Skin fibrosis in systemic sclerosis (SSc) leads to significant morbidity resulting from disfiguring, painful and itchy skin, and joint contractures. We have recently shown by single cell RNA-sequencing (scRNA-seq) that SSc dermal fibroblasts (expressing increased THBS1, PRSS23) and dermal myofibroblasts (also expressing increased SFRP4, ADAM12, TNFSF18 and CTGF) arise from SFRP2-expressing progenitors found in healthy control skin. These studies provide a framework for understanding the profibrotic drivers of these cell states. Transcription factors (TFs) are pivotal in regulating gene expression and provide a powerful landmark for these cell states. Using SCENIC, a computational method developed for detecting TF-associated regulatory networks (regulons) in single cell datasets, we identified putative TFs driving myofibroblast differentiation: FOXP1, HIF1A, IRF7, STAT1 and FOSL2. Additionally, in preliminary results we employed Assay for Transposase Accessible Chromatin by Sequencing (scATAC-seq) data supporting the importance of these TFs in SSc myofibroblast differentiation. In our first aim, we will assess the importance of these TFs in further multiome studies, and confirm their roles in myofibroblast differentiation by measuring the effects of TF knock-down on fibroblast transcriptome and epigenome. Markers of macrophage activation correlate strongly with the main clinical measure of skin disease severity, the modified Rodnan skin score (MRSS), suggesting that macrophages deliver profibrotic signals to drive myofibroblast differentiation. Recent studies in SSc-ILD have confirmed IL-6 in pathogenesis of lung disease and we see its downstream target CCL18 also upregulated in skin macrophages. In our second aim, we will use a novel system biology methodology, CausER, to analyze latent factors regulating the macrophage-fibroblast interaction and generate snRNA-seq data before and after tocilizumab to better understand the role of IL-6 in activating profibrotic macrophages in SSc skin. We expect that this will inform the similar process occurring in SSc-interstitial lung disease. Localized scleroderma (LS) continues to cause disfiguring and functional disabilities in children as well as adults. Our preliminary results implicate IFNg as activating macrophages and fibroblasts in LS skin. In aim 3, using similar approaches to study of SSc, we will compare the immune and non-immune cell populations in LS to SSc skin. First, we will combine our existing LS (n=14), SSc (n=27) and healthy control (n=14) scRNA-seq datasets, and examine differences in fibroblast and myeloid cell transcriptome-phenotypes and differentially expressed genes. Then as in aim 2, we will employ CausER to identify latent factors regulating the interaction between these cells. We will then identify TFs regulating myeloid and fibroblast phenotypes using SCENIC and multiome. We expect these studies of LS to provide new insights into the cytokines and intracellular pathways activatin...