AB0221 Estrogen modulation of endosome-associated toll-like receptors and ZAS3: An underlying mechanism of gender-BIAS in systemic lupus erythematosus?

Background Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease characterized by significant gender bias. Previous studies have demonstrated the contributions of hormones, particularly estrogen (E2), to SLE pathogenesis. E2 has been shown to regulate the expression of multiple genes through ligand activation of estrogen receptors (ER) α and β. While many ER-target genes have been previously described, many others remain to be characterized. In this work, we examined ZAS3 and toll-like receptors (TLRs) as potential targets of E2-mediated regulation. ZAS3 is a member of the ZAS family of zinc-finger transcription factors and is thought to be involved in controlling cellular growth and inflammation (1). Furthermore, recent work with large patient cohorts has shown that TLRs appear to have a strong association as SLE susceptibility markers. TLRs have also been of interest as potential autoimmune response triggers (2). Objectives Since sex hormones have been shown to play a significant role in SLE pathogenesis, we examined the effects of estrogen on gene expression in peripheral blood mononuclear cells (PBMCs) from premenopausal healthy individuals and SLE patients. Methods RNA was isolated from the peripheral blood of SLE patients and healthy donors. In addition, PBMCs were isolated and treated with a physiological dose of testosterone, E2, and/or TLR agonists. Western blotting and real-time RT-PCR of protein and mRNA expression were then analyzed, respectively. Nuclear extracts were isolated for EMSA analysis from E2-treated lymphocyte cell lines. Results In whole blood, SLE patients expressed higher levels of ZAS3 and all endosome-associated TLRs: TLR3, TLR7, TLR8, and TLR9. Also, E2-induced expression of endosomal TLRs and ZAS3 was confirmed in healthy PBMCs in vitro , while no effects were observed with testosterone treatment. EMSA analysis with intronic estrogen receptor response element probes within the ZAS3 region revealed that ERα binds directly and that E2 stimulation enhanced complex formation. Using TLR agonists to treat these cells, we identified an autocrine loop with positive feedback over TLR expression. This mechanism was observed in all endosomal TLRs. Furthermore, we discovered that TLR8 agonist stimulation of PBMCs accentuates TLR8 expression in the presence of estrogen. Not only was an enhanced response with E2 and TLR8 agonist observed, but female PBMCs were more sensitive to this effect. Conclusions Thus, the E2/ERα-mediated stimulation of ZAS3 expression occurs through direct, genomic interactions with intragenic enhancing elements. Furthermore, primary human PBMCs display heightened expression of endosome-associated TLRs in response to E2. Taken together, we propose that estrogen-induced upregulation of ZAS3 and endosomal TLRs, along with the TLR autocrine loop, lowers the inflammatory threshold. These novel mechanisms could explain, in part, the gender bias in SLE. 1. Hong JW, Allen CE, Wu LC. Inhibition of NF-kappaB by ZAS3, a zinc-finger protein that also binds to the kappaB motif. Proc Natl Acad Sci U S A 2003;100(21):12301-6. 2. Li M, Zhou Y, Feng G, Su SB. The critical role of Toll-like receptor signaling pathways in the induction and progression of autoimmune diseases. Curr Mol Med 2009;9(3):365-74. Disclosure of Interest None Declared