Anti-inflammatory Effects Of Estrogen Research Articles

17β-estradiol replacement therapy induces eNOS, nNOS and estrogen receptor β in hypophysectomized rats with inflamed footpads.

Nitric oxide (NO) plays a key role in inflammation. It is partly produced by three forms of NOS: eNOS of inflammatory cells, nNOS of neural cells and iNOS (inducible isoform). Estrogens can cause an anti-inflammatory effect, although it is not yet clear through which NOS isoforms. The aim of this study was to evaluate the role of the different NOS isoforms, as well as estrogen receptors (ERs) α and β, on the anti-inflammatory effects of estrogens. To avoid the influence of endogenous glucocorticoids or sexual hormones, male rats were hypophysectomized. Animals were segregated into two control groups (no-treatment control group and SHAM-operated animals) and three hypophysectomized groups (no-hormonal treatment, with estradiol-17β, or with testosterone replacement treatment). Freund's complete adjuvant (1 mg) was administered to the footpad of all animals. Measurements were made based on footpad inflammation (with a plethysmometer) such as eNOS, nNOS, iNOS and ER α and β protein expression (by immunohistochemistry principle/method) on days 1, 7 and 14. Only estradiol decreased inflammation, accompanied by increased levels of eNOS and nNOS and differential expression of ERs α and β in the inflammatory infiltrate. The higher levels of estradiol-induced eNOS and nNOS ocurred perhaps through the activation of ER β.

ER Alpha Rapid Signaling Is Required for Estrogen Induced Proliferation and Migration of Vascular Endothelial Cells.

Estrogen promotes the proliferation and migration of vascular endothelial cells (ECs), which likely underlies its ability to accelerate re-endothelialization and reduce adverse remodeling after vascular injury. In previous studies, we have shown that the protective effects of E2 (the active endogenous form of estrogen) in vascular injury require the estrogen receptor alpha (ERα). ERα transduces the effects of estrogen via a classical DNA binding, “genomic” signaling pathway and via a more recently-described “rapid” signaling pathway that is mediated by a subset of ERα localized to the cell membrane. However, which of these pathways mediates the effects of estrogen on endothelial cells is poorly understood. Here we identify a triple point mutant version of ERα (KRR ERα) that is specifically defective in rapid signaling, but is competent to regulate transcription through the “genomic” pathway. We find that in ECs expressing wild type ERα, E2 regulates many genes involved in cell migration and proliferation, promotes EC migration and proliferation, and also blocks the adhesion of monocytes to ECs. ECs expressing KRR mutant ERα, however, lack all of these responses. These observations establish KRR ERα as a novel tool that could greatly facilitate future studies into the vascular and non-vascular functions of ERα rapid signaling. Further, they support that rapid signaling through ERα is essential for many of the transcriptional and physiological responses of ECs to E2, and that ERα rapid signaling in ECs, in vivo, may be critical for the vasculoprotective and anti-inflammatory effects of estrogen.

Transcript analysis of laser capture microdissected white matter astrocytes and higher phenol sulfotransferase 1A1 expression during autoimmune neuroinflammation.

BackgroundAstrocytes, the most abundant cell population in mammal central nervous system (CNS), contribute to a variety of functions including homeostasis, metabolism, synapse formation, and myelin maintenance. White matter (WM) reactive astrocytes are important players in amplifying autoimmune demyelination and may exhibit different changes in transcriptome profiles and cell function in a disease-context dependent manner. However, their transcriptomic profile has not yet been defined because they are difficult to purify, compared to gray matter astrocytes. Here, we isolated WM astrocytes by laser capture microdissection (LCM) in a murine model of multiple sclerosis to better define their molecular profile focusing on selected genes related to inflammation. Based on previous data indicating anti-inflammatory effects of estrogen only at high nanomolar doses, we also examined mRNA expression for enzymes involved in steroid inactivation.MethodsExperimental autoimmune encephalomyelitis (EAE) was induced in female C57BL6 mice with MOG35–55 immunization. Fluorescence activated cell sorting (FACS) analysis of a portion of individual spinal cords at peak disease was used to assess the composition of immune cell infiltrates. Using custom Taqman low-density-array (TLDA), we analyzed mRNA expression of 40 selected genes from immuno-labeled laser-microdissected WM astrocytes from lumbar spinal cord sections of EAE and control mice. Immunohistochemistry and double immunofluorescence on control and EAE mouse spinal cord sections were used to confirm protein expression in astrocytes.ResultsThe spinal cords of EAE mice were infiltrated mostly by effector/memory T CD4+ cells and macrophages. TLDA-based profiling of LCM-astrocytes identified EAE-induced gene expression of cytokines and chemokines as well as inflammatory mediators recently described in gray matter reactive astrocytes in other murine CNS disease models. Strikingly, SULT1A1, but not other members of the sulfotransferase family, was expressed in WM spinal cord astrocytes. Moreover, its expression was further increased in EAE. Immunohistochemistry on spinal cord tissues confirmed preferential expression of this enzyme in WM astrocytic processes but not in gray matter astrocytes.ConclusionsWe described here for the first time the mRNA expression of several genes in WM astrocytes in a mouse model of multiple sclerosis. Besides expected pro-inflammatory chemokines and specific inflammatory mediators increased during EAE, we evidenced relative high astrocytic expression of the cytoplasmic enzyme SULT1A1. As the sulfonation activity of SULT1A1 inactivates estradiol among other phenolic substrates, its high astrocytic expression may account for the relative resistance of this cell population to the anti-neuroinflammatory effects of estradiol. Blocking the activity of this enzyme during neuroinflammation may thus help the injured CNS to maintain the anti-inflammatory activity of endogenous estrogens or limit the dose of estrogen co-regimens for therapeutical purposes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-015-0348-y) contains supplementary material, which is available to authorized users.

