Deena Khan

Suppression of LPS-induced Interferon-γ and nitric oxide in splenic lymphocytes by select estrogen-regulated microRNAs: a novel mechanism of immune modulation

MicroRNAs (miRNAs), recently identified noncoding small RNAs, are emerging as key regulators in homeostasis of the immune system. Therefore, aberrant expression of miRNAs may be linked to immune dysfunction, such as in chronic inflammation and autoimmunity. In this study, we investigated the potential role of miRNAs in estrogen-mediated regulation of innate immune responses, as indicated by up-regulation of lipopolysaccharide (LPS)-induced interferon-gamma (IFNgamma), inducible nitric oxide synthase (iNOS), and nitric oxide in splenic lymphocytes from estrogen-treated mice. We found that miR-146a, a negative regulator of Toll-like receptor (TLR) signaling, was decreased in freshly isolated splenic lymphocytes from estrogen-treated mice compared with placebo controls. Increasing the activity of miR-146a significantly inhibited LPS-induced IFNgamma and iNOS expression in mouse splenic lymphocytes. Further, miRNA microarray and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed that estrogen selectively up-regulates/down-regulates the expression of miRNAs in mouse splenic lymphocytes. miR-223, which is markedly enhanced by estrogen, regulates LPS-induced IFNgamma, but not iNOS or nitric oxide in splenic lymphocytes. Inhibition of miR-223 activity decreased LPS-induced IFNgamma in splenic lymphocytes from estrogen-treated mice. Our data are the first to demonstrate the selective regulation of miRNA expression in immune cells by estrogen and are indicative of an important role of miRNAs in estrogen-mediated immune regulation.

Identification of a common lupus disease-associated microRNA expression pattern in three different murine models of lupus.

Background Recent reports have shown that microRNAs (miRNAs) regulate vital immunological processes and have emerged as key regulators of immune system development and function. Therefore, it is important to determine miRNA dysregulation and its pathogenic contribution in autoimmune diseases, an aspect not adequately addressed thus far. Methodology/Principal Findings In this study, we profiled miRNA expressions in splenic lymphocytes from three murine lupus models (MRL-lpr, B6-lpr and NZB/WF1) with different genetic background by miRNA microarray assays and Real-time RT-PCR. Despite the genetic differences among these three lupus stains, a common set of dysregulated miRNAs (miR-182-96-183 cluster, miR-31, and miR-155) was identified in splenocytes when compared with age-matched control mice. The association of these miRNAs with the disease was highlighted by our observation that this miRNA expression pattern was evident in NZB/W mice only at an age when lupus disease is manifested. Further, we have shown that the miRNA dysregulation in MRL-lpr mice was not simply due to the activation of splenocytes. By Real-time RT-PCR, we confirmed that these miRNAs were upregulated in both purified splenic B and T cells from MRL-lpr mice. miR-127 and miR-379, which were greatly upregulated in splenocytes from lpr mice, were moderately increased in diseased NZB/W mice. In addition, Real-time RT-PCR revealed that miR-146a, miR-101a, and miR-17-92 were also markedly upregulated in splenic T, but not B cells from MRL-lpr mice. Conclusions/Significance The identification of common lupus disease-associated miRNAs now forms the basis for the further investigation of the pathogenic contribution of these miRNAs in autoimmune lupus, which will advance our knowledge of the role of miRNAs in autoimmunity. Given that miRNAs are conserved, with regard to both evolution and function, our observation of a common lupus disease-associated miRNA expression pattern in murine lupus models is likely to have significant pathogenic, diagnostic, and/or therapeutic implications in human lupus.

The Immune System Is a Natural Target for Estrogen Action: Opposing Effects of Estrogen in Two Prototypical Autoimmune Diseases.

