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Inhibition of Pyruvate Dehydrogenase Kinase 4 in CD4+ T Cells Ameliorates Intestinal Inflammation

  • Author Footnotes
    ∗ Authors share co-first authorship.
    Hoyul Lee
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    ∗ Authors share co-first authorship.
    Affiliations
    Research Institute of Aging and Metabolism, Kyungpook National University, Daegu, Republic of Korea
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  • Author Footnotes
    ∗ Authors share co-first authorship.
    Jae Han Jeon
    Footnotes
    ∗ Authors share co-first authorship.
    Affiliations
    Research Institute of Aging and Metabolism, Kyungpook National University, Daegu, Republic of Korea

    Department of Internal Medicine, School of Medicine, Kyungpook National University, Kyungpook National University Chilgok Hospital, Daegu, Republic of Korea
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  • Yu-Jeong Lee
    Affiliations
    Cell & Matrix Research Institute, Kyungpook National University, Daegu, Republic of Korea
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  • Mi-Jin Kim
    Affiliations
    Research Institute of Aging and Metabolism, Kyungpook National University, Daegu, Republic of Korea
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  • Woong Hee Kwon
    Affiliations
    Leading-Edge Research Center for Drug Discovery and Development for Diabetes and Metabolic Disease, Kyungpook National University Hospital, Daegu, Republic of Korea
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  • Dipanjan Chanda
    Affiliations
    Research Institute of Aging and Metabolism, Kyungpook National University, Daegu, Republic of Korea
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  • Themis Thoudam
    Affiliations
    Research Institute of Aging and Metabolism, Kyungpook National University, Daegu, Republic of Korea
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  • Haushabhau S. Pagire
    Affiliations
    Department of Chemistry, Gwangju Institute of Science and Technology, Gwangju, Republic of Korea
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  • Suvarna H. Pagire
    Affiliations
    Department of Chemistry, Gwangju Institute of Science and Technology, Gwangju, Republic of Korea
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  • Jin Hee Ahn
    Affiliations
    Department of Chemistry, Gwangju Institute of Science and Technology, Gwangju, Republic of Korea
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  • Robert A. Harris
    Affiliations
    Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas
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  • Author Footnotes
    § Authors share co-senior authorship.
    Eun Soo Kim
    Correspondence
    Correspondence Address correspondence to: Eun Soo Kim, MD, PhD, Division of Gastroenterology, Department of Internal Medicine, School of Medicine, Kyungpook National University, 130 Dongdeok-ro, Jung-gu, Daegu, Republic of Korea 41944. fax: +82-53-200-5879.
    Footnotes
    § Authors share co-senior authorship.
    Affiliations
    Division of Gastroenterology, Department of Internal Medicine, Kyungpook National University, Kyungpook National University Hospital, Daegu, Republic of Korea
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  • Author Footnotes
    § Authors share co-senior authorship.
    In-Kyu Lee
    Correspondence
    In-Kyu Lee, MD, PhD, Department of Internal Medicine, School of Medicine, Kyungpook National University, 130 Dongdeok-ro, Jung-gu, Daegu, Republic of Korea 41944.
    Footnotes
    § Authors share co-senior authorship.
    Affiliations
    Research Institute of Aging and Metabolism, Kyungpook National University, Daegu, Republic of Korea

    Department of Internal Medicine, School of Medicine, Kyungpook National University, Kyungpook National University Hospital, Daegu, Republic of Korea
    Search for articles by this author
  • Author Footnotes
    ∗ Authors share co-first authorship.
    § Authors share co-senior authorship.
Open AccessPublished:October 10, 2022DOI:https://doi.org/10.1016/j.jcmgh.2022.09.016

      Background & Aims

      Despite recent evidence supporting the metabolic plasticity of CD4+ T cells, it is uncertain whether the metabolic checkpoint pyruvate dehydrogenase kinase (PDK) in T cells plays a role in the pathogenesis of colitis.

      Methods

      To investigate the role of PDK4 in colitis, we used dextran sulfate sodium (DSS)-induced colitis and T-cell transfer colitis models based on mice with constitutive knockout (KO) or CD4+ T-cell–specific KO of PDK4 (Pdk4fl/flCD4Cre). The effect of PDK4 deletion on T-cell activation was also studied in vitro. Furthermore, we examined the effects of a pharmacologic inhibitor of PDK4 on colitis.

      Results

      Expression of PDK4 increased during colitis development in a DSS-induced colitis model. Phosphorylated PDHE1α, a substrate of PDK4, accumulated in CD4+ T cells in the lamina propria of patients with inflammatory bowel disease. Both constitutive KO and CD4+ T-cell–specific deletion of PDK4 delayed DSS-induced colitis. Adoptive transfer of PDK4-deficient CD4+ T cells attenuated murine colitis, and PDK4 deficiency resulted in decreased activation of CD4+ T cells and attenuated aerobic glycolysis. Mechanistically, there were fewer endoplasmic reticulum–mitochondria contact sites, which are responsible for interorganelle calcium transfer, in PDK4-deficient CD4+ T cells. Consistent with this, GM-10395, a novel inhibitor of PDK4, suppressed T-cell activation by reducing endoplasmic reticulum–mitochondria calcium transfer, thereby ameliorating murine colitis.

      Conclusions

      PDK4 deletion from CD4+ T cells mitigates colitis by metabolic and calcium signaling modulation, suggesting PDK4 as a potential therapeutic target for IBD.

      Graphical abstract

      Keywords

      Abbreviations used in this paper:

      ATP (adenosine triphosphate), CD (Crohn’s disease), DEG (differentially expressed gene), DSS (dextran sodium sulfate), ECAR (extracellular acidification rate), ELISA (enzyme-linked immunosorbent assay), ER (endoplasmic reticulum), GSEA (Gene Set Enrichment Analysis), IBD (inflammatory bowel disease), IEC (intestinal epithelial cells), IFN (interferon), IHC (immunohistochemistry), IL (interleukin), IP3R (inositol trisphosphate receptor), KO (knockout), LP (lamina propria), MAM (mitochondria-associated membrane), mTOR (mechanistic target of rapamycin), NFAT (nuclear factor of an activated T cell), OCR (oxygen consumption rate), OXPHOS (oxidative phosphorylation), PBS (phosphate-buffered saline), PCR (polymerase chain reaction), PDHE1α (pyruvate dehydrogenase E1α), PDK (pyruvate dehydrogenase kinase), SOCE (store-operated calcium entry), STIM1 (stromal interaction molecule 1), TCR (T-cell receptor), TNF (tumor necrosis factor), UC (ulcerative colitis), VDAC (voltage-dependent anion channel), WT (wild-type)
      Metabolic reprograming and calcium signaling are critical to pathogenic CD4+ T cells. Here, we demonstrate that PDK4 deletion mitigates aerobic glycolysis and calcium signaling by hindering mitochondria-associated membranes in activated CD4+ T cells. Consequently, PDK4 inhibition attenuates colitis in mice.
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      Here, we show increased PDK4 and p-PDHE1α expression in T cells infiltrating the colonic lamina propria (LP) of patients with IBD and in the LP of dextran sulfate sodium (DSS)-challenged mice. Genetic and pharmacologic inhibition of PDK4 prevented DSS-induced colitis. Of note, ablation of PDK4 in CD4+ T cells, but not in intestinal epithelial cells (IEC), is required for amelioration of colitis. In vitro studies revealed that PDK4-deficient CD4+ T cells were less activated than wild-type (WT) CD4+ T cells. In addition, PDK4 deficiency restored the Th17/Treg balance both in vitro and in vivo. Mechanistically, PDK4 ablation led to bioenergetic dysfunction, accompanied by decreased MAM formation/interorganelle calcium transfer and decreased aerobic glycolysis/mechanistic target of rapamycin (mTOR) signaling pathway activation in CD4+ T cells, leading to suppression of T-cell activation and effector functions.

