Regulation of IL-17 in autoimmune diseases by transcriptional factors and microRNAs

In recent years, IL-17A (IL-17), a pro-inflammatory cytokine, has received intense attention of researchers and clinicians alike with documented effects in inflammation and autoimmune diseases. IL-17 mobilizes, recruits and activates different cells to increase inflammation. Although protective in infections, overproduction of IL-17 promotes inflammation in autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, psoriasis, among others. Regulating IL-17 levels or action by using IL-17-blocking antibodies or IL-17R antagonist has shown to attenuate experimental autoimmune diseases. It is now known that in addition to IL-17-specific transcription factor, RORγt, several other transcription factors and select microRNAs (miRNA) regulate IL-17. Given that miRNAs are dysregulated in autoimmune diseases, a better understanding of transcriptional factors and miRNA regulation of IL-17 expression and function will be essential for devising potential new therapies. In this review, we will overview IL-17 induction and function in relation to autoimmune diseases. In addition, current findings on transcriptional regulation of IL-17 induction and plausible interplay between IL-17 and miRNA in autoimmune diseases are highlighted.


IL-17 and Autoimmune Diseases
In healthy homeostatic conditions, the levels of IL-17A in human sera are undetectable, however, the levels are markedly increased in inflammatory bowel disease, psoriasis, systemic lupus erythematosus (SLE), multiple sclerosis (MS), and rheumatoid arthritis (RA; Wang et al., 2012;Jiang et al., 2014;Babaloo et al., 2015;Mease, 2015;Talaat et al., 2015). IL-23 has been shown to be critical for expansion, and/or survival and stabilization of Th17 cells by activating STAT3 and partially STAT4 (Aggarwal et al., 2003;Harrington et al., 2005). Interaction of IL-23-producing APCs and Th17 cells has been shown to have a role in many autoimmune diseases. In support of this view, targeting IL-23 pathway, IL-17 production or action by using IL-17R antagonist and IL-17A-blocking antibodies have been shown to attenuate autoimmune diseases (Hueber et al., 2010;Yeilding et al., 2011;Leonardi et al., 2012;Papp et al., 2012;Sofen et al., 2014). In this regard, several clinical trials are underway to treat psoriasis. These include, ustekinumab anti-p40-IL-23 mAb and guselkumab, an anti-IL-23-specific mAb, ixekizumab and secukinumab (anti-IL-17A mAbs) and brodalumab (an anti-IL-17RA mAb) are currently under clinical trials. A recent study has demonstrated that a combination of inhibition of IL-23 and IL-17 is more efficacious in treating Th17-mediated autoimmunity in mouse models (Mangan et al., 2015). In addition, human recombinant IL-37 has also been shown to decrease IL-17 expression and Th17 cell frequency in PBMCs and CD4 + T cells from RA patients (Ye et al., 2015). Together these studies have shown promising results of targeting IL-17 induction and signaling pathways in the treatment of chronic autoimmune diseases.

Molecular Aspects of IL-17 Induction
Although initial reports showed that TGFβ1 inhibits IL-17A production in a dose-dependent manner in human naïve CD4 + T cells (Acosta-Rodriguez et al., 2007), other studies have shown that low concentrations of TGFβ1 in combination with either IL-21 (Yang et al., 2008a), or IL-1β and IL-23 (Manel et al., 2008;Duhen and Campbell, 2014) or IL-1β, IL-23, and IL-6 (Volpe et al., 2008) promote differentiation of human CD4 + T cells into Th17 cells. Interestingly, it was recently shown that TGFβ3induced Th17 cells have pathogenic effector signature when compared to TGFβ1-induced Th17 cells (Lee et al., 2012). Studies have also shown that IL-17 expression may be transient and not a terminal/end-stage Th cell differentiation (Kurschus et al., 2010;Hirota et al., 2011). Further, there is dynamic plasticity among Th subsets and Th17 differentiation is highly dependent on the kind of stimuli (polarizing conditions) received from the local tissues.
Sentinel cells of innate immune system (neutrophils, γδ T, Lti, Paneth, and iNKT cells) also secrete IL-17 (Cua and Tato, 2010). These cells constitutively express transcriptional regulators for IL-17 induction (discussed in the next section), therefore, upon activation produce IL-17 within hours of stimuli (Sutton et al., 2009;Cua and Tato, 2010). The next section will focus on multiple transcription factors involved in the positive and negative regulation of IL-17 (Figure 1).