Estrogen-mediated protection of experimental autoimmune encephalomyelitis: Lessons from the dissection of estrogen receptor-signaling in vivo.

A growing body of evidence from basic and clinical studies supports the therapeutic potential of estrogens in multiple sclerosis (MS), originating from the well-established reduction in relapse rates observed among women with MS during pregnancy. The biological effects of estrogens are mediated by estrogen receptors (ERα and ERβ). Estrogens or selective ER-agonists have been shown to exert potent neuroprotective or anti-inflammatory effects in experimental autoimmune encephalomyelitis (EAE), the mouse model of MS. A central question in EAE is to identify the cellular targets that express a functional ER isotype, and the mechanisms underlying the neuroprotective and anti-inflammatory effects of estrogens. Using pharmacological approaches targeting ER-specific functions, and genetic tools such as conditional knockout mice in which ERα or ERβ are selectively deleted in specific cell populations, a clearer picture is now emerging of the various cellular targets and downstream molecules responsible for estrogen-mediated protection against central nervous system autoimmunity.

Suppression of the inflammatory response in experimental arthritis is mediated via estrogen receptor α but not estrogen receptor β

IntroductionThe immune modulatory role of estrogens in inflammation is complex. Both pro- and anti-inflammatory effects of estrogens have been described. Estrogens bind both estrogen receptor (ER)α and β. The contribution of ERα and ERβ to ER-mediated immune modulation was studied in delayed type hypersensitivity (DTH) and in experimental arthritisMethodsER-mediated suppression of rat adjuvant arthritis (AA) was studied using ethinyl-estradiol (EE) and a selective ERβ agonist (ERB-79). Arthritis was followed for 2 weeks. Next, effects of ER agonists (ethinyl-estradiol, an ERα selective agonist (ERA-63) and a selective ERβ agonist (ERB-79) on the development of a tetanus toxoid (TT)-specific delayed type hypersensitivity response in wild type (WT) and in ERα - or ERβ-deficient mice were investigated. Finally, EE and ERA-63 were tested for their immune modulating potential in established collagen induced arthritis in DBA/1J mice. Arthritis was followed for three weeks. Joint pathology was examined by histology and radiology. Local synovial cytokine production was analyzed using Luminex technology. Sera were assessed for COMP as a biomarker of cartilage destruction.ResultsEE was found to suppress clinical signs and symptoms in rat AA. The selective ERβ agonist ERB-79 had no effect on arthritis symptoms in this model. In the TT-specific DTH model, EE and the selective ERα agonist ERA-63 suppressed the TT-specific swelling response in WT and ERβKO mice but not in ERαKO mice. As seen in the AA model, the selective ERβ agonist ERB-79 did not suppress inflammation. Treatment with EE or ERA-63 suppressed clinical signs in collagen induced arthritis (CIA) in WT mice. This was associated with reduced inflammatory infiltrates and decreased levels of proinflammatory cytokines in CIA joints.ConclusionsERα, but not ERβ, is key in ER-mediated suppression of experimental arthritis. It remains to be investigated how these findings translate to human autoimmune disease.

