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.

BACKGROUND Transfusion‐related acute lung injury (TRALI) leads to chest infiltrates and hypoxia after 0.02–1% of transfusions with an associated mortality up to 15%. Sphingolipid ceramide weakens endothelial barriers while sphingosine‐1‐phosphate (S1P) is barrier protective. Aged stored platelets elicit TRALI by acid sphingomyelinase (ASM) dependent ceramide formation, which causes lung injury ( in vivo) and impairs pulmonary endothelial cell (EC) barrier integrity ( in vitro) . Concomitantly, aged platelets form extracellular vesicles (EVs) which can shuttle hydrophobic lipids such as sphingolipids to target cells. Combining these concepts, we hypothesized that 5‐day but not 1‐day stored platelets generate EVs which mediate TRALI in a sphingolipid‐dependent manner. METHODS EVs from male C57BL/6 wild type (WT) or Smpd1 −/− (ASM knockout) platelets stored for 1‐ or 5‐days duration were isolated by differential centrifugation and filtration prior to enumeration (flow cytometry) and sphingolipid analysis (mass spectroscopy). For TRALI induction, BALB/c male mice were primed with 2 mg/kg intraperitoneal lipopolysaccharide (LPS) two hours prior to intravenous infusion of 10 ml/kg EVs (≈ 5×10 6 ) or saline (sham) and were assessed (6 hours later) by measuring lung tissue wet‐to‐dry (W/D) weight ratios, bronchoalveolar lavage fluid protein (BAL‐p) and lung tissue myeloperoxidase activity (MPO). Transendothelial electrical resistance (TEER) was measured in cultured human microvascular pulmonary ECs treated with PBS (sham) or 5×10 5 EVs (from 1‐ vs 5‐day stored platelets) for 4 hours. RESULTS Compared with 1‐day EVs, 5‐day EVs were more numerous, ceramide enriched and S1P depleted. 5‐day EVs from ASM KO platelets were less numerous than 5‐day WT EVs, but had similar sphingolipid profiles. LPS primed mice receiving 5‐day EVs had increased W/D (p < 0.05), MPO and BAL‐p (p<0.05) relative to mice receiving LPS priming in addition to saline (sham), 1‐day EVs or ASM KO 5‐day EVs. 5‐day EVs but not 1‐day EVs led to reductions in TEER (< 2 hours) in vitro in keeping with EC loss of barrier integrity which persisted for > 4 hours. 5‐day EVs could be made to resemble 1‐day EVs (no injury) with addition of exogenous S1P at levels equivalent to those found in 1‐day EVs. CONCLUSIONS Ceramide formation in aged platelets contributes to EV formation and impairs human EC barrier integrity resulting in TRALI. Removal or modification of aged EVs from stored platelets may prove a therapeutic option to reduce the risk of aged cellular blood products from causing TRALI. Support or Funding Information This work was jointly funded by Canadian Blood Services and the Canadian Institute of Health Research (CIHR). MJM is supported by a CIHR Vanier Scholarship This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

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/β-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.

