WO2024071319A1 - Composition comprising isolated or enriched phosphoenolpyruvate or a salt thereof - Google Patents
Composition comprising isolated or enriched phosphoenolpyruvate or a salt thereof Download PDFInfo
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- WO2024071319A1 WO2024071319A1 PCT/JP2023/035468 JP2023035468W WO2024071319A1 WO 2024071319 A1 WO2024071319 A1 WO 2024071319A1 JP 2023035468 W JP2023035468 W JP 2023035468W WO 2024071319 A1 WO2024071319 A1 WO 2024071319A1
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- pep
- salt
- phosphoenolpyruvate
- cells
- phosphate
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Definitions
- the present disclosure relates to a composition
- a composition comprising an isolated or enriched glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof.
- PEP phosphoenolpyruvate
- G6P glucose-6-phosphate
- F6P fructose-6-phosphate
- 3PG 3-phosphoglycerate
- the present disclosure also relates to a pharmaceutical composition
- a pharmaceutical composition comprising an isolated or enriched glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof.
- PEP phosphoenolpyruvate
- G6P glucose-6-phosphate
- F6P fructose-6-phosphate
- 3PG 3-phosphoglycerate
- T cell differentiation and functions are closely associated with T cell differentiation and functions. Differentiation of naive T cells into effector T cell subsets relies on metabolic reprogramming from oxidative phosphorylation to aerobic glycolysis, while differentiation into memory T cells and T regulatory (Treg) cells is largely dependent on mitochondrial fatty acid oxidation (Chapman et al., 2020; Man and Kallies, 2015; Pearce et al., 2013). The primary role of aerobic glycolysis is to fulfill the demand for energy and building blocks for anabolism in rapidly proliferating effector T cells (O'Neill et al., 2016; Palmer et al., 2015; Zhu and Thompson, 2019).
- TCR T cell receptor
- Aerobic glycolysis is required for differentiation and pathogenicity of T helper 17 (Th17) cells, a subset of effector CD4 T cells expressing interleukin-17 (IL-17) (Geltink et al., 2018; Omenetti et al., 2019).
- Th17 T helper 17
- IL-17 interleukin-17
- Th17 cells Differentiation of Th17 cells is induced in the presence of cytokines IL-6 and TGF-b (Bettelli et al., 2006; Mangan et al., 2006; Veldhoen et al., 2006), and its pathogenicity is dependent on the IL-23 signal (Cua et al., 2003; Ghoreschi et al., 2010; Hirota et al., 2013; Lee et al., 2012; McGeachy et al., 2007; McGeachy et al., 2009; Stockinger and Omenetti, 2017).
- Th17 cells that cause autoimmunity exhibit higher expression of glycolytic genes and glycolytic activity than gut-resident homeostatic Th17 cells (Omenetti et al., 2019; Wu et al., 2020).
- High glucose intake promotes Th17 differentiation (Zhang et al., 2019), while loss of glucose transporters decreases Th17 generation and pathogenicity (Hochrein et al., 2022; Macintyre et al., 2014).
- genetic ablation of glycolytic enzymes dramatically impairs clonal expansion of Th17 cells (Wu et al., 2020; Xu et al., 2021).
- the AP-1 transcription factors JunB and basic leucine zipper transcription factor ATF-like (BATF), collaborate with interferon regulatory factor 4 (IRF4) to regulate the transcriptional program for Th17 differentiation (Carr et al., 2017; Hasan et al., 2017; Yamazaki et al., 2017).
- IRF4 interferon regulatory factor 4
- These transcription factors are induced upon TCR stimulation (Iwata et al., 2017; Schraml et al., 2009) and form JunB/BATF/IRF4 trimers that bind to AP-1 IRF composite elements (AICEs) adjacent to the target genes (Ciofani et al., 2012; Glasmacher et al., 2012; Li et al., 2012).
- the BATF and IRF4 are indispensable for differentiation of both pathogenic and non-pathogenic Th17 cells (Brustle et al., 2007; Huber et al., 2008; Schraml et al., 2009). They promote expression of Th17-signature molecules, such as IL-17A, retinoic acid-related orphan receptor (ROR)gt and IL-23 receptor (IL-23R) by enhancing the chromatin accessibility (Ciofani et al., 2012; Pham et al., 2019). In contrast, JunB is selectively required for pathogenic Th17 generation, probably due to functional redundancy among Jun family members (Carr et al., 2017; Hasan et al., 2017). Whether cellular metabolism controls the function of AP-1 transcription factors remains unexplored.
- Th17-signature molecules such as IL-17A, retinoic acid-related orphan receptor (ROR)gt and IL-23 receptor (IL-23R) by enhancing the chromatin accessibility (Ci
- the present disclosure provides a composition comprising an isolated or enriched glycolytic intermediate metabolite or a salt thereof.
- the present disclosure provides a composition comprising an isolated or enriched phosphoenolpyruvate or a salt thereof.
- the present disclosure also provides a pharmaceutical composition comprising an isolated or enriched glycolytic intermediate metabolite or a salt thereof.
- the present disclosure also provides a pharmaceutical composition comprising an isolated or enriched phosphoenolpyruvate or a salt thereof.
- glycolytic intermediate metabolite or a salt thereof can inhibit an activity or induction of T helper 17 (Th17) cells.
- phosphoenolpyruvate or a salt thereof can inhibit an activity or induction of T helper 17 (Th17) cells.
- the inventors identify a glycolytic intermediate metabolite, such as phosphoenolpyruvate (PEP), as a negative regulator of pathogenic Th17 generation.
- PEP phosphoenolpyruvate
- a composition for use in a method of preventing, inhibiting, or reducing activation of T helper 17 (Th17) cell immunity or development of Th17-mediated immune disease or condition in a subject in need thereof, comprising an effective amount of an isolated or enriched glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof.
- PEP phosphoenolpyruvate
- G6P glucose-6-phosphate
- F6P fructose-6-phosphate
- 3PG 3-phosphoglycerate
- composition of (1) above, wherein the isolated or enriched glycolytic intermediate metabolite comprises or a salt thereof is one or more selected from the group consisting of phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), and 3-phosphoglycerate (3PG), and a salt thereof.
- PEP phosphoenolpyruvate
- G6P glucose-6-phosphate
- F6P fructose-6-phosphate
- 3PG 3-phosphoglycerate
- a pharmaceutical composition for use in a method of preventing, inhibiting, or reducing T helper 17 (Th17) cell immunity in a subject in need thereof, comprising an effective amount of an isolated or enriched glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof.
- PEP phosphoenolpyruvate
- G6P glucose-6-phosphate
- F6P fructose-6-phosphate
- 3PG 3-phosphoglycerate
- a pharmaceutical composition for use in a method of treating an inflammation or disease associated with an activated or increased T helper 17 (Th17) cell immunity in a subject in need thereof, comprising an effective amount of an isolated or enriched glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof.
- PEP phosphoenolpyruvate
- G6P glucose-6-phosphate
- F6P fructose-6-phosphate
- 3PG 3-phosphoglycerate
- composition of (5) or (6) above, wherein the isolated or enriched glycolytic intermediate metabolite or a salt thereof comprises or is one or more selected from the group consisting of phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), and 3-phosphoglycerate (3PG), and a salt thereof.
- PEP phosphoenolpyruvate
- G6P glucose-6-phosphate
- F6P fructose-6-phosphate
- 3PG 3-phosphoglycerate
- the composition of (5) or (6) above, wherein the isolated or enriched glycolytic intermediate metabolite or a salt thereof comprises or is phosphoenolpyruvate (PEP).
- a pharmaceutical composition for use in a method of treating an inflammation or an immune-related disease in a subject in need thereof, comprising an effective amount of an isolated or enriched glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof.
- PEP phosphoenolpyruvate
- G6P glucose-6-phosphate
- F6P fructose-6-phosphate
- 3PG 3-phosphoglycerate
- the pharmaceutical composition of (12) above, wherein the immune-related disease is selected from the group consisting of rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, encephalomyelitis, and psoriasis.
- the pharmaceutical composition of (12) above, wherein the inflammation and the immune-related disease are caused by or associated with an activated or increased T helper 17 (Th17) cell immunity.
- the pharmaceutical composition of (14) above, wherein the inflammation and the immune-related disease are caused by or associated with an activated or increased T helper 17 (Th17) cell immunity.
- the present disclosure also provides, for example, inventions described below.
- a method of treating a subject in need thereof comprising administering to the subject an effective amount of a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof.
- a glycolytic intermediate metabolite or a salt thereof preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof.
- a glycolytic intermediate metabolite or a salt thereof preferably, phosphoenolpyruvate (PE
- a method of treating a subject in need thereof comprising intravenously, transdermally or intradermally administering to the subject an effective amount of a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof, wherein the subject has psoriasis.
- a glycolytic intermediate metabolite or a salt thereof preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof, wherein the subject has psoriasis.
- a method of treating a subject in need thereof comprising intravenously or intraarticularly administering to the subject an effective amount of a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof, wherein the subject has rheumatoid arthritis.
- a glycolytic intermediate metabolite or a salt thereof preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof, wherein the subject has rheumatoid arthritis.
- a method of treating a subject in need thereof comprising intravenously or orally administering to the subject an effective amount of a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof, wherein the subject has inflammatory bowel disease.
- a glycolytic intermediate metabolite or a salt thereof preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof, wherein the subject has inflammatory bowel disease.
- a method of treating a subject in need thereof comprising intracerebrospinally, intravenously or orally administering to the subject an effective amount of a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof, wherein the subject has multiple sclerosis.
- a glycolytic intermediate metabolite or a salt thereof preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof, wherein the subject has multiple sclerosis.
- a method of treating a subject in need thereof comprising intracerebrospinally, intravenously or orally administering to the subject an effective amount of a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof, wherein the subject has encephalomyelitis.
- a glycolytic intermediate metabolite or a salt thereof preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof, wherein the subject has encephalomyelitis.
- the present disclosure also provides, for example, inventions described below.
- a glycolytic intermediate metabolite or a salt thereof preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof in the manufacture of a medicament for use in a method of any one of (21) to (29) above.
- a glycolytic intermediate metabolite or a salt thereof preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof for use in a method of any one of (21) to (29) above.
- PEP phosphoenolpyruvate
- G6P glucose-6-phosphate
- F6P fructose-6-phosphate
- 3PG 3-phosphoglycerate
- a pharmaceutical composition for use in a method of any one of (21) to (29) above, comprising a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof.
- PEP glycolytic intermediate metabolite
- G6P glucose-6-phosphate
- F6P fructose-6-phosphate
- 3PG 3-phosphoglycerate
- a composition for use in a method of preventing, inhibiting, or reducing JunB-dependent generation of autoimmune T helper 17 (Th17) cells in a subject in need thereof, comprising an effective amount of an isolated or enriched glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof.
- PEP phosphoenolpyruvate
- G6P glucose-6-phosphate
- F6P fructose-6-phosphate
- 3PG 3-phosphoglycerate
- Fig. 1 shows that intracellular PEP inhibits IL-17A expression.
- Naive CD4 T cells were activated with CD3/CD28 antibodies in the absence (vehicle control (Ctrl)) or presence of glycolytic intermediate metabolites (A-C) or glycolysis inhibitors (D) under npTh17 conditions (with TGF ⁇ and IL-6).
- Cells were activated in the absence or presence of G6P (20mM), F6P (10mM), FBP (1mM), G3P (1mM), 3PG (10mM), or PEP (10mM) for 60 h. Representative plots are shown in Figure 7A.
- Fig. 2 shows that PEP supplementation inhibits differentiation of Th17 and Th2 cells.
- Naive CD4 T cells were activated with CD3/CD28 antibodies in the absence or presence of PEP (5 or 10 mM) under npTh17 (with TGF ⁇ and IL-6) or pTh17 (with IL-6, IL-1 ⁇ , and IL-23) conditions (A-C, F) and Th1, Th2, or iTreg conditions (D, E). Cells were collected at 48 h (C, F) or 60 h (A, B, D, E) after activation.
- A, B Flow cytometry analysis of IL-17A and IFN- ⁇ (A) and ROR ⁇ t (B). Representative plots are shown in the upper panels.
- the p value was calculated by a two-tailed unpaired Student’s t test (**** p ⁇ 0.0001).
- A-E The p values were calculated by one-way ANOVA with Bonferroni’s multiple comparison tests (* p ⁇ 0.05, **** p ⁇ 0.0001, ns: not significant). In all panels, error bars indicate mean ⁇ SD. Data are representative of at least two experiments.
- Fig. 3 shows that PEP suppresses IL-17A expression independently of its role in glycolysis and T cell activation.
- A-E Naive CD4 T cells were activated with CD3/CD28 antibodies in the absence (Ctrl) or presence of PEP (10 mM) under npTh17 (with TGF ⁇ and IL-6) or pTh17 (with IL-6, IL-1 ⁇ , and IL-23) conditions. Cells were collected at 48 h (E) or 72 h (A-D) after activation.
- E Immunoblot analysis of PKM2 in nuclear and cytoplasmic fractions. Nuclear histone H3 and cytoplasmic ⁇ -tubulin were also detected.
- Fig. 4 shows that PEP regulates JunB-dependent transcriptional program in Th17 cells.
- A Principal component analysis of RNA-seq data.
- B Volcano plots showing differentially expressed genes (DEGs) in PEP-treated vs control cells (log2 fold change (FC) > 1, p ⁇ 0.05).
- Fig. 5 shows that PEP inhibits DNA binding of JunB, BATF, and IRF4 at the Il17a locus.
- A, B Naive CD4 T cells were activated with CD3/CD28 antibodies in the absence or presence of PEP (5 or 10 mM) under npTh17 (with TGF ⁇ and IL-6) or pTh17 (with IL-6, IL-1 ⁇ , and IL-23) conditions. Expression of Th17-related transcription factors in cells activated for 60 h were analyzed by immunoblot.
- C Immunoblot analysis (IB) of the immunoprecipitation (IP) of FLAG-tagged JunB together with HA-tagged BATF expressed in HEK293 cells. Cells were harvested at 60 h after transfection of the expression vectors.
- D ChIP-seq peaks for JunB, BATF, and IRF4 detected at Il17a and Irf8 loci. ChIP-seq data were from GSE86535. Schematic diagrams at the tops of panels indicate transcription start sites (arrows) and exons (filled boxes) of each gene. Open boxes represent regions detected by ChIP-PCR in E.
- Fig. 6 shows that PEP inhibits Th17 differentiation in vivo and ameliorates EAE.
- A, B Flow cytometry analysis of expression of IL-17A and IFN- ⁇ (A) or ROR ⁇ t and Foxp3 (B) in OT-II T cells.
- FIG. 7 shows that effects of glycolytic metabolites or inhibitors on IL-17A expression.
- Figure 1A Representative flow cytometry plots for Figure 1A
- B Representative flow cytometry plots for Figure 1D.
- the bar graph shows the percentages of living cells. The p values were calculated by one-way ANOVA with Bonferroni’s multiple comparison tests (ns: not significant). Error bars represent mean ⁇ SD. Data are representative of at least two experiments.
- Fig. 8 shows that PEP supplementation does not affect CD25 expression and proliferation of Th17 cells.
- Naive CD4 T cells were activated with CD3/CD28 antibodies in the absence (Ctrl) or presence of PEP (10 mM) under npTh17 (with TGF ⁇ and IL-6) or pTh17 (with IL-6, IL-1 ⁇ , and IL-23) conditions for 48 h.
- A CD25 expression in control or PEP-treated cells was analyzed by flow cytometry. The bar graph shows CD25 MFI. The p value was calculated by a two-tailed unpaired Student’ s t test (ns: not significant). Error bars represent mean ⁇ SD. Data are representative of at least two experiments.
- B CFSE dilution in control or PEP-treated cells was analyzed by flow cytometry. Data are representative of at least two experiments.
