ABSTRACT
Transfusion-related acute lung injury (TRALI) is a hazardous transfusion complication with an associated mortality of 5% to 15%. We previously showed that stored (5 days) but not fresh platelets (1 day) cause TRALI via ceramide-mediated endothelial barrier dysfunction. As biological ceramides are hydrophobic, extracellular vesicles (EVs) may be required to shuttle these sphingolipids from platelets to endothelial cells. Adding to complexity, EV formation in turn requires ceramide. We hypothesized that ceramide-dependent EV formation from stored platelets and EV-dependent sphingolipid shuttling induces TRALI. EVs formed during storage of murine platelets were enumerated, characterized for sphingolipids, and applied in a murine TRALI model in vivo and for endothelial barrier assessment in vitro. Five-day EVs were more abundant, had higher long-chain ceramide (C16:0, C18:0, C20:0), and lower sphingosine-1-phosphate (S1P) content than 1-day EVs. Transfusion of 5-day, but not 1-day, EVs induced characteristic signs of lung injury in vivo and endothelial barrier disruption in vitro. Inhibition or supplementation of ceramide-forming sphingomyelinase reduced or enhanced the formation of EVs, respectively, but did not alter the injuriousness per individual EV. Barrier failure was attenuated when EVs were abundant in or supplemented with S1P. Stored human platelet 4-day EVs were more numerous compared with 2-day EVs, contained more long-chain ceramide and less S1P, and caused more endothelial cell barrier leak. Hence, platelet-derived EVs become more numerous and more injurious (more long-chain ceramide, less S1P) during storage. Blockade of sphingomyelinase, EV elimination, or supplementation of S1P during platelet storage may present promising strategies for TRALI prevention.
Subject(s)
Extracellular Vesicles/physiology , Platelet Transfusion/adverse effects , Sphingolipids/metabolism , Transfusion-Related Acute Lung Injury/etiology , Animals , Blood Platelets/ultrastructure , Blood Preservation , Ceramides/metabolism , Endothelial Cells/physiology , Endotoxins/toxicity , Humans , Lysophospholipids/physiology , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Models, Biological , Sphingomyelin Phosphodiesterase/antagonists & inhibitors , Sphingomyelin Phosphodiesterase/deficiency , Sphingomyelin Phosphodiesterase/physiology , Sphingosine/analogs & derivatives , Sphingosine/physiology , Transfusion-Related Acute Lung Injury/metabolism , Transfusion-Related Acute Lung Injury/prevention & controlABSTRACT
BACKGROUND: Human umbilical cord mesenchymal stromal cells possess considerable therapeutic promise for acute respiratory distress syndrome. Umbilical cord mesenchymal stromal cells may exert therapeutic effects via extracellular vesicles, while priming umbilical cord mesenchymal stromal cells may further enhance their effect. The authors investigated whether interferon-γ-primed umbilical cord mesenchymal stromal cells would generate mesenchymal stromal cell-derived extracellular vesicles with enhanced effects in Escherichia coli (E. coli) pneumonia. METHODS: In a university laboratory, anesthetized adult male Sprague-Dawley rats (n = 8 to 18 per group) underwent intrapulmonary E. coli instillation (5 × 10 colony forming units per kilogram), and were randomized to receive (a) primed mesenchymal stromal cell-derived extracellular vesicles, (b) naïve mesenchymal stromal cell-derived extracellular vesicles (both 100 million mesenchymal stromal cell-derived extracellular vesicles per kilogram), or (c) vehicle. Injury severity and bacterial load were assessed at 48 h. In vitro studies assessed the potential for primed and naïve mesenchymal stromal cell-derived extracellular vesicles to enhance macrophage bacterial phagocytosis and killing. RESULTS: Survival increased with primed (10 of 11 [91%]) and naïve (8 of 8 [100%]) mesenchymal stromal cell-derived extracellular vesicles compared with vehicle (12 of 18 [66.7%], P = 0.038). Primed-but not naïve-mesenchymal stromal cell-derived extracellular vesicles reduced alveolar-arterial oxygen gradient (422 ± 104, 536 ± 58, 523 ± 68 mm Hg, respectively; P = 0.008), reduced alveolar protein leak (0.7 ± 0.3, 1.4 ± 0.4, 1.5 ± 0.7 mg/ml, respectively; P = 0.003), increased lung mononuclear phagocytes (23.2 ± 6.3, 21.7 ± 5, 16.7 ± 5 respectively; P = 0.025), and reduced alveolar tumor necrosis factor alpha concentrations (29 ± 14.5, 35 ± 12.3, 47.2 ± 6.3 pg/ml, respectively; P = 0.026) compared with vehicle. Primed-but not naïve-mesenchymal stromal cell-derived extracellular vesicles enhanced endothelial nitric oxide synthase production in the injured lung (endothelial nitric oxide synthase/ß-actin = 0.77 ± 0.34, 0.25 ± 0.29, 0.21 ± 0.33, respectively; P = 0.005). Both primed and naïve mesenchymal stromal cell-derived extracellular vesicles enhanced E. coli phagocytosis and bacterial killing in human acute monocytic leukemia cell line (THP-1) in vitro (36.9 ± 4, 13.3 ± 8, 0.1 ± 0.01%, respectively; P = 0.0004) compared with vehicle. CONCLUSIONS: Extracellular vesicles from interferon-γ-primed human umbilical cord mesenchymal stromal cells more effectively attenuated E. coli-induced lung injury compared with extracellular vesicles from naïve mesenchymal stromal cells, potentially via enhanced macrophage phagocytosis and killing of E. coli.