Phosphorylation of IRF4 by ROCK2 regulates IL-17 and IL-21 production and the development of autoimmunity in mice
Partha S. Biswas, … , Govind Bhagat, Alessandra B. Pernis
Partha S. Biswas, … , Govind Bhagat, Alessandra B. Pernis
Published August 9, 2010
Citation Information: J Clin Invest. 2010;120(9):3280-3295. https://doi.org/10.1172/JCI42856.
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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 3

ROCK2 can directly phosphorylate IRF4.

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ROCK2 can directly phosphorylate IRF4. (A–C) CD4+ T cells were purified ...
(A–C) CD4+ T cells were purified from Def6+/+ or Def6trap/trap mice and left unstimulated or restimulated for 48 hours with anti-CD3 and anti-CD28 Abs in the presence or absence of Y27632. (A) Nuclear extracts were analyzed by Western blotting using an IRF4 Ab; the blot was later stripped and reprobed with a Lamin B Ab or with a tubulin Ab to ensure for equivalent loading and purity of the fractions. Lanes were run on the same gel but were noncontiguous (white lines). (B) ChIP assays were carried out with IRF4 or control Ab. Quantification of IRF4 binding to the IL-17A, IL-21, and RORγt promoters was performed using quantitative PCR. White bars, Def6+/+; black bars, Def6trap/trap. *P ≤ 0.004. (C) Nuclear extracts were prepared and subjected to ONP assay (see Methods). Precipitated proteins were analyzed by Western blotting with an IRF4 Ab; IRF4 levels in the input extracts are shown below. (D) 293T cells were transiently transfected with empty vector (mock) or with an expression vector for IRF4ΔDBD mutant. Whole cell lysates were then prepared and immunoprecipitated with an IRF4 Ab. Immunoprecipitates were subjected to in vitro kinase reactions with or without purified CAROCK2 in the presence of [γ-32P]ATP. Y27632 was added in selected reaction samples as indicated. Reaction samples were resolved by 10% SDS-PAGE, and phosphorylated products were subsequently detected by autoradiography. Data are representative of 2 (B and D) or 3 (A and C) independent experiments.