Session Information
Title: Systemic Sclerosis, Fibrosing Syndromes and Raynaud's - Pathogenesis, Animal Models and Genetics
Session Type: Abstract Submissions (ACR)
Background/Purpose
IL6 is a key mediator in activation of extracellular matrix (ECM) in scleroderma (SSc) fibroblasts and via its interplay with chemokines may modulate mononuclear cell recruitment and fibrosis. 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) via Ficoll gradient centrifugation. Dermal fibroblasts were cultured from skin biopsies from healthy controls (n=4) and dcSSc (n=4). Flow cytometry was used to quantify M2 macrophages in PBMCs defined by CD68+CD163+ cells. A magnetic-activated 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 IL6/sIL6R. Flow cytometry was then used to determine the effect of IL6 trans-signalling on macrophage polarisation. Control and dcSSc 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 protein levels induced by CM and the effect of CM on fibroblast contractile response was evaluated with collagen gel contraction assays.
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 years) and disease duration (34.2 ±15.6 months) . Flow cytometry of PBMCs demonstrated significantly higher proportion of circulating M2 macrophages (mean ± SEM %) in dcSSc patients compared to healthy controls (9.3±0.3% vs 1.4±1.2%, p=0.0087) respectively. There is no significant difference in the total number of CD68+macrophages between the two groups. Stimulation of isolated macrophages with IL6/sIL6R polarised the M2 macrophage population by (4-fold, p<0.02) and (1.6-fold, p<0.04) in control and SSc macrophages respectively. Cultured fibroblasts treated with CM generated from SSc M2 macrophages led to increased synthesis of 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 to control CM.
Conclusion
Our data indicate that M2 macrophage phenotype in early stage scleroderma may partly be polarised by IL-6 trans-signalling. This supports the critical role of distinct macrophage subpopulation in regulation of key fibrotic markers, myofibroblastic differentiation and contractile response in SSc. Elucidating these pathways may lead to better understanding of macrophage biology in disease pathogenesis and the potential for targeted specific subpopulation as emerging therapeutics in SSc.
Disclosure:
R. Alade,
None,
9;
S. Xu,
None;
K. Khan,
None;
A. Tam,
None;
C. P. Denton,
Actelion Pharmaceuticals US,
5;
V. H. Ong,
Actelion Pharmaceuticals US,
8.
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/il-6-trans-signalling-activates-m2-macrophage-polarisation-and-mediates-fibrotic-response-in-scleroderma/