- Fig. 9 shows that PEP supplementation does not affect ROS production.
- Naive CD4 T cells were activated with CD3/CD28 antibodies in the absence (Ctrl) or presence of PEP (10 mM) under npTh17 (with TGF ⁇ and IL-6) or pTh17 (with IL-6, IL-1 ⁇ , and IL-23) conditions for 48 h.
- ROS production was analyzed by staining with DCFDA / H2DCFDA Cellular ROS Assay Kit. Cells treated with 0.03% H2O2 were included as positive controls.
- the p values were calculated by one-way ANOVA with Bonferroni’ s multiple comparison tests (**** p ⁇ 0.0001, ns: not significant). Error bars represent mean ⁇ SD. Data are representative of at least two experiments.
- Fig. 10 shows that PEP supplementation does not affect chromatin accessibility at the Il17a locus.
- Naive CD4 T cells were activated with CD3/CD28 antibodies in the absence (Ctrl) or presence of PEP (10 mM) under npTh17 (with TGF ⁇ and IL-6) or pTh17 (with IL-6, IL-1 ⁇ , and IL-23) conditions for 48 h and analyzed by ATAC-seq. The results for the Il17a locus are shown by UCSC Genome Browser (UCSC Genomics Institute).
- Fig. 11 shows that PEP administration inhibits accumulation of IL17A-expressing OT-II T cells in the spleen of mice immunized with OVA.
- Figure 5 Flow cytometry analysis of IL-17A and IFN- ⁇ expression (A) and ROR ⁇ t and Foxp3 (B) in OT-II T cells isolated from the spleen of mice adoptively transferred with OT-II, followed by immunization with OVA as described in Figure 1A.
- the p value was calculated by a two-tailed unpaired Student’ s t test (* p > 0.05, ns: not significant). Error bars represent mean ⁇ SD. Data are representative of at least two experiments.
- subject refers to an animal, for example, a mammal, in particular a dog, a cat, a horse, sheep, cow, or a human.
- treating refers to a therapy or a prevention.
- therapy means therapy, cure, or prevention of a disease or a disorder; improvement of remission; or a reduction in the speed of progress of a disease or a disorder.
- prevention means a reduction in risk of onset of a disease or a pathologic state or a delay of onset of a disease or a pathologic state.
- isolated refers to a separation of a substance from at least one element in a composition the substance was originally contained in.
- enriched refers to making something more concentrated, compared to another substance or the original concentration.
- composition refers to a composition suitable for a pharmaceutical application.
- pharmaceutical composition may also refer to a composition for use in a pharmaceutical application.
- disease means a symptom or illness, preferably a symptom or illness of which therapy is helpful.
- immune-related disease means a disease caused by over-reactivity, disfunction or malfunction of immune system.
- Diseases caused by over-reactivity of immune system includes, for example, but not limited to autoimmune disease.
- autoimmune diseases There are a variety of autoimmune diseases. The most common autoimmune diseases include Graves’ disease, rheumatoid arthritis, Hashimoto’s thyroiditis, type 1 diabetes, systemic lupus erythematosus, and vasculitis.
- autoimmune include Addison’s disease, polymyositis, Sjogren’s syndrome, myositis, dermatomyositis, scleroderma, progressive systemic scleroderma, many forms of glomerulonephritis (kidney inflammation), and some forms of infertility.
- Th17 cell means a subset of helper T cells that can produce interleukin-17 (IL-17).
- Th17 cells are produced from naive CD4 T cells (Th0).
- Th0 cells When Th0 cells are stimulated with interleukin-6 (IL-6) and transforming growth factor- ⁇ (TGF- ⁇ ), Th0 cells express RAR-related orphan receptor ⁇ t (ROR ⁇ t) to differentiate into Th17 cells.
- IL-23 Interleukin-23
- IL-6 and TGF- ⁇ without IL-23 leads to non-pathogenic Th17 cells (npTh17).
- IL-23 and JunB are positively involved in differentiation of pathogenic Th17 cells, and are not required in order to induce non-pathogenic Th17 cells.
- IL-17 is mainly produced by Th17 cells, and acts on fibroblasts, epithelial cells, endothelial cells, or macrophages to produce inflammatory cytokines and chemokines and then induce inflammation.
- Th17 cell relates to autoimmune disease such as rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, encephalomyelitis, and psoriasis.
- autoimmune disease such as rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, encephalomyelitis, and psoriasis.
- Phosphoenolpyruvate refers to a metabolite (or an intermediate) in glycolytic system.
- Phosphoenolpyruvate has the following formula: .
- Phosphoenolpyruvate is generated from 2-phosphoglycerate by phosphoglycerate isomerase in vivo.
- Pyruvate can be generated from phosphoenolpyruvate by pyruvate kinase.
- oxaloacetic acid (OAA) can be generated from phosphoenolpyruvate by phosphoenolpyruvate carboxylase.
- a glycolytic intermediate metabolite and phosphoenolpyruvate is in form of monomer.
- a glycolytic intermediate metabolite or phosphoenolpyruvate can be isolated, enriched, synthesized or purified.
- glycolytic system refers to a metabolic pathway that converts glucose into pyruvate.
- glucose is converted by hexokinase to glucose-6-phosphate.
- glucose-6-phosphate is then converted by glucose-6-phosphate isomerase to fructose-6-phosphate.
- fructose-6-phosphate is converted by phosphofructokinase-1 to fructose 1,6-bisphosphate.
- fructose 1,6-bisphosphate is converted by fructose 1,6-bisphosphate aldolase to dihydroxyacetone phosphate and glyceraldehyde 3-phosphate.
- glycolytic system In glycolytic system, (v) glyceraldehyde 3-phosphate is converted by glyceraldehyde phosphate dehydrogenase to 1,3-bisphosphoglycerate. In glycolytic system, (vi) 1,3-bisphosphoglycerate is converted by phosphoglycerate kinase to 3-phosphoglycerate. In glycolytic system, (vii) 3-phosphoglycerate is converted by phosphoglycerate mutase to 2-phosphoglycerate. In glycolytic system, (viii) 2-phosphoglycerate is converted by enolase to phosphoenolpyruvate.
- glycolytic system (ix) phosphoenolpyruvate is converted by pyruvate kinase to pyruvate.
- Example of metabolites in glycolytic system include, for example, but not limited to, glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), 3-phosphoglycerate (3PG), fructose 1,6 bisphosphate (F1,6BP), glycerol-3-phosphate (G3P), and phosphoenolpyruvate, preferably phosphoenolpyruvate.
- the metabolite or a salt thereof is one or more selected from the group consisting of phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), and 3-phosphoglycerate (3PG), and a salt thereof, more preferably phosphoenolpyruvate (PEP).
- PEP phosphoenolpyruvate
- G6P glucose-6-phosphate
- F6P fructose-6-phosphate
- 3PG 3-phosphoglycerate
- PEP phosphoenolpyruvate
- treatment refers to therapeutic treatment or preventive treatment.
- composition or pharmaceutical composition
- Aerobic glycolysis a metabolic pathway essential for effector T cell survival and proliferation, regulates the differentiation of autoimmune T helper (Th)17 cells, but the mechanism underlying this regulation was largely unknown.
- the inventors identify a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof, as a negative regulator of Th17 differentiation.
- PEP supplementation or inhibition of downstream glycolytic enzymes in differentiating Th17 cells increases intracellular PEP levels and inhibits expression of Th17 signature molecules, such as IL-17A.
- Th17 signature molecules such as IL-17A.
- PEP supplementation does not significantly affect metabolic reprogramming, cell proliferation, and survival of differentiating Th17 cells.
- PEP regulates JunB-mediated pathogenic Th17 transcriptional program by inhibiting DNA-binding activity of the JunB/BATF/IRF4 complex.
- daily administration of PEP to mice inhibits generation of Th17 cells and ameliorates Th17-dependent autoimmune encephalomyelitis.
- the present disclosure provides a composition comprising a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof.
- a glycolytic intermediate metabolite or a salt thereof preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof.
- phosphoenolpyruvate or a salt thereof can inhibit an expression of a Th17 signature molecules such as IL-17A; inhibit production of Th17 cells; and therefore, inhibit pathogenic Th17 cells that are pathogenetically activated, and/or inhibit inflammation and/or immune-related disease or condition such as an excessive immune response, over-reaction of immune system, or autoimmune diseases.
- the composition can be a food composition, a drink composition, or a supplement.
- the composition can be used for treating a condition, preferably a non-pathological condition, associated with pathogenic Th17 cells, or increased or activated Th17 cells.
- the present disclosure provides a pharmaceutical composition
- a glycolytic intermediate metabolite or a salt thereof preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof.
- the pharmaceutical composition can be used for inhibiting an expression of a Th17 signature molecules such as IL-17A; inhibiting production of Th17 cells; and/or therefore, inhibiting pathogenic Th17 cells that are pathogenetically activated, and/or treating inflammation and/or immune-related disease such as an excessive immune response, over-reaction of immune system, or autoimmune diseases.
- an inflammation and an immune-related disease are caused by pathogenic Th17 cells, or activated or increased Th17 cells.
- a regulatory T cell (Treg) can be suitably induced in the presence of a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof.
- the pharmaceutical composition can be used for treating a pathological condition or disease that can be associated with pathogenic Th17 cells, or activated or increased Th17 cells, such as an inflammation and an immune-related disease.
- phosphoenolpyruvate or a salt thereof is a major active ingredient in the composition or the pharmaceutical composition.
- a glycolytic intermediate metabolite or a salt thereof is a major active ingredient in the composition or the pharmaceutical composition.
- 1w/w% or more, 2w/w% or more, 3w/w% or more, 4w/w% or more, 5w/w% or more, 6w/w% or more, 7w/w% or more, 8w/w% or more, 9w/w% or more, 10w/w% or more, 20w/w% or more, 30w/w% or more, 40w/w% or more, 50w/w% or more, 60w/w% or more, 70w/w% or more, 80w/w% or more, 90w/w% or more, 95w/w% or more, or 100w/w% of the active ingredients included in the composition or the pharmaceutical composition may be a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenol
- the composition or the pharmaceutical composition may comprise 0.1w/w% or more, 0.2w/w% or more, 0.3w/w% or more, 0.4w/w% or more, 0.5w/w% or more, 0.6w/w% or more, 0.7w/w% or more, 0.8w/w% or more, 0.9w/w% or more, 1w/w% or more, 2w/w% or more, 3w/w% or more, 4w/w% or more, 5w/w% or more, 6w/w% or more, 7w/w% or more, 8w/w% or more, 9w/w% or more, 10w/w% or more, 20w/w% or more, 30w/w% or more, 40w/w% or more, 50w/w% or more, 60w/w% or more, 70w/w% or more, 80w/w% or more, 90w/w% or more, or 100w/w% of a glycolytic intermediate metabolite or
- a glycolytic intermediate metabolite or a salt thereof preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof is in a free form (i.e., without or outside nano particles).
- a glycolytic intermediate metabolite or a salt thereof preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof is in a form of monomer (i.e., is not polymerized to form an oligomer or a polymer).
- a glycolytic intermediate metabolite or a salt thereof preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof can penetrate the cell membrane and enter into cells (for example, Th17 cell and Th0 cell), particularly Th17 cell or Th0 cell, which has a low intracellular PEP concentration, even without using drug delivery system (DDS).
- DDS drug delivery system
- a glycolytic intermediate metabolite or a salt thereof preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof is encapsulated in a particle, such as nanoparticle, lipid-base nanoparticle (LNP), liposome, and micelle.
- Such a particle may enhance the drug delivery of a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof into cells such as target cells.
- a glycolytic intermediate metabolite or a salt thereof preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof into cells such as target cells.
- nanoparticle refers to a particle having sub-micrometer in its hydrodynamic diameter.
- the particle can be a vesicle that can encompass a substance such as a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof.
- a substance such as a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof.
- PEP phosphoenolpyruvate
- G6P glucose-6-phosphate
- F6P fructose-6-phosphate
- 3PG 3-phospho
- Examples of the salt include a salt with an inorganic base, an ammonium salt, a salt with an organic base, a salt with an inorganic acid, a salt with an organic acid and a salt with a basic or acidic amino acid.
- the salt with an inorganic base include an alkali metal salt such as a sodium salt and a potassium salt; an alkaline earth metal salt such as a calcium salt, a magnesium salt, a barium salt; and an aluminum salt.
- an alkali metal salt such as a sodium salt and a potassium salt
- an alkaline earth metal salt such as a calcium salt, a magnesium salt, a barium salt
- an aluminum salt such as aluminum salt.
- salt with an organic base examples include salts with trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine, dicyclohexylamine and N, N'-dibenzylethylenediamine.
- salt with an inorganic acid examples include salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid and phosphoric acid.
- the salt with an organic acid include salts with formic acid, acetic acid, trifluoroacetic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid and p-toluenesulfonic acid.
- salt with a basic amino acid examples include salts with arginine, lysine and ornithine.
- salt with an acidic amino acid include salts with aspartic acid and glutamic acid.
- a pharmaceutically acceptable salt is preferable.
- a glycolytic intermediate metabolite or a salt thereof preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof may be encapsulated into a vesicle such as lipid nanoparticles.
- lipid nanoparticles include, but are not limited to, the lipid particles described in US8,058,069B, which is incorporated herein by reference in its entirety. Amphiphilic lipids can form lipidic nanoparticles in aqueous solution.
- lipid nanoparticle 50 mol% to 60 mol% of total lipid is cationic lipid such as DLin-MC3-DMA, ALC-0315, and SM-102; 4 mol% to 10 mol% of total lipid is phospholipid such as DSPC; 30 mol% to 40 mol% of total lipid is cholesterol or a derivative thereof; and 0.5 mol% to 2 mol% of total lipid is a conjugated lipid such as PEGylated lipid such as PEG2000-DMG and ALC-0159, which can inhibit aggregation of particles.
- cationic lipid such as DLin-MC3-DMA, ALC-0315, and SM-102
- 4 mol% to 10 mol% of total lipid is phospholipid such as DSPC
- 30 mol% to 40 mol% of total lipid is cholesterol or a derivative thereof
- 0.5 mol% to 2 mol% of total lipid is a conjugated lipid such as PEGylated
- the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, additive, or excipient.
- a pharmaceutically acceptable carrier various organic or inorganic carrier substances routinely used as a drug substance are used and blended as an excipient, a lubricant, a binder or a disintegrator in a solid preparation; a solvent, a solubilizer, a suspending agent, a tonicity agent, a buffer or a soothing agent in a liquid preparation.
- a formulation additive such as a preservative, an antioxidant, a stabilizer, colorants or a sweetener can be used.
- the pharmaceutical composition can be administered orally or parenterally.
- the pharmaceutical composition will be administered by intravenous administration, intramuscular administration, intraperitoneal administration, intradermal administration, transdermal administration, intracerebrospinal fluid administration, intraarticular administration, inhalation administration, instillation administration, or nasal administration.
- the pharmaceutical composition will be formulated such that the pharmaceutical composition will be suitable for at least one of these administration routes.
- the pharmaceutical composition may be either a solid preparation such as a powder, a granule, a tablet or a capsule, or a liquid such as an injection, a syrup or an emulsion.
- the pharmaceutical composition can be produced in accordance with a routine method including mixing, kneading, granulating, tableting, coating, sterilizing and emulsifying, depending on the dosage form of the preparation.
- a routine method including mixing, kneading, granulating, tableting, coating, sterilizing and emulsifying, depending on the dosage form of the preparation.
- each section of General Rules for Preparations of the Japanese Pharmacopoeia for example, can be referred.
- the medicament of the present invention may be formed as a sustained release agent comprising an active ingredient and a biodegradable polymer compound.
