microRNA-122 and microRNA-1247 regulate the pathogenic phenotype of effector CD4+ T cell subsets in (auto)immune responses in vivo

Abstract

Autoimmune diseases are often associated with an imbalance between CD4+ T cell subsets, namely pro-inflammatory effector cells, including T helper 1 (Th)1 and Th17 cells (IFN-γ- and IL-17-producers, respectively), and anti-inflammatory Foxp3+ regulatory cells (Treg). The differentiation of these distinct CD4+ T cell subsets is known to be regulated by microRNAs (miRNAs), small non-coding RNAs that fine-tune gene expression at the post-transcriptional level. While various individual miRNAs have been implicated in this process, a holistic approach focused on in vivo immune responses is missing to better understand how miRNA networks shape the CD4+ T cell compartment in pathophysiology. To address this biological question, we established a triple reporter mouse for Ifng, Il17, and Foxp3, and subjected it to experimental autoimmune encephalomyelitis (EAE), a widely used rodent model of Multiple Sclerosis (MS). We performed miRNA-seq analysis on Th1, Th17, and Treg cells isolated from the spleen and lymph nodes (LNs) at the peak-plateau stage of EAE, and found 110 miRNAs to be differentially expressed between the effector and regulatory subsets. From there, we studied the functional role of 5 candidate miRNAs as they were specifically upregulated in one population versus the others. In vivo miRNA modulation showed that silencing miR-122 (upregulated in Th17 cells) increased the frequency of IL-17A+ cells in the LNs and precipitated the onset of EAE, whereas upregulation of miR-1247 (highly expressed in the Th1 subset) decreased the severity of the disease reducing the number of IFN-γ+ cells in the LNs. We further found that both IL-6 and TGF-β induce miR-122 expression, whereas IL-23 and IL-1β repress its expression. Given that IL-23 and IL-1β are critical to induce Th17-mediated pathogenicity, our data suggests that miR-122 is expressed in a non-pathogenic context. Interestingly, we have observed a pathogenic gene signature in CNS-derived Th17 cells (when compared to peripheral Th17 cells) with concomitantly decreased levels of miR-122, suggesting that miR-122 may regulate Th17 pathogenicity. Similarly, we observed that once Th1 cells infiltrate the CNS, their levels of miR-1247 decrease, and they produce higher levels of IFN-γ. Furthermore, as we found that this miRNA is induced by the anti-inflammatory cytokines IL-10 and TGF-β, we propose that miR-1247 constitutes an auto-regulatory mechanism of Th1 cells in the periphery, which is disrupted upon CNS infiltration. Overall, our results suggest that miR-122 and miR-1247 control the pathogenic phenotype of effector Th17 and Th1 cells, respectively, during CNS autoimmunity. These findings may have important implications for autoimmune diseases, which we are now assessing in samples from MS patients.