Wang, Supeng

Abstract Rationale: We previously reported (PMID: 38325750) that a nebulized exosome product isolated from human platelets (PEP) attenuated murine cigarette smoke (CS)-induced emphysema. To further elucidate the mechanism by which PEP attenuates CS-induced effects in the murine lung, emphysema was induced, and mice were treated with PEP or placebo (saline). Immune and cellular mechanisms affected by PEP were then evaluated in this system. Methods: C57BL6 female black mice were exposed to CS 5 days a week for 3 months and then randomized into PEP-treated or placebo groups. CS-exposed mice were treated with 3 mL of 7.5 mg/mL PEP (∼1 x 1010 extracellular vesicles per mL) or 0.9% saline by nebulization 3 times a week for an additional 4 weeks, using a nose-sealed, pressure and humidity-controlled inhalation tower for 30 minutes. Lung compliance, cutaneous O2 saturation, mean linear intercepts, and lung inflammation were measured during and after the study. Lung tissue was further analyzed by immunostaining, and gene expression analyses to elucidate the molecular mechanisms of PEP's action. Inflammatory and immune regulatory cell markers evaluated included CD4 (CD4+ T lymphocytes), CD4/Foxp3 (T regulatory – Treg) cells, S100A9 (interstitial lung macrophages), and myeloperoxidase (MPO) for neutrophils. Results: Histologic review of lung tissue sections by mean linear intercept counts confirmed prior findings and showed a significant reduction in the free distance of chronic CS exposure-induced alveolar space enlargement in PEP-treated mice (P=0.003). In addition, lung inflammation scores showed a significant overall reduction of the lymphocytic infiltrates in PEP-nebulized lungs compared to the CS-exposed placebo control group (P<0.0001). Further analysis of the immune cell infiltrates revealed increased FOXP3+CD4+ T regulatory cells within the lymphocytic infiltrates (P<0.0001) and decreased S100A9+ interstitial lung macrophages (p=0.0002) and MPO+ neutrophils (p=0.005) in PEP nebulized lungs compared to the placebo controls. PEP-treated mice had similar oxygen saturation levels when compared to the CS control group (94.7±1.47 vs. 95.4±.1.6 respectively). Conclusions: In addition to validating prior findings on the effect of nebulized PEP in mitigating CS-induced murine lung injury and emphysema, the current data suggests an immunoregulatory role for PEP by decreasing inflammatory macrophage and neutrophil infiltration while augmenting anti-inflammatory Treg cell numbers in the lung.