STUDY OF EXTRACELLULAR VESICLES IN PATIENTS WITH IDIOPATHIC INFLAMMATORY MYOPATHIES: DEVELOPMENT OF A NOVEL ISOLATION METHOD, CHARACTERIZATION, miRNA PROFILING AND EVALUATION OF CLINICAL CORRELATES. A CROSS-SECTIONAL COMPARATIVE ANALYSIS FROM A MONOCENTRIC COHORT

Idiopathic inflammatory myopathies (IIM) are a heterogeneous group of autoimmune disorders whose pathogenesis has not yet been completely elucidated and for which biomarkers for early diagnosis have not yet been identified. Extracellular vesicles (EV) are cell-derived nanoparticles involved in intercellular signaling convoying their cargo of proteins, lipids, nucleic acids that act in autoimmune diseases. They also carry microRNAs (miRNAs) whose dysregulation has been associated with the development and progression of several autoimmune diseases. The study aims to investigate the potential role of circulating EV as IIM biomarkers by validating an EV isolation method from blood, characterizing them, evaluating their cellular origin, exploring their miRNA cargo, and correlating clinical characteristics of IIM with EV features. EV were isolated from platelet-free plasma of adult IIM patients and healthy donors (HD) through size-exclusion chromatography (SEC) and ultrafiltration (UF). EV were observed by transmission electron microscopy (TEM), immunocharacterized by imaging-flow cytometry (IFC), quantified by nanoparticles tracking analysis (NTA) and EV-miRNAs cargo investigated by Next-Generation Sequencing (NGS). 65 IIM patients and 65 HD were included in the study. TEM images showed intact small particles with the typical cup-shape morphology of EV. IFC showed EV to be positive for constitutive surface tetraspanins CD63, CD81, CD9, and integrin CD11c, with a prevalence of CD63+ EV (p<0.0001) in both 30 IIM and 30 HD (mean EVs/mL ± SD 2.91x10^8±2.07x10^8 vs. 2.40x10^8±1.85x10^8, respectively). Moreover, IFC highlighted a prevalence of CD3-CD19+ EV compared to CD3+ EVs in both 26 IIM and 25 HD groups (p<0.0001). NTA measurements reported a higher mean concentration of circulating EV in IIM than HD (1.71x10^10±1.29x10^10 vs. 1.31x10^10±7.17x10^9, p=0.0306). The highest EV levels were found in CAM vs. HD (2.35x10^10±2.20x10^10, p=0.0026) and no CAM (1.51x10^10±7.51x10^9, p=0.0206). Patients in clinical remission displayed higher EV levels than active patients (2.13x10^10±1.60x10^10 vs. 1.46x10^10±1.02x10^10, p=0.0452). Patients on glucocorticoids (GC) alone displayed higher EV levels than those receiving GC and immunosuppressants (2.23x10^10±1.99x10^10 vs. 1.49x10^10±7.25x10^9, p=0.0482). EV concentration was decreased in patients receiving rituximab vs. other therapies (9.63x10^9±3.26x10^9 vs. 1.96x10^10±1.46x10^10, p=0.0228). NGS detected 10 EV-miRNAs differently expressed between 21 IIM and 21 HD: hsa- miR-451a (p=0.0010), miR-15a-5p (p=0.0086), miR-486-5p (p=0.0012), miR-222-3p (p=0.0098), miR-32-5p (p=0.0038), miR-185-5p (p=0.0217) were up-regulated; let-7b-5p (p=0.0046), let-7a-5p (p=0.0032), let-7e-5p (p=0.014), let-7f-5p (p=0.0123) down-regulated in IIM vs. HD. Other EV-miRNAs expression varied across IIM subsets: CAM displayed down-regulation of miR-23b-3p (p=0.0303), miR-361-5p (p=0.0468), miR-143-3p (p=0.0312); up-regulation of miR-374a-5p (p=0.0068) and miR-26b-5p (p=0.0468). PM + ASyS showed up-regulation of miR-30c-5p (p=0.0333) and miR-186-5p (p=0.0241). DM displayed up-regulation of miR-125b-5p (p=0.0215), miR-29c-3p (p=0.0275), miR-361-5p (p=0.0394). MiR-122-5p was down-regulated in IIM with a concurrent diagnosis of interstitial-lung disease (p=0.0482). MiR-155-5p resulted up-regulated (p=0.0259) and miR-347a-5p down-regulated (p=0.0420) in patients with active disease than clinical remission. We propose SEC with UF as a reliable approach to isolate intact EV with preserved morphology and good purity from blood, as confirmed by their characterization. The prevalence of B lymphocyte markers on circulating EV surface might indicate their cell origin. Differences in EV concentrations and EV-miRNAs expression tell apart IIM from HD and may provide an early distinction among IIM subtypes, submitting EV as potential biomarkers of disease, differential diagnosis, and treatment response.

