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. 2020 Mar 23;18(3):e3000646. doi: 10.1371/journal.pbio.3000646

Fig 7. IL-23 induces pRLC-S20 through ROCK dependent mechanisms in Th17 cells.

Fig 7

(a) Total lymph node cells from Il23r-gfp reporter mice were processed for analysis of IL-23R (GFP) expression by flow cytometry. Representative contour plots show CD44 and IL-23R/GFP expression in the CD3+CD4+ population, and inset number represents the percentage of IL-23R+ cells within the indicated gate. Graph represents the percentage of nTh17 cells, gated as CD3+CD4+CD44hiIL-23R+ (mean ± sd, n = 13 mice). (b) nTh17 cells were sorted from lymph nodes of Il23r-gfp reporter mice as CD4+CD44hiIL-23R+ and cultured in presence of IL-7 for 7 days. Right graph shows IL-23R/GFP MFI after 7 days of culture, normalized to CD4 naïve cells (sorted in parallel to nTh17 as CD4+CD44lowIL-23R- and cultured for 7 days in IL-7, mean ± sd, n = 3 independent cultures, p = 0.0314). IL-7-cultured nTh17 were stimulated with IL-23 or left untreated for 18 h before assessing pSTAT3-Y705 by flow cytometry. Left graph shows pSTAT3-Y705 MFI, normalized to untreated cells (mean ± sd, n = 3 independent cultures, p = 0.0132). (c) IL-7-cultured nTh17 were stimulated with IL-23 in presence or absence of Y27632 or left untreated for 18 h before assessing pRLC-S20 by flow cytometry. Left graph shows pRLC-S20 MFI, normalized to untreated cells (n = 3 independent cultures, *p = 0.0121, **p = 0.0012). (d) Il23r-gfp reporter mice were immunized with CFA/MOG to induce EAE; 12 days after EAE induction, total splenic cells were processed for analysis of IL-23R/GFP expression by flow cytometry. Representative contour plots show CD44 and IL-23R/GFP expression in the CD3+CD4+ population, and inset numbers represent the percentage of IL-23R+ cells within the indicated gate. Graph represents the percentage of iTh17 cells, gated as CD3+CD4+CD44hiIL-23R+ (mean ± sd, n = 14 mice from 2 independent experiments). (e) iTh17 cells were sorted from lymph nodes and spleens of EAE-treated Il23r-gfp reporter mice as CD4+CD44hiIL-23R+ and cultured in presence of IL-7 for 7 days. Right graph shows IL-23R/GFP MFI after 7 days of culture, normalized to CD4 naïve cells (sorted as CD4+CD44lowIL-23R- and cultured for 7d in IL-7, mean ± sd, n = 6 independent cultures, p < 0.0001). IL-7-expanded iTh17 were stimulated with IL-23 or left untreated for 18 h before assessing pSTAT3-Y705 by flow cytometry. Left graph shows pSTAT3-Y705 MFI, normalized to untreated cells (mean ± sd, n = 10 independent cultures, p < 0.0001). (f) IL-7-expanded iTh17 were stimulated with IL-23 in presence or absence of Y27632 or left untreated for 18 h before assessing pRLC-S20 by flow cytometry. Left graph shows pRLC-S20 MFI, normalized to untreated cells (mean ± sd, n = 4–8 independent cultures, ****p < 0.0001). Statistical analysis: (b, e) One sample t test. (c, f) One-way ANOVA test with Dunnett’s correction for multiple comparisons. Individual numerical values for quantifications presented in Fig 7 can be found in S7 Data. CFA, Complete Freunds´ Adjuvant; MOG, Myelin oligodendrocyte glycoprotein; EAE, experimental autoimmune encephalomyelitis; GFP, Green Fluorescent Protein; IL-23, Interleukin 23; iTh17, induced Th17; MFI, Mean of fluorescence intensity; nTh17, natural Th17; pRLC-S20, phospho-RLC-Serine20; ROCK, Rho-associated protein kinase.