MicroRNA-21 promotes Th17 differentiation and mediates experimental autoimmune encephalomyelitis

Abstract

Accumulation of IL-17-producing Th17 cells is associated with the development of multiple autoimmune diseases; however, the contribution of microRNA (miRNA) pathways to the intrinsic control of Th17 development remains unclear. Here, we demonstrated that miR-21 expression is elevated in Th17 cells and that mice lacking miR-21 have a defect in Th17 differentiation and are resistant to experimental autoimmune encephalomyelitis (EAE). Furthermore, we determined that miR-21 promotes Th17 differentiation by targeting and depleting SMAD-7, a negative regulator of TGF-β signaling. Moreover, the decreases in Th17 differentiation in miR-21-deficient T cells were associated with defects in SMAD-2/3 activation and IL-2 suppression. Finally, we found that treatment of WT mice with an anti-miR-21 oligonucleotide reduced the clinical severity of EAE, which was associated with a decrease in Th17 cells. Thus, we have characterized a T cell-intrinsic miRNA pathway that enhances TGF-β signaling, limits the autocrine inhibitory effects of IL-2, and thereby promotes Th17 differentiation and autoimmunity.

Figure 6. Model of molecular mechanisms involved in miR-21 regulation of Th17 differentiation.

Our study demonstrates that miR-21 is increased in T cells stimulated under Th17-polarizing (TGF-β and IL-6) conditions. miR-21, in turn, promotes TGF-β signaling by targeting the inhibitory SMAD SMAD-7. The enhanced TGF-β signaling in WT T cells suppresses IL-2 expression and promotes their differentiation toward the Th17 phenotype. In the absence of miR-21, SMAD-7 levels are elevated, which in turn results in impaired TGF-β signaling, increased IL-2 expression, and the observed inhibition of Th17 differentiation. Black arrows indicate conventional TGF-β signaling in Th17 differentiation. Blue arrows indicate the modulation of TGF-β signaling in Th17 differentiation in the presence or absence of miR-21. Proposed pathways are shown in WT (Mir21^+/+) and miR-21–deficient (Mir21^–/–) T cells.

Figure 4. miR-21 promotes Th17 differentiation by targeting SMAD-7.

(A and B) Representative immunoblots of phosphorylated and total SMAD-2/3/7 proteins in CD4⁺ T cells from WT and Mir21^–/– mice stimulated with TGF-β (2 ng/ml) for the indicated times. (C) miR-21 aligned with the highly conserved 3′ UTR of Smad7 mRNA. (D and E) Luciferase activity of a reporter carrying a mutant or WT Smad7 3′ UTR cotransfected into HEK-293 T cells with miR-21 or with its control. (F) Western blot analysis of SMAD-7, p–SMAD-2, and p–SMAD-3 in CD4⁺ T cells from WT mice treated with miR-21 inhibitors or with its control. (G) Western blot analysis of SMAD-7, p–SMAD-2, and p–SMAD-3 in CD4⁺ T cells from WT mice treated with miR-21 precursors or with its control. (H and I) SMAD-7 knockdown increased IL-17 and expression of other Th17-related cytokines in CD4⁺ T cells. Numbers represent the frequency of CD4⁺ cells. (J) ELISA of IL-2 in Th17 cells transduced with control or SMAD-7–specific shRNA. (K) SMAD-7 knockdown increased TGF-β–induced SMAD-2/3 phosphorylation, while downregulating SMAD-7 levels. (L) Il17 expression in WT and Smad7^–/– T cells cultured under Th17 conditions. (M) SMAD-7 knockdown increased IL-17, while downregulating IL-2 in Mir21^–/– Th17 cells. (N) ELISA of IL-2 in Th17 cells from WT and Mir21^–/– mice. (O) Neutralization of IL-2 restored IL-17 levels in Mir21^–/– Th17 cells. Data are representative of 2 to 3 independent experiments. Error bars represent the mean ± SEM. **P < 0.01 and ***P < 0.001 by unpaired Student’s t test.

Figure 5. Silencing miR-21 ameliorates the clinical severity of EAE.

