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

肺移植后缺血导致的原发性移植物功能障碍（PGD） 再灌注损伤（IRI）是肺移植结果不佳的主要原因。病理学上，PGD是 以中性粒细胞外渗到肺泡腔为特征。在肺移植的鼠模型中， 中性粒细胞外渗致移植物不可逆损伤。非选择性中性粒细胞耗竭 可以改善PGD，但鉴于其在病原体清除中的重要性，在临床上不可行。因此，委员会认为， 我们集中于了解驱动中性粒细胞外渗到 同种异体移植导致肺移植后PGD。我们发现，在肺移植后，Ly 6ClowCCR 2- 非经典单核细胞（NCM），保留在供体肺中，和Ly 6C高CCR 2+经典单核细胞（CM）， 招募到同种异体移植物中，这两者对于中性粒细胞外渗到间质空间中是必需的， 造成的肺损伤NCM和CM均响应于IRI产生IL-1β，具有不同的后果。我们 目前的初步数据表明，由NCM产生的IL-1β激活供体肺泡巨噬细胞（AM）， 诱导它们分泌单核细胞趋化蛋白1（MCP-1），这导致CM的募集。 我们的数据还表明，CM募集到同种异体移植物进一步产生IL-1β，以响应信号转导 通过Toll样受体，其通过下调紧密连接蛋白使内皮透化， 封闭带2（ZO-2），促进中性粒细胞外渗。使用遗传谱系追踪系统， 异时性脾移植我们报道来源于骨髓的CM接受来自 脾对于它们在移植后介导中性粒细胞外渗的功能是必要的， 脾在这种反应中作为活跃的免疫器官。我们提出的初步数据来支持我们的假设， 肺移植后，供体NCM释放的IL-1β诱导供体肺泡分泌MCP-1， 巨噬细胞，这是必要的招募受体CM引发脾红髓巨噬细胞。 这导致同种异体移植物中持续的IL-β信号传导，其促进中性粒细胞外渗和移植物损伤。 我们将从两个方面来检验这一假设。目标1。为了确定供体NCM和肺泡 巨噬细胞依赖性MCP-1分泌招募宿主脾CM介导中性粒细胞外渗 肺移植后目标2.确定脾内皮细胞Fractalkine是否保留CM 由从红髓巨噬细胞释放的TGF-β募集以引发NLRP 3炎性体。我们使用 小鼠模型中的因果遗传策略确定了疾病机制，并将这些实验与 可以在人体中测试的适用疗法。此外，我们还注意将我们的结果与 对之前和多次从供体肺活检中获得的人肺组织进行无偏分析 在肺移植期间再灌注后，以创建人缺血再灌注损伤的分子图谱。 这些研究将促进我们的发现迅速转化为临床实践。

PROJECT SUMMARY 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-1β in response to IRI, with differing consequences. We present preliminary data suggesting that IL-1β 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-1β 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-1β 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-β 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-β 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.