mil 2  (Miltenyi Biotec)

Journal: iScience

Article Title: CCL20-CCR6 signaling alters the metabolic reprogramming to promote the pathogenic Th17 cell differentiation

doi: 10.1016/j.isci.2025.114385

Figure Lengend Snippet: CCR6 + CD4 + T cells show an increased Th1-like Th17 phenotype during inflammatory colitis (A and B) Acute colitis was induced in wild-type C57BL/6 mice by administering 2% DSS in their drinking water, and body weight loss was monitored. On day 10, spleen (SP), mesenteric lymph nodes (mLN), Peyer’s patches (PPs), and lamina propria (LP) cells were harvested, and cells were analyzed using flow cytometry. (A) Body weight was monitored and plotted (upper panel). CCL20 expression in colon tissue sections was analyzed by immunofluorescence staining (lower panel). Original magnification 400x, scale bars 100 μm. (B) The dot plot represents the expression of RORγt on CD4 + CCR6 - and CD4 + CCR6 + T cells (upper left), and IL-17A + and IFN-γ+ expression (upper right). Mean percentages of RORγt + cells (lower left), mean percentages of IL-17A + cells (lower middle), and IL-17 and IFN-γ expression (lower right) on CD4 + CCR6 - and CD4 + CCR6 + T cells. (C) Representative immunofluorescence images of PP of wild-type mice treated with or without DSS are shown (upper right). Original magnification 400x, scale bars 100 μm. (D) CCR6 −/− or CCR6 +/+ mice were given 2% DSS in the drinking water. The body weight changes in mice were monitored (left), and RORγt + T-bet + cells were analyzed in the indicated organs by flow cytometry (right). (E) CCR6 −/− splenocytes (CD45.2 congenic; 10 × 10 6 cells/mouse) were adoptively transferred into CD45.1 congenic mice (CCR6 +/+ genotype), and the mice were administered 2% DSS in their drinking water. On day 12, the mice were sacrificed. (F) The immunofluorescence staining of CD45.2 and CD4 markers was performed on the colon tissue. Original magnification 400x, scale bars 100 μm. (G and H) RORγt + and RORγt + T-bet + cells were analyzed after gating on CD45.2 + CD4 + or CD45.2 − CD4 + T cells. (I) IL-17A + IFN-γ + cells were analyzed after gating on CD45.2 + CD4 + or CD45.2 − CD4 + T cells. (J) Naive CD4 + T cells (CD4 + CD25 − CD44 lo CD45RB hi CD62L hi Foxp3rfp − T cells; 0.5 × 10 6 cells/mouse) from CCR6 gfp/+ Foxp3 rfp/rfp mice were adoptively transferred into RAG1 −/− mice. The mean percentage in body weight change from the initial weight was plotted. ( n = 4 mice/group). The error bar represents ±SD. (K) On day 21, RORγt + T-bet + cells were analyzed after gating on CCR6gfp + and CCR6gfp − CD4 + T cells in the SP, mLN, and lamina propria (LP) (left panel). The mean percentage and MFI of RORγt + cells were analyzed after gating on CD4 + CCR6gfp − T cells or CD4 + CCR6gfp + cells (right panel). The bar represents the mean ± SEM; each symbol represents data from an individual mouse, with n = 5–6 mice/group (A–K). The p -value for the comparison between the two groups is indicated in the graphs. The data shown are representative of three independent experiments (A–K). Student’s t test was used, not significant (ns): p > 0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001.

Article Snippet: pGL4 plasmid containing mouse 2 Kbp IL-17 promoter , Adgene , Cat# 20127.

Techniques: Flow Cytometry, Expressing, Immunofluorescence, Staining, Comparison

Journal: iScience

Article Title: CCL20-CCR6 signaling alters the metabolic reprogramming to promote the pathogenic Th17 cell differentiation

doi: 10.1016/j.isci.2025.114385

Figure Lengend Snippet: CCR6 intrinsic signaling induces the phosphorylation of the PI3K/Akt/mTORC1/STAT3 pathway and promotes RORγt binding on IL-17A regulatory elements (A) CCR6 + and CCR6 - CD4 T cells were sorted based on the expression of eGFP and stimulated with 100 ng/mL CCL20 at different time points (0, 5, 10, 15, 30, and 60 min). They were then immunoblotted with antibodies against various kinases in the downstream signaling pathways of Akt/mTOR/STAT3. (B) Sorted CCR6 + cells were rested overnight with or without rapamycin (50 ng/mL) and, the next day, stimulated with recombinant CCL20 (100 ng/mL) at two time points, 0 and 30 min. The phosphorylation status of STAT3 was analyzed by immunoblotting. Cyclophilin B and total STAT3 were used as loading controls. Blots are from 2 to 3 independent experiments. (C) The schematic presentation of IL-17A promoter (IL-17AP), IL-17F promoter (IL-17FP), conserved non-coding sequences (CNSs), and binding of RORγt. (D) CCR6gfp + Jurkat cells were transfected with the pGL4 plasmid vector containing the mouse IL-17 promoter (2 Kbp) and CNS5 enhancer elements. Transfected cells were stimulated with purified recombinant CCL20 (100 ng/mL) or TGF-β1 plus IL-6 at 37°C for 72 h. Then, cells were lysed, and luciferase activity was measured. The data shown are normalized to the Renilla luciferase activity. Each dot represents an independent experiment. Student’s t test. p -values are shown. (E) CCR6gfp + Jurkat cells were transfected with the pGL4 plasmid containing the IL-17 promoter (2 Kb) or IL-17 promoter (2 Kb) having a mutation in the RORγt binding site. Cells were cultured as given above, and luciferase activity was measured and plotted. The data shown are representative of one of the two independent experiments. The error bar represents ±SD. (F) Naive CD4 T cells were differentiated into the Th17 lineage condition (TGF-β + IL-6) in the presence or absence of CCL20 and with or without rapamycin (25 ng/mL) for 4 days. Expression of RORγt was analyzed after gating on CD4 + T cells. n = 3 experiments. (G) The schematic CCR6-CCL20 signaling pathways are shown. The p -value for the comparison between the two groups is indicated in the graphs. not significant (ns): p > 0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001.

Article Snippet: pGL4 plasmid containing mouse 2 Kbp IL-17 promoter , Adgene , Cat# 20127.

Techniques: Phospho-proteomics, Binding Assay, Expressing, Protein-Protein interactions, Recombinant, Western Blot, Transfection, Plasmid Preparation, Purification, Luciferase, Activity Assay, Mutagenesis, Cell Culture, Comparison