Immune cells are fundamental regulators of extracellular matrix (ECM) production by fibroblasts and have important roles in determining extent of fibrosis in response to inflammation. Although much is known about fibroblast signaling in fibrosis, the molecular signals between immune cells and fibroblasts that drive its persistence are poorly understood. We therefore analyzed skin and lung samples of patients with diffuse cutaneous systemic sclerosis, an autoimmune disease that causes debilitating fibrosis of the skin and internal organs. Here, we define a critical role of epiregulin-EGFR signaling between dendritic cells and fibroblasts to maintain elevated ECM production and accumulation in fibrotic tissue. We found that epiregulin expression marks an inducible state of DC3 dendritic cells triggered by type I interferon and that DC3-derived epiregulin activates EGFR on fibroblasts, driving a positive feedback loop through NOTCH signaling. In mouse models of skin and lung fibrosis, epiregulin was essential for persistence of fibrosis in both tissues, which could be abrogated by epiregulin genetic deficiency or a neutralizing antibody. Therapeutic administration of epiregulin antibody reversed fibrosis in patient skin and lung explants, identifying it as a previously unexplored biologic drug target. Our findings reveal epiregulin as a crucial immune signal that maintains skin and lung fibrosis in multiple diseases and represents a promising antifibrotic target.

Objective Systemic sclerosis (SSc) is a rheumatic autoimmune disease affecting roughly 20 000 people worldwide and characterized by excessive collagen accumulation in the skin and internal organs. Despite the high morbidity and mortality associated with SSc, there are no approved disease-modifying agents. Our objective in this study was to explore transcriptomic and model-based drug discovery approaches for systemic sclerosis. Methods In this study, we explored the molecular basis for SSc pathogenesis in a well-studied mouse model of scleroderma. We profiled the skin and lung transcriptomes of mice at multiple timepoints, analyzing the differential gene expression that underscores the development and resolution of bleomycin-induced fibrosis. Results We observed shared expression signatures of upregulation and downregulation in fibrotic skin and lung tissue, and observed significant upregulation of key pro-fibrotic genes including GDF15, Saa3, Cxcl10, Spp1, and Timp1. To identify changes in gene expression in responses to anti-fibrotic therapy, we assessed the effect of TGF-β pathway inhibition via oral ALK5 (TGF-β receptor I) inhibitor SB525334 and observed a time-lagged response in the lung relative to skin. We also implemented a machine learning algorithm that showed promise at predicting lung function using transcriptome data from both skin and lung biopsies. Conclusion This study provides the most comprehensive look at the gene expression dynamics of an animal model of systemic sclerosis to date, provides a rich dataset for future comparative fibrotic disease research, and helps refine our understanding of pathways at work during SSc pathogenesis and intervention.

BackgroundScleroderma (SSc) is an autoimmune connective tissue disease involving complex interactions between various cell types leading to organ-based tissue fibrosis. Emergence of the monocytes (Mo)/macrophages (Mφ) lineage(s) as key contributors to inflammation, vascular dysfunction and scarring in scleroderma1,2 have led to increased scrutiny of their phenotype and function.ObjectivesTo determine the circulating Mo subpopulations and phenotypes of Mφ in SSc.MethodsPBMC were collected from healthy (HC) and SSc donors, and analysed by flow cytometry using Mo phenotypic antibodies or purified and cultured in vitro. For flow cytometry immunophenotyping, Mo were gated on CD3-CD19-CD56-HLA-DR+populations, and subsets defined by CD14, CD16, CD163 and CD206 expression. For Mφ cultures, Mo were negatively selected from PBMCs, cultured for 7 days, and treated with IFN-γ(5 ng/ml) or IL-4(20 ng/ml) for 24 hours. Cytokine levels in the conditioned media were evaluated by MSD analyses and normalised to total protein levels.ResultsThe frequency of circulating CD163+ non-classical Mo (CD14loCD16hi) was 2-fold higher in SSc patients than in HC (unpaired t-test, p=0.026). No difference was found in the frequency of CD206+ monocyte subsets between HC and SSc. In vitro, unstimulated SSc Mφ (M0) secreted higher levels of classically-activated pro-inflammatory (M1) and alternatively-activated pro-regenerative (M2) cytokines. Compared to HC cells, SSc Mφ were more readily polarised towards an M1 phenotype or an M2 phenotype, when cultured in the presence of IFN-γ or IL-4, respectively. Th17 markers and MMPs were significantly increased in SSc Mφ (table 2).Abstract FRI0406 – Table 1Demographics.Mo (flow cytometry)Mφ supernatant (cytokine assay) nHC n=9SSc n=10HC n=13SSc n=27 Age (years)56.7±14.350.7±5.760.6±16.752.1±13.0Female : Male7:28:26:726:5SSc subtype-dcSSc(10-dcSSc(27Disease duration-≤5 years(10-≤5 years(,15>5 year(12Abstract FRI0406 – Table 2Cytokines significantly increased in SSc vs control. Unpaired t-tests, *p<0.05, **P<0.01.ConclusionsStudies exploring Mo have revealed distinct populations with selective biological functions. Our observation of an increased number of CD163+ non-classical Mo in SSc suggests that this subpopulation may play a key role in inflammatory-driven fibrosis and act as an important source of pro-fibrotic cytokines. This data is consistent with previous reports of elevated serum levels of CD163 and increased CD163 secretion by SSc PBMCs3. SSc Mφ showed a pronounced and enhanced dual M1 and M2 polarisation basally compared to HC, indicating cells were ‘primed’ to undergo phenotypic polarisation. Our studies support the notion that Mφ cytokine secretion generates a pro-fibrotic milieu in scleroderma tissues, playing a prominent role in dysregulated tissue repair in fibrosis.References[1] Chia JJ, Lu TT. Curr Opin Rheumatol2015Nov;27(6):530–536.[2] Christmann RB, Lafyatis R. Arthritis Res Ther. 2010;12(5):146.[3] Hassan WASE, et al. Eur J Rheumatol2016Sep;3(3):95–100.Disclosure of InterestNone declared