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

项目摘要肺移植后因缺血导致的原发性移植物功能障碍(PGD) 再灌注损伤(IRI)是导致肺移植效果不佳的主要原因。从病理上讲，PGD是 以中性粒细胞渗入肺泡腔为特征。在小鼠肺移植模型中， 中性粒细胞渗出导致不可逆的同种异体移植损伤。中性粒细胞的非选择性耗竭 可以改善PGD，但考虑到它们在病原体清除中的重要性，在临床上并不可行。因此， 我们一直专注于了解促使中性粒细胞外溢进入血管内的具体机制。 同种异体肺移植后发生PGD。我们发现肺移植后，Ly6ClowCCR2- 供体肺保留的非经典单核细胞(NCM)和Ly6ChighCCR2经典单核细胞(CM)， 被招募到同种异体移植物中，既是中性粒细胞外溢到间质间隙所必需的，也是 由此造成的肺损伤。NCM和CM均产生IL-1β以响应IRI，但后果不同。我们 目前的初步数据表明，由NCM产生的IL-1β可以激活供者肺泡巨噬细胞(AM)， 诱导其分泌单核细胞趋化蛋白1(MCP-1)，从而导致CM的募集。 我们的数据还表明，被招募到同种异体移植物上的CM进一步产生IL-1β以响应信号 通过Toll样受体，通过下调紧密连接蛋白使内皮通透性， 封闭带2(ZO-2)，促进中性粒细胞外渗。使用遗传血统追踪系统和 异时脾移植我们报告起源于骨髓的CMS从 脾在调节移植后中性粒细胞外渗中的作用 在这种反应中，脾作为一个活跃的免疫器官。我们提供了初步数据来支持我们的假设 肺移植后供体肺巨噬细胞释放IL-1β诱导供体肺泡分泌单核细胞趋化蛋白-1 巨噬细胞，这是由脾红髓巨噬细胞启动的受体CM募集所必需的。 这导致同种异体移植物中持续的IL-β信号，促进中性粒细胞外溢和移植物损伤。 我们将在两个目标上检验这一假设。目的1.确定供体NCM和牙槽骨 巨噬细胞依赖的单核细胞趋化蛋白-1分泌招募宿主脾CM介导中性粒细胞外渗 肺移植后。目的2.确定脾内皮细胞碎裂因子是否保留CM 由转化生长因子-β从红髓巨噬细胞释放，以启动NLRP3炎症小体。我们用 小鼠模型中的因果遗传策略确定疾病机制并将这些实验与 可以在人体上进行测试的适用疗法。此外，我们还注意将我们的结果与 供体肺活检前和多次取人肺组织的无偏分析 肺移植再灌流后建立人肺缺血再灌注损伤分子图谱。 这些研究将促进我们的研究成果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.