Two Forms of the Estrogen Receptor Play Opposing Roles in Lupus Inflammation

Systemic lupus erythematosus has long baffled researchers with its striking bias: roughly nine in ten patients are women, and the gap is widest during the reproductive years, when circulating estrogen is at its highest. For decades, scientists have suspected that the female sex hormone estrogen helps drive this autoimmune disease, in which the immune system mistakenly attacks the kidneys, brain, joints, and other tissues. A new study published in Biology of Sex Differences adds a crucial layer of nuance to that hypothesis. Rather than estrogen acting through a single receptor with a single effect, the research shows that two naturally occurring variants of the estrogen receptor alpha, the full-length ERα66 and a shorter splice variant called ERα46, can push immune-relevant cells in dramatically different directions depending on the cell type involved and, in some cases, the sex of the donor from whom the cells were obtained.

The study, led by Zhilan Li of Xiangya Hospital at Central South University in collaboration with Jena R. Wirth, C. Alex. Colvert, Rachael J. Werner, and Melissa A. Cunningham of the Medical University of South Carolina, set out to address a persistent gap in lupus research. Most work on estrogen signaling in autoimmunity has focused on lymphocytes, the adaptive immune cells that produce antibodies. Far less is known about how estrogen receptor variants behave in innate immune cells, the first responders of the immune system, or in structural cells such as those lining the kidney’s filtering units, which also participate in inflammatory signaling during lupus nephritis. Understanding these cells matters because innate immune activation, driven in part by immune complexes containing nucleic acids sensed through Toll-like receptor 7, is central to the pathology of systemic lupus erythematosus.

To dissect the contributions of the two receptor variants, the team used a transfection strategy in which cultured cells were given plasmids encoding either ERα66, ERα46, both variants in equal proportion, or an empty control vector. The full-length ERα66 contains all of the functional domains of the receptor, including the two activation function regions that recruit co-regulators to hormone-responsive genes. ERα46, by contrast, lacks the amino-terminal AF-1 domain entirely, which means it cannot engage the same set of transcriptional co-activators and may instead act as a modulator or even a dominant inhibitor of ERα66-driven transcription. Because both proteins are encoded by the same gene and differ only through alternative splicing, any divergent effects between them can be attributed to the presence or absence of specific protein domains rather than to different genes altogether.

The first cellular model was HMC3, a human microglial cell line derived from a male donor. Microglia are the resident immune cells of the central nervous system, and they are believed to play a critical role in neuropsychiatric or central nervous system lupus, one of the most devastating manifestations of the disease. When the researchers overexpressed ERα46 in these cells, they observed a clear promotion of cell proliferation, measured by EdU incorporation assays that detect cells in the act of DNA synthesis, alongside CCK-8 assays that track metabolic viability. Notably, both receptor variants ultimately decreased the levels of inflammatory cytokines, but the mechanism was sobering rather than protective: the apparent anti-inflammatory effect stemmed from reduced cell viability, meaning fewer surviving cells were available to churn out inflammatory mediators. This distinction between a genuine immunomodulatory effect and one that simply reflects cell loss is a technical point with major interpretive consequences for the field.

The picture shifted substantially when the team moved to a second myeloid model, the murine macrophage line RAW 264.7, also male-derived. Here, overexpressing ERα46 promoted cell viability but did not spur proliferation, a subtle but important dissociation between survival and division. More strikingly, when the macrophages were stimulated with a Toll-like receptor 7 agonist, both ERα66 and ERα46 promoted the expression of inflammatory cytokines. TLR7 is a sensor of single-stranded RNA, and its overactivity is tightly linked to lupus pathogenesis, since self-RNA contained in immune complexes can chronically activate this pathway in susceptible individuals. The finding that both receptor variants amplified TLR7-driven inflammation in macrophages directly contradicts the anti-inflammatory picture seen in microglia and underscores that estrogen receptor biology cannot be generalized across the innate immune compartment.

