Association of fibrotic-related extracellular vesicle microRNAs with lung involvement in systemic sclerosis

Background There is a pressing need to identify early biomarkers of lung involvement in systemic sclerosis to start antifibrotic therapy as soon as possible. We aimed to identify extracellular vesicle-derived microRNAs (miRNAs) that are differentially expressed between systemic sclerosis patients with and without interstitial lung disease, and to explore their diagnostic value and functional properties. Methods Small extracellular vesicles derived from plasma were isolated from 91 well-characterised patients with systemic sclerosis with (n=45) and without (n=46) interstitial lung disease and 43 matched healthy subjects. Small RNA-sequencing followed by quantitative reverse transcriptase PCR were used to identify and validate small extracellular vesicle miRNAs associated with systemic sclerosis-associated interstitial lung disease. Correlations between systemic sclerosis-associated interstitial lung disease miRNAs and clinical parameters were assessed, as well as the effect of related miRNAs/small extracellular vesicles on fibrosis. Results We identified a four-miRNA signature associated with interstitial lung disease in the context of systemic sclerosis (miR-584-5p, miR-744-5p, miR-1307-3p and miR-10b-5p) (area under the receiver operating characteristic curve 0.85, 95% CI 0.76–0.94; p<0.0001). Deeper analysis revealed a correlation of these candidates with pulmonary function tests (diffusing capacity of the lung for carbon monoxide and forced vital capacity), highlighting their use in monitoring lung fibrosis progression in systemic sclerosis patients. Furthermore, small extracellular vesicle miRNAs associated with systemic sclerosis-associated interstitial lung disease are positively correlated with and enriched in circulating lymphocytes, suggesting that these immune cells are their cellular source. Finally, functional studies highlighted altered functional properties of small extracellular vesicles in the context of systemic sclerosis-associated interstitial lung disease, mainly due to the transfer of profibrotic miR-584-5p in lung fibroblasts. Conclusions Our small extracellular vesicle-based biomarker approach identified a promising four‑miRNA signature characteristic of interstitial lung disease in systemic sclerosis patients. Furthermore, the profibrotic properties of small extracellular vesicles associated with systemic sclerosis-associated interstitial lung disease suggest a prominent role of these vesicles in systemic sclerosis severity.