RORγt and RUNX1
It is now well established that for Th17 differentiation, it is critical to have TGFβ1 in the presence of IL-1, IL-6, or IL-21 to decrease suppressive FoxP3 and upregulate RORc gene encoded unique lineage-specific transcription factor, RORγt, an retinoic acid related-orphan nuclear receptor (Bettelli et al., 2006;Ivanov et al., 2006;McGeachy et al., 2007;Yang et al., 2008b;Biswas et al., 2010;Ikeda et al., 2014). Runt-related transcription factor 1 (Runx1) regulates Th17 differentiation by upregulating RORγt expression and by direct binding to RORγt (Zhang et al., 2008;Liu et al., 2015). Interestingly, a study has shown that IL-17-secreting Treg cells (Tr17) have co-expression of FoxP3, RORγt, Runx1, and Runx3 . A recent study has shown that RORγt-transcriptional activity, and subsequent IL-17 induction is increased by Sirtuin 1 (SIRT1), a protein deacetylase. Inhibition of SIRT1 by chemical Ex-527 based inhibition or T cell specific deletion strongly suppressed the development of experimental autoimmune encephalitis (EAE) in mice (Lim et al., 2015). In addition, a selective RORγt inverse agonist (TMP778) has been shown to inhibit Th17 signature gene expression, and IL-17 production from Tc17 and γδ T cells indicating the therapeutic potential of targeting RORγt in inflammatory conditions (Skepner et al., 2014).

STAT3
Activation of IL-6R (ligand binding IL-6Rα and signal transducing gp130) by IL-6 results in activation of JAK-2/STAT3 pathway. Activated STAT3 binds to the promoter of IL-17A and IL-17F (Chen et al., 2006). Depletion of either STAT3 or gp130 in CD4 + T cells by utilizing Cre-loxP recombination results in decreased RORγt expression and Th17 differentiation, suggesting that IL-6-gp130-STAT3 regulate IL-17 induction at least in part by regulating RORγt levels (Nishihara et al., 2007). In vivo inhibition of JAK2-STAT3 pathway by AG490 was recently shown to decrease Th17 but increase Tregs in collagen-induced arthritis mice (Park et al., 2014).

NF-κB
It has recently been shown that members of NF-κB family RelA (p65) and c-Rel bind to promoters of RORγ and RORγt, respectively (Ruan et al., 2011). The positive role of NF-κB in IL-17 induction was further substantiated by the findings that activation of NF-κB increases secretion of IL-17 (Huang FIGURE 1 | Positive and negative transcriptional tegulators of IL-17 induction: Different cytokines and antigen specific stimuli trigger (black arrows and lines) different signaling cascades for activation of RORc and consequently Il17 gene. Negative regulators (red arrows and lines), T-bet or FoxP3 interaction with RUNX1 prevents RORγt-RUNX1 interaction, which prevents RORγt-mediated IL-17 induction. Def6 binding to IRF4 prevents ROCK2-mediated IRF4 phosphorylation and subsequent IL-17 induction.

Negative IL-17 Regulators
Both IFNγ and IL-4 markedly inhibit Th17 differentiation (Harrington et al., 2005;Yeh et al., 2014). In addition, IL-25 and IL-27 have been found to negatively regulate Th17 cells (Batten et al., 2006;Kleinschek et al., 2007;Tang et al., 2015). Suppressor of cytokine signaling (SOCS3) also negatively regulates IL-6-gp160 signal transduction resulting in decreased IL-17 (Babon et al., 2014;Vartoukian et al., 2014). FoxP3 interacts directly with RORγt through the exon 2 region and forkhead domain of FoxP3 and suppresses the activation of the IL-17 promoter (Ichiyama et al., 2008). Moreover, it has also been shown that Runx1 interacts with FoxP3 and negatively regulates Th17 differentiation (Zhang et al., 2008). T-bet, a Th1-lineage specific transcription factor, also suppresses Th17 development by binding to Runx1 via tyrosine 304 of T-bet. This T-bet-Runx1 binding has been shown to block the transactivation of RORc gene and therefore IL-17 induction (Lazarevic et al., 2011). These studies indicate that Runx1 associates either with RORγt to upregulate IL-17, or with T-bet /FoxP3 to suppress Th17 differentiation (Gocke et al., 2007). Interestingly, a recent report has shown that in EAE, pathogenic IFNγ-secreting Th17 cells have co-expression of T-bet, Runx-1 or -3 . These findings suggest a critical role of Runx1 in differentiation of Th cells into different specific Th cell lineages.

