magcellect mouse cd4 cd25 regulatory t cell isolation kit  (R&D Systems)

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Hypercholesterolemic Dysregulation of Calpain in Lymphatic Endothelial Cells Interferes With Regulatory T-Cell Stability and Trafficking

doi: 10.1161/atvbaha.122.317781

Figure Lengend Snippet: Figure 2. TGF (transforming growth factor)-β1–mediated stabilization of regulatory T cells (Tregs) on lymphatic endothelial cells is limited by lysophosphatidic acid (LPA)-induced disinhibition of conventional calpains. A, Treg stability on SVEC4-10 cells. Isolated splenic CD4+ cells were seeded on SVEC4-10 cell monolayer and cultured for 5 days in the presence or absence of LPA at 1 µmol/L (n=6 experiments). B, Expression levels of Treg-stabilizing factors in SVEC4-10 cells. Control and Capns1-knockdown cells were stimulated with LPA at 1 µmol/L for 6 hours. (Ctl: n=7 experiments, Ctl+LPA: n=8 experiments, siCapns1-1+LPA: n=8 experiments). C, Pharmacological assessment of Treg stabilization. Splenic CD4+ cells were cocultured with SVEC4-10 cells in the presence of a TGF-β type-I receptor antagonists SB431542 (3 µmol/L), SB525334 (3 µmol/L), LY-364947 (3 µmol/L), or IDO1 inhibitor INCB 024360 (1 µmol/L) for 5 days, concomitant with LPA at 1 µmol/L (Ctl: n=13 experiments, siCapns1-1: n=13 experiments, SB4: n=8 experiments, SB5: n=8 experiments, Ly36: n=5 experiments, INCB: n=5 experiments). D, Immunoblot analysis of intracellular TGF-β1 protein levels in SVEC4-10 cells (n=6 experiments) and human dermal lymphatic endothelial cells (HDLECs; n=4 experiments). Control and siRNA-transfected cells were stimulated with LPA at 1 µmol/L for 16 hours. E, Impact of LPA- induced calpain activation on phorbol 12-myristate 13-acetate (PMA)–induced TGF-β1 production in LECs. SVEC4-10 cells were treated with PMA at 200 nmol/L. LPA at 1 µmol/L was added as required (n=3 experiments). F, Pharmacological assessment of TGF-β1 synthesis pathways in SVEC4-10 cells. Cells were treated with an ERK inhibitor PD98059 at 50 µmol/L, JNK inhibitor SP600125 at 50 µmol/L, p38MAPK (mitogen-activated protein kinase) inhibitor SB203580 at 5 µmol/L, or a c-fos/activator protein 1 inhibitor T5224 at 50 µmol/L for 24 hours in the presence of LPA at 1 µmol/L (n=3 experiments). G, Phosphorylation of ERK and JNK in SVEC4-10 cells. Cells were cultured with LPA at 1 µmol/L for 24 hours (n=3 experiments). H, Stability of MEKK1 (mitogen-activated protein kinase kinase kinase 1) in SVEC4-10 cells. Cells were stimulated with LPA at 1 µmol/L for 24 hours (n=6 experiments). Data were expressed as median with 95% confidence limits. P values were determined by a Kruskal-Wallis test with post hoc Bonferroni test (A–H). ACTB indicates beta-actin; Ctl, control; ERK, extracellular signal-regulated kinase; FOXP3, forkhead box P3; INCB, INCB024360; JNK, c-Jun N-terminal kinase; MEKK, mitogen-activated protein kinase kinase kinase 1; PD, PD98059; SB, SB203580; SB4, SB431542; SB5, SB525334; Si, siRNA; siCapns1, siRNA against Capns1; SP, SP600125; T, T5224; pERK, phospho ERK; pJNK, phospho JNK; and Veh, vehicle.

Article Snippet: Then the cells were labeled by using a MagCellect Mouse CD4+ T-Cell Isolation Kit (R&D Systems, Inc) and were separated by magnetic fields using negative selection.

Techniques: Isolation, Cell Culture, Expressing, Control, Knockdown, Western Blot, Transfection, Activation Assay, Phospho-proteomics

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Hypercholesterolemic Dysregulation of Calpain in Lymphatic Endothelial Cells Interferes With Regulatory T-Cell Stability and Trafficking

