The density of each band was quantified with ImageJ software (National Institutes of Health). ChIP assay The ChIP assay was performed using an EpiQuik ChIP kit (Epigentek). the systemic autoimmunity and selective organ fibrosis in SSc. This study uncovers unidentified functions of dysregulated epithelial cells in SSc pathogenesis. Introduction Systemic sclerosis (SSc), or scleroderma, is usually a chronic connective tissue disease characterized by three cardinal features: autoimmunity/inflammation, vasculopathy, and BMPR1B fibrosis in the skin and numerous internal organs (Asano, 2010; Asano and Sato, 2015). Although SSc pathogenesis RO-9187 remains elusive, genetic studies have demonstrated that most of the susceptibility genes for SSc are HLA haplotypes and non-HLA genes related to immunity and inflammation, suggesting the central role of immune abnormalities in SSc development (Agarwal and Reveille, 2010). Indeed, during the early and sclerotic phases, the infiltration of activated T cells and macrophages and the degranulation of mast cells are observed in the affected skin, which correlate with the severity of skin thickening (Fleischmajer et al., 1977). With regard to CD4+ T cells, the T helper type 1 cell (Th1 cell)/Th2 cell and Th17 cell/regulatory T cell (T reg cell) balances shift to Th2 and Th17 lineage dominance, respectively (OReilly et al., 2012). In particular, the increased expression of several Th2 cytokines, such as IL-6 and IL-13, contributes to fibroblast activation (Khan et al., 2012). In addition, despite the rarity of B cell infiltration in the skin, a cluster of B cellCrelated genes is usually strongly expressed in lesional and nonlesional skin of SSc patients (Whitfield et al., 2003). SSc B cells are constitutively activated, as represented by the increased expression of CD19, a critical positive response regulator (Sato et al., 2004), and produce numerous autoantibodies, including disease-specific antinuclear antibodies (ANAs) and other pathogenic antibodies against disease-related molecules (Sato et al., 2000; Hamaguchi, 2010). In interstitial lung disease (ILD) associated with SSc (SSc-ILD), activated B cells characteristically form aggregates in the lungs (Lafyatis et al., 2007). Supporting the critical role of B cells in SSc-ILD, rituximab, an anti-CD20 antibody, has confirmed efficacious in controlling ILD in a subset of patients (Lafyatis et al., 2009; Jordan et al., 2015). The initial immune activation and autoimmunity lead to the structural and functional abnormalities of vasculature and the constitutive activation of fibroblasts of SSc in various organs (Asano and Sato, 2015). However, the initial triggers of the dysregulated immune homeostasis and the origin of autoimmunity in this disease remain obscure (Harris and Rosen, 2003; Boin and Rosen, 2007; Joseph et al., 2014). Furthermore, although tissue fibrosis most commonly affects the skin, esophagus, and lungs in RO-9187 SSc (Gabrielli et al., 2009), a convincing explanation for this unique target organ specificity is currently lacking. Hence, these unresolved important questions in this disease remain to be resolved. Reflecting the main disease manifestations, the majority of previous studies on SSc have centered on immune cells, vascular endothelial cells, and fibroblasts. However, more recent studies have exhibited anomalous phenotypes of the skin epithelium, or keratinocytes, in SSc (Leask, 2009; Aden et al., 2010; Nikitorowicz-Buniak et al., 2014, 2015; Suwara et al., 2014; Assassi et al., 2015). For example, SSc keratinocytes persistently express wound-associated keratins keratin 6 (K6) and K16 not only in the sclerotic skin, but also in the nonlesional skin (Aden et al., 2010), suggesting that the altered epithelial phenotype manifests early in this disease. Besides, SSc keratinocytes stimulate fibroblasts in cell culture with excessively secreted IL-1 (Aden et al., 2010), which is a major alarmin released from your epithelial cells triggering an inflammatory response in fibroblasts (Suwara et al., 2014). Increased expression of the key profibrotic growth factor connective tissue growth factor (CTGF) is also obvious in SSc epidermis (Leask, 2009; Nikitorowicz-Buniak et al., 2014). Additionally, epithelialCmesenchymal transition (EMT), a central mechanism in fibrosis development driven by TGF-1 (Nieto et al., 2016), is usually enhanced in SSc epidermis with the increased expression of its cardinal regulator SNAI1 (Nakamura and Tokura, 2011; Wei et al., 2011; Nikitorowicz-Buniak et al., 2015). Of particular relevance is usually a recent study on global gene profiling of SSc lesional skin describing a correlation between specific keratin expression signatures and the presence of ILD (Assassi et al., 2015). Therefore, it seems that the epithelial phenotype is not merely related directly to dermal fibrosis but, more profoundly, associated with SSc development itself. Numerous studies from our laboratory and others have demonstrated the crucial role of Friend leukemia computer virus integration 1 (Fli1), a member of the Ets RO-9187 transcription factor family, in SSc pathogenesis. Fli1 is usually constitutively suppressed in dermal fibroblasts, dermal microvascular endothelial cells, and perivascular inflammatory cells not only in the lesional skin, but.