Characterization of immune pleiotropy of ESR1 gene in pigs.

It is well known that the estrogen receptor alpha gene (ESR1) affects the reproductive traits of pigs; however, the immune role of ESR1 gene has not yet been resolved. Here, we characterized the pleiotropic aspects of ESR1 gene in immunity using the pig model. Tissue expression profile showed that the ESR1 gene had a broad ectopic expression in multiple reproductive and immune-related tissues/organs, which provided the tissue-level spatial fundamental of ESR1 gene that might function as a pleiotropic immune regulator. Using the peripheral blood cell model, a coupling transcriptome analytical strategy was proposed and verified that there existed strong positive or negative correlations of ESR1 gene with hundreds of differentially expressed genes that were involved in the immune regulation, indicating that the ESR1 gene might affect or be affected by, directly or indirectly, dozens of immune-related genes in the peripheral blood cells. Furthermore, the results of genetic association analysis showed that the SmaI-polymorphism of ESR1 gene had significant or highly significant associations with multiple immune traits, including platelet (PLT), hematocrit (HCT), the number of CD4-CD8-CD3- cells, plateletcrit (PCT), mean corpuscular volume (MCV), and mean corpuscular hemoglobin concentration (MCHC). Multiple evidences supported the immune pleiotropic roles of ESR1 gene in pigs. The study advances our understanding of the cross-species immune pleiotropic landscape of ESR1 gene and also provides a potential pleiotropic molecular marker for disease-resistant breeding in pigs.

Antibody-mediated delivery of chimeric protein degraders which target estrogen receptor alpha (ERα).

Chimeric molecules which effect intracellular degradation of target proteins via E3 ligase-mediated ubiquitination (e.g., PROTACs) are currently of high interest in medicinal chemistry. However, these entities are relatively large compounds that often possess molecular characteristics which may compromise oral bioavailability, solubility, and/or in vivo pharmacokinetic properties. Accordingly, we explored whether conjugation of chimeric degraders to monoclonal antibodies using technologies originally developed for cytotoxic payloads might provide alternate delivery options for these novel agents. In this report we describe the construction of several degrader-antibody conjugates comprised of two distinct ERα-targeting degrader entities and three independent ADC linker modalities. We subsequently demonstrate the antigen-dependent delivery to MCF7-neo/HER2 cells of the degrader payloads that are incorporated into these conjugates. We also provide evidence for efficient intracellular degrader release from one of the employed linkers. In addition, preliminary data are described which suggest that reasonably favorable in vivo stability properties are associated with the linkers utilized to construct the degrader conjugates.

Parallel factor ChIP provides essential internal control for quantitative differential ChIP-seq.

A key challenge in quantitative ChIP combined with high-throughput sequencing (ChIP-seq) is the normalization of data in the presence of genome-wide changes in occupancy. Analysis-based normalization methods were developed for transcriptomic data and these are dependent on the underlying assumption that total transcription does not change between conditions. For genome-wide changes in transcription factor (TF) binding, these assumptions do not hold true. The challenges in normalization are confounded by experimental variability during sample preparation, processing and recovery. We present a novel normalization strategy utilizing an internal standard of unchanged peaks for reference. Our method can be readily applied to monitor genome-wide changes by ChIP-seq that are otherwise lost or misrepresented through analytical normalization. We compare our approach to normalization by total read depth and two alternative methods that utilize external experimental controls to study TF binding. We successfully resolve the key challenges in quantitative ChIP-seq analysis and demonstrate its application by monitoring the loss of Estrogen Receptor-alpha (ER) binding upon fulvestrant treatment, ER binding in response to estrodiol, ER mediated change in H4K12 acetylation and profiling ER binding in patient-derived xenographs. This is supported by an adaptable pipeline to normalize and quantify differential TF binding genome-wide and generate metrics for differential binding at individual sites.

In vitro impact of bisphenol A on maturation and function of monocyte-derived dendritic cells in patients with primary Sjögren's syndrome.

