Jessica Bryon1, Christopher Wasson1, Rebecca Ross1, Elton Zeqiraj1 and Francesco Del Galdo2, 1University of Leeds, Leeds, United Kingdom, 2Leeds Institute of Rheumatic and Musculoskeletal Medicine, Faculty of Medicine and Health and NIHR Biomedical Research Centre, University of Leeds, Leeds, United Kingdom
Background/Purpose: Elevated Type I IFN response is present in blood and affected tissues in systemic sclerosis (SSc) and correlates with disease activity and response to therapy. It is unknown whether the tissue-specific Type I IFN activation is a consequence of the response observed in blood or rather its source. SSc fibroblasts share many cellular and molecular features with cancer-associated fibroblasts (CAF), and a wealth of evidence supports the role of CAFs in inducing Type I IFN activation in epithelial cancer cells. Here, we aim to determine if keratinocytes in SSc display Type I IFN activation and the potential role of dermal fibroblasts in the process.
Methods: Skin biopsies were obtained from healthy and SSc patients' forearms and processed for immunohistochemistry and subculture of primary fibroblasts and keratinocytes. Human skin keratinocyte cell lines (HaCats) were stimulated with conditioned media from healthy (n=3) and SSc (n=3) fibroblasts. Exosomes were isolated from supernatants of healthy and SSc cultured patient fibroblasts using a combination of ultracentrifugation and polymer-based precipitation (Total Exosome Isolation Kit; Invitrogen). Isolated cellular exosomes were visualised and assessed by immunoblotting, electron microscopy and dynamic light scattering, and these were used to stimulate HaCats. TANK-binding kinase (TBK) was inhibited using a small molecule inhibitor (GSK8612). RT-PCR based IFN array and western blot for pSTAT1 (Tyr701) were used to assess Type I IFN activation in these cells.
Results: Consistent with previous studies we found high pSTAT1 levels in the keratinocyte layer of SSc biopsies. Primary keratinocytes isolated from SSc skin biopsies lost IFN activation after cultured in-vitro. Conditioned media from SSc patient fibroblasts triggered ISG induction: MX1 (2.7 fold, p< 0.05), CXCL10 (2.8 fold, p< 0.01) and CXCL11 (1.9 fold, p< 0.05). Media fractionation by ultracentrifugation indicated that the effect on keratinocytes was driven by pelleted micro-vesicles, suggesting a potential role for cellular exosomes. Fibroblasts from SSc patients shed cellular exosomes of similar quantities and biophysical qualities compared to healthy fibroblasts. SSc exosomes induced a 2.2 fold increase in STAT1 phosphorylation (p< 0.05) and an induction in ISGs, compared to HC exosomes: MX1 (1.45 fold, p< 0.05), CXCL10 (1.84 fold, p< 0.01), CXCL11 (2.11 fold, p< 0.01) and OAS (1.24 fold, p< 0.05). TBK inhibition by GSK8612 suppressed induction by SSc exosomes of pSTAT1 by 83%. Consistent with these findings, IFN array of HaCats stimulated with SSc exosomes showed a significant upregulation of 16 ISGs by at least 1.2 fold compared to healthy control exosomes (p< 0.05).
Conclusion: We show for the first time that scleroderma keratinocytes IFN activation observed in skin biopsies is lost in cells cultured in-vitro. We also show that fibroblast-derived exosomes can re-induce IFN activation though a TBK-dependent mechanism. Exosomes from SSc fibroblasts may carry the "signal zero" of local Type I IFN activation, potentially though nucleic acid induced activation of toll-like receptors, STING or pattern recognition receptors, such as RIG-I.
Disclosures: J. Bryon, None; C. Wasson, None; R. Ross, None; E. Zeqiraj, None; F. Del Galdo, AbbVie/Abbott, AstraZeneca, Boehringer-Ingelheim, Mitsubishi-Tanabe, Capella biosciences, Chemomab LTD, Kymab.
