306. IL6 Trans-Signalling Mediates M2 Macrophage Polarization and Fibrotic Response in SSc

Abstract

Background: IL6 is implicated in activation of extracellular matrix (ECM) in scleroderma (SSc) fibroblasts and via its interplay with chemokines may modulate mononuclear cell recruitment. We have explored the role of IL6 in macrophage differentiation in SSc and its regulation of fibrotic response Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from patients with early stage diffuse cutaneous SSc (dcSSc) (n=12) and healthy controls (n=6). Dermal fibroblasts were cultured from skin biopsies from healthy controls (n=4) and dcSSc (n=4). Flow cytometry was used to quantify alternatively activated M2 macrophages in PBMCs defined by CD68+CD163+ cells. A magneticactivated cell sorting (MACS) based protocol was used to select CD14+ cells. CD14+ Cells were stimulated for 7 days with M-CSF before further stimulation with M-CSF alone or with IL6/sIL6R. Flow cytometry was then used to determine the effect of IL6 trans-signalling on macrophage polarization. Control fibroblasts were stimulated with conditioned media (CM) from cultured M2 macrophages. Western blot analysis for Collagen type-1 (Col-1) and alpha smooth muscle actin (αSMA) were used to assess ECM levels induced by CM and the effect of CM on fibroblast contractile response was evaluated with a collagen gel contraction assay. Results: PMBCs were isolated from healthy controls (n=6) mean age (37±13.8 months), and dcSSc patients (n=12), mean age (42.2±15.6) and disease duration (34.2±15.6). Flow cytometry of PBMCs quantified (mean±SEM %) subpopulation of M2 macrophages (9.3±0.3%) and (1.4±1.2%) P=0.0087 in SSc patients compared with controls respectively, with SSc M2 macrophages significantly higher compared with controls . Stimulation of isolated macrophages with IL6/sIL6R polarized the M2 macrophage population by (4-fold, P<0.02) and (1.6-fold, P<0.04) in control and SSc macrophages respectively. CM generated from SSc M2 macrophages led to increased ECM proteins αSMA (3-fold, P<0.04) and Col-I (3.2- fold, P<0.04) compared with control macrophages in the presence of control fibroblasts. CM from SSc M2 macrophages induced contraction of control fibroblasts leading to (4mm±0.3, P<0.05) reduction in collagen gel diameter compared with control CM. Conclusion: Our results indicate alternatively activated macrophage phenotype in early stage scleroderma may partly be polarized by IL-6 trans-signalling. This supports the critical role of distinct macrophage subpopulation in regulation of myofibroblastic differentiation and contractile response in SSc. Elucidating these pathways may lead to better understanding of pathogenesis and new options for therapy.