Stimulation experiments extended beyond TLR7. The researchers challenged the transfected cells with interferon-alpha, interferon-gamma, and lipopolysaccharide, three inflammatory triggers that represent distinct arms of innate immune activation: type I interferon signaling, which is a hallmark of lupus; type II interferon signaling; and bacterial endotoxin sensing through TLR4. Cytokine output was quantified at both the protein level, by enzyme-linked immunosorbent assay, and the messenger RNA level, by quantitative reverse transcription polymerase chain reaction, providing complementary confirmation of the transcriptional and secretory responses. The convergent message across these assays was that the two receptor variants are not interchangeable, and that their influence on inflammatory gene expression is highly dependent on which stimulus is applied and which cell is listening.

The most provocative results emerged from human renal mesangial cells, primary cells obtained from both male and female donors. Mesangial cells occupy a unique position in lupus nephritis. Although they are structural components of the glomerulus, the kidney’s blood-filtering unit, they display innate immune-like activities during injury, secreting cytokines and chemokines that recruit and amplify inflammatory attack on the kidney tissue. In mesangial cells from male donors, both ERα variants inhibited proliferation but had only a minimal effect on the inflammatory phenotype. In mesangial cells from female donors, however, ERα66 and ERα46 modulated the expression of multiple inflammatory cytokines, hinting at genuinely sex-specific functions of the receptor variants in the kidney. The authors are careful to note that these observations require validation with additional donors, since primary human cells carry inherent inter-individual variability, but the pattern is consistent with the broader hypothesis that estrogen signaling contributes to the female predominance of lupus.

Taken together, the findings dismantle the simple narrative in which estrogen is uniformly bad, or uniformly good, for people with lupus. Instead, the two major splice variants of estrogen receptor alpha act as context-dependent regulators whose effects on proliferation, survival, and cytokine production flip sign depending on the cellular setting. In microglia, the shorter variant encourages division; in macrophages, both variants fan the flames of TLR7-driven inflammation; in male mesangial cells, both variants restrain growth while leaving inflammation largely untouched; and in female mesangial cells, both variants reshape the cytokine landscape. For clinicians and drug developers, this heterogeneity is both a warning and an opportunity. Therapies that broadly block estrogen receptor signaling could produce very different outcomes in the brain, the blood, and the kidney, while strategies that selectively target ERα46, or exploit its missing AF-1 domain, might one day allow tissue-specific tuning of the immune response.

The study was supported by the National Institutes of Health through the National Institute of Arthritis and Musculoskeletal and Skin Diseases, with additional support from the China Scholarship Council, and the authors acknowledge Professor Geoffrey Greene of the University of Chicago for providing the human ERα plasmids. While the work remains at the in vitro stage, and the authors themselves caution that the sex-associated differences in mesangial cells await confirmation across larger donor panels, the research provides a mechanistic framework for a question that has shadowed lupus medicine for generations: why a disease of dysregulated immunity strikes women so disproportionately. The answer, it now appears, is written not just in the hormone itself but in the alternative splice forms of its receptor, and in the distinct ways that microglia, macrophages, and mesangial cells interpret the same molecular message.

Subject of Research: Differential effects of estrogen receptor alpha splice variants ERα66 and ERα46 on proliferation and inflammatory responses in in vitro models of systemic lupus erythematosus inflammation.

Article Title: Estrogen receptor alpha variants differentially influence proliferation and inflammatory responses in in vitro models of lupus inflammation

Article References: Li, Z., Wirth, J. R., Colvert, C. A., Werner, R. J., & Cunningham, M. A. (2026). Estrogen receptor alpha variants differentially influence proliferation and inflammatory responses in in vitro models of lupus inflammation. Biology of Sex Differences. https://doi.org/10.1186/s13293-026-00990-x

Image Credits: AI Generated

DOI: 10.1186/s13293-026-00990-x

Keywords: estrogen receptor alpha, ERα46, lupus, systemic lupus erythematosus, autoimmune disease, microglia, macrophages, mesangial cells, TLR7, inflammatory cytokines, sex differences, splice variants