      Results

      Increased Expression of PDK4 by Colitogenic CD4+ T Cells From Humans and Mice

      To identify the PDK isoform responsible for intestinal inflammation, we first examined the effect of DSS-induced inflammation on expression of PDK in mouse colon. DSS administration led to a significant time-dependent increase in colonic expression of PDK4, as well as enhanced PDHE1α phosphorylation (Figure 1A). This finding led us to focus on the role of PDK4 in the pathogenesis of IBD.
      Figure thumbnail gr1
      Figure 1PDK4 or p-PDHE1α is enriched in CD4+ T cells from a mouse model of DSS-induced colitis. (A and B) WT mice received 2.5% DSS in drinking water for 0, 2, 4, 6, or 8 days. The experiment was repeated twice. (A) Representative Western blots of PDK1-4, p-PDHE1α, and β-actin expression in colon tissues (n = 2–3). (B) Representative images of immunohistochemistry staining of p-PDHE1α in colon tissues from mice after treatment with DSS for 6 days (n = 3). Scale bar, 100 μm. (C) Gating strategy used to examine expression of p-PDHE1α in gut neutrophils, macrophages, dendritic cells, CD8+ T cells, and CD4+ T cells. (D–G) p-PDHE1α levels in cells isolated from the colon of mice at day 0 (Ctrl) and day 6 (DSS-6d) of DSS treatment were analyzed by flow cytometry (n = 5, Ctrl; n = 6, DSS-6d). Expression of p-PDHE1α by (D) CD45+ (hematopoietic cells) and (F) CD45- (non-hematopoietic cells) in the lamina propria single-cell population isolated from DSS-treated mice. (E) CD3+ T cells were further gated into CD3+CD4+ and CD3+CD8+ T-cell populations. (G) Ly6G+ (neutrophils), CD11b+ (macrophages), CD11c+ (dendritic cells), and CD45+Ly6G-CD11b-CD11c- cells. The experiment was repeated twice. Mean ± standard error; ∗P < .05, ∗∗P < .01 (Student t test).
      Immunohistochemistry (IHC) staining was performed to identify the location of phosphorylated PDHE1α in tissue sections from mice at 6 days after DSS challenge. Although there was no difference in p-PDHE1α expression in IECs between before and after DSS challenge, the level of p-PDHE1α–positive cells was markedly higher in the severely inflamed LP (DSS-6d) than in the DSS-unchallenged LP (control) (Figure 1B).
      Next, we profiled p-PDHE1α–expressing cells using flow cytometry (Figure 1C). After DSS challenge, we found increased expression of p-PDHE1α (Figure 1D). In line with the IHC staining results, there was no difference in p-PDHE1α levels in nonhematopoietic cells (CD45-) at DSS0 or DSS6 (Figure 1F), whereas p-PDHE1α levels were elevated in leukocytes (CD45+ cells) (Figure 1D); these data suggest that increased expression of p-PDHE1α upon DSS challenge is a phenomenon confined to leukocytes. Colonic CD3+ T cells, neutrophils, and macrophages, but not dendritic cells, showed higher expression of p-PDHE1α (Figure 1E and G), suggestive of overall immunometabolic reprograming under inflammatory conditions. Under conditions of intestinal inflammation (DSS6), CD4+ T cells showed higher expression of p-PDHE1α than under physiological conditions (DSS0) (Figure 1E). However, altered expression in CD8+ T cells was not as prominent (Figure 1E).
      Consistent with these findings, IHC staining clearly showed that expression of PDK4/p-PDHE1α (Figure 2A and B) was markedly enriched in the LP, but not in the IEC, of inflamed tissue from patients with IBD. This suggests that infiltrating immune cells are likely to be responsible for augmented expression of PDK4/p-PDHE1α in the inflamed area. Because aberrant activation of CD4+ T cells and macrophages is the key contributor to the pathogenesis of IBD,
      • Neurath M.F.
      Cytokines in inflammatory bowel disease.
      we hypothesized that altered expression of PDK4/p-PDHE1α would be associated with accumulation of macrophages or CD4+ T cells in the gut. Therefore, we performed co-immunofluorescence staining of inflamed biopsy tissue collected from patients with IBD and from healthy controls to determine which infiltrating immune cells express p-PDHE1α under inflammatory conditions (Figure 2C and D). In normal controls, the percentage of p-PDHE1α–positive CD4+ T cells was significantly higher than that of CD64+ macrophages (Figure 2E), suggesting that CD4+ T cells are the likely source of augmented PDK expression. Moreover, the p-PDHE1α–expressing CD4+ T-cell population was significantly enriched in inflamed biopsy tissue from patients with IBD when compared with that in controls (Figure 2C). Expression of phosphorylated PDHE1α in macrophages from patients with IBD was not different from that in the normal control (Figure 2D), despite augmented expression of p-PDHE1α by macrophages under conditions of intestinal inflammation observed in the murine colitis model (Figure 1G). Collectively, these results highlight the colitogenic role of the PDK4/p-PDHE1α axis in CD4+ T cells during intestinal inflammation in both patients with IBD and mouse colitis models.
      Figure thumbnail gr2
      Figure 2Augmented expression of p-PDHE1α in lamina propria immune cells from patients with IBD. (A and B) Representative IHC staining of PDK4 and p-PDHE1α in inflamed (I) or noninflamed (N) colonic biopsies from patients with CD (n = 13) and UC (n = 11). Scale bar, 100 μm. (C and D) Co-immunofluorescence staining of p-PDHE1α (magenta) and (C) CD4 (yellow) or (D) CD64 (yellow) in inflamed tissues from patients with IBD (n = 14) and controls (n = 6). Co-staining is indicated by green arrow. Nuclei are stained with DAPI (cyan). (E) Ratios of p-PDHE1α–positive cells to CD4+ T cells and CD64+ macrophages. Mean ± standard error; ∗P < .05, ∗∗P < .01, ∗∗∗P < .001 (Student t test).

      PDK4-Deficient Mice Are Protected From DSS-Induced Colitis

      Next, we tested the anticolitogenic effects of PDK4 deficiency in a DSS-treated colitis mouse model. Before induction of experimental colitis, we confirmed that colonic IEC from PDK4 KO mice display normal histology, proliferation, and apoptosis (Figure 3A–C). Specific gene expression of IEC markers Reg3g (Paneth cells), Muc1 and Muc2 (goblet cells), Kit (crypt cells), and Lgr5 (stem cells) in PDK4 KO mice was also comparable with that in WT mice (Figure 3D and E).
      Figure thumbnail gr3
      Figure 3PDK4-deficient mice have normal colon epithelial functions. (A) Functions of intestinal epithelial cells (IECs) from PDK4-deficient mice were comparable with those of IECs from WT mice (n = 3). Representative images showing H&E staining of colon sections. (B) Representative immunohistochemistry images showing staining of Ki-67. (C) Representative images of TUNEL staining (green); nuclei are stained with DAPI (blue). (D) Representative images showing Alcian blue staining. (E) Expression of mRNA transcripts encoding Reg3g (Paneth cell marker), Muc1 and Muc2 (goblet cell markers), Kit (crypt cell marker), and Lgr5 (stem cell marker). Mean ± standard error; Student t test.
      Next, we challenged WT or PDK4 KO mice with DSS. PDK4 KO mice lost less weight than WT mice upon DSS challenge (Figure 4A). Also, disease was less severe in PDK4 KO mice (Figure 4B). Histologic analysis revealed that WT mice exhibited extensive loss of the epithelial lining, accompanied by severe inflammation of the LP, whereas PDK4 KO mice showed a relatively well-retained intestinal epithelial layer and minimal infiltration of the LP by inflammatory cells (Figure 4C and D). The colon length in PDK4 KO mice was better preserved than that in WT mice (Figure 4E and F). In addition, DSS-induced intestinal permeability was attenuated in PDK4 KO mice (Figure 4G). We also observed down-regulation of mRNA encoding cytokines (Ifng, Il1b, and Il6) in the colon of PDK4 KO mice (Figure 4H). These data clearly show that PDK4 KO mice are resistant to colitis.
      Figure thumbnail gr4
      Figure 4PDK4 deficiency protects against DSS-induced colitis. C57BL/6J PDK4 KO or WT mice received 2.5% DSS in drinking water for 6 days (n = 6). The experiment was repeated 3 times. (A) Weight changes. (B) Disease activity scores. (C) Histologic scores. (D) Representative H&E staining. Scale bars: upper, 500 μm; lower, 100 μm. (E) Gross image of the colon. (F) Colon length. (G) In vivo intestinal permeability test. (H) Relative levels of mRNA transcripts encoding Ifng, Il1b, Il6, Il12b, Il17a, and Tnfa in colon tissues (n = 6). (I) Gating strategy to identify Th1 (IFN-γ), Th17 (IL17α), and Tregs (Foxp3+CD25+) cells. (J) Percentage of helper CD4+ T cells (CD4) among CD3+ T-cell population in the lamina propria was measured by flow cytometry. (K) Flow cytometry analysis of percentage of Th1 (IFN-γ), Th17 (IL17α), and Treg (Foxp3+CD25+) cells among gut-infiltrating CD4+ T cells. (L) Ex vivo cytokine production (IFN-γ, IL1β, IL6, IL12, IL17, and TNF-α) of colon organ cultures was measured by ELISA (n = 6). Mean ± standard error; ∗P < .05, ∗∗P < .01, ∗∗∗P < .001 (Student t test).
      Flow cytometry analysis (Figure 4I) revealed that PDK4 deletion reduced the absolute number and percentage of CD4+ T cells significantly (Figure 4J), including Th1 (interferon gamma [IFN-γ] secreting) and Th17 (interleukin [IL] 17α secreting) cells in the LP (Figure 4K). By contrast, the numbers of immunosuppressive Treg cells in PDK4 KO mice increased significantly (Figure 4K). Colonic cytokine production (IFN-γ, IL1, IL12, IL17, and tumor necrosis factor alpha (TNF-α]) was reduced in response to colitis during ex vivo organ culture (Figure 4L). Taken together, these findings suggest that PDK4 deficiency protects mice against DSS-induced colitis.

