The role of miRNA in inflammation and autoimmunity

Abstract

miRNAs are small non-coding RNA molecules that modulate the expression of multiple protein-encoding genes at the post-transcriptional level. They have recently been recognized as powerful regulators of numerous genes and pathways in the pathogenesis of inflammatory and autoimmune diseases. The targets of most miRNAs remain unknown and their roles in biological processes such as cell differentiation, proliferation, and death (apoptosis) are not clearly understood. In this review we will discuss how certain candidate miRNAs affect inflammatory and immune mediated diseases by regulating their cellular and molecular targets. We focused the influence of gender and sex hormones on miRNA. We believe that understanding the role of miRNAs could shed light on the cause and progression of many inflammatory and autoimmune diseases and eventually lay the groundwork for therapeutic options.

Introduction

MicroRNAs (miRNAs) have emerged as novel molecular regulators of numerous genes and pathways involved in normal immune responses, in the pathogenesis of cancer, and of inflammatory and autoimmune diseases [1], [2], [3], [4] that include rheumatoid arthritis (RA), multiple sclerosis (MS), and systemic lupus erythematosus (SLE). miRNAs are a novel class of small, non-coding RNAs of 18–25 nucleotides that regulate the expression of multiple protein-encoding genes at the post-transcriptional level by inhibiting translation or inducing mRNA degradation [5], [6]. In normal homeostasis, miRNAs participate in cell differentiation, proliferation, apoptosis, hematopoiesis, fat metabolism and limb morphogenesis. In pathological conditions, aberrant expression of miRNA maintains disease states through the regulation of multiple genes. Although miRNAs constitute only 3% of the human genome, they regulate approximately 90% of genes [7]. A single miRNA can regulate hundreds to thousands of target genes, thus playing a significant role in the regulation of a large percentage of the genome including activities such as controlling the development of a normal and functional innate and adaptive arm of the immune system. The innate immune system is non-specific and serves as the body's first line of defense against pathogens without the capacity to generate memory responses. On the other hand, the adaptive immune system elicits specific responses to antigenic stimuli and confers long-term immunological memory that protects from future antigenic challenges. Thus, dysregulated miRNA expression can cause serious complications for the immune system.

Autoimmunity results when the immune system fails to recognize self and directs immune responses against self-antigens, leading to cellular and tissue destruction. This occurs in diseases such as RA and SLE. miRNA expression is modulated by hormones, growth factors, and cytokines. For example, widespread estrogen-dependent suppression of miRNAs influences breast tumor cell growth [8]. Estrogen also regulates the expression of miR21 in SLE patients [9], [10]. Therefore the relationship between female sex hormones and miRNA expression may explain at least in part why some autoimmune diseases are more prevalent in females. Similar relationships between sex hormones and miRNA expression are being defined for several autoimmune conditions and unique miRNA signatures are being identified. In this review, we will focus our discussion on how sex hormones and gender affect miRNA expression in RA, SLE, cancer, MS and Sjögren syndrome.