- the dosage of the pharmaceutical composition varies depending on, e.g., the symptom; the age, sex, body weight and difference in sensitivity of the subject to be administered; timing and interval of administration, the feature, prescription and type of the medicament; and type of active ingredient and is not particularly limited.
- the present pharmaceutical composition may further comprise an immunosuppressant or an anti-inflammatory agent such as steroid and non-steroidal anti-inflammatory drugs (NSAIDs).
- an immunosuppressant or an anti-inflammatory agent such as steroid and non-steroidal anti-inflammatory drugs (NSAIDs).
- NSAIDs non-steroidal anti-inflammatory drugs
- the present pharmaceutical composition may be used in a combination with an immunosuppressant such as tacrolimus or an anti-inflammatory agent such as steroids and non-steroidal anti-inflammatory drugs (NSAIDs).
- an immunosuppressant such as tacrolimus
- an anti-inflammatory agent such as steroids and non-steroidal anti-inflammatory drugs (NSAIDs).
- NSAIDs non-steroidal anti-inflammatory drugs
- the present disclosure provides a method of treating a subject in need thereof.
- the subject may have an activated and/or increased Th17 cells in its body or body part.
- the subject may have an inflammation and/or an immune-related disease such as an excessive immune response, over-reaction of immune system, or autoimmune diseases.
- the subject may have an inflammation and/or immune-related disease that is caused by overexpression of IL-17, or an activated and/or increased Th17 cells.
- the subject may have a Th17 cell-mediated immune disease.
- Th17 cell-mediated immune diseases include, for example, but not limited to, autoimmune disease such as rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, encephalomyelitis, and psoriasis.
- the method comprises administering to the subject an effective amount of a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof.
- autoimmune disease such as rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, encephalomyelitis, and psoriasis.
- the method comprises administering to the subject an effective amount of a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (
- the present disclosure provides a method of treating an activated and/or increased Th17 cells in a subject in need thereof.
- the present disclosure provides a method of treating an inflammation and/or an immune-related disease such as an excessive immune response, over-reaction of immune system, or autoimmune diseases in a subject in need thereof.
- the present disclosure provides a method of treating an inflammation and/or immune-related disease that is caused by overexpression of IL-17, or an activated and/or increased Th17 cells.
- the present disclosure provides a method of treating Th17 cell-mediated immune disease.
- Th17 cell-mediated immune diseases include, for example, but not limited to, autoimmune disease such as rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, encephalomyelitis, and psoriasis.
- the method comprises administering to the subject an effective amount of a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof.
- a glycolytic intermediate metabolite or a salt thereof preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof.
- the subject may be treated in combination with an immunosuppressant such as tacrolimus or an anti-inflammatory agent such as steroids and non-steroidal anti-inflammatory drugs (NSAIDs).
- an immunosuppressant such as tacrolimus or an anti-inflammatory agent such as steroids and non-steroidal anti-inflammatory drugs (NSAIDs).
- the subject may be treated with an immunosuppressant such as tacrolimus or an anti-inflammatory agent such as steroids and non-steroidal anti-inflammatory drugs (NSAIDs) after the treatment with a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof.
- an immunosuppressant such as tacrolimus or an anti-inflammatory agent such as steroids and non-steroidal anti-inflammatory drugs (NSAIDs)
- NSAIDs non-steroidal anti-inflammatory drugs
- the dose of an immunosuppressant such as tacrolimus or an anti-inflammatory agent such as steroids and non-steroidal anti-inflammatory drugs (NSAIDs) can be reduced by administering a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof to the subject.
- a glycolytic intermediate metabolite or a salt thereof preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof to the subject.
- the dose of an immunosuppressant such as tacrolimus or an anti-inflammatory agent such as steroids and non-steroidal anti-inflammatory drugs (NSAIDs) can be reduced by administering an increased amount of a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof.
- a glycolytic intermediate metabolite or a salt thereof preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof.
- the present disclosure provides phosphoenolpyruvate or a salt thereof for use in a method of the present disclosure.
- the present disclosure provides a pharmaceutical composition, for use in a method of the present disclosure, comprising a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof.
- PEP glycolytic intermediate metabolite
- G6P glucose-6-phosphate
- F6P fructose-6-phosphate
- 3PG 3-phosphoglycerate
- the present disclosure provides use of a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof in the manufacture of a medicament for use in a method of the present disclosure.
- PEP phosphoenolpyruvate
- G6P glucose-6-phosphate
- F6P fructose-6-phosphate
- 3PG 3-phosphoglycerate
- the present disclosure provides a method of producing a medicament for use in a method of the present disclosure (for example, preventing or treating Th17-related or Th17-mediated immune disease or condition), comprising mixing at least a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof (for example, isolated or purified phosphoenolpyruvate or a salt thereof) and an additive to form the medicament.
- a glycolytic intermediate metabolite or a salt thereof preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyru
- the additive may be a pharmaceutically acceptable carrier, additives, or excipient.
- the examples of the pharmaceutically acceptable carrier, additives, or excipient include, but not limited to, various organic or inorganic carrier substances routinely used as a drug substance that are used and blended as an excipient, a lubricant, a binder or a disintegrator in a solid preparation; a solvent, a solubilizer, a suspending agent, a tonicity agent, a buffer or a soothing agent in a liquid preparation.
- a formulation additive such as a preservative, an antioxidant, a stabilizer, colorants or a sweetener can be used.
- the present disclosure provides a method of producing a supplement, food or drink for use in a method of the present disclosure (for example, preventing or treating Th17-related or Th17-mediated immune disease or condition), comprising mixing at least a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof (for example, isolated or purified phosphoenolpyruvate or a salt thereof) and an additive to form a supplements, food or drink, preferably a phosphoenolpyruvate-enriched supplements, food or drink.
- a glycolytic intermediate metabolite or a salt thereof preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F
- the additive may be edible.
- the examples of the edible additives may include supplements, drinks and foods.
- the additive may be a food additive.
- the supplement may comprise a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof (for example, isolated or purified phosphoenolpyruvate or a salt thereof) and a food additive.
- PEP phosphoenolpyruvate
- G6P glucose-6-phosphate
- F6P fructose-6-phosphate
- 3PG 3-phosphoglycerate
- the phosphoenolpyruvate-enriched food or drink may comprise a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof (for example, isolated or purified phosphoenolpyruvate or a salt thereof) and a food or drink.
- PEP phosphoenolpyruvate
- G6P glucose-6-phosphate
- F6P fructose-6-phosphate
- 3PG 3-phosphoglycerate
- the present composition may be a food or drink.
- the present composition may be a food or drink where a glycolytic intermediate metabolitea glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof is enriched.
- a glycolytic intermediate metabolitea glycolytic intermediate metabolite or a salt thereof preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof is enriched.
- the present composition may be a food or drink supplemented with a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof.
- a glycolytic intermediate metabolite or a salt thereof preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof.
- mice C57BL/6 mice were obtained from Clea (Tokyo, Japan), and OT-II and B6SJL mice were from the Jackson Laboratory (ME, USA). All mice were maintained under specific pathogen-free conditions. Gender-matched 6-12-week-old mice were utilized for experiments. All animal experimental protocols were approved by the Animal Care and Use Committee at Okinawa Institute of Science and Technology graduate University.
- anti-IFN ⁇ XMG1.2; Biolegend
- anti-IFN ⁇ MP6-XT22; Biolegend
- anti-IL4 11B11; Biolegend
- anti-IL13 eBio13A; eBioscience
- anti-IL10 JES5-16E3; Biolegend
- anti-IL17A TC11-18H10.1; Biolegend
- anti-ROR ⁇ t B2D; eBioscience
- anti-FOXP3 150D; Biolegend
- anti-T-bet 4B10; Biolegend
- anti-GATA3 (16E10A23; Biolegend)
- anti-CD4 GK1.5; Biolegend
- anti-CD62L MEL-14; Biolegend
- anti-CD25 PC61; Biolegend
- anti-CD44 IM7; Biolegend
- anti-IL2 JES6-5H4; Biolegend
- anti-CD3 17A2
- Antibodies for western blotting were as below: anti-BATF (WW8; Santa Cruz, USA), anti-IRF4 (4964; CST, USA), anti-JUND (329; Santa Cruz), anti-JUNB (C11; Santa Cruz), anti-STAT3 (79D7; CST), anti-phospho STAT3 (Tyr705) (D3A7; CST), anti-HIF1 ⁇ (28b; Santa Cruz), anti- ⁇ Actin (6D1; MBL, Japan), anti-PKM2 (D78A4, CST); anti-Histone H3 (D2B12; CST), anti- ⁇ tubulin (PM054; MBL), anti-Flag (M2; Sigma-Aldrich), anti-6X His-tag (RM146, Invitrogen), anti-mouse IgG, HRP-linked (7076; CST), and anti-rabbit IgG, HRP-linked (7074; CST).
- Naive murine CD4 T cells were isolated from spleens using MojoSort mouse CD4 naive T cell isolation kit (Biolegend) for most of in vitro T cell culture experiments.
- CD4 T cells were first enriched with MACS magnetic cell sorting system with anti-CD4 microbeads (Miltenyi), and then naive CD4 T cells (CD4 + CD25 - CD62L hi CD44 lo ) were sorted by FACS AriaII or AriaIII (BD).
- Isolated naive CD4 + T cells were cultured in Iscove’s modified Dulbecco’s medium (IMDM) (Invitrogen) supplemented with 10% FBS, 1X streptomycin-penicillin (Sigma-Aldrich), ⁇ -mercaptoethanol (55 ⁇ M; Invitrogen, USA), and anti-CD28 antibody (1 ⁇ g/mL; 37.51, Biolegend) in 24-well (2x10 5 cells / well) or 48-well (1x10 5 cells / well) plates coated with anti-CD3 ⁇ antibody (5 ⁇ g/mL; 145-2C11, Biolegend).
- IMDM modified Dulbecco’s medium
- the medium was further supplemented with IL-2 (20ng/mL, Biolegend), IL-12 (20 ng/mL; Biolegend), and anti-IL-4 (1 ⁇ g/mL; 11B11, Biolegend) for Th1; IL-2 (20 ng/mL), IL-4 (100 ng/mL; Biolegend), and anti-IFN- ⁇ (1 ⁇ g/mL; R4-6A2, Biolegend) for Th2; IL-6 (20 ng/mL; Biolegend) and TGF- ⁇ 1 (3 ng/mL; Miltenyi) for TH17(B); IL-6 (20 ng/mL), IL-1 ⁇ (20 ng/mL; Biolegend) and IL-23 (40 ng/mL; Biolegend) for TH17(23); TGF- ⁇ 1 (15 ng/mL), IL-2 (20 ng/mL), anti-IL-4 (1 ⁇ g/mL), and anti-IFN- ⁇ (1 ⁇ g/mL) for iT
- Glycolytic metabolites were added to the culture medium. Glycolytic metabolites were dissolved in water or PBS first, adjusted the pH value to 7.3, then added to the culture medium to reach the desired concentration.
- PMA phorbol 12- myristate 13-acetate
- ionomycin 500 ng/mL; Sigma-Aldrich
- brefeldin A 5 mg/mL; Biolegend
- Naive CD4 T cells activated under Th17-polarizing conditions at indicated time points were washed twice with PBS, snap-frozen in liquid nitrogen, and then stored at -80°C until further processing.
- the PEP fluorometric assay was performed with PEP colorimetric/fluorometric assay kits (Sigma-Aldrich) and a SpectraMax M2 96-well reader (Molecular Devices, USA) according to the manufacturer’s instructions.
- Naive CD4 T cells activated under Th17( ⁇ )- and Th17(23)-polarizing conditions for 72 h were harvested for measurements of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) with mito stress (Agilent Technologies, USA) and glycolysis stress kits (Agilent Technologies), respectively.
- OCR oxygen consumption rate
- ECAR extracellular acidification rate
- Cells were washed twice with PBS, transferred to an analysis plate (2 x 10 5 cells per well) coated with 2% gelatin (Sigma-Aldrich), and incubated at 37 o C for 1 h.
- OCR and ECAR were measured using a Seahorse XFe96 analyzer (Seahorse Bioscience, USA) following the manufacturer’s instructions.
- RNA samples were then mixed with ERCC RNA spike-in control mixes (Thermo), and mRNA was isolated with NEBNext poly(A) mRNA magnetic isolation module (E7490; NEB, USA).
- the sequencing Library was prepared with a Collibri Stranded RNA Library Prep Kit for Illumina Systems with Human/Mouse/Rat rRNA Depletion Kits (Thermo), according to the manufacturer’s instructions.
- RNA and ATAC sequencing were performed on an Illumina NovaSeq 6000 to generate 150-nucleotide, paired-end reads with a read depth of ⁇ 20 million reads per sample.
- Raw data processing was performed using nfcore/atacseq (v.1.2.1), a bioinformatics analysis pipeline used for ATAC-seq data at the National Genomics Infrastructure at SciLifeLab Sweden.
- adapters and low-quality reads were removed with Trim Galore!.
- Trimmed fastq files were mapped to the GRCm38 mouse reference genome with BWA, and narrow peaks were called with MACS2.
- the normalized BigWig files, scaled to 1 million mapped reads, were created with BEDTools and bedGraphToBigWig and were uploaded to the UCSC genome browser. Tool versions and full details of the pipeline are available at https://nf-co.re/atacseq.
- Motif enrichment within 2 kb upstream and downstream of the transcriptional start sites of DEGs in PEP-treated vs. control cells was analyzed using the findMotifs function of Homer (version v4.11). Binding motifs for AP-1 (ATGACTCATC), JunB (RATGASTCAT), BATF (DATGASTCAT), and IRF4 (ACTGAAACCA), and AICE (NAGTTTCABTHTGACTNW) within 10 kb upstream and downstream of transcriptional start sites of DEGs were identified using the mouse mm10 genome with the scanMotifGenomeWide.pl function of Homer v4.11.
- Membranes were blocked with 5% skim milk (Wako) or bovine albumin (Wako) in Tris-buffered saline with 0.1% Tween-20 (Sigma-Aldrich). Then they were hybridized with described antibodies at 4 o C overnight, followed by incubation with HRP-conjugated secondary antibodies at room temperature for 2 h. Reactive proteins were detected with Clarity Western ECL (Bio-Rad) or SuperSignal West Femto detection reagents (Thermo) on a Las-3000 imaging system (Fuji film, Japan) or iBright CL1500 Imaging System (Thermo).
- BATF-HA C-terminally HA-tagged BATF (gene ID: 53314)
- BATF-HA C-terminally Flag-tagged JunB (gene ID: 16477)
- gblock Integrated DNA Technology
- JunB-Flag C-terminally Flag-tagged JunB (gene ID: 16477)
- Opti-MEM 5 ⁇ g of pcDNA3.1-BATF-HA and pcDNA3.1-JunB-Flag in 250ul Opti-MEM were mixed with 25 ⁇ L of polyethylenimine (1 mg/mL) (Cosmobio, Japan) in 250 ⁇ L of Opti-MEM, incubated at room temperature for 30 min, and then added to the cell culture. Cells were harvested after 60 h for co-immunoprecipitation assay.
- HEK293 cells transfected with pcDNA3.1-BATF-HA and pcDNA3.1-JunB-Flag were washed with PBS twice, freeze-thawed by liquid nitrogen twice, and lysed in Triton X-100 lysis buffer (1% Triton X-100, 150 mM NaCl, 20 mM Tris (pH 7.5) containing complete protease inhibitor cocktail (Roche) ) on ice for 30 min (briefly vortexed every 10 min). Then, cellular debris was removed by centrifugation at 13,000 g for 10 min.
- Triton X-100 lysis buffer 1% Triton X-100, 150 mM NaCl, 20 mM Tris (pH 7.5) containing complete protease inhibitor cocktail (Roche)
- Lysates (3 mg total protein) were incubated with 3 ⁇ g of anti-Flag (M2; Sigma-Aldrich) or anti-mouse IgG (G3A1; CST) antibodies together with or without PEP (50, 200, and 500 ⁇ M) on a rotator at 4°C for 16 h, followed by incubation with 15 ⁇ L of Dynabeads protein G (Invitrogen) on a rotator at 4°C for another hour.
