In some experiments, TGF-1 receptor kinase inhibitor (SB43152, 10?M; Selleckchem), JNK inhibitor (SP600125, 10?M; EMD Millipore), MEK inhibitor (PD98059, 10?M; Invivogen), p38 inhibitor (SB203580, 10?M; Invivogen), PI3 kinase inhibitor (LY294002, 5?M; Invivogen), SMAD3 inhibitor (SIS3, 10?M; Sigma) or ROCK inhibitor (Y27632, 10?M; Sigma) were added to the culture media. show that N-Bis(2-hydroxypropyl)nitrosamine this AP-1 transcription factor JunB is critical for TH17 pathogenicity. JunB, which is usually induced by IL-6, is essential for expression of RORt and IL-23 receptor by facilitating DNA binding of BATF at the locus in IL-23-dependent pathogenic TH17 cells, but not in TGF-1-dependent non-pathogenic TH17 cells. (encoding RORt) and under TH17(23)-polarizing conditions. Furthermore, we show that JunB is essential for pathogenicity of TH17 cells in EAE and colitis models, but it is not required for generation of non-pathogenic, gut-resident TH17 cells. These data suggest that the JunB-dependent pathway is required for IL-23-dependent pathogenicity of TH17 cells. Results Identification of JunB as a regulator of IL-23 signalling TGF-1 signalling is usually associated with non-pathogenic TH17 differentiation, whereas IL-23 signalling facilitates pathogenicity of TH17 cells13,15,16,17,19. However, transcriptional mechanisms underlying control of TH17 pathogenicity in the presence of these cytokines remain to be fully determined. To better understand IL-23-dependent transcriptional regulation, we attempted to identify transcription factors responsible for expression of genes promoted by IL-23 signalling in TH17 cells. Based on published microarray data13,23, we selected 263 transcription factors that are highly expressed in TH17 cells (Supplementary Data 1). Retroviruses expressing shRNAs N-Bis(2-hydroxypropyl)nitrosamine against these transcription factors were individually transduced into TH17 cells generated under pathogenic TH17(23)-polarizing conditions (in the presence of IL-6, IL-1 and IL-23). To evaluate the effect of transcription factor knockdown on IL-23-dependent signalling, we measured levels of mRNA because we found that induction of was significantly facilitated by IL-23 stimulation (Supplementary Fig. 1a). induction was most heavily diminished by knockdown of an AP-1 transcription factor, JunB (Supplementary Fig. 1b). RNAi ablation of JunB also significantly reduced expression of a TH17 signature molecule, (Supplementary Fig. 1c). JunB interacts with another AP-1 family member, BATF, an essential transcription factor for TH17 differentiation31,33,34, suggesting that JunB might be involved in TH17 differentiation; however, the physiological functions of JunB in TH17 differentiation remain unknown. JunB is usually induced in TH17 cells We first examined JunB expression in TH17 cells differentiated mRNA levels in both TH17() and TH17(23) cells (Fig. 1b). We also KLF11 antibody found that IL-6 stimulation was sufficient to augment JunB expression, whereas neither IL-1 nor IL-23 signalling significantly affected JunB expression in activated CD4+ T cells (Fig. 1c,d). IL-6 signalling is usually mediated by STAT3 (refs 35, 36). Indeed, JunB induction was severely impaired in mRNA. Error bars indicate s.d. (mice were activated, infected with Cre-expressing retrovirus, then cultured under TH17()-polarizing conditions for 60?h. JunB was detected by immunoblot analysis. (a,c,e) Data represent two impartial experiments. (b,d) Data represent three impartial experiments. JunB regulates TH17 differentiation and control mice were activated under TH17()- or TH17(23)-polarizing conditions for 3 days, and expression of IL-17A and IFN- was analysed by flow cytometry (a). RORt was detected by immunoblot analysis (b). (c) Naive CD4+ T cells from and control mice were activated in the presence of TGF-3 and IL-6 (TH17(3)) for 3 days, and expression of IL-17A and IFN- was analysed by flow cytometry. (d) and control cells were activated under TH17()-polarizing conditions for 3 days, and IL-17-high and IL-17-low populations were sorted using the IL-17-capture method. Enrichment of IL-17-high cells was confirmed by detecting IL-17A expression on re-stimulation immediately after sorting. Cells were then cultured in the presence of IL-23 alone for another 2 days. Production of IL-17A and IFN- was assessed by flow cytometry. (a,c) Data represent three N-Bis(2-hydroxypropyl)nitrosamine impartial experiments. (b,d) Data represent two impartial experiments. Remarkably, expression of the Treg grasp transcription factor, forkhead box protein 3 (Foxp3)37, significantly increased in and genes, such as and in TH17() cells, JunB deficiency also resulted in dysregulation of expression of the TH1 grasp transcription factor, (Fig. 3b), which is usually consistent with the reported JunB-dependent suppression of induction in TH2 cells40,41. qRTCPCR data confirmed the defective induction of TH17 signature genes and mRNAs in and under TH17() or TH17(3) conditions (Fig. 3c and Supplementary Fig. 5b,c). Open in a separate window Physique 3 JunB-dependent transcriptional regulation in TH17 cells.(a,b) Microarray analysis of and control cells activated under TH0-, TH17()- or TH17(23)-polarizing conditions for 60?h. Heat map data show fold changes of expression in (KO) versus ((control)) cells for common genes (a), and genes categorized as transcription factors (b). Only genes that showed.