LAUSR

Infection, either direct (pulmonary) or indirect (extrapulmonary), is the most common precipitating factor for the development of acute respiratory distress syndrome (ARDS). Since the coronavirus disease (COVID-19) pandemic, we have started to develop pharmacotherapies specifically for patients with ARDS; however, our knowledge of the underlying pathogenic mechanisms remains incomplete. Determining the pathways that lead from sepsis to ARDS will allow identification of novel therapeutic strategies. Extracellular vesicles (EVs) are membrane-bound anuclear structures that constitute an intercellular communication mechanism, allowing transfer of biologic cargo (including microRNA, mRNA, proteins, andmitochondria) between cell types (1). Cells release EVs during both health and disease states. EVs have been shown to play a pathogenic (and at times protective) role across various inflammatory diseases and cancers, in which EVs act as biomarkers and/or potential therapeutic targets (2). There is an increasing body of evidence to support the role of EVs and their cargo in the pathogenesis of acute lung injury (ALI) and ARDS. EVs are released when human ex vivo perfused lungs are injured with Escherichia coli; these EVs then mediate inflammatory lung injury when isolated and administered to the perfusate of uninjured human lungs (3). Cellular infiltration, elevated inflammatory cytokine release, and reduced alveolar fluid clearance are observed. In this issue of the Journal (pp. 140–149), Tan and colleagues (4) describe their investigation of the role of EVs in mediating indirect ALI; in vivo–generated circulating EVs were extracted from LPStreated mice and administered to the perfusate of ex vivo isolated perfused lungs (IPL). The authors found that circulating LPS-induced total EVs can induce indirect ALI, as characterized by pulmonary edemameasured as an increase in the lung wet/dry ratio and elevated concentrations of a biomarker of alveolar epithelial type 1 cell injury, RAGE (receptor for advanced glycation end products), in the IPL model. This EV-mediated lung injury is dependent on the presence of pulmonary intravascular monocytes, as depletion of monocytes via use of clodronate liposomes in the IPLmodel nullified EV-mediated injury. The major strength of this study is that the authors used an innovative murine in vivo–to–ex vivo adoptive transfer approach to demonstrate the role of EVs in mediating indirect ALI. The authors subsequently found that the myeloid-derived cluster of differentiation molecule (CD11b) subpopulation of EVs predominantly mediates this injurious effect. Indirect lung injury commences with inflammatory injury to the pulmonary endothelium. The authors subsequently undertook in vitro studies showing that human myeloid-derived EVs induce inflammatory injury in a human lung microvascular endothelial cell–peripheral bloodmononuclear cell coculture, with increased cell adhesion molecule and inflammatory cytokine expression. The injurious effect of myeloid-derived EVs was dependent on the presence of peripheral bloodmononuclear cells. Together, these studies provide convincing evidence that in indirect ALI, the injurious effect of myeloid-derived EVs on the pulmonary vasculature is monocyte dependent (Figure 1). Granulocyte-derived EVs have previously been shown to have a proinflammatory effect on monocytes in vitro (5). Previous murine studies have similarly shown that depletion of alveolar macrophages via clodronate liposomes attenuated both ventilatorand LPS-induced lung injury (6–8). The profile of LPS-induced circulating EVs differs between murine and human studies. Murine in vivomodels showed an increase in both neutrophiland monocyte-derived EVs after LPS stimulation. However, healthy volunteer whole blood showed an increase in neutrophil-derived, but not monocyte-derived, EVs after in vitro LPS stimulation, which indicates a limitation of this model. A recent analysis of pulmonary EVs revealed that both monocyteand neutrophil-derived EVs are elevated in patients with sepsis (9). Comparison of patients with sepsis, with and without ARDS, revealed that monocyte-derived EVs were elevated in patients with ARDS; however, the majority of these patients had direct lung injury. Circulating monocyte-derived EVs from patients with sepsis-related ARDS have been found to contain caspase-1 and gasdermin D; after uptake, these EVs induce pulmonary endothelial cell death (10). Thus, the role of monocyte-derived EVs within the myeloid EV population should also be considered. The authors address previous work, which partly contradicts their findings, showing that activated neutrophil-derived EVs have an antiinflammatory effect on macrophages (11, 12). Neutrophil-derived EVs from patients with ARDS and murine models of ALI also have a protective effect on airway epithelial cells; transfer of microRNA-223 (miR-223) cargo downregulates PARP-1 (poly[adenosine diphosphate–ribose] polymerase–1) expression, reducing permeability and inflammatory cytokine release. The authors hypothesize that neutrophil-derived EVs may have a differential impact on monocytes (inflammatory) versus macrophages (antiinflammatory). This differential response may be due to the increased expression of integrin and scavenger receptors on the surface of macrophages that bind EVs. It has previously been shown that during inflammatory conditions, airway epithelial cells take up more EVs and undergo activation, whereas in contrast alveolar macrophages take up fewer EVs and show no change in activation (13); a similar dichotomymay occur with monocytes and macrophages. Studies directly comparing the biological effect of neutrophil-derived EV uptake by monocytes and macrophages are required to confirm this. Further studies to characterize neutrophilderived EV cargo will also help extend our understanding of their effect on target cells and, consequently, the initiation and resolution of inflammation in ARDS. Previous in vitro and in vivomodels of sepsis-associated ARDS have recently reported that EVs containing miR-146a-5p activate TLR-7 (Toll-like Receptor 7) in macrophages, resulting in inflammatory injury to the pulmonary endothelium with increased