Analogous to other physiological systems, the immune system also demonstrates remarkable sex differences. Although the reasons for sex differences in immune responses are not precisely understood, it potentially involves differences in sex hormones (estrogens, androgens, and differential sex hormone receptor-mediated events), X-chromosomes, microbiome, epigenetics among others. Overall, females tend to have more responsive and robust immune system compared to their male counterparts. It is therefore not surprising that females respond more aggressively to self-antigens and are more susceptible to autoimmune diseases. Female hormone (estrogen or 17β-estradiol) can potentially act on all cellular subsets of the immune system through estrogen receptor-dependent and -independent mechanisms. This minireview highlights differential expression of estrogen receptors on immune cells, major estrogen-mediated signaling pathways, and their effect on immune cells. Since estrogen has varied effects in female-predominant autoimmune diseases such as multiple sclerosis and systemic lupus erythematosus, we will mechanistically postulate the potential differential role of estrogen in these chronic debilitating diseases.

Estrogen regulates transcription factors STAT-1 and NF-kappaB to promote inducible nitric oxide synthase and inflammatory responses.

Estrogen regulation of inflammatory responses has broad physiological and pathological consequences. However, the molecular mechanism of estrogen regulation of inflammation is still poorly understood. In this study, we report that activation of both STAT-1 and NF-κB signaling is essential for Con A-induced inducible NO synthase (iNOS) and NO in murine splenocytes. Estrogen enhances STAT-1 DNA-binding activity without increasing the expression of phosphorylated and total STAT-1 protein. We have recently reported that estrogen blocks the nuclear expression of NF-κB p65 and modifies nuclear NF-κBp50. Here, we demonstrated that both nuclear STAT-1 and NF-κB are modified by serine protease-mediated proteolysis, which resulted in altered STAT-1 and NF-κB activity/signaling in splenocytes from estrogen-treated mice. Inhibition of serine protease activity with 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) restores the nuclear expression of full-length STAT-1 and NF-κB proteins, and resulted in decreased STAT-1 DNA-binding activity and formation of NF-κB p65/p50 binding complexes in nuclei of splenocytes from estrogen-treated mice. Consequently, there is significantly decreased iNOS and IFN-γ production in AEBSF-treated splenocytes from estrogen-treated mice, which suggests a positive regulatory role of truncated STAT-1 and/or NF-κB. Interestingly, there is increased production of MCP-1 in STAT-1 or NF-κB small interfering RNA-transfected cells, as well as in AEBSF-treated splenocytes from estrogen-treated mice. These data suggest a differential role of truncated STAT-1 and NF-κB in regulation of various inflammatory molecules in splenocytes from estrogen-treated mice. Together, our data reveal a novel molecular mechanism of estrogen-mediated promotion of inflammatory responses, which involves posttranslational modification of STAT-1 and NF-κB proteins.

Estrogen increases, whereas IL-27 and IFN-γ decrease, splenocyte IL-17 production in WT mice

Estrogen‐mediated regulation of Th1, Th2 and Treg effector functions are well documented but, surprisingly, there is little information whether estrogen modulates IL‐17, a powerful proinflammatory cytokine that plays a pivotal role in several inflammatory and autoimmune diseases. Therefore in the current study, we determined whether estrogen regulates the expression levels of IL‐17 in WT C57BL/6 mice. By ELISA, ELISPOT and/or flow cytometric analyses, we found that estrogen upregulated the levels of not only IL‐17, but also the IL‐17‐specific transcription factor retinoic acid‐related orphan receptor γ t (RORγt), in activated splenocytes. IL‐17 levels were further enhanced by exposure of activated splenocytes to IL‐23, particularly in cells from estrogen‐treated mice. Exposure of splenocytes to IL‐27 or IFN‐γ at the time of activation markedly inhibited the levels of IL‐17 and RORγt. Interestingly, a delay of 24 h in exposure of activated splenocytes to IL‐27 or IFN‐γ decreased IL‐17 levels (albeit less profoundly) but not RORγt. These findings imply that the suppressive effects of IL‐27 and IFN‐γ are more effective prior to the differentiation and commitment of IL‐17‐secreting cells. Furthermore, inhibition of JAK‐2 by AG490 suppressed IL‐17 but not RORγt expression, suggesting that other transcription factors are also critical in estrogen‐mediated upregulation of IL‐17.

