrophils and dendritic cells
  • the inventors were next interested in determining if AM isolated from CP infected Caspl "7" mice exhibited an immune defect compared to those in infected WT mice.
  • Ex vivo Caspl "7" AM did not induce iNOS following CP infection compared to wt AM ( Figure IE).
  • nitric oxide (NO) produced by macrophages after cell activation by IFN- ⁇ hampers the growth of CP (Nathan and Hibbs, 1991; Rottenberg et al., 1999).
  • Macrophage TLR2/MyD88 and NLRP3/ASC/Caspase-l are required for
  • UVCP was unable to induce IL- ⁇ secretion by BMDM, while not wishing to be bound by any particular theory, the inventors believe that UVCP induces pro-IL- ⁇ .
  • BMDM were treated with UVCP (for either 8 or 24 hours) and then exposed to a high extracellular concentration of ATP (5 mM), a stimulus known to activate Caspl via the
  • ATP treatment also increased IL- ⁇ secretion in BMDM after a 6 hour (but not 24 hour) infection with live CP ( Figure 3C and 3E).
  • BMDM treated with UVCP for 24 hours and then given ATP did not secrete IL- ⁇ ( Figure 3C), suggesting that pro-IL- ⁇ is degraded if Caspl is not activated within a finite time window.
  • NLRP3 inflammasome activates Caspl in response to a wide array of stimuli
  • the inventors predicted that CP also induces IL- ⁇ secretion via NLRP3.
  • NLRP3 ⁇ ⁇ or ASC /_ BMDM infected with live CP were found to secrete dramatically reduced levels of IL-
  • Mitochondrial dysfunction is involved in IL- ⁇ secretion
  • CsA cyclosporin A
  • PTP mitochondrial permeability transition pore
  • LPS lipopolysaccharide
  • CsA cyclosporin A
  • PTP permeability transition pore
  • ⁇ loss of which is typically used as a surrogate marker for apoptosis 27 .
  • STS a pro-apoptotic compound
  • Annexin V staining a technique which detects phosphatidylserine (PS) externalized during apoptotic membrane flipping, was first attempted. But this method was not suitable for use in CP infected cells, as CP infection causes transient externalization of PS in the absence of apoptosis (Goth and Stephens, 2001).
  • apoptotic cells were stained with DAPI and cells were scored for nuclear condensation.
  • the nuclei of apoptotic cells unlike those of necrotic cells, exhibit highly condensed chromatin that uniformly stains with DAPI.
  • Increased apoptosis in CP infected macrophages but not UVCP treated cells was observed, consistent with LDH release assays ( Figure 6A).
  • apoptosis induced by CP is linked to NLRP3 inflammasome activation.
  • the pro-apoptotic molecule STS not only leads to loss of ⁇ (Figure 5D), LDH release (Figure 6A) and nuclear condensation (Figure 6B) but also to Caspl activation and subsequent IL- ⁇ ⁇ maturation in LPS-primed BMDM ( Figure 6C).
  • Addition of STS at 6 hr after UVCP or LPS treatment induced IL- ⁇ ⁇ secretion and treatment with STS after 6 hrs of CP infection enhanced IL- ⁇ secretion (Figure 6D).
  • IL- ⁇ secretion in response to STS depends on NLRP3/ASC/Caspl ( Figure 6E). But STS treatment did not affect TNFa production ( Figure 6D).
  • Antimycin A a compound that binds to mitochondrial complex III, inhibits mitochondrial respiration, prevents cytochrome c release (Rieske et al, 1967), and blocks apoptosis (Dairaku et al, 2004) was used.
  • UVCP and LPS-primed BMDM treated with antA displayed a dose-dependent attenuation of IL- ⁇ secretion in response to ATP or STS ( Figure 6G).
  • antA treatment also inhibited IL- ⁇ secretion (without affecting TNFa secretion) in response to live CP infection ( Figure 6G).
  • Annexin V staining a technique which detects phosphatidylserine (PS) externalized during apoptotic membrane flipping. While this method is not suitable for use in CP infected cells because CP infection causes transient externalization of PS in the absence of apoptosis 30 , the inventors found that LPS+ATP, or LPS+NIG induced apoptosis in BMDM as measured by Annexin V flow cytometry ( Figure 17). As an alternative approach, the inventors stained host cell nuclei with DAPI and scored cells for nuclear condensation.
  • apoptotic cells unlike those of necrotic cells, exhibit highly condensed chromatin that uniformly stains with DAPI. Consistent with data from LDH release assays ( Figure 6a), the inventors observed increased apoptosis in CP infected macrophages but not in UVCP treated cells ( Figure 6b). Thus, apoptosis induced by CP is linked to NLRP3 inflammasome activation.
  • the pro-apoptotic molecule STS not only leads to loss of ⁇ ⁇ 1 ( Figure 12d), LDH release (Figure 6a) and nuclear condensation (Figure 6b) but also to caspase-1 activation and subsequent IL- ⁇ maturation in LPS-primed BMDM ( Figure 6c).
  • Addition of STS at 6 h after UVCP or LPS treatment induced IL- ⁇ secretion and treatment with STS after 6 h of CP infection enhanced IL- ⁇ secretion (Figure 6d).
  • IL- ⁇ secretion in response to STS depends on NLRP3/ASC/caspase-l ( Figure 6e).
  • STS treatment did not affect TNFa production ( Figure 6d).
  • pyroptosis 31 caspase-1 -dependent form of programmed cell death.
  • the inventors assessed ⁇ reduction in response to CP, ATP, STS, NIG, and alum in caspase-1 deficient (Casp ⁇ ) BMDM.
  • mitochondrial depolarization was independent of caspase-1 ( Figure 18a), suggesting that pyroptosis cannot account for the inventors' observations.
  • the inventors also assessed LDH release due to CP infection in BMDM and found that caspase-1 also did not play a role in that system ( Figure 18b). Additionally, LPS primed BMDM treated with necrotic stimuli did not secrete IL- ⁇ ( Figure 18c).
  • a macrophage cell line (MCL) was stably transfected with a construct that overexpresses the anti-apoptotic protein Bcl-2 and assessed for IL- ⁇ secretion in response to NLRP3 triggers.
  • this Bcl-2 MCL was found to be resistant both to changes in mitochondrial potential ( ⁇ ) and to apoptosis (as measured by nuclear condensation and LDH release) in response to LPS+ATP and to CP.
  • the Bcl-2 overexpressing MCL was found to secrete significantly less IL-lb (compared to an empty vector control) in response to LPS + ATP, LPS + Alum and CP.
  • MCL 32 immortalized macrophage cell line
  • Figure 13a overexpression vector coding for the anti-apoptotic protein Bcl-2
  • Figure 13b The Bcl-2 overexpressing line secreted significantly less IL- ⁇ compared to cells with an empty vector control (or vs. cells expressing an unrelated gene; Figure 19) in response to CP, LPS+ATP, LPS+STS, or LPS+NIG ( Figure 13b).
  • Salmonella typhimurium infection-induced apoptosis causes IL- ⁇ secretion
  • S. typhimurium is recognized by both the NLRP3 and NLRC4 inflammasome pathways 33 . Indeed, NLRP3 plays an important role in IL- ⁇ secretion ( Figure 14a). Salmonella typhimurium is also known to induce Type III secretion system (T3SS)-dependent apoptosis in macrophages 34 ' 35 . The inventors investigated whether S. typhimurium activation of the NLRP3 inflammasome was licensed by apoptosis. S. typhimurium infection of BMDM for 8 h resulted in marked LDH release (90% of control) ( Figure 14b). However, T3SS-1 defective strains of S.
  • T3SS-1 Type III secretion system
  • IL- ⁇ secretion was only induced by the S. typhimurium strains that caused LDH release and A x V m reduction ( Figure 14d), and IL- ⁇ was not secreted by BMDM infected with strains that failed to induce apoptosis.
  • the inventors infected the Bcl-2 stably transfected MCL cell line with wild type S. typhimurium. Bcl-2 overexpression resulted in significantly reduced IL- ⁇ secretion following S. typhimurium infection with the wild-type strain ( Figure 14e). TNFa levels remained similar between-groups ( Figure 14e).
  • BMDM were grown in the presence of BrdU and then treated with LPS+ATP or LPS+NIG in the presence of the autophagy inhibitor 3-MA to enhance inflammasome activation.
  • NLRP3 was immunoprecipitated and used as input material for BrdU dot-blot.
  • BrdU incorporated DNA was bound to NLRP3 in cells treated with LPS+ATP or LPS+NIG ( Figure 15c), whereas LPS treatment alone did not produce NLRP3-BrdU binding, indicating that secondary stimulation was necessary for this interaction.
  • Oxidized DNA was generated by PCR against the mtCOXl template in presence of 8-OH-dGTP, and PCR product was transfected into BMDM. Data showed increased IL- ⁇ production vs. normal DNA (Figure 12f). Importantly, oxidized DNA did not impact TNFa production. However, the same experiment performed in Nlrp3 ⁇ ' ⁇ BMDM resulted in significantly decreased IL- ⁇ , but not TNFa production ( Figure 15g). Furthermore, DNA containing 8-OH-dGTP could still induce IL- ⁇ secretion in Aim2 ⁇ ' ⁇ BMDM ( Figure 15h), despite the inventors' findings that these cells were refractive to mtDNA transfection-induced IL- ⁇ release . Taken together, these data indicate that oxidized DNA can induce inflammasome activation via preferential activation of NLRP3.
  • Oxygen consumption rates were measured using an XF24 Extracellular Flux Analyzer (Seahorse Bioscience).
  • OCR Oxygen consumption rates
  • XF24 Extracellular Flux Analyzer
  • cells were equilibrated with DMEM lacking bicarbonate at 37°C for 1 h in an incubator without C0 2 . Mixing, waiting, and measurement times were 0.5, 2, and 3 min, respectively (an extra 30 s was added after each injection).
  • Oligomycin which blocks phosphorylation of ADP to ATP, was utilized to prevent mitochondrial respiration and to provide basal 0 2 consumption during the assay.
  • FCCP was used as an uncoupling agent to allow maximal 0 2 consumption under a given condition.
  • Rotenone was employed as an mt respiratory chain complex 1 inhibitor.
  • Mitochondria were isolated using by Mitochondria Isolation Kit (Thermo Scientific, Rockford, IL). Mitochondrial DNA was purified using by QIAamp DNA mini kit (QIAGEN). For detection of mtDNA, PCR was carried out using following primers: mtCOXl, sense primer 5 '-TTCGGAGCCTGAGCGGGAAT-3 ' (SEQ ID NO: l), and antisense primer 5 ' -ATGCCTGCGGCTAGCACTGG-3 ' (SEQ ID NO:2) (Product length: 554 bp).
  • the dGTP analog 8-OH-dGTP was purchased from TriLink BioTechnologies (San Diego, CA).
  • the mtCOXl gene fragment (554 bp) was amplified with unmodified dNTPs and 8-OH-dGTP using Taq DNA polymerase and isolated mtDNA. Amplified DNA was purified by StrataPrepR PCR Purification kit (Agilent Technologies, Santa Clara, CA).
  • BMDM BMDM were preloaded with BrdU (10 ⁇ ) for 48 h and treated as indicated.
  • the rabbit anti-NLRP3 polyclonal Ab (LifeSpan Biosciences Inc., Seattle, WA) was incubated with the cell lysates for 2 h or overnight at 4°C. Subsequently, Trueblot IgG beads (eBioscience, San Diego, CA) were added and the samples were incubated at 4°C for 1 h.
  • mouse anti-Flag mAb M2, Sigma
  • Immunoblotting was performed using anti-BrdU mAb (BU33; Sigma) or mouse anti-80H-dG mAb (15 A3; Rockland Immunochemicals Inc., Gilbertsville, PA).
  • Bcl2 overexpression plasmid named pSFFV-neo-Bcl-2 57, Addgene plasmid 8776, or an empty vector control plasmid pSFFV-neo 58, kindly provided by Dr. Gabriel Nunez (Univ.
  • MCL cells were maintained in the presence of 50 ng/ml ethidium bromide for more than 20 passages and media was supplemented with uridine (50 g/ml) and sodium pyruvate (120 g/ml). Depletion of mtDNA was confirmed by PCR for the mitochondrial genes Coxl and Nd6 compared to a nuclear gene (Gapdh).
  • BMDM were fixed in 4% paraformaldehyde (PFA) for 10 min at 4°C and permeabilized with BD Cytofix/Cytoperm solution for 30 min at 4°C.
  • PFA paraformaldehyde
  • cells were treated with 10 U/ml DNase I (Roche) for 30 min at 37 °C.
  • DNase I Roche
  • cells were incubated overnight at 4°C with primary antibody against BrdU.
  • cells were incubated with appropriate secondary antibodies conjugated with Alexa Fluor 594 for 1 hr at ambient temperature.
  • DAPI ProLong Gold
  • NALP infiammasomes a central role in innate immunity. Semin Immunopathol 29, 213-229.
  • Chlamydia pneumoniae stimulates IFN-gamma synthesis through MyD88-dependent, TLR2- and TLR4-independent induction of IL-18 release. J Immunol 173, 1477-1482.
  • the NOD/RIP2 pathway is essential for host defenses against Chlamydophila pneumoniae lung infection.
  • Greten, F.R. et al. NF-kappaB is a negative regulator of IL-lbeta secretion as revealed by genetic and pharmacological inhibition of IKKbeta. Cell 130, 918-931 (2007). Brough, D. & Rothwell, N.J. Caspase-1 -dependent processing of pro-interleukin- lbeta is cytosolic and precedes cell death. J Cell Sci 120, 772-781 (2007).

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Abstract

Disclosed is a method of treating inflammation, an inflammatory disease, an inflammatory disease condition and/or an autoimmune disease in a subject in need thereof. The method can comprise providing a composition comprising a mitochondrial apoptosis inhibitor or an oxidative nucleotide and administering the composition to the subject to treat the inflammation, the inflammatory disease, the inflammatory disease condition and/or the autoimmune disease. Said method is based on the mitochondria actingg as a unifying point that integrates diverse NLRP3 stimuli and that mitochondrial dysfunction and apoptosis of macrophages are critical steps in various danger signal-induced NLRP3 inflammasome activation.

Description

MITOCHONDRIAL APOPTOSIS-INDUCED INFLAMMATION
STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH
The U.S. Government has a paid-up license in this invention and the right in limited circumstances to require the patent owner to license others on reasonable terms as provided for by the terms of Grant No. NAID-AI 067997-01, awarded by the National Institutes of Health.
FIELD OF INVENTION
This invention relates to the treatment and diagnosis of inflammation, inflammatory diseases and inflammatory disease conditions.
BACKGROUND
All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
Chlamydophila pneumoniae {CP) is a widely prevalent (Blasi et al, 2009) intracellular Gram-negative pathogen that causes upper respiratory infections and contributes to the development of chronic inflammatory conditions such as asthma (Sutherland and Martin, 2007), atherosclerosis (Watson and Alp, 2008), arthritis (Gerard et al, 2009), and chronic obstructive pulmonary lung disease (COPD) (Papaetis et al, 2009).
In a mouse model of CP lung infection, productive host defense requires signaling through TLR/MyD88 (Naiki et al, 2005) and NOD/RIP2 (Shimada et al, 2009). Toll-like receptor (TLR) 2 and TLR4 both use MyD88 to sense CP (Joyee and Yang, 2008), yet TLR2_/" and TLR4_/" mice clear CP and recover from infection (Rodriguez et al., 2006). This finding suggests redundant roles for TLR2 and TLR4 and also implicates the IL-1 receptor— a MyD88-dependent cytokine receptor— in host defense against CP. Indeed, CP infection elicits robust IL-Ιβ secretion in a number of experimental models (Kaukoranta-Tolvanen et al., 1996; Netea et al, 2004; Netea et al., 2000) and aberrant IL-1R modulation plays a role in the pathogenesis of CP infection in COPD patients (Rupp et al, 2003). Secretion of IL-Ιβ, a potent pyrogen that elicits a strong pro-inflammatory response (Dinarello, 2009), is tightly controlled by a diverse class of cytosolic complexes known as inflammasomes (Latz, 2010). The NOD-like Receptor (NLR) family member NLRP3 forms cytosolic oligomers with apoptosis-associated speck like protein (ASC) in dendritic cells (Ghiringhelli et al., 2009) and macrophages (Franchi et al, 2009), triggering autocatalytic activation of caspase-1 (Martinon et al, 2009). Caspase-1, in turn, cleaves pro-IL-Ιβ, producing mature IL-1 β.
Under normal circumstances, NLRP3 undergoes bipartite activation (Latz, 2010). The first signal, often NF-κΒ activation, induces pro-IL-Ιβ and NLRP3 expression. The second signal, any one of a variety of unrelated entities— particulate matter (Dostert et al., 2008), crystals (Duewell et al, 2010), aggregated β-amyloid (Halle et al., 2008), extracellular ATP (Hogquist et al., 1991; Mariathasan et al., 2006) and microbial toxins (Meixenberger et al., 2010)— activates NLRP3. Exactly how the NLRP3 inflammasome responds to such wide range of danger signals was heretofore unclear.
Three models for the activation of the NLRP3 inflammasome have been proposed: reactive oxygen species (ROS) generation (Tschopp and Schroder, 2010), lysosomal damage (Hornung and Latz, 2010) and cytosolic potassium ion (K+) efflux (Petrilli et al., 2007). Though each mechanism has been characterized, reports often contradict concerning the relative contribution of each model to NLRP3 activation and a unifying model that integrates the three has not emerged.
Accordingly, there is a need in the art to elucidate the mechanisms of action and for therapies targeting the inflammation, inflammatory diseases, inflammatory disease conditions, and autoimmune diseases.
SUMMARY OF THE INVENTION
The following embodiments and aspects thereof are described and illustrated in conjunction with compositions and methods which are meant to be exemplary and illustrative, not limiting in scope.
Various embodiments of the present invention provide a method, comprising: providing a composition comprising a mitochondrial apoptosis inhibitor; and administering the composition to a subject in need of treatment for inflammation to treat the inflammation.
