Role of Spleen educated monocytes in mediating ischemia-reperfusion injury following lung transplant

Primary graft dysfunction (PGD) affects over 50% of patients within 24 hours of transplantation and is associated with pneumonitis and hypoxemic respiratory failure. PGD is characterized by the recruitment of neutrophils into the allograft which, upon extravasation into the alveolar space, undergo NETosis to cause irreversible lung injury. Depleting neutrophils can ameliorate PGD, but is not clinically feasible given their importance in pathogen clearance. We discovered that after lung transplantation, Ly6ClowCCR2- non-classical monocytes (NCM), retained in the donor lung, and Ly6ChighCCR2+ classical monocytes (CM), recruited to the allograft, are both necessary for the extravasation of neutrophils into the interstitial space and the resulting lung injury. Both NCM and CM produce IL-1β with differing consequences. IL-1β produced by NCM activates donor alveolar macrophages (AM), inducing their secretion of monocyte chemoattractant protein 1 (MCP-1) and recruitment of CM. The recruited CM then produce more IL-1β in response to signaling through toll-like receptors, which permeabilizes the endothelium to promote neutrophil extravasation. In our recent paper, we employed a genetic lineage tracing system and spleen transplants to show that CMs originating in the bone marrow receive signals from the spleen necessary for their function in mediating neutrophil extravasation after transplantation. These data fundamentally change the understanding of the spleen from a monocyte reservoir into an active immune organ necessary for a coordinated response to injury. Our new preliminary data suggest that CMs are retained in the red pulp of the spleen by endothelial membrane-anchored fractalkine where they interact with red pulp macrophages (RPM). The RPM release TGF- to recruit CM to the spleen, and prime them to release IL-1β through the activation of NLRP3 inflammasome. Collectively, our data strongly support our hypotheses that after lung transplantation, IL-1 released by donor NCM induces the secretion of MCP-1 from donor alveolar macrophages, which is necessary for the recruitment of recipient CM primed by splenic red pulp macrophages. This results in sustained IL- signaling in the allograft that promotes neutrophil extravasation and graft injury. Accordingly, we will determine whether: 1) donor NCM and alveolar macrophage-dependent MCP-1 secretion recruits host splenic classical monocytes to mediate neutrophil extravasation after lung transplantation, and 2) endothelial fractalkine retains CM recruited by TGF- released from red pulp macrophages to prime the NLRP3 inflammasome. Our experimental design takes advantage of our models of lung and spleen transplantation to facilitate the rapid completion of causal genetic studies in mouse models. All of our murine experiments are tied to clinically applicable strategies that could be applied to humans. In addition, we have taken care to pair our results with unbiased analyses of human lung tissue obtained during lung transplantation to create a molecular atlas of human ischemia reperfusion injury. These studies will facilitate the rapid translation of our findings to clinical practice.