Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Fatty Acid Oxidation Promotes Apoptotic Resistance and Proinflammatory Phenotype of CD4 + Tissue-resident Memory T cells in Crohn’s Disease

doi: 10.1016/j.jcmgh.2024.02.014

Figure Lengend Snippet: Transcriptomic features of ex vivo CD4 + T RM cells from CD patients. ( A ) Representative flow cytometry of CD4 + CD45RO + CD69 + CD103 + lamina propria T cells in control and CD patients; percentages in right upper quadrant indicate CD69 + CD103 + T RM cells frequency. ( B ) Statistical analysis results for frequency of CD69 + CD103 + T RM cells among CD4 + CD45RO + lamina propria T cells. ( C ) Immunofluorescence staining of CD103 ( green ) and CD69 ( red ) in intestinal lamina propria tissues from control and CD patients; white arrows indicate double-positive cells. Scale bars represent 50 μm (left panel) and 25 μm (right zoom-in panel). ( D ) Representative flow cytometry of CD69 + CD103 + cells among isolated CD4 + T RM cells in control and CD patients; percentages in right upper quadrant indicate CD69 + CD103 + cell frequency. ( E ) Statistical analysis results for frequency of CD69 + CD103 + cells among isolated CD4 + T RM cells from control and CD patients. ( F ) PCA of RNA sequencing data from ex vivo CD4 + T RM cells in control and CD patients. ( G ) Volcano plot of differentially expressed genes in ex vivo CD4 + T RM cells from control and CD patients ( P < .05, ∣Log 2 (fold change)∣> 1). All data were presented as mean values ± SD of minimum of 3 biological replicates. Statistical analysis was performed with unpaired two-sided t test in ( B ). ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001.

Article Snippet: CD4 + T RM cells were isolated from the LPMCs by negative selection using human CD4 + T-cell memory effector cell isolation kit (#130-094-125, Miltenyi Biotec).

Techniques: Ex Vivo, Flow Cytometry, Control, Immunofluorescence, Staining, Isolation, RNA Sequencing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Fatty Acid Oxidation Promotes Apoptotic Resistance and Proinflammatory Phenotype of CD4 + Tissue-resident Memory T cells in Crohn’s Disease

doi: 10.1016/j.jcmgh.2024.02.014

Figure Lengend Snippet: Transcriptomic features of ex vivo CD4 + T RM cells from CD patients. ( A ) Heatmap of top 40 genes that were up-regulated in CD4 + T RM cells from CD patients; color scale demonstrates Log 2 (fold change of gene expression level in CD versus control). ( B and C ) Top 10 up-regulated Gene Ontology and KEGG pathway clusters of differentially expressed genes in CD4 + T RM cells from CD and control patients; enriched pathways are shown as gene number and - Log 10 (false discovery rate q value). ( D ) Representative GSEA results of differentially expressed genes in CD4 + T RM cells from CD and control patients. ( E and F ) Representative GSEA results of differentially expressed genes in CD4 + T RM cells from CD and control patients. All data were presented as mean values ± SD of minimum of 3 biological replicates. Statistical analysis was performed with unpaired two-sided t test in ( B ). ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001.

Article Snippet: CD4 + T RM cells were isolated from the LPMCs by negative selection using human CD4 + T-cell memory effector cell isolation kit (#130-094-125, Miltenyi Biotec).

Techniques: Ex Vivo, Gene Expression, Control

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Fatty Acid Oxidation Promotes Apoptotic Resistance and Proinflammatory Phenotype of CD4 + Tissue-resident Memory T cells in Crohn’s Disease

doi: 10.1016/j.jcmgh.2024.02.014

Figure Lengend Snippet: Metabolomic analysis of ex vivo CD4 + T RM Cells in CD patients. Heatmap of metabolomic data from ex vivo CD4 + T RM cells in CD and control patients (n = 8).

Article Snippet: CD4 + T RM cells were isolated from the LPMCs by negative selection using human CD4 + T-cell memory effector cell isolation kit (#130-094-125, Miltenyi Biotec).

Techniques: Ex Vivo, Control

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Fatty Acid Oxidation Promotes Apoptotic Resistance and Proinflammatory Phenotype of CD4 + Tissue-resident Memory T cells in Crohn’s Disease

doi: 10.1016/j.jcmgh.2024.02.014

Figure Lengend Snippet: Metabolomic analysis of ex vivo CD4 + T RM cells in CD patients. ( A and B ) The 2-dimensional and 3-dimensional PCA results of metabolomic data from ex vivo CD4 + T RM cells in CD and control patients (n = 8). ( C and D ) Enrichment analysis and KEGG pathway analysis of metabolites in CD4 + T RM cells from CD and control patients. ( E ) Volcano plot of metabolites in ex vivo CD4 + T RM cells from CD and control patients ( P < .05, ∣fold change∣> 1.5). ( F ) Heatmap of metabolites in fatty acid degradation pathway from CD4 + T RM cells of CD and control patients.