(A) qRT-PCR analysis of miR-21 in naive CD4⁺ T cells cultured for 24 hours under Th0 conditions and transfected by nucleofection with the miR inhibitor LNA–anti–miR-21 or scrambled control antisense oligonucleotide (LNA control), followed by Th17 differentiation for another 24 hours. (B) qRT-PCR and flow cytometric analysis of IL-17 in naive CD4⁺ T cells cultured for 24 hours in Th0 conditions and transfected by nucleofection with the miRNA inhibitor LNA–anti–miR-21 or scrambled control antisense oligonucleotide (LNA control), followed by Th17 differentiation for another 4 days. Numbers represent the frequencies of CD4⁺ T cells. (C) qRT-PCR analysis of Th17-related cytokines in Th17 cells polarized in the presence of control and anti–miR-21 inhibitors. (D) Disease course of adoptive EAE in WT recipients (n = 6), reconstituted with Th17 cells polarized in the presence of control and miR-21 inhibitors. (E) Silencing miR-21 ameliorated the clinical severity of EAE. Clinical EAE scores for WT mice (n = 6) treated with anti–miR-21 or scrambled control on days 5, 7, 9, 11, and 13 after immunization. (F) Splenocytes obtained from anti–miR-21 and scrambled control–treated mice were restimulated with MOG_35–55 (20 μg/ml) for 72 hours. Cell-free supernatants were assayed for IL-17 by ELISA. (G) qRT-PCR analysis of Il17 in CNS-derived CD4⁺ T cells isolated from mice treated with or without anti–miR-21. Results are representative of 2 independent experiments. Error bars represent the mean ± SEM. **P < 0.01 and ***P < 0.001 by unpaired Student’s t test.

Figure 3. miR-21–deficient mice are resistant to EAE.

(A) Mir21 mRNA was determined by RT-PCR analysis in CD4⁺ T cells from the spleen and then the CNS of naive and EAE mice at peak disease stage. (B) Clinical scores of WT and Mir21^–/– mice (n = 8) after immunization with MOG_33–55 in CFA. (C and D) Histopathological analysis of spinal cord sections from WT and Mir21^–/– mice (n = 3) 20 days after immunization. Original magnification, ×20 (C) and ×40 (D). (E) Mononuclear cell numbers pooled from the CNS of WT and Mir21^–/– mice (n = 3) 20 days after immunization. (F) Intracellular staining of IFN-γ and IL-17 in CNS-derived CD4⁺ T cells from WT and Mir21^–/– mice with EAE. Numbers indicate the percentage of CD4⁺ cells. (G) mRNA expression of Th17-related cytokines, transcription factors, and surface receptors from CD4⁺ T cells within the CNS as determined by RT-PCR. (H) FACS analysis of frequencies of IL-17–producing CD4⁺ T cells from the CNS of the indicated groups. (I) WT and Mir21^–/– mice (n = 5) were injected i.v. with WT CD4⁺ T cells 5 days before EAE induction. (J) FACS analysis of frequencies of IFN-γ– and IL-17–producing CD4⁺ T cells from the CNS of the indicated groups. (K) Disease course of adoptive EAE in WT recipients, reconstituted with Th17-polarized cells from WT or Mir21^–/– mice (n = 5). (L) FACS analysis of frequencies of GM-CSF–, IL-17–, and IFN-γ–producing CD4⁺ T cells from the CNS of the indicated groups. Data are representative of 3 independent experiments. Error bars represent the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 by unpaired Student’s t test.

Figure 2. miR-21 promotes Th17 differentiation.

(A) Il17a expression in WT and Mir21^–/– T cells cultured under Th17 conditions determined by qRT-PCR. (B) WT and Mir21^–/–-derived naive CD4⁺ T cells cultured under Th17 conditions were restimulated with PMA plus ionomycin on day 5 and stained for IL-17 and IFN-γ. Numbers represent the frequency of CD4⁺ T cells. (C–E) Naive CD4⁺ T cells from WT and Mir21^–/– mice were differentiated into Th17 cells. mRNA expression levels of Th17-related cytokines (Il17a, Il17f, Il21, Il22, and Gmcsf), transcription factors (Rorgt, Rora, Irf4, Hif1a, and Batf), and surface receptors (Il23r and Ccr6) were analyzed by qRT-PCR. Data are representative of 3 independent experiments. Error bars represent the mean ± SEM. **P < 0.01, and ***P < 0.001 by unpaired Student’s t test.

Figure 1. miR-21 is specifically induced in Th17 cells.

(A and B) Naive CD4⁺CD62L^hiCD44^lo T cells were activated with plate-bound anti-CD3 (2 μg/ml) and anti-CD28 (2 μg/ml) in the presence of Th1-, Th2-, Th17-, and Treg-polarizing conditions in vitro. Twenty-four hours later, miR-21 expression was analyzed by quantitative RT-PCR (qRT-PCR) using the snRNAs snoRNA135 and U6 snRNA as endogenous controls. (C) qRT-PCR analysis of miR-21 expression in flow cytometric–sorted CCR6⁺CD4⁺ and CCR6^–CD4⁺ T cells from in vitro Th17 cultures. (D) CD4⁺ T cells from C57BL/6 mice were sorted into IL-17A⁺ and IL-17A^– cell populations and analyzed for expression of miR-21 by qRT-PCR. Data are representative of 3 independent experiments. Error bars represent the mean ± SEM. **P < 0.01 and ***P < 0.001 by unpaired Student’s t test. Med, medium.