Characterization of TH2 Asthma phenotypes by Serum Cytokines and Extracellular Vesicles Analysis

Extracellular vesicles (EVs) are membrane-bound nanoparticles secreted by nearly all cell types, plays a pivotal role in intercellular communication through the transfer of proteins, lipids and nucleic acids. In recent years, EVs have emerged as powerful biomarkers capable of reflecting the cellular state, immune activation and pathological remodeling occurring in various disease contexts. Their role in respiratory disorders, particularly in T2 inflammatory airway diseases, has gained attention. The T2 inflammatory diseases including asthma, asthma with nasal polyps, Eosinophilic granulomatosis with polyangiitis (EGPA), and Hypereosinophilic syndrome (HES) share overlapping immune features such as eosinophilic inflammation and IL-5-driven responses, but differ in clinical expression and systemic involvement. Conventional biomarkers like serum IgE, blood eosinophil counts and FeNO offer limited insight into these subtle differences or response to biologic therapy. This prospective cohort study aimed to characterize EVs profile and serum cytokine dynamics in patients with T2 inflammatory diseases following treatment with mepolizumab, an anti-IL-5 monoclonal antibody. In this study, a total of 71 patients were enrolled, from whom 164 serum samples were obtained across different time points (T0, T6, T12) and diagnostic groups for analysis. We comprehensively profiled serum-derived EVs in patients with T2 airway diseases at baseline and during follow-up after mepolizumab therapy. The methodological approach included flow cytometric profiling of EV surface markers to assess their cellular origin and immune activation status, untargeted quantitative proteomics to characterize functional protein cargo and serum cytokine analysis to evaluate systemic inflammatory responses. The assessment of clinical parameters revealed that patients at baseline across all diagnostic groups exhibited elevated eosinophil counts, high FeNO levels and impaired lung function (FEV1%). Following 12 months of mepolizumab treatment, eosinophil levels significantly decreased across all groups, accompanied by reductions in FeNO and oral corticosteroid (OCS) use, improved Asthma Control Test (ACT) levels along with notable improvements in lung function parameters. EVs were isolated from serum samples using ultracentrifugation, then characterized by Transmission Electron microscopy (TEM) and Nanoparticle Tracking Analysis (NTA). The EVs surface antigen analysis revealed dynamic regulation of surface markers such as CD14, CD146, CD24, CD29, CD41b, and CD42a at follow-ups. Proteomic profiling of serum EVs revealed disease-specific and therapy-related molecular signatures. In asthma, upregulation of KRT86 and EPX was observed at T6 post-treatment, while PIGR was notably downregulated in asthma with nasal polyps. EGPA exhibited upregulated cluster at T12 post treatment, and HES was marked by increased expression of KRTAP13-2. Furthermore, the cytokine analysis across diagnostic groups and time points revealed that the IL-5 levels significantly declined across asthma and EGPA at T12. In particular, IL-10 showed a modest increase in EGPA at T6, while IL-13 and IL-6 remained relatively stable. The IL-25 levels were variable but generally reduced in asthma with nasal polyps and EGPA at follow-up. The TGFB1 concentrations, decreased in all groups at T12. These cytokine trends further supported the immunomodulatory effects observed through EV profiling and proteomic shifts. In conclusion, this study highlights the utility of EVs as dynamic, non-invasive biomarkers that integrate immunological and molecular signatures in T2 inflammatory airway diseases. Their combined surface antigen and proteomic features offer valuable insight into disease pathogenesis and treatment response, supporting the application of EV profiling in precision medicine approaches for T2 airway inflammatory disorders.