Primary graft dysfunction (PGD) after lung transplantation, resulting from ischemia reperfusion injury (IRI), is the predominant cause of poor lung transplant outcomes. Pathologically, PGD is characterized by neutrophil extravasation into the alveolar space. In murine models of lung transplantation, NETosis of extravasated neutrophils induces irreversible allograft injury. Non-selective depletion of neutrophils can ameliorate PGD, but is not clinically feasible given their importance in pathogen clearance. Accordingly, we have focused on understanding the specific mechanisms that drive neutrophil extravasation into the allograft leading to PGD after lung transplantation. 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-1? in response to IRI, with differing consequences. We present preliminary data suggesting that IL-1? produced by NCM activates donor alveolar macrophages (AM), inducing their secretion of monocyte chemoattractant protein 1 (MCP-1) which results in recruitment of CM. Our data also demonstrates that CM recruited to the allograft further produce IL-1? in response to signaling through toll-like receptors, which permeabilizes the endothelium by downregulating the tight junction protein, Zona Occludens 2 (ZO-2), to promote neutrophil extravasation. Using a genetic lineage tracing system and heterochronic spleen transplants we reported that CMs originating in the bone marrow receive signals from the spleen necessary for their function in mediating neutrophil extravasation after transplantation revealing the spleen as an active immune organ in this response. We present preliminary data to support our hypothesis 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. We will test this hypothesis in two aims.
Aim 1. To determine whether donor NCM and alveolar macrophage-dependent MCP-1 secretion recruits host splenic CM to mediate neutrophil extravasation after lung transplantation.
Aim 2. To determine whether splenic endothelial fractalkine retains CM recruited by TGF-? released from red pulp macrophages to prime the NLRP3 inflammasome. We use causal genetic strategies in mouse models identify disease mechanisms and tie these experiments to applicable therapies that can be tested in humans. In addition, we have taken care to pair our results with unbiased analyses of human lung tissue obtained from biopsies of the donor lung before and at several times after reperfusion 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.
While lung transplantation is increasingly being utilized as a life-saving treatment for a variety of end-stage lung diseases, both short- and long-term outcomes of patients undergoing this intervention are limited due to the development of the syndrome of primary graft dysfunction resulting from severe injury to the new lungs from host neutrophils. In this proposal, we will expand on our recently published findings and determine the mechanisms through which classical monocytes are educated in the host spleen and subsequently recruited to the transplanted lungs where they enable neutrophils to leave the blood vessels and mediate lung injury. Since all the experiments in this proposal have a direct clinical relevance, we believe that our results will introduce novel therapies to ameliorate primary graft dysfunction and improve lung transplant outcomes.
