mouse naive cd4 t cells  (R&D Systems)

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

Journal: mBio

Article Title: An Antibiotic-Impacted Microbiota Compromises the Development of Colonic Regulatory T Cells and Predisposes to Dysregulated Immune Responses

doi: 10.1128/mBio.03335-20

Figure Lengend Snippet: Perinatal maternal antibiotic exposure alters offspring colonic lamina propria CD4 + T-cell population frequencies and absolute numbers. Dams and their offspring were exposed to ampicillin via the drinking water from day 5 to 10 of the pups’ lives, and colonic lamina propria leukocyte populations of offspring were isolated at the age of 24, 33, or 45 days and analyzed by flow cytometry. (A and B) Absolute counts per organ, and frequencies among live cells, of CD4 + T cells in the colonic lamina propria. Representative fluorescence-activated cell sorting (FACS) plots are shown in panel A, and summary plots are shown in panel B. (C to E) Absolute counts, and frequencies among all CD4 + T cells, of Foxp3 + Tregs (D) and Foxp3 − effector T cells (E). Representative FACS plots are shown in panel C. (F to H) Absolute counts per organ, and frequencies among all Foxp3 + Tregs, of neuropilin-1-positive (Nrp-1 + ) Tregs (G) and Nrp-1 − Tregs (H). Representative FACS plots are shown in panel F. (I and J) Absolute counts per organ, and frequencies among all Foxp3 + Tregs, of RORγt + Nrp-1 − Tregs. Representative FACS plots are shown in panel I. Two independent studies were pooled for the day 33 and 45 time points, and one study is shown for the day 24 time point. Horizontal lines indicate medians throughout; statistical analyses used the Mann-Whitney test. *, P < 0.05; **, P < 0.01; ***, P < 0.005; ****, P < 0.001. Only statistically significant differences are indicated with asterisks.

Article Snippet: Naive CD4 + T cells were isolated by negative selection from splenic single-cell suspensions using the MagCellect mouse naive CD4 + T-cell isolation kit (R&D Systems).

Techniques: Isolation, Flow Cytometry, Fluorescence, FACS, MANN-WHITNEY

Journal: mBio

Article Title: An Antibiotic-Impacted Microbiota Compromises the Development of Colonic Regulatory T Cells and Predisposes to Dysregulated Immune Responses

doi: 10.1128/mBio.03335-20

Figure Lengend Snippet: Dendritic cells isolated from the MLNs of dysbiotic offspring of antibiotic-exposed dams are less capable of inducing Foxp3 expression in cocultured naive T cells than DCs from control offspring. Immunomagnetically isolated MLN CD11c + DCs from 45-day-old offspring of antibiotic-exposed dams and age-matched control offspring were cultured at 1:5 ratios with naive splenic T cells (20,000 DCs and 100,000 T cells). Cocultures were stained for CD4, and Foxp3 and Treg populations were quantified by flow cytometry. (A) Representative FACS plots of the indicated conditions. (B) Summary plot of all cocultures; data are pooled from two independent experiments. Each symbol represents one coculture; two cultures were assessed per mouse. Horizontal lines indicate medians; statistical analyses used the Mann-Whitney test. *, P < 0.05.

Article Snippet: Naive CD4 + T cells were isolated by negative selection from splenic single-cell suspensions using the MagCellect mouse naive CD4 + T-cell isolation kit (R&D Systems).

Techniques: Isolation, Expressing, Control, Cell Culture, Staining, Flow Cytometry, MANN-WHITNEY

