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-kappaB 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-kappaB p65 and modifies nuclear NF-kappaBp50. Here, we demonstrated that both nuclear STAT-1 and NF-kappaB are modified by serine protease-mediated proteolysis, which resulted in altered STAT-1 and NF-kappaB 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-kappaB proteins, and resulted in decreased STAT-1 DNA-binding activity and formation of NF-kappaB p65/p50 binding complexes in nuclei of splenocytes from estrogen-treated mice. Consequently, there is significantly decreased iNOS and IFN-gamma production in AEBSF-treated splenocytes from estrogen-treated mice, which suggests a positive regulatory role of truncated STAT-1 and/or NF-kappaB. Interestingly, there is increased production of MCP-1 in STAT-1 or NF-kappaB 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-kappaB 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-kappaB proteins.

Signal transducer and activation of transcription (STAT) 4beta, a shorter isoform of interleukin-12-induced STAT4, is preferentially activated by estrogen.

Estrogen, a natural immunomodulatory compound, has been shown to promote the induction of a prototype T helper 1 cytokine, interferon (IFN)-gamma, as well as to up-regulate IFNgamma-mediated proinflammatory molecules (nitric oxide, cyclooxygenase 2, monocyte chemoattractant protein 1). Because IL-12 is a major IFNgamma-inducing cytokine, in this study we investigated whether estrogen treatment of wild-type C57BL/6 mice alters IL-12-mediated signaling pathways. A recent study has shown that IL-12 activates two isoforms of signal transducer and activation of transcription (STAT) 4, a normal-sized (full-length STAT4alpha) and a truncated form (STAT4beta). Interestingly, we found that estrogen treatment preferentially up-regulates the phosphorylation of STAT4beta in splenic lymphoid cells. Time kinetic data showed the differential activation of STAT4beta in splenic lymphoid cells from estrogen-treated mice, but not in cells from placebo controls. The activation of STAT4beta was mediated by IL-12 and not IFNgamma because deliberate addition or neutralization of IL-12, but not IFNgamma, affected the activation of STAT4beta. In contrast to IL-12-induced activation of STAT4beta in cells from estrogen-treated mice, STAT4alpha was not increased, rather it tended to be decreased. In this context, STAT4alpha-induced p27(kip1) protein was decreased in concanavalin A + IL-12-activated lymphocytes from estrogen-treated mice only. By using the in vitro DNA binding assay, we confirmed the ability of pSTAT4beta to bind to the IFNgamma-activated sites (IFNgamma activation sequences)/STAT4-binding sites in estrogen-treated mice. Our data are the first to show that estrogen apparently has selective effects on IL-12-mediated signaling by preferentially activating STAT4beta. These novel findings are likely to provide new knowledge with regard to estrogen regulation of inflammation.

Suppression of LPS-induced Interferon-gamma 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.

Despite inhibition of nuclear localization of NF-kappa B p65, c-Rel, and RelB, 17-beta estradiol up-regulates NF-kappa B signaling in mouse splenocytes: the potential role of Bcl-3.

NF-kappaB plays a major role in regulating the immune system. Therefore, alterations in NF-kappaB activity have profound effects on many immunopathologies, including inflammation, autoimmunity, and lymphoid neoplasia. We investigated the effects of estrogen (17beta-estradiol) on NF-kappaB in C57BL/6 mice since estrogen is a natural immunomodulator and we have recently reported that estrogen up-regulates several NF-kappaB-regulated proteins (inducible NO synthase, IFN-gamma, and MCP-1). We found that in vivo estrogen treatment had differential effects on NF-kappaB family members. Estrogen profoundly blocked the nuclear translocation of p65, c-Rel, and Rel-B, partially blocked p52, but permitted translocation of p50. Despite blockade of both the classical (p65/p50) and alternative (RelB/p52) NF-kappaB activation pathways, estrogen induced constitutive NF-kappaB activity and increased the levels of cytokines regulated by NF-kappaB (IL-1 alpha, IL-1 beta, IL-10, and IFN-gamma). Studies involving a NF-kappaB inhibitor confirmed a positive regulatory role of NF-kappaB on these cytokines. Remarkably, estrogen selectively induced B cell lymphoma 3 (Bcl-3), which is known to associate with p50 to confer transactivation capabilities, thereby providing a potential link between observed p50 DNA-binding activity and estrogen up-regulation of NF-kappaB transcriptional activity. Chromatin immunoprecipitation assays confirmed that Bcl-3 bound to the promoter of the NF-kappaB-regulated inducible NO synthase gene in cells from estrogen-treated mice. Estrogen appeared to act at the posttranscriptional level to up-regulate Bcl-3 because mRNA levels in splenocytes from placebo- and estrogen-treated mice were comparable. The novel findings of differential regulation of NF-kappaB proteins by estrogen provide fresh insight into potential mechanisms by which estrogen can regulate NF-kappaB-dependent immunological events.