Effects of estrogen on temporal expressions of IL-1β and IL-1ra in rat organotypic hippocampal slices exposed to oxygen–glucose deprivation

Anti-inflammatory action of estrogen is involved in neuroprotection but the effects of estrogen on IL-1beta and its endogenous antagonist (IL-1 ra) have not been clearly defined in the ischemic brain. This study was performed to evaluate whether estrogen affects the expression of IL-1beta or IL-1ra and the ratio of the two in the ischemic hippocampus. Rat organotypic hippocampal slices were treated with 17beta estradiol (E2, 1 nM) for 7 days, exposed to oxygen-glucose deprivation (OGD) for 30 min, and then reperfused for 72 h. CA1 neuronal death quantified by propidium iodide (PI) staining and expressions of IL-1beta and IL-1ra in slices measured by real-time PCR and Western blotting were examined. PI intensities in CA1 in slices treated with E2 were significantly reduced at 24 h and 72 h post-OGD, and IL-1beta mRNA expressions were reduced at 6 h and 24 h post-OGD. In addition, IL-1ra mRNA was significantly overexpressed and the ratio of IL-1beta to IL-1ra mRNA expression was reduced by E2 especially at 24 h. In terms of protein levels, E2 downregulated IL-1beta but upregulated IL-1ra and thereby decreased the IL-1beta/IL-1 ra ratio at 24h. These findings demonstrate that estrogen-induced protection is associated with a decrease in IL-1beta and an increase in IL-1ra expression in the ischemic hippocampus during early reperfusion periods, which suggests that modulation of IL-1beta/IL-1ra might be a part of anti-inflammatory effects of estrogen.

Hormonal Replacement Therapy After Menopause Is Protective of Disease Activity in Women With Inflammatory Bowel Disease

The nature of inflammatory bowel disease (IBD) following menopause has not been previously studied. The aim of this study was to characterize the effect of menopause on disease activity and identify possible modifiers of disease activity. This was a retrospective study of women followed at the University of Chicago IBD Clinic. Disease activity was assessed using clinical scoring systems during the pre- and postmenstrual periods of subjects. Variables of interest included: history of smoking, use of oral contraceptives (OCP) prior to onset of menopause, and use of hormone replacement therapy (HRT). Sixty-five women were included, 20 with ulcerative colitis and 45 with Crohn's disease. The median age of menopause was similar to historical controls. Twenty-three patients (35%) experienced active symptoms in the premenopausal time period and 25 patients (38%) had disease indices consistent with a flare within the first 2 yr after menopause (P > 0.05). There was no relation between those who had a pre- versus postmenstrual flare as a group (P > 0.05). However, there was a significant protective effect on disease activity with postmenopausal HRT use (hazard ratio [HR] 0.18, 95% confidence interval [CI] 0.04-0.72). There was also a dose-response effect noted with an HR with longer duration of use (0.20, 0.07-0.65). The likelihood of having a flare postmenopause is not different from having it premenopause. HRT, however, may provide a protective effect for disease activity in the postmenopausal period. The anti-inflammatory effects of estrogen may be the mechanism for this observation.

Identification of Non-Genomic Mechanisms for Estrogen Effects on Monocyte Responses to Inflammatory and Osteoclastogenic Stimuli.

In previous studies we found that estradiol can act directly on primary human monocytes to inhibit RANKL stimulated osteoclastic differentiation, a potential mechanism for anti-osteoporotic effects of estrogen. Estrogen effects were rapid, suggesting involvement of non-genomic mechanisms in the inhibition of osteoclastogenic signals. Direct effects of estradiol on monocytic cells have also been implicated in anti-inflammatory effects of estrogen. It was recently shown that in murine monocytic cells, estrogen could acutely inhibit signaling events downstream from the lipopolysaccharide receptor, Toll-like receptor (TLR) 4 (Ghisletti et al. Mol Cell Biol 25 (2005) 2957). A critical component of TLR effects is the activation of the transcription factor NF-kB, resulting in increased expression of cytokines and chemokines that initiate the inflammatory response to LPS and other stimuli. NF-kB is also a critical mediator of the effects of TNF family members including RANKL. RANKL differs from most TNF family members in relying primarily on a signaling pathway through Traf6 for activation of NF-kB. Traf6 is also a key component of the pathway from TLR to NF-kB, and signaling events downstream from Traf6 appear to be common to RANKL and TLR4 signaling. We used CD14-selected primary human monocytes to examine the role of estrogen effects on NF-kB in its inhibition of monocyte response to RANKL and to determine whether common molecular mechanisms underlie estrogen effects on inflammation and osteoclastogenesis. Estradiol was added to CD14 cell cultures that, after 5 minutes, were treated with RANKL or lipopolysaccharide. After 30 minutes, cells were fixed and NF-kB localization determined by immunofluorescence microscopy using antibodies to p65. The nuclear translocation of NF-kB, evident in parallel cultures treated with RANKL or LPS but not estrogen, was markedly inhibited when cells were cotreated with estradiol. (Estrogen alone showed no effect on NF-kB localization.) Alternatively, after treatment with or without estrogen, LPS or RANKL, nuclear and cytosolic extracts were prepared and evaluated by SDS-PAGE and DNA binding, to confirm estrogen inhibition of the nuclear translocation of NF-kB. Nuclear localization of NF-kB follows the phosphorylation and degradation of IkB. Cells stimulated with RANKL or LPS for 5 or 20 minutes showed inhibition of IkB degradation if cells were cotreated with estradiol. This suggested involvement of the kinase cascade initiated by a signaling protein complex including Traf6, which is the most proximal signaling protein common to the TLR and RANKL pathways. We investigated the possibility that estrogen inhibited TLR and RANKL signal transduction through effects on the Traf6 protein complex. After 5 minutes stimulation with ligands, coimmunoprecipitation studies revealed the association of Traf6 with estrogen receptor alpha (ER) when cells were cotreated with estradiol. Coimmunoprecipitation further revealed the association of the scaffolding protein BCAR1 (p130 Cas), a protein previously implicated in non-genomic estrogen effects in breast cancer cells. We evaluated the role of BCAR1 using siRNA to inhibit its expression. In cells lacking BCAR1, the inhibitory effect of estradiol on NF-kB nuclear translocation was abrogated. The results overall suggest that estradiol may inhibit both TLR and RANKL signaling through ER protein interactions, potentially mediated by BCAR1, that disrupt the Traf6 signaling complex critical for activation of NF-kB.

