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Primary graft dysfunction after lung transplantation, a consequence of ischemia-reperfusion injury (IRI), is a major cause of morbidity and mortality. IRI involves acute inflammation and innate immune cell activation, leading to rapid infiltration of neutrophils. Formyl peptide receptor 1 (FPR1) expressed by phagocytic leukocytes plays an important role in neutrophil function. The cell surface expression of FPR1 is rapidly and robustly upregulated on neutrophils in response to inflammatory stimuli. Thus, we hypothesized that use of [99mTc]cFLFLF, a selective FPR1 peptide ligand, would permit in vivo neutrophil labeling and noninvasive imaging of IRI using single-photon emission computed tomography (SPECT). A murine model of left lung IRI was utilized. Lung function, neutrophil infiltration, and SPECT imaging were assessed after 1 h of ischemia and 2, 12, or 24 h of reperfusion. [99mTc]cFLFLF was injected 2 h before SPECT. Signal intensity by SPECT and total probe uptake by gamma counts were 3.9- and 2.3-fold higher, respectively, in left lungs after ischemia and 2 h of reperfusion versus sham. These values significantly decreased with longer reperfusion times, correlating with resolution of IRI as shown by improved lung function and decreased neutrophil infiltration. SPECT results were confirmed using Cy7- cFLFLF-based fluorescence imaging of lungs. Immunofluorescence microscopy confirmed cFLFLF binding primarily to activated neutrophils. These results demonstrate that [99mTc]cFLFLF SPECT enables noninvasive detection of lung IRI and permits monitoring of resolution of injury over time. Clinical application of [99mTc]cFLFLF SPECT may permit diagnosis of lung IRI for timely intervention to improve outcomes after transplantation. [ABSTRACT FROM AUTHOR]

Ischemia-reperfusion (I/R) injury (IRI), which involves inflammation, vascular permeability, and edema, remains a major challenge after lung transplantation. Pannexin-1 (Panx1) channels modulate cellular ATP release during inflammation. This study tests the hypothesis that endothelial Panx1 is a key mediator of vascular inflammation and edema after I/R and that IRI can be blocked by Panx1 antagonism. A murine hilar ligation model of IRI was used whereby left lungs underwent 1 h of ischemia and 2 h of reperfusion. Treatment of wild-type mice with Panx1 inhibitors (carbenoxolone or probenecid) significantly attenuated I/R-induced pulmonary dysfunction, edema, cytokine production, and neutrophil infiltration versus vehicle-treated mice. In addition, VE-Cad-CreERT2+/Panx1fl/fl mice (tamoxifen-inducible deletion of Panx1 in vascular endothelium) treated with tamoxifen were significantly protected from IRI (reduced dysfunction, endothelial permeability, edema, proinflammatory cytokines, and neutrophil infiltration) versus vehicle-treated mice. Furthermore, extracellular ATP levels in bronchoalveolar lavage fluid is Panx1-mediated after I/R as it was markedly attenuated by Panx1 antagonism in wild-type mice and by endothelial-specific Panx1 deficiency. Panx1 gene expression in lungs after I/R was also significantly elevated compared with sham. In vitro experiments demonstrated that TNF-α and/or hypoxia-reoxygenation induced ATP release from lung microvascular endothelial cells, which was attenuated by Panx1 inhibitors. This study is the first, to our knowledge, to demonstrate that endothelial Panx1 plays a key role in mediating vascular permeability, inflammation, edema, leukocyte infiltration, and lung dysfunction after I/R. Pharmacological antagonism of Panx1 activity may be a novel therapeutic strategy to prevent IRI and primary graft dysfunction after lung transplantation. [ABSTRACT FROM AUTHOR]