Background: Epidemiological studies have shown that environmental factors accelerate the progress of primary Sjögren's syndrome (pSS). Bisphenol A (BPA), a classic endocrine disrupting chemical, affects the immune system. However, the impact of BPA on pSS has not yet been reported. The present study aimed to evaluate the potential relationship between BPA, estrogen receptor (ER), and pSS.Methods: We studied the impact of BPA on monocyte-derived dendritic cells (moDCs) from pSS patients and age-matched healthy controls (HCs). Morphological effects were observed under inverted microscope. Surface markers were analyzed by flow cytometry. ER and cytokine profiles were assessed using real-time polymerase chain reaction. The ability of moDCs to stimulate CD4+ T cells activation was assessed by mixed lymphocyte reaction (MLR).Results: moDCs from both pSS patients and HCs expressed ERα as well as ERß. After BPA-exposure, expression of ERα increased significantly in pSS patients, while that of ERß remained unchanged. moDCs from BPA-exposed pSS patients showed irregular morphology and reduction in cell aggregation. BPA increased HLA-DR on moDCs of pSS patients via ERα, and promoted the secretion of IL6 and IL12. When co-cultured with BPA-treated moDCs, cytokines (IFN-γ, IL4, IL17, IL10) and transcription factors (T-bet, Gata3, RoR-γt, Foxp3) of CD4+ T cells showed imbalance of Th1/Th2/Th17/Treg polarization, with Th1 and Th17 dominating.Conclusions: BPA altered the function of moDCs through ERα, including antigen capture, secretion of inflammatory factors, and ability to stimulate T cells, as well as accelerated the progression and further deterioration of pSS.

Metformin inhibits stromal aromatase expression and tumor progression in a rodent model of postmenopausal breast cancer.

BACKGROUND: Obesity and type II diabetes are linked to increased breast cancer risk in postmenopausal women. Patients treated with the antidiabetic drug metformin for diabetes or metabolic syndrome have reduced breast cancer risk, a greater pathologic complete response to neoadjuvant therapy, and improved breast cancer survival. We hypothesized that metformin may be especially effective when targeted to the menopausal transition, as this is a lifecycle window when weight gain and metabolic syndrome increase, and is also when the risk for obesity-related breast cancer increases. METHODS: Here, we used an 1-methyl-1-nitrosourea (MNU)-induced mammary tumor rat model of estrogen receptor (ER)-positive postmenopausal breast cancer to evaluate the long-term effects of metformin administration on metabolic and tumor endpoints. In this model, ovariectomy (OVX) induces rapid weight gain, and an impaired whole-body response to excess calories contributes to increased tumor glucose uptake and increased tumor proliferation. Metformin treatment was initiated in tumor-bearing animals immediately prior to OVX and maintained for the duration of the study. RESULTS: Metformin decreased the size of existing mammary tumors and inhibited new tumor formation without changing body weight or adiposity. Decreased lipid accumulation in the livers of metformin-treated animals supports the ability of metformin to improve overall metabolic health. We also found a decrease in the number of aromatase-positive, CD68-positive macrophages within the tumor microenvironment, suggesting that metformin targets the immune microenvironment in addition to improving whole-body metabolism. CONCLUSIONS: These findings suggest that peri-menopause/menopause represents a unique window of time during which metformin may be highly effective in women with established, or at high risk for developing, breast cancer.

Natural Anti-Estrogen Receptor Alpha Antibodies Able to Induce Estrogenic Responses in Breast Cancer Cells: Hypotheses Concerning Their Mechanisms of Action and Emergence.

The detection of human anti-estrogen receptor α antibodies (ERαABs) inducing estrogenic responses in MCF-7 mammary tumor cells suggests their implication in breast cancer emergence and/or evolution. A recent report revealing a correlation between the titer of such antibodies in sera from patients suffering from this disease and the percentage of proliferative cells in samples taken from their tumors supports this concept. Complementary evidence of the ability of ERαABs to interact with an epitope localized within the estradiol-binding core of ERα also argues in its favor. This epitope is indeed inserted in a regulatory platform implicated in ERα-initiated signal transduction pathways and transcriptions. According to some experimental observations, two auto-immune reactions may already be advocated to explain the emergence of ERαABs: one involving probably the idiotypic network to produce antibodies acting as estrogenic secretions and the other based on antibodies able to abrogate the action of a natural ERα inhibitor or to prevent the competitive inhibitory potency of released receptor degradation products able to entrap circulating estrogens and co-activators. All of this information, the aspect of which is mainly fundamental, may open new ways in the current tendency to combine immunological and endocrine approaches for the management of breast cancer.