In various embodiments, the mitochondrial apoptosis inhibitor can be selected from the group consisting of Bcl-2, Mcl-l, Bcl-xL, Bcl-w, Bfl-l, nfh, XIAP, Boo/Diva, Nrl3, BH4 domain, Inositol 1,4,5-trisphosphate receptor (IP3R) peptide, GSK-3 , cyclosporine A, viral mitochondrial inhibitor of apoptosis (vMIA) peptide/protein, MitoQ and combinations thereof. In various embodiments, the treatment for inflammation can treat an inflammatory disease, an inflammatory disease condition, an autoimmune disease, or combinations thereof. In certain embodiments, the inflammatory disease, inflammatory disease condition, or autoimmune disease can be where IL-1 beta plays a role. In certain embodiments, the inflammatory disease, inflammatory disease condition, or autoimmune disease where IL-1 beta can play a role can be selected from the group consisting of type 2 diabetes, rheumatoid arthritis, psoriasis, Alzheimer's disease, silicosis and asbestosis, gout, pseudogout, familial cold autoinflammatory syndrome (FCAS), Muckel-Wells syndrome (MWS), neonatal-onset multisystem inflammatory disease (NOMID), and combinations thereof. In certain embodiments, the inflammatory disease or inflammatory disease condition can be selected from the group consisting of arthritis, Crohn's disease, inflammatory bowel disease, Alzheimer's disease, diabetes, gout, atherosclerosis, asbestosis/silicosis induced lung fibrosis and combinations thereof. In certain embodiments, the autoimmune disease can be selected from the group consisting of Hashimoto's thyroiditis, Pernicious anemia, Addison's disease, type I diabetes, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, Sjogren syndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis, reactive arthritis, Grave's disease, celiac disease, and combinations thereof.
Other embodiments of the present invention provide a method, comprising: providing a composition comprising an oxidative nucleotide; and administering the composition to a subject in need of treatment for inflammation to treat the inflammation.
In various embodiments, the oxidative nucleotide can be selected from the group consisting of (i) 8-Hydroxy-2'-deoxyguranosine, (ii) 8-hydroxy Guanosine, (iii) 8-Oxo-2'- deoxyadenosine, (iv) 5-formyl-2'-deoxycytidine, (v) 5-formyl-2'-deoxyuridine, (vi) 5- hydroxymethyl-2'-deoxyuridine, (vii) 5-Hydroxymethyl-2'-deoxycytidine, (viii) 5-hydroxy- 2'-deoxyuridine, (ix) 5-hydroxy-2'-deoxycytidine, and (x) combinations thereof. In various embodiments, the treatment for inflammation can treat an inflammatory disease, an inflammatory disease condition, an autoimmune disease, or combinations thereof. In certain embodiments, the inflammatory disease, inflammatory disease condition, or autoimmune disease can be where IL-1 beta plays a role. In certain embodiments, the inflammatory disease, inflammatory disease condition, or autoimmune disease where IL-1 beta can play a role can be selected from the group consisting of type 2 diabetes, rheumatoid arthritis, psoriasis, Alzheimer's disease, silicosis and asbestosis, gout, pseudogout, familial cold autoinflammatory syndrome (FCAS), Muckel-Wells syndrome (MWS), neonatal-onset multisystem inflammatory disease (NOMID), and combinations thereof. In certain embodiments, the inflammatory disease or inflammatory disease condition can be selected from the group consisting of arthritis, Crohn's disease, inflammatory bowel disease, Alzheimer's disease, diabetes, gout, atherosclerosis, asbestosis/silicosis induced lung fibrosis and combinations thereof. In certain embodiments, the autoimmune disease can be selected from the group consisting of Hashimoto's thyroiditis, Pernicious anemia, Addison's disease, type I diabetes, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, Sjogren syndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis, reactive arthritis, Grave's disease, celiac disease, and combinations thereof.
Other features and advantages of the invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, various features of embodiments of the invention.
BRIEF DESCRIPTION OF THE FIGURES
Exemplary embodiments are illustrated in referenced figures. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive.
Figure 1 shows that Caspl plays a critical protective role during CP lung infection in accordance with various embodiments of the present invention. (A) Caspl deficiency confers higher mortality. Caspl"7" mice or WT were infected intratracheally with 2xl06, 1.5xl06 or lxl 06 inclusion forming units (IFU) of CP. The Kaplan-Meier survival curve is shown. Statistical significance was determined by Fisher's exact test. (B) Caspl"7" mice show increased lymphocytic infiltration in the lung during late-stage infection. BALF was harvested at days 1, 3, 5 and 12 following CP infection (lxl 06 IFU/mouse) and the number of macrophages (MAC), polymorphonuclear cells (PMN) and lymphocytes (LYM), as well as the total cell number, was determined (n=5~9). Data shown are representative of three independent experiments. (C) Caspl deficiency results in delayed cytokine production and reduced bacterial clearance. Cytokine (IL-6, IFNy, IL-12p40 and IL-1 β) levels in both BALF and lung homogenates were determined using ELISA. (D) Bacterial burden in infected WT and Caspl"7" lung homogenates was also quantitated. Data shown are representative of three independent experiments. (E) Ex vivo Caspl"7" macrophages are defective in iNOS activation. iNOS expression in alveolar macrophages (CDl lc+, F4/80+) 2 days post-infection. Representative histograms are shown and the mean fluorescence intensity (MFI) is indicated. Data for all experiments shown represent at least two independent experiments. (F) Caspl"7" mice exhibit significant tissue damage after CP infection. Lungs were harvested 12 days after infection (lxl 06 IFU/mouse), fixed in 10% buffered formalin, embedded in paraffin, sectioned, stained with hematoxylin and eosin (H&E) and scored for tissue damage. Note on statistical significance: * p<0.05, ** p< 0.01, *** p<0.001 (Student's t test used unless otherwise noted).
Figure 2 shows that IL-1 signaling is crucial for host survival and bacterial clearance during CP lung infection in accordance with various embodiments of the present invention. (A and B) Blocked IL-1 signaling leads to higher mortality and reduced bacterial clearance. WT mice were daily given an IL-IRA (500 μg) or vehicle control and then infected with CP (1.5xl06 IFU/mouse). The Kaplan-Meier survival curve is shown. Bacterial burden in lung homogenates was determined for IL-IRA and vehicle control treated mice 5 days post infection with lxl 06 IFU. (C and D) Caspl"7" mice given recombinant rIL-Ιβ exhibit reduced mortality and enhanced bacterial clearance. Caspl"7" mice were treated with rIL-Ιβ (8 ng) daily for 3 days or vehicle control and then infected with CP (1.5xl06 IFU/mouse). The Kaplan-Meier survival curve is shown. Bacterial burden in lung homogenates of rIL-Ιβ and vehicle control treated mice 5 days after infection with lxl 06 IFU. Note on statistical significance: one-way ANOVA with Tukey's post-hoc test - * p<0.05, ** p< 0.01, *** pO.001.
Figure 3 depicts macrophage TLR2/MyD88 signaling and the NLRP3/ASC/Caspase-l inflammasome are required for IL-Ιβ secretion in response to CP in accordance with various embodiments of the present invention. (A and B) TLR2/MyD88 signaling is necessary for IL-Ιβ secretion in response to live CP. IL-Ιβ concentration in the culture supernatant was determined using ELISA after a 24 h treatment with various stimuli— UVCP (MOI 2.5, 5 or 10), live CP (MOI 2.5, 5 or 10), LPS or PGN— in WT, MyD88_/", TRIF_/~, and RIP2_/" BMDM and in WT, TLR2_/", TLR4_/~, and TLR9_/~ BMDM. Data shown are representative of at least three independent experiments. (C, D, E and F) Live CP, not UVCP, stimulates maximal IL- 1β secretion from macrophages 24 hours after infection. IL-Ιβ and TNFa concentrations were measured in the culture supernatants of WT and Caspl"7" BMDM treated for 24 h with the aforementioned stimuli. A proportion of cells were also treated with 5 mM ATP for the final 2 h of culture. Data shown are representative of at least three independent experiments. (G and H) The NLRP3/ASC inflammasome is required for macrophage IL-Ιβ secretion in response to live CP. IL-1 β and TNFa concentrations in the culture supernatants of WT, AS /_, and NLRP3 " " BMDM were measured 24 hours after treatment with UVCP (MOI 2.5, 5, 10), live CP (MOI 2.5, 5, 10), LPS or PGN. Data shown are representative of at least three independent experiments.
Figure 4 shows that phagocytosis and bacterial de novo protein synthesis are necessary to activate the NLRP3 inflammasome in CP infected macrophages in accordance with various embodiments of the present invention. (A) Bacterial internalization by macrophages is necessary for IL-Ιβ secretion in response to CP. IL-Ιβ (black bars) and TNFa (gray bars) secretion by CP infected (MOI 10, 24 h) BMDM in the presence or absence of cytochalasin D was quantified using ELISA. Data shown are representative of two or more independent experiments. (B) CP activation of the NLRP3 inflammasome in macrophages is cathepsin independent. Using ELISA, IL-Ιβ concentration in culture supematants of CP infected (MOI 10, 24 h) BMDM was determined in the presence of increasing amounts of Ac-LLM. Also, LPS-primed (1 μg/ml, 8 h) BMDM treated with alum (ALM, 130 μg/ml, final 2 h of culture) were given increasing amounts of Ac-LLM. Data shown are representative of two or more independent experiments. (** p< 0.01, *** p<0.001; Student's t test). (C and D) Antioxidant (N-acetylcysteine, NAC) treatment does not specifically inhibit IL-Ιβ secretion. ELISA was used to determine IL-Ιβ and TNFa concentration in the culture supematants of CP (MOI 10, 24 h) infected BMDM and LPS- primed (8 h), ATP treated (5 mM, final 2 h culture) BMDM in the presence of increasing doses of NAC. (E) CP activation of the NLRP3 inflammasome is reactive oxygen species (ROS) independent. A fluorometric assay was used to quantitate ROS production by BMDM in response to UVCP or live CP. Data shown are representative of two or more independent experiments. (F) Chlamydophilal de novo protein synthesis is required for CP induced IL-Ιβ secretion. ELISA was used to determine IL-Ιβ and TNFa concentration in the culture supematants of CP (MOI 10, 24 h) infected BMDM in the presence of increasing doses of chloramphenicol. Data shown are representative of two or more independent experiments.
Figure 5 shows that mitochondrial dysfunction is linked to NLRP3 inflammasome activation in accordance with various embodiments of the present invention. (A) Mitochondrial protein synthesis is important in the NLRP3 inflammasome activation cascade. ELISA was used to determine IL-Ιβ and TNFa concentration in the culture supematants LPS-primed (1 μg/ml, 8 h), ATP treated (5 mM, final 2 h culture) BMDM in the presence of increasing doses of chloramphenicol. Also shown is an LDH release assay of the same BMDM. Data shown are representative of two or more independent experiments. (B) The mitochondrial permeability transition pore (mPTP) plays a key role in CP and LPS+ATP induced NLRP3 activation. IL-Ιβ and TNFa secretion by CP and LPS+ATP treated BMDM were measured using ELISA in the presence of increasing doses of cyclosporin A (CsA). Data shown are representative of two or more independent experiments. (C and D) Loss of mitochondrial membrane potential (ΔΨιη) correlates with NLRP3 inflammasome activation. BMDM were treated with UVCP or infected with live CP (MOI 2.5, 5, or 10; 24 h) and then examined for TMRM incorporation. Likewise, LPS-primed BMDM (1 μg/ml, 8 h) were treated with ATP (5mM) or staurosporine (STS, 5 μΜ) and examined for TMRM incorporation. The kinetics of ΔΨιη loss were monitored over time using a similar TMRM incorporation assay. BMDM were treated with live CP (MOI 10), ATP (5 mM), STS (5 μΜ), alum (ALM, 130 μg/ml), or nigericin (NIG, 10μΜ). Data shown are representative of two or more independent experiments (** p< 0.01, *** p<0.001; Student's t test). (E) Extracellular ATP, a potent NLRP3 activator, causes rapid, irreversible loss of ΔΨιη. BMDM were stimulated with LPS (1 μg/ml, 3 h) and then loaded with 200 nM TMRM in Ringer's solution for 30 min. Following probe loading, 5 mM ATP was added and the live cell kinetics of ΔΨιη were analyzed every 20 seconds thereafter. The kinetic profile shown depicts the mean value +/- the SEM at each time point. Representative image of mitochondrial TMRM fluorescence and associated 3 dimensional intensity map are shown. Data are representative of two or more independent experiments.
Figure 6 depicts apoptotic stimuli activate the NLRP3 inflammasome in macrophages in accordance with various embodiments of the present invention. (A) NLRP3 activators cause cell death. BMDM were treated with UVCP (MOI 10), live Cpn (MOI 10), ATP (5 mM), STS (5 μΜ) or alum (ALM, 130 μg/ml) and LDH release was determined at the indicated time points. Data shown are representative of three or more independent experiments. (B) NLRP3 activators lead to apoptosis. BMDM were treated with UVCP (MOI 10), live CP (MOI 10), ATP (5 mM) or STS (5 μΜ) for the indicated times. Cells were then fixed in paraformaldehyde, stained with DAPI and scored for the presence of condensed nuclei. Data shown are representative of three or more independent experiments. (C) Staurosporine induces caspase-1 activation, IL- 1β maturation and IL-Ιβ secretion in primed macrophages. Immunoblotting was used to analyze mouse caspase-1, pro-IL-Ιβ, and IL-Ιβ in culture supernatants and lysates of BMDM treated with (left to right) UVCP (MOI 10, 8 h), live CP (MOI 10, 8 h), LPS (1 8 h), LPS + ATP (5 mM, final 2 h of culture), ATP alone, LPS + STS (5 μΜ, final 2 h of culture), or STS alone. Data shown are representative of three or more independent experiments. (D) Staurosporine induces IL-Ιβ secretion from primed macrophages without affecting TNFa secretion. IL-Ιβ and TNFa secretion by WT and Caspl ' BMDM was measured using ELISA. BMDM were primed with UVCP (MOI 10, 8 h), live Cpn (MOI 10, 8 h) or LPS (1 μ§/ηι1, 8 h). Cells were then treated with STS (2.5 μΜ or 5 μΜ) for the final 2 h of culture. Data shown are representative of three or more independent experiments. (E) Staurosporine-induced IL-Ι β secretion by macrophages depends on the NLRP3/ASC inflammasome. WT, ASC/_ or NLRP3 " " BMDM were given UVCP (MOI 10, 8 h), live CP (MOI 10, 8 h) or LPS (1 8 h) and then treated with STS
(5 μΜ, final 2 h of culture). Data shown are representative of three or more independent experiments. (F) Staurosporine induces NLRP3 activation only if it is the second signal. ELISA was used to quantify IL-Ιβ secretion by BMDM primed with UVCP (MOI 10, 8 h), live CP (MOI 10, 8 h) or LPS (1 μg/ml, 8 h) and then treated with ATP (5 mM, 0 h or 6 h after priming) or STS (5 μΜ, 0 h or 6 h after priming). Data shown are representative of three or more independent experiments. (G) Antimycin A (AntA) dose dependently reduces IL-Ιβ secretion without affecting TNFa production. BMDM were treated with UVCP (MOI 10, 8 h), live CP (MOI 10, 8 h), LPS (1 μg/ml, 8 h), UVCP + ATP (5 mM, final 2 h of culture), live CP + ATP, LPS + ATP, UVCP + STS (5 μΜ, final 2 h of culture), live CP + STS, or LPS + STS and then exposed to various concentrations of AntA (0, 2.5, 5, 10 μΜ). IL-Ιβ and TNFa secretion were then measured using ELISA. Data shown are representative of three or more independent experiments. (H) Apoptotic cells release IL-Ιβ in a Caspl -dependent manner. BMDM were treated with live CP (MOI 10, 8 h), LPS (1 μg/ml, 5 h), LPS + ATP (5 mM, final 2 h of culture), or ATP alone. Then, cells were fixed in paraformaldehyde and stained with an antibody specific for intracellular IL-Ιβ and with DAPI. Data shown are representative of three or more independent experiments.
Figure 7 shows that Bcl2 overexpression reduced IL-Ι β secretion, mitochondrial collapse and apoptosis, but not TNFa production in accordance with various embodiments of the present invention. (A and B) IL-Ιβ and TNFa in supernatant was measured in Bcl2- overexpressed macrophages. (C) ΔΨιη was determined by TMRM incorporation assay. (D) Apoptotic cell was determined by DAPI staining.
Figure 8 depicts Alveolar macrophages are reservoir for C. pneumoniae in Caspl-/- mice in accordance with various embodiments of the present invention. (A, B and C) Compared to WT, Caspl -/-phagocytes contain more Chlamydophila and macrophages are the principal C. pneumoniae harboring cell type. Single-cell suspensions from C. pneumoniae infected lungs of WT and Caspl -/-were prepared 12 days following infection. Cells were then stained for characteristic leukocyte markers and stained intracellular C. pneumoniae with a FITC conjugated anti-Chlamydia monoclonal antibody (mAb), and analyzed by flow cytometry to determine which cell types contain C. pneumoniae. Representative flow cytometry histograms of CD45+ cells, F4/80+ cells (CDl lc+ gated), and Ly6G+ (CDl lc+ gated) cells are shown. Also shown are the proportions of total lung leukocytes that contain C. pneumoniae in WT and Caspl-/-mice and the absolute numbers of leukocytes in C. pneumoniae infected lungs from WT and Caspl-/-mice. Data for all experiments shown represent at least two independent experiments.
Figure 9 shows that TLR2/MyD88 is indispensable for C. pneumoniae-induced TNF- α production by macrophages in accordance with various embodiments of the present invention. (A) WT, MyD88-/-, TRIF-/-, and RIP2-/-BMDM were treated with live C. pneumoniae (MOI 2.5, 5, 10), LPS (1 μg/ml), PGN, poly I:C, muramyldipeptide (MDP), and CpG DNA and assessed for TNFa production by ELISA. (B) WT, TLR2-/-, TLR4-/-, and TLR9-/-BMDM were treated with live C. pneumoniae (MOI 2.5, 5, 10), LPS (1 μg/ml), PGN, and CpG DNA and assessed for TNFaproduction by ELISA.