- M2 anti-Flag
- G3A1 anti-mouse IgG
- CST CST
- chromatin immunoprecipitation was performed using SimpleChIP kits (CST) and Dynabeads protein G (Invitrogen) according to the manufacturer’s instructions, except for two modifications: (1) the amount of micrococcal nuclease was reduced to 0.05 ⁇ L per million cells; (2) chromatin-bound beads were washed with low-salt wash solution 4 times, followed by high-salt wash solution 2 times at 4 o C for 5 min.
- CST SimpleChIP kits
- Dynabeads protein G Invitrogen
- anti-BATF ww8; Santa Cruz
- anti-JunB C-11; Santa Cruz
- anti-IRF4 4964; CST
- anti-mouse IgG G3A1; CST
- anti-Rabbit IgG 2729; CST
- mice 8-9-week-old, gender-matched B6SJL mice were subcutaneously (s.c.) injected with vehicle (200 ⁇ L PBS) or PEP (1g/kg body weight, dissolved in 200 ⁇ L PBS, pH adjusted to 7.3), followed 6 h later by intravenous injection of naive CD4 T cells isolated from OT-II mice (CD45.1 + CD45.2 + ).
- mice were immunized with OVA 323-339 (50 ⁇ g per mouse; ISQAVHAAHAEINEAGR, GL Biochem, China) emulsified in CFA (200 ⁇ L per mouse) supplemented with or without PEP (10 mg/mouse).
- mice were s.c. injected with vehicle or PEP as described above.
- mice were euthanized, and cells isolated from inguinal lymph nodes and spleens were analyzed as described above.
- mice 8-week-old, female C57BL/6 mice were s.c. injected with MOG 35-55 peptides (300 mg per mouse) emulsified in complete Freund’s adjuvant (CFA) (200 ⁇ L per mouse) containing dead Mycobacterium tuberculosis (1 mg per mouse) on day 0.
- CFA complete Freund’s adjuvant
- pertussis toxin 400 ng per mouse was intraperitoneally injected into mice. From day 0 (8 h prior to MOG immunization) until the end of experiments, mice were s.c. injected with vehicle (200 ⁇ L PBS) or PEP (1g/kg per mice, dissolved in 200 ⁇ L PBS, pH adjusted to 7.3) daily.
- RNA-seq and ATAC-seq data that support the finding of this study have been deposited to DDBJ database (DRA014503).
- naive CD4 T cells to Th17 cells with TGF- ⁇ and IL-6 (known as non-pathogenic Th17 (npTh17) cells) in media supplemented with individual glycolytic intermediate metabolites.
- npTh17 non-pathogenic Th17
- G6P glucose-6-phosphate
- F6P fructose-6-phosphate
- 3PG 3-phosphoglycerate
- PEP significantly decreased IL-17A expression without affecting cell viability
- Figure 1B fructose 1,6 bisphosphate
- G3P glycerol-3-phosphate
- HA heptelidic acid
- GPDH glyceraldehyde phosphate dehydrogenase
- OXA oxalate
- PEP inhibits differentiation of Th17 and Th2 cells, but not Th1 and iTreg cells
- PEP inhibits IL-17A expression independently of its role in glycolysis
- PKM2 the glycolytic enzyme catalyzing the conversion of PEP to pyruvate, is upregulated upon TCR stimulation, and a portion of PKM2 translocates to the nucleus to promote the transcriptional program for Th1 and Th17 differentiation (Angiari et al., 2020; Damasceno et al., 2020; Kono et al., 2019).
- PEP supplementation did not influence PKM2 expression or nuclear translocation in Th17 cells (Figure 3E).
- PEP supplementation inhibits Th17 differentiation without affecting glycolysis or PKM2 activity.
- PEP supplementation significantly inhibited IL-17 expression even in 2-DG-treated cells ( Figure 3F), suggesting that PEP can inhibit IL-17 expression independently of its role in glycolysis.
- PEP suppresses DNA-binding of JunB, BATF, and IRF4 at the Il17 locus
- ovalbumin ovalbumin
- CFA complete Friend’s adjuvant
- PEP an immunoregulatory metabolite that inhibits pathogenic Th17 differentiation. Metabolites can link cellular metabolism to regulation of gene expression at epigenetic and transcriptional levels (Chapman et al., 2020; Shyer et al., 2020; Zhang et al., 2018), but the role of glycolytic metabolites in T cells remains largely uncharacterized, despite a close relationship between glycolysis and T cell biology (Palmer et al., 2015).
- Our data demonstrate that PEP inhibits expression of Th17 signature molecules, including IL-17A, both in vitro and in vivo. Notably, PEP administration to mice suppresses Th17 generation and EAE, a Th17-dependent autoimmune neuroinflammatory disease. Mechanistically, PEP suppresses JunB-dependent pathogenic Th17 transcriptional program by inhibiting the DNA-binding activity of the transcription complex of JunB, BATF and IRF4.
- a transcription factor complex composed of a BATF-containing AP-1 dimer and IRF4 plays a central role in Th17 differentiation by acting as a pioneering transcription factor that promotes chromatin accessibility of various Th17-related genes (Ciofani et al., 2012; Pham et al., 2019).
- JunB one of the BATF dimeric partners, is required for transcriptional control of a subset of genes regulated by BATF and IRF4 (Carr et al., 2017; Hasan et al., 2017; Yamazaki et al., 2017).
- JunB loss of JunB inhibits IL-17A expression in both pathogenic and non-pathogenic Th17 cells, but it decreases ROR ⁇ t and IL-23R expression in pathogenic Th17 cells but not in non-pathogenic Th17 cells (Hasan et al., 2017).
- JunB also regulates Th2 differentiation, but not Th1 and iTreg differentiation (Hsieh et al., 2022).
- our data show a similar context-dependent inhibitory effect on the differentiating Th2 and Th17 cells by PEP supplementation.
- PEP inhibits binding of JunB, BATF, and IRF4 at the Il17a locus, without affecting chromatin accessibility.
- RNA-seq data reveal that PEP supplementation inhibits expression of Il-17a, but not Irf8, although JunB directly promotes transcription of both Il17a and Irf8 in npTh17 cells (Carr et al., 2017; Hasan et al., 2017). Consistent with this, our ChIP-PCR data indicate that PEP regulates DNA-binding of JunB, BATF, and IRF4 at the Il17a locus, but not at the Irf8 locus. This locus-specific effects on DNA-binding of JunB, BATF, and IRF4 may account for the effect of PEP supplementation on the expression of a specific subset of genes regulated by JunB in Th17 cells. Our results show that PEP supplementation does not impair the expression of JunB, BATF, and IRF4 and the interaction between JunB and BATF. Thus, PEP likely regulates activity of JunB/BATF/IRF complex in a locus-specific manner.
- PEP-dependent inhibition of JunB activity mediates the crosstalk between metabolic reprogramming of glycolysis and the Th17 transcriptional program.
- PEP can inhibit Th17-dependent autoimmunity by controlling JunB-dependent pathogenic Th17 transcriptional program without significantly affecting glycolysis and T cell activation. Since JunB is required for differentiation of pathogenic Th17, but not non-pathogenic Th17 cells, a new approach utilizing PEP or its derivatives to modulate JunB function might have potential for selective therapy of autoimmunity.
- Interleukin-23 rather than interleukin-12 is the critical cytokine for autoimmune inflammation of the brain. Nature 421, 744-748. 10.1038/nature01355.
- Metabolites can regulate stem cell behavior through the STAT3/AKT pathway in a similar trend to that under hypoxic conditions.
- BATF-JUN is critical for IRF4-mediated transcription in T cells. Nature 490, 543-546. 10.1038/nature11530. Macintyre, A.N., Gerriets, V.A., Nichols, A.G., Michalek, R.D., Rudolph, M.C., Deoliveira, D., Anderson, S.M., Abel, E.D., Chen, B.J., Hale, L.P., and Rathmell, J.C. (2014).
- the glucose transporter Glut1 is selectively essential for CD4 T cell activation and effector function.
- TGF-beta and IL-6 drive the production of IL-17 and IL-10 by T cells and restrain T(H)-17 cell-mediated pathology. Nat Immunol 8, 1390-1397. 10.1038/ni1539.
- the interleukin 23 receptor is essential for the terminal differentiation of interleukin 17-producing effector T helper cells in vivo. Nat Immunol 10, 314-324. 10.1038/ni.1698. O'Neill, L.A., Kishton, R.J., and Rathmell, J. (2016). A guide to immunometabolism for immunologists. Nat Rev Immunol 16, 553-565. 10.1038/nri.2016.70.
- HIF1alpha-dependent glycolytic pathway orchestrates a metabolic checkpoint for the differentiation of TH17 and Treg cells.
- TGFbeta in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing T cells.
- the AP-1 transcription factor JunB is required for Th17 cell differentiation.
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Abstract
The present disclosure provides a composition comprising a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof. The composition can be a pharmaceutical composition for use in treating immune-related disease, such as autoimmune disease, and disease associated with an activated or increased T helper 17 cell.
Description
The present disclosure relates to a composition comprising an isolated or enriched glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof. The present disclosure also relates to a pharmaceutical composition comprising an isolated or enriched glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof.
Cellular metabolism is closely associated with T cell differentiation and functions. Differentiation of naive T cells into effector T cell subsets relies on metabolic reprogramming from oxidative phosphorylation to aerobic glycolysis, while differentiation into memory T cells and T regulatory (Treg) cells is largely dependent on mitochondrial fatty acid oxidation (Chapman et al., 2020; Man and Kallies, 2015; Pearce et al., 2013). The primary role of aerobic glycolysis is to fulfill the demand for energy and building blocks for anabolism in rapidly proliferating effector T cells (O'Neill et al., 2016; Palmer et al., 2015; Zhu and Thompson, 2019). However, aerobic glycolysis also promotes T cell receptor (TCR) signaling and expression of genes related to effector T cell differentiation through non-glycolytic functions of glycolytic enzymes and metabolites (Geltink et al., 2018; Zhu and Thompson, 2019). Importantly, manipulation of metabolic activity can alter T cell fate decisions and functions, providing new therapeutic approaches for controlling inflammation (Palsson-McDermott and O’Neill, 2020; Patel et al., 2019).
Aerobic glycolysis is required for differentiation and pathogenicity of T helper 17 (Th17) cells, a subset of effector CD4 T cells expressing interleukin-17 (IL-17) (Geltink et al., 2018; Omenetti et al., 2019). Differentiation of Th17 cells is induced in the presence of cytokines IL-6 and TGF-b (Bettelli et al., 2006; Mangan et al., 2006; Veldhoen et al., 2006), and its pathogenicity is dependent on the IL-23 signal (Cua et al., 2003; Ghoreschi et al., 2010; Hirota et al., 2013; Lee et al., 2012; McGeachy et al., 2007; McGeachy et al., 2009; Stockinger and Omenetti, 2017). Pathogenic Th17 cells that cause autoimmunity exhibit higher expression of glycolytic genes and glycolytic activity than gut-resident homeostatic Th17 cells (Omenetti et al., 2019; Wu et al., 2020). High glucose intake promotes Th17 differentiation (Zhang et al., 2019), while loss of glucose transporters decreases Th17 generation and pathogenicity (Hochrein et al., 2022; Macintyre et al., 2014). Furthermore, genetic ablation of glycolytic enzymes dramatically impairs clonal expansion of Th17 cells (Wu et al., 2020; Xu et al., 2021). Paradoxically, however, blockade of glycolysis by pharmacological inhibitors can enhance IL-17 expression in Th17 cells (Brucklacher-Waldert et al., 2017), although contradictory results have also been reported (Shi et al., 2011).
The AP-1 transcription factors, JunB and basic leucine zipper transcription factor ATF-like (BATF), collaborate with interferon regulatory factor 4 (IRF4) to regulate the transcriptional program for Th17 differentiation (Carr et al., 2017; Hasan et al., 2017; Yamazaki et al., 2017). These transcription factors are induced upon TCR stimulation (Iwata et al., 2017; Schraml et al., 2009) and form JunB/BATF/IRF4 trimers that bind to AP-1 IRF composite elements (AICEs) adjacent to the target genes (Ciofani et al., 2012; Glasmacher et al., 2012; Li et al., 2012). The BATF and IRF4 are indispensable for differentiation of both pathogenic and non-pathogenic Th17 cells (Brustle et al., 2007; Huber et al., 2008; Schraml et al., 2009). They promote expression of Th17-signature molecules, such as IL-17A, retinoic acid-related orphan receptor (ROR)gt and IL-23 receptor (IL-23R) by enhancing the chromatin accessibility (Ciofani et al., 2012; Pham et al., 2019). In contrast, JunB is selectively required for pathogenic Th17 generation, probably due to functional redundancy among Jun family members (Carr et al., 2017; Hasan et al., 2017). Whether cellular metabolism controls the function of AP-1 transcription factors remains unexplored.
The present disclosure provides a composition comprising an isolated or enriched glycolytic intermediate metabolite or a salt thereof. The present disclosure provides a composition comprising an isolated or enriched phosphoenolpyruvate or a salt thereof. The present disclosure also provides a pharmaceutical composition comprising an isolated or enriched glycolytic intermediate metabolite or a salt thereof. The present disclosure also provides a pharmaceutical composition comprising an isolated or enriched phosphoenolpyruvate or a salt thereof. According to the present disclosure, glycolytic intermediate metabolite or a salt thereof can inhibit an activity or induction of T helper 17 (Th17) cells. According to the present disclosure, phosphoenolpyruvate or a salt thereof can inhibit an activity or induction of T helper 17 (Th17) cells.
The inventors identify a glycolytic intermediate metabolite, such as phosphoenolpyruvate (PEP), as a negative regulator of pathogenic Th17 generation. We found that PEP inhibits the generation of Th17 cells in vitro and in vivo, and administration of PEP to mice ameliorates Th17-dependent autoimmune encephalomyelitis. Mechanistically, PEP regulates JunB-dependent pathogenic Th17 transcriptional program by inhibiting DNA-binding activity of JunB, BATF, and IRF4. These findings shed light on glycolysis-dependent negative regulation of pathogenic Th17 differentiation, which might be a novel therapeutic target for autoimmune diseases.
The present disclosure provides, for example, inventions described below.
(1) A composition, for use in a method of preventing, inhibiting, or reducing activation of T helper 17 (Th17) cell immunity or development of Th17-mediated immune disease or condition in a subject in need thereof, comprising an effective amount of an isolated or enriched glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof.
(2) The composition of (1) above, wherein the isolated or enriched glycolytic intermediate metabolite comprises or a salt thereof is one or more selected from the group consisting of phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), and 3-phosphoglycerate (3PG), and a salt thereof.
(3) The composition of (1) or (2) above, wherein the isolated or enriched glycolytic intermediate metabolite or a salt thereof comprises or is phosphoenolpyruvate (PEP).
(4) The composition of any one of (1) to (3) above, wherein the composition is a food composition, a drink composition, a supplement, or a pharmaceutical composition.
(5) A pharmaceutical composition, for use in a method of preventing, inhibiting, or reducing T helper 17 (Th17) cell immunity in a subject in need thereof, comprising an effective amount of an isolated or enriched glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof.
(6) A pharmaceutical composition, for use in a method of treating an inflammation or disease associated with an activated or increased T helper 17 (Th17) cell immunity in a subject in need thereof, comprising an effective amount of an isolated or enriched glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof.
(7) The composition of (5) or (6) above, wherein the isolated or enriched glycolytic intermediate metabolite or a salt thereof comprises or is one or more selected from the group consisting of phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), and 3-phosphoglycerate (3PG), and a salt thereof.
