Systemic lupus erythematosus (SLE) is an autoimmune disease associated with pathogenic autoantibodies. Genetic predisposition and epigenetic changes/regulation play a significant role in disease pathogenesis in addition to a host of other factors (e.g., gender, hormonal, environmental). Failure to maintain immune tolerance and epigenetic homeostasis may lead to aberrant and/or dysregulated gene expression that can result in loss of immune tolerance, inflammation and development of systemic autoimmunity.

Recent studies have provided further evidence that microRNAs contribute to inflammation in lupus pathogenesis (1–5). MicroRNAs (miRNAs) are small single-stranded (18-25 nucleotides), non-protein-coding RNA molecules that modulate gene expression at the post-transcriptional level. Indeed, recently, miRNAs have emerged as powerful regulators of many genes and pathways involved in the pathogenesis of inflammatory diseases (6–12). The precise role and targets of miRNAs, including their influence on disease pathogenesis, are poorly understood. Dysregulated expression of miRNAs has been reported in SLE patients (10, 13–16). Under-expression of certain miRNAs has been shown to be negatively correlated with SLE disease activity (SLEDAI) and IFN scores (14, 17, 18).

In this study, specific miRNAs were chosen based on literature, their expression pattern, and probable role in lupus pathogenesis. Since miRNAs are involved in many biological functions including inflammation and abnormal expression of some miRNAs is associated with SLE, we examined whether miR21, miR25, miR146a, and miR186 expression profiles in SLE patients’ PBMCs are different from those in healthy controls. The aim of the manuscript is to determine specific miRNAs role and relationship with estradiol, pro-inflammatory cytokines, and disease activity in SLE patients. Recent evidence also suggests that estrogens may contribute to gender bias in SLE by modulating selected miRNAs expression (19–21). Thus, miRNAs play important roles in the pathogenesis of SLE, and estrogens may regulate these miRNAs and their functions. However, an intricate balance/interaction between miRNAs and disease pathogenesis and SLE disease activity are beginning to be explored and not yet completely clear. Moreover, how pro-inflammatory cytokines and miRNAs are interconnected is not fully understood. In this translational study, we provide novel insights regarding candidate miRNAs and their interaction and mechanistic correlation with sex hormones (17β-estradiol), pro-inflammatory cytokines, and SLEDAI in SLE patients, and compare the data with healthy controls. Our data shows a dynamic interplay between SLE disease pathogenesis and miRNA expression and pro-inflammatory cytokines.

The present study was designed to identify, validate candidate microRNAs, and to decipher their relationship with pro-inflammatory cytokines and 17β-estradiol levels that play an important role in SLE pathology. Recent studies indicate miRNAs have a role in SLE pathogenesis (1, 5, 41). In this study, the specific miRNAs tested were chosen based on their expression and probable role in lupus pathogenesis. Both T cell and B cell-related miRNAs were described recently. B cell-related circulating miRNAs with a potential role in differential diagnosis and disease activity in lupus nephritis were described in SLE (42); in addition, miRNA-mediated control of B cell responses was also shown recently (42, 43). Several miRNAs involved in regulation of B cells, which play an important role in lupus pathogenesis, and other B cell hyperactivation and functions have been described (44–50). Similarly, T cell-related miRNAs have been described that function in inflammation and SLE (15, 51–57). We provide evidence herein that miR21, miR25, miR186 expression levels were significantly increased in SLE patients’ PBMCs compared to healthy controls. In addition, we showed that the miR146a (anti-inflammatory miRNA) expression level was significantly decreased in SLE patients ( Figure 1). We also demonstrated a significant positive correlation between candidate miR21, miR25, miR186 and SLEDAI score in SLE patients ( Figure 2). In addition, we showed that miR146a expression levels were negatively correlated with SLEDAI score ( Figure 2). Our data agrees with other investigators that found that miR146a expression levels were significantly decreased in lupus nephritis patients compared to healthy controls (18, 58). Recently it was described that miR146a targets TRAF6 (TNF Receptor-Associated Factor 6) (18). This study postulated that miR146a reduction and TRAF6 upregulation increased the progress of ESRD (end stage renal disease). Earlier it was shown that miR146a suppressed NFκB activation and subsequent cytokine production by targeting signaling adaptor proteins, e.g., TNF receptor-associated family (TRAF)-6 and IL-1 receptor-associated kinase (IRAK)-1 (59). It was further suggested that miR146a can inhibit type 1 interferon by targeting TLR7 (Toll-like receptor-7), STAT1 (Signal transducer and activator of transcription 1), and RIG-1(Retinoic acid-inducible gene I) pathways (14, 60). In agreement with our study, the study found a reverse correlation between type 1 interferon and SLE disease activity (14). In addition, recent meta-analyses showed that miR146a expression is associated with SLE risk and there were further differences in the expression of miR146a in Asian versus Caucasian populations (61, 62). These differences in the expression level could be due to sample size, medications and active versus inactive disease status in SLE patients.

Further, we showed that protein level of IL-12 and IL-21 are positively correlated with miR21, miR25, miR186 expression levels. Our data for miR21 agreed with other investigators who have found similar increased expression level in SLE patients (13, 58, 63–69). Previously it was shown that miR21 contributes to DNA hypo-methylation in lupus CD4⁺ T cells by targeting DNA methyltransferase 1 (13). Another study provided evidence that miR21 positively regulates FoxP3 expression and negatively regulates T regulatory cell (T_reg) development (70). Previously, we and others have demonstrated deficiency of FoxP3 expression and T_regs in SLE patients (24, 71). Further it was shown that silencing of miR21 in vivo ameliorates autoimmune splenomegaly in lupus mice (49). Thus, miR21 plays an important role in SLE disease pathogenesis.

