CHARACTERIZATION OF MONOCYTE/MACROPHAGE CELL POPULATIONS IN PERIPHERAL BLOOD AND IN THE LUNG TISSUE OF SYSTEMIC SCLEROSIS PATIENTS

BACKGROUND Peripheral monocytes (PMo) and lung tissue macrophages are actively involved in systemic sclerosis interstitial lung disease (SSc-ILD) [1]. SSc PMo significantly express M2 markers and the hybrid subpopulation of M1/M2 PMo correlates with severity of SSc-ILD [2]. AIMS To characterize macrophage surface markers in lung samples (LS) of SSc-ILD patients by immunohistochemistry, in comparison with normal lung controls (NLC). To compare the distribution of surface markers in LS from SSc-ILD patients with their expression on PMo, from SSc patients (with and without ILD). MATERIALS AND METHODS SSc-ILD LS were collected from SSc patients that underwent lung transplantation at the University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania (USA). NLC were obtained from subjects that underwent lung biopsy for diagnostic purposes (i.e. recurrent pneumothorax) at the IRCCS Ospedale Policlinico San Martino, Genova, Italy. PMo were obtained from blood of SSc patients with and without associated ILD from the same institution in Genova, Italy. Masson’s trichrome staining and single immunostaining with primary antibodies (CD68/pan macrophages, CD80/M1, CD86/M1, TLR4/M1, CD163/M2, CD204/M2, CD206/M2) were performed. Cells positive for M1, M2 or M1/M2 surface markers were quantified by Leica Image Scope 12.3 Software. Flow cytometry analysis included markers for monocyte lineage (CD45, CD14, CD16), M1 phenotype (CD80, TLR4), M2 phenotype (CD163, CD204, CD206) and dendritic lineage (CD1c, Slan). RESULTS LS were obtained from 9 SSc-ILD patients (50±9 years old) and 5 non-SSc patients (58±23 years old). CD68+ cells (lung tissue macrophages) were significantly more frequent in SSc-ILD than NLC (p<0.001), as well as M1 and M2 markers analyzed (CD80=p<0.05, CD86=p<0.01, TLR4-CD163-CD204-CD206=p<0.0001). CD80 and CD86 were significantly less expressed than TLR4 as well CD163, CD204, CD206 (p<0.001) in SSc-ILD LS. A further cohort of 39 SSc patients (62±16 years old, SSc-ILD positive n=24, SSc-ILD negative n=15) was recruited for flow cytometry analysis and once again the percentage of TLR4+M2 PMo (SLAN-CD1c-CD80-TLR4+CD163+CD204+CD206+) was found significantly higher in SSc-ILD positive patients than SSc-ILD negative (p<0.05). CONCLUSIONS Hybrid (TLR4+M2) monocytes/macrophages seem significantly prevalent in SSc-ILD patients (both in lung tissue and peripheral blood). Multiple immunohistochemical LS tissue analyses from SSc-ILD patients are ongoing to implement present results. REFERENCES 1. Soldano S, et al. Ann Rheum Dis. 2018. 2. Trombetta AC, et al. Respir Res. 2018.