Comparative analysis of murine Th17 cell transcriptomes in the presence of Smo inhibitor cyclopamine or carrier control

T helper 17 (Th17) cells play a key role in barrier protection against fungal and bacterial pathogens but are also pathological drivers of many inflammatory diseases. We find that Hh signaling inhibition through Smoothened inhibitor cyclopamine selectively inhibits Th17 lineage differentiation but not the development of Th1, Th2, or iTreg CD4+ Th cells. In these experiments we analysed the transcriptomes of Th17 cells that had been polarized in the presence of cyclopamine or carrier control to profile the global transcriptome of Th17 cells in which the Hh pathway had been blocked. The aim of this analysis was to identify the target genes downstream of Smo that may be important for Th17 polarisation. Naïve CD4+ T cells were purified from spleen and peripheral lymph nodes of C57BL/6 mice and stimulated with plate-bound anti-CD3e/CD28 antibodies in the presence of polarizing cytokines (IL-6, IL-23, IL-1b, TGFb) to generate Th17 cells. Th17 cells were polarized as described above, stimulated in the presence of the indicated doses of cyclopamine (5uM or 7.5uM) or carrier control for three days and harvested at day 3. Six samples/group. Libraries were generated using the TruSeq stranded mRNA kit (Illumina) following the manufacturer’s instructions and sequenced using single-read sequencing with the HiSeq4000 platform (Illumina). Reads were aligned to the mouse genome version GRCm38 using STAR v2.5.3a. Read counts were obtained using feature Counts function in Subread v1.5.267 and read counts were normalized and tested for differential gene expression using the DESeq2 workflow. Multiple testing correction was applied using the Benjamini–Hochberg method.