Characterization of the cargo of circulating extracellular vesicles in patients affected with idiopathic inflammatory myopathies and evaluation of clinical correlates in a cross-sectional comparative analysis from a monocentric cohort.

Background Idiopathic inflammatory myopathies (IIM) are heterogeneous autoimmune disorders that comprise different clinical entities, characterized by different features. Although several myositis-specific and myositis-associated antibodies have been characterized, the molecular mechanisms underlying these conditions require further exploration. The research field on extracellular vescicles (EVs) is rapidly evolving, highlighting their role in intercellular communication. EVs convoy a cargo of proteins and nucleic acids, such as microRNA (miRNAs), that mediate immune-response regulation in autoimmune diseases. miRNAs regulate gene-expression post-transcriptionally and are involved in multiple molecular pathways of human disease. Evidence of EVs and miRNAs in IIM is still limited and undefined. Aim of the study This study aims to quantify the circulating EVs and characterize their cargo, with a specific focus on miRNAs content to propose novel biomarkers of IIM. Materials and methods A monocentric study was conducted including adult IIM patients (≥18 years old) followed at the Rheumatology Unit of Padua University Hospital, and age- and sex- matched healthy controls (HD). EVs were isolated from platelet-free plasma through size exclusion chromatography followed by ultrafiltration. EVs were quantified by nanoparticle tracking analysis (NTA) and. EV-miRNA cargo was investigated through Next-Generation Sequencing (NGS). Statistical analysis was performed with parametric Student-T test and one-way Anova (Bonferroni correction). Results Sixty-four consecutive IIM patients and sixty-five HDs were included in the study. NTA measurements of EVs concentration showed a significantly higher mean concentration of circulating EVs in IIM patients than in HD (p=0.0073). Across IIM subsets, patients affected with cancer associated myositis (CAM) displayed the highest levels of circulating EVs compared to no CAM patients (p=0.0060) and to HD (p=0.0004). Patients with circulating myositis-associated autoantibodies displayed significantly higher EV levels than HD (p=0.0363). Patients in clinical remission displayed higher levels of circulating EVs compared to those with active disease (p=0.0087). EVs levels were significantly reduced in IIM patients treated with rituximab (RTX) than in patients receiving other treatments (p<0.0001). NGS analysis detected EV-miRNAs with different expression profiles between IIM (n=47) and HDs (n=49): miR-223-3p (p=0.019), miR-15a-5p (p=0.0189), miR-451a (p=0.0074), miR-486-5p (p=0.0052), miR-32-5p (p=0.0146), and miR-222-3p (p=0.0282) were up-regulated in IIM, while miR-141-3p (p=0.0313), miR-142-3p (p=0.0244), and let-7a-5p (p=0.0003) were down-regulated in IIM patients vs. HDs. Other EV-miRNAs expression varied across IIM subsets: CAM patients displayed up-regulated expression of miR-143-3p compared to non-CAM patients (p=0.0085), while miR-148a-3p (p=0.0171) and miR-335-5p (p=0.0171) were up-regulated in dermatomyositis vs. polymyositis/ inclusion body myositis/anti-synthetase syndrome patients. Patients characterized by active disease displayed an up-regulated expression of miR-222-3p (p=0.002) and miR-151-3p (p=0.0233) and down-regulated expression of miR-363-3p (p=0.0001), miR-374a-5p (p=0.0258), miR-144-3p (p=0.0170), miR-181a-5p (p=0.0037) compared to those in clinical remission. Moreover, IIM patients receiving only glucocorticoids (GC) reported up-regulated expression of miR-4433b-5p (p=0.0439), miR-92a-3p (p=0.0111), let-7f-5p (p=0.0304), and down-regulated expression of miR-27a-3p (p=0.0486) compared to patients receiving GC in combination with immunosuppressants (IS). Conclusions Our results showed significantly increased concentration of circulating EVs in IIM patients. That is confirmed within specific disease phenotypes and pharmacological treatments. EV miRNAs exhibited a differential expression profile between IIM and HD, and significant differences were outlined among IIM subsets.