Role of spleen educated monocytes in mediating ischemia-reperfusion injury followinglung transplant surgery

由缺血再灌注损伤(IRI)引起的原发性移植物功能障碍(PGD)发生率极高(&gt；50%)是 肺移植后预后受损的主要原因导致短期死亡以及 慢性肺移植排斥反应。PGD是由中性粒细胞募集到同种异体移植物中驱动的 渗入肺泡腔，引起不可逆的肺损伤。我们和其他人有 发现去除中性粒细胞可以改善PGD，但考虑到它们在临床上的重要性，这在临床上是不可行的 病原体清除。因此，在上一个供资期间，我们侧重于了解 促进中性粒细胞在移植后向肺内运输。我们的工作帮助发现了一个令人着迷的 PGD背后的多细胞信号通路：A)供体来源的Ly6ClowCCR2-非经典单核细胞 (NCM)滞留在供体肺中，释放中性粒细胞趋化因子CXCL12以招募受体中性粒细胞，B)骨- 储存在脾中的骨髓来源受体Ly6ChighCCR2+经典单核细胞(CM)被动员到 通过供体NCM依赖的供体肺泡巨噬细胞(AM，C)进入同种异体肺移植 同种异体移植后，CM通过依赖IL-1β下调小带对肺内皮细胞的通透性 阻滞剂-2和紧密连接蛋白Claudin-5可使受体中性粒细胞外渗。实验 在前一个周期中执行，额外的初步数据进一步表明，CM是通过 Toll受体信号通路和IL-1β的释放是NLRP3炎症体依赖的。令人兴奋的是，我们的数据 揭示了脾在启动骨髓来源的CM以激活NLRP3炎症体中的一种新功能。 单细胞转录和高分辨率活体空间成像显示脾嗜金属 巨噬细胞释放转化生长因子-将CM募集到不依赖CCR2-CCL2轴的脾内，而红髓 巨噬细胞通过激活NLRP3炎症小体释放IL-1β，这一过程需要 3天。这些数据从根本上改变了对脾的理解，从单核细胞储存库转变为单核细胞储存库 对损伤作出协调反应所必需的活跃的免疫器官。总的来说，我们的数据有力地支持了 肺移植后，脾刺激的CM释放的IL-1被招募到肺中的假设 依赖于TLR信号和NLRP3炎症小体的激活。我们将使用两个例子来验证我们的假设 目的：目的：1.确定CM从脾向移植肺迁移后的活化机制。 目的2.确定脾是否既是单核细胞预置的贮存库又是单核细胞介导肺移植的场所 受伤。我们的实验设计利用了我们的序贯脾和肺移植模型 以及最先进的技术，如空间活体成像和单细胞/空间转录。我们 将使用互补的遗传和药理学技术进行因果实验，以证明我们的 假设，同时创建丰富的人类缺血再灌注损伤的分子图谱。这些研究将有助于 将我们的发现迅速转化为临床实践。

Extremely high rates (>50%) of primary graft dysfunction (PGD) due to ischemia reperfusion injury (IRI) are the predominant cause of impaired post-lung transplant outcomes resulting in both short-term mortality as well as chronic lung allograft rejection. PGD is driven by the recruitment of neutrophils into the allograft which, upon extravasation into the alveolar space, undergo NETosis to cause irreversible lung injury. We and others have found that depleting neutrophils can ameliorate PGD but is not clinically feasible given their importance in pathogen clearance. Accordingly, in the prior funding period, we focused on understanding the mechanisms that drive neutrophil trafficking to the lung following transplantation. Our work facilitated the discovery of a fascinating multicellular signaling pathway that underlies PGD: A) donor-origin Ly6ClowCCR2- non-classical monocytes (NCM) retained in donor lungs release neutrophil chemokine CXCL12 to recruit recipient neutrophils, B) bone- marrow derived recipient Ly6ChighCCR2+ classical monocytes (CM) stored in the spleen are mobilized to the allograft through donor NCM-dependent activation of donor alveolar macrophages (AM), C) upon entry into the allograft, the CM permeabilize the pulmonary endothelium through IL-1β dependent downregulation of zona occludens-2 and tight junction protein Claudin-5 to enable extravasation of recipient neutrophils. Experiments performed in the prior cycle and additional preliminary data further suggested that the CM are activated through toll receptor signaling (TLR) and the release of IL-1β is NLRP3 inflammasome dependent. Excitingly, our data uncovers a novel function of the spleen in priming bone marrow derived CM to activate the NLRP3 inflammasome. Single cell transcriptomics and high resolution spatial intravital imaging indicated that the splenic metallophilic macrophages released TGF- to recruit CM to the spleen independent of CCR2-CCL2 axis while the red pulp macrophages primed them release IL-1β through the activation of NLRP3 inflammasome, a process that required 3 days. 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. Collectively, our data strongly support the hypothesis that after lung transplantation, IL-1 released by spleen-primed CM recruited to the lung is dependent on TLR signaling and activation of NLRP3 inflammasome. We will test our hypothesis using two aims: Aim 1. Determine the mechanisms of activation of CM after migration from spleen to the transplanted lung. Aim 2. Determine if spleen functions as both a reservoir and site for monocyte priming to mediate lung allograft injury. Our experimental design takes advantage of our models of sequential spleen and lung transplantation as well as state-of-the-art techniques such as spatial intravital imaging and single cell/spatial transcriptomics. We will use complementary genetic and pharmacological techniques to perform causal experiments to prove our hypothesis while creating a rich molecular atlas of human ischemia reperfusion injury. These studies will facilitate the rapid translation of our findings to clinical practice.