Figure 10 shows that Caspl deficiency does not affect macrophage phagocytic activity or Chlamydophilal infectivity in accordance with various embodiments of the present invention. (A) Caspl -/-macrophages are as effective as WT macrophages in internalizing C. pneumoniae. BMDMs were infected with labeled C. pneumoniae (solid line histogram, MOI 2.5, 5, 10, and 20) or vehicle control (gray-filled histogram). The mean fluorescence intensity (MFI) and percentage of labeled C. pneumoniae internalized cells are indicated. (B) WT and Caspl -/-BMDMs were infected with C. pneumoniae (MOI 10). Cell lysates were harvested at indicated time points and viable bacteria were quantified by infecting HEp2 cells.
Figure 11 shows C. pneumoniae-induced ΔΨιη loss in macrophages is NLRP3 inflammasome independent. (A) WT, ASC-/-, NLRP3-/-and Caspl -/-BMDM were treated with UVCP (MOI 2.5, 5, 10), live C. pneumoniae (MOI 2.5, 5, 10), or STS (5 μΜ) and then examined for TMRM incorporation. Statistical significance was determined by Student's t test in comparison to non-treated cells (* p<0.05, ** p< 0.01, *** p<0.001). The LDH release was determined in C. pneumoniae-mfected BMDM at 24hs in WT and Caspl-/-BMDM (B). Data shown are representative of three or more independent experiments.
Figure 12 also shows that mitochondrial dysfunction is linked to NLRP3 inflammasome activation in accordance to various embodiments of the present application, (a-c) ELISA was used to determine IL-Ιβ and TNFa concentrations in BMDM culture supernatants treated with (a) various NLRP3 inflammasome pathway-activating stimuli indicated or (b) LPS (1 μg/ml) plus ATP (5 mM, final 2h culture) in the presence of increasing doses of chloramphenicol. Also shown is an LDH release assay of the same BMDM. (c) Cyclosporin A (CsA) inhibits LPS+ATP-induced IL-Ι β in WT BMDM and cyclophilin D (CypD) deficient BMDM in a dose-dependent manner, (d) Loss of mitochondrial membrane potential (ΔΨιη) correlates with NLRP3 inflammasome activation. BMDM were treated with UV-killed CP (UVCP, MOI=10) or infected with live CP (MOI=10), treated with ATP (5 mM) or staurosporine (STS, 5 μΜ) for 24 h and examined for TMRM incorporation, (e) The kinetics of ΔΨιη loss were monitored over time using a similar TMRM incorporation assay. BMDM were treated with live CP (MOI=10), ATP (5 mM), STS (5 μΜ), nigericin (NIG, ΙΟμΜ), or alum (130 μg/ml) (f,g) Extracellular ATP causes rapid, irreversible loss of AXVM by TMRM microscopy. A representative image of mitochondrial TMRM fluorescence and associated 3D intensity map are shown in (f). The kinetic profile shown in (g) depicts the mean value +/- the SEM at each time point. (h,i) High concentrations of extracellular K+ (h) block IL-Ιβ secretion and (i) prevent ΔΨι^ reduction as measured by TMRM incorporation. Four h after LPS priming (1 μg/ml) or after CP infection (MOI=10), BMDM were transferred to culture media containing either 5 mM or 100 mM K+. Six h after LPS priming, BMDM were then treated with ATP (5 mM), STS (5 μΜ) or NIG (10 μΜ) and cultured for an additional 2 h before culture supernatants were collected and IL- 1 β concentration was measured. CP infected BMDM were cultured for an additional 20 h before supernatants were collected for IL-Ι β measurement, (j) Mitochondrial oxygen consumption is linked to NLRP3 inflammasome activation. Oxygen consumption rate (OCR) was measured in macrophages. The base line OCR was significantly reduced 2 h after ATP (5 mM), STS (5 μΜ) or NIG (10 μΜ) treatment, (k) The change of OCR was monitored after Ι μΜ oligomycin, 1 μΜ FCCP, and Ι μΜ Rotenone sequential additions. (1) The kinetics of OCR was monitored in macrophages in response to NLRP3 activator. Data shown are representative of two or more independent experiments. Note on statistical significance: * p< 0.05, ** p<0.01 , ***p<0.001.
Figure 13 also shows that Bcl-2 inversely regulates mitochondrial dysfunction and NLRP3 inflammasome activation in accordance to various embodiments of the present application, (a) BCI-2-UQOX or empty vector (EV, neor) was stably expressed in MCL macrophages, and Bcl2 expression was determined by immunoblot. (b,c) Bcl-2 overexpressing macrophages were treated with live CP (MOI=10, 24 h), LPS (1 μg/ml, 5 h), ATP (5 mM, final 2 h of culture), LPS+ATP, STS (5 μΜ, final 2 h of culture), LPS+STS, NIG (10 μΜ, final 2 h), or LPS+NIG and then (b) IL-Ι β or TNFa secretion was measured by ELISA or (c) immunob lotting was used to analyze mouse capsase-1 , pro-IL-Ι β, or IL-Ι β in culture supernatants or cell lysates. (d) Delta XVM and (e) apoptotic cells were measured after NLRP3 inflammasome stimulation at 8 h for CP, or at 4 h for ATP, STS and NIG . (f) BMDM from Bcl-2 transgenic mice were treated with live CP, LPS, LPS+ATP, LPS+STS, LPS+NIG, or LPS+alum (130 μg/ml, final 8 h). IL-Ιβ and TNFa secretion were then measured using ELISA. (g) Validation of shRNA targeting Bcl-2 in stably-expressing MCL macrophages by immunoblot. (h,i) shBcl-2 macrophages were primed with LPS and treated with ATP (5mM for 4 h), and LDH release, IL-Ι β and TNFa were then measured in supernatants. Data shown are representative of three or more independent experiments. Note on statistical significance: * p<0.05, ** p< 0.01, *** p<0.001.
Figure 14 shows that Salmonella type III secretion protein and invasion factor SipB induces mitochondrial depolarization and caspase-1 dependent IL-Ιβ secretion in accordance to various embodiments of the present application, (a) Three h LPS-primed Nlrp3-/-BMDM were exposed to S. typhimurium (MOI=5) and nonflagellated mutant, and IL-Ιβ in the culture supernatant was quantified by ELISA. Significant differences are indicated; *** p<0.001, by one-way ANOVA and Tukey's HSD post-hoc test. (b,c) Three h LPS-primed BMDM were exposed to various strains of S. typhimurium (MOI=5) and (b) cell viability was determined by LDH release assay or (c) mitochondrial depolarization was determined by TMRM incorporation assay. (d,e) IL-Ιβ in the culture supernatant was quantified by ELISA. (f) Bcl-2 overexpressing macrophages were exposed to S. typhimurium (MOI=5), and IL-Ιβ or TNFa secretion was then measured by ELISA. Data shown are representative of three or more independent experiments. Note on statistical significance: * p<0.05, ** p< 0.01.
Figure 15 shows that oxidized mitochondrial DNA binds to NLRP3 and activates the inflammasome in accordance to various embodiments of the present application. (a) Flag- NLRP3 and empty vector (EV) stably-expressing 293 cells were exposed to BrdU-labeled mitochondrial DNA (mtDNA) with lipofectamine. Cell lysates were collected 3 h later and immunoprecipitated. mtDNA was detected by BrdU dot-blot, and NLRP3 Ab was used for immunoblotting as a loading control, (b) BMDM were preloaded with BrdU (10 μΜ), and cells were treated with LPS (1 μg/ml, 3 h), followed by 3-MA (2.5 mM, 1 h), ATP (5 mM, 1 h) or NIG (10 μΜ, 1 h). Cell lysates were collected and immunoprecipitated with anti- NLRP3 Ab, then detected by dot-blots probed with anti-BrdU or anti-80H-dG Abs. As a loading control, NLRP3 immunoblot was performed, (c) Mitochondrial COX1 DNA associates with NLRP3 immunoprecipitates. COX1 DNA was amplified from the NLRP3 immunoprecipitates by PCR. (d) Mitochondrial DNA was extracted from BMDM after NLRP3 inflammasome stimulation, and levels of 8-OH-dG were quantified by ELISA. (e,f) LPS-primed (e) WT or (f) Nlrp3^ BMDM were treated with exogenous 8-OH-dG incorporated DNA (oxDNA, 2 μ /ηι1) or control DNA for 8 h. IL-Ιβ and TNFa levels were quantified in supematants by ELISA. (g) LPS-primed Aim2~f~ BMDM were treated with exogenous oxDNA (2 μ /ηι1) and IL-Ιβ and TNFa levels were determined in culture supematants by ELISA. (h,i) BMDM were stimulated with LPS (1 μ§/ι 1) in the presence of deoxyguanosine (dG) and 8-OH-dG, then treated with (h) ATP, NIG or (i) poly(dA:dT) (2 μ§/ι 1), and IL-Ιβ release was quantified by ELISA. (j) BMDM were primed with LPS (1 μ^πύ, 3 h) in the presence of dG or 8-OH-dG (200 μΜ), pretreated with 3-MA (2.5 mM for lh), and then treated with ATP (5 mM, 1 h). Immunoprecipitation with anti-NLRP3 Ab was performed, and BrdU or 8-OH-dG dot-blotting was performed with immunoblotting for NLRP3 as a loading control. Data shown are representative of three or more independent experiments. Note on statistical significance: * p<0.05.
Figure 16 shows that macrophage NLRP3, ASC, CASP1 deficiency does not alter TNFa secretion in accordance to various embodiments of the present application. BMDM were exposed to UV-killed CP or live CP (MOI 10, 24hrs), or treated with LPS (1 μg/ml, 5 h), LPS + ATP (5 mM, final 2 h of culture), ATP. IL-Ιβ and TNFa secretion were then measured using ELISA. Data shown are representative of three independent experiments.
Figure 17 shows that Bcl2 overexpression suppresses apoptosis in accordance to various embodiments of the present application. BMDM were stimulated with LPS (1 μg/ml, 3 h) and then treated with ATP (5 mM, 4h) and NIG (10 μΜ, 4h). Cells were labeled with annexin V and 7AAD. Apoptotic cell was analyzed by flow cytometry. Data shown are three independent experiments.
Figure 18 shows that pyroptosis and necrosis do not play a role in NLRP3 inflammasome activation in accordance to various embodiments of the present application, (a) CASP1-/- BMDM were infected with CP (MOI 10), treated with ATP (5 mM), STS (5 μΜ), NIG (10 μΜ) or Alum (130 μg/ml) for 4 h. and examined for TMRM incorporation. The change of ΔΨιη were examined using a TMRM incorporation assay, (b) The LDH release was determined in CP-infected BMDM at 24hs in WT and CASP1-/- BMDM. (c) Necrotic stimulus of 7BIO does not induce IL-Ιβ in LPS-primed BMDM. BMDM were exposed to 7-bromoindimbin-3'-oxime (7BIO) for 18 h in LPS (1 μg/ml, 6 h) -primed BMDM. LDH release and IL-Ι β level in the supernatant was determined. Data shown are three independent experiments.
Figure 19 shows Bcl2 overexpression suppressed IL-Ιβ secretion not TNFa secretion in accordance to various embodiments of the present application. The stably expressing Bcl2- neor, Empty vector (EV, neor), and YFP-neor macrophages were stimulated as indicated. IL- 1β and TNFa level in the supernatant were determined. Data shown are three independent experiments. * p< 0.05, Student's t test.
Figure 20 shows that mitochondrial DNA contributes NLRP3 inflammasome. (a) pOMCL was generated with 50 μg/ml of EtBr for more than 20 passages. Mitochondrial DNA contents were determined by qPCR of mitochondria specific gene mtCOXl compared to nuclear DNA (GAPDH). (b-c) Metabolic and non-metabolic mitochondrial labeling were compared in pOMCL. (d) pOMCL is attenuated IL-Ιβ secretion in response to NLRP3 stimuli. pOMCL and control macrophages were treated with LPS (1 μg/ml, 5 h), LPS + ATP (5 mM, final 2 h of culture), ATP, LPS + NIG (10 μΜ, final 2 h of culture), or NIG. IL-Ιβ and TNFa secretion were then measured using ELISA. (e) LPS-primed BMDM were treated with ATP (5 mM) and NIG (10 μΜ) for 15 and 30 min. Cytosolic fraction was collected from homogenates. Mitochondrial DNA was detected by qPCR of mtCOXl . Data shown are representative of three or more independent experiments. Note on statistical significance: *** p<0.001 (Student's t test used unless otherwise noted).
Figure 21 shows that mitochondrial DNA is colocalized with NLRP3. (a) YFP-tagged
NLRP3 stably expressed 293 cells were exposed to BrdU-labeled mitochondrial DNA with lipofectamin in accordance to various embodiments of the present application. 3hrs after exposure, Cell were fixed with 2% PF A/PBS, permeabilized, treated with 10 U/ml for 30 min, and then stained with anti-BldU mAb. The co-localization of YFP-NLRP3 and mtDNA were analyzed by confocal microscopy, (b) The representative 3 dimensional analysis for co- localization of YFP-NLRP3 and mtDNA.
DESCRIPTION OF THE INVENTION
All references cited herein are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, 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 belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 3rd ed., J. Wiley & Sons (New York, NY 2001); March, Advanced Organic Chemistry Reactions,
Mechanisms and Structure 5th ed., J. Wiley & Sons (New York, NY 2001); and Sambrook and Russel, Molecular Cloning: A Laboratory Manual 3rd ed., Cold Spring Harbor
Laboratory Press (Cold Spring Harbor, NY 2001), provide one skilled in the art with a general guide to many of the terms used in the present application.
The present invention provides for methods to treat inflammation and inflammatory diseases and disease conditions; particularly, inflammatory diseases and disease conditions wherein the role of IL-Ι β has been established. Examples of inflammatory diseases and disease conditions that can be treated by the present invention include but are not limited to autoimmune diseases, arthritis, Crohn's disease, inflammatory bowel disease, Alzheimer's disease, diabetes, gout, atherosclerosis, and asbestosis/silicosis induced lung fibrosis. Examples of autoimmune diseases include but are not limited to Hashimoto's thyroiditis, Pernicious anemia, Addison's disease, type I diabetes, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, Sjogren syndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis, reactive arthritis, Grave's disease, and celiac disease - sprue (gluten sensitive enteropathy).
The inventors discovered that the mitochondrial apoptotic process is an upstream mechanism of NLRP3 inflammasome activation. IL-Ιβ is a highly inflammatory cytokine and inhibitors, such as mitochondrial apoptosis inhibitors, can be used to block the IL-Ιβ induced inflammatory pathway in multiple inflammatory diseases where the role of IL-Ιβ has been established. Examples mitochondrial apoptosis inhibitors include but are not limited to Bcl-2, Mcl-1, Bcl-xL, Bcl-w, Bfl-1, nfh, XIAP, Boo/Diva, Nrl3, BH4 domain, Inositol 1,4,5- trisphosphate receptor (IP3R) peptide, ϋ8Κ-3β, cyclosporine A, viral mitochondrial inhibitor of apoptosis (vMIA) peptide/protein, and MitoQ.
Additionally, apoptosis is thought to be a 'silent death' that fails to provoke inflammation. As discussed herein, the inventors report that in the presence of signal 1, the NLRP3 inflammasome is activated by mitochondrial apoptotic signaling that licenses production of the pro-inflammatory cytokine, interleukin-ΐ β (IL-Ιβ). NLRP3 secondary signal activators such as ATP induce mitochondrial dysfunction and apoptosis, which results in the release of oxidized mitochondrial DNA (mtDNA). The inventors show that cytosolic mtDNA binds to and activates the NLRP3 inflammasome. The anti-apoptotic protein Bcl-2 inversely regulates mitochondrial dysfunction and NLRP3 inflammasome activation. Mitochondrial DNA directly induces NLRP3 inflammasome activation, because macrophages lacking mtDNA have severely attenuated IL-Ιβ production, yet still undergo apoptosis. Both binding of oxidized mtDNA to the NLRP3 inflammasome and IL-Ι β secretion can be competitively inhibited by the oxidized nucleoside, 8-OH-dG. Thus, the inventors' data reveal that oxidized mtDNA released during programmed cell death causes activation of the NLRP3 inflammasome. These results provide a missing link between apoptosis and inflammasome activation, via binding of cytosolic oxidized mtDNA to the NLRP3 inflammasome. Shown herein, caspase-1 dependent IL-Ιβ secretion is critical for host defense during CP lung infection and that CP infection alone triggers caspase-1 activation via the NLRP3 inflammasome. CP infected macrophages secreted less IL-Ιβ in the presence of inhibitors of apoptotic signals. Closer examination of this phenomenon revealed that mitochondrial damage which specifically resulted in apoptotic signals induced NLRP3 -dependent IL-Ιβ secretion in primed macrophages. Given that both mitochondrial dysfunction and apoptosis are inextricably linked to ROS, intracellular K+ levels and lysosomal degradation, while not wishing to be bound by any particular theory, the inventors believe that the mitochondria as a central hub for the diverse signals sensed by NLRP3. Taken together, the inventors' results provide evidence for a novel innate immune defense program by which macrophages, in response to danger signals and cytosolic stress, activate a mitochondria-dependent apoptotic cascade necessary for activation of the NLRP3 inflammasome.
Also shown herein is that Caspl -dependent IL-Ιβ secretion is required for host defense against CP lung infection. Caspl"7" mice displayed delayed pulmonary bacterial clearance leading to increased mortality compared to WT mice. Macrophages play a key role in this process, responding to CP via the NLRP3 inflammasome. Caspl"7" mice showed delayed IFN-γ production and defective iNOS activation in Caspl"7" AM, consistent with reports showing the critical role of IFN-γ and iNOS in clearing CP infection (Rottenberg et al, 1999).
The inventors discovered evidence that the mitochondria play a role within the
NLRP3 activation cascade. Closer examination of this phenomenon led the inventors to discover a novel mode of NLRP3 activation whereby the mitochondria sense cellular danger and disseminate apoptotic signals that trigger the NLRP3 inflammasome.