RA, an inflammatory autoimmune disease, is characterized by synovial hyperplasia and bone erosion, often leading to joint damage. The major cellular contributors in RA are T- and B-lymphocytes, neutrophils, monocyte/macrophages, and proliferating synovial fibroblast-like cells. Modification of the synovial microenvironment by proinflammatory cytokines and chemokines attract T, B, and mononuclear antigen presenting cells (APCs), and promote secretion of proteases that promote joint destruction. A unique miRNA signature consisting of differential expressions of miR15a, miR16, miR124a, miR146a, miRNA155, miR203 and miR-346 has been identified in RA tissues [11], [12], [13], [14]. For example, over expression of miR155 in RA synovial fibroblasts suppresses production of the metalloproteinases MMP-1(Matrix metalloproteinase-1) and MMP-3, which arise in response to induction of proinflammatory cytokines and activation of TLR ligands [14]. MMP-1 and -3 promote the disease state. Toll-like receptors (TLRs) play an important role in the identification and clearance of invading pathogens. TLRs 1–6 are expressed by RA synovial fibroblasts and products of their activation are thought to provoke joint inflammation [15], [16]. Studies in miR155-deficient mice show that these mice are protected from collagen-induced arthritis [17], suggesting the importance of this miRNA in the regulation of RA. A recent study has shown that polymorphisms in the candidate miRNAs or their gene targets could be associated with susceptibility to RA [18]. miRNA146a targets both IRAK1 (IL-1R-associated kinase 1) and TRAF6 (TNFR-associated factor 6) [13], [19], [20] that modulate IL-1 induced gene MMP-13, which is involved in the degradation of cartilage in arthritis [20]. Furthermore miR146a expression in CD4 T cells from synovial fluid positively correlates with TNFα (tumor necrosis factors-α) levels and negatively with Fas-associated factor 1 (measured in serum and synovial fluid) that regulates T-cell apoptosis [21]. Lu et al. [22] demonstrated that miR146a plays an important role in suppressor functions of T-regulatory cells. Similarly, Curtale et al. [23] showed that miR146a is an anti-apoptotic factor that is involved in T-cell activation by modulating activation induced cell death. A positive correlation between high miR146a expression levels and high disease activity based on CRP (C-reactive protein) and ESR (erythrocyte sedimentation rate) values, suggests the potential utility of miRNA-146a as a RA disease activity biomarker [13]. The aberrant expression of miR16 in plasma and synovial fluid has been shown to correlate with RA disease activity. Murata et al. showed that plasma miRNA16 inversely correlates with DAS28 (disease activity score: a widely used measure of disease activity in RA) [24]. In recent studies Nagata et al. have shown that injection of miRNA15a induces apoptosis in the synovium of mice by modulating Bcl-2 levels [25]. Thus miRNA15 and miRNA16 have regulatory roles in cell proliferation and apoptosis by targeting cell cycle proteins and anti-apoptotic genes. Similarly, miRNA-203, miRNA-346, and miRNA124a are upregulated in RA synovial cells compared to cells from osteoarthritic joints or healthy joints [26], [27], [28], [29]. miRNA-346 is involved in the secretion of MMP-1 and IL-6 via the NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) pathway [29] and suppresses the IL-18 response of fibroblast-like synoviocytes by inhibiting Bruton's tyrosine kinase gene transcription [26]. In addition, miR346 controls the release of TNFα- in RA fibroblast-like cells (FLS) by stabilizing tristetraprolin (TTP) [28]. The decrease of miR124a is associated with an increased proliferation of RA synovial cells, with CDK2 (cyclin-dependent kinase 2) and CCL2/MCP1 being identified as its targets [27]. We have found that in a subset of female RA patients, levels of plasma estradiol positively correlate with expression of miRNA21, miRNA25, and miRNA124a in peripheral blood cells. The influence of estradiol on miRNA124a is particularly interesting, since this miRNA has major effects on synovial proliferation as reviewed above. At present, the targets of most miRNAs remain to be defined and the relationship between candidate miRNAs and estradiol levels requires further investigation. Future mechanistic studies are necessary to fully understand the biological processes regulated by candidate miRNAs.

