IL-23-mediated ROCK activation promotes Tγδ17 cell migration.

(a) IL-7-expanded Tγδ17 cells were stimulated with IL-23 for 6 h in the presence or absence of Y27632 inhibitor or left unstimulated (uns.). Representative histogram shows pSTAT3-Y705 measured by flow cytometry. Graph shows pSTAT3-Y705 MFI in TCRγδ+CD44hi cells, relative to unstimulated cells (mean ± sd, n = 3–5 independent cell cultures). (b) IL-7-expanded Tγδ17 cells were stimulated with IL-23 for 18 h in the presence or absence of the indicated inhibitors or left unstimulated. Cytokine production was assessed by flow cytometry. Graphs show the percentage of IL-17a producers (left) and IL-22 (right) among TCRγδ+CD44hi cells (mean ± sd, n = 3–6 independent cell cultures). Left graph, **p = 0.001, ^##p = 0.0028. Right graph *p = 0.0106, ^#p = 0.0157. (c) IL-7-expanded Tγδ17 cells were shorted as CD27 negative and stimulated with IL-23 for 18 h or left unstimulated. Cells were allowed to settle onto fibronectin/poly-L-Lysine-coated wells for 1 h in presence of 20 μM Y27632 inhibitor before imaging. Graph shows the frequency of cells with polarized morphology (***p = 0.0001, ****p < 0.0001). Images and quantification are representative of n = 2 independent cell cultures. (d) IL-7-expanded Tγδ17 cells from Il23r-gfp reporter mice were stimulated with IL-23 for 18 h or left unstimulated. Next day cells were pretreated for 1 h with AZD1480 or Y27632 and placed into a 3-μM transwell insert. Cells were left to migrate into the lower chamber containing medium with the same conditions as the upper chamber (IL-7, ±IL-23, and ±inhibitors) for 6 h. Cell numbers in the lower chamber were determined by flow cytometry. Graph represents migrated cells (IL-23R+/GFP), relative to untreated cells (migration index, mean ± sd, n = 4–9 independent cultures, ****p < 0.0001). (e) IL-7-expanded Tγδ17 cells from Il23r-gfp reporter mice were stimulated with IL-23 for 18 h or left unstimulated. Next day, IL-23-stimulated cells were placed into a 3-μM transwell insert and left to migrate into the lower chamber containing medium with the same conditions as the upper chamber (IL-7 and IL-23). For chemotactic assays, unstimulated cells were placed in transwell inserts and left to migrate for 6 h into the lower chamber containing the indicated concentrations of chemokine ligands CCL2 or CCL20. Cell numbers in the lower chamber were determined by flow cytometry. Graph represents migrated cells (IL-23R+/GFP) relative to untreated cells (migration index, mean ± sd, n = 3–10 independent cultures, **p = 0.0035). (f) TCRγδ cells were isolated from 5 d IMQ-treated mice, labeled with CellTrace Violet, and adoptively transferred into recipient mice that were previously ear-treated with IMQ for 5 days. Recipient animals received 2 intraperitoneal injections of ROCK inhibitor Y27632 or control vehicle (DMSO/PBS) prior to adoptive transfer of CellTrace-labeled TCRγδ cells; 12 h after transfer, ear skin and LN from recipient mice were harvested and processed for analysis by flow cytometry in presence of Accucheck counting beads to determine numbers and frequencies of adoptively transferred Tγδ17. In skin, the adoptively transferred Tγδ17 cells were gated as dermal TCRγδ cells (CD3+TCRγδintCD44hi and CellTrace+). In LN, adoptively transferred Tγδ17 cells were gated as CD3+TCRγδ+CD44hi and CellTrace+. Left graph represents the number of recruited Tγδ17 cells to the inflamed ear skin in IMQ-sensitized mice. Right graph represents the frequency of recruited Tγδ17 cells in inflamed ear skin and inguinal lymph nodes (nondraining) of IMQ-treated mice among total Tγδ17 cells. Data are pooled from 2 experiments (n = 3–5 mice per group, *p = 0.03, **p = 0.002). (g) IL-7-cultured nTh17 cells from Il23r-gfp reporter mice were stimulated with IL-23 for 18 h or left untreated. Next day cells were pretreated for 1 h with Y27632 and placed into a 3-μM transwell insert. Cells were left to migrate into the lower chamber containing medium with the same conditions as the upper chamber (IL-7, ±IL-23, and ±inhibitor) for 2 h. Cell numbers in the lower chamber were analyzed by flow cytometry in presence of Accucheck counting beads. Graph represents migrated cells (CD4+CD44^hiIL-23R+/GFP) relative to untreated cells (migration index, mean ± sd, n = 3 independent cultures, *p = 0.013). (h) IL-7-expanded iTh17 from EAE-treated Il23r-gfp reporter mice were stimulated with IL-23 for 18 h or left untreated. Next day cells were pretreated for 1 h with Y27632 and placed into a 3-μM transwell insert. Cells were left to migrate into the lower chamber containing medium with the same conditions as the upper chamber (IL-7, ±IL-23, and ±inhibitor) for 2 h. Cell numbers in the lower chamber were analyzed by flow cytometry in presence of Accucheck counting beads. Graph represents migrated cells (CD4+CD44^hiIL-23R+/GFP) relative to untreated cells (migration index, mean ± sd, n = 8 independent cultures, ***p = 0.0002, ****p < 0.0001). (i) Proposed model of the role of IL-23 signaling pathway in the development of inflammatory diseases. Statistical analysis: (b, c, d, g, h) One-way ANOVA test with Dunnett´s correction for multiple comparisons. (e) One-sample t test. (f) t test with Welch´s correction. Individual numerical values for quantifications presented in Fig 8 can be found in S8 Data. EAE, experimental autoimmune encephalomyelitis; GFP, Green Fluorescent Protein; IL-23, Interleukin 23; IMQ, Imiquimod; LN, lymph nodes; nTh17, natural Th17; ROCK, Rho-associated protein kinase.