Ab0150 isolation and Phenotypic Characterization of Circulating Extracellular Vesicles in Patients With Idiopathic Inflammatory Myopathies

Abstract

BackgroundIdiopathic Inflammatory myopathies (IIM) encompass a group of heterogeneous systemic autoimmune diseases for which biomarkers are needed. Extracellular vesicles (EVs) are cell-derived nanoparticles regulating several biological functions[1]and likely involved in the pathogenesis of autoimmune diseases[2], yet their role in IIM was not investigated thoroughly.Objectives1) to develop an effective and reproducible laboratory method to isolate EVs from venous blood; 2) to trace any prominent EVs cellular origin; 3) to perform a quantitative and comparative evaluation of EVs between IIM patients and healthy donors (HD) and across distinct IIM subsets.MethodsWe enrolled adult (≥18 years) consecutive IIM patients (EULAR 2017 criteria) and sex- and age-matched HD. Clinical evaluation of IIM patients was performed by an expert rheumatologist. A venous blood sample (5 mL) was collected from each participant and processed through size exclusion chromatography (SEC) and subsequent ultra-filtration (UF). Particles morphology was observed via transmission electron microscopy (TEM). Nanoparticle tracking analysis (NTA) was performed to evaluate the concentration of EV. The characterization of EV surface markers was conducted through imaging flow cytometry (IFC). Comparisons were carried out using Student’s t-test or one-way ANOVA with multiple comparisons.ResultsSixty-five consecutive IIM patients (dermatomyositis (DM) n=19; polymyositis (PM) n=8; inclusion body myositis (IBM) n=2; anti-synthetase syndrome (ASyS) n=17; cancer-associated myositis (CAM) n=16; unspecified n=3) and 65 HDs were included.Isolated EVs concentration values reached 1010EV/mL. IFC showed surface expression of EVs tetraspanins CD63, CD81, CD9, and integrin CD11c and highlighted a predominance of CD19+ EVs vs. CD3+ EVs in both IIM and HDs (p<0.0001 for both). TEM images showed intact small particles with the typical EVs-“cup-shape” morphology. NTA showed a significantly higher mean concentration of circulating EVs in IIM vs. HD (mean [EVs/mL] ± SD 1.71x1010± 1.29x1010vs. 1.31x1010± 7.17x109, p=0.031).Across IIM subsets, the highest EVs levels were found in CAM vs. HD (2.35x1010± 2.20x1010vs. 1.31x1010± 7.17x109, p=0.0026) and vs. no-CAM patients (2.35x1010± 2.20x1010vs. 1.51x1010± 7.51x109, p=0.0206). Patients in clinical remission (n=26, 40%) displayed higher EVs levels than active patients (2.13x1010± 1.60x1010vs. 1.46x1010± 1.02x1010, p=0.0452) (Figure 1).Concerning treatment, patients on glucocorticoids (GC) alone (n=20, 30.7%) displayed higher EVs levels than patients receiving GC plus immunosuppressants (IS) (2.23x1010± 1.99x1010vs. 1.49x1010± 7.25x109, p=0.0341). EVs concentration was significantly decreased in patients receiving rituximab (RTX) vs. other IS (9.63x109± 3.26x109vs. 1.96x1010± 1.46x1010, p<0.001).ConclusionThe combination of SEC and UF is a reliable approach to obtain intact EVs with preserved morphology and good purity from blood, as confirmed by diverse characterization techniques. The prevalence of CD19+ circulating EVs might indicate B cells as their main origin. Different EVs concentrations discriminate IIM from HD and may provide distinction among specific IIM subsets and treatment response.References[1]Buzas, E.I. The roles of extracellular vesicles in the immune system.Nat Rev Immunol(2022).https://doi.org/10.1038/s41577-022-00763-8[2]Wu WC, ert al. Role of Extracellular Vesicles in Autoimmune Pathogenesis. Front Immunol. 2020;11:579043. doi: 10.3389/fimmu.2020.579043Figure 1.Representation of EVs shape (TEM), surface markers (IFC) and quantitative analysis (NTA) across IIM susbetsAcknowledgements:NIL.Disclosure of InterestsNone Declared.