Fig. 8: Canonical and non-canonical Hh signaling contribute to Th17 polarization.
From: Cell-autonomous Hedgehog signaling controls Th17 polarization and pathogenicity
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.
