Background. Normothermic ex vivo lung perfusion (EVLP) increases the pool of donor lungs by requalifying marginal lungs refused for transplantation through the recovery of macroscopic and functional properties. However, the cell response and metabolism occurring during EVLP generate a nonphysiological accumulation of electrolytes, metabolites, cytokines, and other cellular byproducts which may have deleterious effects both at the organ and cell levels, with impact on transplantation outcomes. Methods. We analyzed the physiological, metabolic, and genome-wide response of lungs undergoing a 6-h EVLP procedure in a pig model in 4 experimental conditions: without perfusate modification, with partial replacement of fluid, and with adult or pediatric dialysis filters. Results. Adult and pediatric dialysis stabilized the electrolytic and metabolic profiles while maintaining acid-base and gas exchanges. Pediatric dialysis increased the level of IL-10 and IL-6 in the perfusate. Despite leading to modification of the perfusate composition, the 4 EVLP conditions did not affect the gene expression profiles, which were associated in all cases with increased cell survival, cell proliferation, inflammatory response and cell movement, and with inhibition of bleeding. Conclusions. Management of EVLP perfusate by periodic replacement and continuous dialysis has no significant effect on the lung function nor on the gene expression profiles ex vivo. These results suggest that the accumulation of dialyzable cell products does not significantly alter the lung cell response during EVLP, a finding that may have impact on EVLP management in the clinic.
Lung transplantation is the only curative option for end-stage chronic respiratory diseases. However the survival rate is only about 50% at 5 years. Although experimental evidences have shown that innate allo-responses impact on the clinical outcome, the knowledge of the involved mechanisms involved is limited. We established a cross-circulatory platform to monitor the early recruitment and activation of immune cells in an extracorporeal donor lung by coupling blood perfusion to cell mapping with a fluorescent marker in the pig, a commonly-used species for lung transplantation. The perfusing pig cells were easily detectable in lung cell suspensions, in broncho-alveolar lavages and in different areas of lung sections, indicating infiltration of the organ. Myeloid cells (granulocytes and monocytic cells) were the dominant recruited subsets. Between 6 and 10 h of perfusion, recruited monocytic cells presented a strong upregulation of MHC class II and CD80/86 expression, whereas alveolar macrophages and donor monocytic cells showed no significant modulation of expression. This cross-circulation model allowed us to monitor the initial encounter between perfusing cells and the lung graft, in an easy, rapid, and controllable manner, to generate robust information on innate response and test targeted therapies for improvement of lung transplantation outcome.
Lung transplantation is the only curative option of end-stage chronic respiratory diseases. However the survival rate is only about 50% at 5 years. Whereas experimental evidences support that innate allo-responses impact on the clinical outcome, the knowledge of the involved mechanisms is limited. Here, we evaluate a cross-circulatory platform for monitoring the early recruitment and activation of immune cells in an extracorporeal donor lung by coupling blood perfusion to cell mapping with a fluorescent marker in the pig, a commonly-used species for lung transplantation. The perfusing pig cells were easily detectable in lung cell suspensions, in broncho-alveolar lavages and in different areas of lung sections, indicating infiltration of the organ. Myeloid cells (granulocytes and monocytic cells) were the dominantly recruited subsets. Between 6 and 10 h of perfusion, recruited monocytic cells presented a strong upregulation of MHC class II and CD80/86 expression, whereas alveolar macrophages and donor monocytic cells showed no significant modulation of expression. Altogether the cross-circulation model permits to monitor the initial encounter between perfusing cells and lung graft, in an easy, rapid, and controllable manner, for generating robust information on innate response and testing targeted therapies for improvement of lung transplantation outcome.
In response to the increasing demand for lung transplantation, ex vivo lung perfusion (EVLP) has extended the number of suitable donor lungs by rehabilitating marginal organs. However despite an expanding use in clinical practice, the responses of the different lung cell types to EVLP are not known. In order to advance our mechanistic understanding and establish a refine tool for improvement of EVLP, we conducted a pioneer study involving single cell RNA-seq on human lungs declined for transplantation. Functional enrichment analyses were performed upon integration of data sets generated at 4 h (clinical duration) and 10 h (prolonged duration) from two human lungs processed to EVLP. Pathways related to inflammation were predicted activated in epithelial and blood endothelial cells, in monocyte-derived macrophages and temporally at 4 h in alveolar macrophages. Pathways related to cytoskeleton signaling/organization were predicted reduced in most cell types mainly at 10 h. We identified a division of labor between cell types for the selected expression of cytokine and chemokine genes that varied according to time. Immune cells including CD4+ and CD8+ T cells, NK cells, mast cells and conventional dendritic cells displayed gene expression patterns indicating blunted activation, already at 4 h in several instances and further more at 10 h. Therefore despite inducing inflammatory responses, EVLP appears to dampen the activation of major lung immune cell types, what may be beneficial to the outcome of transplantation. Our results also support that therapeutics approaches aiming at reducing inflammation upon EVLP should target both the alveolar and vascular compartments.
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