It is currently not clear how oligomeric NLRP3 inflammasome complexes sense such a tremendous range of cytosolic danger signals. NLRP3 contains a leucine rich repeat (LRR) domain, a central NACHT domain and a pyrin domain (PYD) (Martinon et al, 2007). Hoffman and colleagues showed that BMDM from mice lacking the LRR domain of NLRP3 exhibit attenuated but not ablated IL-Ιβ secretion (Hoffman et al., 2010). As such, it is thought that, in a mechanism distinct from pattern recognition receptors (PRR), NLRP3 does not sense each of these diverse ligands directly with its LRR. Instead, it is believed that three broad physiological changes— reactive oxygen species (ROS) generation, potassium cation (K+) efflux, or lysosomal leakage— activate the NLRP3 inflammasome (Stutz et al., 2009). Currently, though, these three proposed models of NLRP3 activation are not reconciled with one another and no model provides the unifying paradigm.
The inventors' data argue against the universality of ROS in activating the NLRP3 inflammasome. Even though UVCP and CP induced similar amounts of ROS in macrophages, UVCP did not elicit IL-Ιβ secretion while live CP did. Moreover, these results call in to question results from other studies that use the antioxidant N-acetylcysteine (NAC). Though this agent was found to reduce IL-Ιβ secretion, it also caused a concomitant reduction in TNFa production, indicating that NAC likely affects pro-IL-Ιβ production via NF-KB.
The lysosome rupture model also does not seem to be universal, as it does not agree well with the CP induced NLRP3 activation observed in the present study. CP is small (infectious elementary body (EB); 300-600 nm diameter) compared to other intracellular bacteria, and so internalization of the EB is unlikely to exceed the capacity of the phagolysosome. As part of the CP life cycle, the infectious EB converts to the vegetative reticulate body (RB), which forms inclusion bodies in the host cell phagosome (6.0-7.4 μιη diameter). Though these inclusions might be large enough to cause vesicle rupture, Chlamydophila are known to inhibit actively the process of phagolysosomal fusion. So even if CP-containing phagosomes ruptured, lysosomal enzymes would not be present. Also, it was found that CP is able to induce IL-Ιβ secretion in the presence of a specific cathepsin B inhibitor, further arguing against lysosomal degradation as the means by which CP activates the NLRP3 inflammasome.
It is of major interest that the three models of NLRP3 inflammasome activation— ROS, lysosome stability and intracellular K+ concentration— are all inextricably linked to the mitochondrial dysfunction leading to apoptosis. As both sensors and robust sources of ROS, mitochondria respond rapidly to elevated ROS by releasing cytochrome c and inducing apoptosis (Ott et al, 2007). Cathepsins (and ROS, for that matter) released during lysosomal rupture can also profoundly impact mitochondrial membrane integrity, causing mitochondrial membrane permeabilization and subsequent initiation of apoptosis (Boya et al., 2003; Ferri and Kroemer, 2001). The mitochondrial K+ cycle, inexorably linked to intracellular K+ concentration, is absolutely central to maintaining mitochondrial volume, controlling metabolic ROS (Garlid and Paucek, 2003) and induction of apoptosis (Park and Kim, 2002). Perhaps the mitochondria have been overlooked as the control station for NLRP3 activation because all three physiological perturbations not only lead to but also result from mitochondrial apoptotic cascades.
It is shown herein that NLRP3 triggers, such as alum, ATP, nigericin and live CP cause mitochondrial dysfunction and cell death in macrophages. Moreover, it is demonstrated that antimycin A (an anti-apoptotic) and cyclosporin A (an inhibitor of mitochondrial pore formation) dose-dependently inhibit IL-Ιβ release but do not affect TNFa secretion caused by these agents. Given the connection between the physiological triggers of NLRP3, the mitochondria and apoptosis, and the causal relationship between pro-apoptotic signals and NLRP3, and while not wishing to be bound by any particular theory, the inventors believe that apoptosis as an indispensable and unifying step in NLRP3 inflammasome activation.
Though the role of the mitochondria in host defense and pathogenesis in response to microorganisms has been well studied (Arnoult et al, 2009; Carneiro et al, 2009), and IL-Ιβ release during apoptosis was reported nearly two decades ago (Hogquist et al, 1991), this is the first study to identify a direct link between mitochondrial apoptotic signals and activation of the NLRP3 inflammasome in macrophages. Greten et al. demonstrated that LPS-primed ΙΚ β knockout macrophages, which undergo more spontaneous apoptosis than wild-type counterparts, secrete considerable amounts of IL-Ιβ in the absence of a second signal (Greten et al, 2007), which supports this model. Further, Brough and Rothwell found that macrophages exhibit concomitant induction of cell death pathways and IL-Ι β secretion (Brough and Rothwell, 2007). Studies have also identified a connection between apoptotic processes and N ALP 1 inflammasome: NALP1 interacts with Caspase-9 and the pro-apoptotic protein Apaf-1 (Chu et al, 2001), the anti-apoptotic protein Bcl-2 suppresses NALP1 activation (Bruey et al., 2007) and Bcl-xL and Bcl-2 gene transfection attenuates muramyl dipeptide induced caspase-1 activity (Faustin et al, 2009). It is also interesting that many inflammasome associated proteins have homologs which act as apoptotic factors in C. elegans (Miura et al, 1993).
According to this model, both the apoptosome and the NLRP3 inflammasome share common upstream activating factors derived from the mitochondria. Yet, the apoptosome and the inflammasome are functionally distinct. Mice deficient in Apaf-1, Caspase-9 or Caspase- 3 exhibit infertility, abnormal brain development, and lethality (Honarpour et al., 2000; Kuida et al, 1998; Kuida et al, 1996) whereas no such phenotype has been found for Caspase-1, NLRP3 or ASC deficient mice. These findings reveal that NLRP3 activation occurs concomitantly with apoptosis and that many physiological NLRP3 triggers are likely governed by the mitochondrial apoptotic cascades. But further investigation must be done to reveal which mitochondrial factor associated with apoptosis, such as cytochrome c, Apaf-1, Bcl-2 and Caspase-9 play a role in NLRP3 activation and regulation.
An implication of the present study is that inhibition of mitochondrial apoptosis by intracellular microbes serves a dual role: inhibition of IL-Ιβ secretion and maintenance of a viable host cell for intracellular growth.
Secretion of IL-Ιβ, a potent pyrogen that elicits a strong pro-inflammatory response l, is tightly controlled by a diverse class of cytosolic complexes known as the inflammasome 2. The NOD-like Receptor (NLR) family member NLRP3 forms cytosolic oligomers with apoptosis-associated speck like protein (ASC) in dendritic cells 3 and macrophages 4, triggering autocatalytic activation of caspase-1 5. Caspase-1, in turn, cleaves pro-IL-Ιβ, producing mature IL-Ιβ. Under normal circumstances, NLRP3 undergoes bipartite activation 2. The first signal, often NF-κΒ activation, induces pro-IL-Ιβ and NLRP3 expression. The second signal, any one of a variety of unrelated entities— particulate matter 6, crystals 7, aggregated β-amyloid 8, extracellular ATP 9' 10, or microbial toxins 11 activates the NLRP3 inflammasome.
Mitochondria are exquisitely complex regulators of cytosolic homeostasis. These organelles sense and respond to changes in intracellular K+ 17 and ROS 18, and have an important relationship with the lysosome 19. Perturbation of intracellular K+, ROS or lysosomal stability can result in mt dysfunction and apoptosis 20~22. Therefore, these cellular organelles are well positioned to regulate the NLRP3 activation pathway. The inventors' initial investigation focusing on NLRP3 activation by Chlamydia pneumoniae (CP) infection led the inventors to a result that implicated mt in NLRP3 activation 23. Described herein, the inventors reveal that mt damage, which specifically triggers apoptotic signals, induces NLRP3 -dependent IL-Ιβ secretion in primed macrophages. Evidence is provided herein for a novel innate immune defense program by which primed macrophages, in response to danger signals and cytosolic stress, activate an mt-dependent apoptotic cascade, which is necessary and sufficient for caspase-1 activation by the NLRP3 inflammasome in the presence of signal 1. This apoptotic cascade activates the NLRP3 inflammasome through cytosolic release of oxidized mtDNA, which binds to and activates the NLRP3 inflammasome. Given that both mt dysfunction and apoptosis are inextricably linked to ROS, intracellular K+ levels and lysosomal degradation, it would seem that mt are positioned as the central hub for integration of the diverse signals sensed by NLRP3.
The NLRP3 inflammasome has become a critical nexus mediating IL-Ιβ and IL-18 responses to pathogens and innate immune stimuli. Because inflammasome stimuli are diverse and often unrelated, discerning the mechanism of NLRP3 activation has proven difficult and remains elusive. Recent evidence has begun to indicate mt as key players in NLRP3 inflammasome signaling 15' 16. During the analysis of C. pneumoniae infection, the inventors uncovered evidence that mt play an important role in activating the NLRP3 inflammasome. Herein, the inventors provide a mechanistic explanation for NLRP3 activation. In this model, mt sense cellular danger that results in apoptosis, during which mtDNA is released into the cytosol and binds to NLRP3. This cascade of events results in activation of the NLRP3 inflammasome and casapse-1 maturation.
Identifying mt as key players in NLRP3 inflammasome induction unifies the three physiological perturbations under the single umbrella of apoptosis. As both sensors and robust sources of ROS, mt rapidly respond by releasing cytochrome c and inducing apoptosis 39. Moreover, cathepsins and ROS released during lysosomal rupture can also profoundly impact mt membrane integrity, causing membrane permeabilization and subsequent initiation of apoptosis 40' 41. The mt K+ cycle, inexorably linked to intracellular K+ concentration, is obligatorily central to maintaining mt volume, controlling metabolic ROS 17 and inducing
42
apoptosis .
As discussed, NLRP3 triggers, such as alum, ATP, NIG and live CP cause mt dysfunction and cell death in macrophages. It is also shown herein that STS, a pro-apoptotic compound, is sufficient to act as a second signal for NLRP3 activation. Moreover, LPS- primed macrophages treated with NLRP3 activators secrete less IL-Ι β, but the same amount of TNFa in the presence of cyclosporin A. Apoptosis is critical for NLRP3 inflammasome induction, because overexpression of anti-apoptotic Bcl2 attenuates IL-Ιβ secretion by LPS- primed macrophages and Bcl2 knock-down results in the converse. The inventors also find that S. typhimurium type III secretion mutants that cannot induce apoptosis are unable to induce IL-Ιβ secretion, and Bcl2 overexpression inhibits wild-type S. typhimurium infection- induced IL-Ιβ secretion. Given that the physiological triggers of NLRP3 are inextricably linked to mt and apoptosis, and that a causal relationship exists between pro-apoptotic signals and NLRP3, it seems that apoptosis is an indispensable step in NLRP3 inflammasome activation. While apoptosis is often portrayed as 'silent' cell death, the inventors' data suggest that in the presence of proinflammatory signal 1, the apoptotic machinery activates the NLRP3 inflammasome. Thus, intrinsic apoptosis has the capability of activating the NLRP3 inflammasome, but remains a silent death unless in the context of signal 1.
It was not clear how oligomeric NLRP3 inflammasome complexes sense such a wide range of cytosolic danger signals, including ATP, K+ efflux, alum, uric acid crystals, β- amyloid, and various microbial infections 43' 44. As such, in a mechanism distinct from pattern recognition receptors, NLRP3 does not seem to sense each of these diverse ligands directly with its leucine-rich repeat (LRR) domain. Instead, it was believed that three broad physiological changes— ROS generation, K+ efflux, or lysosomal leakage— activate the NLRP3 inflammasome 45. The inventors' model of oxidized mtDNA binding to NLRP3 as the activation step neatly assembles and unifies previous models of NLRP3 activation. While the inventors found that oxidized mtDNA associates with the NLRP3 inflammasome after stimulation, the inventors' data utilizing 293 cells transfected with mtDNA suggest that the DNA could bind directly to NLRP3. However, these results do not rule out the association of the mtDNA with other members of the NLRP3 inflammasome complex.
Two recent investigations linked autophagy and inflammasome activation with mt activity. The first report found that blocking autophagy resulted in accumulation of mt-driven ROS formation, which in turn activated the NLRP3 inflammasome 15. The inventors' results presented here generally agree with the basic findings of the Zhou and colleagues report. Specifically, those authors concluded that apoptosis was not involved in their system based on lack of LDH release after various stimuli, and were unable to detect LDH release after exposure to NIG; a proton inophore that causes cytosol acidification and has been shown to decrease intracellular pH and induce apoptosis in other models 46' 47. Indeed, the inventors found that NIG induced nuclear condensation, and another group reported LDH release after NIG exposure 48. Interestingly, while Zhou et al. did not detect LDH release after monosodium urate treatment, this compound may have off-target effects as it has been shown to inhibit neutrophil apoptosis at low concentrations, but causes LDH release at higher levels 49. Nonetheless, when considering the preponderance of data present in this study linking apoptosis and NLRP3 activation, it seems that initiation of apoptotic events is required for proper NLRP3 activation.
Both Zhou et al. and Nakahira and coworkers found that mt-derived ROS were required for NLRP3 activation 15' 16. However, Nakahira et al. also found that mtDNA release was critical for NLRP3 activation, and this was dependent on ROS generation. The inventors also found that mtDNA was released into the cytosol and that its presence was absolutely required for NLRP3 activation. However, Nakahira and colleagues concluded that NLRP3 itself was required for mtDNA release, as they did not detect mtDNA in the cytosol of NLRP3 deficient macrophages. Importantly, the inventors' results suggest that transfected mtDNA can bind to NLRP3 overexpressed in 293 cells and that DNA released from mt during apoptosis binds to the NLRP3 inflammasome. Therefore, a likely explanation for the lack of cytosolic mtDNA in NLRP3 deficient macrophages is that NLRP3 itself stabilizes mtDNA in the cytosol by binding to it. The direct binding of mtDNA to NLRP3 could therefore be the triggering mechanism for NLRP3 activation.
Mitochondrial ROS are now appreciated to play a central role in NLRP3 inflammasome activation 15' 16. During apoptosis, ROS are generated and oxidize mtDNA 36. The inventors' data indicate that it is this oxidized form of mtDNA that binds to and activates the NLRP3 inflammasome and that this interaction can be competitively inhibited by oxidized dG. Therefore the inventors' data are in agreement with these previous studies regarding the importance of mtROS, and now provide the likely mechanism for NLRP3 inflammasome activation. Additionally, while it appears that transfected oxidized mtDNA can activate both NLRP3 and AIM2, AIM2 is preferentially activated by normal DNA, while NLRP3 , by oxidized DNA.
Though the role of mt in host defense and pathogenesis in response to microorganisms has been well studied 29' 50, and IL-Ιβ release during apoptosis was reported nearly two decades ago 9, the inventors' is the first study to identify a direct link between mt apoptotic signals and activation of the NLRP3 inflammasome in macrophages. In support of the inventors' model, Greten et al. demonstrated that LPS-primed ΙκΒ kinase β deficient macrophages, which undergo more spontaneous apoptosis than wild-type cells, secrete considerable amounts of IL-Ιβ in the absence of a second signal 51. Further, Brough and Rothwell found that macrophages exhibit concomitant induction of cell death pathways and IL-Ιβ secretion 52.
While the inventors' data clearly show that induction of apoptosis is required for
NLRP3 inflammasome activation, it was not known at what point in the signaling cascade the two events diverge. According to the inventors' model, both the apoptosome and the NLRP3 inflammasome share common upstream activating factors derived from mt. However, it is a byproduct of apoptosis, oxidized mtDNA released into the cytosol, which seems to be the activating factor for the NLRP3 inflammasome. The divergence of the two pathways after initiation of apoptosis is evidenced by the fact that mice deficient in Apaf-1, caspase-9 or caspase-3 exhibit infertility, abnormal brain development, and lethality 53 55 whereas no such phenotypes have been found for caspase-1, NLRP3- or ASC deficient mice. Thus, while the two pathways share a common origin, their downstream effects are different.
The inventors show that mt dysfunction leading to apoptosis occurs with, and is necessary and sufficient for, NLRP3 activation in the presence of signal 1. A key implication of the present study is that inhibition of apoptosis by intracellular microbes serves a dual role: attenuation of IL-Ιβ secretion and maintenance of a viable host cell for intracellular growth. Moreover, the inventors' results suggest that evolution has developed an innate immune strategy that relies on mt to determine the right time to sacrifice a jeopardized host cell for the sake of initiating a strong inflammatory cascade via IL-Ιβ. Therefore, apoptosis is not always 'silent'; rather, it can be a powerful voice to instruct nearby cells of imminent danger in the presence of NF-KB-activating signal 1.
Additionally, oxidative mitochondrial DNA (mtDNA) is released into cytosol upon apoptosis then which binds to NRLP3 inflammasome and triggers IL-lbeta release. Furthermore, oxidative nucleotides can competitively inhibit the oxidized mtDNA from binding and activating NLRP3, therefore inducing IL-lbeta.
Embodiments of the present invention are based, at least in part, on these findings.
Accordingly, various embodiments of the present invention provide a method of treating inflammation in a subject in need thereof. The method can comprise providing a composition comprising a mitochondrial apoptosis inhibitor and administering the composition to the subject to treat the inflammation. In various embodiments, the mitochondrial apoptosis inhibitor can be selected from the group consisting of Bcl-2, Mcl-1, Bcl-xL, Bcl-w, Bfl-1, nfh, XIAP, Boo/Diva, Nrl3, BH4 domain, Inositol 1,4,5-trisphosphate receptor (IP3R) peptide, GSK-3 , cyclosporine A, viral mitochondrial inhibitor of apoptosis (vMIA) peptide/protein, MitoQ and combinations thereof.