Estrogen receptor alpha promotes lupus in (NZB×NZW)F1 mice in a B cell intrinsic manner.

Lupus is a systemic autoimmune disease characterized by the production of autoreactive antibodies against nuclear antigens. Women are disproportionately affected by lupus, and this sex bias is thought to be due, in large part, to the ability of estrogens to promote lupus pathogenesis. Previously, we have shown that global deletion of estrogen receptor alpha (ERα) significantly attenuated loss of tolerance, immune cell activation, autoantibody production, and the development of lupus nephritis. Here we show that targeted deletion of ERα specifically in B cells retards production of pathogenic autoantibodies and the development of nephritis in lupus-prone (NZB×NZW)F1 mice. Furthermore, we observed that ERα deletion in B cells was associated with decreased B cell activation in young, pre-autoimmune (NZB×NZW)F1 females. Altogether, these data suggest that ERα acts in a B cell-intrinsic manner to control B cell activation, autoantibody production, and lupus nephritis.

Complete knockout of estrogen receptor alpha is not directly protective in murine lupus.

Systemic lupus erythematosus (SLE) is a chronic and potentially severe autoimmune disease that disproportionately affects women. Despite a known role for hormonal factors impacting autoimmunity and disease pathogenesis, the specific mechanisms of action remain poorly understood. Our laboratory previously backcrossed "estrogen receptor alpha knockout (ERαKO)" mice onto the NZM2410 lupus prone background to generate NZM/ERαKO mice. This original ERαKO mouse, developed in the mid-1990s and utilized in hundreds of published studies, is not in fact ERα null. They express an N-terminally truncated ERα, and are considered a functional KO. They have physiologic deficiencies including infertility due to disruption of a critical activation domain (AF-1) at the N terminus of ERα, required for most classic estrogen (E2) actions. We demonstrated that female NZM/ERαKO mice had significantly less renal disease and significantly prolonged survival compared to WT littermates despite similar serum levels of autoantibodies and glomerular immune complex deposition. Herein, we present results of experiments using a lupus prone true ERα-/- mice (deletional KO mice on the NZM2410 background), surprisingly finding that these animals were not protected if they were ovariectomized, suggesting that another hormonal component confers protection, possibly testosterone, rather than the absence of the full-length ERα.

IL-1ß induced methylation of the estrogen receptor ERα gene correlates with EMT and chemoresistance in breast cancer cells.

Inflammation has been recently acknowledged as a key participant in the physiopathology of oncogenesis and tumor progression. The inflammatory cytokine IL-1ß has been reported to induce the expression of markers associated with malignancy in breast cancerous cells through Epithelial-Mesenchymal Transition (EMT). Aggressive breast cancer tumors classified as Triple Negative do not respond to hormonal treatment because they lack three crucial receptors, one of which is the estrogen receptor alpha (ERα). Expression of ERα is then considered a good prognostic marker for tamoxifen treatment of this type of cancer, as the binding of this drug to the receptor blocks the transcriptional activity of the latter. Although it has been suggested that inflammatory cytokines in the tumor microenvironment could regulate ERα expression, the mechanism(s) involved in this process have not yet been established. We show here that, in a cell model of breast cancer cells (6D cells), in which the inflammatory cytokine IL-1ß induces EMT by activation of the IL-1ß/IL-1RI/ß-catenin pathway, the up regulation of TWIST1 leads to methylation of the ESR1 gene promoter. This epigenetic modification produced significant decrease of the ERα receptor levels and increased resistance to tamoxifen. The direct participation of IL-1ß in these processes was validated by blockage of the cytokine-induced signaling pathway by wortmannin inactivation of the effectors PI3K/AKT. These results support our previous reports that have suggested direct participation of the inflammatory cytokine IL-1ß in the transition to malignancy of breast cancer cells.

FOXO3, estrogen receptor alpha, and androgen receptor impact tumor growth rate and infiltration of dendritic cell subsets differentially between male and female mice.

