Background/Purpose: Global population data has indicated that post-pubertal females have decreased rates of infectious diseases when compared to male counterparts and preadolescent or postmenopausal females.  In contrast, many autoimmune diseases like Systemic Lupus Erythematosus (SLE) have a significant bias towards post-pubertal women. ZAS3 is an important immuno-regulatory transcription factor involved in both B and T cell maturation and function.  We hypothesized that post-pubertal females have more robust immune responses in part through estrogen’s regulation of ZAS3.  In this study, we examine the influence of estrogen over ZAS3 expression and the functional implication of ZAS3 deficiency on the immune response.       

Methods: Wild type (WT) mice were injected subcutaneously with estrogen (E2) daily for 5 days and lymphoid tissues were harvested.  Transgenic mice with a luciferase reporter under the control of kB binding sites were bred into ZAS3 knockouts and luciferase activity in males and females was measured by IVIS.  Peripheral blood mononuclear cells (PBMCs) isolated from healthy subjects and SLE patients were subjected to experimental analyses of gene expression and assayed for proliferation or cytokine production in the presence of either anti-CD3 or tetanus toxoid with or without estrogen.  Nuclear extracts were isolated for EMSA analysis from E2-treated lymphocyte cell lines.

Results: Estrogen treated mice had elevated ZAS3 expression in the spleen, thymus, bone marrow, and lymph nodes when compared to PBS-injected controls.  NFkB-mediated luciferase reporter activity in female ZAS3 knockout mice was significantly lower than WT controls, whereas males exhibited no significant differences.  Further, ZAS3 knockout mice expressed significantly lower peroxisome-proliferator activated receptor gamma (PPARγ) expression. ZAS3 was also found to be significantly elevated at baseline in SLE patients when compared to age and sex-matched healthy controls.  PBMCs from healthy females incubated in vitro with estrogen or estrogen receptor alpha (ERα) agonist showed significant upregulation of ZAS3.  Similarly, ZAS3 expression was upregulated in response to estrogen both on the protein and RNA level in T Cells, B Cells, monocyte, and NK cell lines.  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. Furthermore, estrogen significantly enhanced the cellular proliferation responses in primary PBMCs cultured with anti-CD3 or Tetanus toxoid.  When these cells were stimulated with E2, we also demonstrated significant production of MIP-1β.

Conclusion: Primary human PBMCs display a heightened ZAS3 response to E2 and this effect is mediated at least in part through NFkB and leads to PPARγ upregulation. Furthermore, The E2/ERα-mediated stimulation of ZAS3 expression occurs through direct, genomic interactions with intragenic enhancing elements.  Taken together, this data suggests that E2 lowers the threshold of activation by priming the immune system of females.  While this may be beneficial in the defense against foreign antigens, it can be detrimental in the development of autoimmunity.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/estrogen-modulation-of-zas3-is-mediated-through-estrogen-receptor-%ce%b1-an-underlying-mechanism-of-gender-bias-in-systemic-lupus-erythematosus/