      CD4+ T-Cell–Specific PDK4-Deficient Mice Are Protected From DSS-Induced Colitis

      Next, we used a CD4+ T-cell–specific PDK4 KO mouse (PDK4CD4) model to show that PDK4 deficiency-mediated protection against DSS-induced colitis is regulated by CD4+ T cells (Figure 5A–C). As observed in PDK4 KO mice, PDK4CD4 mice showed significant attenuation of DSS-induced colitis, as judged by less weight loss, lower disease severity scores, and milder sequelae on histologic analysis (Figure 5D–G). We observed marked infiltration of the LP by immune cells, as well as loss of IEC, in WT mice; these pathologies were profoundly attenuated in PDK4CD4 mice (Figure 5G). Furthermore, colon length and intestinal barrier function (Figure 5H–J) were better in PDK4CD4 mice than in WT controls. Expression of mRNA encoding all proinflammatory cytokines (Ifng, Il1b, Il6, Il12b, Il17a, and Tnfa) tested in this study was attenuated in PDK4CD4 mice (Figure 5K). Flow cytometry analysis (Figure 5L) revealed that the total number of LP CD4+ T cells in PDK4CD4 mice was significantly lower than that in WT mice (Figure 5M), and that PDK4CD4 mice had lower percentages of Th1 and Th17 (Figure 5N). By contrast, PDK4CD4 mice had higher numbers of immunosuppressive Tregs than WT controls (Figure 5N). Inflamed colon tissues from PDK4CD4 mice secreted substantially lower amounts of cytokines (IL1, IL12, IL17, and TNF-α) than the control (Figure 5O). Collectively, these data suggest that expression of PDK4 in CD4+ T cells is required for development of DSS-induced colitis.
      Figure thumbnail gr5
      Figure 5PDK4 deletion by CD4-Cre reduced intestinal inflammation in mice. (A–C) Validation of PDK4CD4 mouse line. (A) Expression of PDK4 protein in kidney, liver, and spleen samples from PDK4 WT PDK4fl/fl or PDK4CD4 mice. (B) Relative protein expression was normalized to that of HSP90. (C) Pdk4 (exon2) mRNA transcript levels in MACS-sorted CD4+ or CD4- cells, as measured by quantitative PCR. (D–K) PDK4fl/fl (denoted as WT) and PDK4CD4 (denoted as KO) mice (n = 6–8/group) received 2.5% DSS in drinking water for 6 days. The experiment was repeated 3 times. (D) Weight changes. (E) Disease activity scores. (F) Histologic scores. (G) Representative H&E staining of tissues from PDK4fl/fl and PDK4CD4 mice. Scale bars: upper, 500 μm; lower, 100 μm. (H) Gross image of the colon. (I) Colon length. (J) In vivo intestinal permeability test. (K) Relative expression of mRNA transcripts encoding Ifng, Il1b, Il6, Il12b, Il17a, and Tnfa in colonic tissues. (L) Gating strategy to identify Th1 (IFN-γ), Th17 (IL17α), and Tregs (Foxp3+CD25+) cells (n = 6 mice/group). (M) Percentage of helper CD4+ T cells (CD4) among the CD3+ T-cell population in the lamina propria was measured by flow cytometry. (N) Flow cytometry analysis of percentage of Th1 (IFN-γ), Th17 (IL17α), and Treg (Foxp3+CD25+) cells among gut-infiltrating CD4+ T cells. (O) Ex vivo cytokine production (IFN-γ, IL1β, IL6, IL12, IL17, and TNF-α) of colon organ cultures was measured by ELISA (n = 5). Mean ± standard error; ∗P < .05, ∗∗P < .01, ∗∗∗P < .001 (Student t test).

      PDK4 Deletion From IEC Does Not Protect Against Experimental Colitis

      To rule out the possibility that PDK4 deficiency in IECs contributes to protection against intestinal inflammation despite its redundant role in IECs under normal conditions (Figure 3), we challenged mice lacking PDK4 specifically in IECs (PDK4villin) (Figure 6A and B) with DSS. Weight loss and disease severity scores after DSS challenge were similar between DSS-treated and control mice (Figure 6C and D). Histologic analysis revealed that inflammation was comparable between the test and control groups (Figure 6E and F). Although colon length was modestly shorter in PDK4villin mice (Figure 6G and H), DSS-induced intestinal permeability (Figure 6I) was not different between groups. These observations indicate that PDK4 in IECs plays a dispensable role in pathogenesis of colitis.
      Figure thumbnail gr6
      Figure 6Deletion of PDK4 from IECs is not essential for protection against DSS-induced colitis. (A and B) Validation of PDK4villin mouse line. (A) Expression of PDK4 protein in intestinal epithelial cells (IECs) isolated from the small intestine (SI) and colon. (B) Levels of Pdk4 transcripts in the liver, kidney, muscle, white adipose tissue (WAT), SI, and colon, as measured by qPCR. (C–I) PDK4flox (indicated as WT) and PDK4Villin (indicated as KO) mice (n = 6) received 2.5% DSS in drinking water for 6 days. The experiment was repeated twice. (C) Weight changes. (D) Disease activity scores. (E) Representative images showing H&E staining of PDK4flox and PDK4Villin after onset of DSS-induced colitis. (F) Histologic scores. (G) Gross images of the colon. (H) Colon length. (I) In vivo permeability test results after oral administration of FD4. Mean ± standard error, Student t test: ∗P < .05.

      PDK4-deficient T Cells Ameliorate Adoptive T-Cell Transfer Colitis

      Because the chemically induced colitis model has limitations with respect to the pathologic mechanisms that cause human disease, as well as colitis-associated gene expression,
      • te Velde A.A.
      • de Kort F.
      • Sterrenburg E.
      • Pronk I.
      • ten Kate F.J.
      • Hommes D.W.
      • van Deventer S.J.
      Comparative analysis of colonic gene expression of three experimental colitis models mimicking inflammatory bowel disease.
      we adoptively transferred natural Treg-depleted naive CD4+ T cells (CD4+CD45RBhiCD25-) from WT or PDK4 KO mice to Rag1-/- recipient mice. These mice, which lack Treg cells, are prone to developing colitis spontaneously.
      • Mottet C.
      • Uhlig H.H.
      • Powrie F.
      Cutting edge: cure of colitis by CD4+CD25+ regulatory T cells.
      ,
      • Mizoguchi E.
      • Low D.
      • Ezaki Y.
      • Okada T.
      Recent updates on the basic mechanisms and pathogenesis of inflammatory bowel diseases in experimental animal models.
      Unlike mice receiving WT T cells, mice injected with PDK4 KO naive T cells developed less severe colitis, as assessed by weight changes and disease severity scores (Figure 7A and B). Histologic examination revealed a near absence of inflammation in PDK4 KO T-cell–transferred mice (Figure 7C and D). Despite comparable colon lengths (Figure 7E and F), in vivo intestinal permeability was also significantly less severe in these mice (Figure 7G). Expression of mRNA encoding cytokines Il1b, Il12, and Il17a in the colon was significantly lower in PDK4 KO T-cell–transferred mice (Figure 7H).
      Figure thumbnail gr7
      Figure 7PDK4-deficient T cells delay intestinal homing and induction of colitis after adoptive transfer. Rag1-/- mice received 4 × 105 CD4+CD45RBhiCD25- T cells sorted from splenocytes isolated from WT or PDK4 KO mice (n = 9). The experiment was repeated 3 times. (A) Weight changes. (B) Disease activity scores. (C) Histologic scores. (D) Representative H&E staining of tissues from PDK4fl/fl and PDK4CD4 mice. Scale bars: upper, 500 μm; lower, 100 μm. (E) Gross image of the colon. (F) Colon length. (G) In vivo intestinal permeability test. (H) Relative expression of mRNA transcripts encoding Ifng, Il1b, Il6, Il12b, Il17a, and Tnfa in colon tissues (n = 6). (I) Gating strategy to identify Th1 (IFN-γ), Th17 (IL17α), and Tregs (Foxp3+CD25+) cells (n = 6 mice/group). (J) Percentage of helper CD4+ T cells (CD4) among the CD3+ T-cell population in the lamina propria was measured by flow cytometry. (K) Flow cytometry analysis of percentage of Th1 (IFN-γ), Th17 (IL17α), and Treg (Foxp3+CD25+) cells among gut-infiltrating CD4+ T cells. (L) Ex vivo cytokine production (IFN-γ, IL1β, IL6, IL12, IL17, and TNF-α) of colon organ cultures was measured by ELISA (n = 6). Mean ± standard error; ∗P < .05, ∗∗P < .01, ∗∗∗P < .001 (Student t test). (M and N) Natural Treg-depleted naive CD4+ T cells (CD4+CD45RBhiCD25-) were transferred to Rag1-/- mice. Samples were collected from the spleen and mesenteric lymph nodes and subjected to flow cytometry analysis to investigate whether PDK4 KO T cells show normal survival, proliferation, and tissue-homing capacity in secondary lymphoid organs [(M) spleen and (N) mesenteric lymph nodes] in vivo.
      Flow cytometry analysis (Figure 7I) indicated that the number of PDK4 KO CD4+ T cells in the LP was lower than that in control mice, whereas the percentage of CD4+ within the CD3+ T-cell population was comparable, suggesting that PDK4 is likely important for intestinal homing (Figure 7J). The percentage of Th1 and Th17 cells in the LP of mice receiving PDK4 KO T cells was significantly lower than that in controls (Figure 7K), whereas the percentage of iTregs in the LP of PDK4 KO T-cell–transferred mice was higher than that in the controls (Figure 7K), despite the absence of adoptive natural Treg cells. Cytokine production (IL1, IL12, and IL17) by ex vivo colon explant cultures was significantly down-regulated (Figure 7L).
      Because PDK4-deficient naive CD4+ T cells failed to induce intestinal inflammation, we assessed the reconstitution of CD4+ T cells in lymphoid organs in Rag1-/- mice. Flow cytometry analysis revealed a comparable distribution of CD3+ T cells and CD4+ T cells in spleen and mesenteric lymph nodes (Figure 7M and N) between WT and PDK4 KO T-cell–transferred mice, which indicates intact cell survival and lymphoid tissue homing in vivo. These results demonstrate that protection from colitis mediated by PDK4-deficient naive CD4+ T cells was due to diminished local immune responses in the gut. Taken together, the results indicate that CD4-specific PDK4 deficiency is sufficient to trigger anticolitogenic mechanisms in mice without disturbing immune integrity in other organs.