Article Snippet: CD4 + T RM cells were isolated from the LPMCs by negative selection using human CD4 + T-cell memory effector cell isolation kit (#130-094-125, Miltenyi Biotec).

Techniques: Ex Vivo, Control

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Fatty Acid Oxidation Promotes Apoptotic Resistance and Proinflammatory Phenotype of CD4 + Tissue-resident Memory T cells in Crohn’s Disease

doi: 10.1016/j.jcmgh.2024.02.014

Figure Lengend Snippet: Metabolite and gene expression level in fatty acid uptake and oxidation pathway from CD4 + T RM cells. ( A ) Labeling pattern of acetyl-CoA, aspartate, and TCA cycle intermediates (M+2) in the isotope tracing ( 13 C 16 -palmitic acid) experiment. Schematic model shows 13 C 16 -palmitic acid experiment in ex vivo CD4 + T RM cells from CD and control patients. Red circles depict carbons from 13 C 16 -palmitic acid, and black circles indicate unlabeled carbons. ( B ) Uptake of 13 C 16 -palmitic acid in ex vivo CD4 + T RM cells from CD and control patients. ( C ) FPKM value of CD36 and FABP5 from RNA sequencing data in ex vivo CD4 + T RM cells. ( D and E ) Protein level of CD36 and FABP5 in ex vivo CD4 + T RM cells from CD and control patients. ( F ) FPKM value of key enzymes involved in fatty acid biosynthesis from RNA sequencing data in ex vivo CD4 + T RM cells. All data were presented as mean values ± SD of minimum of 3 biological replicates. Statistical analysis was performed with unpaired two-sided t test in A , B , C , E , and F . ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001.

Article Snippet: CD4 + T RM cells were isolated from the LPMCs by negative selection using human CD4 + T-cell memory effector cell isolation kit (#130-094-125, Miltenyi Biotec).

Techniques: Gene Expression, Labeling, Ex Vivo, Control, RNA Sequencing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Fatty Acid Oxidation Promotes Apoptotic Resistance and Proinflammatory Phenotype of CD4 + Tissue-resident Memory T cells in Crohn’s Disease

doi: 10.1016/j.jcmgh.2024.02.014

Figure Lengend Snippet: Lipidomic analysis of ex vivo CD4 + T RM cells in CD patients. ( A ) Heatmap of lipidomic data from ex vivo CD4 + T RM cells in CD and control patients (n = 4). ( B and C ) The 2-dimensional and 3-dimensional PCA plots of lipidomic data from ex vivo CD4 + T RM cells in CD and control patients (n = 4).

Article Snippet: CD4 + T RM cells were isolated from the LPMCs by negative selection using human CD4 + T-cell memory effector cell isolation kit (#130-094-125, Miltenyi Biotec).

Techniques: Ex Vivo, Control

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Fatty Acid Oxidation Promotes Apoptotic Resistance and Proinflammatory Phenotype of CD4 + Tissue-resident Memory T cells in Crohn’s Disease

doi: 10.1016/j.jcmgh.2024.02.014

Figure Lengend Snippet: Lipidomic analysis of ex vivo CD4 + T RM cells in CD patients. ( A and B ) Bar chart depicts relative abundance of DAG and TAG levels in ex vivo CD4 + T RM cells from control and CD patients (n = 4). ( C ) Representative images of lipid droplets by Bodipy staining ( green ) in CD4 + T RM cells from CD and control patients. Scale bar, 25 μm. ( D and E ) Representative flow cytometry analysis of lipid droplets by Bodipy staining in CD4 + T RM cells from CD and control patients. ( F ) FPKM value of key enzymes involved in lipid lipolysis and lipophagy from RNA sequencing data in ex vivo CD4 + T RM cells. ( G ) The qPCR results of genes from lipid lipolysis and lipophagy pathway in CD4 + T RM cells. All data were presented as mean values ± SD of minimum of 3 biological replicates. Statistical analysis was performed with unpaired two-sided t test in A , B , E , F , and G . ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001.

Article Snippet: CD4 + T RM cells were isolated from the LPMCs by negative selection using human CD4 + T-cell memory effector cell isolation kit (#130-094-125, Miltenyi Biotec).