(8) The composition of (5) or (6) above, wherein the isolated or enriched glycolytic intermediate metabolite or a salt thereof comprises or is phosphoenolpyruvate (PEP).
(9) The pharmaceutical composition of (5) or (8) above, wherein the subject is suffered from an autoimmune disease.
(10) The pharmaceutical composition of any one of (5) to (9) above, wherein the pharmaceutical composition is formulated as a parenteral formulation.
(11) The pharmaceutical composition of any one of (5) to (9) above, wherein the pharmaceutical composition is formulated as a transdermal formulation.
(1) A composition, for use in a method of preventing, inhibiting, or reducing activation of T helper 17 (Th17) cell immunity or development of Th17-mediated immune disease or condition in a subject in need thereof, comprising an effective amount of an isolated or enriched glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof.
(2) The composition of (1) above, wherein the isolated or enriched glycolytic intermediate metabolite comprises or a salt thereof is one or more selected from the group consisting of phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), and 3-phosphoglycerate (3PG), and a salt thereof.
(3) The composition of (1) or (2) above, wherein the isolated or enriched glycolytic intermediate metabolite or a salt thereof comprises or is phosphoenolpyruvate (PEP).
(4) The composition of any one of (1) to (3) above, wherein the composition is a food composition, a drink composition, a supplement, or a pharmaceutical composition.
(5) A pharmaceutical composition, for use in a method of preventing, inhibiting, or reducing T helper 17 (Th17) cell immunity in a subject in need thereof, comprising an effective amount of an isolated or enriched glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof.
(6) A pharmaceutical composition, for use in a method of treating an inflammation or disease associated with an activated or increased T helper 17 (Th17) cell immunity in a subject in need thereof, comprising an effective amount of an isolated or enriched glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof.
(7) The composition of (5) or (6) above, wherein the isolated or enriched glycolytic intermediate metabolite or a salt thereof comprises or is one or more selected from the group consisting of phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), and 3-phosphoglycerate (3PG), and a salt thereof.
(8) The composition of (5) or (6) above, wherein the isolated or enriched glycolytic intermediate metabolite or a salt thereof comprises or is phosphoenolpyruvate (PEP).
(9) The pharmaceutical composition of (5) or (8) above, wherein the subject is suffered from an autoimmune disease.
(10) The pharmaceutical composition of any one of (5) to (9) above, wherein the pharmaceutical composition is formulated as a parenteral formulation.
(11) The pharmaceutical composition of any one of (5) to (9) above, wherein the pharmaceutical composition is formulated as a transdermal formulation.
The present disclosure also provides, for example, inventions described below.
(12) A pharmaceutical composition, for use in a method of treating an inflammation or an immune-related disease in a subject in need thereof, comprising an effective amount of an isolated or enriched glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof.
(13) The pharmaceutical composition of (12) above, wherein the immune-related disease is an autoimmune disease.
(14) The pharmaceutical composition of (12) above, wherein the immune-related disease is selected from the group consisting of rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, encephalomyelitis, and psoriasis.
(15) The pharmaceutical composition of (12) above, wherein the inflammation and the immune-related disease are caused by or associated with an activated or increased T helper 17 (Th17) cell immunity.
(16) The pharmaceutical composition of (13) above, wherein the inflammation and the immune-related disease are caused by or associated with an activated or increased T helper 17 (Th17) cell immunity.
(17) The pharmaceutical composition of (14) above, wherein the inflammation and the immune-related disease are caused by or associated with an activated or increased T helper 17 (Th17) cell immunity.
(12) A pharmaceutical composition, for use in a method of treating an inflammation or an immune-related disease in a subject in need thereof, comprising an effective amount of an isolated or enriched glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof.
(13) The pharmaceutical composition of (12) above, wherein the immune-related disease is an autoimmune disease.
(14) The pharmaceutical composition of (12) above, wherein the immune-related disease is selected from the group consisting of rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, encephalomyelitis, and psoriasis.
(15) The pharmaceutical composition of (12) above, wherein the inflammation and the immune-related disease are caused by or associated with an activated or increased T helper 17 (Th17) cell immunity.
(16) The pharmaceutical composition of (13) above, wherein the inflammation and the immune-related disease are caused by or associated with an activated or increased T helper 17 (Th17) cell immunity.
(17) The pharmaceutical composition of (14) above, wherein the inflammation and the immune-related disease are caused by or associated with an activated or increased T helper 17 (Th17) cell immunity.
The present disclosure also provides, for example, inventions described below.
(21) A method of treating a subject in need thereof, comprising administering to the subject an effective amount of a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof.
(22) The method of (21) above, wherein the subject has an activated or increased T helper 17 (Th17) cell immunity.
(23) The method of (21) above, wherein the subject has an inflammation or an immune-related disease.
(24) The method of (22) above, wherein the subject has an inflammation or an immune-related disease.
(25) The method of any one of (21) above, wherein the inflammation and the immune-related disease are caused by or associated with an activated or increased T helper 17 (Th17) cell immunity.
(26) The method of any one of (23) above, wherein the inflammation and the immune-related disease are caused by or associated with an activated or increased T helper 17 (Th17) cell immunity.
(27) The method of any one of (24) above, wherein the inflammation and the immune-related disease are caused by or associated with an activated or increased T helper 17 (Th17) cell immunity.
(28) The method of any one of (21) to (27) above, wherein the immune-related disease is an autoimmune disease.
(29) The method of (28) above, wherein the immune-related disease is selected from the group consisting of rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, encephalomyelitis, and psoriasis.
(30) A method of treating a subject in need thereof, comprising intravenously, transdermally or intradermally administering to the subject an effective amount of a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof, wherein the subject has psoriasis.
(31) A method of treating a subject in need thereof, comprising intravenously or intraarticularly administering to the subject an effective amount of a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof, wherein the subject has rheumatoid arthritis.
(32) A method of treating a subject in need thereof, comprising intravenously or orally administering to the subject an effective amount of a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof, wherein the subject has inflammatory bowel disease.
(33) A method of treating a subject in need thereof, comprising intracerebrospinally, intravenously or orally administering to the subject an effective amount of a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof, wherein the subject has multiple sclerosis.
(34) A method of treating a subject in need thereof, comprising intracerebrospinally, intravenously or orally administering to the subject an effective amount of a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof, wherein the subject has encephalomyelitis.
(21) A method of treating a subject in need thereof, comprising administering to the subject an effective amount of a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof.
(22) The method of (21) above, wherein the subject has an activated or increased T helper 17 (Th17) cell immunity.
(23) The method of (21) above, wherein the subject has an inflammation or an immune-related disease.
(24) The method of (22) above, wherein the subject has an inflammation or an immune-related disease.
(25) The method of any one of (21) above, wherein the inflammation and the immune-related disease are caused by or associated with an activated or increased T helper 17 (Th17) cell immunity.
(26) The method of any one of (23) above, wherein the inflammation and the immune-related disease are caused by or associated with an activated or increased T helper 17 (Th17) cell immunity.
(27) The method of any one of (24) above, wherein the inflammation and the immune-related disease are caused by or associated with an activated or increased T helper 17 (Th17) cell immunity.
(28) The method of any one of (21) to (27) above, wherein the immune-related disease is an autoimmune disease.
(29) The method of (28) above, wherein the immune-related disease is selected from the group consisting of rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, encephalomyelitis, and psoriasis.
(30) A method of treating a subject in need thereof, comprising intravenously, transdermally or intradermally administering to the subject an effective amount of a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof, wherein the subject has psoriasis.
(31) A method of treating a subject in need thereof, comprising intravenously or intraarticularly administering to the subject an effective amount of a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof, wherein the subject has rheumatoid arthritis.
(32) A method of treating a subject in need thereof, comprising intravenously or orally administering to the subject an effective amount of a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof, wherein the subject has inflammatory bowel disease.
(33) A method of treating a subject in need thereof, comprising intracerebrospinally, intravenously or orally administering to the subject an effective amount of a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof, wherein the subject has multiple sclerosis.
(34) A method of treating a subject in need thereof, comprising intracerebrospinally, intravenously or orally administering to the subject an effective amount of a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof, wherein the subject has encephalomyelitis.
The present disclosure also provides, for example, inventions described below.
(51) Use of a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof in the manufacture of a medicament for use in a method of any one of (21) to (29) above.
(52) A glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof for use in a method of any one of (21) to (29) above.
(53) A pharmaceutical composition, for use in a method of any one of (21) to (29) above, comprising a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof.
(54) A composition, for use in a method of preventing, inhibiting, or reducing JunB-dependent generation of autoimmune T helper 17 (Th17) cells in a subject in need thereof, comprising an effective amount of an isolated or enriched glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof.
(51) Use of a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof in the manufacture of a medicament for use in a method of any one of (21) to (29) above.
(52) A glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof for use in a method of any one of (21) to (29) above.
(53) A pharmaceutical composition, for use in a method of any one of (21) to (29) above, comprising a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof.
(54) A composition, for use in a method of preventing, inhibiting, or reducing JunB-dependent generation of autoimmune T helper 17 (Th17) cells in a subject in need thereof, comprising an effective amount of an isolated or enriched glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof.
Definition
The term “subject” as used herein refers to an animal, for example, a mammal, in particular a dog, a cat, a horse, sheep, cow, or a human.
The term “treating” as used herein refers to a therapy or a prevention. The term “therapy” as used herein means therapy, cure, or prevention of a disease or a disorder; improvement of remission; or a reduction in the speed of progress of a disease or a disorder. The term “prevention” as used herein means a reduction in risk of onset of a disease or a pathologic state or a delay of onset of a disease or a pathologic state.
The term “isolated” as used herein refers to a separation of a substance from at least one element in a composition the substance was originally contained in. The term “enriched” as used herein refers to making something more concentrated, compared to another substance or the original concentration.
The term “pharmaceutical composition” as used herein refers to a composition suitable for a pharmaceutical application. The term “pharmaceutical composition” may also refer to a composition for use in a pharmaceutical application.
The term “disease” means a symptom or illness, preferably a symptom or illness of which therapy is helpful.
The term “immune-related disease” means a disease caused by over-reactivity, disfunction or malfunction of immune system. Diseases caused by over-reactivity of immune system includes, for example, but not limited to autoimmune disease. There are a variety of autoimmune diseases. The most common autoimmune diseases include Graves’ disease, rheumatoid arthritis, Hashimoto’s thyroiditis, type 1 diabetes, systemic lupus erythematosus, and vasculitis. Other diseases considered autoimmune include Addison’s disease, polymyositis, Sjogren’s syndrome, myositis, dermatomyositis, scleroderma, progressive systemic scleroderma, many forms of glomerulonephritis (kidney inflammation), and some forms of infertility.
The term “Th17 cell” means a subset of helper T cells that can produce interleukin-17 (IL-17). Th17 cells are produced from naive CD4 T cells (Th0). When Th0 cells are stimulated with interleukin-6 (IL-6) and transforming growth factor-β (TGF-β), Th0 cells express RAR-related orphan receptor γt (RORγt) to differentiate into Th17 cells. Further addition of Interleukin-23 (IL-23) to IL-6 and IL-1β upon Th0 cell stimulation leads to pathogenic Th17 cells (pTh17), while IL-6 and TGF-β without IL-23 leads to non-pathogenic Th17 cells (npTh17). IL-23 and JunB are positively involved in differentiation of pathogenic Th17 cells, and are not required in order to induce non-pathogenic Th17 cells. IL-17 is mainly produced by Th17 cells, and acts on fibroblasts, epithelial cells, endothelial cells, or macrophages to produce inflammatory cytokines and chemokines and then induce inflammation. Th17 cell relates to autoimmune disease such as rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, encephalomyelitis, and psoriasis. Thus, it can be thought that inhibiting or decreasing Th17 cell will result in a treatment of inflammations and immune-related diseases such as autoimmune diseases, including Th17-related diseases.
The term “phosphoenolpyruvate” (PEP) as used herein refers to a metabolite (or an intermediate) in glycolytic system. Phosphoenolpyruvate has the following formula:
.
Phosphoenolpyruvate is generated from 2-phosphoglycerate by phosphoglycerate isomerase in vivo. Pyruvate can be generated from phosphoenolpyruvate by pyruvate kinase. Also, oxaloacetic acid (OAA) can be generated from phosphoenolpyruvate by phosphoenolpyruvate carboxylase. A glycolytic intermediate metabolite and phosphoenolpyruvate is in form of monomer. A glycolytic intermediate metabolite or phosphoenolpyruvate can be isolated, enriched, synthesized or purified.
.
Phosphoenolpyruvate is generated from 2-phosphoglycerate by phosphoglycerate isomerase in vivo. Pyruvate can be generated from phosphoenolpyruvate by pyruvate kinase. Also, oxaloacetic acid (OAA) can be generated from phosphoenolpyruvate by phosphoenolpyruvate carboxylase. A glycolytic intermediate metabolite and phosphoenolpyruvate is in form of monomer. A glycolytic intermediate metabolite or phosphoenolpyruvate can be isolated, enriched, synthesized or purified.
The term “glycolytic system” refers to a metabolic pathway that converts glucose into pyruvate. In glycolytic system, (i) glucose is converted by hexokinase to glucose-6-phosphate. In glycolytic system, (ii) glucose-6-phosphate is then converted by glucose-6-phosphate isomerase to fructose-6-phosphate. In glycolytic system, (iii) fructose-6-phosphate is converted by phosphofructokinase-1 to fructose 1,6-bisphosphate. In glycolytic system, (iv) fructose 1,6-bisphosphate is converted by fructose 1,6-bisphosphate aldolase to dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. In glycolytic system, (v) glyceraldehyde 3-phosphate is converted by glyceraldehyde phosphate dehydrogenase to 1,3-bisphosphoglycerate. In glycolytic system, (vi) 1,3-bisphosphoglycerate is converted by phosphoglycerate kinase to 3-phosphoglycerate. In glycolytic system, (vii) 3-phosphoglycerate is converted by phosphoglycerate mutase to 2-phosphoglycerate. In glycolytic system, (viii) 2-phosphoglycerate is converted by enolase to phosphoenolpyruvate. In glycolytic system, (ix) phosphoenolpyruvate is converted by pyruvate kinase to pyruvate. Example of metabolites in glycolytic system include, for example, but not limited to, glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), 3-phosphoglycerate (3PG), fructose 1,6 bisphosphate (F1,6BP), glycerol-3-phosphate (G3P), and phosphoenolpyruvate, preferably phosphoenolpyruvate. In a preferable embodiment, the metabolite or a salt thereof is one or more selected from the group consisting of phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), and 3-phosphoglycerate (3PG), and a salt thereof, more preferably phosphoenolpyruvate (PEP).
The term “treatment” or related words as used herein refers to therapeutic treatment or preventive treatment.
The present composition or pharmaceutical composition
Aerobic glycolysis, a metabolic pathway essential for effector T cell survival and proliferation, regulates the differentiation of autoimmune T helper (Th)17 cells, but the mechanism underlying this regulation was largely unknown. Here, the inventors identify a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof, as a negative regulator of Th17 differentiation. PEP supplementation or inhibition of downstream glycolytic enzymes in differentiating Th17 cells increases intracellular PEP levels and inhibits expression of Th17 signature molecules, such as IL-17A. However, PEP supplementation does not significantly affect metabolic reprogramming, cell proliferation, and survival of differentiating Th17 cells. Mechanistically, PEP regulates JunB-mediated pathogenic Th17 transcriptional program by inhibiting DNA-binding activity of the JunB/BATF/IRF4 complex. Furthermore, daily administration of PEP to mice inhibits generation of Th17 cells and ameliorates Th17-dependent autoimmune encephalomyelitis. These data demonstrate that PEP links aerobic glycolysis to the JunB-dependent pathogenic Th17 transcriptional program, suggesting the therapeutic potential of PEP for autoimmune diseases.