Journal: mBio

Article Title: An Antibiotic-Impacted Microbiota Compromises the Development of Colonic Regulatory T Cells and Predisposes to Dysregulated Immune Responses

doi: 10.1128/mBio.03335-20

Figure Lengend Snippet: Fecal transplantation of antibiotic-perturbed microbiota into germfree mice recapitulates the colonic CD4 + T-cell and Treg phenotypes of the donors. Fecal microbiota from 3-week-old offspring of control or ampicillin-exposed dams (two donors each) was collected and transferred by oral gavage to 6- to 8-week-old germfree recipients. Microbial community structure and CD4 + T cell populations were analyzed 28 days posttransfer. (A) Unweighted UniFrac distance rarefied at 5,000 reads with the 95% confidence intervals around each recipient group; the Adonis test was used to determine statistical significance. (B) Taxonomic abundance in the now-conventionalized recipient mice compared to the fecal inocula transferred from antibiotic-perturbed or control donors. (C to H) Colonic lamina propria T-cell populations in the now-conventionalized, formerly GF mice. (C) Absolute counts per organ, and frequencies among live cells, of CD4 + T cells in the colonic lamina propria. (D and E) Absolute counts per organ, and frequencies among all CD4 + T cells, of Foxp3 + Tregs (D) and Foxp3 − effector T cells (E). (F and G) Absolute counts per organ, and frequencies among all Foxp3 + Tregs, of Nrp-1 + Tregs (F) and Nrp-1 − Tregs (G). (H) Absolute counts per organ, and frequencies among all Foxp3 + Tregs, of RORγt + Nrp-1 − Tregs. Data in panels A and B are from two independent studies; data in panels C to H are from one study. GF Ctrl and GF Amp , conventionalized, previously germfree recipients of control and ampicillin-impacted fecal inocula; Inoculum Ctrl and Inoculum Amp , fecal inocula used for conventionalization. Horizontal lines indicate medians throughout; statistical analyses used the Mann-Whitney test. **, P < 0.01.

Article Snippet: Naive CD4 + T cells were isolated by negative selection from splenic single-cell suspensions using the MagCellect mouse naive CD4 + T-cell isolation kit (R&D Systems).

Techniques: Transplantation Assay, Control, MANN-WHITNEY

Journal: mBio

Article Title: An Antibiotic-Impacted Microbiota Compromises the Development of Colonic Regulatory T Cells and Predisposes to Dysregulated Immune Responses

doi: 10.1128/mBio.03335-20

Figure Lengend Snippet: Antibiotic exposure of dams results in dysregulated Th1 responses of dysbiotic offspring to Citrobacter rodentium infection and in differential food allergy severity. (A to D) Offspring of control and ampicillin-exposed dams were intragastrically infected with Citrobacter rodentium at 7 weeks of age and sacrificed 2 weeks later. (A) C. rodentium colonization of the cecum, colon, and MLNs as determined by plating and colony counting. (B) Absolute counts per organ, and frequencies among all live cells, of CD4 + T cells in the colonic lamina propria as determined by flow cytometry. (C and D) Frequencies of IFN-γ + CD4 + T cells among all CD4 + T cells, as assessed by ex vivo restimulation with PMA/ionomycin. Representative FACS plots are shown in panel C. Data in panels A to D are pooled from two independent experiments. (E to H) Offspring of control and ampicillin-exposed dams were intraperitoneally immunized twice with alum-adjuvanted ovalbumin at 5 and 7 weeks of age and challenged on four consecutive days with orally administered ovalbumin 4 weeks after the first sensitization (s/c). Control mice were sensitized with ovalbumin but mock challenged with PBS only. Additional controls were mock sensitized and mock challenged with PBS only. (E) Cumulative anaphylaxis score over 4 days. (F) Frequencies of Foxp3 + Tregs among all CD4 + T cells in MLNs. (G) Frequencies of Nrp-1 − cells among all Foxp3 + Tregs in MLNs. (H) Frequencies of Ki67 + cells among all Nrp-1 − Foxp3 + Tregs in MLNs. Data in panels E to H are pooled from two experiments. Horizontal lines indicate medians throughout; statistical analyses were done using the Mann-Whitney test. *, P < 0.05; **, P < 0.01; ***, P < 0.005. Only statistically significant differences are indicated with asterisks.

Article Snippet: Naive CD4 + T cells were isolated by negative selection from splenic single-cell suspensions using the MagCellect mouse naive CD4 + T-cell isolation kit (R&D Systems).

Techniques: Infection, Control, Flow Cytometry, Ex Vivo, MANN-WHITNEY