Tumors evade immune recognition and destruction in many ways including the creation of an immune-suppressive tumor microenvironment (TME). Dendritic cells (DC) that infiltrate the TME are tolerogenic, and suppress effector T cells and anti-tumor activity. Previous reports demonstrated that a key regulator of tolerance in DC is the transcription factor FOXO3. Gender disparity has been studied in cancer in relation to incidence, aggressiveness, and prognosis. Few studies have touched on the importance in relation to impact on the immune system. In the current study, we show that there are significant differences in tumor growth between males and females. Additionally, frequencies and the function of FOXO3 expressed by DC subsets that infiltrate tumors vary between genders. Our results show for the first time that DC FOXO3 expression and function is altered in females. In vitro results indicate that these differences may be the result of exposure to estrogen. These differences may be critical considerations for the enhancement of immunotherapy for cancer.

Estradiol inhibits Th17 cell differentiation through inhibition of RORγT transcription by recruiting the ERα/REA complex to estrogen response elements of the RORγT promoter.

The symptoms of vaginal candidiasis exacerbate in the second half of the menstrual cycle in premenopausal women when the serum estradiol level is elevated. Estradiol has been shown to inhibit Th17 differentiation and production of antifungal IL-17 cytokines. However, little is known about the mechanisms. In the present study, we used mouse splenocytes and found that estradiol inhibited Th17 differentiation through downregulation of Rorγt mRNA and protein expression. Estradiol activated estrogen receptor (ER)α to recruit repressor of estrogen receptor activity (REA) and form the ERα/REA complex. This complex bound to three estrogen response element (ERE) half-sites on the Rorγt promoter region to suppress Rorγt expression. Estradiol induced Rea mRNA and protein expression in mouse splenocytes. Using Rea small interfering RNA to knock down Rea expression enhanced Rorγt expression and Th17 differentiation. Alternatively, histone deacetylase 1 and 2 bound to the three ERE half-sites, independent of estradiol. Histone deacetylase inhibitor MS-275 dose- and time-dependently increased Rorγt expression and subsequently enhanced Th17 differentiation. In 15 healthy premenopausal women, high serum estradiol levels are correlated with low RORγT mRNA levels and high REA mRNA levels in the vaginal lavage. These results demonstrate that estradiol upregulates REA expression and recruits REA via ERα to the EREs on the RORγT promoter region, thus inhibiting RORγT expression and Th17 differentiation. This study suggests that the estradiol/ERα/REA axis may be a feasible target in the management of recurrent vaginal candidiasis.

Triptorelin and cetrorelix induce immune responses and affect uterine development and expressions of genes and proteins of ESR1, LHR, and FSHR of mice.

CONTEXT: GnRH immunity can reduce the expression of pituitary GnRH levels, and cause the changes in reproductive behaviors. It is unclear whether triptorelin (TRI) and cetrorelix (CET) immunity influences uterine development and expression of follicle-stimulating hormone receptor (FSHR), luteinizing hormone receptor (LHR), and estradiol receptor 1 (ERS1) in the uterus. OBJECTIVE: The study investigated the effects of active immunity of GnRH agonist and antagonist on uterine development, microstructures, expression of hormone receptors mRNAs, and proteins in uteri. MATERIALS AND METHODS: One hundred and five mice were assigned into CET, TRI, and control groups (CG). Mice in CET-1, CET-2, and CET-3 (n = 15) were subcutaneously injected with 10, 20, and 40 µg CET antigens for seven days, respectively. Mice in TRI-1, TRI-2, and TRI-3 were injected with 10, 20, and 40 µg TRI antigens for seven days, respectively. The qPCR and Western blot were implemented to determine expressions of ESR1, LHR and FSHR mRNAs, and proteins. RESULTS: Compared with CG, the uterine weights of CET-1, CET-2, and CET-3 increased by 42.86, 62.86, and 10.00% on day 35 (p < 0.05), respectively. Uterine weights of TRI-2, TRI-3 reduced by 28.57% and 11.43% (p < 0.05), respectively. The uterine cavity in CET-1, CET-2, and CET-3 increased; the uterine wall became thick. The cytoplasm of endometrial epithelial cells (EEC) increased slightly. In TRI group, the uterine wall thinned. Uterine cavity became narrow slightly in TRI-1. Numbers of uterine glands reduced. The endometrium epithelial thickness (EET) in CET-1 and CET-2 increased by 68.21% and 79.46% (p < 0.05), respectively. EET in TRI-1 was decreased by 13.69%. Uterine wall thicknesses (UWT) in CET-1 and CET-2 were higher than CG, with the increment of 28.59% and 30.72%. UWT of TRI-1, TRI-2, and TRI-3 reduced by 29.35, 15.36, and 14.41%, respectively. Expressions of ESR1, FSHR, and LHR mRNAs in CET and TRI mice increased. ESR1 and FSHR protein levels increased in all experimental mice (p < 0.05), with a maximum of TRI-3. LHR protein levels of the CET decreased. LHR protein levels of TRI group increased, with a maximum of TRI-3 (p < 0.05). ESR1 protein level had significant negative correlations to mRNA expressions of ESR1, LHR, and FSHR. CONCLUSIONS: CET immunity promoted the uterine development, improved EET and UWT, and also promoted the expressions of ESR1 and FSHR protein levels. It lessened the LHR protein levels. TRI immunity blocked EET and UWT, inhibited uterine growth and development. The efficacy of CET immunity was more obvious than TRI.