The present disclosure provides a composition comprising a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof. According to the present disclosure, phosphoenolpyruvate or a salt thereof can inhibit an expression of a Th17 signature molecules such as IL-17A; inhibit production of Th17 cells; and therefore, inhibit pathogenic Th17 cells that are pathogenetically activated, and/or inhibit inflammation and/or immune-related disease or condition such as an excessive immune response, over-reaction of immune system, or autoimmune diseases. In an embodiment, the composition can be a food composition, a drink composition, or a supplement. The composition can be used for treating a condition, preferably a non-pathological condition, associated with pathogenic Th17 cells, or increased or activated Th17 cells.
The present disclosure provides a pharmaceutical composition comprising a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof. The pharmaceutical composition can be used for inhibiting an expression of a Th17 signature molecules such as IL-17A; inhibiting production of Th17 cells; and/or therefore, inhibiting pathogenic Th17 cells that are pathogenetically activated, and/or treating inflammation and/or immune-related disease such as an excessive immune response, over-reaction of immune system, or autoimmune diseases. In an embodiment, an inflammation and an immune-related disease are caused by pathogenic Th17 cells, or activated or increased Th17 cells. A regulatory T cell (Treg) can be suitably induced in the presence of a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof. In an embodiment, the pharmaceutical composition can be used for treating a pathological condition or disease that can be associated with pathogenic Th17 cells, or activated or increased Th17 cells, such as an inflammation and an immune-related disease.
In an embodiment, phosphoenolpyruvate or a salt thereof is a major active ingredient in the composition or the pharmaceutical composition. In an embodiment, a glycolytic intermediate metabolite or a salt thereof is a major active ingredient in the composition or the pharmaceutical composition.
In an embodiment, 1w/w% or more, 2w/w% or more, 3w/w% or more, 4w/w% or more, 5w/w% or more, 6w/w% or more, 7w/w% or more, 8w/w% or more, 9w/w% or more, 10w/w% or more, 20w/w% or more, 30w/w% or more, 40w/w% or more, 50w/w% or more, 60w/w% or more, 70w/w% or more, 80w/w% or more, 90w/w% or more, 95w/w% or more, or 100w/w% of the active ingredients included in the composition or the pharmaceutical composition may be a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof.
In an embodiment, the composition or the pharmaceutical composition may comprise 0.1w/w% or more, 0.2w/w% or more, 0.3w/w% or more, 0.4w/w% or more, 0.5w/w% or more, 0.6w/w% or more, 0.7w/w% or more, 0.8w/w% or more, 0.9w/w% or more, 1w/w% or more, 2w/w% or more, 3w/w% or more, 4w/w% or more, 5w/w% or more, 6w/w% or more, 7w/w% or more, 8w/w% or more, 9w/w% or more, 10w/w% or more, 20w/w% or more, 30w/w% or more, 40w/w% or more, 50w/w% or more, 60w/w% or more, 70w/w% or more, 80w/w% or more, 90w/w% or more, or 100w/w% of a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof.
In an embodiment, a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof is in a free form (i.e., without or outside nano particles). In an embodiment, a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof is in a form of monomer (i.e., is not polymerized to form an oligomer or a polymer). A glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof can penetrate the cell membrane and enter into cells (for example, Th17 cell and Th0 cell), particularly Th17 cell or Th0 cell, which has a low intracellular PEP concentration, even without using drug delivery system (DDS). In an embodiment, a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof is encapsulated in a particle, such as nanoparticle, lipid-base nanoparticle (LNP), liposome, and micelle. Such a particle may enhance the drug delivery of a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof into cells such as target cells. The term “nanoparticle” refers to a particle having sub-micrometer in its hydrodynamic diameter. The particle can be a vesicle that can encompass a substance such as a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof.
Examples of the salt include a salt with an inorganic base, an ammonium salt, a salt with an organic base, a salt with an inorganic acid, a salt with an organic acid and a salt with a basic or acidic amino acid.
Preferable examples of the salt with an inorganic base include an alkali metal salt such as a sodium salt and a potassium salt; an alkaline earth metal salt such as a calcium salt, a magnesium salt, a barium salt; and an aluminum salt.
Preferable examples of the salt with an organic base include salts with trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine, dicyclohexylamine and N, N'-dibenzylethylenediamine.
Preferable examples of the salt with an inorganic acid include salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid and phosphoric acid.
Preferable examples of the salt with an organic acid include salts with formic acid, acetic acid, trifluoroacetic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid and p-toluenesulfonic acid.
Preferable examples of the salt with a basic amino acid include salts with arginine, lysine and ornithine.
Preferable examples of the salt with an acidic amino acid include salts with aspartic acid and glutamic acid.
Of these salts, a pharmaceutically acceptable salt is preferable.
In an embodiment, a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof may be encapsulated into a vesicle such as lipid nanoparticles. Examples of lipid nanoparticles include, but are not limited to, the lipid particles described in US8,058,069B, which is incorporated herein by reference in its entirety. Amphiphilic lipids can form lipidic nanoparticles in aqueous solution. In a preferable lipid nanoparticle, 50 mol% to 60 mol% of total lipid is cationic lipid such as DLin-MC3-DMA, ALC-0315, and SM-102; 4 mol% to 10 mol% of total lipid is phospholipid such as DSPC; 30 mol% to 40 mol% of total lipid is cholesterol or a derivative thereof; and 0.5 mol% to 2 mol% of total lipid is a conjugated lipid such as PEGylated lipid such as PEG2000-DMG and ALC-0159, which can inhibit aggregation of particles.
The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, additive, or excipient. As the pharmaceutically acceptable carrier, various organic or inorganic carrier substances routinely used as a drug substance are used and blended as an excipient, a lubricant, a binder or a disintegrator in a solid preparation; a solvent, a solubilizer, a suspending agent, a tonicity agent, a buffer or a soothing agent in a liquid preparation. If necessary, a formulation additive such as a preservative, an antioxidant, a stabilizer, colorants or a sweetener can be used.
The pharmaceutical composition can be administered orally or parenterally. The pharmaceutical composition will be administered by intravenous administration, intramuscular administration, intraperitoneal administration, intradermal administration, transdermal administration, intracerebrospinal fluid administration, intraarticular administration, inhalation administration, instillation administration, or nasal administration. The pharmaceutical composition will be formulated such that the pharmaceutical composition will be suitable for at least one of these administration routes. The pharmaceutical composition may be either a solid preparation such as a powder, a granule, a tablet or a capsule, or a liquid such as an injection, a syrup or an emulsion. The pharmaceutical composition can be produced in accordance with a routine method including mixing, kneading, granulating, tableting, coating, sterilizing and emulsifying, depending on the dosage form of the preparation. Here, as to the production of the preparation, each section of General Rules for Preparations of the Japanese Pharmacopoeia, for example, can be referred. The medicament of the present invention may be formed as a sustained release agent comprising an active ingredient and a biodegradable polymer compound.
The dosage of the pharmaceutical composition varies depending on, e.g., the symptom; the age, sex, body weight and difference in sensitivity of the subject to be administered; timing and interval of administration, the feature, prescription and type of the medicament; and type of active ingredient and is not particularly limited.
In an embodiment, the present pharmaceutical composition may further comprise an immunosuppressant or an anti-inflammatory agent such as steroid and non-steroidal anti-inflammatory drugs (NSAIDs).
In an embodiment, the present pharmaceutical composition may be used in a combination with an immunosuppressant such as tacrolimus or an anti-inflammatory agent such as steroids and non-steroidal anti-inflammatory drugs (NSAIDs).
The present method
The present disclosure provides a method of treating a subject in need thereof. In an embodiment, the subject may have an activated and/or increased Th17 cells in its body or body part. The subject may have an inflammation and/or an immune-related disease such as an excessive immune response, over-reaction of immune system, or autoimmune diseases. In an embodiment, the subject may have an inflammation and/or immune-related disease that is caused by overexpression of IL-17, or an activated and/or increased Th17 cells. In an embodiment, the subject may have a Th17 cell-mediated immune disease. Th17 cell-mediated immune diseases include, for example, but not limited to, autoimmune disease such as rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, encephalomyelitis, and psoriasis. The method comprises administering to the subject an effective amount of a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof.
The present disclosure provides a method of treating an activated and/or increased Th17 cells in a subject in need thereof. The present disclosure provides a method of treating an inflammation and/or an immune-related disease such as an excessive immune response, over-reaction of immune system, or autoimmune diseases in a subject in need thereof. The present disclosure provides a method of treating an inflammation and/or immune-related disease that is caused by overexpression of IL-17, or an activated and/or increased Th17 cells. The present disclosure provides a method of treating Th17 cell-mediated immune disease. Th17 cell-mediated immune diseases include, for example, but not limited to, autoimmune disease such as rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, encephalomyelitis, and psoriasis. The method comprises administering to the subject an effective amount of a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof.
In an embodiment, the subject may be treated in combination with an immunosuppressant such as tacrolimus or an anti-inflammatory agent such as steroids and non-steroidal anti-inflammatory drugs (NSAIDs). In an embodiment, the subject has been treated with an immunosuppressant such as tacrolimus or an anti-inflammatory agent such as steroids and non-steroidal anti-inflammatory drugs (NSAIDs). In an embodiment, the subject may be treated with an immunosuppressant such as tacrolimus or an anti-inflammatory agent such as steroids and non-steroidal anti-inflammatory drugs (NSAIDs) after the treatment with a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof. In an embodiment, the dose of an immunosuppressant such as tacrolimus or an anti-inflammatory agent such as steroids and non-steroidal anti-inflammatory drugs (NSAIDs) can be reduced by administering a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof to the subject. In an embodiment, the dose of an immunosuppressant such as tacrolimus or an anti-inflammatory agent such as steroids and non-steroidal anti-inflammatory drugs (NSAIDs) can be reduced by administering an increased amount of a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof.
The present medical use of phosphoenolpyruvate or a salt thereof
The present disclosure provides phosphoenolpyruvate or a salt thereof for use in a method of the present disclosure. The present disclosure provides a pharmaceutical composition, for use in a method of the present disclosure, comprising a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof.
The present disclosure provides use of a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof in the manufacture of a medicament for use in a method of the present disclosure.
The present disclosure provides a method of producing a medicament for use in a method of the present disclosure (for example, preventing or treating Th17-related or Th17-mediated immune disease or condition), comprising mixing at least a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof (for example, isolated or purified phosphoenolpyruvate or a salt thereof) and an additive to form the medicament. In an embodiment, the additive may be a pharmaceutically acceptable carrier, additives, or excipient. The examples of the pharmaceutically acceptable carrier, additives, or excipient, include, but not limited to, various organic or inorganic carrier substances routinely used as a drug substance that are used and blended as an excipient, a lubricant, a binder or a disintegrator in a solid preparation; a solvent, a solubilizer, a suspending agent, a tonicity agent, a buffer or a soothing agent in a liquid preparation. If necessary, a formulation additive such as a preservative, an antioxidant, a stabilizer, colorants or a sweetener can be used.
The present disclosure provides a method of producing a supplement, food or drink for use in a method of the present disclosure (for example, preventing or treating Th17-related or Th17-mediated immune disease or condition), comprising mixing at least a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof (for example, isolated or purified phosphoenolpyruvate or a salt thereof) and an additive to form a supplements, food or drink, preferably a phosphoenolpyruvate-enriched supplements, food or drink. In an embodiment, the additive may be edible. The examples of the edible additives may include supplements, drinks and foods. In an embodiment, the additive may be a food additive. In an embodiment, the supplement may comprise a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof (for example, isolated or purified phosphoenolpyruvate or a salt thereof) and a food additive. In an embodiment, the phosphoenolpyruvate-enriched food or drink may comprise a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof (for example, isolated or purified phosphoenolpyruvate or a salt thereof) and a food or drink.
In an embodiment, the present composition may be a food or drink. In an embodiment, the present composition may be a food or drink where a glycolytic intermediate metabolitea glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof is enriched. In an embodiment, the present composition may be a food or drink supplemented with a glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof.
Methods
Mice
C57BL/6 mice were obtained from Clea (Tokyo, Japan), and OT-II and B6SJL mice were from the Jackson Laboratory (ME, USA). All mice were maintained under specific pathogen-free conditions. Gender-matched 6-12-week-old mice were utilized for experiments. All animal experimental protocols were approved by the Animal Care and Use Committee at Okinawa Institute of Science and Technology Graduate University.
Antibodies
The following antibodies were used for flow cytometry analysis and fluorescence-activated cell sorting, with 1:200 dilution: anti-IFNγ (XMG1.2; Biolegend), anti-IFNγ (MP6-XT22; Biolegend), anti-IL4 (11B11; Biolegend), anti-IL13 (eBio13A; eBioscience), anti-IL10 (JES5-16E3; Biolegend), anti-IL17A (TC11-18H10.1; Biolegend), anti-RORγt (B2D; eBioscience), anti-FOXP3 (150D; Biolegend), anti-T-bet (4B10; Biolegend), anti-GATA3 (16E10A23; Biolegend), anti-CD4 (GK1.5; Biolegend), anti-CD62L (MEL-14; Biolegend), anti-CD25 (PC61; Biolegend), anti-CD44 (IM7; Biolegend), anti-IL2 (JES6-5H4; Biolegend), anti-CD3 (17A2; Biolegend), anti-CD45.1 (A20; Biolegend), and anti-CD45.2 (104; Biolegend). Antibodies for western blotting were as below: anti-BATF (WW8; Santa Cruz, USA), anti-IRF4 (4964; CST, USA), anti-JUND (329; Santa Cruz), anti-JUNB (C11; Santa Cruz), anti-STAT3 (79D7; CST), anti-phospho STAT3 (Tyr705) (D3A7; CST), anti-HIF1α (28b; Santa Cruz), anti-β Actin (6D1; MBL, Japan), anti-PKM2 (D78A4, CST); anti-Histone H3 (D2B12; CST), anti-β tubulin (PM054; MBL), anti-Flag (M2; Sigma-Aldrich), anti-6X His-tag (RM146, Invitrogen), anti-mouse IgG, HRP-linked (7076; CST), and anti-rabbit IgG, HRP-linked (7074; CST).
in vitro CD4 T-cell differentiation
Naive murine CD4 T cells were isolated from spleens using MojoSort mouse CD4 naive T cell isolation kit (Biolegend) for most of in vitro T cell culture experiments. For RNA sequencing, ATAC sequencing, and ChIP PCR, CD4 T cells were first enriched with MACS magnetic cell sorting system with anti-CD4 microbeads (Miltenyi), and then naive CD4 T cells (CD4+CD25-CD62LhiCD44lo) were sorted by FACS AriaII or AriaIII (BD). Isolated naive CD4+ T cells were cultured in Iscove’s modified Dulbecco’s medium (IMDM) (Invitrogen) supplemented with 10% FBS, 1X streptomycin-penicillin (Sigma-Aldrich), β-mercaptoethanol (55 μM; Invitrogen, USA), and anti-CD28 antibody (1 μg/mL; 37.51, Biolegend) in 24-well (2x105 cells / well) or 48-well (1x105 cells / well) plates coated with anti-CD3ε antibody (5 μg/mL; 145-2C11, Biolegend). The medium was further supplemented with IL-2 (20ng/mL, Biolegend), IL-12 (20 ng/mL; Biolegend), and anti-IL-4 (1 μg/mL; 11B11, Biolegend) for Th1; IL-2 (20 ng/mL), IL-4 (100 ng/mL; Biolegend), and anti-IFN-γ (1 μg/mL; R4-6A2, Biolegend) for Th2; IL-6 (20 ng/mL; Biolegend) and TGF-β1 (3 ng/mL; Miltenyi) for TH17(B); IL-6 (20 ng/mL), IL-1β(20 ng/mL; Biolegend) and IL-23 (40 ng/mL; Biolegend) for TH17(23); TGF-β1 (15 ng/mL), IL-2 (20 ng/mL), anti-IL-4 (1 μg/mL), and anti-IFN-γ (1 μg/mL) for iTreg differentiation. In several experiments, additional inhibitors or metabolites were added to the culture medium. Glycolytic metabolites were dissolved in water or PBS first, adjusted the pH value to 7.3, then added to the culture medium to reach the desired concentration. For analysis of cytokine expression, cells were harvested at indicated time points, re-stimulated with phorbol 12- myristate 13-acetate (PMA; 50 ng/mL; Sigma-Aldrich) and ionomycin (500 ng/mL; Sigma-Aldrich), and brefeldin A (5 mg/mL; Biolegend) for 4 hours. Cells were then fixed with 4% paraformaldehyde, permeabilized in permeabilization/wash buffer (421002, Biolegend), and stained with antibodies against cytokines. For analysis of expression of transcription factors, Foxp3 staining buffer set (00-5253-00, eBioscience) was used according to the manufacturer’s instructions.