Techniques: Ex Vivo, Control, Staining, Flow Cytometry, RNA Sequencing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Fatty Acid Oxidation Promotes Apoptotic Resistance and Proinflammatory Phenotype of CD4 + Tissue-resident Memory T cells in Crohn’s Disease

doi: 10.1016/j.jcmgh.2024.02.014

Figure Lengend Snippet: Transcription factor NF-κB regulates expression levels of lipid metabolism genes in CD4 + T RM cells. ( A and B ) Heatmap of genes in FAO pathway and NF-κB signaling pathway from RNA sequencing data in ex vivo CD4 + T RM cells of CD and control patients. ( C and D ) The qPCR results of genes in lipid metabolism and NF-κB signaling pathway from CD4 + T RM cells. All data were presented as mean values ± SD of minimum of 3 biological replicates. Statistical analysis was performed with unpaired two-sided t test in C and D . ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001.

Article Snippet: CD4 + T RM cells were isolated from the LPMCs by negative selection using human CD4 + T-cell memory effector cell isolation kit (#130-094-125, Miltenyi Biotec).

Techniques: Expressing, RNA Sequencing, Ex Vivo, Control

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Fatty Acid Oxidation Promotes Apoptotic Resistance and Proinflammatory Phenotype of CD4 + Tissue-resident Memory T cells in Crohn’s Disease

doi: 10.1016/j.jcmgh.2024.02.014

Figure Lengend Snippet: Transcription factor NF-κB regulates expression levels of lipid metabolism genes in CD4 + T RM cells. ( A ) Western blot for nuclear and cytoplasmic p65 expression in CD4 + T RM cells from CD and control patients, tubulin, and lamin A were used as cytoplasmic and nuclear internal control. ( B ) Statistical analysis was performed with Mann-Whitney test. ( C ) Immunofluorescence staining of nuclear and cytoplasmic p65 in CD4 + T RM cells from CD and control patients (p65, green ; DAPI, blue ). Scale bar, 25 μm. ( D ) The mRNA levels of lipid metabolism genes in ex vivo CD4 + T RM cells from CD patients treated with NF-κB inhibitor (BAY/JSH). GAPDH and β-actin served as unmodified negative control, and ICAM-1 was the positive control of NF-kB target gene. ( E ) The mRNA levels of lipid metabolism genes in ex vivo control and RELA/p65 knockdown CD4 + T RM cells from CD patients. GAPDH and β-actin served as unmodified negative control, and ICAM-1 was the positive control of NF-kB target gene. All data were presented as mean values ± SD of minimum of 3 biological replicates. Statistical analysis was performed with unpaired two-sided t test and Mann-Whitney test in B and one-way analysis of variance in D and E . ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001.

Article Snippet: CD4 + T RM cells were isolated from the LPMCs by negative selection using human CD4 + T-cell memory effector cell isolation kit (#130-094-125, Miltenyi Biotec).

Techniques: Expressing, Western Blot, Control, MANN-WHITNEY, Immunofluorescence, Staining, Ex Vivo, Negative Control, Positive Control, Knockdown

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Fatty Acid Oxidation Promotes Apoptotic Resistance and Proinflammatory Phenotype of CD4 + Tissue-resident Memory T cells in Crohn’s Disease

doi: 10.1016/j.jcmgh.2024.02.014

Figure Lengend Snippet: Transcription factor NF-κB regulates expression levels of lipid metabolism genes in CD4 + T RM cells. ( A ) Consensus binding motif of RELA/p65 identified in promoter regions of lipid metabolism genes by JASPAR. ( B ) ChIP-qPCR analysis of RELA/p65 binding at promoter regions of lipid metabolism genes in ex vivo CD4 + T RM cells from CD and control patients. ( C ) ChIP-qPCR analysis of RELA/p65 binding at promoter regions of lipid metabolism genes in ex vivo control and RELA/p65 knockdown CD4 + T RM cells from CD patients. GAPDH and β-actin served as unmodified negative control, and ICAM-1 was the positive control of NF-kB target gene. ( D ) Labeling pattern of acetyl-CoA and TCA cycle intermediates (M+2) in isotope tracing ( 13 C 16 -palmitic acid) experiment of ex vivo CD4 + T RM cells from CD patients treated with NF-κB inhibitor (BAY/JSH). All data were presented as mean values ± SD of minimum of 3 biological replicates. Statistical analysis was performed with one-way analysis of variance in B–D . ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001.

Article Snippet: CD4 + T RM cells were isolated from the LPMCs by negative selection using human CD4 + T-cell memory effector cell isolation kit (#130-094-125, Miltenyi Biotec).