SLE is an autoimmune disease mostly afflicting women of childbearing age and is caused by autoantibodies, autoreactive immune complexes and self-reacting lymphocytes. Dysregulated immune responses cause inflammation damaging many different cell types, including endothelial cells, mesangial cells, and podocytes in the kidneys. SLE is a prominent cause of morbidity and mortality in young women. More than two million people in the US suffer from this disease. SLE is caused by autoreactive autoantibodies (e.g. anti-DNA) and deposition of complement-fixing immune complexes, contributing to the inflammation and damage of multiple organs [30], [31], [32]. Although much has been learned about the disease, the cellular and molecular role of miRNAs in the regulation and pathogenesis of SLE remains to be elucidated. Dysregulation of miR21, miR25, miR125a, miR146a, miR148, and miR186 has recently been reported in SLE patients [33], [34], [35], [36], [37]. We have found significant increases in the expression of miR21, miR25, and miR186 in PBMCs (peripheral blood mononuclear cells) of SLE patients compared to healthy controls. In addition, we found that the plasma concentration of estradiol positively correlates with expression levels of miR21, miR25, miR146a, and miR186, and, more importantly, expression of miR21 and miR186 positively correlates with the SLE Disease Activity Index (SLEDAI) score in SLE patients (unpublished). The SLEDAI is used to quantify the lupus disease activity in patients, and is one of the major scoring systems used to evaluate disease activity during clinical studies. Similar to our findings, Dai et al. showed ~ 16 dysregulated miRNAs in PBMCs of SLE patients [38]. Furthermore Tang et al. identified 42 differentially expressed miRNAs in PBMCs of SLE patients compared to healthy controls [35]. In their study, miR146a expression was observed to be decreased (> 6-fold) in comparison to healthy controls. miR146a is a negative regulator of Type1 interferon and TLR7 signaling pathways and targets STAT1 and the interferon regulatory factor 5 (IRF-5) in innate immune cells; it also influences T-lymphocytes. Chan et al. discussed the role of candidate miRNA and IFNα regulation in plasmacytoid dendritic cells (pDC) from lupus patients [39]. There are also reports that indicate altered expression of miRNA in specific cellular lymphocytic subsets such as CD4 T-and B-cells and in kidneys from SLE patients [34], [35], [36], [37], [40], [41]. Pan et al. [34] found that the expressions of miR21 and miR148a were significantly increased in CD4 T cells derived from lupus prone MRL/lpr mice and from SLE patients. Divekar et al. [42] found that expression of Dicer was decreased and expression of miR155 was increased in regulatory T cells in MRL/lpr mice compared to C3H mice. The study further identified that miR155 targets CD62L. Another study found decreased expression of miR155 in serum and urine of SLE patients [43]. In vivo silencing of miR21 has recently been shown to ameliorate autoimmune splenomegaly in lupus mice [9]. miR16 and miR146a have recently been shown to target Bcl2 and regulate apoptosis in NZB/NZW F1 mice [44]. In pediatric SLE patients the expression of miR181a was decreased and it was shown that it modulates B and T cell differentiation, maturation and function [45]. Similarly, a unique miRNA signature involving miR1224-3p in lupus nephritis patients [41] has been identified. Taken together, these studies strongly suggest the involvement of specific miRNAs in SLE pathogenesis (Table 1).

Sjögren's syndrome (SS) is a systemic autoimmune disease primarily characterized by chronic inflammation of the exocrine glands, in particular the salivary and lacrimal glands. Extra-glandular manifestations occur in many patients and may involve almost any organ. Tandon et al. employed RNA sequencing of minor salivary glands of SS patients, revealing a number of novel microRNAs [46]. Two miRNAs, hsa-miR-4524b-3p and hsa-miR-4524b-5p, lie within an intron of the gene for ATP-binding cassette subfamily A member 6 (ABCA6). This gene lies within a cluster of five ABCA family genes on chromosome 17, revealing two important features: first, intronic regions play a more significant role than exonic regions during the evolutionary development of all the genes in this cluster and, secondly, ABCA6 was one of only two genes within the cluster that was under strong positive selection [47]. As miRNAs are important in translational regulation, it is likely that they are selected for, supporting the idea that hsa-miR-4524b-3p and hsa-miR-4524b-5p are likely to be genuine miRNAs. Another interesting, previously unidentified miRNA is hsa-miR-5100. Not only has this miRNA amplified by qPCR in all tested samples, but it also showed different expression patterns between patients and healthy volunteers. An additional interesting observation is that this miRNA increases drastically as salivary flow decreases [46]. Similarly Alevizos et al. have shown the potential role of miRNA as putative diagnostic and prognostic biomarker candidates in Sjögren's syndrome [48]. Jimenez et al. have recently presented that miRNA and other non-coding RNA regulatory elements may participate in the development of systemic sclerosis and primary Sjögren's syndrome [49].