Estrogen modulation of endosome-associated toll-like receptor 8: an IFNα-independent mechanism of sex-bias in systemic lupus erythematosus.

Females of child-bearing age are more resistant to infectious disease and have an increased risk of systemic lupus erythematosus (SLE). We hypothesized that estrogen-induced gene expression could establish an immunoactivated state which would render enhanced defense against infection, but may be deleterious in autoimmune development. Using peripheral blood mononuclear cells (PBMCs), we demonstrate enhanced responses with immunogen stimulation in the presence of 17ß-estradiol (E2) and gene array analyses reveal toll-like receptor 8 (TLR8) as an E2-responsive candidate gene. TLR8 expression levels are up-regulated in SLE and PBMCs stimulated with TLR8 agonist display a female sex-biased, E2-sensitive response. Moreover, we identify a putative ERα-binding region near the TLR8 locus and blocking ERα expression significantly decreases E2-mediated TLR8 induction. Our findings characterize TLR8 as a novel estrogen target gene that can lower the inflammatory threshold and implicate an IFNα-independent inflammatory mechanism that could contribute to higher SLE incidence in women.

Estrogen modulates ß2-adrenoceptor-induced cell-mediated and inflammatory immune responses through ER-α involving distinct intracellular signaling pathways, antioxidant enzymes, and nitric oxide.

Sympathetic noradrenergic neuronal activity in the lymphoid organs regulates immunity through the release and binding of norepinephrine to ß2-adrenergic receptors (AR) on lymphocytes. In women, estrogen modulates immune responses during menstrual cycles, and in aging and age-associated diseases. The intent of the present study is to characterize the extent of immunomodulation by ß2-AR in the presence of estrogen and the involvement of intracellular signaling mechanisms including the role of antioxidant enzymes (AOE) in lymphocytes. In vitro effects of terbutaline, ß2-AR agonist, either alone or in combination with 17ß-estradiol (E2) were examined on splenocyte proliferation, cytokine (IFN-γ, IL-2, and IL-6) production, intracellular signaling molecules (p-ERK, p-CREB, p-Akt, and p-NF-κB) expression, NO production, and AOE activities [superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx)]. The specificity of their actions was investigated using ß-AR antagonist, and inhibitors of signaling targets and inducible nitric oxide synthase (iNOS). Terbutaline suppressed T cell proliferation and IL-6 production and increased AOE activities involving ERK, PKA, PKC, and NF-κB pathways and NO production. E2 alone enhanced T cell proliferation and decreased IL-6 production and NF-κB expression through ER-α. E2 in the presence of terbutaline reversed terbutaline-induced effects on T cell proliferation, IL-6 production, p-ERK and p-CREB expression, AOE activities, NO production, and NF-κB expression. Estrogen through ER-α differentially modulates ß2-AR-induced immune responses involving ERK, PKA, PKC, and NF-κB pathways, and NO that may be responsible for estrogen-induced immunosenescence and development of female-specific diseases.