IFN-gamma-inducing transcription factor, T-bet is upregulated by estrogen in murine splenocytes: role of IL-27 but not IL-12.

Estrogen is believed to be involved in regulation of the differentiation, survival, or function of diverse immune cells as well as in many autoimmune and inflammatory diseases. However, the mechanisms behind the immunomodulatory effects of estrogen are poorly understood. Previously, we have shown that natural estrogen can upregulate IFN-gamma and IFN-gamma-mediated-inflammatory events (iNOS, nitric oxide, COX-2). Since IFN-gamma is regulated by T-bet, in this study, we investigated whether estrogen induces T-bet expression in primary murine splenocytes. We found that in vivo estrogen treatment primes splenocytes for early upregulation of T-bet upon activation by T cell stimulants, Concanavalin-A (Con-A) or anti-CD3 antibodies. The expression of T-bet protein was not altered by IL-12 while IFN-gamma had partial effects on T-bet in splenocytes from estrogen-treated mice. Notably, T-bet expression increased in Con-A-activated splenocytes from estrogen-treated mice in the presence of IL-27. Together, our studies show that in vivo estrogen exposure primes lymphocytes towards Th1 type development by promoting/upregulating T-bet expression, which is upregulated in part by IFN-gamma and IL-27. Given that T-bet is a potent inducer of IFN-gamma, these studies may lead to new lines of investigation in relation to many female-predominant autoimmune diseases and inflammatory disorders.

Estrogen selectively regulates chemokines in murine splenocytes.

Estrogen has striking effects on immunity and inflammatory autoimmune conditions. One potential mechanism of estrogen-induced regulation of immunity and inflammatory autoimmune conditions is by altering the secretion of chemokines by lymphocytes, an aspect not well addressed thus far. We found that estrogen has marked, but differential, effects on the secretion of chemokines from activated splenocytes. Estrogen treatment significantly increased the secretion of MCP-1, MCP-5, eotaxin, and stromal cell-derived factor 1beta from Con A-activated splenocytes when compared with placebo-treated controls, and it had no effects on the levels of RANTES, thymus and activation-regulated chemokine, and keratinocyte-derived chemokine (KC) at 24 h. A kinetic analysis showed that chemokines tended to increase with stimulation time, but only MCP-1 and MCP-5 showed a biological trend of increasing in splenocytes from estrogen-treated mice, and KC was decreased significantly in estrogen-treated splenocytes at 18 h. Estrogen did not affect the protein levels of chemokine receptors CCR1 or CCR2 at 24 h. Estrogen-induced alterations in the levels of MCP-1 and MCP-5 are mediated, in part, by IFN-gamma, as estrogen treatment of IFN-gamma null mice, unlike wild-type mice, did not up-regulate these chemokines. However, addition of recombinant IFN-gamma resulted in markedly increased secretion of MCP-1 and MCP-5 only in the cells derived from estrogen-treated mice. These studies provide novel data indicating that estrogen may promote inflammatory conditions by altering the levels of chemokines, providing evidence for an additional mechanism by which estrogens can regulate inflammation.

17beta-estradiol downregulates interferon regulatory factor-1 in murine splenocytes.