      PDK4 Deficiency Suppresses T-Cell Activation and Th17 Differentiation

      To gain insight into the molecular mechanisms underlying the anticolitic effects of PDK4 ablation, we measured expression of PDKs in activated CD4+ T cells (Figure 8A and B). Unstimulated basal PDK4 protein levels were relatively low but peaked after 2 hours of activation, whereas PDK2 was suppressed, and PDK3 reached a peak at a later time (48 hours) (Figure 8A). Pdk4 transcription peaked at 4 hours after activation, whereas pdk3 transcription increased at a later time (Figure 8B). Publicly available CD4+ T-cell activation RNA-sequencing data from the GEO database (GSE96538) also revealed increased pdk4 transcription at 2 hours (Figure 8C),
      • Schmidt A.
      • Marabita F.
      • Kiani N.A.
      • Gross C.C.
      • Johansson H.J.
      • Elias S.
      • Rautio S.
      • Eriksson M.
      • Fernandes S.J.
      • Silberberg G.
      • Ullah U.
      • Bhatia U.
      • Lahdesmaki H.
      • Lehtio J.
      • Gomez-Cabrero D.
      • Wiendl H.
      • Lahesmaa R.
      • Tegner J.
      Time-resolved transcriptome and proteome landscape of human regulatory T cell (Treg) differentiation reveals novel regulators of FOXP3.
      which was consistent with our own data. These data illustrate that PDK4 is induced early during T-cell activation.
      Figure thumbnail gr8
      Figure 8PDK4 differentially regulates expression of genes associated with T-cell activation. (A and B) WT naive CD4+ T cells were treated with αCD3 and αCD28 for 0, 1, 2, 4, 8, 24, and 48 hours. The fold changes in protein and mRNA expression of each PDK1-4 isotype were measured by Western blotting and qPCR, respectively. (C) Meta-analysis of PDK transcript levels in human WT naive CD4+ T cells after TCR-induced activation based on previously published gene expression data sets (GSE96538). Expression levels were normalized to T = 2 hours. (D) WT or PDK4 KO naive CD4+ T cells were activated for 48 hours under Th0 conditions. Scatter plot of Gene Set Enrichment Analysis (GSEA) using a gene set of interest (immune response on C2:KEGG or C2:BIOCARTA pathway database). (E) Next-generation sequencing identified 425 differentially expressed genes (DEGs). Volcano plot showing DEGs based on the following cutoff setting (P value <.05; fold change >1.5). Overall, 210 genes were down-regulated and 215 were up-regulated in KO T cells. DEGs associated with lymphocyte activation (GO:0046649) were labeled. (F) GSEA of genes involved in regulation of lymphocyte activation (GO:0051249). (G) PDK4 WT or KO naive CD4+ T cells were stimulated for 3 days under Th0 conditions in presence/absence of IL2 (n = 4). T-cell activation makers (CD62L-, CD25+, CD69+) were analyzed by flow cytometry. The experiment was repeated twice. (H–M) Naive CD4+ PDK4 WT or KO T cells were activated for 72 hours (n = 2–3). (H) Cell size, as assessed by forward scatter (FSC), and (I) cell granularity, as assessed by side scatter (SSC). (J) Secreted IFN-γ and IL17α were measured in sandwich ELISAs. Viability, proliferation, and apoptosis were evaluated by (K) Live/Dead staining, (L) CFSE staining, and (M) PI/Annexin V staining. This experiment was repeated at least twice. Mean ± standard error, Student t test: ∗P < .05, ∗∗P < .01, ∗∗∗P < .001.
      Next, we analyzed the gene expression profile in activated CD4+ T cells using unbiased RNA-sequencing. In total, 425 genes were differentially regulated, of which 215 were up-regulated and 210 were down-regulated, in PDK4 KO CD4+ T cells (Figure 8E). Notably, Gene Set Enrichment Analysis (GSEA) revealed that the calcium signaling pathway, inositol lipid mediated signaling, and PI3K signaling were down-regulated in PDK4 KO T cells (Figure 8D).
      Because enrichment of lymphocyte activation was evident (P = .03) (Figure 8F), we examined whether PDK4 ablation affects activation of CD4+ T cells under Th0 conditions (Figure 8G). Because IL2 is a potent enhancer of T-cell function, we also tested whether IL2 production is a limiting factor. Consistent with this, we observed that PDK4 deficiency resulted in reduced T-cell activation (Figure 8G), as well as reduced cell granularity and size (Figure 8H and I). Secretion of proinflammatory cytokines (IFN-γ and IL17α) decreased significantly under PDK4-deficient conditions (Figure 8J), whereas cell viability, proliferation, and apoptosis were affected modestly by PDK4 deficiency (Figure 8K–M). Addition of exogenous IL2 restored neither T-cell activation (Figure 8G) nor IL17α secretion (Figure 8J), suggesting that IL2 secretion is not a notable cause of T-cell activation.
      Metabolic reprograming toward aerobic glycolysis is a hallmark of T-cell activation.
      • Chi H.
      Regulation and function of mTOR signalling in T cell fate decisions.
      ,
      • Pearce E.L.
      • Poffenberger M.C.
      • Chang C.H.
      • Jones R.G.
      Fueling immunity: insights into metabolism and lymphocyte function.
      We found that PDK4 deficiency altered expression of glycolytic genes and mTOR signaling pathways in CD4+ T cells (Figure 9A–C), implying differential bioenergetics profiles. PDK4 KO CD4+ T cells had reduced basal glycolysis and glycolytic capacity (Figure 8D–F). At the same time, we observed decreased basal, maximal, and adenosine triphosphate (ATP)–linked oxygen consumption rate (OCR) and reserve capacity in PDK4-deficient CD4+ T cells (Figure 8G–K). Metabolic responsiveness to exogenous IL2 was significantly lower in PDK4-deficient CD4+ T cells (Figure 8D–K). Nevertheless, the degree of reduction in basal glycolysis was greater than that of basal OCR, resulting in a relatively higher OCR/extracellular acidification rate (ECAR) ratio (Figure 8L). Consistent with this, the increase in cytosolic lactate upon T-cell activation was attenuated significantly in PDK4-deficient CD4+ T cells (Figure 8M), indicating reduced aerobic glycolysis. Despite addition of IL2, phosphorylation of ribosomal protein S6 (p-RPS6), a readout of mTOR signaling, was also attenuated (Figure 8N). Taken together, these findings suggest that deletion of PDK4 from CD4+ T cells rewires metabolism toward down-regulated mTOR signaling and reduced aerobic glycolysis.
      Figure thumbnail gr9
      Figure 9PDK4 mediates metabolic reprograming of activated CD4+ T cells and T-cell differentiation. (A) Scatter plot of Gene Set Enrichment Analysis (GSEA) results using a gene set of interest (metabolic process on C2:KEGG or C2:BIOCARTA pathway database). (B) Heatmap showing expression of genes associated with glycolytic enzymes and the mTOR signaling pathway. (C) GSEA plot showing genes involved in positive regulation of glycolytic processes (Gene set GO:0045821). (D–F) WT or PDK4 KO CD4+ T cells were stimulated for 3 days under Th0 conditions. (D) Glycolysis stress test assessed using an XF96 analyzer. The experiment was repeated 3 times. (E) Basal glycolysis measured in glycolysis stress test. (F) Glycolytic capacity was measured in glycolysis stress test. (G) Oxygen consumption rate was measured in mitochondrial stress test. (H–K) Basal OCR, maximal OCR, reserve capacity OCR, and ATP-linked OCR were calculated on basis of the mitochondrial stress test. (L) Ratio of basal OCR to basal ECAR. (M) Relative amounts of cytosolic lactate were measured by liquid chromatography-mass spectrometry. (N) Frequency of phosphorylated ribosomal protein S6 (p-RPS6) was analyzed by flow cytometry (n = 3). (O–R) PDK4 WT or KO naive CD4+ T cells were differentiated for 2 days under (O) Th1, (P) Th2, (Q) Th17, or (R) Treg polarizing conditions. Each T-cell subsets were identified by flow cytometry analysis. The experiment was repeated 3 times. Mean ± standard error, Student t test: ∗P < .05, ∗∗P < .01, ∗∗∗P < .001.
      Imbalance between effector T and regulatory T cells contributes to tissue damage during colitis. Therefore, we examined the ability of PDK4 KO naive CD4+ T cells to differentiate into Th1, Th2, Th17, and Treg cells. Although IFN-γ secretion by PDK4 KO CD4+ T cells activated under Th0 conditions was differentially regulated (Figure 7J), we did not observe any differences in Th1 differentiation (Figure 8O). Naive CD4+ T cells from both groups polarized into Th2 cells (Figure 8P). Notably, expression of IL17a by PDK4-deficient Th17 cells was significantly lower than that in the WT (Figure 8Q), whereas the proportion of Foxp3+CD25+ Tregs differentiated from PDK4 KO naive CD4+ T cells increased markedly (Figure 8R). Collectively, these observations demonstrate that inhibition of PDK4 suppresses T-cell effector functions and Th17/Treg differentiation.