Background Sphingosine-1-phosphate regulates endothelial barrier integrity and promotes cell survival and proliferation. We hypothesized that upregulation of sphingosine-1-phosphate during ex vivo lung perfusion would attenuate acute lung injury and improve graft function. Methods C57BL/6 mice (n = 4-8/group) were euthanized, followed by 1 hour of warm ischemia and 1 hour of cold preservation in a model of donation after cardiac death. Subsequently, mice underwent 1 hour of ex vivo lung perfusion with 1 of 4 different perfusion solutions: Steen solution (Steen, control arm), Steen with added sphingosine-1-phosphate (Steen + sphingosine-1-phosphate), Steen plus a selective sphingosine kinase 2 inhibitor (Steen + sphingosine kinase inhibitor), or Steen plus both additives (Steen + sphingosine-1-phosphate + sphingosine kinase inhibitor). During ex vivo lung perfusion, lung compliance and pulmonary artery pressure were continuously measured. Pulmonary vascular permeability was assessed with injection of Evans Blue dye. Results The combination of 1 hour of warm ischemia, followed by 1 hour of cold ischemia created significant lung injury compared with lungs that were immediately harvested after circulatory death and put on ex vivo lung perfusion. Addition of sphingosine-1-phosphate or sphingosine kinase inhibitor alone did not significantly improve lung function during ex vivo lung perfusion compared with Steen without additives. However, group Steen + sphingosine-1-phosphate + sphingosine kinase inhibitor resulted in significantly increased compliance (110% ± 13.9% vs 57.7% ± 6.6%, P < . 0001) and decreased pulmonary vascular permeability (33.1 ± 11.9 μg/g vs 75.8 ± 11.4 μg/g tissue, P = .04) compared with Steen alone. Conclusions Targeted drug therapy with a combination of sphingosine-1-phosphate + sphingosine kinase inhibitor during ex vivo lung perfusion improves lung function in a murine donation after cardiac death model. Elevation of circulating sphingosine-1-phosphate via specific pharmacologic modalities during ex vivo lung perfusion may provide endothelial protection in marginal donor lungs leading to successful lung rehabilitation for transplantation. [ABSTRACT FROM AUTHOR]

Background: Lung ischemia-reperfusion (IR) injury after transplantation as well as acute shortage of suitable donor lungs are two critical issues impacting lung transplant patients. This study investigates the anti-inflammatory and immunomodulatory role of human mesenchymal stromal cells (MSCs) and MSC-derived extracellular vesicles (EVs) to attenuate lung IR injury and improve of ex-vivo lung perfusion (EVLP)-mediated rehabilitation in donation after circulatory death (DCD) lungs.Methods: C57BL/6 wild-type (WT) mice underwent sham surgery or lung IR using an in vivo hilar-ligation model with or without MSCs or EVs. In vitro studies used primary iNKT cells and macrophages (MH-S cells) were exposed to hypoxia/reoxygenation with/without co-cultures with MSCs or EVs. Also, separate groups of WT mice underwent euthanasia and 1 h of warm ischemia and stored at 4 °C for 1 h followed by 1 h of normothermic EVLP using Steen solution or Steen solution containing MSCs or EVs.Results: Lungs from MSCs or EV-treated mice had significant attenuation of lung dysfunction and injury (decreased edema, neutrophil infiltration and myeloperoxidase levels) compared to IR alone. A significant decrease in proinflammatory cytokines (IL-17, TNF-α, CXCL1 and HMGB1) and upregulation of keratinocyte growth factor, prostaglandin E2 and IL-10 occurred in the BAL fluid from MSC or EV-treated mice after IR compared to IR alone. Furthermore, MSCs or EVs significantly downregulated iNKT cell-produced IL-17 and macrophage-produced HMGB1 and TNF-α after hypoxia/reoxygenation. Finally, EVLP of DCD lungs with Steen solution including MSCs or EVs provided significantly enhanced protection versus Steen solution alone. Co-cultures of MSCs or EVs with lung endothelial cells prevents neutrophil transendothelial migration after exposure to hypoxia/reoxygenation and TNF-α/HMGB1 cytomix.Conclusions: These results suggest that MSC-derived EVs can attenuate lung inflammation and injury after IR as well as enhance EVLP-mediated reconditioning of donor lungs. The therapeutic benefits of EVs are in part mediated through anti-inflammatory promoting mechanisms via attenuation of immune cell activation as well as prevention of endothelial barrier integrity to prevent lung edema. Therefore, MSC-derived EVs offer a potential therapeutic strategy to treat post-transplant IR injury as well as rehabilitation of DCD lungs. [ABSTRACT FROM AUTHOR]

