Systemic sclerosis (SSc) is a chronic autoimmune disease characterized by inflammation, vascular injury, and fibrosis. One in three patients with SSc dies within 10 years of diagnosis, making it one of the most deadly autoimmune diseases. It affects women 5-10 times more frequently than men, yet relatively little is known about the role that sex hormones, particularly estrogen, play in disease development and/or progression. Recent work implicates macrophages (MØs) in the pathogenesis of SSc, as we have shown that MØs induce SSc fibroblast activation, resulting in upregulation of pathways that regulate extracellular matrix synthesis and inflammation. While the sex bias in SSc is clear and estrogen is a known modulator of MØ immune function, a potential role for estrogen in the regulation of pro-fibrotic MØ activation in SSc has not been addressed. This is a fundamental gap in our knowledge of disease pathogenesis that has significant clinical implications for the treatment of SSc and potentially other sex-biased autoimmune diseases. Based on preliminary findings, we hypothesize that estrogen induces pro-fibrotic MØ activation, enhancing inflammation and exacerbating fibrosis. Results of this work may be used to inform the utilization of estrogen pathway-targeting therapeutics in SSc patients, for whom current treatment options are significantly limited. The aims that will be tested in this application are: 1. Determine how estrogen regulates SSc MØ activation and subset heterogeneity. SSc MØs express estrogen receptors (ER) and estrogen mediates effects on MØ immune function, although the effect of estrogen on SSc MØ activation is unknown. Studies in this aim will address how 17β estradiol and estrone alter SSc MØ pathway activation and subset heterogeneity using CITE-seq. In addition, we will specifically interrogate the contribution of estrogen (and estrogen inhibition) to STAT3 activation, which have shown is basally induced in SSc MØs. 2. Determine how estrogen-mediated changes in MØ activation promote inflammation and fibrosis in SSc. Co- culture studies demonstrate that SSc MØs induce activation of SSc fibroblasts, implicating a role for MØs in the induction and maintenance of fibrosis and inflammation. This aim will determine how estrogen-directed changes in SSc MØ activation impact fibroblast activation using a novel 3D fabricated skin model of SSc.