Fig 1: Canonical and non-canonical Hh signaling contribute to Th17 polarization.a Schematic overview of canonical/non-canonical Hedgehog signaling. b Naïve CD4+ T cells were purified from spleen/lymph nodes of Gli1eGFP+/+ (WT), Gli1eGFP+/- (HET) or Gli1eGFP-/- (KO) mice. Cells were polarized to Th0, Th1, Th2, Th17 or iTregs. Cells were assessed for of Gli1 mRNA expression on day 3 (Th17, left) or harvested for flow cytometry on day 5 (right). n = 4 mice per genotype. c Th17 cells were polarized from tamoxifen-treated CD4CreERT2+ Ihh+/fl (HET) or CD4CreERT2+ Ihhfl/fl (KO) mice and analysis of Gli1 and Gli3 expression was performed by qRT-PCR at day 3. n = 2–6 mice. d Th17 cells were polarized from C57BL/6 mice in the presence of the indicated dose of cyclopamine or carrier control for three days. Data are normalized to Tbp as a reference gene. n = 4 independent experiments. e Expression of all putative Gli3 target genes predicted by Miraldi et al.20 in RNA-Seq analysis from Fig. 7c. Th17 cells were polarized in the presence of the indicated doses of cyclopamine or carrier control for three days and harvested at day 3. Six samples/group. f–h Naïve CD4+ T cells were purified from spleen/lymph nodes of C57BL/6 mice and stimulated under Th17 polarizing conditions. f After 24 h CRISPR/Cas9-RNP complexes targeting Gli3 were electroporated. Cells were harvested for qRT-PCR analysis of Gli3 at day 3 (left panel) and for flow cytometry analysis on day 5 (middle panel). Right: Quantitation of IL-17a expression. n = 3 independent experiments. g Naïve CD4+ T cells were stimulated in the presence of the indicated doses of cyclopamine or carrier control for three days. Immunoblot analysis of Th17 cells on day 3 for pAMPK, AMPK, CaMKK2, LKB1 and Tubulin is shown. n = 3 independent experiments. h Naïve CD4+ T cells were stimulated in the presence of 100 ng/ml pertussis toxin or carrier control for five days. Cells were analyzed by flow cytometry on day 5. n = 3 independent experiments. Data are means +/- SD. p-values were calculated in (b, d) using a one-way/two-way ANOVA with Tukey's multiple comparison test or an unpaired two-tailed Student’s t test (c, f). *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided in the source data file.
Fig 2: Hedgehog signaling components are expressed in human CD4+ T cells in the intestine and are upregulated in ulcerative colitis patients.Analysis of human RNA-Seq datasets summarized in panel (a). b Expression of IHH, SMO and GLI3 mRNA from FACS-sorted CD4+ T cells isolated from blood as well as intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs) isolated from the terminal ileum from healthy human donors in a study from Raine et al. (2015)31. n = 6. Error bars show SD. c, d Expression of SMO and GLI3 mRNA from human rectal biopsy samples from patients with male (?) or female (?) ulcerative colitis (UC) or healthy controls recruited as part of the PROTECT study (GSE109142). Each datapoint represents an individual patient. c Shows expression of SMO and GLI3 across all groups. p-values were calculated using a limma based moderated t test. *p < 0.05, **p < 0.01, ***p < 0.001. d Shows correlation analysis of SMO, GLI3 as well as Th17-related transcripts CCR6 and IL17A respectively with simple linear regression analysis. Source data are provided in the source data file.
Fig 3: Cathelicidin induces Th17 cells via AHR up-regulation.CD4+ T cells isolated from C57BL/6 J mice were cultured in Th17-driving conditions for 24 h in the presence or absence of 2.5 µM cathelicidin. A–C gene expression differences were assessed by a Nanostring mouse immunology chip. D Representative flow cytometry plots showing aryl hydrocarbon receptor staining which was assessed over time in culture (E) and over increasing concentrations of cathelicidin (F). G, H IL-17A and F production was assessed by flow cytometry following blockade of AHR with the antagonist CH223191 (ant.). I Th17 cultures were repeated using IMDM as well as RPMI medium. Data shown are individual mice with line at medium. Statistical significance was assessed using (B, C) a paired two-tailed t-test with Bonferroni post-test, E repeated measures ANOVA with Sidak’s post-test, F Kruskal–Wallis test, G, H two-way ANOVA with Tukey’s post-test and I two-tailed t-test with no correction. N values: B- 3, C - 3, E - 3, F - 3, G - 5, H - 5, I - 3. The error bars in I show standard error of the mean. Black symbols represent untreated samples and open symbols are samples treated with cathelicidin. Red symbols show samples treated with AHR antagonist.
Fig 4: Analysis of skin-infiltrating T cells in IMQ-treated K14-I?B?–KO mice.All analyses were performed after 7 days of IMQ treatment. (A) Flow cytometry analysis of T cell subsets in the ears of IMQ-treated Ctrl and K14-KO mice. T cell subsets were detected as CD45+ and CD3+, aßTCR+, or ?dTCR+ cells. Single data points derive from 2 ears. Shown is the mean of 4–12 mice per group ± SEM. (B) Gene expression analysis of Il22 and Il17a in skin tissue of untreated and IMQ-treated control and K14-KO mice, normalized to the reference gene Actin. n = 4–14 ± SEM. (C) Determination of the percentage of IL-17A– and IL-22–producing aß and ?d T cells in IMQ-treated control and K14-KO mice. After fixation and permeabilization, cells were gated as in A, except for an additional gating on either IL-17A+ or IL-22+ cells. n = 3 ± SEM. (D) Gene expression analysis of Il1b and Il23a in untreated and IMQ-treated mice, similar as in B. (E) mRNA levels of Ccl2, Ccl17, and Ccl20 in untreated mice, similar as in B. (F) mRNA levels of Ccr2, Ccr4, and Ccr6 in the skin of untreated control and K14-KO mice, similar as in B. n = 5–14 ± SEM. P values were calculated using 2-tailed Student’s t test (*P < 0.05, **P < 0.01, and ***P < 0.001).
Fig 5: Cathelicidin induces IL-17F-producing but not IL-22-producing T cells.Splenocytes isolated from C57BL/6 J mice were cultured in Th17 conditions for 48 h in the presence or absence of 2.5 µM cathelicidin. A–D Expression of IL-17A and F was determined by intracellular flow cytometry. E IL-17F and F IL-22 production was quantified by ELISA of cell culture supernatant after 48 h in culture. Data shown are individual mice in separate experiments. B–F are analysed by paired two-tailed t-tests. SP = single positive. N values: B - 9, C - 9, D - 9, E - 5, F - 4. Black symbols represent untreated samples and open symbols are samples treated with cathelicidin.
Supplier Page from BioLegend for ELISA MAX(TM) Deluxe Set Mouse IL-17A