Estradiol blocks the induction of CD40 and CD40L expression on endothelial cells and prevents neutrophil adhesion: An ERα-mediated pathway

Interferon gamma (IFN-gamma) was shown to induce CD40 and CD40L expression on endothelial cells (ECs) and consequently to promote neutrophil adhesion. The pro- and anti-inflammatory effects of estrogens are well recognized but their role on the regulation of CD40 and CD40L expression on ECs remains undefined. Treatment of porcine aortic endothelial cells (PAEC) with IFN-gamma for 24 h enhanced CD40 and CD40L expression by 97% and 78%, respectively. Pretreatment of PAEC with 17-beta-estradiol (17betaE) for 24 h prevented the latter expression of CD40/CD40L. Treatment of PAEC with antisense oligomers targeting ERalpha mRNA attenuated the ability of 17betaE to inhibit the IFN-gamma-induced CD40 and CD40L protein expression. The IFN-gamma activation pathway of CD40 is known to involve the phosphorylation of the Janus activated kinase (JAK) and the signal transducer and activator of transcription 1 (Stat1). 17betaE, acting via the estrogen receptor alpha (ERalpha), abrogated IFN-gamma-mediated effects on Stat1 but failed to inhibit Jak1 and Jak2 phosphorylation. Furthermore, 17betaE prevented neutrophil adhesion induced by IFN-gamma. In summary, 17betaE binding to ERalpha blocked IFN-gamma-induced Stat1 phosphorylation, CD40 and CD40L protein expression, and neutrophil adhesion onto ECs.

Estrogen and progestagens differentially modulate vascular proinflammatory factors

The potential benefit of ovarian hormone replacement therapy in cerebrovascular disease is well supported by experimental observations but not by recent large, randomized clinical trials. This discrepancy points out the need for better understanding of the vascular actions of ovarian hormones as well as medroxyprogesterone acetate (MPA), a synthetic analog of progesterone (P) widely prescribed in combination with estrogens. Therefore, we investigated whether in vivo exposure to 17beta-estradiol (E) and/or P or MPA modifies inflammation in the cerebral vasculature, a key process in the evolution of ischemic brain injury. Female rats were injected (ip) with LPS to induce inflammation, and 6 h later brains were taken for blood vessel isolation and Western blot analysis of the inflammatory enzymes inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2). In ovariectomized (O) females, LPS induced cerebrovascular iNOS and COX-2; however, this effect was significantly decreased when O animals were treated for 3 wk with E. In contrast, treatment of O females with either MPA or P exacerbated the cerebrovascular inflammatory response to LPS. In intact females, LPS induction of iNOS and COX-2 in cerebral vessels was found to vary with the stage of the estrous cycle: LPS had the greatest effect during estrus, when circulating estrogen is low and progesterone is high. Thus exposure to endogenous or exogenous ovarian hormones appears to modulate cerebrovascular inflammation. Anti-inflammatory effects of estrogen would attenuate ischemic brain injury; however, this vasoprotective benefit may be diminished in the presence of progestagens.

Focus Issue: Women’s Health

This week’s issues of Science and Science’ s STKE concern health-related issues that are specifically pertinent to women. Topics at STKE include cell signaling pathways through which human papillomaviruses promote the development of cervical cancer and the role of membrane-associated signaling pathways in the anti-inflammatory effects of estrogen and the beneficial actions of estrogen on cardiovascular function.