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

脾脏单核细胞在介导肺移植术后缺血再灌注损伤中的作用

• 批准号: 10661445
• 负责人: Ankit Bharat
• 金额: $ 79.74万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2027-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10661445
• 关键词: Affect; Allografting; Alveolar; Alveolar Macrophages; Atlases; Biology; Bone Marrow; CCL2 gene; Cells; Chronic; Clinical; Complex; Credentialing; Data; Donor person; Down-Regulation; Endothelium; Experimental Designs; Extravasation; Funding; Genetic; Genetic study; Herpes zoster disease; Hour; Human; Hypoxemic Respiratory Failure; Image; Immune; Impairment; Inflammasome; Injury; Interleukin-1 beta; Laboratories; Lung; Lung Transplantation; Macrophage; Mediating; Modeling; Molecular; Mus; Mutate; Neutrophil Infiltration; Organ; Outcome; Pathogenesis; Pathway interactions; Patients; Permeability; Process; Proteins; Publishing; Pulmonary Inflammation; Receptor Signaling; Reperfusion Injury; Research; Resolution; Role; Sampling; Signal Pathway; Signal Transduction; Site; Spleen; Splenic Red Pulp; Stromal Cell-Derived Factor 1; Structure of parenchyma of lung; Surgical Models; Techniques; Testing; Tight Junctions; Translations; Transplantation; Transplantation Surgery; Variant; Vascularization; Work; allograft rejection; chemokine; clinical application; clinical practice; clinical translation; clinically relevant; experimental study; fascinate; graft dysfunction; human tissue; intravital imaging; lung allograft; lung injury; migration; monocyte; mortality; mouse model; neutrophil; novel; pathogen; pharmacologic; receptor; reconstitution; recruit; response to injury; single-cell RNA sequencing; spleen transplantation; tool; trafficking; transcriptomics; two-photon; Affect; Allografting; Alveolar; Alveolar Macrophages; Atlases; Biology; Bone Marrow; CCL2 gene; Cells; Chronic; Clinical; Complex; Credentialing; Data; Donor person; Down-Regulation; Endothelium; Experimental Designs; Extravasation; Funding; Genetic; Genetic study; Herpes zoster disease; Hour; Human; Hypoxemic Respiratory Failure; Image; Immune; Impairment; Inflammasome; Injury; Interleukin-1 beta; Laboratories; Lung; Lung Transplantation; Macrophage; Mediating; Modeling; Molecular; Mus; Mutate; Neutrophil Infiltration; Organ; Outcome; Pathogenesis; Pathway interactions; Patients; Permeability; Process; Proteins; Publishing; Pulmonary Inflammation; Receptor Signaling; Reperfusion Injury; Research; Resolution; Role; Sampling; Signal Pathway; Signal Transduction; Site; Spleen; Splenic Red Pulp; Stromal Cell-Derived Factor 1; Structure of parenchyma of lung; Surgical Models; Techniques; Testing; Tight Junctions; Translations; Transplantation; Transplantation Surgery; Variant; Vascularization; Work; allograft rejection; chemokine; clinical application; clinical practice; clinical translation; clinically relevant; experimental study; fascinate; graft dysfunction; human tissue; intravital imaging; lung allograft; lung injury; migration; monocyte; mortality; mouse model; neutrophil; novel; pathogen; pharmacologic; receptor; reconstitution; recruit; response to injury; single-cell RNA sequencing; spleen transplantation; tool; trafficking; transcriptomics; two-photon

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.

由于缺血再灌注损伤（IRI）引起的原发性移植功能障碍（PGD）的极高率（> 50％）是 肺部移植后结果受损的主要原因，导致短期死亡率和 慢性肺同种异体移植排斥。 PGD​​是由嗜中性粒细胞募集到同种异体移植的驱动的。 向肺泡空间渗出，会导致脂肪症引起不可逆的肺损伤。我们和其他人有 发现枯竭的中性粒细胞可以改善PGD，但鉴于它们在 病原体清除。根据以前的资助期，我们专注于理解的机制 移植后，将中性粒细胞运输到肺部。我们的工作准备了一个有趣的发现 基于PGD的多细胞信号通路：a）供体 - 原始ly6clowccr2-非经典单核细胞 保留在供体肺中的（NCM）释放中性粒细胞趋化因子CXCL12，以招募受体中性粒细胞，b）骨 - 骨髓衍生的受体Ly6chighccr2+经典单核细胞（CM）存储在脾脏中 通过供体NCM依赖型肺泡巨噬细胞（AM），C）的同种异体移植物进入 同种异体移植物，CM通过IL-1β的依赖性下调Zona透化肺​​部内皮 Occludens-2和紧密连接蛋白Claudin-5可以使受体中性粒细胞渗出。实验 在先前的周期中执行，并进一步表明CM通过 收费接收器信号（TLR）和IL-1β的释放是NLRP3炎性体依赖性的。令人兴奋的是，我们的数据 发现袖子在启动骨髓中的新功能，以激活NLRP3炎性体。 单细胞转录组学和高分辨率的空间浸润成像表明脾气金属粒子 巨噬细胞释放了TGF-，将CM招募到脾脏，而与CCR2-CCL2轴无关 巨噬细胞通过激活NLRP3炎性体释放IL-1β，这一过程需要 3天。这些数据从根本上改变了对脾脏的理解，从单核细胞储存到 对损伤的协调反应所需的主动免疫器官。总的来说，我们的数据强烈支持 假设在肺移植后，由脾脏招募到肺部释放的IL-1是 取决于TLR信号传导和NLRP3炎症体的激活。我们将使用两个检验我们的假设 目的：目标1。确定从索伦到移植肺迁移后CM激活的机理。 AIM 2。确定Sleen是否功能作为储层和单核细胞启动的位点以介导肺同种异体移植物 受伤。我们的实验设计利用了我们的虚假吊带和肺移植模型 以及最先进的技术，例如空间内部内部成像和单细胞/空间转录组学。我们 将使用完整的遗传和药物技术进行因果实验来证明我们 假设在创造人类缺血再灌注损伤的丰富分子图中。这些研究将有助于 我们发现的快速转化为临床实践。