Techniques: Expressing, Binding Assay, ChIP-qPCR, Ex Vivo, Control, Knockdown, Negative Control, Positive Control, Labeling

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Fatty Acid Oxidation Promotes Apoptotic Resistance and Proinflammatory Phenotype of CD4 + Tissue-resident Memory T cells in Crohn’s Disease

doi: 10.1016/j.jcmgh.2024.02.014

Figure Lengend Snippet: Targeting FAO reverses apoptosis-resistant and proinflammatory phenotype of CD4 + T RM cells in CD patients. ( A ) Apoptosis induced by anti-CD3 in ex vivo CD4 + T RM cells from CD and control patients as determined by flow cytometry. ( B ) Anti-CD3–induced apoptosis rates of ex vivo CD4 + T RM cells from CD and control patients. ( C and D ) Endogenous apoptosis (without anti-CD3 induction) rates in ex vivo CD4 + T RM cells from CD patients treated with FAO inhibitor (etomoxir [ETO], 2 μmol/L; ranolazine [Ran], 5 μmol/L). All data were presented as mean values ± SD of minimum of 3 biological replicates. Statistical analysis was performed with unpaired two-sided t test in B and one-way analysis of variance in D . ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001.

Article Snippet: CD4 + T RM cells were isolated from the LPMCs by negative selection using human CD4 + T-cell memory effector cell isolation kit (#130-094-125, Miltenyi Biotec).

Techniques: Ex Vivo, Control, Flow Cytometry

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Fatty Acid Oxidation Promotes Apoptotic Resistance and Proinflammatory Phenotype of CD4 + Tissue-resident Memory T cells in Crohn’s Disease

doi: 10.1016/j.jcmgh.2024.02.014

Figure Lengend Snippet: Targeting FAO reverses apoptosis-resistant and proinflammatory phenotype of CD4 + T RM Cells in CD patients and migration inhibition effect of CD4 + T RM cells on normal intestinal epithelial cell lines. ( A and B ) Anti-CD3–induced apoptosis rates in ex vivo CD4 + T RM cells from CD patients treated with FAO inhibitor (etomoxir, ETO; ranolazine, Rano). ( C and D ) The mRNA and protein levels of indicated cytokines from ex vivo CD4 + T RM cells in CD patients treated with FAO inhibitor. ( E ) Normal intestinal epithelial cell lines were co-cultured with control medium and CM from CD4 + T RM cells treated with vehicle or FAO inhibitors etomoxir in Transwell inserts. Scale bar, 200 μm. ( F ) Quantitative statistical results from Transwell assay. ( G ) Monolayers of normal intestinal epithelial cells were cultured in control medium and CM from CD4 + T RM cells treated with vehicle or FAO inhibitors etomoxir. Scale bar, 400 μm. ( H ) Quantitative statistical results from wound healing assay. All data were presented as mean values ± SD of minimum of 3 biological replicates. Statistical analysis was performed with one-way analysis of variance. ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001.

Article Snippet: CD4 + T RM cells were isolated from the LPMCs by negative selection using human CD4 + T-cell memory effector cell isolation kit (#130-094-125, Miltenyi Biotec).

Techniques: Migration, Inhibition, Ex Vivo, Cell Culture, Control, Transwell Assay, Wound Healing Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Fatty Acid Oxidation Promotes Apoptotic Resistance and Proinflammatory Phenotype of CD4 + Tissue-resident Memory T cells in Crohn’s Disease

doi: 10.1016/j.jcmgh.2024.02.014

Figure Lengend Snippet: Targeting FAO reverses migration inhibition effect of CD4 + T RM cells on normal intestinal epithelial cell lines. ( A ) Normal human intestinal epithelial cell lines were co-cultured with control medium and CD4 + T RM cells treated with isotype antibody, anti-TNFα, anti-IL-17, anti-IFN-γ, or their combination in Transwell inserts. Scale bar, 200 μm. ( B and C ) Quantitative statistical results from Transwell assay. ( D and E ) Monolayers of human normal intestinal epithelial cells were co-cultured with control medium and CD4 + T RM cells treated with isotype antibody, anti-TNFα, anti-IL-17, anti-IFN-γ, or their combination. Scale bar, 400 μm. ( F and G ) Quantitative statistical results from wound healing assay. All data were presented as mean values ± SD of minimum of 3 biological replicates. Statistical analysis was performed with one-way analysis of variance in B and C and F and G . ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001.

Article Snippet: CD4 + T RM cells were isolated from the LPMCs by negative selection using human CD4 + T-cell memory effector cell isolation kit (#130-094-125, Miltenyi Biotec).

Techniques: Migration, Inhibition, Cell Culture, Control, Transwell Assay, Wound Healing Assay