In addition, Liang et al. identified Sjögren syndrome antigen B (SSB)/La as a pre-miRNA-binding protein that regulates miRNA processing in vitro. All three RNA-binding motifs (LAM, RRM1, and RRM2) of La/SSB are required for efficient pre-miRNA binding [50]. La/SSB recognizes the characteristic stem-loop structure of pre-miRNAs, of which the majority lacks a 3′ UUU terminus. Moreover, La/SSB associates with endogenous pri-/pre-miRNAs and promotes miRNA biogenesis by stabilizing pre-miRNAs from nuclease (e.g. MCPIP1)-mediated decay in mammalian cells. Accordingly, they observed positive correlations between the expression status of La/SSB and Dicer in human cancer transcriptome and prognosis. These studies identified an important function of La/SSB as a global regulator of miRNA expression, and implicate stem-loop recognition as a major mechanism that mediates association between La/SSB and diverses RNA molecules [50].

MS is an autoimmune disease that afflicts two million people worldwide [51]. It is usually diagnosed between the ages of 20 and 40 and is a primary cause of non-traumatic neurological disability in young adults [52]. It affects the brain and spinal cord. Immune reactions and inflammation lead to impairment of the myelin sheath that slows or stops the transmission of nerve impulses. A recent study analyzed the expression of 365 miRNA in lymphocytes from patients diagnosed with relapsing-remitting MS (RRMS), demonstrating the first evidence for distinct miRNA expression profiles in CD4 T, CD8 T cells and B cells in MS patients when compared to healthy volunteers [53]. The study reported that miR17-5p, which is involved in autoimmunity, is upregulated in the peripheral blood CD4 T cells of MS patients, targeting phosphatase and the tensin homology and phosphatidyl-inositol-3-kinase regulatory subunit 1[53]. Functional experiments with a synthetic inhibitor of miR-17 supported the link between miRNA expression and the altered target gene expression. On the contrary, the expression of miR-20a and miR17 are significantly reduced in MS. Both of these miRNAs regulate T cell activation genes in knock-in and knock-down T cell models [54]. In active MS lesions miR-155, miR-326, and miR-34a are all upregulated. These three miRs target CD47, a membrane glycoprotein and arbitrator of macrophage inhibition, through its receptor-regulatory protein alpha on myeloid cells [55]. CD47 is downregulated in active MS lesions compared to healthy individuals. miR-326 has been shown to regulate Th17 cell differentiation, which is involved in the pathogenesis of chronic autoimmune diseases like MS, by targeting Ets-1, a negative regulator of Th17 differentiation [56]. In addition, another recent study by Thamilarasan et al. has shown that miRNAs play an important diagnostic role in multiple sclerosis and experimental autoimmune encephalomyelitis [57].

Circulating miRNAs might serve as biomarkers for disease staging in MS as suggested recently [58]. The study found two important candidate miRNAs: miR92 and let7. miR92 target genes are involved in cell cycle regulation and cell signaling, while let7 regulates stem cell differentiation, T cell activation, and activation of TLR-7, all of which are linked to neurodegeneration. All these studies emphasize that miRNAs play a pivotal role in MS pathobiology.

As miRNA is involved in normal cellular functions, malfunctioning miRNAs have a role in the pathological events underlying cancer etiology. The principle of co-option of pathways that function during development to promote aberrant tumor cell behavior also applies to the role of miRNAs in cancer. Examples of miRNAs that modify tumor phenotypes include the miRNA let-7, a key regulator of developmental timing in C. elegans, and the miR-17-92 cluster, which conducts essential regulatory functions during mammalian development. The oncogenic role of the estrogen receptor (ER) α and its correlation with miRNA let-7 has been studied and confirmed in breast tumors. miR let-7 acts as a tumor suppressor and regulates the ERα signaling pathway in human breast tumors and breast cancer stem cells [59]. The miR-17-92 miRNA cluster is often activated in cancer cells, and a recent study showed that miR-17-92 is a potent inhibitor of TGF-β signaling. By functioning both upstream and downstream of pSMAD2, miR-17-92 activation triggers down regulation of multiple key effectors along the TGF-β signaling cascade, while also leading to direct inhibition of TGF-β-responsive genes [60]. Other noncoding RNAs like miRNA-21 expression promote growth, metastasis, and chemo- or radioresistance in non-small cell lung cancer cells by targeting PTEN [61]. miR-29, one of the more interesting miRNA families in humans, consists of three mature members, miR-29a, miR-29b, and miR-29c, which are encoded in two genetic clusters. Members of this family have been shown to be silenced or down-regulated in many different types of cancer and have subsequently been attributed with predominantly tumor-suppressing properties [62]. However, exceptions have been described where miR-29s may also have tumor-promoting functions [63], [64]. miR-29 targets expression of diverse proteins like collagens, transcription factors, methyl transferases, and others which may partake in abnormal migration, invasion or proliferation of cells and may favor development of cancer. Furthermore, members of the miR-29 family can be activated by interferon signaling, which suggests a role for the immune system [62]. Currently, we do not have a complete understanding of how the diverse classes of miRNA function in cancer. An understanding of miRNA-regulated pathways can be applied in the development of novel therapeutic strategies for cancer. Discussions on how to move ahead have led to the consensus that further investigations are necessary to define the roles of miRNA in the development of cancer with the broad goals of: 1) Finding commonalities between developmental and cancer pathways controlled by miRNA; 2) unraveling the mechanisms by which developmental and cancer pathways influence noncoding RNA processing and activity; 3) defining the molecular mechanisms through which miRNAs function in these pathways; 4) defining the mechanisms through which mRNAs induce and maintain immune suppression; and 5) utilizing our understanding of miRNA-regulated pathways in the development of novel therapeutic strategies for cancer and other diseases.