Interferon regulatory factor-1 (IRF-1) is an important transcription factor that mediates interferon-gamma (IFN-gamma)-induced cell-signaling events. In this study, we examined whether 17beta-estradiol alters IRF-1 in splenic lymphocytes, in view of the immunomodulatory effects of this natural female sex hormone including its ability to alter IFN-gamma levels. We find that IRF-1 expression is markedly downregulated in splenocytes or purified T-cells from estrogen-treated mice at all time points studied when compared with their placebo counterparts. This decrease in IRF-1 in splenocytes from estrogen-treated mice is neither due to upregulation of IRF-1-interfering proteins (nucleophosmin or signal transducer and activator of transcription (STAT)-5) nor due to alternatively spliced IRF-1 mRNA. Given that IFN-gamma is a potent inducer of IRF-1, direct addition of recombinant IFN-gamma to splenocytes from either wild-type or IFN-gamma-knockout mice, or the addition of recombinant IFN-gamma to purified T-cells, was expected to stimulate IRF-1 expression. However, robust expression of IRF-1 in cells from estrogen-treated mice was not seen, unlike what was observed in cells from placebo-treated mice. Diminished IFN-gamma induction of IRF-1 in cells from estrogen-treated mice was noticed despite comparable phosphorylated STAT-1 activation. These studies are the first to show that estrogen regulates IFN-gamma-inducible IRF-1 in lymphoid cells, a finding that may have implications to IFN-gamma-regulated immune and vascular diseases.

Estrogen up-regulates inducible nitric oxide synthase, nitric oxide, and cyclooxygenase-2 in splenocytes activated with T cell stimulants: role of interferon-gamma.

Estrogen is implicated in many autoimmune diseases and is a robust immunomodulator. For example, it regulates interferon (IFN)-gamma, a cytokine believed to up-regulate inducible nitric oxide synthase (iNOS). A notable gap in the literature is a lack of information on the regulation of nitric oxide in immune tissues by estrogen. We now show that activation of splenocytes with T cell stimulants [concanavalin-A (Con-A) or anti-CD3 antibodies] results in copious release of nitric oxide in splenocyte cultures from estrogen-treated but not placebo-treated mice. Moreover, even a low dose of T cell stimulants induced nitric oxide in splenocytes from estrogen-treated, but not placebo-treated, mice. Con-A-activated splenocytes from estrogen-treated mice also have up-regulated iNOS mRNA, iNOS protein, and cyclooxygenase-2 (a nitric oxide-regulated downstream proinflammatory protein) when compared with controls. Our studies suggest that the induction of nitric oxide by activated splenocytes from estrogen-treated mice is mediated in part by IFNgamma. First, blocking costimulatory signals mediated through interactions of CD28 and B7 molecules by CTLA-4Ig markedly decreased not only IFNgamma but also nitric oxide. Second, estrogen treatment of IFNgamma-knockout (IFNgamma(-)/(-)) mice did not induce iNOS protein or nitric oxide. Finally, in vitro addition of recombinant IFNgamma to Con-A-activated splenocytes from IFNgamma((-)/(-)) mice induced iNOS protein primarily in estrogen-treated mice. Overall, this is the first report to show that estrogen treatment up-regulates IFNgamma-inducible-iNOS gene expression, iNOS protein, nitric oxide, and cyclooxygenase-2 as an indirect consequence of activation of T cells. These findings may have wide implications to immunity and inflammatory disorders including female-predominant autoimmune diseases.

The relationship between proinflammatory mediators and heat stress induced rhabdomyolysis in exercising marines.

The problem of heat stress continues to plague military combat units deployed worldwide. Heat stroke is the most severe form of heat illness and carries 10% mortality despite intervention. Proteolytic and pyrogenic pro-inflammatory mediators, such as IL-6, are released into peripheral circulation in heat illness. It is not known how these mediators affect the outcome of heat illness. Identifying relationships between release of proinflammatory mediators and extent of rhabdomyolysis may lead to an intervention to reduce severity of outcome. This article presents the most current knowledge regarding the physiologic and pathophysiologic manifestations of heat illness. Based on this current state of the science the article introduces a study that will be conducted to address military unique heat stress research.