doi: 10.1161/atvbaha.122.317781

Figure Lengend Snippet: Figure 5. Conventional calpains in lymphatic endothelial cells decelerate lymphatic trafficking through induction of VCAM1 (vascular cell adhesion molecule 1). A, Lymphocyte homing assay. Following 12 weeks of diet feeding, CFDA (5(6)-carboxyfluorescein diacetate)-labeled splenic CD4+ cells were injected into the footpad of mice. Sixteen hours after the injection, CD4+ T cells and CD4+FOXP3+ regulatory T cells (Tregs) in popliteal lymph nodes (LNs) were monitored (LNL [loxP-flanked Neo cassette]: n=9 animals, cTg: n=8 animals). B, Gene expression profiles in mesenteric LNs. The genes exhibiting high expression levels with a statistically significant difference are indicated in mean-average plot (left; n=3 animals). C, VCAM1 neutralization inhibits afferent lymphatic transportation of Tregs. VCAM1 antibody (2.5 µg/site) or nonimmune IgG (2.5 µg/site) was injected together with CD4+ cells (Cont IgG: n=5 animals, VCAM1 mAb: n=6 animals). D, Quantification of VCAM1 immunofluorescent intensity in lymphatic endothelial cells (LECs) in human coronary artery disease (CAD) and non-CAD cases (non-CAD case: n=77 cells from 5 cases, CAD case: n=63 cells from 3 cases). E, Immunohistochemical localization of VCAM1 in gp38+PROX1+ (prospero-related homeobox 1) lymphatic vessels in brachial LNs in normal laboratory diet (NLD)–fed or high cholesterol diet (HCD)–fed Ldlr–/– mice. Arrows represent LECs. Bar: 20 µm. VCAM1 fluorescence intensity in individual gp38+PROX1+ LECs was measured (LNL-CAST, n=34 cells from 3 animals; CAST cTg, n=32 cells from 3 animals; Capns1 flox, n=36 cells from 3 animals; Capns1 cKO, n=30 cells from 3 animals). Bar: 10 µm. Data in C and D were expressed as median with 95% confidence limits. Others were indicated as mean±SEM. P values were determined by Mann-Whitney test (C and D), Student t test (A [CD4+ cells], B, E [CAST]), or Welch test (A [Tregs] and E [Capns1]). A.U. indicates arbitrary unit; CAST, calpastatin; CFSE, 5(6)-carboxyfluorescein diacetate succinimidyl ester; FSC, forward scatter; mAb, monoclonal antibody; Neg Cont, negative control; and pLN, popliteal LNs.

Article Snippet: Then the cells were labeled by using a MagCellect Mouse CD4+ T-Cell Isolation Kit (R&D Systems, Inc) and were separated by magnetic fields using negative selection.

Techniques: Labeling, Injection, Gene Expression, Expressing, Neutralization, Immunohistochemical staining, Fluorescence, MANN-WHITNEY, Negative Control

Journal: Cellular and Molecular Gastroenterology and Hepatology

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

doi: 10.1016/j.jcmgh.2022.09.016

Figure Lengend Snippet: PDK4 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).

Article Snippet: 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.

Techniques: Western Blot, Expressing, Immunohistochemistry, Staining, Isolation, Flow Cytometry

Journal: Cellular and Molecular Gastroenterology and Hepatology

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

doi: 10.1016/j.jcmgh.2022.09.016

Figure Lengend Snippet: Augmented 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).

Article Snippet: 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.

Techniques: Expressing, Immunohistochemistry, Immunofluorescence, Staining

Journal: Cellular and Molecular Gastroenterology and Hepatology

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

doi: 10.1016/j.jcmgh.2022.09.016

Figure Lengend Snippet: PDK4 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).

Article Snippet: 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.

Techniques: Activity Assay, Staining, In Vivo, Permeability, Flow Cytometry, Ex Vivo, Enzyme-linked Immunosorbent Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

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

doi: 10.1016/j.jcmgh.2022.09.016

Figure Lengend Snippet: PDK4 deletion by CD4-Cre reduced intestinal inflammation in mice. ( A–C ) Validation of PDK4 CD4 mouse line. ( A ) Expression of PDK4 protein in kidney, liver, and spleen samples from PDK4 WT PDK4 fl/fl or PDK4 CD4 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) PDK4 fl/fl (denoted as WT) and PDK4 CD4 (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 PDK4 fl/fl and PDK4 CD4 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).

Article Snippet: 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.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Activity Assay, Staining, In Vivo, Permeability, Flow Cytometry, Ex Vivo, Enzyme-linked Immunosorbent Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

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

doi: 10.1016/j.jcmgh.2022.09.016

Figure Lengend Snippet: PDK4-deficient T cells delay intestinal homing and induction of colitis after adoptive transfer. Rag1 -/- mice received 4 × 10 5 CD4 + CD45RB hi CD25 - 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 PDK4 fl/fl and PDK4 CD4 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 + CD45RB hi CD25 - ) 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.

Article Snippet: 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.

Techniques: Adoptive Transfer Assay, Isolation, Activity Assay, Staining, In Vivo, Permeability, Expressing, Flow Cytometry, Ex Vivo, Enzyme-linked Immunosorbent Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

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

doi: 10.1016/j.jcmgh.2022.09.016

Figure Lengend Snippet: PDK4 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.

Article Snippet: 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.

Techniques: Expressing, Activation Assay, Western Blot, Next-Generation Sequencing, Labeling, Flow Cytometry, Staining

Journal: Cellular and Molecular Gastroenterology and Hepatology

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

doi: 10.1016/j.jcmgh.2022.09.016

Figure Lengend Snippet: PDK4 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.

Article Snippet: 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.

Techniques: Cell Differentiation, Expressing, Liquid Chromatography, Mass Spectrometry, Flow Cytometry

Journal: Cellular and Molecular Gastroenterology and Hepatology

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

doi: 10.1016/j.jcmgh.2022.09.016

Figure Lengend Snippet: MAM/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).

Article Snippet: 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.

Techniques: Immunofluorescence, Staining, In Situ, Proximity Ligation Assay, MANN-WHITNEY, Binding Assay, Sandwich ELISA

Journal: Cellular and Molecular Gastroenterology and Hepatology

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

doi: 10.1016/j.jcmgh.2022.09.016

Figure Lengend Snippet: Pharmacologic 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).

Article Snippet: 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.

Techniques: Inhibition, Activation Assay, De-Phosphorylation Assay, Cell Culture, Flow Cytometry, Positive Control, MANN-WHITNEY, Staining, Permeability, Expressing, In Vivo