AB0659 Recruitment and prevalence of mixed phenotype macrophages is prominent in Bronchoalveolar Lavage (BAL) of Systemic Sclerosis (SSc) patients with Interstitial Lung Disease (ILD).

Abstract

BackgroundILD is a major problem in SSc for which no disease-modifying therapies are available. Many observations suggest monocyte/macrophage involvement in SSc and might be a key component in the development of ILD. Macrophages polarize into M1 and M2 phenotypes and thereby orchestrate inflammation and subsequent fibrosis by responding to and producing a vast array of cytokines and chemokines. Differences in the levels of the chemokines might explain variations in the recruitment and activation of macrophages in affected organs. In SSc, monocytes with a mixed phenotype (expressing both M1 and M2 markers) are prominent in peripheral blood. No previous study has investigated the mixed phenotype macrophages locally on the lung level.Objectivesa-to measure macrophage-related chemokines in BAL fluid obtained from SSc-ILD and SSc-no-ILD patients.b-to investigate levels of M1 and M2 macrophage markers in BAL cells obtained from SSc-ILD and SSc-no-ILD patients.MethodsA cross-sectional study in which BAL procedure was performed on 15 treatment-naïve SSc patients divided into two groups according to lung involvement determined by HRCT and lung function tests: SSc-no-ILD group and SSc-ILD group (Table 1). Levels of chemokines (CCL18, CXCL-10 and CCR2) were analysed using ELISA. mRNA expression levels of CD86 (M1; inflammatory macrophage marker) and CD206 (M2; fibrogenic macrophage marker) in cells isolated from BAL were assessed using RT-qPCR. In addition, immunofluorescence studies and flow cytometry analyses were performed to evaluate the expression of M1 (CD86) or M2 (CD206) markers in BAL macrophages.Table 1.Patient characteristicsno ILDILDNumber of patients87Age57 (51-68)60 (53-73)Female75%57%Raynauds phenomenon100%85%Age start of Raynauds36 (22-53)56 (41-66)Age start of non-Raynauds50 (33-60)49 (36-68)Skin thickening50%43%Digital ulcer at this moment0%29%Digital ulcer in the past38%29%Pitting scars38%43%Telangiectasia50%43%Calcinosis13%29%Cardiac25%14%Gastro-intestinal88%43%Kidney0%0%ResultsMacrophages were the predominant immune cell population in BAL of SSc patients (73%). The levels of CCL18, CCR2 and CXCL10 were slightly elevated in the SSc-ILD group compared to the SSc-no-ILD (Figure 1.1). RT-qPCR data showed that CD86 expression was elevated in the SSc-ILD group while no difference was observed in CD206 expression between the two groups (data not shown). The SSc-ILD group had higher proportions of double-positive (CD86+, CD206+) macrophages than SSc-no-ILD which, in contrast, had higher proportions of CD86+, CD206- macrophages (Figure 1.2). These results were supported by fluorescent microscopy images in which the SSc-ILD group had higher CD86 and CD206 expression than the SSc-no-ILD (Figure 1.3). Double staining clearly showed a more prevalent mixed activation of macrophages in the SSc-ILD group.ConclusionIn this pilot study, recruitment and activation of mixed phenotype macrophages was more prominent in SSc-ILD than in SSc-no-ILD. Although levels of chemokines were not profoundly different between both groups, studying extensively the macrophage activation patterns in BAL provided novel findings regarding differences between the two groups. Since SSc is characterised by inflammatory and fibrotic phases, the mixed polarization of macrophages we showed for the first time in SSc-ILD patients on an organ level is a key aspect in such disease and could potentially serve as a target for therapeutics.