In the current study, we investigated the expression levels of miR21, miR25, and miR186 in SLE patients and the impact on pro-inflammatory cytokines and SLE disease activity. Herein, we showed that pro-inflammatory cytokine protein levels (IL-12p40 and IL-21) were positively correlated with miR21, miR25, and miR186 expression levels ( Figure 4) and thus play an important role in SLE pathogenesis. Enhanced immune responses of PBMCs with 17β-estradiol (E2) treatment and further gene array analyses demonstrated toll-like receptor 8 (TLR8) as an E2-responsive candidate gene (72). TLR8 expression levels are up-regulated in vivo in SLE and in PBMCs stimulated in vitro with TLR8 agonists. Further, it was demonstrated that estrogen-regulated STAT1 activation promotes TLR8 expression to facilitate signaling via miR21 in SLE (73). It was also shown that treatment with liposomal encapsulated miR21 significantly stimulated IL-12, IL-13, and TLR8 expression and this stimulation was suppressed with MyD88 (Myeloid differentiation primary response 88) inhibition, which suggests a direct association with the TLR8-mediated signaling pathways. Further it was shown that a miR21 antagonist, chloroquine, significantly reduced TLR8 expression more than by blocking miR21 alone. Collectively, these data suggest that chloroquine may be binding to other miRNAs to prevent TLR8 induction within endosomal compartments and that multiple miRNAs are involved in TLR8 activation. Additionally, a potential role for estrogen in transcriptionally modulating many miRNAs, including miR21 has been described (74).

The potential role of miR25 and miR186 in SLE has not been explored as extensively as that of miR21. We demonstrated in this study that miR25 is upregulated in SLE patients compared to healthy controls. Similar to our study, others have used next generation sequencing and found an increased expression of miR25 in SLE patients compared to healthy controls (75). Further, it was shown that miR25 inhibits AMPD2 (adenosine monophosphate deaminase 2) in peripheral blood mononuclear cells of SLE patients (75). Over-expression of miR25 down-regulated the protein expression of AMPD2. Additionally, recent computational analysis identified the TNF-related apoptosis-inducing ligand (TRAIL) Death Receptor-4 (DR4) as a potential novel target of miR25 (76).

We also demonstrated increased expression of miR186 in SLE patients, in agreement with others (77, 78). miR186 is predicted to target major lupus susceptibility genes and is strongly associated with the predisposition to SLE disease (57, 78). In addition, it was shown that miR186 targets ROCK-1 (Rho-associated protein kinase 1) and IGF1 (Insulin-like growth factor 1) in tumor cells (79–81). Although, we were not able to study candidate miRNAs’ targets and gene ontology (GO) analyses in relation to the estradiol/hormone system (which will be investigated in greater detail in a future manuscript), we have listed candidate miRNA targets and their functions in SLE (see Table 2) based on current literature, and further demonstrated that four of these miRNAs are significant in inflammation and disease pathogenesis in SLE. We also demonstrated in this study that pro-inflammatory miRNA (miR21, miR25, and miR186) expression levels positively correlated with the plasma levels of 17β-estradiol in SLE patients ( Figure 5). Previously, we showed that 17β-estradiol levels were significantly increased in SLE patients compared to healthy controls (24). Further, we showed that anti-inflammatory miR146a expression levels were negatively correlated with 17β-estradiol levels. Thus, 17β-estradiol plays a dynamic role based on candidate miRNAs’ expression levels from pro-inflammatory to anti-inflammatory pathways and vice versa. Thus, our data suggests that increased levels of pro-inflammatory miRNA together with 17β-estradiol may contribute to the female predisposition to SLE partly due to effects on SLE-susceptible genes and on pro-inflammatory pathway activation. In the present study, we also showed that 17β-estradiol, miRNAs, and pro-inflammatory cytokines are interrelated, and their deep interaction causes and influences SLE pathogenesis (Figures 2, 4, 5).

Earlier we showed the effect of 17β-estradiol treatment on PBMCs from healthy individuals and found that 17β-estradiol significantly increased production of IL-12 (25). The role of IL-12 and the IL-23/Th17 axis has been recently demonstrated in lupus (82). Higher levels of the IL-12p40 subunit and circulating frequencies of Th17 cells were found to be correlated with SLE disease activity index (SLEDAI) including lupus nephritis (83, 84). In addition, genetic polymorphisms within the IL-12/IL12R pathways have been associated with SLE pathogenesis (85, 86). It was shown previously that several miRNAs are influenced by the estrogen levels and female sex hormone affected several lupus specific miRNA (21, 87–90). Thus, pro-inflammatory cytokines and 17β-estradiol and miRNAs are influenced by each other. The molecular interaction between miRNAs, pro-inflammatory pathways, and 17β-estradiol in SLE remains to be fully elucidated. Moreover, the molecular mechanisms by which 17β-estradiol interacts with candidate miRNAs, and pro-inflammatory cytokines in SLE are not completely clear. Future studies to delineate the detailed molecular mechanisms are required to address this interaction including target genes and inflammatory pathways.

The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

The study was approved by the Institutional Review Board of the University of California Los Angeles. The patients/participants provided their written informed consent to participate in this study.

RPS contributed to the experimental design, obtaining funding, conducting experiments, analyzing data, preparing figures, and writing of the manuscript. BHH contributed to funding and editing of the manuscript. DSB contributed to figure and manuscript editing. All authors contributed to the article and approved the submitted version.

This work was supported by the NIH grants AR54034, AI 083894, AI65645 to RPS; UCLA Senate Core Grant to BHH and RPS; UCLA Oppenheimer Clinical Seed Grant and American Autoimmune Related Disease Association grant to RPS.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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