      Calcium Homeostasis and Metabolic Reprograming in PDK4-Deficient CD4+ T Cells Are Impaired Through Disrupted MAM

      Intracellular calcium flux is essential for lymphocyte activation; thus, any biological processes that impede calcium homeostasis can impact lymphocyte activation.
      • Bertin S.
      • Aoki-Nonaka Y.
      • de Jong P.R.
      • Nohara L.L.
      • Xu H.
      • Stanwood S.R.
      • Srikanth S.
      • Lee J.
      • To K.
      • Abramson L.
      • Yu T.
      • Han T.
      • Touma R.
      • Li X.
      • Gonzalez-Navajas J.M.
      • Herdman S.
      • Corr M.
      • Fu G.
      • Dong H.
      • Gwack Y.
      • Franco A.
      • Jefferies W.A.
      • Raz E.
      The ion channel TRPV1 regulates the activation and proinflammatory properties of CD4(+) T cells.
      ,
      • Chi H.
      Regulation and function of mTOR signalling in T cell fate decisions.
      ,
      • Martinvalet D.
      The role of the mitochondria and the endoplasmic reticulum contact sites in the development of the immune responses.
      RNA-sequencing analysis revealed that PDK4 deficiency led to compromised calcium signaling signatures (Figure 10A and B), including Plcg2, which is a key mediator that triggers SOCE (Figure 10B). To investigate whether PDK4 deletion affects calcium homeostasis, we induced SOCE by ER calcium depletion using ATP or αCD3.
      • Ferris C.D.
      • Huganir R.L.
      • Snyder S.H.
      Calcium flux mediated by purified inositol 1,4,5-trisphosphate receptor in reconstituted lipid vesicles is allosterically regulated by adenine nucleotides.
      ,
      • Yang R.
      • Liang B.T.
      Cardiac P2X(4) receptors: targets in ischemia and heart failure?.
      Deletion of PDK4 significantly and simultaneously compromised both SOCE and mitochondrial calcium levels in CD4+ T cells (Figure 10C–F).
      Figure thumbnail gr10
      Figure 10MAM/SOCE/NFAT pathway is compromised by PDK4 deletion in activated CD4+ T cells. (A) GSEA plot of genes involved in the calcium signaling pathway (KEGG). (B) Heatmap of DEGs associated with the KEGG calcium signaling pathways. (C–F) Cytosolic and mitochondrial calcium were measured using Fura-2 and Rhod-2, respectively (n = 3). This experiment was repeated twice. IP3R-mediated calcium release from ER of naive CD4+ T cells from PDK4 WT or KO mice was induced by ATP (C and D) or αCD3 (E and F). Later, extracellular calcium (2 mmol/L) was added to induce SOCE. (G) Mitochondria-ER colocalization was visualized by co-immunofluorescence staining. CD4+ T cells from PDK4 WT or KO naive were activated for 3 days under Th0 conditions. Co-immunofluorescence staining of ER (PDI, magenta), mitochondria (VDAC, cyan), and nuclei (DAPI, blue). Scale bar, 10 μm. Mander’s coefficient was measured by ImageJ. (H and I) Mitochondria-ER colocalization was also visualized by in situ proximity ligation assay (PLA) (red dots). WT or PDK4 KO CD4+ T cells activated by αCD3+αCD28+IL2 for 72 hours. Nuclei were stained with DAPI (blue). (H) Representative in situ PLA z-stacked images of VDAC1-IP3R, IP3R-GRP75, or GRP75-VDAC1. (I) Representative confocal images of in situ PLA (red dots) of VDAC1/IP3R. Mean ± standard error; ∗P < .05, ∗∗P < .01, ∗∗∗P < .001 (Mann-Whitney test). (J) MAM is enriched in gut-infiltrating CD4 T cells from patients with UC (n = 10) compared with normal controls (n = 5); in situ PLA of VDAC1/IP3R (red) co-stained with hCD4 (green). Nuclei are stained with DAPI (blue). Mean ± standard error, Student t test: ∗∗∗P < .001. (K) Localization of NFATc1 (green) upon αCD3/αCD28 stimulation for 0.5 or 12 hours. Nuclei are stained with DAPI (blue and dashed line). (L) Relative transcription of glycolytic enzymes or transcriptional regulators bearing an NFAT-binding motif. (M) Secreted IL2 was measured in sandwich ELISA. Mean ± standard error; ∗P < .05, ∗∗P < .01, ∗∗∗P < .001 (Mann-Whitney test).
      One plausible hypothesis for altered calcium homeostasis in PDK4 KO CD4+ T cells is that PDK4 may reinforce MAM formation.
      • Thoudam T.
      • Ha C.M.
      • Leem J.
      • Chanda D.
      • Park J.S.
      • Kim H.J.
      • Jeon J.H.
      • Choi Y.K.
      • Liangpunsakul S.
      • Huh Y.H.
      • Kwon T.H.
      • Park K.G.
      • Harris R.A.
      • Park K.S.
      • Rhee H.W.
      • Lee I.K.
      PDK4 augments ER-mitochondria contact to dampen skeletal muscle insulin signaling during obesity.
      Consistent with this hypothesis, co-immunofluorescence staining of disulfide isomerase and VDAC (markers of the ER and mitochondria, respectively) revealed less colocalization in PDK4 KO CD4+ T cells (Figure 10G). In situ proximity ligation assays, a more reliable tool for detecting MAM formation, showed that the interaction between MAM components VDAC1, IP3R, and GRP75 was compromised in PDK4 KO CD4+ T cells (Figure 10H and I). Next, we investigated formation of MAM in pathogenic CD4+ T cells of patients with UC. A proximity ligation assay revealed that MAM was significantly augmented in CD4+ T cells from patients with UC (Figure 10J). Therefore, we speculate that PDK4 inhibition may be useful to buffer pathogenic CD4+ T cells in both mouse and human pathogenic T cells via MAM disorganization.
      SOCE and calcium signaling induce immediate calcineurin-mediated translocalization of NFATc1.
      • Vaeth M.
      • Maus M.
      • Klein-Hessling S.
      • Freinkman E.
      • Yang J.
      • Eckstein M.
      • Cameron S.
      • Turvey S.E.
      • Serfling E.
      • Berberich-Siebelt F.
      • Possemato R.
      • Feske S.
      Store-operated Ca(2+) entry controls clonal expansion of T cells through metabolic reprogramming.
      Therefore, we tested whether PDK4 deficiency impairs nuclear translocation of NFATc1 in T cells. The intensity of nuclear NFATc1 staining in PDK4-deficient CD4+ T cells was reduced markedly after TCR stimulation (Figure 10K). Accordingly, the transcription levels of transcriptional regulators (Myc, Hif1a, and Irf4) or glycolytic genes (Slc2a3 and Pgk1) that bear NFAT-binding motifs in the regulatory regions
      • Vaeth M.
      • Maus M.
      • Klein-Hessling S.
      • Freinkman E.
      • Yang J.
      • Eckstein M.
      • Cameron S.
      • Turvey S.E.
      • Serfling E.
      • Berberich-Siebelt F.
      • Possemato R.
      • Feske S.
      Store-operated Ca(2+) entry controls clonal expansion of T cells through metabolic reprogramming.
      were significantly down-regulated (Figure 10L). In line with this, secretion of IL2, which is transcriptionally regulated by NFAT, was markedly reduced in PDK4 KO T cells compared with the control (Figure 10M). Collectively, these data demonstrate that PDK4 seems to regulate metabolic reprograming in pathogenic CD4+ T cells via MAM/SOCE/calcium signaling pathways and NFATc1-dependent transcriptional expression of glycolytic genes.