Ischemia-reperfusion (I/R) injury leads to increased mortality and morbidity in lung transplant patients. Lung I/R injury involves inflammation contributed by innate immune responses. IL-17 and TNF-α, from iNKT cells and alveolar macrophages, respectively, contribute importantly to lung I/R injury. This study tests the hypothesis that IL-17 and TNF-α synergistically mediate CXCL1 (a potent neutrophil chemokine) production by alveolar type II epithelial (ATII) cells via an NADPH oxidase-dependent mechanism during lung I/R. Using a hilar clamp model, wild-type and p47phox-/- (NADPH oxidase-deficient) mice underwent left lung I/R, with or without recombinant IL-17 and/or TNF-α treatment. Wild-type mice undergoing I/R treated with combined IL-17 and TNF-α had significantly enhanced lung dysfunction, edema, CXCL1 production, and neutrophil infiltration compared with treatment with IL-17 or TNF-α alone. However, p47phox-/- mice had significantly less pulmonary dysfunction, CXCL1 production, and lung injury after I/R that was not enhanced by combined IL-17-TNF-α treatment. Moreover, in an acute in vitro hypoxia-reoxygenation model, murine ATII cells showed a multifold synergistic increase in CXCL1 expression after combined IL-17-TNF-α treatment compared with treatment with either cytokine alone, which was significantly attenuated by an NADPH oxidase inhibitor. Conditioned media transfer from hypoxia-reoxygenation-exposed iNKT cells and macrophages, major sources of IL-17 and TNF-α, respectively, to ATII cells significantly enhanced CXCL1 production, which was blocked by NADPH oxidase inhibitor. These results demonstrate that IL-17 and TNF-α synergistically mediate CXCL1 production by ATII cells after I/R, via an NADPH oxidase-dependent mechanism, to induce neutrophil infiltration and lung I/R injury. [ABSTRACT FROM AUTHOR]

Dysregulation of inflammation-resolution pathways leads to postlung transplant (LTx) ischemia-reperfusion (IR) injury and allograft dysfunction. Our hypothesis is that combined treatment with specialized pro-resolving lipid mediators, that is, Resolvin D1 (RvD1) and Maresin-1 (MaR1), enhances inflammation-resolution of lung IR injury. Expression of RvD1 and MaR1 was analyzed in bronchoalveolar lavage (BAL) fluid of patients on days 0, 1, and 7 post-LTx. Lung IR injury was evaluated in C57BL/6 (WT), FPR2−/−, and LGR6 siRNA treated mice using a hilar-ligation model with or without administration with RvD1 and/or MaR1. A donation after circulatory death and murine orthotopic lung transplantation model was used to evaluate the protection by RvD1 and MaR1 against lung IR injury. I n vitro studies analyzed alveolar macrophages and type II epithelial cell activation after treatment with RvD1 or MaR1. RvD1 and MaR1 expressions in BAL from post-LTx patients was significantly increased on day 7 compared to days 0 and 1. Concomitant RvD1 and MaR1 treatment significantly mitigated early pulmonary inflammation and lung IR injury in WT mice, which was regulated via FPR2 and LGR6 receptors. In the murine orthotopic donation after cardiac death LTx model, RvD1 and MaR1 treatments significantly attenuated lung IR injury and increased PaO 2 levels compared to saline-treated controls. Mechanistically, RvD1/FPR2 signaling on alveolar macrophages attenuated HMGB1 and TNF-α secretion and upregulated uptake of macrophage-dependent apoptotic neutrophils (efferocytosis), whereas MaR1/LGR6 signaling mitigated CXCL1 secretion by epithelial cells. Bioactive proresolving lipid mediator-dependent signaling that is, RvD1/FPR2 and MaR1/LGR6- offers a novel therapeutic strategy in post-LTx injury. [ABSTRACT FROM AUTHOR]

Our hypothesis is that the immunomodulatory capacities of mesenchymal stem cell‒derived extracellular vesicles (EVs) can be enhanced by specific microRNAs (miRNAs) to effectively attenuate post-transplant lung ischemia‒reperfusion (IR) injury. The expression of miR-206 was analyzed in bronchoalveolar lavage (BAL) fluid of patients on Days 0 and 1 after lung transplantation. Lung IR injury was evaluated in C57BL/6 mice using a left lung hilar-ligation model with or without treatment with EVs or antagomiR-206‒enriched EVs. Murine lung tissue was used for miRNA microarray hybridization analysis, and cytokine expression, lung injury, and edema were evaluated. A donation after circulatory death and murine orthotopic lung transplantation model was used to evaluate the protection by enriched EVs against lung IR injury. In vitro studies analyzed type II epithelial cell activation after coculturing with EVs. A significant upregulation of miR-206 was observed in the BAL fluid of patients on Day 1 after lung transplantation compared with Day 0 and in murine lungs after IR injury compared with sham. Treatment with antagomiR-206‒enriched EVs attenuated lung dysfunction, injury, and edema compared with treatment with EVs alone after murine lung IR injury. Enriched EVs reduced lung injury and neutrophil infiltration as well as improved allograft oxygenation after murine orthotopic lung transplantation. Enriched EVs significantly decreased proinflammatory cytokines, especially epithelial cell‒dependent CXCL1 expression, in the in vivo and in vitro IR injury models. EVs can be used as biomimetic nanovehicles for protective immunomodulation by enriching them with antagomiR-206 to mitigate epithelial cell activation and neutrophil infiltration in the lungs after IR injury. [ABSTRACT FROM AUTHOR]