Abstract
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.
Highlights
Interleukin 23 (IL-23) is a pro-inflammatory cytokine implicated in the resolution of infections caused by particular extracellular pathogens
Co-cultures were supplemented with Th17 polarizing cytokines (TGFβ, IL-6, IL-1β), together with IL-21 and IL-23 to induce maximal expression of IL-23R [9]
We explored the expression of the IL-23R in these cultures, and detected an increase of IL-23R mRNA expression with maximal levels of IL-23R detected at day 8 (Fig 1F)
Summary
Interleukin 23 (IL-23) is a pro-inflammatory cytokine implicated in the resolution of infections caused by particular extracellular pathogens. Several reports have established a solid connection between excessive IL-23 production and the development of inflammatory diseases in murine models of experimental autoimmune encephalomyelitis (EAE), psoriasis, and inflammatory bowel disease [1,2,3]. Increased levels of IL-23 have been found in biopsies from patients with Crohn disease, ulcerative colitis, and psoriasis [3,4]. IL-23 signaling promotes cell migration via ROCK. Institutional grants from the Fundacion Ramon Areces and Banco de Santander to the CBMSO are acknowledged. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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