miR-146a modulates autoreactive Th17 cell differentiation and regulates organ-specific autoimmunity
Bo Li, … , Mark P. Boldin, Lili Yang
Bo Li, … , Mark P. Boldin, Lili Yang
Published September 5, 2017
Citation Information: J Clin Invest. 2017;127(10):3702-3716. https://doi.org/10.1172/JCI94012.
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Research Article Autoimmunity Immunology

miR-146a modulates autoreactive Th17 cell differentiation and regulates organ-specific autoimmunity

Abstract

Autoreactive CD4 T cells that differentiate into pathogenic Th17 cells can trigger autoimmune diseases. Therefore, investigating the regulatory network that modulates Th17 differentiation may yield important therapeutic insights. miR-146a has emerged as a critical modulator of immune reactions, but its role in regulating autoreactive Th17 cells and organ-specific autoimmunity remains largely unknown. Here, we have reported that miR-146a–deficient mice developed more severe experimental autoimmune encephalomyelitis (EAE), an animal model of human multiple sclerosis (MS). We bred miR-146a–deficient mice with 2D2 T cell receptor–Tg mice to generate 2D2 CD4 T cells that are deficient in miR-146a and specific for myelin oligodendrocyte glycoprotein (MOG), an autoantigen in the EAE model. miR-146a–deficient 2D2 T cells induced more severe EAE and were more prone to differentiate into Th17 cells. Microarray analysis revealed enhancements in IL-6– and IL-21–induced Th17 differentiation pathways in these T cells. Further study showed that miR-146a inhibited the production of autocrine IL-6 and IL-21 in 2D2 T cells, which in turn reduced their Th17 differentiation. Thus, our study identifies miR-146a as an important molecular brake that blocks the autocrine IL-6– and IL-21–induced Th17 differentiation pathways in autoreactive CD4 T cells, highlighting its potential as a therapeutic target for treating autoimmune diseases.

Authors

Bo Li, Xi Wang, In Young Choi, Yu-Chen Wang, Siyuan Liu, Alexander T. Pham, Heesung Moon, Drake J. Smith, Dinesh S. Rao, Mark P. Boldin, Lili Yang

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Figure 8

TRAF6 and IRAK1 are possible mediators of miR-146a–regulated Th17 differentiation of autoreactive CD4 T cells.

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TRAF6 and IRAK1 are possible mediators of miR-146a–regulated Th17 differ...
All experiments were repeated 3 times, and representative results are presented. (A–E) 2D2-KO T cells were stimulated for 4 days with plate-bound anti-CD3 (5 μg/ml) and soluble anti-CD28 (1 μg/ml) and transfected with nonsilencing control siRNA (siCtrl) or siRNAs specific for mouse IRAK1 (siIRAK1) or mouse TRAF6 (siTRAF6), or both (siIRAK1 + siTRAF6). On day 4, cells were collected for analysis. (A) Western blot analysis of the indicated protein levels in siRNA-transfected 2D2-KO T cells. (B–E) qPCR analysis of IL-6 (B), IL-21 (C), RORγt (D), and IL-17A (E) mRNA expression in siRNA-transfected 2D2-KO T cells. Data are presented as the mean ± SEM of triplicate cultures. *P < 0.05, **P < 0.01, and ***P < 0.001, by 1-way ANOVA. (F and G) 2D2-KO T cells were stimulated with plate-bound anti-CD3 (5 μg/ml) and soluble anti-CD28 (1 μg/ml) and transfected with the indicated siRNAs for 4 days under the indicated Th17-polarizing culture conditions. On day 4, cells were pulsed with PMA plus ionomycin for 4 hours in the presence of GolgiStop, followed by intracellular cytokine staining. (F) Representative FACS plots showing intracellular IL-17A and IFN-γ staining of siRNA-transfected 2D2-KO T cells. (G) Quantification of the FACS plots presented in F. Data are presented as the mean ± SEM of triplicate cultures. **P < 0.01, by 1-way ANOVA.