The method can also comprise providing a composition comprising an oxidative nucleotide and administering the composition to the subject to treat the inflammation. In various embodiments, the inflammation is where IL-1 beta plays a role. In various embodiments, the oxidative nucleotide is selected from the group consisting of
(i) 8-Hydroxy-2'-deoxyguranosine (CAS 88847-89-6; also known as 8-OH-dG, 8- OHdG, 8-Oxo-7,8-dihydro-2'-deoxyguanosine, 8-Oxo-7,8-dihydrodeoxyguanosine, 8-Oxo- dG, 8-hydroxydeoxyguanosine, oh8dG),
(ii) 8-hydroxy Guanosine (CAS 3868-31-3; also known as 8-OHG, 8-OH-guanosine, 8-Oxoguanosine, 7,8-dihydro-8-oxo-Guanosine, 8-Oxoguanosine, 8-hydroxyguanosine, Purine-6,8(lH,9H)-dione), (iii) 8-0x0-2 '-deoxyadenosine (CAS 62471-63-0; also known as 2'-Deoxy-7,8- dihydro-8-oxoadenosine, 62471-63-0, 6-amino-9-[(4S,5R)-4-hydroxy-5- (hydroxymethyl)oxolan-2-yl]-7H-purin-8-one, 6-amino-9-[(4S,5R)-4-hydroxy-5- (hydroxymethyl)tetrahydrofuran-2-yl]-7H-purin-8-one, 6-amino-9-[(4S,5R)-4-hydroxy-5- methylol-tetrahydrofuran-2-yl]-7H-purin-8-one, 6-azanyl-9-[(4S,5R)-4-hydroxy-5- (hydroxymethyl)oxolan-2-yl]-7H-purin-8-one, 8-Oxo-2' -deoxyadenosine, 8-Oxo-7,8- dihydro-2' -deoxyadenosine, 8-Oxo-dA, 8-Oxo-dado, Adenosine 2'-deoxy-7,8-dihydro-8- oxo-, C10H13N5O4),
(iv) 5-formyl-2'-deoxycytidine (CAS 137017-45-9; also known as 5-CHO-dC, (v) 5-formyl-2'-deoxyuridine (CAS 4494-26-2; also known as 5-Formyl-2'- deoxyuridine, 5-formyl-20-deoxyuridine, CCRIS 6343, Uridine, 2'-deoxy-5-formyl-, CHEBI:448445, AIDS048624, NSC 148297, AIDS-048624, CID98561, NSC148297, LS- 134709, l,2,3,4-Tetrahydro-l-(2-deoxy-beta-D-ribofuranosyl)-2,4-dioxo-5- pyrimidinecarboxaldehyde, 5-Pyrimidinecarboxaldehyde, l,2,3,4-tetrahydro-l-(2-deoxy- beta-D-ribofuranosyl)-2,4-dioxo- 1 -((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)- tetrahydrofuran-2-yl)-2,4-dioxo-l ,2,3,4-tetrahydropyrimidine-5-carbaldehyde 4494-26-2, 5- Pyrimidinecarboxaldehyde, 1 -(2-deoxy-beta-D-erythro-pentofuranosyl)- 1 ,2,3 ,4-tetrahydro- 2,4-dioxo-),
(vi) 5 -hydroxymethyl-2 '-deoxyuridine (CAS 5116-24-5; also known as a- Hy droxythymidine ; 5 -hy droxymethy 1-2 ' -D ; alpha-hy droxythymidine; 5 - hydroxymethyldeoxyuridine;5 -hydroxymethyl-2 ' -deoxyuridine;2 ' -deoxy-5 - (hy droxymethy l)uridine;2 ' -deoxy-5 -(hydroxymethyl)-uridin;2 ' -Deoxy-5 - hydroxymethyluridine, 98%; 1 -[(2R,4S,5R)-4-hydroxy-5-methylol-tetrahydrofuran-2-yl]-5- methylol-pyrimidine-2,4-quinone;l-[(2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]- 5-(hydroxymethyl)pyrimidine-2,4-dione),
(vii) 5 -Hydroxymethyl-2 '-deoxycytidine (CAS 7226-77-9),
(viii) 5-hydroxy-2'-deoxyuridine (CAS 5168-36-5; also known as 5-hydroxy-2-dU; 5- hydroxy-2 ' -deoxyuridine;Oh-5 -du;Uridine, 2 ' -deoxy-5 -hydroxy-
(ix) 5 -hydroxy-2' -deoxycytidine (also known as 5-hydroxy-dC; 5- Hydroxydeoxycytidine; 5 -Hydroxy deoxycytidine'; 5-OHdC; 5-OH-dC; 5-Hydroxy-2'- deoxycytidine; CHEBI:460515; AIDS088694;AIDS-088694 ;CID477413 ;52278-77-0), and
(x) combinations thereof. Other embodiments of the present invention provide a method of treating an inflammatory disease, an inflammatory disease condition, or an autoimmune disease in a subject in need thereof. The method can comprise providing a composition comprising a mitochondrial apoptosis inhibitor and administering the composition to the subject to treat the inflammatory disease, the inflammatory disease condition or the autoimmune disease.
The method can also comprise providing a composition comprising an oxidative nucleotide and administering the composition to the subject to treat the inflammatory disease, the inflammatory disease condition or the autoimmune disease. In various embodiments, inflammatory disease, the inflammatory disease condition or the autoimmune disease is one where IL-1 beta plays a role. In various embodiments, inflammatory disease, the inflammatory disease condition or the autoimmune disease where IL-1 beta plays a role is selected from the group consisting of type 2 diabetes, rheumatoid arthritis, psoriasis, Alzheimer's disease, silicosis and asbestosis, gout/pseudogout, familial cold autoinflammatory syndrome (FCAS), Muckel- Wells syndrome (MWS), neonatal-onset multisystem inflammatory disease (NOMID), an combinations thereof.
In various embodiments, the inflammatory disease or the inflammatory disease condition can be selected from the group consisting of an autoimmune disease, arthritis, Crohn's disease, inflammatory bowel disease, Alzheimer's disease, diabetes, gout, atherosclerosis, asbestosis/silicosis induced lung fibrosis and combinations thereof. In various embodiments the autoimmune disease can be selected from the group consisting of Hashimoto's thyroiditis, Pernicious anemia, Addison's disease, type I diabetes, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, Sjogren syndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis, reactive arthritis, Grave's disease, celiac disease - sprue (gluten sensitive enteropathy) and combinations thereof.
In various embodiments, the mitochondrial apoptosis inhibitor can be selected from the group consisting of Bcl-2, Mcl-1, Bcl-xL, Bcl-w, Bfl-1, nfh, XIAP, Boo/Diva, Nrl3, BH4 domain, Inositol 1,4,5-trisphosphate receptor (IP3R) peptide, GSK-3 , cyclosporine A, viral mitochondrial inhibitor of apoptosis (vMIA) peptide/protein, MitoQ and combinations thereof.
In various embodiments, the oxidative nucleotide is selected from the group consisting of (i) 8-Hydroxy-2'-deoxyguranosine (CAS 88847-89-6; also known as 8-OH-dG, 8- OHdG, 8-Oxo-7,8-dihydro-2'-deoxyguanosine, 8-Oxo-7,8-dihydrodeoxyguanosine, 8-Oxo- dG, 8-hydroxydeoxyguanosine, oh8dG),
(ii) 8-hydroxy Guanosine (CAS 3868-31-3; also known as 8-OHG, 8-OH-guanosine, 8-Oxoguanosine, 7,8-dihydro-8-oxo-Guanosine, 8-Oxoguanosine, 8-hydroxyguanosine,
Purine-6,8(lH,9H)-dione),
(iii) 8-0x0-2 '-deoxyadenosine (CAS 62471-63-0; also known as 2'-Deoxy-7,8- dihydro-8-oxoadenosine, 62471-63-0, 6-amino-9-[(4S,5R)-4-hydroxy-5- (hydroxymethyl)oxolan-2-yl]-7H-purin-8-one, 6-amino-9-[(4S,5R)-4-hydroxy-5- (hydroxymethyl)tetrahydrofuran-2-yl]-7H-purin-8-one, 6-amino-9-[(4S,5R)-4-hydroxy-5- methylol-tetrahydrofuran-2-yl]-7H-purin-8-one, 6-azanyl-9-[(4S,5R)-4-hydroxy-5- (hydroxymethyl)oxolan-2-yl]-7H-purin-8-one, 8-Oxo-2' -deoxyadenosine, 8-Oxo-7,8- dihydro-2' -deoxyadenosine, 8-Oxo-dA, 8-Oxo-dado, Adenosine 2'-deoxy-7,8-dihydro-8- oxo-, C10H13N5O4),
(iv) 5-formyl-2'-deoxycytidine (CAS 137017-45-9; also known as 5-CHO-dC,
(v) 5-formyl-2'-deoxyuridine (CAS 4494-26-2; also known as 5-Formyl-2'- deoxyuridine, 5-formyl-20-deoxyuridine, CCRIS 6343, Uridine, 2'-deoxy-5-formyl-, CHEBL448445, AIDS048624, NSC 148297, AIDS-048624, CID98561, NSC148297, LS- 134709, l,2,3,4-Tetrahydro-l-(2-deoxy-beta-D-ribofuranosyl)-2,4-dioxo-5- pyrimidinecarboxaldehyde, 5-Pyrimidinecarboxaldehyde, l,2,3,4-tetrahydro-l-(2-deoxy- beta-D-ribofuranosyl)-2,4-dioxo-l-((2R,4S,5R)-4-hydroxy-5-(hydroxyrnethyl)- tetrahydrofuran-2-yl)-2,4-dioxo-l ,2,3,4-tetrahydropyrimidine-5-carbaldehyde 4494-26-2, 5- Pyrimidinecarboxaldehyde, 1 -(2-deoxy-beta-D-erythro-pentofuranosyl)- 1 ,2,3 ,4-tetrahydro- 2,4-dioxo-),
(vi) 5 -hydroxymethyl-2 '-deoxyuridine (CAS 5116-24-5; also known as a-
Hy droxythymidine ; 5 -hy droxymethy 1-2 ' -D ; alpha-hy droxythymidine; 5 - hydroxymethyldeoxyuridine;5 -hydroxymethyl-2 ' -deoxyuridine;2 ' -deoxy-5 - (hy droxymethy l)uridine;2 ' -deoxy-5 -(hydroxymethyl)-uridin;2 ' -Deoxy-5 - hydroxymethyluridine, 98%; 1 -[(2R,4S,5R)-4-hydroxy-5-methylol-tetrahydrofuran-2-yl]-5- methylol-pyrimidine-2,4-quinone; 1 -[(2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]- 5-(hydroxymethyl)pyrimidine-2,4-dione),
(vii) 5 -Hydroxymethyl-2 '-deoxycytidine (CAS 7226-77-9),
(viii) 5-hydroxy-2'-deoxyuridine (CAS 5168-36-5; also known as 5-hydroxy-2-dU; 5- hydroxy-2 ' -deoxyuridine;Oh-5 -du;Uridine, 2 ' -deoxy-5 -hydroxy- (ix) 5-hydroxy-2'-deoxycytidine (also known as 5-hydroxy-dC; 5- Hydroxydeoxycytidine; 5-Hydroxydeoxycytidine'; 5-OHdC; 5-OH-dC; 5-Hydroxy-2'- deoxycytidine; CHEBL460515; AIDS088694; AIDS-088694; CID477413; 52278-77-0), and
(x) combinations thereof.
In various embodiments, the present invention provides pharmaceutical compositions including a pharmaceutically acceptable excipient along with a therapeutically effective amount of the mitochondrial apoptosis inhibitor or the oxidative nucleotide. "Pharmaceutically acceptable excipient" means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients may be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous.
In various embodiments, the pharmaceutical compositions according to the invention may be formulated for delivery via any route of administration. "Route of administration" may refer to any administration pathway known in the art, including but not limited to aerosol, nasal, oral, transmucosal, transdermal or parenteral. "Transdermal" administration may be accomplished using a topical cream or ointment or by means of a transdermal patch. "Parenteral" refers to a route of administration that is generally associated with injection, including intraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal. Via the parenteral route, the compositions may be in the form of solutions or suspensions for infusion or for injection, or as lyophilized powders. Via the enteral route, the pharmaceutical compositions can be in the form of tablets, gel capsules, sugar-coated tablets, syrups, suspensions, solutions, powders, granules, emulsions, microspheres or nanospheres or lipid vesicles or polymer vesicles allowing controlled release. Via the parenteral route, the compositions may be in the form of solutions or suspensions for infusion or for injection. Via the topical route, the pharmaceutical compositions based on compounds according to the invention may be formulated for treating the skin and mucous membranes and are in the form of ointments, creams, milks, salves, powders, impregnated pads, solutions, gels, sprays, lotions or suspensions. They can also be in the form of microspheres or nanospheres or lipid vesicles or polymer vesicles or polymer patches and hydrogels allowing controlled release. These topical-route compositions can be either in anhydrous form or in aqueous form depending on the clinical indication. Via the ocular route, they may be in the form of eye drops.
The pharmaceutical compositions according to the invention can also contain any pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" as used herein refers to a pharmaceutically acceptable material, composition, or vehicle that is involved in carrying or transporting a compound of interest from one tissue, organ, or portion of the body to another tissue, organ, or portion of the body. For example, the carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or a combination thereof. Each component of the carrier must be "pharmaceutically acceptable" in that it must be compatible with the other ingredients of the formulation. It must also be suitable for use in contact with any tissues or organs with which it may come in contact, meaning that it must not carry a risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that excessively outweighs its therapeutic benefits.
The pharmaceutical compositions according to the invention can also be encapsulated, tableted or prepared in an emulsion or syrup for oral administration. Pharmaceutically acceptable solid or liquid carriers may be added to enhance or stabilize the composition, or to facilitate preparation of the composition. Liquid carriers include syrup, peanut oil, olive oil, glycerin, saline, alcohols and water. Solid carriers include starch, lactose, calcium sulfate, dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, acacia, agar or gelatin. The carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax.
The pharmaceutical preparations are made following the conventional techniques of pharmacy involving milling, mixing, granulation, and compressing, when necessary, for tablet forms; or milling, mixing and filling for hard gelatin capsule forms. When a liquid carrier is used, the preparation will be in the form of a syrup, elixir, emulsion or an aqueous or non-aqueous suspension. Such a liquid formulation may be administered directly p.o. or filled into a soft gelatin capsule.
The pharmaceutical compositions according to the invention may be delivered in a therapeutically effective amount. The precise therapeutically effective amount is that amount of the composition that will yield the most effective results in terms of efficacy of treatment in a given subject. This amount will vary depending upon a variety of factors, including but not limited to the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication), the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and the route of administration. One skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount through routine experimentation, for instance, by monitoring a subject's response to administration of a compound and adjusting the dosage accordingly. For additional guidance, see Remington: The Science and Practice of Pharmacy (Gennaro ed. 20th edition, Williams & Wilkins PA, USA) (2000).
Typical dosages of an effective the mitochondrial apoptosis inhibitor can be in the ranges recommended by the manufacturer where known therapeutic compounds are used, and also as indicated to the skilled artisan by the in vitro responses or responses in animal models. Such dosages typically can be reduced by up to about one order of magnitude in concentration or amount without losing the relevant biological activity. Thus, the actual dosage will depend upon the judgment of the physician, the condition of the patient, and the effectiveness of the therapeutic method based, for example, on the in vitro responsiveness of the relevant primary cultured cells or histocultured tissue sample, such as biopsied malignant tumors, or the responses observed in the appropriate animal models, as previously described.
The present invention is also directed to a kit to treat inflammation, inflammatory disease and/or inflammatory disease condition. The kit is useful for practicing the inventive method of treating inflammation, inflammatory disease and inflammatory disease condition. The kit is an assemblage of materials or components, including at least one of the inventive compositions. Thus, in some embodiments the kit contains a composition including a mitochondrial apoptosis inhibitor as described above. In other embodiments, the kit contains a composition including an oxidative nucleotide as described above.
The exact nature of the components configured in the inventive kit depends on its intended purpose. For example, some embodiments are configured for the purpose of treating inflammation; other embodiments are configured for the purpose of treating inflammatory diseases; other embodiments are configured for the purpose of treating inflammatory disease conditions; and other embodiments are configured for the purpose of treating autoimmune diseases. In one embodiment, the kit is configured particularly for the purpose of treating mammalian subjects. In another embodiment, the kit is configured particularly for the purpose of treating human subjects. In further embodiments, the kit is configured for veterinary applications, treating subjects such as, but not limited to, farm animals, domestic animals, and laboratory animals. Instructions for use may be included in the kit. "Instructions for use" typically include a tangible expression describing the technique to be employed in using the components of the kit to effect a desired outcome, such as to treat inflammation, inflammatory disease and/or inflammatory disease condition. Optionally, the kit also contains other useful components, such as, diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring tools, bandaging materials or other useful paraphernalia as will be readily recognized by those of skill in the art.
The materials or components assembled in the kit can be provided to the practitioner stored in any convenient and suitable ways that preserve their operability and utility. For example the components can be in dissolved, dehydrated, or lyophilized form; they can be provided at room, refrigerated or frozen temperatures. The components are typically contained in suitable packaging material(s). As employed herein, the phrase "packaging material" refers to one or more physical structures used to house the contents of the kit, such as inventive compositions and the like. The packaging material is constructed by well-known methods, preferably to provide a sterile, contaminant- free environment. The packaging materials employed in the kit are those customarily utilized in treating inflammation, inflammatory disease and/or inflammatory disease condition. As used herein, the term "package" refers to a suitable solid matrix or material such as glass, plastic, paper, foil, and the like, capable of holding the individual kit components. Thus, for example, a package can be a glass vial used to contain suitable quantities of an inventive composition containing a mitochondrial apoptosis inhibitor. The packaging material generally has an external label which indicates the contents and/or purpose of the kit and/or its components.
One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described.
EXAMPLES
The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention. Example 1
Mice
Caspl"7" mice (Kuida et al., 1995) were kindly provided by Dr. Richard Flavell (Yale Univ, New Haven, CT). NLRP3 ~ ~ mice and ASC ~ ~ mice (Mariathasan et al., 2006), and Aim2_/_ mice were kindly provided by Dr. Katherine Fitzgerald (University of Massachusetts Medical School, Worcester, MA). C57BL/6 mice, TRIF ~ ~ mice and Ppif ( ypD) were obtained from Jackson Labs. MyD88_/", RIP2_/", TLR2_/", TLR4_/", and TLR9_/" (Naiki et al, 2005; Shimada et al., 2009) mice were maintained according to Cedars-Sinai Medical Center Institutional Animal Care and Use Committee guidelines. All mice were used at 8-12 weeks of age.
Example 2
Infection and Bacterial Quantification
BALF and lung homogenates from C. pneumoniae (CM-1, ATCC, Manassa, VA) infected mice were propagated in HEp2 cells and counted as previously described (Shimada et al, 2009).