Response to HER-2 pulsed DC1 vaccines is predicted by both HER-2 and estrogen receptor expression in DCIS.

BACKGROUND: Patients with estrogen-independent (ER(neg)) human epidermal growth factor receptor-2 (HER-2)-positive ductal carcinoma in situ (DCIS) treated with lumpectomy alone or lumpectomy and radiation are at increased risk of developing subsequent breast cancer events. METHODS: Thirty-eight patients with HER-2 expressing DCIS received a HER-2 pulsed autologous dendritic cell (DC1) vaccine administered over 4-6 weeks before surgical resection. HER-2 and estrogen receptor (ER) expression were determined by immunohistochemistry. In 35 patients, CD4(pos) T-cell sensitization to HER-2 peptides was identified by ELISPOT. In 19 patients, CD8(pos) T-cell responses were identified by ELISA. Clinical and immune responses postvaccination were compared between intermediate-expressing HER-2 (2+) and high-expressing HER-2 (3+) patients, as well as ER(neg) and estrogen-dependent (ER(pos)) patients. RESULTS: There was no significant difference in immune response after HER-2 vaccination in patients with HER-2 (2+) and (3+) tumors or ER(neg) and ER(pos) tumors. Complete tumor regression rates were similar in patients with HER-2 (2+) and (3+) DCIS. Overall, clinical response rates were similar in patients with ER(neg) and ER(pos) DCIS, but complete tumor regression was significantly more common in patients with ER(neg) DCIS. CONCLUSIONS: Despite equivalent immune responses after vaccination in patients with HER-2 (2+), HER-2 (3+), ER(neg) and ER(pos) DCIS, HER-2 pulsed DC1 induces more complete responses in patients with ER(neg) DCIS. These data provide a rationale for developing vaccinations to reduce recurrence in patients with ER(neg) DCIS for whom there are currently limited adjuvant options.

Autoantibodies to estrogen receptor α interfere with T lymphocyte homeostasis and are associated with disease activity in systemic lupus erythematosus.

OBJECTIVE: Estrogens influence many physiologic processes and are also implicated in the development or progression of numerous diseases, including autoimmune disorders. Aberrations of lymphocyte homeostasis that lead to the production of multiple pathogenic autoantibodies, including autoantibodies specific to estrogen receptor (ER), have been detected in the peripheral blood of patients with systemic lupus erythematosus (SLE). This study was undertaken to assess the presence of both anti-ERα and anti-ERß antibodies in sera from patients with SLE, to analyze the effect of these antibodies on peripheral blood T lymphocyte homeostasis, and to evaluate their role as determinants of disease pathogenesis and progression. METHODS: Anti-ER antibody serum immunoreactivity was analyzed by enzyme-linked immunosorbent assay in samples from 86 patients with SLE and 95 healthy donors. Phenotypic and functional analyses were performed by flow cytometry and Western blotting. RESULTS: Anti-ERα antibodies were present in 45% of the patients with SLE, whereas anti-ERß antibodies were undetectable. In healthy donors, anti-ERα antibodies induced cell activation and consequent apoptotic cell death in resting lymphocytes as well as proliferation of anti-CD3-stimulated T lymphocytes. A significant association between anti-ERα antibody values and clinical parameters, i.e., the SLE Disease Activity Index and arthritis, was found. CONCLUSION: Our data suggest that anti-ERα autoantibodies interfere with T lymphocyte homeostasis and are significantly associated with lupus disease activity.

Estrogen receptor α signaling in T lymphocytes is required for estradiol-mediated inhibition of Th1 and Th17 cell differentiation and protection against experimental autoimmune encephalomyelitis.

