AB0420 CIRCULATING MONOCYTES HAVE DISTINCT PHYSICAL PROPERTIES THAT CORRELATE WITH DISEASE ACTIVITY AND SEVERITY AND PREDICT PROGRESSION IN SYSTEMIC SCLEROSIS

Background:Systemic sclerosis (SSc) is associated with high morbidity and is one of the autoimmune rheumatic diseases with the highest mortality. However, tools to evaluate disease activity, response to treatment or to predict disease progression are scarce. Dysregulated immune responses are major pathogenic players at the onset and in the progression of SSc. Recent evidence demonstrates that mechanical properties of circulating leukocytes reflect their states and functions, and during activation ensure their adaptation to the changing physical requirements (e.g. softening to extravasate and migrate in the tissues) (1). Real-time fluorescence and deformability cytometry (RT-FDC) is a novel technique that allows the identification of cells from a heterogenous population by marker expression, with their subsequent mechanical phenotyping in a high-throughput manner (2, 3).Objectives:Here we characterized the physical properties of circulating immune cells in SSc patients, aiming to identify disease-related changes in their phenotypes, clinical correlates of these changes and their potential to predict disease progression.Methods:51 patients fulfilling the 2013 ACR/EULAR classification criteria for SSc and 17 age- and sex-matched healthy controls were included in the study. Blood was collected from the donors between 05.2019 and 10.2020. Peripheral blood mononuclear cells (PBMCs) were isolated and stained with antibodies against major circulating lymphoid (CD8+, CD4+ T cells, B cells, NK cells, NKT-like cells) and myeloid subpopulations (classical, intermediate and inflammatory monocytes, conventional dendritic cells and plasmacytoid dendritic cells). Each subpopulation was identified in RT-FDC by standard gating based on its marker expression and its area, deformation and apparent Young’s modulus (a measure of cell stiffness) were determined. The analysis was conducted using a custom Python script. For the patients included, demographic and clinical data were collected at every visit. Correlations with clinical parameters were analyzed in R.Results:All three subpopulations of monocytes identified by expression of HLA-DR, CD14 and/or CD16 had higher deformation and cross-sectional area in SSc patients as compared to healthy controls. From the SSc patients, monocytes had higher deformation and area in those with diffuse cutaneous SSc, extensive lung fibrosis and active disease as compared to those with limited cutaneous SSc, limited lung fibrosis and stable disease, respectively. Moreover, monocyte deformation and area significantly correlated with the EUSTAR activity index, with mRSS, with the extent of lung involvement on HR-CT (positive correlation), with DLCO and FVC (negative correlation). Follow-up data collected one year after the measurements showed that a higher monocyte deformation and cross-sectional area at baseline predicts future progression of lung disease, defined according to the INBUILD study, as well as future progression of skin fibrosis.Conclusion:We demonstrated that circulating subsets of monocytes in SSc patients show an increase in deformation and cross-sectional area, that these changes correlate with current disease activity and can identify patients with high risk of future progression of skin or lung fibrosis. These changes might reflect an activated state of circulating monocytes in SSc that facilitate their tissue migration. Mechanical phenotyping of monocytes by RT-FDC might thus serve as a useful tool for clinical evaluation of SSc patients.References:[1]Bashant KR, Toepfner N, Day CJ, Mehta NN, Kaplan MJ, Summers C, et al. The mechanics of myeloid cells. Biol Cell. 2020;112(4):103-12.[2]Otto O, Rosendahl P, Mietke A, Golfier S, Herold C, Klaue D, et al. Real-time deformability cytometry: on-the-fly cell mechanical phenotyping. Nat Methods. 2015;12(3):199-202, 4 p following.[3]Rosendahl P, Plak K, Jacobi A, Kraeter M, Toepfner N, Otto O, et al. Real-time fluorescence and deformability cytometry. Nat Methods. 2018;15(5):355-8.Disclosure of Interests:Alexandru-Emil Matei: None declared, Kubánková Markéta: None declared, Andrea-Hermina Györfi: None declared, Evgenia Boxberger: None declared, Despina Soteriou: None declared, Maria Papava: None declared, Julia Muth: None declared, Martin Kräter: None declared, Georg Schett: None declared, Jochen Guck: None declared, Jörg H.W. Distler Consultant of: Actelion, Active Biotech, Anamar, ARXX, Bayer Pharma, Boehringer Ingelheim, Celgene, Galapagos, GSK, Inventiva, JB Therapeutics, Medac, Pfizer, RuiYi and UCB, Grant/research support from: Anamar, Active Biotech, Array Biopharma, aTyr, BMS, Bayer Pharma, Boehringer Ingelheim, Celgene, Galapagos, GSK, Inventiva, Novartis, Sanofi-Aventis, RedX, UCB, Employee of: Stock owner of 4D Science and Scientific head of FibroCure