Example 3
Histopathological analysis
Lungs were fixed in formalin buffer, paraffin-embedded, and hematoxylin and eosin
(H&E)-stained sections were scored by a trained pathologist blinded to the genotypes as previously described (Shimada et al, 2009).
Example 4
Flow cytometric analysis
Isolated single cells were stained with anti-F4/80 mAb (clone BM8), anti-CDl lc mAb (clone HL3). For intracellular iNOS staining, cells were permeabilized using Cytofix/Cytoperm kit (BD Biosciences) and stained with -conjugated anti-mouse iNOS mAb (clone 6/iNOS/NOS Type II, BD Bioscienses). Flow cytometric analysis was performed by CyAn™ Flow cytometer (Beckman Coulter) and the data was analyzed by Summit (Dako, Carpinteria, CA, USA). Example 5
Measurement of mitochondrial membrane potential (Aym) Cells were stained with the cationic dye TMRM (AnaSpec, Fremont, CA, USA) as described in the manufacturer's protocol. Cells were loaded with 200 nm TMRM for 30 min, washed three times with PBS and fluorescence was measured using a SpectraMaX M2 Microplate Reader (Molecular Devices Corp., Sunnyvale, CA, USA) or by fluorescence microscopy (Nikon Eclipse T2000).
Example 6
Measurement of ROS production
Cells were incubated in phenol red- free RPMI1640 medium containing 10 μΜ 6- carboxy-2', 7 '-dichlorodihydro fluorescein diacetate (Molecular Probes, Eugene, OR) for 30 min and then infected with CP. The loading buffer was removed, washed and fluorescent intensity was measured using a microplate reader.
Example 7
LDH release assay
LDH release was assessed in cell-free medium at indicated times following the manufacturer's instructions (Cytotoxicity Detection Kit, Roche Diagnostics). The data are expressed as percentage of maximum LDH release for the particular treatment at each time point.
Example 8
Immunoblot
BMDM were stimulated for indicated time, supernatants were collected and proteins were precipitated by methanol-chloroform extraction, and cell lysates were collected. Immunoblot analysis was done with described antibodies; anti mouse caspase-1 plO (sc-514; Santa Cruz Biotechnology), anti-mouse IL-Ιβ (AF-401-NA; R&D Systems), anti-GAPDH (6C5; Santa Cruz Biotechnology).
Example 9
Reagents
Recombinant IL-1 receptor antagonist (IL-IRA) (Kineret, Amgen), N-Acetyl-L- leucyl-L-leucyl-L-methional (Tocris bioscience, Ellisville, MO), adenosine 5 "-triphosphate, chloramphenicol, cytochalasin D, staurosporine, cyclosporine A, antimycin A and N-Acetyl- L-cysteine, nigericin, Alum, 2'-deoxyguanocine (Sigma, St. Louis, MO), LPS from E. coli (InvivoGen, San Diego, CA), 8-Hydroxy-2'-deoxyguanosine, 7-Bromoindirubin-3'- monoxime (Enzo life sciences, Plymouth Meeting, PA) and 8-OHdG quantitation kit (Cell Biolabs, Inc., San Diego, CA) were purchased commercially. NLRP3 cDNA was kindly provided by Dr. Eicke Latz (University of Massachusetts Medical School, Worcester, MA). Salmonella typhimurium (IR715, ΔίηνΑ, AhilA, AorgA, AspiB, AprgH, AmsbB, AfljB/fliC, AsspB) was kindly provided by Dr. Andreas Baumler (University of California in Davis, Davis, CA). Chlamydia pneumoniae (CM-1, ATCC, Manassa, VA) was propagated in Hep2 cells.
Example 10
Detection of cytokines
The cytokine concentrations in the BALF, lung homogenates or culture supernatant were determined using by OptiEIA Mouse IL-6 ELISA Set (BD Biosciences, San Jose, CA, USA) and Mouse ΙΚΝγ ELISA, Mouse IL-12p40 ELISA, Mouse IL-Ιβ ELISA and Mouse TNFa ELISA (eBioscience, San Diego, CA). The assays were performed as described in manufacturers' protocols. Example 11
Tissue damage scoring
Tissue damage was assigned as arbitrary score of 0 (normal = no inflammation), 1 (minimal = perivascular, peribronchial, or patchy interstitial inflammation involving less than 10% of lung volume), 2 (mild = perivascular, peribronchial, or patchy interstitial inflammation involving 10-20% of lung volume), 3 (moderate = perivascular, peribronchial, patchy interstitial, or diffuse inflammation involving 20-50% of lung volume), and 4 (severe = diffuse inflammation involving more than 50% of lung volume).
Example 12
Flow cytometric analysis
The lymphocytic makeup in the lungs after infection was analyzed by flow cytometry of lung homogenates. Briefly, lymphocytes were isolated by digesting the lung tissue at 37°C for lh with HANKS' containing 100 μg/ml Blenzyme (Roche Diagnostics, Indianapolis, IN, USA) and 50 units/ml DNase I (Roche Diagnostics) and filtering through a 70 μηι cell strainer (BD Biosciences). Erythrocytes were depleted by lysis buffer before staining. Isolated single cells were stained with following specific mAbs: CD 16/32 (clone 93), Ly6G (clone 1A8), CDl lb (clone Ml/70), F4/80 (clone BM8), CDl lc (clone HL3), CD45 (clone 30-F11), CD4 (clone RM4-5), CD8 (clone 53-6.7), NK1.1 (clone PK136) and B220 (clone RA3-6B2) were purchased from eBioscience as direct conjugates to FITC, PE or PECy5. Cells were identified based on expression of following antigens: pulmonary macrophages (F4/80+ and CD1 lc+), DC (F4/80- and CD1 lc+), Neutrophils (Ly6G+ and CD1 lb+), T cells (CD3+), NK cells (NK1.1+), B cells (B220+ and CD19+). For intracellular Chlamydophila staining cells were permeabilized using Cytofix/Cytoperm kit (BD Biosciences) and stained with FITC-conjugated anti-Chlamydia LPS mAb (Accurate Chemical and Scientific Corporation, Westbury, NY, USA). Flow cytometric analysis was performed by CyAn™ Flow cytometer (Beckman Coulter) and the data was analyzed by Summit (Dako, Carpinteria, CA, USA).
Example 13
Preparation of bone marrow-derived macrophages (BMDM) and infection
Femora and tibiae of mice were rinsed with cell culture medium (RPMI 1640 media). Bone marrow cells were cultured in RPMI 1640 medium containing 10% FBS and 15% L929 cell conditioned medium. BMDM were harvested at day 7 and infected with C. pneumoniae by centrifugation at 500x g for 30 min. BMDM were infected with S. typhimurium (MOI=5) and spun at 1800 rpm, RT for 5 min, then incubated for 30 min in the absence of antibiotics. Cells were then washed with PBS to remove non-phagocytosed bacteria, replaced in 100 g/ml gentamicin-containing media and incubated for additional 7.5 h.
Example 14
Caspase-1 and IL-Ιβ are critical in host innate immune responses against pulmonary CP infection
To determine the role of the Caspase-1 (Caspl) in host defense against CP, Caspl"7" mice were infected intratracheally with CP (2xl06, 1.5xl06 or lxlO6 IFU). Caspl deficiency resulted in greater mortality at 1.5xl06 IFU, compared to WT mice (Figure 1A). Next, the bronchoalveolar lavage fluid (BALF) and lung homogenates from Caspl"7" mice exhibited significantly higher leukocyte recruitment, particularly macrophages and lymphocytes, at days 5 and 12 post infection compared to WT mice (Figure IB). Histological examination of Caspl"7" lungs 12 days after infection mice revealed higher inflammation and tissue damage scores compared to WT lungs (Figure IF).
Given that innate immune defenses limit CP replication and colonization and that Caspl is required for processing of pro-IL-Ιβ (Li et al, 1995), but while not wishing to be bound by any particular theory, the inventors believe that cytokine production would differ in Caspl"7" and WT mice. IL-Ιβ was undetectable in the BALF (at all time points) and in homogenates (days 5 and 12) in Caspl"7" mice (Figure 1C). Of note, some IL-Ιβ was detected by ELISA on day 1 in Caspl"7" lungs. This is most likely pro-IL-Ιβ released as a result of tissue homogenizing.
At days 1 and 3 post-infection, it was observed that a significant delay in IFN-γ and IL-6 production in CP infected Caspl"7" mice compared to WT animals early, but these cytokines were significantly elevated on days 5 and 12 (Figure 1C). Consistent with increased mortality, impaired IL-Ι β secretion, and delayed cytokine production, Caspl"7" mice displayed a greater bacterial burden on days 5 and 12 (Figure ID). Notably, CP infected Caspl"7" and WT mice exhibited no significant differences in IL-12p40 secretion at any time point examined (Figure 1C). Thus, the inventors conclude that Caspl plays a key role in initiation of early inflammatory responses that lead to bacterial clearance and survival from infection.
To verify that Caspl activation exerts its protective role via IL-Ιβ secretion, wild-type mice were injected with either IL-1 receptor antagonist (IL-IRA) or a control vehicle. Treatment of WT mice with IL-IRA on days -1, 0 and 1 relative to CP infection caused a dramatic increase in mortality (Figure 2A) and in bacterial load (Figure 2B) in the lung (at day 5, p < 0.001 compared to vehicle control), much akin to what is observed in infected Caspl"7" mice. Timing was crucial, as early treatment (days -1 to 1 or days -1 to 4) with IL- IRA, but not later treatment (days 2 to 4) resulted in impaired bacterial clearance at day 5 (Figure 2B).
To further verify the role of IL-1 β signaling in host defense against CP infection, a complementary experiment was performed in which CP infected Caspl"7" mice were injected with either recombinant IL-Ιβ (rIL-Ιβ) or a control vehicle. Early rIL-Ιβ treatment during infection (i.e. days 0, 1, and 2 post-infection) rescued Caspl"7" mice, restoring survival (Figure 2C) and reducing lung bacterial load (Figure 2D). But mice treated later (days 2, 3 and 4 post-infection) showed significantly increased bacterial counts (p < 0.05) relative to early-treated mice (Figure 2D), indicating that IL-Ιβ secretion is critical for initial host immune responses that limit bacterial proliferation in the lung. The inventors next sought to determine the predominant cell type infected by CP in the lungs. Since CP is an obligate intracellular pathogen, the cell types infected by CP in Caspl"7" mice compared to wild-type mice (Figure 8) were analyzed by flow cytometry. CP was found predominantly in alveolar macrophages (AM) and, to a lesser degree, in neutrophils and dendritic cells (DC) on day 12 (Figure 8). There were relatively more infected Caspl"7" AM than their wild-type counterparts, revealing that AM are likely a reservoir of bacterial replication after pulmonary infection in Caspl"7" mice.
The inventors were next interested in determining if AM isolated from CP infected Caspl"7" mice exhibited an immune defect compared to those in infected WT mice. Ex vivo Caspl"7" AM did not induce iNOS following CP infection compared to wt AM (Figure IE). Indeed, nitric oxide (NO) produced by macrophages after cell activation by IFN-γ hampers the growth of CP (Nathan and Hibbs, 1991; Rottenberg et al., 1999). The inventors' findings are consistent with the delayed IFN-γ production observed in Caspl"7" mice (Figure 1C), supporting a model whereby IL-Ιβ secretion by AM induces the early IFN-γ that, in turn, activates AM at the infection site.
Example 15
Macrophage TLR2/MyD88 and NLRP3/ASC/Caspase-l are required for
IL-Ιβ secretion in response to C pneumoniae
As AM are the principal CP-harboring cell in vivo, the inventors sought to elucidate the mechanism of CP induced IL-Ιβ production and secretion by macrophages in vitro. To determine which signaling pathway plays a role in CP induced pro-IL-Ιβ production, MyD88-, TRIF- or RIP2-deficient BMDM were infected with CP for 24 hours and then assayed IL-Ιβ secretion. MyD88, but neither TRIF nor Rip2 signaling, was required for IL- 1β secretion in response to CP (Figure 3 A). Further investigation revealed that pathogen sensing by TLR2 is of central importance in induction of IL-Ιβ secretion during CP infection (Figure 3B). TLR2/MyD88 was also required for CP induced TNFa production in BMDM (Figure 9).
8 hours post-infection and, more so, at 24 hours post-infection, cultured bone marrow derived macrophages (BMDM) infected with CP secreted IL-Ιβ in a Caspl -dependent manner (Figure 3C and 3E). On the contrary, neither 8 hour nor 24 hour treatment of BMDM with UV-killed CP (UVCP) stimulated IL-Ιβ secretion (Figure 3C and 3E), indicating that active CP infection is required to induce IL-Ιβ secretion. Bacterial replication in Caspl"7" BMDM was normal, and Caspl" BMDM demonstrated wild-type phagocytic capability and
TNFa production (Figure 10).
Though UVCP was unable to induce IL-Ιβ secretion by BMDM, while not wishing to be bound by any particular theory, the inventors believe that UVCP induces pro-IL-Ιβ. To test this, BMDM were treated with UVCP (for either 8 or 24 hours) and then exposed to a high extracellular concentration of ATP (5 mM), a stimulus known to activate Caspl via the
NLRP3 inflammasome. Interestingly, BMDM treated with UVCP for 6 hours and then exposed to ATP for 2 additional hours secreted IL-Ιβ (Figure 3E). ATP treatment also increased IL-Ιβ secretion in BMDM after a 6 hour (but not 24 hour) infection with live CP (Figure 3C and 3E). But BMDM treated with UVCP for 24 hours and then given ATP did not secrete IL-Ιβ (Figure 3C), suggesting that pro-IL-Ιβ is degraded if Caspl is not activated within a finite time window.
Since the NLRP3 inflammasome activates Caspl in response to a wide array of stimuli, the inventors predicted that CP also induces IL-Ιβ secretion via NLRP3. NLRP3 ~ ~ or ASC/_ BMDM infected with live CP were found to secrete dramatically reduced levels of IL-
1β (Figure 3G). Nevertheless, these BMDM retained the ability to secrete TNFa at WT levels
(Figure 3H). This indicates that CP infection activates the NLRP3/ASC inflammasome in macrophages to induce IL-Ιβ secretion.
Example 16
Mitochondrial dysfunction is involved in IL-Ιβ secretion
To further understand the mechanism by which live CP activates the NLRP3 inflammasome in macrophages, it was tested whether CP cell entry is required for IL-Ιβ secretion. In the presence of cytochalasin D, an inhibitor of actin polymerization and phagocytosis, IL-Ιβ secretion (but not TNFa secretion) by BMDM in response to CP was significantly reduced in a dose dependent manner suggesting that NLRP3 activation requires bacterial uptake by the host cell (Figure 4A).
Next, whether phagosomal cathepsin B activity plays a role in IL-Ιβ secretion in response to CP infection was examined. An inhibitor of Cathepsins B and L, N-Acetyl-Leu- Leu-Met-al (Ac-LLM), did not alter IL-Ιβ secretion induced by CP, but did reduce Alum induced IL-Ιβ secretion from LPS-primed BMDM (Figure 4B). Therefore, Cathepsin B activity likely does not play a significant role in CP-induced inflammasome activation. To test whether ROS play a role in inflammasome activation during CP infection, CP infected BMDM was treated with the antioxidant N-acetyl-L-cysteine (NAC). Although it was observed that NAC attenuates CP induced IL-Ιβ secretion, it was also found that this antioxidant reduces TNFa production (Figure 4C). A concomitant reduction of both IL-Ιβ and TNFa secretion was also observed when LPS-primed BMDM were pre-treated with NAC before ATP addition (Figure 4D). Indeed, others have also observed NF-κΒ inhibition by antioxidants (Fox and Leingang, 1998); therefore, the inventors believe this non-specific effect precludes using NAC to reach any meaningful conclusions about ROS in CP induced NLRP3 activation.
In an effort to clarify further the role of ROS in CP induced IL-Ιβ production, the amount of ROS produced during live CP infection and during treatment with UVCP was measured. Remarkably, UVCP displayed an ROS generation profile nearly identical to that of live CP in macrophages (Figure 4E). Since live CP but not UVCP treatment induces IL-Ιβ secretion, it was concluded that ROS generated during CP infection are not required for NLRP3 inflammasome activation.
To shed more light on exactly how CP activates NLRP3, the determination of the role of bacterial protein synthesis in CP induced IL-Ιβ secretion was made. Treatment of CP infected BMDM with the antimicrobial chloramphenicol, an inhibitor of the bacterial ribosome, led to nearly complete inhibition of IL-Ιβ secretion, without affecting TNFa production (Figure 4F). This result indicates that de novo protein synthesis by CP is necessary for Caspl activation in BMDM. As a control, LPS-primed BMDM was treated with chloramphenicol before addition of ATP. This led to an unexpected attenuation of IL-Ιβ secretion, albeit with a lower sensitivity than compared to IL-Ιβ inhibition in CP infected cells (Figure 5 A). Further, in these BMDM it was found that TNFa production increased reciprocally in proportion to IL-Ιβ inhibition (Figure 5 A). To rule out the possibility that chloramphenicol might be toxic to BMDM, LDH release in BMDM treated with chloramphenicol was examined. It was found that chloramphenicol caused a dose-dependent reduction of LDH release (Figure 5A), indicating that the observed increase in TNFa production is indeed related to the increase in BMDM survival.
Considering the high level of homology between the bacterial ribosome and the mitochondrial ribosome, and in light of previous studies showing that chloramphenicol can inhibit mitochondrial ribosomal protein synthesis (Riesbeck et al., 1990), the inventors predicted that chloramphenicol's effect on CP-induced IL-Ιβ secretion might in part be due to attenuation of a signal emanating from the mitochondria.
Since mitochondria are highly sensitive regulators of cellular physiology (McBride et al., 2006), the inventors predicted that a signal emanating from the mitochondria activates the NLRP3 inflammasome. To address this, CP infected BMDM was treated with cyclosporin A (CsA), an inhibitor of the mitochondrial permeability transition pore (PTP). CsA inhibited IL- 1β secretion and increased TNFa production in a dose-dependent manner (Figure 5B). Similar attenuation of IL-Ιβ secretion by CsA was observed in BMDM treated with LPS and ATP (Figure 5B).