Estrogen treatment exerts a protective effect on experimental autoimmune encephalomyelitis (EAE) and is under clinical trial for multiple sclerosis therapy. Estrogens have been suspected to protect from CNS autoimmunity through their capacity to exert anti-inflammatory as well as neuroprotective effects. Despite the obvious impacts of estrogens on the pathophysiology of multiple sclerosis and EAE, the dominant cellular target that orchestrates the anti-inflammatory effect of 17ß-estradiol (E2) in EAE is still ill defined. Using conditional estrogen receptor (ER) α-deficient mice and bone marrow chimera experiments, we show that expression of ERα is critical in hematopoietic cells but not in endothelial ones to mediate the E2 inhibitory effect on Th1 and Th17 cell priming, resulting in EAE protection. Furthermore, using newly created cell type-specific ERα-deficient mice, we demonstrate that ERα is required in T lymphocytes, but neither in macrophages nor dendritic cells, for E2-mediated inhibition of Th1/Th17 cell differentiation and protection from EAE. Lastly, in absence of ERα in host nonhematopoietic tissues, we further show that ERα signaling in T cells is necessary and sufficient to mediate the inhibitory effect of E2 on EAE development. These data uncover T lymphocytes as a major and nonredundant cellular target responsible for the anti-inflammatory effects of E2 in Th17 cell-driven CNS autoimmunity.

Estrogen receptor alpha modulates Toll-like receptor signaling in murine lupus.

Systemic lupus erythematosus (SLE) is a disease that disproportionately affects females. Despite significant research effort, the mechanisms underlying the female predominance in this disease are largely unknown. Previously, we showed that estrogen receptor alpha knockout (ERαKO) lupus prone female mice had significantly less pathologic renal disease and proteinuria, and significantly prolonged survival. Since autoantibody levels and number and percentage of B/T cells were not significantly impacted by ERα genotype, we hypothesized that the primary benefit of ERα deficiency in lupus nephritis was via modulation of the innate immune response. Using BMDCs and spleen cells/B cells from female wild-type or ERαKO mice, we found that ERαKO-derived cells have a significantly reduced inflammatory response after stimulation with TLR agonists. Our results indicate that the inflammatory response to TLR ligands is significantly impacted by the presence of ERα despite the absence of estradiol, and may partially explain the protective effect of ERα deficiency in lupus-prone animals.

Estrogen receptor signaling promotes dendritic cell differentiation by increasing expression of the transcription factor IRF4.

During inflammation, elevated granulocyte macrophage-colony-stimulating factor (GM-CSF) directs the development of new dendritic cells (DCs). This pathway is influenced by environmental factors, and we previously showed that physiologic levels of estradiol, acting through estrogen receptor alpha (ERalpha), promote the GM-CSF-mediated differentiation of a CD11b(+) DC subset from myeloid progenitors (MPs). We now have identified interferon regulatory factor 4 (IRF4), a transcription factor induced by GM-CSF and critical for CD11b(+) DC development in vivo, as a target of ERalpha signaling during this process. In MPs, ERalpha potentiates and sustains GM-CSF induction of IRF4. Furthermore, retroviral delivery of the Irf4 cDNA to undifferentiated ERalpha(-/-) bone marrow cells restored the development of the estradiol/ERalpha-dependent DC population, indicating that an elevated amount of IRF4 protein substitutes for ERalpha signaling. Thus at an early stage in the MP response to GM-CSF, ERalpha signaling induces an elevated amount of IRF4, which leads to a developmental program underlying CD11b(+) DC differentiation.

Immune cell-mediated neuroprotection is independent of estrogen action through estrogen receptor-alpha.

It has been well documented that both estrogen and immune cells (CD4+ T cells) mediate neuroprotection in the mouse facial nerve axotomy model. Estrogen has been shown to play an important role in regulating the immune response. However, it is unclear whether immune cell-mediated neuroprotection is dependent on estrogen signaling. In this study, using FACS staining, we confirmed that the majority of CD4+ T cells express high levels of estrogen receptor-alpha (ERα), suggesting that CD4+ T cell-mediated neuroprotection may be modulated by estrogen signaling. We previously found that immunodeficient Rag-2KO mice showed a significant increase in axotomy-induced facial motoneuron death compared to immunocompetent wild-type mice. Therefore, we investigated axotomy-induced facial motoneuron loss in immunodeficient Rag-2KO mice that received 17ß-estradiol treatment or adoptive transfer of immune cells from mice lacking functional ERα. Our results indicate that while estradiol treatment failed to rescue facial motoneurons from axotomy-induced cell death in Rag-2KO mice, immune cells lacking ERα successfully restored facial motoneuron survival in Rag-2 KO mice to a wild-type level. Collectively, we concluded that CD4+ T cell-mediated neuroprotection is independent of estrogen action through ERα.