      Small Molecule Targeting of PDK4 Prevents DSS-Induced Colitis

      Recently, we synthesized a novel small molecule, GM-10395, that targets PDK4. This compound successfully dephosphorylated PDHE1α in a dose-dependent manner, thereby suppressing PDK4 activity in CD4+ T cells (Figure 11A). PDK4 inhibition by GM-10395 decreased expression of T-cell activation markers CD25, CD69, and CD44 in CD4+ T cells (Figure 11B), which is consistent with the previous findings in genetically PDK4-deleted CD4+ T cells (Figure 11C). In addition, treatment with GM-10395 decreased cytosolic calcium influx triggered by ATP/calcium-induced SOCE, as well as simultaneous mitochondrial calcium influx, in a dose-dependent manner (Figure 11C–F), which is consistent with the previous findings in PDK4 KO CD4+ T cells (Figure 10). Taken together with the absence of PDK4 (Figure 8, Figure 9, Figure 10), these data support the findings of compromised T-cell activation via abrogated intraorganelle calcium transfer between the ER and mitochondria upon PDK4 inhibition.
      Figure thumbnail gr11
      Figure 11Pharmacologic inhibition of PDK4 inhibits T-cell activation and SOCE in CD4+ T cells and prevents acute/chronic DSS-induced colitis. (A) Effects of GM-10395 (0.5, 1, 5, and 10 μmol/L) on dephosphorylation of PDHE1α in activated CD4+ T cells cultured for 6 hours under Th0/IL-2 conditions. The experiment was repeated twice. (B) Naive CD4+ T cells were activated under Th0/IL2 conditions and co-treated with GM-10395 (1, 5, 10, 20, and 50 μmol/L) for 72 hours. T-cell activation markers (CD25+, CD69+, and CD44+) were measured by flow cytometry. Cyclosporin A (CsA; 10 μg/mL) was used as a positive control for T-cell inactivation. The experiment was repeated twice. (C–F) Effects of GM-10395 (0.1, 0.3, 1, and 3 μmol/L) on ATP-triggered SOCE. Cytosolic and mitochondrial calcium levels were measured by Fura-2 (C and D) and Rhod-2 (E and F), respectively. The experiment was repeated twice. Mean ± standard error; ∗P < .05, ∗∗P < .01, ∗∗∗P < .001 (Mann-Whitney test). (G) C57BL/6J JmsSlc mice (n = 6) received 4% DSS in drinking water for 9 days. GM-10395 (0.5 or 1 mg per kg) was administered orally. The experiment was repeated twice. (H) Body weight. (I) Representative images of H&E staining. (J) Histologic scores. (K) Gross image of the colon. (L) Colon length. (M) Permeability test results after oral administration of FD4. (N) Relative expression of mRNA transcripts encoding Ifng, Il1b, Il6, Il12b, Il17a, and Tnfa in colonic tissues. (O) C57BL6J JmsSlc mice (n = 7–8) were treated with 3 cycles of DSS. GM-10395 (1 mg/kg) was administered orally for 1 week, beginning after 3 days of DSS treatment. Mice were anesthetized for colitis examination. (P) H&E staining. (Q) Histologic scores were evaluated to determine disease severity. (R) Disease scores. (S) Gross image of the colon. (T) Colon length. (U) In vivo intestinal permeability. (V) Weight changes. Mean ± standard error; ∗P < .05, ∗∗P < .01, ∗∗∗P < .001 (Student t test).
      Remarkably, GM-10395 ameliorated both acute and chronic DSS-induced colitis, as evidenced by improved histology, preserved colon length, ameliorated intestinal permeability, and more rapid recovery of weight after DSS withdrawal (Figure 11G–V). Moreover, GM-10395–treated mice had significantly lower colonic transcription levels of Ifng, Il6, Il12b, Il17a, and Tnfa (Figure 11N). Collectively, these data demonstrate that pharmacologic inhibition of PDK4 by GM-10395 has therapeutic potential for IBD through compromised T-cell activation and mitochondria-ER calcium transfer in CD4+ T cells.

      Discussion

      We observed marked accumulation of PDK4- and p-PDHE1α–positive CD4+ T cells in colon biopsy samples from patients with IBD. Furthermore, both genetic and pharmacologic approaches showed that PDK4 in T cells, but not in IECs, is required for colitis induction in an experimental colitis model. In vitro studies revealed that PDK4-deficient CD4+ T cells were less activated, with slowed metabolism, as indicated by decreased OCR and ECAR, as well as by transcriptome analysis showing down-regulation of the glycolytic pathway,
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      PDK4 deficiency resulted in a significant defect in Th17 differentiation but expanded Treg polarization, both in vitro and in vivo. Mechanistically, PDK4 deletion reduced SOCE, resulting in diminished cytosolic and mitochondrial calcium concentrations. Notably, consistent with our previous study in myotubes,
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      However, the role of MAM in mitochondrial calcium and SOCE during T-cell activation is unclear. Previously, the effect of ER-mitochondrial MAM dissociation on SOCE was demonstrated in fibroblasts. Knockdown of neuronal calcium sensor 1 impairs IP3R-induced mitochondrial calcium uptake by fibroblasts through ER-mitochondrial calcium transport, resulting in modest impairment of both SOCE and mitochondrial respiration.
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      In this study, we showed that PDK4 inhibition significantly compromised transcription of glycolytic enzymes containing an NFAT-binding motif (Figure 5), thereby decreasing aerobic glycolysis in activated T cells (Supplementary Figure 8D). In addition, PDK4 inhibition mitigated Th17 differentiation in vivo and in vitro. Collectively, the data suggest that PDK4 inhibition is a viable strategy for resolving immune responses by pathogenic CD4+ T cells.
      We also demonstrated robust metabolic reprograming toward aerobic glycolysis by measuring p-PDHE1α in some of LP-infiltrating leukocytes using a DSS-induced colitis model. These cells include not only CD4+ T cells but also neutrophils and macrophages (Supplementary Figure 1C). Although we focused on the role of PDK4 in CD4+ T-cell activation by metabolic reprograming and mitochondria-ER calcium transfer, we cannot rule out the possibility that PDK4 deficiency in macrophages or neutrophils has anticolitogenic effects in mice. Previous studies show that expression of PDK4 is up-regulated significantly in LPS-treated
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      • Hyeon D.Y.
      • Hwang D.
      • Kim Y.H.
      • Lee C.H.
      • Lee T.
      • Kim J.W.
      • Choi Y.K.
      • Park K.G.
      • Chawla A.
      • Lee J.
      • Harris R.A.
      • Lee I.K.
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      ,
      • Na Y.R.
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      • Song J.
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      Moreover, neutrophils depend substantially on aerobic glycolysis and on the pentose phosphate pathway for their immune functions,
      • Jeon J.H.
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      Current understanding on the metabolism of neutrophils.
      as shown by several inhibitor studies,
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      ,
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      despite lack of direct evidence that PDKs are involved.
      Collectively, our findings demonstrate that suppressing PDK4 attenuates activation of pathogenic CD4+ T cells by metabolic reprograming via MAM/calcium-dependent pathways. These results provide convincing evidence that PDK4 is a potential novel therapeutic target for management of IBD. Future studies examining whether PDK4 inhibitors can be applied to human IBD are anticipated.

      Materials and Methods

      Mice

      PDK4 KO mice (B6.129-Pdk4tm1Rhar/J), provided by Dr Robert A. Harris (Indiana University School of Medicine), were backcrossed to WT C57BL/6J mice for at least 8 generations. Littermates were used as controls in all experiments. Rag1-/- mice (B6.129S7-Rag1tm1Mom/J) were purchased from the Jackson Laboratory. CD4Cre/+ mice (B6.Cg-TgCd4-cre1Cwi/BfluJ), provided by Dr Gap R. Lee (Sogang University, South Korea), and VillinCre/+ mice (B6.Cg-TgVil1-cre997Gum/J), provided by Dr Mi N. Kweon (University of Ulsan College of Medicine/Asan Medical Center, South Korea), were used to generate PDK4CD4 and PDK4Villin mice, respectively, by crossing with PDK4fl/fl mice (Cyagen Biosciences, Suzhou, China). C57BL/6J JmsSlc mice were used for the pharmacologic inhibition test. All mice were housed in a specific pathogen-free and temperature-controlled room (12:12 hours light/dark cycle).

