Proteomic and functional characterization of serum extracellular vesicles In progressive scleroderma interstitial lung disease

Background Systemic sclerosis (SSc) is the disease associated with the highest mortality among rheumatic conditions, mainly as a direct or indirect consequence of interstitial lung disease (ILD). Reliable biomarkers to timely predict the progression of lung fibrosis and guide the treatment are lacking. Extracellular vesicles (EVs) are newly discovered mediators of intercellular communication providinga cell-specific exchange of functionally active molecules. Aim of the study The main objective of the study was to compare circulating EV proteome profiles of SSc patients with progressive ILD, non-progressive ILD, and without ILD. The exploratory objective was to assess the capacity of SSc serum EV lysates to induce epithelial-to-mesenchymal transition (EMT) of alveolar epithelial cells (AECs) or activation of lung fibroblasts (HLFs) in vitro. Methods Serum EVs were enriched from selected SSc patients classified into three clinical groups according to ILD status and 24-month follow-up (patients with progressive ILD, non-progressive ILD, and without ILD). The EV enrichment process result was evaluated by Western blot (WB) characterization of EV markers and transmission electronic microscopy (TEM). The proteomic profile was assessed though proximity extension assay (PEA) on EVs lysate, combined with new generation sequencing (NGS) of about 3000 proteins. A Gene Set Enrichment Analysis (GSEA) was performed to determine whether a priori-defined sets of functionally related proteins were differentially represented between the clinical groups. The biological activity of EV lysates was explored though exposure of human A549 AEC and HLF cell cultures for 48 hours in standard conditions. EMT and HLF activity marker expression was assessed by real-time polymerase chain reaction (RT-PCR) and WB of the cell culture extracted samples. Results Of the 52 patients included in the final analysis (age of 57.0 ± 12.5 years, males 21.2%), 53.8% presented the diffuse cutaneous variant (dcSSc) with a median disease duration of 6.0 years; 80.8% and 65.4% of the patients were respectively exposed to vasoactive and immunosuppressive treatments during the observation period. Of 38 (73.1%) patients presenting ILD, 13 were progressors and 25 non-progressors, while the remaining 14 did not present ILD. A total of 1161 proteins were detected in all the EV-lysate samples and 1456 in at least one sample. Among the cell-specific EV-related markers evaluated on PEA, the most abundant suggested a prevalent platelet, endothelial, or immune-cell origin for circulating EVs. Two-hundred-twenty-one single proteins differed between SSc patients with and without ILD, and 189 between progressive and non-progressive SSc-ILD patients. The GSEA revealed for both comparisons some differences in the abundance of proteins involved in EMT-related processes (such as epithelial carcinogenesis, organogenesis, and expression of surface cellular markers), extracellular matrix (ECM) metabolism, angiogenesis, and vesicle trafficking. Differences in adaptive immune response-related proteins were specifically reported in the comparison between SSc patients with and without ILD, while EV lysates from progressive and non-progressive SSc-ILD patients differed in the abundance of proteins involved in the metabolism of reactive oxygen species and xenobiotics. The exploratory functional assessment finally revealed an EMT induction after the exposure of A549 AEC cell cultures with EV-lysate from SSc-ILD patients but not from SSc patients without ILD. Significant reactions were not reported after exposure of HLFs to EV-lysates. Conclusions Serum EV-lysates derived from SSc patients differ in proteomic profiles according to the presence of ILD and the occurrence of ILD progression over time. The differently abundant proteins are related to key pathways involved in SSc pathogenesis, such as EMT, ECM deposition and remodelling, angiogenesis, immune response, and the reaction to oxidative stress and xenobiotics. The observation of AEC EMT after EV lysate exposure supports the hypothesis of a direct pathogenetic role for circulating EVs in SSc-related lung fibrosis.