Pulmonary extracellular vesicles drive alveolar macrophage dysfunction in Acute Respiratory Distress Syndrome

Acute Respiratory Distress Syndrome (ARDS) is a hyper-inflammatory disorder of the lungs which commonly develops following sepsis. We previously found alveolar macrophages (AMs) from ARDS patients have impaired efferocytosis compared to control ventilated sepsis patients (PMID 34112730). This defect is replicated when healthy AMs are treated with ARDS patient broncho-alveolar lavage fluid (BAL) (PMID 34660643). Impaired AM efferocytosis contributes to alveolar inflammation in ARDS via secondary necrosis of persistent apoptotic neutrophils. Models of lung injury implicate extracellular vesicles (EVs) in ARDS pathogenesis. We hypothesised that EVs in ARDS patient BAL drive AM dysfunction via metabolic reprogramming. EVs were isolated from ARDS patient BAL via ultracentrifugation, prior to characterisation by Exoview, TEM & nanoparticle tracking analysis. Human AMs were isolated from lung tissue resections of non-smoking patients. AMs were treated with BAL EVs 24 hours prior to flow cytometric assessment of efferocytosis and phagocytosis, and Seahorse XF analysis. ARDS BAL EV treatment impaired AM efferocytosis of apoptotic neutrophils (fold change 0.541, p=0.0029, n=9) and phagocytosis of S. Pneumoniae, to a greater degree than EV-depleted ARDS BAL. AMs treated with ARDS BAL EVs showed an increase in total ATP generated, with rises in oxygen consumption and proton efflux rates compared to untreated AMs. Transcriptional analysis reveals enrichment of microRNAs which modulate mitochondrial activity in ARDS BAL EVs compared to post-operative controls. Targeting EV cargo may attenuate AM dysfunction and inflammation, offering a therapeutic strategy for ARDS patients.