Le miopatie infiammatorie idiopatiche (MII) sono un gruppo eterogeneo di malattie autoimmuni la cui patogenesi non è stata ancora completamente chiarita e per cui non sono stati identificati biomarcatori di diagnosi precoce. Le vescicole extracellulari (EV) sono nanoparticelle di origine cellulare coinvolte nella comunicazione intercellulare che trasportano proteine, lipidi, acidi nucleici con un ruolo nelle malattie autoimmuni. Le EV trasportano microRNA (miRNA) la cui deregolazione è associata a malattie autoimmuni. Lo studio ha l’obiettivo di indagare il potenziale ruolo delle EV circolanti come biomarcatori di MII validando una metodologia per isolarle da sangue, caratterizzandole, valutando l’origine cellulare e EV-miRNA e correlando le caratteristiche cliniche con le EV. Le EV erano isolate da plasma privo di piastrine di pazienti MII adulti e donatori sani (DS) mediante cromatografia ad esclusione dimensionale (SEC) ed ultrafiltrazione (UF). Le EV erano osservate in microscopia elettronica a trasmissione (MET), immunocaratterizzate in citofluorimetria per immagini (IFC), quantificate con nanoparticles tracking analysis (NTA) e il cargo EV-miRNA studiato con Next-Generation Sequencing (NGS). Sono stati reclutati 65 pazienti MII e 65 DS. Le immagini MET mostravano piccole particelle intatte con tipica morfologia. L’IFC riportava EV positive per le tetraspanine costitutive di superficie CD63, CD81, CD9 e integrina CD11c, con prevalenza di EV CD63+ (p<0,0001) nei 30 MII e 30 DS (media EV/mL ± SD 2,91x10^8±2,07x10^8 vs 2,40x10^8±1,85x10^8). IFC evidenziava una prevalenza di EV CD3-CD19+ vs CD3+ nei 26 MII e 25 DS (p<0,0001). Le misurazioni NTA riportavano una concentrazione media maggiore di EV circolanti in MII vs DS (1,71x10^10±1,29x10^10 vs 1,31x10^10±7,17x10^9, p=0,0306). I livelli più elevati erano trovati nei CAM vs DS (2,35x10^10±2,20x10^10, p=0,0026) e vs no CAM (1,51x10^10±7,51 x10^9, p=0,0206). I pazienti in remissione clinica mostravano livelli di EV più elevati vs pazienti attivi (2,13x10^10±1,60x10^10 vs 1,46x10^10±1,02x10^10, p=0,0452). I pazienti trattati solo con glucocorticoidi (GC) mostravano livelli più alti vs pazienti trattati con GC e immunosoppressori (2,23x10^10±1,99x10^10 vs 1,49x10^10±7,25x10^9, p=0,0482). La concentrazione di EV risultava ridotta nei pazienti trattati con rituximab vs altre terapie (9,63x10^9±3,26x10^9 vs 1,96x10^10±1,46x10^10, p=0,0228). NGS ha rilevato 10 EV-miRNA differenzialmente espressi tra 21 MII e 21 DS: hsa- miR-451a (p=0,0010), miR-15a-5p (p=0,0086), miR-486-5p (p=0,0012), miR-222-3p (p=0,0098), miR-32-5p (p=0,0038), miR-185-5p (p=0,0217) erano sovra-espressi; let-7b-5p (p=0,0046), let-7a-5p (p=0,0032), let-7e-5p (p=0,014), let-7f-5p (p=0,0123) sotto-espressi in MII vs. DS. L'espressione di altri EV-miRNA variava tra sottogruppi: CAM mostrava una sotto-regolazione di miR-23b-3p (p=0,0303), miR-361-5p (p=0,0468), miR-143-3p (p=0,0312) e sovra-espressione di miR-374a-5p (p=0,0068), miR-26b-5p (p=0,0468). PM + ASyS mostrava una sovra-espressione di miR-30c-5p (p=0,0333) e miR-186-5p (p=0,0241). DM riportava una sovra-regolazione di hsa-miR-125b-5p (p=0,0215), hsa-miR-29c-3p (p=0,0275), hsa-miR-361-5p (p=0,0394). MiR-122-5p era sotto-espresso in MII con malattia polmonare interstiziale. MiR-155-5p è risultato sovra-espresso (p=0,0259) e hsa-miR-347a-5p sotto-espresso (p=0,0420) in pazienti con malattia attiva vs. remissione clinica. Proponiamo SEC con UF come approccio affidabile per isolare EV intatte con morfologia preservata da sangue, confermato dalle caratterizzazioni. La prevalenza dei marcatori di linfociti B sulle EV circolanti potrebbe indicare l’origine cellulare. Differenze in concentrazioni di EV e espressione di EV-miRNA distinguono i pazienti MII dai DS e potrebbero distinguere precocemente sottotipi di MII, presentando le EV come potenziali biomarcatori di malattia, diagnosi differenziale e risposta al trattamento.