Next, the effect of CP infection on inner mitochondrial membrane potential (ΔΨιη) was determined. Infection with live CP, but not UVCP treatment, resulted in a significant decrease in ΔΨηι (Figure 5C) as measured by a tetramethyl rhodamine methyl ester (TMRM) incorporation assay, further implicating the mitochondria in NLRP3 inflammasome activation. Examining the kinetics of ΔΨηι reduction (Figure 5D), the inventors found that CP infection, ATP, and staurosporine (STS) led to a rapid, irreversible decrease in the ΔΨηι (Figure 5D). Interestingly, incubation of BMDM with nigericin or alum (both induce the NLRP3 inflammasome) also led to reduced ΔΨηι (Figure 5D). By TMRM fluorescence microscopy, it was observed that ATP treatment of LPS-primed BMDM resulted in a rapid, irreversible decrease in ΔΨηι (Figure 5G). Live imaging analysis of mitochondrial depolarizationin response to exogenous ATP was also performed. Extracellular ATP causes rapid, irreversible loss of ΔΨιη. BMDM were stimulated with LPS (1 μg/ml, 3 h) and then loaded with 200 nMTMRM in Ringer's solution for 30 min. Following probe loading, the live cell kinetics of ΔΨιη were monitored every 20 seconds thereafter. ATP (5 mM) was added 120 seconds after monitoring (data not shown). Of note, the kinetic differences observed between the incorporation assay (Figure 6e) and TMRM fluorescence microscopy (Figure 5G and Figure 18) may be due to the higher sensitivity of live cell fluorescence microscopy. Since live CP, ATP, and alum (but not UVCP) provide a signal required for Caspl activation by NLRP3, these results strongly associate mitochondrial dysfunction with NLRP3 inflammasome activation.
During the course of investigating inflammasome activation after mouse CP infection, the inventors found that infection-induced IL-Ιβ secretion in macrophages required live bacteria and was dependent on NLRP3, ASC, and caspase-1 (Figure 12a). On the other hand, tumor necrosis factor alpha (TNFa) secretion was NLRP3 inflammasome -independent (Figure 16). While investigating the effects of chloramphenicol on CP infection-induced activation of the NLRP3 inflammasome, the inventors discovered that TNFa production was enhanced in high dose chloramphenicol-treated bone marrow-derived macrophages (BMDM; data not shown). Intrigued by this result, lipopolysaccharide (LPS)-primed BMDM was treated with chloramphenicol before addition of ATP. This led to an unexpected attenuation of IL-Ιβ secretionlb. Further, the inventors found that TNFa production increased reciprocally in proportion to IL-Ιβ inhibition in these BMDM (Figure 12b). To rule out the possibility that chloramphenicol might be cytotoxic in this scenario, the inventors examined LDH release in BMDM treated with the compound. The inventors found that chloramphenicol caused a dose-dependent reduction in LDH release (Figure 12b), suggesting that reduced IL-Ιβ production is proportional to BMDM survival.
Considering the high degree of homology between bacterial and mitochondrial (mt) ribosomes, and previous studies showing that chloramphenicol inhibits mt ribosomal protein synthesis 24 , but while not wishing to be bound by any particular theory, the inventors believe that the effect of the compound on CP-induced IL-Ιβ secretion might be due to attenuation of a signal emanating from mt. Since mt are highly sensitive regulators of intracellular physiology 25 , and while not wishing to be bound by any particular theory, the inventors also believe that this mt signal might activate the NLRP3 inflammasome. To test this, the inventors treated BMDM with LPS plus ATP in presence of cyclosporin A (CsA), a mt inhibitor that blocks cytochrome c release 26. CsA attenuated IL-Ιβ secretion but had no effect on TNFa production (Figure 2c). It has been suggested that CsA acts by blocking the permeability transition pore (PTP) 26. However, the inventors used CypD~'~ BMDM as a control in the CsA inhibition assay, and found that CsA was still able to inhibit IL-Ιβ secretion in these cells (Figure 12c). Thus, CsA inhibition of IL-Ιβ secretion is apparently independent of the PTP.
The inventors next determined the effect of CP infection on inner mt membrane potential ( Ψ^, loss of which is typically used as a surrogate marker for apoptosis 27. Live CP infection, ATP, and staurosporine (STS, a pro-apoptotic compound), but not ultra-violet light-killed CP (UVCP) treatment, resulted in decreased ΑΨΏ1 (Figure 12d) as measured by tetramethyl rhodamine methyl ester (TMRM) incorporation assay, further implicating mt in NLRP3 inflammasome activation. CP infection, ATP, STS, alum, and nigericin (NIG, another pro-apoptotic compound) all led to an irreversible decrease in ΔΨιη (Figure 12e). Interestingly, incubation of BMDM with NIG also reduced ΑΨ∞ (Figure 12e). By TMRM fluorescence microscopy, ATP treatment of LPS-primed BMDM resulted in a similar effect on ΔΨιη (Figure 12f). Of note, the kinetic differences observed between the incorporation assay (Figure 12e) and TMRM (Figure 12f) may be due to greater inherent sensitivity of TMRM fluorescence microscopy. The addition of ATP to macrophages resulted in a permanent decrease in Ψ^ and this decrease was not dependent on the presence of NLRP3 (Figure 12g). Because ATP and CP (but not UVCP) all caused reduced ΑΨ^ and since these stimuli both trigger caspase-1 activation by NLRP3, the inventors suggest that mt dysfunction is implicated in NLRP3 inflammasome activation.
Low intracellular K+ concentration has been linked to caspase-1 activation by the NLRP3 inflammasome 14' 28. The evidence for this conclusion emanates from studies showing that cells exposed to a high concentration (> 90 mM) of K+ or treated with pharmacological inhibitors of K+ efflux exhibit attenuated NLRP3 activation in response to a variety of NLRP3 stimuli. To determine if K+ efflux played a role in CP-induced NLRP3 activation, BMDM was infected with CP and then exposed cells to 100 mM of extracellular K+. Results showed that elevated extracellular K+ concentration almost completely blocked IL-Ιβ secretion by CP-infected BMDM (Figure 12h). Fascinatingly, the inventors also found that high extracellular K+ protected BMDM from ΔΨηι reduction (Figure 12i). To further characterize mt dysfunction induced by ATP or other NLRP3 inflammasome stimuli, the inventors analyzed the rate of oxygen consumption under these conditions. The addition of ATP, STS, or NIG to macrophages all resulted in reduced oxygen consumption and hence mt dysfunction (Figures 12j-l). Together, these results further link mt with NLRP3 activation, and also shed light on the mechanism by which cytosolic K+ efflux triggers NLRP3.
Example 17
Mitochondrial apoptotic signals activate the NLRP3 inflammasome
In order to establish a niche, intracellular pathogens subvert, activate or otherwise manipulate host cell death signaling pathways to further their own survival and proliferation (Arnoult et al., 2009). Since the mitochondria are known to play a crucial role in the initiation of cell death, and because these data have shown that mitochondrial dysfunction correlates with inflammasome activation, the role of mitochondrial cell death pathways in activating the NLRP3 inflammasome was examined next. It was found that live CP, but not UVCP, caused LDH release from infected BMDM, although at a slower rate compared to ATP, STS or Alum (Figure 6A). To analyze which cell death pathway is induced by live CP infection, the use of Annexin V staining, a technique which detects phosphatidylserine (PS) externalized during apoptotic membrane flipping, was first attempted. But this method was not suitable for use in CP infected cells, as CP infection causes transient externalization of PS in the absence of apoptosis (Goth and Stephens, 2001).
As an alternative, host cell nuclei were stained with DAPI and cells were scored for nuclear condensation. The nuclei of apoptotic cells, unlike those of necrotic cells, exhibit highly condensed chromatin that uniformly stains with DAPI. Increased apoptosis in CP infected macrophages but not UVCP treated cells (Figure 6B) was observed, consistent with LDH release assays (Figure 6A). Thus, apoptosis induced by CP is linked to NLRP3 inflammasome activation. Intriguingly, the pro-apoptotic molecule STS not only leads to loss of ΔΨηι (Figure 5D), LDH release (Figure 6A) and nuclear condensation (Figure 6B) but also to Caspl activation and subsequent IL-Ι β maturation in LPS-primed BMDM (Figure 6C). Addition of STS at 6 hr after UVCP or LPS treatment induced IL-Ι β secretion and treatment with STS after 6 hrs of CP infection enhanced IL-Ιβ secretion (Figure 6D). Indeed, IL-Ιβ secretion in response to STS depends on NLRP3/ASC/Caspl (Figure 6E). But STS treatment did not affect TNFa production (Figure 6D). Collectively, these results indicate that an apoptotic stimulus suffices to activate Caspl and induce IL-Ιβ secretion via the NLRP3 inflammasome.
To investigate the specific role of apoptosis in NLRP3 activation, Antimycin A (antA), a compound that binds to mitochondrial complex III, inhibits mitochondrial respiration, prevents cytochrome c release (Rieske et al, 1967), and blocks apoptosis (Dairaku et al, 2004) was used. UVCP and LPS-primed BMDM treated with antA displayed a dose-dependent attenuation of IL-Ιβ secretion in response to ATP or STS (Figure 6G). Moreover, antA treatment also inhibited IL-Ιβ secretion (without affecting TNFa secretion) in response to live CP infection (Figure 6G). These results reveal that suppression of apoptotic signals specifically leads to a corresponding suppression of IL-Ιβ secretion. Though antA itself causes mitochondrial dysfunction, LPS-primed BMDM treated with antA did not release IL-Ιβ (data not shown). This indicates that only those mitochondrial perturbations that lead to apoptosis induce IL-Ιβ secretion via the NLRP3 inflammasome.
To further substantiate the observation that apoptotic processes act as the second signal for NLRP3 activation, the timing of NLRP3 activation with respect to LPS, UVCP and CP was examined. If STS or ATP was added before or simultaneously with UVCP or LPS, IL-Ιβ secretion was undetectable (Figure 6F). Likewise, when STS or ATP was added concomitantly with CP infection, IL-Ιβ secretion was drastically reduced (Figure 6F). If instead STS or ATP was added 6 hr after LPS or UVCP priming, IL-Ιβ secretion was robust (Figure 6F). Using immunohistochemistry, the inventors also found that the secretion of IL- 1β (indicated by a disappearance of cytosolic IL-Ιβ staining) in response to CP or LPS+ATP is Caspl dependent and is more frequent in apoptotic cells (Figure 6H). These results demonstrate that activation of the NLRP3 inflammasome by a putative apoptotic stimulus must occur after, but not before or concomitant with, priming.
Recent studies have described a Caspl -dependent form of programmed cell death called pyroptosis (Fink and Cookson, 2005). To rule out pyroptosis as the source of the observed mitochondrial dysfunction and cell death, ΔΨηι reduction and LDH release in response to CP infection in NLRP3" ", ASC_/", and Caspl"7" BMDM were assessed. It was found that CP induced mitochondrial depolarization and LDH release independently of Caspl, NLRP3 or ASC (Figure 1).
To further their own survival, intracellular pathogens subvert, activate or otherwise manipulate host cell death signaling pathways 29. Since mt play a crucial role as initiators of cell death, and because the data thus far showed that mt dysfunction correlates with inflammasome activation, the inventors next examined the putative role of mt cell death pathways in activating the NLRP3 inflammasome. The inventors found that live CP, but not UVCP, caused LDH release from infected BMDM, albeit with slower kinetics vs. ATP, STS or alum (Figure 6a). To analyze which cell death pathway was induced by live CP infection, the inventors first attempted to use Annexin V staining, a technique which detects phosphatidylserine (PS) externalized during apoptotic membrane flipping. While this method is not suitable for use in CP infected cells because CP infection causes transient externalization of PS in the absence of apoptosis 30, the inventors found that LPS+ATP, or LPS+NIG induced apoptosis in BMDM as measured by Annexin V flow cytometry (Figure 17). As an alternative approach, the inventors stained host cell nuclei with DAPI and scored cells for nuclear condensation. The nuclei of apoptotic cells, unlike those of necrotic cells, exhibit highly condensed chromatin that uniformly stains with DAPI. Consistent with data from LDH release assays (Figure 6a), the inventors observed increased apoptosis in CP infected macrophages but not in UVCP treated cells (Figure 6b). Thus, apoptosis induced by CP is linked to NLRP3 inflammasome activation. Intriguingly, the pro-apoptotic molecule STS not only leads to loss of ΑΨΏ1 (Figure 12d), LDH release (Figure 6a) and nuclear condensation (Figure 6b) but also to caspase-1 activation and subsequent IL-Ιβ maturation in LPS-primed BMDM (Figure 6c). Addition of STS at 6 h after UVCP or LPS treatment induced IL-Ιβ secretion and treatment with STS after 6 h of CP infection enhanced IL-Ιβ secretion (Figure 6d). Indeed, IL-Ιβ secretion in response to STS depends on NLRP3/ASC/caspase-l (Figure 6e). As an important off-pathway control, STS treatment did not affect TNFa production (Figure 6d). Collectively, these results indicate that an apoptotic stimulus suffices to activate caspase-1 and induce IL-Ιβ secretion via the NLRP3 inflammasome in primed macrophages.
To further substantiate the inventors' observation that apoptotic processes act as the second signal for NLRP3 activation, the inventors examined the timing of NLRP3 activation with respect to LPS, UVCP and CP. If STS or ATP was added before or simultaneously with UVCP or LPS, IL-Ιβ secretion was undetectable (Figure 6f). Likewise, when STS or ATP was added concomitantly with CP infection, IL-Ιβ secretion was drastically reduced (Figure 6f). Instead, if STS or ATP was added 6 hr after LPS or UVCP priming, IL-Ιβ secretion was strikingly increased (Figure 6f). These data further substantiate that ATP and STS rapidly induce apoptosis (as initially shown in Figures 6a and 6b), which would prevent the cleavage and secretion of IL-Ιβ because signal 1 has not had enough time to prime the cells for induction of pro-IL-Ιβ. To show that the apoptotic cells themselves are releasing IL-Ιβ, the inventors used immunohistochemistry. Indeed, secretion of IL-Ιβ (indicated by a disappearance of cytosolic IL-Ιβ staining) in response to CP or LPS+ATP was caspase-1- dependent and was more frequent in apoptotic cells (those with condensed nuclei, Figure 6g). This argues against the possibility that ATP released from dying cells activated the NLRP3 inflammasome of neighboring cells in a paracrine manner. Further ruling out this bystander effect, the inventors observed that CP-infected or alum-treated macrophages given apyrase, an enzyme which rapidly hydrolyzes extracellular ATP, exhibited no significant difference in IL-Ιβ secretion compared to untreated cells (data not shown). These results also demonstrate that activation of the NLRP3 inflammasome by an apoptotic stimulus must occur after, but not before or concomitant with, innate immune cell priming.
Recent studies have described a caspase-1 -dependent form of programmed cell death called pyroptosis 31. To rule out pyroptosis as the source of mt dysfunction and cell death, the inventors assessed ΔΨιη reduction in response to CP, ATP, STS, NIG, and alum in caspase-1 deficient (Casp^) BMDM. In all cases studied, mitochondrial depolarization was independent of caspase-1 (Figure 18a), suggesting that pyroptosis cannot account for the inventors' observations. The inventors also assessed LDH release due to CP infection in BMDM and found that caspase-1 also did not play a role in that system (Figure 18b). Additionally, LPS primed BMDM treated with necrotic stimuli did not secrete IL-Ιβ (Figure 18c). Example 18
To verify that macrophage apoptosis is required for NLRP3 activation, a macrophage cell line (MCL) was stably transfected with a construct that overexpresses the anti-apoptotic protein Bcl-2 and assessed for IL-Ιβ secretion in response to NLRP3 triggers. Importantly, this Bcl-2 MCL was found to be resistant both to changes in mitochondrial potential (ΔΨ) and to apoptosis (as measured by nuclear condensation and LDH release) in response to LPS+ATP and to CP. The Bcl-2 overexpressing MCL was found to secrete significantly less IL-lb (compared to an empty vector control) in response to LPS + ATP, LPS + Alum and CP. It is crucial to note that in all cases TNF-a secretion was unaffected. This shows that inhibition of apoptosis at the level of the mitochondria is sufficient to cause specific reduction of IL-Ιβ secretion triggered by NLRP3 stimuli. The inventors conclude, therefore, that apoptotic processes play a crucial role in the NLRP3 activation cascade.
Example 19
Bcl-2 expression regulates NLRP3 inflammasome activation and IL-Ιβ secretion To verify that macrophage apoptosis was required for NLRP3 activation, the inventors stably transfected an immortalized macrophage cell line, MCL 32, with an overexpression vector coding for the anti-apoptotic protein Bcl-2 (Figure 13a) and assessed IL-Ιβ secretion in response to NLRP3 triggers. The Bcl-2 overexpressing line secreted significantly less IL-Ιβ compared to cells with an empty vector control (or vs. cells expressing an unrelated gene; Figure 19) in response to CP, LPS+ATP, LPS+STS, or LPS+NIG (Figure 13b). Importantly, in all cases TNFa secretion was unaffected (Figure 13b). Western blot analyses revealed similar results with reduced levels of both intracellular caspase-1 plO or mature (secreted) IL-Ιβ (Figure 13c). Importantly, the Bcl-2 overexpressing MCL line was resistant to both changes in ΑΨΏ1 (Figure 13 d) and to apoptosis in response to LPS+ATP, LPS+STS, or LPS+NIG, and to CP (Figure 13e and S3). The inventors also derived BMDM from Bcl-2 transgenic (Bcl-2-tg) mice and performed similar analysis. Indeed, these macrophages secreted significantly less IL-Ιβ in response to a host of NLRP3 stimuli (Figure 13f) while TNFa secretion remained constant. These data clearly show that inhibition of apoptosis at the level of mt is sufficient to cause reduction of IL-Ιβ maturation and secretion triggered by NLRP3 -activating stimuli. Finally, the inventors stably transfected MCL cells with a vector encoding shRNA for Bcl-2 that efficiently reduced Bcl-2 protein abundance (Figure 13g). Attenuated Bcl-2 protein should result in increased apoptosis and, as expected, the inventors found elevated LDH release (Figure 13h). Using LPS+ATP to stimulate the NLRP3 inflammasome, the inventors found these cells secreted more IL-Ιβ but not TNFa (Figure 13i). Based on these results, the inventors conclude that apoptotic processes blocked by Bcl-2 are necessary for NLRP3 activation. Importantly, LPS+Fas ligand treatment did not result in NLRP3 activation as determined by IL-Ιβ secretion (data not shown), suggesting that NLRP3 activation involves only the intrinsic apoptosis pathway.
