Aryl hydrocarbon receptor-mediated induction of the microRNA-132/212 cluster promotes interleukin-17-producing T-helper cell differentiation

Abstract

Aryl hydrocarbon receptor (AHR) plays critical roles in various autoimmune diseases such as multiple sclerosis by controlling interleukin-17 (IL-17)-producing T-helper (TH17) and regulatory T cells. Although various transcription factors and cytokines have been identified as key participants in TH17 generation, the role of microRNAs in this process is poorly understood. In this study, we found that expression of the microRNA (miR)-132/212 cluster is up-regulated by AHR activation under TH17-inducing, but not regulatory T-inducing conditions. Deficiency of the miR-132/212 cluster prevented the enhancement of TH17 differentiation by AHR activation. We also identified B-cell lymphoma 6, a negative regulator of TH17 differentiation, as a potential target of the miR-212. Finally, we investigated the roles of the miR-132/212 cluster in experimental autoimmune encephalomyelitis, a murine model of multiple sclerosis. Mice deficient in the miR-132/212 cluster exhibited significantly higher resistance to the development of experimental autoimmune encephalomyelitis and lower frequencies of both TH1 and TH17 cells in draining lymph nodes. Our findings reveal a unique mechanism of AHR-dependent TH17 differentiation that depends on the miR-132/212 cluster.

Keywords: autoimmunity; dioxin receptor; immune regulation.

Fig. 1.

The miR-132/212 cluster is induced by AHR agonists under T_H17-polarizing conditions. Isolated naïve T cells from control or AHR KO mice were cultured with anti-CD3/CD28 beads and stimulated with the indicated cytokines in the presence or absence of TCDD (A and B) or FICZ (C) for 3 d. Expression of miR-132 and -212 was examined by using quantitative PCR (qPCR). Data show means ± SD of at least three independent experiments. *P < 0.05; **P < 0.01.

Fig. 2.

The miR-132/212 cluster participates in promotion of T_H17 generation by FICZ. (A) Purified naïve T cells from control or miR-132/212 DKO mice were stimulated with plate-bound anti-CD3 and soluble anti-CD28 in the presence of IL-6 and TGF-β, with or without FICZ, for 5 d. Frequencies of IFN-γ– and IL-17–positive cells were determined by flow cytometry. Dot plots and values are representative of at least three independent experiments. (B) Bar charts show means ± SD of the frequency of IL-17–positive cells from at least three independent experiments. *P < 0.05.

Fig. 3.

Deficiency of the miR-132/212 cluster suppresses EAE development. Control and miR-132/212 DKO mice were immunized with MOG emulsified with CFA at the base of the tail on day 0. (A) The mean clinical score and incidence were recorded until day 21 (control, n = 17; miR-132/212 DKO, n = 15). Data are pooled from five independent experiments. (B–D) Draining lymph-node cells obtained from mice 21 d after immunization were counted after staining for CD4 in combination with IFN-γ, IL-17, or Foxp3, as indicated (control, n = 8; miR-132/212 DKO, n = 5). (E and F) On day 21, RNA was isolated from draining lymph-node cells, and expression levels of proinflammatory cytokines IL-6 (E) and TNF-α (F) were determined by using qPCR (control, n = 8; miR-132/212 DKO, n = 5). *P < 0.05; **P < 0.01.

Fig. 4.

The miR-132/212 cluster participates in T_H17 differentiation via a Stat1- and Stat5-independent pathway. Purified naïve T cells were transfected with miR-132 mimic, miR-212 mimic, or negative-control mimic and then stimulated with anti-CD3/CD28 beads in the presence of IL-6 and TGF-β for 3 d. The cultures were supplemented with anti–IFN-γ and anti–IL-4 antibodies. (A) IL-17 levels in the supernatant were measured by ELISA (n = 8 in each condition). (B) The expression of T_H17-related genes was determined by qPCR (n = 7 in each condition). Data show means ± SD of at least three independent experiments. *P < 0.05; **P < 0.01. (C) Transfected T cells were lysed 24 h after stimulation and subjected to Western blotting. Data are from one representative of at least three experiments.

Fig. 5.

The miR-132/212 cluster negatively regulates expression of Bcl-6. Isolated naïve T cells from control or miR-132/212 DKO mice were stimulated with anti-CD3/CD28 beads in the presence of IL-6, TGF-β, and FICZ for 3 d (DNA microarray) or 4 d (Western blotting). Neutralizing antibodies against IFN-γ and IL-4 were added in the cultures. (A) The expression of genes previously reported as negative regulators of T_H17 differentiation was investigated by DNA microarray. (B) Bcl-6 protein levels were determined by Western blotting. Data are from one representative of at least three experiments. (C) The free energy of miR-212 binding to its candidate targeting site in the Bcl-6 3′ UTR was calculated, and the predicted structure was determined by using RNAhybrid. Jurkat cells were cotransfected with a luciferase reporter vector containing the Bcl-6 target site along with miR-132 mimic, miR-212 mimic, or negative-control mimic. (D) Luciferase activity was measured 48 h after transfection; normalized levels of luciferase activity are shown. Data show means ± SD of at least three independent experiments. **P < 0.01; N.S., not significant.