Mathieu Glorion , Florentina Pascale ,Jérôme Estephan,Maxime Huriet,Carla Gouin, Céline Urien, Fany Blanc, Julie Rivière, Christophe Richard, Valérie Gelin, Julien De Wolf, Morgan Le Guen, Antoine Magnan, Antoine Roux, Isabelle Schwartz-Cornil , Edouard Sage
Abstract
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.
Introduction
Allogeneic lung transplantation (LT) is the sole therapeutic option for terminal chronic respiratory diseases. While LT practice is increasing worldwide, the outcomes are disappointing with a median survival of 6 years [1]. The major non-infectious early complication of successful surgery is severe primary graft dysfunction (PGD), a non-cardiogenic pulmonary edema syndrome that occurs in the first 3 days post LT, in about 11–25% of patients [2]. In most patients, chronic lung allograft dysfunction (CLAD) develops following a series of rejection and reparation events and stands as a major impediment to long term survival [3].
Material and methods
We basically followed the procedure described in [12, 15]. The circuit was filled with 1.5 L ringer lactate (Vetivex®, Dechra Northwich, UK). As shown in Fig 1, the circuit consists in a main console (SCPC centrifugal pump console, LivaNova, London, UK), a disposable pump (Revolution centrifugal pump, LivaNova), a hard-shell reservoir (LivaNova) and three 8-inch tubing (Smart coated tubing, LivaNova). An EOS® oxygenator (LivaNova) was used to heat the circuit. The gas connection was occluded. The PA and pulmonary vein (PV) pressures, the PA flow, and the temperature data were continuously monitored. The perfusing pig was maintained on a continuous heparin infusion (100 U/Kg/h). The activated clotting time was measured using a IStat® kit (Abbott, Chicago, IL, USA) and the heparin drip was adjusted to maintain a target clotting time value of 150–200 sec. The donor lungs were placed in dorsal position on an XVIVO® chambers (XVIVO Perfusion) and the trachea was cannulated with a 7.5 mm diameter cuffed endotracheal tube (Mallinckrodt, Staines-upon-Thames, UK). The tubing was spliced to connect the perfusing pig to the dedicated circuit, marking the start of cross-circulation.
Results
We performed a similar analysis on the CFSE- MoCs. CFSE- MoCs originate from the donor and possibly from some CFSE- recruited cells. On CFSE- MoCs, the upregulation of MHC class II and of CD80/86 was non-statistically significant between 6 h and 10 h (Fig 5, middle panels, and S5 Fig), although some non-statistically significant upregulation related to inter-animal variability was observed. This finding indicates that donor MoCs do not consistently upregulate the antigen presentation molecules, differently from the recruited CFSE+ MoCs. In the AMs (SSChiCD172AhiCD163hi, S6 Fig) all express MHC class II and CD80/86, therefore geometric mean intensities were used for illustration and their expression was not modified by cross-circulation (Fig 5, right panel).
Discussion
The cross-circulatory platform coupled to cell mapping permitted us to show that many cell types were recruited to the allogeneic lung, mainly PMNs and MoCs and also NK cells, T-cells, B-cells and cDC1. We proceeded to cell labeling exclusively on freshly isolated cells in order to avoid bias related to cell freezing. Therefore our antibody panel was adjusted for reasons of feasibility and for instance, it did not include the complex mAb combination for cDC2 detection [18]. Importantly, the single cell RNA-seq technique could be particularly suitable to further study the cell recruitment and activation in the first encounter between donor and recipient cells. Indeed, as we used female perfusing pigs and male donors, the donor origin can be deduced from genes expressed by the Y chromosome. Using scRNA-seq, discrete cell types could be identified and well characterized with much larger signatures that what can be done with classical cytometry. In addition modulated functions and pathways in the recruited cells and donor cells could be inferred from differentially expressed genes, thereby providing a powerful system to finely investigate ischemia-reperfusion response in the allogeneic context not only in immune cell types but also in the epithelial, fibroblastic, lymphatic and endothelial cells of the graft.
Acknowledgments
The work has benefited from the facilities and expertise of @BRIDGe (GABI, INRA-AgroParisTech, Paris-Saclay University, France) for the histology slide preparations and the scanner usage with the valuable assistance of Marthe Vilotte. We warmly thank the “Installation expérimentale porcine” of the GABI unit and in particular Pascal Lafaux and Giorgia Egidy as well as the Pig Physiology and Phenotyping Experimental Facility (https://doi.org/10.15454/1.5573932732039927E12) in particular Nelly Muller and Eloïse Delamaire. We thank Justine Cohen of the Foch Hospital for anatomopathological assessments. We are grateful to Sebastien Jacqmin, Frederic Harvengt and Nicolas Lavole for their help in managing pig perfusions. The surgery was done thanks to the Surgery platform facility CIMA, DOI: MIMA2, INRAE, 2018. Microscopy and Imaging Facility for Microbes, Animals and Foods, https://doi.org/10.15454/1.5572348210007727E12.
Citation: Glorion M, Pascale F, Estephan J, Huriet M, Gouin C, Urien C, et al. (2023) A cross-circulatory platform for monitoring innate allo-responses in lung grafts. PLoS ONE 18(5): e0285724. https://doi.org/10.1371/journal.pone.0285724
Editor: Lourdes Chacon Alberty, Texas Heart Institute, UNITED STATES
Received: January 18, 2023; Accepted: April 28, 2023; Published: May 30, 2023
Copyright: © 2023 Glorion et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: ES received funding from Association Chirurgicale Pour Le Développement et L'Amélioration des Techniques de Dépistage et de Traitement des Maladies Cardio-vasculaires (ADETEC-Coeur) and from la « Chaire Universitaire de Transplantation Université de Versailles-Saint-Quentin en Yvelines, Hôpital Foch et Fondation Foch, AR received a grant from the Association Gregory Lemarchal and the association Vaincre la Mucoviscidose (project number RF20220503016) and ISC received funding from INRAE institutional support. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Abbreviations: Lung transplantation, LT; alveolar macrophages, AMs; monocytic cells, MoCs; dendritic cells, DCs; polymorphonuclear neutrophils, PMNs; primary graft dysfunction, PGD; carboxyfluorescein succinimidyl ester, CFSE; pulmonary artery, PA; pulmonary vein, PV; swine, sw; human, hu; broncho-alveolar lavage, BAL; isotype control, ISC; single cell RNA-seq, scRNA-seq; hematoxylin-eosin-saffron, HES
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0285724#abstract0