      Patients

      Mucosal biopsies were collected from 24 patients with newly diagnosed IBD (11, UC; 13, CD) and from 6 non-IBD healthy controls at Kyungpook National University Hospital. No patients were taking corticosteroids or biologic treatments. Forceps biopsies of inflamed and noninflamed mucosa, as assessed by colonoscopy, were taken.

      DSS-induced Colitis Model

      Acute colitis was induced in male mice to exclude possible effects of hormonal differences or menstrual cycle. Eight-week-old male mice received 2.5% DSS (MP Biomedicals, Irvine, CA) in sterile drinking water ad libitum for 6 days, followed by normal drinking water for 5 days unless otherwise indicated.

      Adoptive T-Cell Transfer in the Colitis Model

      CD4+ T cells were enriched from splenocytes using an EasySep Mouse CD4+ T Cell Isolation Kit (cat. no. 19852; STEMCELL Technologies, Vancouver, Canada). To induce colitis, male Rag1-/- mice (6–7 weeks old) were infused (intraperitoneally) with 4 × 105 naive CD4+ T cells (CD4+CD45RBhiCD25-) in sterile phosphate-buffered saline (PBS). CD4+CD45RBhiCD25- cells were sorted using a MoFlo XDP cell sorter (Beckman Coulter, Brea, CA) or FACSAria III (BD Biosciences).

      Assessment of Colitis

      Mice were monitored daily or weekly for body weight loss and signs of intestinal inflammation. The disease activity score is a semiquantitative score calculated as the sum of stool consistency scores, occult blood scores, and weight loss scores.
      • Xu Y.
      • Hunt N.H.
      • Bao S.
      The role of granulocyte macrophage-colony-stimulating factor in acute intestinal inflammation.
      To analyze intestinal epithelial permeability in vivo, mice were administrated oral fluorescein isothiocyanate–dextran 4 kDa (FD4) 46944 (Sigma-Aldrich, St Louis, MO) in sterile saline. Blood was collected after 4 hours by cardiac puncture, and serum FD4 was measured with a fluorescence spectrophotometer at λem 488 nm/λex 520 nm. For some experiments, colon length was measured and compared between groups. Disease severity was examined histopathologically using a previously published scoring system,
      • Chinen T.
      • Komai K.
      • Muto G.
      • Morita R.
      • Inoue N.
      • Yoshida H.
      • Sekiya T.
      • Yoshida R.
      • Nakamura K.
      • Takayanagi R.
      • Yoshimura A.
      Prostaglandin E2 and SOCS1 have a role in intestinal immune tolerance.
      for which the criteria include loss of goblet cells, crypt abscesses, cellular infiltration, mucosa elongation, and epithelial erosion.

      In Vitro Culture of Naive T Cells

      Naive CD4+ T cells were isolated using a mouse CD4+CD62L+ T Cell Isolation Kit 130-106-643 (Miltenyi Biotec, Bergisch Gladbach, Germany). The purity was checked by flow cytometry analysis (>92%). Naive CD4+ T cells were cultured in RPMI medium (Welgene, Gyeongsan, South Korea) at 37°C for 2–3 days with plate-bound anti-CD3 and anti-CD28 antibodies under Th0 conditions (10% fetal bovine serum, 1% penicillin-streptomycin, and 2.5 μmol/L mercaptoethanol, in the presence or absence of 50 U/mL IL2).

      RNA-Sequencing

      Isolated naive CD4+ T cells were activated for 2 days in vitro, as described above. Total mRNA was extracted from 2,000,000 cells using Trizol (Qiagen, Hilden, Germany). Samples with RNA integrity number >6.1 and DV200 >50% were used to generate a library of reasonable quality using Quant-Seq 3´ mRNA-Seq Library Prep Kits. Because Quant-Seq specifically targets the 3´ end of transcripts, even RNAs with a lower RIN are suitable as input material. The amplified library was sequenced using a HiSeq 2500 System (Illumina, San Diego, CA). To compare differential gene expression, DESeq2 (Bioconductor v3.3.0) in R studio was used.
      • Love M.I.
      • Huber W.
      • Anders S.
      Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.
      GSEA was performed using the Broad Institute tool GSEA v.4.0.3.
      • Subramanian A.
      • Tamayo P.
      • Mootha V.K.
      • Mukherjee S.
      • Ebert B.L.
      • Gillette M.A.
      • Paulovich A.
      • Pomeroy S.L.
      • Golub T.R.
      • Lander E.S.
      • Mesirov J.P.
      Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles.
      Test gene sets included the C2.CP.KEGG and C5.BP gene sets from the Molecular Signatures Database. All data, including expression profiles generated in this study, are available through the Gene Expression Omnibus (accession no. GSE161653).

      Measurement of Intracellular Calcium

      Naive CD4+ T cells were incubated for 40 minutes with 2.5 mmol/L Fura-2-AM or Rhod-2-AM (Thermo Fisher Scientific, Waltham, MA). After washing, cells were kept in calcium-free Hanks balanced salt solution. Intracellular or mitochondrial calcium levels were measured using FlexStation 3 (Molecular Devices, San Jose, CA). After 1 minute, ATP (300 μmol/L) or CD3 (10 μg/mL) was added to induce depletion of ER calcium stores. Calcium influx was initiated by addition of calcium-containing Hanks balanced salt solution (final concentration, 2 mmol/L).

      Flow Cytometry Analysis

      For intracellular staining of phosphor-proteins, cells were fixed with 2% paraformaldehyde, followed by permeabilization in 95% methanol. The following antibodies were used: anti-p-PDHE1α (AP1062; Calbiochem, Darmstadt, Germany) and a fluorescein isothiocyanate conjugated antirabbit secondary antibody (Santa Cruz Biotechnology, Dallas, TX). For staining of intracellular cytokines/transcription factors, either the Mouse Foxp3 Buffer Set 560409 (BD Biosciences, Franklin Lakes, NJ) or the Foxp3/Transcription Factor Staining Buffer Set 00-5523-00 (eBioscience, San Diego, CA) was used along with the following antibodies: CD45-BV421, CD3-PECy7, CD4-APC, CD8-Alexafluor700, NK1.1-BV711, IFN-γ-BV510, IL-4-BV605, IL-17-PerCP-Cy5.5, CD25-BV785, Foxp3-FITC, CD69-PerCPCy5.5, and CD62L-PerCPCy5.5 (all from BioLegend, San Diego, CA). Cell viability was measured using a Live/Dead staining kit (Thermo Fisher). Cell death was measured using an FITC/Annexin V Apoptosis Detection Kit (556547; BD Biosciences).

      Western Blot Analysis

      Protein samples (20 μg) were separated in premade NuPAGE Bis-Tris gels (Thermo Fisher) and transferred to polyvinylidene difluoride membranes. The following antibodies were used: anti-PDK1 ADI-KAP-PK112-D (Enzo Biochem, New York, NY), anti-PDK2 sc-100534 (Santa Cruz Biotechnology), anti-PDK3 serum (rabbit antiserum provided by Dr Robert. A Harris), anti-PDK4 ab214938 (Abcam, Cambridge, MA), anti-p-PDHE1α (Calbiochem, San Diego, CA), anti-HSP90 #4874 (Cell Signaling Technology, Danvers, MA), and anti-β-actin A5441 (Sigma-Aldrich). Proteins were visualized using an LAS-4000 (BD Biosciences) or iBright1500 (Thermo Fisher).

      Quantitative Polymerase Chain Reaction

      Total RNA was extracted using Trizol reagent (Qiagen), and a RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher) was used to generate a cDNA library. Quantitative real-time polymerase chain reaction (qPCR) was performed using Power SYBR Green PCR Master Mix (Applied Biosystems, Waltham, MA) and a ViiA 7 Real-time PCR system (Applied Biosystems). The primers used in this study are listed in Table 1.
      Table 1List of Primers Used in This Study
      GeneForwardReverse
      Ifng3´-CAGCAACAGCAAGGCGAAAA-5´3´-TGGACCTGTGGGTTGTTGAC-5´;
      Tnfa3´-AGCCGATGGGTTGTACCTTG-5´3´-ATAGCAAATCGGCTGACGGT-5´;
      Il63´-CTTGGGACTGATGCTGGTGA-5´3´-GGTCTGTTGGGAGTGGTATCC-5´;
      Il1b3´-GAGCACCTTCTTTTCCTTCATCTT-5´3´-TCACACACCAGCAGGTTATCATC-5´;
      Il12b3´-TTGCCATCGTTTTGCTGGTG-5´3´-CACTGTTTCTCCAGGGGCAT-5´;
      Il173´-TCCTGGTCCTGAAGAGGGAG-5´3´-CACACCCACCAGCATCTTCT-5´;
      Reg3g3´-TTCCTGTCCTCCATGATCAAAA-5´3´-CATCCACCTCTGTTGGGTTCA-5´;
      Muc13´-TCTTTCCAACCCAGGACACC-5´3´-ACTGCCATTACCTGCCGAAA-5´
      Muc23´-AGAAGCCAGATCCCGAAACC-5´3´-GGGAATCGGTAGACATCGCC-5´;
      Kit3´-GTCCTGTTGGTCCTGCTCC-5´3´-TGTGCTGGATGGATGGATGG-5´;
      Lgr53´-ATCTCCTGTCGTCCTTCCCT-5´3´-CTGTAAGGCTCGGTTCCCTG-5´.
      Myc3´-TGTGGAGAAGAGGCAAACCC-5´3´-GTTGTGCTGGTGAGTGGAGA-5´
      Hif1a3´-TGTTCCAGAGGGAGCCAAAA-5´3´-GTTATGAACCTGCTGGGCTG-5´
      Irf43´-TATGCCACGATTGGAGCAGG-5´3´-GAAAACGGAGCAAACAGCCA-5´
      Slc2a43´-GTCGTGATGAGGGTGGACTT-5´3´-TTGGCTCCATTGTCCAAGCA-5´
      Pgk13´-TGAAGTGTGGGAAAACGGGG-5´3´-AGCCAGAAATCGGTGGTGTT-5´

