FRI0406 Increased frequency of circulating cd163+ non-classical monocytes in scleroderma and enhanced dual polarisation towards m1 and m2-like phenotypes in monocyte-derived macrophages
Authors: A. Tam, L. Reinke-Breen, G. Trujillo, S. Xu, C. P. Denton, D. J. Abraham, G. Jarai, V. H. Ong
Journal: Annals of the Rheumatic Diseases (2018)

<h3>Background</h3> Scleroderma (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 scleroderma<sup>1,2</sup> have led to increased scrutiny of their phenotype and function. <h3>Objectives</h3> To determine the circulating Mo subpopulations and phenotypes of Mφ in SSc. <h3>Methods</h3> PBMC were collected from healthy (HC) and SSc donors, and analysed by flow cytometry using Mo phenotypic antibodies or purified and cultured <i>in vitro</i>. For flow cytometry immunophenotyping, Mo were gated on CD3<sup>-</sup>CD19<sup>-</sup>CD56<sup>-</sup>HLA-DR<sup>+</sup>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. <h3>Results</h3> The frequency of circulating CD163<sup>+</sup> non-classical Mo (CD14<sup>lo</sup>CD16<sup>hi</sup>) 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<sup>+</sup> monocyte subsets between HC and SSc. <i>In vitro</i>, 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). <h3>Conclusions</h3> Studies exploring Mo have revealed distinct populations with selective biological functions. Our observation of an increased number of CD163<sup>+</sup> 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 PBMCs<sup>3</sup>. 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. <h3>References</h3> [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. <h3>Disclosure of Interest</h3> None declared