In patients and most animal models of autoimmunity the female gender is far more likely to develop most autoimmune diseases [52], [53]. RA is more common in women with a female to male ratio of 5 to 1. Similarly, MS is more prevalent in women than in men (3:1). The vast majority of patients with Sjogren's syndrome (SS), SLE, etc. are females, and in SLE, the female to male ratio is 9:1 [65], [66]. It is thus believed that sex hormones, especially estrogen, play an important role in autoimmune diseases (Fig. 1). Previous studies showed that in vivo estrogen treatment of non-autoimmune mice induces secretion of IFN-γ [67], [68]. Estrogen promotes the secretion of proinflammatory cytokines such as IFN-γ and IL-17 [69], [70] either through the modification of key transcription factors in inflammation or through the regulation of miRNA expression. Recent studies by David Baltimore's group [71] showed that T-cells lacking miRNA-146a are hyperactive in chronic inflammatory autoimmune responses, and they further postulated that miRNA146a controls T-cell receptor signaling via NF-κB [71]. Another study found that altered expression of miRNA146a correlates with the development of chronic renal failure [72]. miRNA146a has an inhibitory effect on LPS-induced IFNγ in splenic lymphocytes [73]. In this study, when splenic lymphocytes were treated with estrogen, miRNA146a expression was decreased but miRNA18 and miRNA708 were increased. In the human breast cancer cell line (MCF-7) an estrogen-dependent suppression of miRNAs was previously shown; in particular, the expression of miR181a [8]. Similarly, estrogen and estrogen receptor alpha (ER-α) inhibited the expression of miR125a and miR-145 in MCF-7 cells [74]. miR22, miR206, miR221, miR222, miR18a, miR18b, miR194b and miR302c were regulated by ER-α [75]. Estrogen also regulates miR21, and recently it was shown that miR21 binds to and targets PDCD4 (programmed cell death 4) gene and Bcl2 [76], [77]. A recent study [78] indicates the protective role of estrogen induced miR29 expression in the carbon tetrachloride-induced mouse liver injury model. In this model the levels of miR-29a and miR-29b expression were significantly decreased in the livers of male, but not in female mice following 4 weeks of carbon tetrachloride treatment. The down-regulation of miR-29a and miR-29b in male mice livers correlated with the early development of liver fibrosis, as indicated by increased expressions of fibrotic markers in male mice relative to female mice. In another study [79] it was shown that estrogen directly and indirectly activates activation-induced deaminase (AID) expression. AID is a key regulator of B cell development, and its molecular mechanism of cytosine deamination induces immunoglobulin affinity maturation and antibody class switching, which then can lead to hyper-stimulation. Estrogen's influence on AID also regulates candidate miRNAs including miRNA-155 and miRNA-181b. Thus estrogen regulates numerous miRNAs and participates in disease pathogenesis. Its ability to modulate miRNA that regulate genes involved in autoimmunity may explain in part why females are more susceptible to these diseases.