      Immunohistochemistry

      Formalin-fixed paraffin-embedded sections were rehydrated by immersing the slides in xylene for 4 minutes (twice), 100% ethanol for 2 minutes (twice), 95% ethanol for 2 minutes (twice), 80% ethanol for 2 minutes (twice), 70% ethanol for 2 minutes (twice), and PBS for 5 minutes (twice). Epitope retrieval was performed in IHC-Tek Epitope Retrieval Solution IW-1100 (IHC World, Ellicott, MD). The slides were blocked with 5% bovine serum albumin and stained with the following antibodies: PDK4 (Novus), p-PDHE1α, Ki-67, anti-CD4-PECy7, anti-CD3-PECy7 (all BioLegend), and goat antirabbit immunoglobulin G conjugated to Alexa Fluor 647 (A27040; Invitrogen, Carlsbad, CA).

      Measurement of Secreted Cytokines

      Secreted IFN-γ and IL17 were measured using sandwich Quantikine enzyme-linked immunosorbent assay (ELISA) kits (all from R&D systems, Minneapolis, MN). Secreted IL2 was measured using an IL-2 Mouse ELISA kit (Invitrogen).

      Ex Vivo Intestinal Organ Culture

      The colon was placed in sterile ice-cold PBS, and luminal contents were removed by flushing with ice-cold PBS. The colon tissues were then opened longitudinally and washed thoroughly with ice-cold PBS. Approximately 1 cm of the middle part of the colon was excised using a scissors and incubated in RPMI medium supplemented with 10% fetal bovine serum, 1% P/S, and protease inhibitor cocktail P1860 (Sigma-Aldrich) for 24 hours at 37°C. The amounts of secreted cytokines were measured using ELISA kits (Invitrogen).

      In Situ Proximity Ligation Assay

      In situ proximity ligation assay was performed according to the instructions for the Duolink in situ PLA kit (Sigma-Aldrich). Anti-VDAC1, anti-IP3R1, or anti-GRP75 antibodies were used to visualize MAM formation. Fluorescent blobs were detected using a FV1000 confocal laser scanning biological microscope (Olympus, Tokyo, Japan) or an ImageXpress Micro Confocal Imaging System (Molecular Devices). The number of detected blobs per nucleus was calculated by ImageJ software or MetaMorph Microscopy Software (Molecular Devices).

      Cellular Bioenergetics Respiration Analysis Using the Extracellular Flux Analyzer

      Activated naive CD4+ T cells were added to CellTak-coated XF96-well plates at 200,000 cells/well. Mitochondrial stress and glycolysis stress tests were performed using an eXF96 analyzer (Agilent Technologies, Santa Clara, CA) according to the manufacturer’s instructions.

      In Vitro T-Cell Differentiation

      Mouse naive CD4+ T cells were differentiated for 3 days into Th1, Th2, Th17, or Treg cells using CellXVivo Lymphocyte Differentiation Kits CDK018, CDK019, CDK017, or CDK007 (R&D Systems), respectively.

      Pharmacologic Inhibition of PDK4

      C57BL/6J JmsSlc WT male mice were exposed to 4% DSS in drinking water for 9 days ad libitum. GM-10395, a novel inhibitor of PDK4, was administered at 0.5 or 1 mg/kg daily by oral gavage from day –2 to 10 (13 days in total). No adverse effects were observed at this dose (data not shown). In vitro assay of GM-10395 was performed as described previously.
      • Lee D.
      • Pagire H.S.
      • Pagire S.H.
      • Bae E.J.
      • Dighe M.
      • Kim M.
      • Lee K.M.
      • Jang Y.K.
      • Jaladi A.K.
      • Jung K.Y.
      • Yoo E.K.
      • Gim H.E.
      • Lee S.
      • Choi W.I.
      • Chi Y.I.
      • Song J.S.
      • Bae M.A.
      • Jeon Y.H.
      • Lee G.H.
      • Liu K.H.
      • Lee T.
      • Park S.
      • Jeon J.H.
      • Lee I.K.
      • Ahn J.H.
      Discovery of novel pyruvate dehydrogenase kinase 4 inhibitors for potential oral treatment of metabolic diseases.
      Cyclosporin A (Enzo Life Sciences), a calcineurin inhibitor, was used as a positive control. To induce chronic colitis, we administered 3 cycles of 3.5% DSS in sterile drinking water ad libitum for 1 week, followed by normal drinking water for 2 weeks. After the last cycle, mice were killed 1 week after return to normal drinking water. GM-10395 (1 mg/kg daily) was administered from day 3 after DSS challenge (and continued for 1 week).

      Statistical Analysis

      The Student t test or the Mann-Whitney test was performed in Prism5 (GraphPad, La Jolla, CA) or Excel (Microsoft, Redmond, WA). The Pearson coefficient and Mander’s coefficient were calculated by ImageJ (National Institutes of Health, Bethesda, MD). Error bars represent the mean ± standard error of the mean. P values of <.05, <.01, or <.001 are indicated by 1, 2, or 3 asterisks, respectively, in all figures.

      Study Approval

      All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Daegu-Gyeongbuk Medical Innovation Foundation, South Korea (DGMIF-19070201-00) and by the IACUC of Kyungpook National University (KNU-2021-0080). The present human study was reviewed and approved by the Institutional Review Board of Kyungpook National University Hospital (KNUH-2017-10-034). All patients provided informed consent.

      Acknowledgments

      The authors thank Gap R. Lee (Sogang University, South Korea), Mi N. Kweon (University of Ulsan College of Medicine/Asan Medical Center, South Korea), and Robert A. Harris (Indiana University School of Medicine) for providing genetically modified animals.

      CRediT Authorship Contributions

      Hoyul Lee (Conceptualization: Equal; Methodology: Lead; Software: Lead; Formal analysis: Lead; Investigation: Equal; Data Curation: Lead; Writing – original draft preparation: Equal; Visualization: Lead; Funding acquisition: Supporting)
      Jae Han Jeon (Conceptualization: Equal; Formal analysis: Supporting; Writing – original draft preparation: Equal; Funding acquisition: Supporting)
      Yu-Jeong Lee (Formal analysis: Supporting; Investigation: Equal; Data Curation: Supporting)
      Mi-Jin Kim (Formal analysis: Supporting; Investigation: Supporting)
      Woong Hee Kwon (Investigation: Supporting)
      Dipanjan Chanda (Writing – original draft preparation: Supporting)
      Themis Thoudam (Writing – original draft preparation: Supporting)
      Haushabhau S. Pagire (Resources: Equal)
      Suvarna H. Pagire (Resources: Equal)
      Jin Hee Ahn (Resources: Equal)
      Robert A. Harris (Resources: Equal)
      Eun Soo Kim (Conceptualization: Equal; Writing – original draft preparation: Equal; Supervision: Equal; Resources: Equal; Funding acquisition: Equal)
      In-Kyu Lee (Conceptualization: Equal; Supervision: Equal; Funding acquisition: Equal)

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      Linked Article

      • Metabolic Fire-Up T Cell Induction of Intestinal Inflammation
        Cellular and Molecular Gastroenterology and Hepatology
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          CD4 T-cell responses to gut microbiota are crucial in regulating intestinal inflammation. Whereas T helper (Th)1 and Th17 cells reactive to gut microbiota are central to the pathogenesis of inflammatory bowel disease (IBD), regulatory T cells (Treg) inhibit it. CD4 T-cell metabolism is highly dynamic. T-cell activation demands biosynthetic precursors and adequate energy for effector function.1 Metabolic reprogramming of T effector cells and Treg cells leads to distinct metabolic programs. If activated T cells fail to induce appropriate metabolic pathways, effector function and the ability to induce or inhibit inflammatory disease are impaired.
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