Proteomic profiling of urinary sEVs reveals signature proteins in T2hi versus T2lo asthma

Proteomics techniques are suited to find endotype-associated biomarkers in heterogeneous diseases like asthma. In the present study, we used LC-MS/MS to analyse the proteome of urinary small extracellular vesicles (usEVs) isolated from healthy controls (HC; n=4) and asthma patients with T2^hi (n=4) and T2^lo (n=4) endotypes. Additionally, we examined the phenotype of circulating eosinophils and lymphocytes (CD44, CD48, CD62L, IL-3Rα, IL-5Rα, and CD16) across the same groups (HC, n=6; T2^hi, n=9; T2^lo, n=6) via flow cytometry. Proteomic analysis (SWATH-DDA) revealed lower levels of cellular adhesion, platelet/coagulation (e.g., VCL, FLNA, FERMT3, CEACAM1), and innate immune system (e.g., TTR, SERPINB1) proteins in asthma compared with HC usEVs. Additionally, within asthma population, T2^lo usEVs showed increased levels of proteins involved in endocytosis and cellular response to stress (e.g., CLU, B2M, and SFMRP1), while T2^hi usEVs presented upregulated levels of T-cell and/or eosinophils activation proteins (e.g., CD44, CD7). As CD44 was recently shown crucial for recruiting leukocytes to inflamed tissues in an HDM-model of acute asthma, the upregulation of CD44 was tested in circulating lymphocytes and eosinophils. Thus, we found CD44 downregulated in lymphocytes from T2^lo compared with HC (<italic>p</italic> = 0.0423). In addition, eosinophils from T2^lo patients presented lower levels of this protein compared with T2^hi (<italic>p</italic> = 0.0217). Other eosinophil activation markers followed the same trend (CD48, CD62L, and IL-3Rα) but without statistical significance. Overall, our preliminary findings suggest that usEVs are a promising source of biomarkers for asthma characterization.