Intracellular PEP Quantification
Naive CD4 T cells activated under Th17-polarizing conditions at indicated time points were washed twice with PBS, snap-frozen in liquid nitrogen, and then stored at -80℃ until further processing. The PEP fluorometric assay was performed with PEP colorimetric/fluorometric assay kits (Sigma-Aldrich) and a SpectraMax M2 96-well reader (Molecular Devices, USA) according to the manufacturer’s instructions.
qRT-PCR
Total RNA was isolated from cells using an RNeasy Plus Mini kit (Qiagen, Germany). cDNA was synthesized with a Revertra Ace qPCR Kit (Toyobo, Japan). PCR was performed with KAPA SYBR fast qPCR kit master mix (Kapa Biosystems) and StepOnePlus Real-Time PCR (Applied Biosystems, USA). Primers used for qPCR are listed in Table 1.
Seahorse assay
Naive CD4 T cells activated under Th17(β)- and Th17(23)-polarizing conditions for 72 h were harvested for measurements of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) with mito stress (Agilent Technologies, USA) and glycolysis stress kits (Agilent Technologies), respectively. Cells were washed twice with PBS, transferred to an analysis plate (2 x 105 cells per well) coated with 2% gelatin (Sigma-Aldrich), and incubated at 37oC for 1 h. OCR and ECAR were measured using a Seahorse XFe96 analyzer (Seahorse Bioscience, USA) following the manufacturer’s instructions.
Sample preparation for RNA and ATAC sequencing
Cells activated under Th17(β)- and Th17(23)-polarizing conditions for 48 h were harvested, and total RNA was extracted using RNeasy Plus Mini kits (Qiagen). RNA samples were then mixed with ERCC RNA spike-in control mixes (Thermo), and mRNA was isolated with NEBNext poly(A) mRNA magnetic isolation module (E7490; NEB, USA). The sequencing Library was prepared with a Collibri Stranded RNA Library Prep Kit for Illumina Systems with Human/Mouse/Rat rRNA Depletion Kits (Thermo), according to the manufacturer’s instructions. The quality of the cDNA library was checked using Qubit dsDNA HS and BR Assay Kits (Thermo) and High Sensitivity DNA Reagents kits (Agilent, USA) with a Qubit 4 fluorometer (Thermo) and a 2100 Bioanalyzer instrument (Agilent), respectively. For ATAC sequencing, naive CD4 T cells were activated under Th17(β)-polarizing conditions for 48 h. Then cells were harvested, snap-frozen in liquid nitrogen, and submitted to the OIST Sequencing Section (SQC) for further preparation and sequencing. Both RNA and ATAC sequencing were performed on an Illumina NovaSeq 6000 to generate 150-nucleotide, paired-end reads with a read depth of ≧20 million reads per sample.
RNA-seq data analysis
Data quality was assessed using FastQC (v.0.11.9) (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Reads were further processed to remove adaptor and low-quality sequences using Trimmomatic1 (v.0.39) software with the options (SLIDINGWINDOW:4:20 LEADING:20 TRAILING:20 MINLEN:35). HISAT23 (v2.2) was utilized to align reads to the GRCm38 reference genome (Mus_musculus.GRCm38.dna.primary_assembly.fa file, downloaded from Ensembl2). We counted the number of reads overlapping the genes in the reference transcriptome annotations (Mus_musculus.GRCm38.98.gtf downloaded from Ensemb4l) with featureCounts from Subread5 (v2.0.1). To detect differentially expressed genes, transcripts with zero expression were first filtered out, and statistical significance was analyzed with the Wald test using DESeq26 (v.1.34.0). Gene set enrichment analysis based on Kyoto Encyclopedia of Genes and Genomes (KEGG) was performed using the clusterProfiler7 R package.
ATAC-seq data processing
Raw data processing was performed using nfcore/atacseq (v.1.2.1), a bioinformatics analysis pipeline used for ATAC-seq data at the National Genomics Infrastructure at SciLifeLab Stockholm, Sweden. In brief, adapters and low-quality reads were removed with Trim Galore!. Trimmed fastq files were mapped to the GRCm38 mouse reference genome with BWA, and narrow peaks were called with MACS2. The normalized BigWig files, scaled to 1 million mapped reads, were created with BEDTools and bedGraphToBigWig and were uploaded to the UCSC genome browser. Tool versions and full details of the pipeline are available at https://nf-co.re/atacseq.
Motif analysis
Motif enrichment within 2 kb upstream and downstream of the transcriptional start sites of DEGs in PEP-treated vs. control cells was analyzed using the findMotifs function of Homer (version v4.11). Binding motifs for AP-1 (ATGACTCATC), JunB (RATGASTCAT), BATF (DATGASTCAT), and IRF4 (ACTGAAACCA), and AICE (NAGTTTCABTHTGACTNW) within 10 kb upstream and downstream of transcriptional start sites of DEGs were identified using the mouse mm10 genome with the scanMotifGenomeWide.pl function of Homer v4.11.
Immunoblot analysis
Cells were lysed with RIPA buffer (Wako, Japan) with a complete protease inhibitor cocktail (Roche, Switzerland). Clear lysates were mixed with 5X sample loading buffer (250 mM Tris-HCl pH 6.8, 10% SDS, 30% glycerol, 5%β-mercaptoethanol, and 0.02% bromophenol blue) and subjected to SDS polyacrylamide gel electrophoresis. Blotting was performed with Immobilon P transfer membranes (Millipore) using a Trans-blot electrophoretic transfer system (Bio-Rad). Membranes were blocked with 5% skim milk (Wako) or bovine albumin (Wako) in Tris-buffered saline with 0.1% Tween-20 (Sigma-Aldrich). Then they were hybridized with described antibodies at 4oC overnight, followed by incubation with HRP-conjugated secondary antibodies at room temperature for 2 h. Reactive proteins were detected with Clarity Western ECL (Bio-Rad) or SuperSignal West Femto detection reagents (Thermo) on a Las-3000 imaging system (Fuji film, Japan) or iBright CL1500 Imaging System (Thermo).
Cell transfection
To overexpress mouse BATF and JunB, BATF-HA (C-terminally HA-tagged BATF (gene ID: 53314)) (BATF-HA) and JunB-Flag (C-terminally Flag-tagged JunB (gene ID: 16477)) were amplified from gblock (Integrated DNA Technology) and cloned into pCDNA3.1 (Thermo). HEK293 cells cultured in Dulbecco’s modified eagle medium (DMEM) (Thermo) supplemented with 10% FBS and MEM non-essential amino acids (Thermo) were seeded in 10-cm culture dishes 24 h before transfection with 80% confluency. 5 μg of pcDNA3.1-BATF-HA and pcDNA3.1-JunB-Flag in 250ul Opti-MEM were mixed with 25 μL of polyethylenimine (1 mg/mL) (Cosmobio, Japan) in 250 μL of Opti-MEM, incubated at room temperature for 30 min, and then added to the cell culture. Cells were harvested after 60 h for co-immunoprecipitation assay.
Co-immunoprecipitation
HEK293 cells transfected with pcDNA3.1-BATF-HA and pcDNA3.1-JunB-Flag were washed with PBS twice, freeze-thawed by liquid nitrogen twice, and lysed in Triton X-100 lysis buffer (1% Triton X-100, 150 mM NaCl, 20 mM Tris (pH 7.5) containing complete protease inhibitor cocktail (Roche) ) on ice for 30 min (briefly vortexed every 10 min). Then, cellular debris was removed by centrifugation at 13,000 g for 10 min. Lysates (3 mg total protein) were incubated with 3 μg of anti-Flag (M2; Sigma-Aldrich) or anti-mouse IgG (G3A1; CST) antibodies together with or without PEP (50, 200, and 500 μM) on a rotator at 4°C for 16 h, followed by incubation with 15 μL of Dynabeads protein G (Invitrogen) on a rotator at 4°C for another hour. Beads were then washed four times (10 min incubation in each wash) with buffer containing 0.2% Triton X-100, 150 mM NaCl, 20 mM Tris (pH 7.5), and a complete protease inhibitor cocktail (Roche) at 4°C. Immunoprecipitates were eluted by heating the beads in sample buffer (0.05% Bromophenol blue, 2%β-mercaptoethanol, 10% glycerol, 2% sodium dodecyl sulfate in Tris-Cl (pH=6.8)) at 70oC for 15 min.
Chromatin immunoprecipitation
Cells were harvested at the indicated timepoint, and chromatin immunoprecipitation was performed using SimpleChIP kits (CST) and Dynabeads protein G (Invitrogen) according to the manufacturer’s instructions, except for two modifications: (1) the amount of micrococcal nuclease was reduced to 0.05 μL per million cells; (2) chromatin-bound beads were washed with low-salt wash solution 4 times, followed by high-salt wash solution 2 times at 4oC for 5 min. The following antibodies (2 μg per sample) were used for immunoprecipitation: anti-BATF (ww8; Santa Cruz), anti-JunB (C-11; Santa Cruz), anti-IRF4 (4964; CST), anti-mouse IgG (G3A1; CST), and anti-Rabbit IgG (2729; CST).
OVA immunization
8-9-week-old, gender-matched B6SJL mice were subcutaneously (s.c.) injected with vehicle (200 μL PBS) or PEP (1g/kg body weight, dissolved in 200 μL PBS, pH adjusted to 7.3), followed 6 h later by intravenous injection of naive CD4 T cells isolated from OT-II mice (CD45.1+ CD45.2+). One day later, mice were immunized with OVA323-339 (50 μg per mouse; ISQAVHAAHAEINEAGR, GL Biochem, China) emulsified in CFA (200 μL per mouse) supplemented with or without PEP (10 mg/mouse). From day 1 to day 7 after immunization, mice were s.c. injected with vehicle or PEP as described above. On day 7 after immunization, mice were euthanized, and cells isolated from inguinal lymph nodes and spleens were analyzed as described above.
EAE induction
8-week-old, female C57BL/6 mice were s.c. injected with MOG35-55 peptides (300 mg per mouse) emulsified in complete Freund’s adjuvant (CFA) (200 μL per mouse) containing dead Mycobacterium tuberculosis (1 mg per mouse) on day 0. On days 0 and 2, pertussis toxin (400 ng per mouse) was intraperitoneally injected into mice. From day 0 (8 h prior to MOG immunization) until the end of experiments, mice were s.c. injected with vehicle (200 μL PBS) or PEP (1g/kg per mice, dissolved in 200 μL PBS, pH adjusted to 7.3) daily. Disease severity was evaluated on a scale of 1-5 as follows: 1, limp tail; 2, limp tail and weakness of hind legs; 3, limp tail with paralysis of one hind leg; 4, limp tail with paralysis of both hind legs; 5, complete hind and front leg paralysis. Mice with a disease score of 5 were euthanized.
Statistical analysis
Unpaired two-tailed Student’s t tests and one-way ANOVA followed by Tukey’s post-hoc tests were performed with Prism (GraphPad). P values < 0.05 were considered statistically significant.
Unpaired two-tailed Student’s t tests and one-way ANOVA followed by Tukey’s post-hoc tests were performed with Prism (GraphPad). P values < 0.05 were considered statistically significant.
Data availability
The RNA-seq and ATAC-seq data that support the finding of this study have been deposited to DDBJ database (DRA014503).
Results
PEP suppresses IL-17A expression
The role of glycolytic intermediate metabolites in T helper differentiation is largely uncharacterized. To address this question, we polarized naive CD4 T cells to Th17 cells with TGF-β and IL-6 (known as non-pathogenic Th17 (npTh17) cells) in media supplemented with individual glycolytic intermediate metabolites. Interestingly, supplementation with glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), 3-phosphoglycerate (3PG), and PEP significantly decreased IL-17A expression without affecting cell viability (Figure 1A, B, and Figure 7A), while fructose 1,6 bisphosphate (F1,6BP) and glycerol-3-phosphate (G3P) induced cell death (Figure 1B). Given the significant reduction of IL-17A expression in PEP-treated npTh17 cells, we decided to further investigate the function of PEP in Th17 differentiation. PEP permeates the plasma membrane of several cell types, including red blood cells and human mesenchymal stem cells (Hamasaki and Kawano, 1987; Jeong et al., 2019), but whether this is the case in T cells was unknown. Therefore, we analyzed intracellular PEP levels in PEP-treated differentiating Th17 cells. PEP supplementation significantly increased intracellular PEP in cells activated under npTh17-polarizing conditions within 2 h (Figure 1C), indicating that the plasma membrane of differentiating Th17 cells is permeable to PEP. These results suggest that an increase in intracellular PEP is associated with the reduction of IL-17A expression.
We next sought to examine the relationship between PEP produced by glycolysis and IL-17 expression. Inhibition of glycolysis by a hexokinase inhibitor, 2-deoxy-D-glucose (2-DG), can enhance IL-17 expression in Th17 cells (Brucklacher-Waldert et al., 2017). This, taken together with the observation that 2-DG suppresses PEP production (Ho et al., 2015), supports the notion that PEP generated by glycolysis can inhibit IL-17A expression. To further examine the effects of manipulation of PEP levels, we treated differentiating npTh17 cells with other pharmacological inhibitors of glycolysis that can modulate PEP levels in T cells: heptelidic acid (HA) decreases PEP by inhibiting glyceraldehyde phosphate dehydrogenase (GAPDH), while oxalate (OXA) increases PEP levels by inhibiting pyruvate kinase (Ho et al., 2015). We found that HA, as well as 2-DG, increased IL-17A expression, while OXA decreased it (Figure 1D and Figure 7B). These results suggest that endogenous PEP generated by glycolysis can suppress IL-17A expression.
PEP inhibits differentiation of Th17 and Th2 cells, but not Th1 and iTreg cells
We found that PEP supplementation significantly reduced the frequency of IL-17A-expressing cells, not only under npTh17-polarizing conditions, but also under pathogenic Th17 (pTh17)-polarizing conditions (cultured with IL-6, IL-1β, and IL-23) in a dose-dependent manner (Figure 2A). Expression of the Th17-lineage-specifying transcription factor, RORγt, was dramatically decreased by PEP treatment under pTh17-polarizing conditions, but not under npTh17-polarizing conditions (Figure 2B). We confirmed that PEP supplementation decreased mRNA expression of Il17a and Rorc (encoding RORγt) (Figure 2C). We also assessed the effect of PEP supplementation on differentiation of Th1, Th2, and induced T regulatory (iTreg) cells. PEP supplementation significantly reduced IL-4 and IL-13 expression without affecting GATA3 expression in Th2 cells, while it slightly increased T-bet and IFN-γ expression in Th1 cells (Figure 2D, E). However, PEP supplementation did not affect Foxp3 expression in iTreg cells (Figure 2 E). These data indicate that PEP supplementation inhibits differentiation of Th17 and Th2 cells, but not Th1 and iTreg cells.