Effects of Sex Steroids on Innate and Adaptive Immunity

Estrogens and androgens are classically recognized as reproductive sex steroid hormones because of their well-documented effects on reproductive tissues. However, extensive research in diverse biological disciplines have clearly established that reproductive hormones have broad physiological effects on nonreproductive tissues, including the immune, central nervous, cardiovascular, and skeletal systems. Thus, the term “sex/reproductive hormones” describes only a narrow (albeit important) aspect of biological effects of sex steroids. In this chapter, the effects of sex hormones on the innate and adaptive immune system are highlighted. Generally, estrogens upregulate proinflammatory cytokines (e.g., IFNγ) and IFNγ-inducible molecules (nitric oxide, NOS2, and COX2), whereas androgens suppress proinflammatory responses. Immunomodulation by sex steroids may have both physiological and pathological implications (e.g., sex differences in immune capabilities and in inflammatory diseases, respectively).

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.

Epigenetic Regulation of Non-Lymphoid Cells by Bisphenol A, a Model Endocrine Disrupter: Potential Implications for Immunoregulation.

Endocrine disrupting chemicals (EDC) abound in the environment since many compounds are released from chemical, agricultural, pharmaceutical, and consumer product industries. Many of the EDCs such as Bisphenol A (BPA) have estrogenic activity or interfere with endogenous sex hormones. Experimental studies have reported a positive correlation of BPA with reproductive toxicity, altered growth, and immune dysregulation. Although the precise relevance of these studies to the environmental levels is unclear, nevertheless, their potential health implications remain a concern. One possible mechanism by which BPA can alter genes is by regulating epigenetics, including microRNA, alteration of methylation, and histone acetylation. There is now wealth of information on BPA effects on non-lymphoid cells and by comparison, paucity of data on effects of BPA on the immune system. In this mini review, we will highlight the BPA regulation of estrogen receptor-mediated immune cell functions and in different inflammatory conditions. In addition, BPA-mediated epigenetic regulation of non-lymphoid cells is emphasized. We recognize that most of these studies are on non-lymphoid cells, and given that BPA also affects the immune system, it is plausible that BPA could have similar epigenetic regulation in immune cells. It is hoped that this review will stimulate studies in this area to ascertain whether or not BPA epigenetically regulates the cells of the immune system.

Sex differences and estrogen regulation of miRNAs in lupus, a prototypical autoimmune disease

Unique dysregulated expression patterns of microRNAs (miRNAs) have been reported in many disease conditions including autoimmune diseases such as systemic lupus erythematosus (SLE). We have recently reported that miRNAs are differentially expressed not only between autoimmune and control mice, but also between male and female lupus-prone mice. This important observation is of potential clinical and experimental significance since females have higher incidence, earlier expression or severity of lupus when compared to their male counterparts. Further, estrogen administration to orchiectomized males accelerates the expression of lupus-related miRNAs and induces unique miRNA signature profile. Understanding the basis of altered miRNAs in autoimmune diseases offers a new paradigm to understand autoimmune diseases, including sex-differential susceptibility. In this review, we summarize miRNA biogenesis and function, and focus on miRNA dysregulation in SLE in the context of sexual bias. Furthermore, the effect of estrogen on epigenetic miRNA regulation in relation to SLE is highlighted.

Estrogen and Signaling in the Cells of Immune System

Hypothalamus-pituitary-gonadal axis regulation of estrogen, which acts on reproductive tissues, is well established. However, it is also evident that estrogens physiologically act on not only reproductive tissues but also on a broad range of tissues such as immune system. It is well documented that estrogen regulates all facets of the immunoregulation thereby affecting the outcome of autoimmune and inflammatory immune responses. Given the broader role of estrogen in immunobiology, it is important to understand how estrogens act on the cells of the immune system. Estrogens act in estrogen receptor dependent and/or independent manner to affect the regulation of cytokines and chemokines. This review focuses on sources and biosynthesis of estrogens, differential expression of estrogen receptors on the cells of immune system, key signaling pathways and its effect on the induction of key pro- and anti-inflammatory cytokines. Since estrogen has contrasting effects in female-predominant autoimmune diseases such as multiple sclerosis (MS) and systemic lupus erythematosus (SLE), we briefly overview the mechanistic role of estrogen on these chronic diseases. Recent evidence suggests that estrogens also alter microRNAs, which regulate a broad range of transcription of genes. The review also addresses this and other newly discovered mechanisms of estrogen-induced immunomodulation in health and disease.