The Role of Estrogen Receptor Alpha Variant Size and Localization in Modulating TLR7-Induced Inflammation

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Systemic lupus erythematosus (SLE) is one of many autoimmune diseases that disproportionately affects females. Although many risk factors for lupus are identified: >170 genes including GPR174, myriad environmental exposures, and aberrant X chromosome inactivation, none of these sufficiently explain the steep rise in incidence of ADs at the time of puberty in a female-specific manner. Epidemiology suggests a major role for sex hormones and their receptors in autoimmune diseases. We previously showed that female lupus-prone mice, expressing only a short form of estrogen receptor alpha (ERα short), have significantly reduced renal disease and increased survival. Determining the mechanism of this protective effect, which is estrogen dependent, is the primary goal of this proposal. Of note, ERα-/- (null) lupus prone mice were not similarly protected. Combined, our data suggest that the presence of the short form of ERα confers protection, not the absence of full-length ERα. Others and we demonstrated a critical role for ERα in dendritic cell (DC) development and endosomal Toll-like receptor (TLR) responsiveness. Interestingly, the ERα expressed in ERα short mice is similar in structure to an endogenous ERα variant (ERα46) that lacks the same AF-1 domain, and differentially regulates gene transcription compared to full length ERα. Overexpressing ERα46 in vitro also modulates TLR- induced responses, relevant to this proposal. In the proposed study, we will further investigate the role of ERα short variants in modulating TLR7-induced immune responses, and determine whether genomic and/or non- genomic mechanisms of ERα short variant action are protective. Our overall hypothesis is that increasing expression of ERα short or ERα46 in immune cells will be anti-inflammatory, and that the ratio of ERα46 to ERα66 is decreased in lupus patients versus healthy controls. We also hypothesize that targeting immune cells with novel anti-inflammatory selective estrogen receptor modulators (SERMs) that alter ERα membrane signaling and/or ERα-induced transcription will uncouple estrogen-mediated anti-inflammatory responses from those impacting reproductive tissues. We will test our hypotheses by accomplishing these Specific Aims: 1) Overexpress ERα short or treat immune cells with novel SERMs (OBHS, PaPE) that select for anti-inflammatory properties of ERα and determine the effect on known TLR7-induced inflammatory endpoints, 2) Investigate effects of membrane-only ERα signaling vs. nuclear only ERα expression on TLR7-induced pathways in mice, and 3) Identify ERα variants in human monocyte-derived dendritic cells (mo-DCs) and B cells using droplet digital PCR and Iso-Seq technology, to determine whether ERα46 is differentially expressed in lupus patients vs. controls, potentially explaining a biologic difference in females predisposed to autoimmunity. These aims will allow us to determine if we can separate ERα's reproductive effects from its potentially modifiable immune effects as a therapeutic strategy, which if successful, will provide novel approaches to immune modulation in lupus and other immune mediated diseases, especially those with a significant sex bias.