Small extracellular vesicles in bronchial secretions from mechanically ventilated patients: when to collect and where from?

Small extracellular vesicles (sEVs) released by cells during lung injury, act as cell-to-cell messengers involved in inflammation. Blood circulating sEVs are candidates as diagnostic biomarkers and therapeutic target in mechanically ventilated(MV) patients. However, their cell source heterogeneity is a significant challenge. This study aimed to develop a new protocol to isolate sEVs from bronchial secretions obtained by bronchoscopy or tracheal tube blind aspiration in MV patients. We have investigated the relevance of variables as post-intubation sampling time(day-0,day-7) and sampled lung region(upper, lower). Samples from diagnostic bronchoscopies of non-intubated patients were used as controls. 2ml of bronchial liquid, collected from patients(approval 55366/23-12-22) were exported to the Imperial College Tissue Bank (HTA-12275 R23038)for sEV analyses. sEVs were isolated by ultrafiltration and size exclusion chromatography, using Exospin-EXO350 columns. sEV concentration and size distribution was evaluated by nanoparticle tracking analysis (NTA) using Nanosight-NS3000. sEV size distribution was similar in all samples (p=ns). Regardless of sample region sEVs increased at day-0 (3285*10⁶±299particles/ml, p<0.05)in MV patients, compared to controls (1344*10⁶±245particles/ml). More specifically, sEVs increased in the aspiration samples (4392*10⁶±337particles/ml, p<0.01), or samples from upper lung (3942*10⁶±539particles /ml, p<0.05) of MV patients compared to controls (1344*10⁶±245particles/ml). In summary sEVs increase in the MV bronchial secretions. Further studies should investigate the sEV diagnostic and prognostic potential at admission in Intensive Care Units.