Donor nonclassical monocytes initiate lung injury following transplantation

肺移植的适应症显著扩大，导致临床增长超过50% 在过去的十年里。然而，与其他实体器官相比，肺移植后的存活率最差 分别只有80%和50%的患者存活1年和5年。原发性移植物功能障碍（PGD）， 由缺血-再灌注损伤引起，在移植后24小时内影响超过50%的受体， 已经成为短期死亡率和长期移植物丢失的最重要的危险因素， 慢性排斥因此，针对改善PGD的疗法具有改善肺损伤的最大潜力。 移植结果先前的报道已经证明，中性粒细胞的募集和外渗可能与中性粒细胞的增殖有关。 同种异体移植物是PGD发展所必需的。消耗中性粒细胞可以改善PGD，但不能 鉴于其在病原体清除中的重要性，临床上是可行的。因此，我们专注于 了解移植后中性粒细胞向肺募集的机制。我们 发现孤儿核受体（NR 4A 1）依赖性非经典单核细胞（NCM），其特征在于 在人中通过CD 14 dimCD 16 ++和在小鼠中通过Ly 6ClowCX 3CR 1 highCCR 2-，与肺内皮结合 通过白细胞整合素（淋巴细胞因子相关抗原-1），并保留在供体肺中，尽管 目前的器官灌注技术。在小鼠和人肺移植后，活化的供体来源的 NCM介导中性粒细胞募集和外渗到同种异体移植物中的机制， 未知我们已发表的和初步的数据表明，在供体来源的细胞中，Toll样受体的激活可能是一个重要的因素。 NCM导致中性粒细胞化学引诱物巨噬细胞炎性蛋白2（MIP-2）的产生。的 供体NCM也释放IL-1 β并有助于增强同种异体移植物中的血管通透性。重要的是， 使用遗传谱系追踪系统，我们发现供体来源的NCM也迁移到远处器官， 移植的地方可以募集中性粒细胞。总的来说，这些数据支持我们的假设，即供体来源的 NCM在肺缺血-再灌注后被激活，驱动中性粒细胞流入同种异体移植物，并促进 对远处器官的继发性损伤。我们建立了一种小鼠原位肺血管化模型 移植，其中来自选择性缺乏NCM的NR 4A 1缺陷型供体小鼠的肺可以被移植， 用荧光野生型或遗传突变的NCM重建，以研究在细胞中的信号传导途径。 供体来源的NCM及其对肺IRI的贡献。因此，我们将剖析分子 在同种异体移植物和旁观者组织中的NCM介导的损伤的潜在机制。我们将确定：1） 负责激活供体NCM的toll样受体，2）供体NCM 迁移到远处器官并介导旁观者损伤，3）炎性小体依赖性的 NCM介导的IL-1介导中性粒细胞外渗通过这些实验，我们将确定 可以在移植前临床靶向改善PGD而不引起受体毒性。

The indications for lung transplantation have expanded significantly, resulting in a clinical growth of over 50% in the last decade. However, the survival following lung transplant is the worst compared to other solid organs with only 80% and 50% of patients alive at 1 and 5 years, respectively. Primary graft dysfunction (PGD), resulting from ischemia-reperfusion injury, affects over 50% of recipients within 24 hours of transplantation and has emerged as the most important risk factor for both short-term mortality as well as long-term graft loss from chronic rejection. As such, therapies directed to ameliorate PGD have the highest potential for improving lung transplant outcomes. Prior reports have demonstrated that recruitment and extravasation of neutrophils into the allograft is necessary for the development of PGD. Depleting neutrophils can ameliorate PGD, but is not clinically feasible given their importance in pathogen clearance. Accordingly, we have focused on understanding the mechanisms that drive neutrophil recruitment to the lung following transplantation. We discovered that orphan nuclear receptor (NR4A1)-dependent non-classical monocytes (NCM), characterized by CD14dimCD16++ in humans and Ly6ClowCX3CR1highCCR2- in mice, are bound to the pulmonary endothelium via a leukocyte integrin (lymphocyte factor associated antigen-1) and retained in the donor lungs despite the current techniques for organ perfusion. Following murine and human lung transplant, activated donor-derived NCM mediate neutrophil recruitment and extravasation into the allograft through mechanisms that remain unknown. Our published and preliminary data suggest that activation of toll-like receptors in donor-derived NCM leads to the production of neutrophil chemoattractant, macrophage inflammatory protein 2 (MIP-2). The donor NCM also release IL-1 and contribute to enhanced vascular permeability in the allograft. Importantly, using a genetic lineage tracing system we found that donor-derived NCM also migrate to distant organs after transplant where they recruit neutrophils. Collectively, these data support our hypothesis that donor-derived NCM are activated following lung ischemia-reperfusion, drive neutrophil influx into the allograft, and promote secondary injury to distant organs. We have developed a model of murine vascularized orthotopic lung transplantation where lungs from NR4A1-deficient donor mice, which selectively lack NCM, can be reconstituted with fluorescent wild-type or genetically mutated NCM to study the signaling pathways in donor-derived NCM and their contribution to lung IRI. Accordingly, we will dissect the molecular mechanisms underlying NCM-mediated injury in the allograft and bystander tissues. We will determine: 1) The toll-like receptors responsible for the activation of donor NCM, 2) Mechanisms through which donor NCM migrate to distant organs and mediate bystander injury, 3) The role of inflammasome-dependent production of IL-1 by NCM in mediating neutrophil extravasation. Through these experiments, we will identify pathways that could be clinically targeted, prior to transplantation, to ameliorate PGD without causing recipient toxicity.