Systemic sclerosis (SSc) is a complex disease involving multiple cell types, leading to a plethora of clinical manifestations. SSc-associated interstitial lung disease (SSc-ILD) is currently the most lethal complication of the disease. We have recently reported results of single cell RNA-sequencing (scRNA-seq) of lung explants from patients with SSc-ILD, as well as from patients with idiopathic pulmonary fibrosis (IPF), showing alterations in gene expression in fibroblast, macrophage (Mφ) and other populations associated with disease. Although the cellular and molecular changes seen in IPF are largely paralleled in SSc-ILD, SSc-ILD is not associated with any of the genetic markers of senescence seen in IPF. Instead, patients with SSc harbor SNPs in genes involved in immune regulation, and develop autoantibodies against a select series of autoantigens, implicating immune dysregulation. Thus, we hypothesize that immune and inflammatory cells drive the pathological fibrosis seen in SSc-ILD and that inflammatory interleukins and cytokines activate differentiation of myofibroblasts. Several cytokines have been implicated in SSc-ILD pathogenesis. Our and other studies have implicated transforming growth factor-beta (TGF-b) and platelet derived growth factor (PDGF) in SSc-fibrosis. Interleukin-6 inhibition is an approved for SSc therapy that appears to block Mφ activation in the skin. Inhibition of IL-13 has recently shown promise as a therapeutic target and expansion of profibrotic SPP1 Mφs suggest that IL-13 and CSF-1 might block fibrosis. Unfortunately, currently available murine and in vitro models for SSc fall short of fully and accurately modeling the disease, and thus do not permit convincing assessments for the roles of these and other soluble mediators in disease pathogenesis. We propose to test the role of these cytokines in healthy control (HC) and SSc-ILD explant cultures. As single cell studies of SSc-ILD explant lungs have led to many of our current insights, we propose that single cell analyses of explant culture of SSc-ILD lungs will provide unprecedented insight into the cellular interactions and soluble mediators that lead to lung fibrosis. In aim 1 we will refine explant culture methodologies and examine the effect of the cytokines described above on HC lung cells in explant cultures. We will compare the effect of these cytokines to the changes in gene expression by fibroblast and Mφ populations seen in our previous scRNA-seq studies of SSc-ILD. In aim 2 we will establish the effect of soluble mediators on SSc-ILD explant cells, in this case testing the effects of therapeutic interventions on gene expression of SSc-ILD lung cell populations. In this setting we anticipate that blocking cytokines or their receptors will normalize gene expression back toward scRNA-seq gene expression seen in HC lungs. We anticipate that these studies will not only shed light on the role of these cytokines in disease pathogenesis but also provide a tracta...