Example 20
Salmonella typhimurium infection-induced apoptosis causes IL-Ιβ secretion
S. typhimurium is recognized by both the NLRP3 and NLRC4 inflammasome pathways 33. Indeed, NLRP3 plays an important role in IL-Ιβ secretion (Figure 14a). Salmonella typhimurium is also known to induce Type III secretion system (T3SS)-dependent apoptosis in macrophages 34' 35. The inventors investigated whether S. typhimurium activation of the NLRP3 inflammasome was licensed by apoptosis. S. typhimurium infection of BMDM for 8 h resulted in marked LDH release (90% of control) (Figure 14b). However, T3SS-1 defective strains of S. typhimurium (invA, hilA, orgA, and prgH) had severely retarded LDH release, while a T3SS-2 defective strain (spiB), and the lipid A non-signaling strain (msbB) induced high levels of LDH release (Figure 14b). Finally, the invasin mutant (sspB) did not induce LDH release. Accordingly, LDH release inversely correlated with ΔΨιη in LDH- inducing strains, while ΔΨιη remained unaltered in non-LDH-re leasing mutants (Figure 14c). As a control, poly(dA:dT), which utilizes AIM2 but not NLRP3, did not induce changes in ΔΨιη. Importantly, IL-Ιβ secretion was only induced by the S. typhimurium strains that caused LDH release and AxVm reduction (Figure 14d), and IL-Ιβ was not secreted by BMDM infected with strains that failed to induce apoptosis. To further test whether IL-Ιβ secretion due to S. typhimurium infection induced apoptosis, the inventors infected the Bcl-2 stably transfected MCL cell line with wild type S. typhimurium. Bcl-2 overexpression resulted in significantly reduced IL-Ιβ secretion following S. typhimurium infection with the wild-type strain (Figure 14e). TNFa levels remained similar between-groups (Figure 14e). It should be noted that Bcl-2 overexpression did not impact LPS+poly(dA:dT) treatment outcomes (Figure 14e), presumably since poly(dA:dT) utilizes AIM2 and not NLRP3. Taken together, these data further generalize the link between apoptosis and NLRP3 inflammasome activation. Example 21
Mitochondrial DNA is required for NLRP 3 inflammasome-mediated IL-1 β secretion Recently, Nakahira et al. suggested that mtDNA was involved in NLRP3 activation 16 , but the mechanism for this was not disclosed. To discern the mechanism connecting mt dysfunction, apoptosis, and NLRP3 inflammasome activation, the inventors created pO (lacking mtDNA) MCL cells (Figure 20a) that had normal ΔΨιη and similar mt content as control cells (Figure 20b-c). Corroborating results of Nakahira et al., the inventors found that pO cells were unable to secrete IL-Ιβ in response to NLRP3 -inducing stimuli, while TNFa secretion remained unaltered (Figure 20d). It is well-known that mtDNA is oxidized during apoptosis by mt-derived ROS 36. While not wishing to be bound by any particular theory, the inventors believe that mtDNA released into the cytosol 37 may act as a trigger for NLRP3 inflammasome activation. To begin testing this, the inventors examined cytosolic DNA content following stimulation with LPS+ATP or LPS+NIG. Shortly (15-30 min) after treatment with ATP or NIG, the mitochondrial genes Coxl and Nd3 were readily detected in the cytosol by PCR, but genomic DNA encoding Gapdh was undetectable in this cellular compartment (Figure 20e). Again, while not wishing to be bound by any particular theory, the inventors also further believed that cytosolic mtDNA might bind to NLRP3 directly. Thus, the inventors co-transfected 293 cells with a NLRP 3 -Flag construct and purified mtDNA previously labeled with bromodeoxyuridine (BrdU). NLRP3 was subsequently immunoprecipitated using anti-Flag M2 antibody, and immunoprecipitates were dot-blotted for BrdU. BrdU signal was clearly detected in NLRP3 immunoprecipitates, indicating that mtDNA directly or indirectly associated with NLRP3 (Figure 15a). To validate these observations, the inventors probed for colocalization of BrdU-labeled mtDNA with NLRP3 by microscopy and found similar results (Figure 21). Similar to Nakahira and colleagues, IL- 1β secretion induced by BMDM-transfected mtDNA was AIM2 dependent (Figure 20f).
To ensure that mtDNA-NLRP3 interaction was not owed to overexpression artifact, the inventors investigated whether endogenous mtDNA interacted with native NLRP3 during inflammasome activation. BMDM were grown in the presence of BrdU and then treated with LPS+ATP or LPS+NIG in the presence of the autophagy inhibitor 3-MA to enhance inflammasome activation. NLRP3 was immunoprecipitated and used as input material for BrdU dot-blot. BrdU incorporated DNA was bound to NLRP3 in cells treated with LPS+ATP or LPS+NIG (Figure 15c), whereas LPS treatment alone did not produce NLRP3-BrdU binding, indicating that secondary stimulation was necessary for this interaction. Because mt-derived ROS play an important role in NLRP3 activation, and it is well-known that ROS oxidize mtDNA, the inventors moved on to probe NLRP3 immunoprecipitates for the oxidized DNA indicator, 8-hydroxy-guanosine (8-OH-dG). Interestingly, the inventors found that under LPS+ATP conditions 8-OH-dG was detected in immunoprecipitates that was also present more weakly with LPS+NIG stimulation (Figure 15c). Finally, the inventors investigated the origin of the immunoprecipitated DNA by performing PCR on the BrdU incorporated, NLRP3 bound DNA. Using primers specific for nuclear vs. mtDNA, the inventors only detected mtDNA (Figure 15d). Thus, oxidized mtDNA released into the cytosol bound NLRP3.
To further assess the role of oxidized mtDNA during NLRP3 activation, the inventors investigated 8-OH-dG induction under NLRP3 activating conditions. Under conditions where BMDM were exposed to either ATP or NIG, increased levels of 8-OH-dG were found (Figure 15e). Interestingly, addition of LPS to either ATP or NIG resulted in further increased 8-OH-dG abundance (Figure 15e). This finding dovetails with a recent report showing that TLR signaling enhances mtROS production 38. The inventors next assessed whether oxidized DNA vs. normal DNA could enhance IL-Ιβ production. Oxidized DNA was generated by PCR against the mtCOXl template in presence of 8-OH-dGTP, and PCR product was transfected into BMDM. Data showed increased IL-Ιβ production vs. normal DNA (Figure 12f). Importantly, oxidized DNA did not impact TNFa production. However, the same experiment performed in Nlrp3~'~ BMDM resulted in significantly decreased IL-Ιβ, but not TNFa production (Figure 15g). Furthermore, DNA containing 8-OH-dGTP could still induce IL-Ιβ secretion in Aim2~'~ BMDM (Figure 15h), despite the inventors' findings that these cells were refractive to mtDNA transfection-induced IL-Ιβ release . Taken together, these data indicate that oxidized DNA can induce inflammasome activation via preferential activation of NLRP3.
The inventors next assessed whether 8-OH-dG might competitively inhibit NLRP3 inflammasome activation. Under NLRP3 activating conditions, the inventors added increasing amounts of either dG or 8-OH-dG into the media at the same time as LPS treatment. While the addition of dG had no effect on IL-Ιβ production, 8-OH-dG was able to significantly reduce IL-Ιβ secretion (Figure 15h). Importantly, the inventors repeated the immunoprecipitation of NLRP3 and BrdU incorporated mtDNA under LPS+ATP conditions, but in the presence of either dG or 8-OH-dG, and found that 8-OH-dG could prevent BrdU- incorporated mtDNA from co-precipitating with NLRP3 (Figure 15i). Finally the inventors probed for the presence of 8-OH-dG in NLRP3 immunoprecipitates and found that instead of BrdU mtDNA, only 8-OH-dG co-precipitated with NLRP3 (Figure 15i). Thus, 8-OH-dG was able to competitively inhibit NLRP3 -mtDNA interaction. Taken together, these data indicate that oxidized mtDNA is the actual binding motif for NLRP3 and the activator of the NLRP3 inflammasome.
Example 22
Measurement of mitochondrial oxygen consumption
Oxygen consumption rates (OCR) were measured using an XF24 Extracellular Flux Analyzer (Seahorse Bioscience). For the XF24 assay, cells were equilibrated with DMEM lacking bicarbonate at 37°C for 1 h in an incubator without C02. Mixing, waiting, and measurement times were 0.5, 2, and 3 min, respectively (an extra 30 s was added after each injection). Oligomycin, which blocks phosphorylation of ADP to ATP, was utilized to prevent mitochondrial respiration and to provide basal 02 consumption during the assay. FCCP was used as an uncoupling agent to allow maximal 02 consumption under a given condition. Rotenone was employed as an mt respiratory chain complex 1 inhibitor.
Example 23
Mitochondrial isolation and DNA extraction
Mitochondria were isolated using by Mitochondria Isolation Kit (Thermo Scientific, Rockford, IL). Mitochondrial DNA was purified using by QIAamp DNA mini kit (QIAGEN). For detection of mtDNA, PCR was carried out using following primers: mtCOXl, sense primer 5 '-TTCGGAGCCTGAGCGGGAAT-3 ' (SEQ ID NO: l), and antisense primer 5 ' -ATGCCTGCGGCTAGCACTGG-3 ' (SEQ ID NO:2) (Product length: 554 bp).
Example 24
8-OH-dG incorporation
The dGTP analog 8-OH-dGTP was purchased from TriLink BioTechnologies (San Diego, CA). The mtCOXl gene fragment (554 bp) was amplified with unmodified dNTPs and 8-OH-dGTP using Taq DNA polymerase and isolated mtDNA. Amplified DNA was purified by StrataPrepR PCR Purification kit (Agilent Technologies, Santa Clara, CA). Example 25
Immunoprecipitation
BMDM were preloaded with BrdU (10 μΜ) for 48 h and treated as indicated. For immunoprecipitations, the rabbit anti-NLRP3 polyclonal Ab (LifeSpan Biosciences Inc., Seattle, WA) was incubated with the cell lysates for 2 h or overnight at 4°C. Subsequently, Trueblot IgG beads (eBioscience, San Diego, CA) were added and the samples were incubated at 4°C for 1 h. For immunoprecipitations from Flag-NLRP3 stably expressing 293 cells, mouse anti-Flag mAb (M2, Sigma) was used. The immune complexes were then washed and the associated proteins were eluted from the beads by boiling and then the supernatant was dot-blotted and UV cross-linked to a nitrocellulose membrane. Immunoblotting was performed using anti-BrdU mAb (BU33; Sigma) or mouse anti-80H-dG mAb (15 A3; Rockland Immunochemicals Inc., Gilbertsville, PA).
Example 26
Macrophage stable transfection
A bone marrow derived macrophage cell line, MCL 32, was transfected with either a
Bcl2 overexpression plasmid named pSFFV-neo-Bcl-2 57, Addgene plasmid 8776, or an empty vector control plasmid pSFFV-neo 58, kindly provided by Dr. Gabriel Nunez (Univ.
Michigan, Ann Arbor, MI), using lipofectamine 2000 (Invitrogen) and selected by G418. For Bc/2 downregulation, MCL were exposed to Bcl2 shRNA or control shRNA (Santa Cruz
Biotechnology, Santa Cruz, CA) and selected by puromycin.
Example 27
Establishment of mitochondrial DNA deficient cells(p0 cells) MCL cells were maintained in the presence of 50 ng/ml ethidium bromide for more than 20 passages and media was supplemented with uridine (50 g/ml) and sodium pyruvate (120 g/ml). Depletion of mtDNA was confirmed by PCR for the mitochondrial genes Coxl and Nd6 compared to a nuclear gene (Gapdh). Example 28
Confocal microscopy
BMDM were fixed in 4% paraformaldehyde (PFA) for 10 min at 4°C and permeabilized with BD Cytofix/Cytoperm solution for 30 min at 4°C. After three rinses in PBS, cells were treated with 10 U/ml DNase I (Roche) for 30 min at 37 °C. After three additional rinses in PBS, cells were incubated overnight at 4°C with primary antibody against BrdU. After three rinses in PBS, cells were incubated with appropriate secondary antibodies conjugated with Alexa Fluor 594 for 1 hr at ambient temperature. After three additional rinses in PBS, cells were then mounted in fluorescence mounting medium with DAPI (ProLong Gold). Images were acquired in independent channels with a Zeiss ApoTome- equipped fluorescence microscope.
Example 29
Statistical analysis
To compare differences, the two-tailed Students t-test (at 95% confidence interval) was used to compare unpaired samples. For experiments involving three groups the inventors used one-way ANOVA with Tukey's post-hoc test. A p value less than 0.05 was considered significant and results are presented as mean±SEM.
Various embodiments of the invention are described above in the Detailed Description. While these descriptions directly describe the above embodiments, it is understood that those skilled in the art may conceive modifications and/or variations to the specific embodiments shown and described herein. Any such modifications or variations that fall within the purview of this description are intended to be included therein as well. Unless specifically noted, it is the intention of the inventors that the words and phrases in the specification and claims be given the ordinary and accustomed meanings to those of ordinary skill in the applicable art(s).
The foregoing description of various embodiments of the invention known to the applicant at this time of filing the application has been presented and is intended for the purposes of illustration and description. The present description is not intended to be exhaustive nor limit the invention to the precise form disclosed and many modifications and variations are possible in the light of the above teachings. The embodiments described serve to explain the principles of the invention and its practical application and to enable others skilled in the art to utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed for carrying out the invention. While particular embodiments of the present invention have been shown and described, it will be obvious to those skilled in the art that, based upon the teachings herein, changes and modifications may be made without departing from this invention and its broader aspects and, therefore, the appended claims are to encompass within their scope all such changes and modifications as are within the true spirit and scope of this invention. It will be understood by those within the art that, in general, terms used herein are generally intended as "open" terms (e.g., the term "including" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," the term "includes" should be interpreted as "includes but is not limited to," etc.).
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Claims

WHAT IS CLAIMED IS:
1. A method, comprising:
providing a composition comprising a mitochondrial apoptosis inhibitor; and administering the composition to a subject in need of treatment for inflammation to treat the inflammation.
2. The method of claim 1, wherein the mitochondrial apoptosis inhibitor is selected from the group consisting of Bcl-2, Mcl-1, Bcl-xL, Bcl-w, Bfl-1, nfh, XIAP, Boo/Diva, Nrl3, BH4 domain, Inositol 1,4,5-trisphosphate receptor (IP3R) peptide, GSK-3 , cyclosporine A, viral mitochondrial inhibitor of apoptosis (vMIA) peptide/protein, MitoQ and combinations thereof.
3. The method of claim 1, wherein the treatment for inflammation treats an inflammatory disease, an inflammatory disease condition, an autoimmune disease, or combinations thereof.
4. The method of claim 3, wherein the inflammatory disease, inflammatory disease condition, or autoimmune disease is where IL-1 beta plays a role.
5. The method of claim 4, wherein the inflammatory disease, inflammatory disease condition, or autoimmune disease where IL-1 beta plays a role is selected from the group consisting of type 2 diabetes, rheumatoid arthritis, psoriasis, Alzheimer's disease, silicosis and asbestosis, gout, pseudogout, familial cold autoinflammatory syndrome (FCAS), Muckel- Wells syndrome (MWS), neonatal-onset multisystem inflammatory disease (NOMID), and combinations thereof.
6. The method of claim 3, wherein the inflammatory disease or inflammatory disease condition is selected from the group consisting of arthritis, Crohn's disease, inflammatory bowel disease, Alzheimer's disease, diabetes, gout, atherosclerosis, asbestosis/silicosis induced lung fibrosis and combinations thereof.
7. The method of claim 3, wherein the autoimmune disease is selected from the group consisting of Hashimoto's thyroiditis, Pernicious anemia, Addison's disease, type I diabetes, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, Sjogren syndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis, reactive arthritis, Grave's disease, celiac disease, and combinations thereof.
8. A method, comprising:
providing a composition comprising an oxidative nucleotide; and administering the composition to a subject in need of treatment for inflammation to treat the inflammation.
9. The method of claim 8, wherein the oxidative nucleotide is selected from the group consisting of (i) 8-Hydroxy-2'-deoxyguranosine, (ii) 8-hydroxy Guanosine, (iii) 8- Oxo-2'-deoxyadenosine, (iv) 5-formyl-2'-deoxycytidine, (v) 5-formyl-2'- deoxyuridine, (vi) 5-hydroxymethyl-2'-deoxyuridine, (vii) 5-Hydroxymethyl-2'- deoxycytidine, (viii) 5-hydroxy-2'-deoxyuridine, (ix) 5-hydroxy-2'-deoxycytidine, and (x) combinations thereof.
10. The method of claim 8, wherein the treatment for inflammation treats an inflammatory disease, an inflammatory disease condition, an autoimmune disease, or combinations thereof.
11. The method of claim 10, wherein the inflammatory disease, inflammatory disease condition, or autoimmune disease is where IL-1 beta plays a role.
12. The method of claim 11, wherein the inflammatory disease, inflammatory disease condition, or autoimmune disease where IL-1 beta plays a role is selected from the group consisting of type 2 diabetes, rheumatoid arthritis, psoriasis, Alzheimer's disease, silicosis and asbestosis, gout, pseudogout, familial cold autoinflammatory syndrome (FCAS), Muckel- Wells syndrome (MWS), neonatal-onset multisystem inflammatory disease (NOMID), and combinations thereof.
13. The method of claim 10, wherein the inflammatory disease or inflammatory disease condition is selected from the group consisting of arthritis, Crohn's disease, inflammatory bowel disease, Alzheimer's disease, diabetes, gout, atherosclerosis, asbestosis/silicosis induced lung fibrosis and combinations thereof.
14. The method of claim 10, wherein the autoimmune disease is selected from the group consisting of Hashimoto's thyroiditis, Pernicious anemia, Addison's disease, type I diabetes, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, Sjogren syndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis, reactive arthritis, Grave's disease, celiac disease, and combinations thereof.
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