Since PEP promotes TCR signaling by controlling intracellular calcium concentration (Ho et al., 2015), we next examined whether PEP supplementation enhances the TCR signaling-dependent production of IL-2, which inhibits IL-17A expression (Laurence et al., 2007). Unexpectedly, we found that IL-2 expression was decreased by PEP supplementation in Th17 cells (Figure 2F). We also observed that expression of T cell activation marker CD25 in PEP-treated Th17 cells was comparable to that in control Th17 cells (Figure 8A). Furthermore, PEP treatment showed no obvious effects on proliferation of differentiating Th17 cells (Figure 8B). These data suggest that PEP-mediated inhibition of Th17 differentiation is not due to promotion of TCR-dependent IL-2 production or enhanced activation of CD4 T cells.
PEP inhibits IL-17A expression independently of its role in glycolysis
We next assessed whether PEP supplementation inhibits IL-17A expression through modulation of metabolic reprogramming of Th17 cells. Our seahorse analysis showed that PEP supplementation did not affect extracellular acidification rates (ECAR) (Figure 3A, B) or oxygen consumption rates (OCR) (Figure 3C, D), suggesting that elevation of PEP levels does not significantly modulate the glycolysis and oxidative phosphorylation pathways in differentiating Th17 cells. Furthermore, PEP supplementation did not affect reactive oxygen species (ROS) levels in Th17 cells (Figure S3). PKM2, the glycolytic enzyme catalyzing the conversion of PEP to pyruvate, is upregulated upon TCR stimulation, and a portion of PKM2 translocates to the nucleus to promote the transcriptional program for Th1 and Th17 differentiation (Angiari et al., 2020; Damasceno et al., 2020; Kono et al., 2019). We found that PEP supplementation did not influence PKM2 expression or nuclear translocation in Th17 cells (Figure 3E). Taken together, these data suggest that PEP supplementation inhibits Th17 differentiation without affecting glycolysis or PKM2 activity. Furthermore, PEP supplementation significantly inhibited IL-17 expression even in 2-DG-treated cells (Figure 3F), suggesting that PEP can inhibit IL-17 expression independently of its role in glycolysis.
PEP controls JunB-dependent gene expression in Th17 differentiation
To investigate the association between PEP and the Th17 transcriptome, we performed an RNA-seq analysis of npTh17 and pTh17 cells induced in the presence or absence of PEP supplementation. Gene expression profiles of control and PEP-treated Th17 subsets were clearly different, and PEP supplementation altered the transcriptome of pTh17 cells more significantly than npTh17 cells (Figure 4A). PEP supplementation significantly affected expression of 300 genes (199 upregulated genes and 101 downregulated genes) and 552 genes (241 upregulated genes and 311 downregulated genes) in npTh17 and pTh17 cells, respectively (Figure 4B). Gene set enrichment analysis revealed that genes downregulated by PEP supplementation were enriched in gene ontology (GO) terms related to chemokine/cytokine signaling and inflammatory responses (Figure 4C). In contrast, there was no enrichment of genes affected by PEP supplementation in GO terms related to cellular metabolism (Figure 4C), which is consistent with largely normal ECAR and OCR in PEP-treated cells (Figure 3A-D). PEP supplementation downregulated expression of several Th17 signature molecules (Il17a, Il17f, Il23r, Rorc, and Rora) and other cytokines and transcription factors related to T cell biology (such as Ifng, Il2, and Tbx21) in pTh17 cells (Figure 4D). In contrast, under npTh17 conditions, PEP supplementation did not significantly decrease expression of Il23r, Rorc, and Rora, although it decreased Il17a and Il17f expression (Figure 4D).
We next sought to identify upstream transcription factors that can regulate expression of genes affected by PEP supplementation. Motif analysis demonstrated that IRF (TTTC/GAAA) and AP-1 motifs (TGAnTCA) were enriched within ±2 kbp of transcription start sites (TSS) of differentially expressed genes (DEGs) in control vs PEP-treated Th17 cells (Figure 4E). Members of the AP-1 and IRF families, JunB, BATF, and IRF4, play a pivotal role in the Th17 transcriptional program by binding to AICEs (Carr et al., 2017; Glasmacher et al., 2012; Hasan et al., 2017; Li et al., 2012). Notably, we found that about 80% of the DEGs contains AICE motifs within ±10 kbp of the TSS (Figure 4F). Furthermore, by cross-referencing DEGs with our previous data set for JunB-regulated genes (Hasan et al., 2017), we found that JunB-regulated genes accounted for 26.7% and 30.3% of genes affected by PEP treatment under npTh17 and pTh17 conditions, respectively (Figure 4G). We also analyzed the effect of PEP supplementation on genome-wide chromatin accessibility by assay for transposase-accessible chromatin sequencing (ATAC-seq), because BATF and IRF4 promote chromatin accessibility in Th17 cells (Ciofani et al., 2012; Pham et al., 2019). However, PEP supplementation affected chromatin accessibility only at a few gene loci, expression of which was affected by PEP supplementation (Table. 2), but not at the Il17a locus (Figure S4), suggesting that PEP regulates Il17a transcription at a step after chromatin opening. Taken together, these data suggest that PEP regulates the Th17 transcriptional program mediated by JunB.
PEP suppresses DNA-binding of JunB, BATF, and IRF4 at the Il17 locus
Our observation that PEP supplementation affects expression of genes regulated by JunB, led us to examine whether PEP regulates expression or activity of JunB and its partner, BATF and IRF4. First, we found that PEP supplementation did not significantly affect expression of JunB, BATF, IRF4, or other transcription factors related to Th17 differentiation, Hif1α, STAT3, and JunD in differentiating npTh17 cells, although it slightly decreased JunB expression in pTh17 differentiation (Figure 5A, B). Furthermore, a co-immunoprecipitation assay showed that PEP supplementation did not affect the interaction between JunB and BATF (Figure 5C). We next evaluated whether PEP supplementation affects the DNA-binding of JunB, BATF, and IRF4 at the Il17a locus (Figure 5D). ChIP-PCR analysis demonstrated a significant reduction of binding of JunB, BATF, and IRF4 at the Il17a locus in CD4 T cells cultured under npTh17 conditions for 36 h (Figure 5E). We also assessed the effect of PEP supplementation on DNA-binding of JunB at the Irf8 locus, because JunB binds to and inhibits Irf8 expression in Th17 cells (Carr et al., 2017). Consistent with normal Irf8 expression observed in npTh17 cells treated with PEP (Figure 3D), PEP did not affect DNA-binding of JunB, BATF, and IRF4 at the Irf8 locus (Figure 5E). These results suggest that PEP inhibits the DNA-binding of JunB, BATF, and IRF4 at specific gene loci, including Il17a.
PEP administration inhibits in vivo Th17 differentiation and ameliorates EAE
To evaluate effects of PEP supplementation on Th17 differentiation in vivo, we adoptively transferred ovalbumin (OVA)-specific OT-II T cells into congenic recipients, followed by immunization with OVA peptides emulsified in complete Friend’s adjuvant (CFA) and daily subcutaneous injection of PEP. Daily injection of PEP resulted in a remarkable decrease of expression of IL-17A, but not IFN-γ in OT-II T cells in the lymph nodes and spleens at 7 days post-immunization (Figure 6A and Figure 11A). Consistent with this, expression of RORγt, but not T-bet, was reduced by PEP administration (Figure 6B and Figure 11B), suggesting that PEP inhibits Th17 generation in vivo. Next, we sought to determine whether PEP administration can inhibit experimental autoimmune encephalomyelitis (EAE), a murine model of human multiple sclerosis, which depends on the generation of JunB-dependent Th17 cells (Carr et al., 2017; Hasan et al., 2017; Yamazaki et al., 2017). For this purpose, we subcutaneously injected mice with PEP or vehicle daily from the day of induction of EAE by immunization with MOG peptides emulsified in CFA followed by pertussis toxin injection. We found that disease symptoms were significantly milder in PEP-injected animals than in controls (Figure 6C). In summary, these data indicate the therapeutic potential of PEP for Th17-mediated autoimmune disorders.
Discussion
In this study, we identified PEP as an immunoregulatory metabolite that inhibits pathogenic Th17 differentiation. Metabolites can link cellular metabolism to regulation of gene expression at epigenetic and transcriptional levels (Chapman et al., 2020; Shyer et al., 2020; Zhang et al., 2018), but the role of glycolytic metabolites in T cells remains largely uncharacterized, despite a close relationship between glycolysis and T cell biology (Palmer et al., 2015). Our data demonstrate that PEP inhibits expression of Th17 signature molecules, including IL-17A, both in vitro and in vivo. Notably, PEP administration to mice suppresses Th17 generation and EAE, a Th17-dependent autoimmune neuroinflammatory disease. Mechanistically, PEP suppresses JunB-dependent pathogenic Th17 transcriptional program by inhibiting the DNA-binding activity of the transcription complex of JunB, BATF and IRF4.
Our discovery of this non-canonical function of PEP, inhibiting Th17 differentiation provides new insight into how glycolysis regulates Th17 differentiation. We observed that hexokinase and GAPDH inhibitors, which downregulate the PEP level, enhance IL-17A expression, whereas pyruvate kinase inhibitor, which upregulates the PEP levels, suppresses IL-17A expression. This supports the model that glycolysis can inhibit Th17 differentiation through PEP production. However, other groups have reported that the same or similar glycolytic inhibitors that downregulate the PEP levels suppress IL-17A expression (Kornberg et al., 2018; Shi et al., 2011). We speculate that this discrepancy might arise due to differences in cellular glycolytic status and efficacy of glycolysis inhibitors in each experiment, which might change the balance between glycolysis-associated positive regulators of Th17 differentiation and PEP. For example, differences in the expression or activity of the glycolytic enzyme, PKM2, which is not only associated with the levels of its substrate PEP but also promotes Th17 differentiation (Angiari et al., 2020; Damasceno et al., 2020; Kono et al., 2019), might affect the effect of glycolysis inhibitors on Th17 differentiation. Taken together, our findings suggest an intricate relationship between glycolysis and Th17 differentiation. Glycolysis is critical for Th17 differentiation and clonal expansion, but it also provides PEP-mediated rheostat control of pathogenic Th17 generation.
Our data highlight PEP-mediated modulation of AP-1 transcription factor activity. A transcription factor complex composed of a BATF-containing AP-1 dimer and IRF4 plays a central role in Th17 differentiation by acting as a pioneering transcription factor that promotes chromatin accessibility of various Th17-related genes (Ciofani et al., 2012; Pham et al., 2019). We and others reported that JunB, one of the BATF dimeric partners, is required for transcriptional control of a subset of genes regulated by BATF and IRF4 (Carr et al., 2017; Hasan et al., 2017; Yamazaki et al., 2017). Of note, loss of JunB inhibits IL-17A expression in both pathogenic and non-pathogenic Th17 cells, but it decreases RORγt and IL-23R expression in pathogenic Th17 cells but not in non-pathogenic Th17 cells (Hasan et al., 2017). JunB also regulates Th2 differentiation, but not Th1 and iTreg differentiation (Hsieh et al., 2022). Notably, our data show a similar context-dependent inhibitory effect on the differentiating Th2 and Th17 cells by PEP supplementation. Furthermore, our results reveal that PEP inhibits binding of JunB, BATF, and IRF4 at the Il17a locus, without affecting chromatin accessibility. Taken together, these observations suggest that JunB may be a major target for PEP in negative regulation of Th17 differentiation.
Our RNA-seq data reveal that PEP supplementation inhibits expression of Il-17a, but not Irf8, although JunB directly promotes transcription of both Il17a and Irf8 in npTh17 cells (Carr et al., 2017; Hasan et al., 2017). Consistent with this, our ChIP-PCR data indicate that PEP regulates DNA-binding of JunB, BATF, and IRF4 at the Il17a locus, but not at the Irf8 locus. This locus-specific effects on DNA-binding of JunB, BATF, and IRF4 may account for the effect of PEP supplementation on the expression of a specific subset of genes regulated by JunB in Th17 cells. Our results show that PEP supplementation does not impair the expression of JunB, BATF, and IRF4 and the interaction between JunB and BATF. Thus, PEP likely regulates activity of JunB/BATF/IRF complex in a locus-specific manner.
In conclusion, PEP-dependent inhibition of JunB activity mediates the crosstalk between metabolic reprogramming of glycolysis and the Th17 transcriptional program. PEP can inhibit Th17-dependent autoimmunity by controlling JunB-dependent pathogenic Th17 transcriptional program without significantly affecting glycolysis and T cell activation. Since JunB is required for differentiation of pathogenic Th17, but not non-pathogenic Th17 cells, a new approach utilizing PEP or its derivatives to modulate JunB function might have potential for selective therapy of autoimmunity.
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Claims (12)
- A composition, for use in a method of preventing, inhibiting, or reducing activation of T helper 17 (Th17) cell immunity or development of Th17-mediated immune disease or condition in a subject in need thereof, comprising an effective amount of an isolated or enriched glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof.
- The composition of claim 1, wherein the isolated or enriched glycolytic intermediate metabolite or a salt thereof comprises or is one or more selected from the group consisting of phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), and 3-phosphoglycerate (3PG), and a salt thereof.
- The composition of claim 1, wherein the isolated or enriched glycolytic intermediate metabolite or a salt thereof comprises or is phosphoenolpyruvate (PEP).
- The composition of any one of claims 1 to 3, wherein the composition is a food composition, a drink composition, a supplement, or a pharmaceutical composition.
- A pharmaceutical composition, for use in a method of preventing, inhibiting, or reducing activation of T helper 17 (Th17) cell immunity in a subject in need thereof, comprising an effective amount of an isolated or enriched glycolytic intermediate metabolite or a salt thereof, preferably, glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), 3-phosphoglycerate (3PG), or phosphoenolpyruvate (PEP), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof.
- A pharmaceutical composition, for use in a method of treating a disease associated with an activated or increased T helper 17 (Th17) cell immunity in a subject in need thereof, comprising an effective amount of an isolated or enriched glycolytic intermediate metabolite or a salt thereof, preferably, glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), 3-phosphoglycerate (3PG), or phosphoenolpyruvate (PEP), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof.
- The composition of claim 5 or 6, wherein the isolated or enriched glycolytic intermediate metabolite or a salt thereof is one or more selected from the group consisting of glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), 3-phosphoglycerate (3PG), and phosphoenolpyruvate (PEP), and a salt thereof.
- The composition of claim 5 or 6, wherein the isolated or enriched glycolytic intermediate metabolite or a salt thereof comprises or is phosphoenolpyruvate (PEP).
- The pharmaceutical composition of any one of claims 5 to 8, wherein the subject is suffered from an autoimmune disease.
- The pharmaceutical composition of any one of claims 5 to 9, wherein the pharmaceutical composition is formulated as a parenteral formulation.
- The pharmaceutical composition of any one of claims 5 to 9, wherein the pharmaceutical composition is formulated as a transdermal formulation.
- A composition, for use in a method of preventing, inhibiting, or reducing JunB-dependent generation of autoimmune T helper 17 (Th17) cells in a subject in need thereof, comprising an effective amount of an isolated or enriched glycolytic intermediate metabolite or a salt thereof, preferably, phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), or 3-phosphoglycerate (3PG), or a salt thereof, more preferably, phosphoenolpyruvate (PEP) or a salt thereof.
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US8058069B2 (en) | 2008-04-15 | 2011-11-15 | Protiva Biotherapeutics, Inc. | Lipid formulations for nucleic acid delivery |
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US8058069B2 (en) | 2008-04-15 | 2011-11-15 | Protiva Biotherapeutics, Inc. | Lipid formulations for nucleic acid delivery |
WO2020232357A1 (en) * | 2019-05-16 | 2020-11-19 | Arizona Board Of Regents On Behalf Of Arizona State University | Metabolite delivery for modulating metabolic pathways of cells |
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