Interleukin 23 (IL-23) triggers pathogenic features in pro-inflammatory, IL-17-secreting T cells (Th17 and Tγδ17) that play a key role in the development of inflammatory diseases. However, the IL-23 signaling cascade remains largely undefined. Here, we used quantitative phosphoproteomics to characterize IL-23 signaling in primary murine Th17 cells. We quantified 6,888 phosphorylation sites in Th17 cells and found 168 phosphorylations regulated upon IL-23 stimulation. IL-23 increased the phosphorylation of the myosin regulatory light chain (RLC), an actomyosin contractibility marker, in Th17 and Tγδ17 cells. IL-23-induced RLC phosphorylation required Janus kinase 2 (JAK2) and Rho-associated protein kinase (ROCK) catalytic activity, and further study of the IL-23/ROCK connection revealed an unexpected role of IL-23 in the migration of Tγδ17 and Th17 cells through ROCK activation. In addition, pharmacological inhibition of ROCK reduced Tγδ17 recruitment to inflamed skin upon challenge with inflammatory agent Imiquimod. This work (i) provides new insights into phosphorylation networks that control Th17 cells, (ii) widely expands the current knowledge on IL-23 signaling, and (iii) contributes to the increasing list of immune cells subsets characterized by global phosphoproteomic approaches.

白介素23（IL-23）会触发促炎，分泌IL-17的T细胞（Th17和Tγδ17）的致病性，而T细胞在炎性疾病的发展中起关键作用。但是，IL-23信号级联在很大程度上仍然不确定。在这里，我们使用定量磷酸化蛋白质组学来表征原代鼠Th17细胞中的IL-23信号传导。我们对Th17细胞中的6,888个磷酸化位点进行了定量，发现在IL-23刺激下有168个磷酸化被调节。IL-23在Th17和Tγδ17细胞中增加了肌球蛋白调节性轻链（RLC）（一种肌动球蛋白收缩性标志物）的磷酸化。IL-23诱导的RLC磷酸化需要Janus激酶2（JAK2）和Rho相关蛋白激酶（ROCK）催化活性，对IL-23 / ROCK连接的进一步研究揭示了IL-23通过ROCK活化在Tγδ17和Th17细胞迁移中的意外作用。此外，ROCK的药理学抑制作用在用炎性药物咪喹莫特（Imiquimod）攻击后可减少Tγδ17对发炎皮肤的募集。这项工作（i）为控制Th17细胞的磷酸化网络提供了新的见解，（ii）广泛扩展了有关IL-23信号传导的现有知识，并且（iii）促进了以全局磷酸化蛋白质组学为特征的免疫细胞亚群的增加。