Parallel Session 6 - Fibrosis in SSc: Cause(S) and Consequences

Abstract

CO.11

Myeloid Specific Deletion of C-REL Protects Against Fibrosis in the Bleomycin Mouse Model of Systemic Sclerosis

J. Worrell, J. Leslie, L. Borthwick, D. Mann, F. Oakley

Newcastle Fibrosis Research Group, Institute of Cellular Medicine, Newcastle University, Newcastle Upon Tyne, UNITED KINGDOM

Introduction: Systemic sclerosis (SSc) is a heterogeneous and debilitating disease with complex interplay between vascular and inflammatory factors. The c-Rel subunit of the transcription factor NF kappaB has been implicated in potentiating fibrogenesis and is known to regulate inflammation and cell survival. Previous studies have shown c-Rel-/- mice are protected from heart, liver and skin fibrosis. The activation of circulating immune cells and recruitment of inflammatory macrophages are known drivers of scleroderma pathogenesis. Focusing on myeloid cells allows us to evaluate the effect of c- Rel deletion in the pathogenesis of systemic sclerosis and further elucidate the role of c-Rel in innate immunity.

Material and Methods: We generated myeloid cell-specific conditional c-Rel knockout mice by crossing c-Rel flox/flox mice with LysM Cre mice. Cell specific deletion of c-Rel was validated using flow cytometry and western blotting. Lung and skin fibrosis were induced independently in vivo using intra-tracheal or subcutaneous bleomycin respectively. Fibrosis was quantified histologically by Masson’s Trichrome and Sirius Red staining. Fibrogenic and inflammatory genes were quantified using qPCR and release of soluble factors measured by ELISA. Bone marrow derived macrophages were generated from c- Rel-/- mice and phenotypically characterized.

Results: Myeloid specific cRel-/- mice have significantly reduced collagen deposition, scar cell activation and reduced neutrophil, T-lymphocyte and macrophage recruitment in the lung. c-Rel expression was absent in resident macrophages, inflammatory macrophages and neutrophils. Significantly decreased dermal thickening and collagen deposition was observed in myeloid specific c-Rel-/- mice in response to bleomycin challenge compared to littermate controls. cRel-/- BMMs fail to activate correctly and have impaired responses to either M1 (LPS + IFNgamma) or M2 (IL-4 + IL-13) polarization signals in vitro.

Conclusions: Our data highlights intrinsic defects in innate immune cell function in c-Rel -/-mice, which may contribute to the fibro-protective phenotype. We conclude that c-Rel is a critical regulator of inflammation and fibrogenesis and represents an attractive therapeutic target in the treatment of systemic sclerosis.

CO.12

IL-31 Promotes Pathogenic Mechanisms in Scleroderma and Induces Skin Fibrosis in Mice

Z. Taki¹, S. Zafar¹, B. Abdi Ahmed¹, A. Tam¹, D. Abraham¹, C. Denton¹, S. Black¹, H. Rosario¹, H. Lopez², R. Stratton¹

¹Centre for Rheumatology and Conective Tissue Diseases, Royal Free Hospital, UCL, London, UNITED KINGDOM, ²Murigenics, Valejo, CA, USA

Introduction: In systemic sclerosis (SSc) T cell derived cytokines provide a mechanistic link between autoimmunity and the fibrotic pathology seen. IL31, a recently described Th2 cytokine which signals via the dimerized oncostatin MR/IL-31RA receptor, is implicated in other pruritic dermopathies. We sought to determine whether IL-31 is elevated within the skin lesions or systemically in SSc, and test whether the IL-31RA is expressed by cells relevant to the disease process. Responses of target cells to IL-31 were evaluated. Possible in vivo pro-fibrotic effects of IL-31 were investigated in a mouse model.

Material and Methods: IL-31 was measured by ELISA of plasma (SSc n=43, healthy control (HC) n=27) and lesional skin blister fluid (BF) (SSc n=44, HC n=22). Itch severity was determined by the 5D pruritus scale. IL31RA mRNA levels were assayed by qPCR of SSc and HC skin and lung fibroblasts, epidermal tissue extracts, as well as fat derived MSCs. Responses of target cells to IL-31 were measured by next generation sequencing (NGS), migration assays, qPCR and Western blotting. In wild type 6 - 8 week old Male Balb/c mice (Charles River Laboratories, CA), IL-31 (Preprotech # 210-31-10) was administered by subcutaneous Alza osmotic minipump 200 ng/day, with or without additional TGFbeta (Prosec # cyt-8585b) 800ng/day for 14 days. Mice were euthanized on day 21 and skin biopsy material taken adjacent to the treated site. Lung tissue and plasma were also sampled. Plasma was assayed by ELISA for IL-6, TGFbeta and PDGF-BB.

Results: Plasma IL-31 was elevated in SSc (HC 0-792, median 55, IQR 0-375, SSc 0-17764, median 193, IQR 13-658 pg/ml, p<0.048), (HC 0-2777, median 0, IQR 0-23, SSc 0-38222, median 0, IQR 0-175 pg/ml, pNS). IL-31RA was expressed by target cells relevant to the fibrotic process, skin and lung fibroblasts, epidermal cells and mesenchymal stem cells (MSCs). Treatment of skin fibroblasts with IL-31 induced gene expression profiles linked to integrin signaling, growth, actin polymerisation, motility, plus Wnt and TGFbeta signaling, and induced phenotypic changes including migration and collagen protein release, resembling the activation state in the disease. In mice, IL-31 induced skin fibrosis more than TGFbeta, and led to increased cytokine and growth factor levels in a scleroderma-like pattern (increased dermis by PSR, P<0.03, raised plasma IL-6 p<0.03).

Conclusions: IL-31 is increased in SSc, induces phenotypic and gene expression changes in target cells, and leads to skin fibrosis in mice, consistent with a pathogenic role in scleroderma, distinct from and additive to TGFbeta.