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 6

ROCK2 haploinsufficiency impairs Th17 differentiation.

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ROCK2 haploinsufficiency impairs Th17 differentiation. (A) Sorted naive ...
(A) Sorted naive CD4+ T cells from either Rock2+/+ or Rock2+/– mice were stimulated under either Th0 or Th17 (TGF-β + IL-6) for 3 days. IL-17 and IL-21 production was determined by ELISA. *P ≤ 0.0005. (B) Sorted naive CD4+ T cells were stimulated as in A. Relative expression of RORγt mRNA was quantified using quantitative PCR. *P = 0.001. (C) Sorted naive CD4+ T cells from either Rock2+/+ or Rock2+/– mice were stimulated under either Th0 or Th17 (TGF-β + IL-6) conditions for 5 days. IL-17 and IL-21 production was determined by ELISA. *P ≤ 0.0009. (D) Sorted naive CD4+ T cells from either Rock2+/+ or Rock2+/– mice were stimulated under either Th0 or Th17 (TGF-β + IL-21) cell conditions for 5 days. IL-17 production was determined by ELISA. *P = 0.007. (E) Sorted naive CD4+ T cells from either Rock2+/+ or Rock2+/– mice were stimulated under either Th0 or Th17 (IL-6 + TGF-β + or IL-21 + TGF-β) conditions for 5 days. After restimulation with PMA/lonomycin, intracellular staining for IL-17 and IFN-γ was performed. (F) Sorted naive CD4+ T cells from Rock2+/+ and Rock2+/– mice were stimulated as in A. Nuclear extracts were analyzed by Western blotting as in Figure 4E. (G) Sorted naive CD4+ T cells from either Rock2+/+ or Rock2+/– mice cultured as in A were subjected to ChIP assays with either IRF4 or control Ab. Quantification of IRF4 binding to the IL-17A, IL-21, and RORγt promoters was performed using quantitative PCR. *P ≤ 0.01. Data are representative of 2 (B, F, and G) or 3 (A, C, D, and E) independent experiments.