Phosphorylation of IRF4 by ROCK2 regulates IL-17 and IL-21 production and the development of autoimmunity in mice
Partha S. Biswas, Sanjay Gupta, Emily Chang, Li Song, Roslynn A. Stirzaker, James K. Liao, Govind Bhagat, Alessandra B. Pernis
Partha S. Biswas, Sanjay Gupta, Emily Chang, Li Song, Roslynn A. Stirzaker, James K. Liao, Govind Bhagat, Alessandra B. Pernis
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Research Article Autoimmunity

Phosphorylation of IRF4 by ROCK2 regulates IL-17 and IL-21 production and the development of autoimmunity in mice

Abstract

Deregulated production of IL-17 and IL-21 plays a key pathogenic role in many autoimmune disorders. A delineation of the mechanisms that underlie the inappropriate synthesis of IL-17 and IL-21 in autoimmune diseases can thus provide important insights into potential therapies for these disorders. Here we have shown that the serine-threonine kinase Rho-associated, coiled-coil–containing protein kinase 2 (ROCK2) becomes activated in mouse T cells under Th17 skewing conditions and phosphorylates interferon regulatory factor 4 (IRF4), a transcription factor that is absolutely required for the production of IL-17 and IL-21. We furthermore demonstrated that ROCK2-mediated phosphorylation of IRF4 regulated the synthesis of IL-17 and IL-21 and the differentiation of Th17 cells. Whereas CD4+ T cells from WT mice activated ROCK2 physiologically under Th17 conditions, CD4+ T cells from 2 different mouse models of spontaneous autoimmunity aberrantly activated ROCK2 under neutral conditions. Moreover, administration of ROCK inhibitors ameliorated the deregulated production of IL-17 and IL-21 and the inflammatory and autoantibody responses observed in these autoimmune mice. Our findings thus uncover a crucial link among ROCK2, IRF4, and the production of IL-17 and IL-21 and support the idea that selective inhibition of ROCK2 could represent an important therapeutic regimen for the treatment of autoimmune disorders.

Authors

Partha S. Biswas, Sanjay Gupta, Emily Chang, Li Song, Roslynn A. Stirzaker, James K. Liao, Govind Bhagat, Alessandra B. Pernis

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Figure 8

Enhanced ROCK2 activation and aberrant IRF4 function in MRL/lpr CD4+ T cells.

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Enhanced ROCK2 activation and aberrant IRF4 function in MRL/lpr CD4+ T c...
(A–C) CD4+ T cells were purified from Def6+/+, Def6trap/trap, and MRL/lpr mice, subjected to primary stimulation, and left unstimulated or restimulated for 48 hours with anti-CD3 and anti-CD28 Abs in the presence or absence of Y27632. (A) ROCK2 kinase activity was assayed as in Figure 1B, with total ROCK2 levels in the input samples shown below. (B) Cells were subjected to ONP assay as in Figure 3C. Lanes in A and B were run on the same gel but were noncontiguous (white lines). (C) Nuclear extracts were analyzed by Western blotting as in Figure 6F. (D) Purified CD4+ T cells from Balb/c (white bars) and MRL/lpr (black bars) mice were stimulated as in A. Supernatants were then collected and assayed for IL-17 and IL-21 production by ELISA. *P ≤ 0.04. Data in A–D are representative of 3 independent experiments. (E–H) MRL/lpr mice were treated or not with Fasudil (n = 7 per group, unless otherwise indicated). (E) Spleens were collected, and IL-17 and IL-21 gene expression was analyzed by real-time RT-PCR (n = 5, untreated MRL/lpr). *P ≤ 0.04. (F) Percentages of splenic B220+sIgG1+ and B220loCD138+ cells. *P ≤ 0.04. (G) Serum levels of anti-dsDNA Abs, analyzed by ELISA. *P = 0.02. In E–G, circles represent individual mice; bars and parenthetical values denote group means. (H) Proteinuria over 9 weeks of treatment beginning at 10 weeks of age. *P ≤ 0.02, 2-tailed Mann Whitney U test.