IL-17 and miRNA
Positive correlations in miRNA expression and IL-17 levels have been observed in different studies ( Table 1). In experimental autoimmune uveoretinitis (EAU), miR-142-5p and miR-21 were increased but miR-182 decreased in eye. The kinetic changes in these miRNA paralleled with increased IL-17 levels (Ishida et al., 2011). Given that IL-17 levels are increased in skin lesions and sera of psoriasis vulgaris patients, it is noteworthy that miR-1266 levels, a putative regulator of IL-17A, were also increased in the sera of these patients (Ichihara et al., 2012). miR-146 is also positively correlated with IL-17A levels in psoriasis (Xia et al., 2012) and in RA disease severity, and is co-expressed with IL-17A in the PBMC and synovium in RA patients (Niimoto et al., 2010). Inverse relationship between select miRNAs and IL-17 has also been observed in autoimmune conditions and other diseases ( Table 1). For example, combined treatment with anti-TNFα/disease-modifying antirheumatic drugs (anti-TNFα/DMARDs) increased miR-16-5p, miR-23-3p, miR125b-5p, miR-126-3p, miRN-146a-5p, miR-223-3pmiRNA with a concomitant decrease in TNFα, interleukin (IL)-6, IL-17, rheumatoid factor (RF), and C-reactive protein (CRP; Castro-Villegas et al., 2015). Imbalance of Th17/Treg in RA patients has been associated with decreased miR-21 levels, increased STAT3 activation and decreased STAT5/pSTAT5 protein and FoxP3 mRNA levels (Dong et al., 2014). In experimental autoimmune myasthenia gravis (EAMG), there is marked downregulation of miR-145 expression. Administration of lentiviral-miR-145 decreased EAMG disease severity by decreased IL-17 production (Wang et al., 2013). miRNA dysregulation has been best studied in cancer and in several cancer studies miRNA regulation of IL-17 has been demonstrated, which may have implications for autoimmune diseases (Matsuyama et al., 2011;Arisawa et al., 2012;Zhang et al., 2013;Li et al., 2014b). A positive correlation in expession of miR-133b and miR-206 and IL-17 in both αβ and γδ T cells in human and inbred mouse strains have also been reported (Haas et al., 2011). It was found that these miRNAs were clustered nearly 45 kb upstream of Il17a/f locus (Haas et al., 2011). On the other hand, in dermatomyositis patients, there is downregulation of miR-206 and upregulation of miR-206 predicted target KLF4, a positive regulator of IL-17 and Th17 cells . It is likely that miR-206 regulation of IL-17 may be context dependent.
A recent report has shown that miR-21 increased Th17 differentiation by targeting and depleting a negative regulator of TGF-β signaling SMAD-7 (Murugaiyan et al., 2015). Treatment of wild type mice with anti miR-21 oligonucleotide decreased EAE clinical severity along with decreased Th17 cells (Murugaiyan et al., 2015). In MS patients, there is downregulation of miR-20b. In EAE, miR-20b overexpression decreased disease severity by decreasing Th17 differentiation by targeting RORγt and STAT3 (Zhu et al., 2014). There is upregulation of miR-873 in brain tissue of EAE mice and in IL-17 activated mouse primary astrocytes (Liu et al., 2014b). In EAE model, miR-873 induced by IL-17 stimulation aggravated disease severity and increased inflammation by targeting A20/NF-κ (Liu et al., 2014b). Importantly, Du et al. (2009) reported that miR-326 expression correlated with MS disease severity in human patients. Further in EAE mice, miR-326 played an important role in pathogenesis by regulating Th-17 cell differentiation through translational inhibition of Ets-1, a negative regulator of Th17 differentiation (). In MS patients there is decreased expression of an IL-6-related miRNA, miR-26a . In vivo silencing of miR-26a increased Th17-related cytokines and EAE severity .
Taken together, these studies indicate that IL-17 is regulated by interplay of multiple transcription factors and miRNAs and vary with different disease condition and cell type studied. It is recognized that in addition to miRNAs, other epigenetic mechanisms such as histone modifications and DNA methylation also contribute to autoimmune diseases. However, this topic is beyond the scope of this concise focused review.

Concluding Comments
Overall, IL-17 is regarded as a potent proinflammatory cytokine that is essential for defense against pathogens. However, dysregulated IL-17 production or response has been associated with tissue damage in various inflammatory diseases. Given that IL-17 has now been associated with many inflammatory and autoimmune diseases, a better understanding of IL-17 induction and regulation is imperative to institute novel effective targeted therapeutic strategies. While RORγt, is considered as a IL-17specific transcription factor, recent collective data clearly show that induction of IL-17 is regulated by multiple transcription factors. Transcription factors that positively and negatively regulate IL-17 have now been identified. In addition, posttranscriptional regulation of IL-17 by specific miRNAs is now increasingly appreciated. Aberrant miRNA expression is reported in several human autoimmune diseases (Dai and Ahmed, 2011;Dai et al., 2013;Khan et al., 2015). Dysregulated miRNA expression profiles have the potential to serve as good diagnostic and prognostic marker and/or therapeutic targets. Signature miRNA profile can be potentially used as novel biomarkers for Th17-mediated immune reactions. However, more in-depth and mechanistic studies are required to further define the role of miRNAs in IL-17 induction and interplay of miRNA with IL-17-related transcription factors and signaling pathways. Since blocking a major cytokine such as IL-17 may have unintended consequences, more refined (tissue-dependent) manipulation of select IL-17-regulating-miRNAs may be a viable therapeutic option in some diseases.

Author Contributions
DK and SA designed the work, drafted and revised the work and finally approved the version to be published and agree to be accountable for all aspects of the work.