BACKGROUNDTransfusion‐related acute lung injury (TRALI) presents as chest infiltrates and hypoxia after 0.02–1% of transfusions with an associated mortality of 5–15%. Aged platelet products elicit TRALI via a mechanism invovling formation of the sphingolipid ceramide (CER) by acid sphingomyelinase (ASM), which causes lung injury in vivo and impairs pulmonary endothelial cell (EC) barrier integrity in vitro. Concomitantly, aged platelets form abundant microparticles (PMPs) which can shuttle nucleic acids, proteins, and ‐ importantly ‐ lipids, to target cells.OBJECTIVETo test the hypothesis that aged (5 days) but not fresh (1 day) platelets will generate PMP that mediate TRALI in a CER‐dependent manner.METHODSPMPs from male C57BL/6 wild type (WT) or Smpd1−/− (ASM knockout) platelets stored for 1 (fresh) or 5 (aged) days duration were isolated by differential centrifugation and filtration before enumeration (flow cytometry) and CER analysis (mass spectroscopy). Cultured human microvascular pulmonary ECs (passage 6–8) were incubated with PBS (sham) or 5×105 fresh or aged PMPs for 4 hours and transendothelial electrical resistance (TEER) was measured every 30 minutes. For TRALI induction, BALB/c male mice primed with 2 mg/kg intraperitoneal LPS two hours prior to intravenous (IV) infusion of 10 ml/kg PMPs (≈ 5×106) or saline (sham) and were assessed (6 hours later) by measuring lung tissue wet‐to‐dry (W/D) ratios, bronchoalveolar lavage fluid protein (BAL‐p) and lung tissue myeloperoxidase activity (MPO).RESULTSCompared with fresh WT PMP, aged WT PMPs were more numerous and CER‐enriched. In contrast, aged ASM KO PMPs were less numerous compared with fresh ASM KO PMP but contained similar CER content as WT aged PMPs. Aged (CER rich) but not fresh (CER poor) PMPs lead to reductions in TEER (< 2 hours) in vitro in keeping with EC loss of barrier integrity which persisted for > 4 hours. Reductions in TEER seen over 4 hours with aged PMPs was prevented with pre‐treatment of WT PMPs with ceramidase (0.75 μg/mL) prior to incubation with ECs. LPS primed mice receiving IV aged WT PMPs exhibited increased W/D (p < 0.05), MPO and BAL‐p (p<0.05) relative to mice receiving LPS priming in addition to saline, fresh PMPs or ASM KO aged PMPs.CONCLUSIONSCeramide formation in aged platelets contributes to PMP formation and impairs human EC barrier integrity resulting in TRALI. Removal or modification of aged PMPs may prove a therapeutic option to reduce the risk of aged cellular blood products from causing TRALI.Support or Funding InformationThis work was jointly funded by Canadian Blood Services and the Canadian Institute of Health Research.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

BACKGROUNDTransfusion‐related acute lung injury (TRALI) has a mortality of 5–15% and an incidence ranging from 0.02–1%. TRALI causes chest infiltrates and hypoxia in part due to lung endothelial cell (EC) barrier failure and edema formation. Aged platelets which have a high risk of causing TRALI, accumulate microparticles (PMPs) throughout storage. PMPs have been shown to mediate pro‐inflammatory processes such as neutrophil priming.RESEARCH QUESTIONConsidering the role of ECs in TRALI, we set out to determine if aged platelet derived PMPs could mediate EC barrier disruption?HYPOTHESISSpecifically, we hypothesized that PMPs from aged but not fresh platelets will lead to EC monolayer disruption.METHODSC57BL/6 mouse platelets were stored for 1 (fresh) or 5 (aged) days duration prior to PMP isolation (centrifugation/filtration) and enumeration (flow cytometry). Human microvascular pulmonary ECs (passage 6–8) were cultured with PBS (sham) or 0.1 mg/ml LPS two hours prior to addition of PBS, 5×105 of fresh or aged PMPs, or PMP depleted supernatants from stored platelet pools. Transendothelial electrical resistance (TEER) readings were obtained every 30 minutes for 8 hours in triplicate. Alternatively aged 10 ml/kg PMPs (≈ 5×106) or saline (sham) was transfused into primed (2 mg/kg LPS intraperitoneal) BALB/c mice for assessment of lung tissue wet‐to‐dry (W/D) ratios 6 hours post transfusion.RESULTSPMPs were found to be increased more than 2 fold in aged as compared to fresh platelet storage pools. ECs exposed to LPS alone did not reveal decreases in TEER relative to PBS sham. When aged PMPs were added 2 hours after PBS there was a marked drop in TEER within 2 hours which became maximal (nearly 50% decrease relative to PBS alone) by 5 hours. Addition of LPS instead of PBS prior to aged PMPs led to similar decreases in TEER readings but with faster onset. Addition of platelet and PMP depleted supernatants (fresh or aged) or fresh PMPs with LPS did not show signs of decreased TEER readings relative to PBS sham. Beyond being more numerous, aged PMPs appear more injurious than fresh PMPs when delivered in our EC model at identical doses. Aged PMPs are able to reduce EC monolayer integrity within 2 hours and sustain this effect for up to 6 hours +/− LPS. LPS primed mice transfused aged PMPs had higher W/D ratios than LPS primed mice transfused saline (p<0.05).CONCLUSIONSPMPs may in part contribute to TRALI when aged platelets which are a rich source of PMPs are transfused. In our model aged PMPs were injurious, impairing EC barrier integrity in vitro and leading to increased W/D ratios in vivo. Targeting aged PMPs may prove a therapeutic option to reduce the risk of aged cellular blood products from causing TRALI.Support or Funding InformationCanadian Institutes of Health Research (CIHR)Canadian Blood Services (CBS)