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

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) 导致原发性移植物功能障碍 (PGD) 的发生率极高 (>50%) 肺移植后结果受损的主要原因，导致短期死亡以及 慢性肺同种异体移植排斥反应。 PGD​​ 是由中性粒细胞募集到同种异体移植物中驱动的， 外渗到肺泡腔，发生 NETosis，导致不可逆的肺损伤。我们和其他人有 发现消耗中性粒细胞可以改善 PGD，但鉴于其重要性，在临床上不可行 病原体清除。因此，在之前的资助期间，我们重点了解了以下机制： 移植后驱动中性粒细胞运输至肺部。我们的工作促进了一个令人着迷的发现 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)保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- 骨髓来源的受体 Ly6ChighCCR2+ 储存在脾脏中的经典单核细胞 (CM) 被动员到 同种异体移植物通过供体肺泡巨噬细胞 (AM) 的供体 NCM 依赖性激活，C) 在进入 同种异体移植物中，CM 通过 IL-1β 依赖性透明带下调使肺内皮细胞透化 occlusionns-2 和紧密连接蛋白 Claudin-5 使受体中性粒细胞外渗。实验 在前一个周期中执行的和额外的初步数据进一步表明 CM 是通过以下方式激活的： Toll 受体信号传导 (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 揭示了脾脏在启动骨髓源性 CM 激活 NLRP3 炎性体方面的新功能。 单细胞转录组学和高分辨率空间活体成像表明脾亲金属 巨噬细胞释放 TGF-β，将 CM 募集至脾脏，独立于 CCR2-CCL2 轴，而红髓 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认识，从单核细胞储存库转变为 活跃的免疫器官对损伤做出协调反应所必需的。总的来说，我们的数据强烈支持 假设肺移植后，脾启动的 CM 释放的 IL-1 被募集到肺中 依赖于 TLR 信号传导和 NLRP3 炎症小体的激活。我们将使用两个来检验我们的假设 目的： 目的 1. 确定 CM 从脾脏迁移到移植肺后的激活机制。 目标 2. 确定脾脏是否既作为单核细胞启动的储存库又作为介导肺同种异体移植的部位 受伤。我们的实验设计利用了我们的连续脾脏和肺移植模型 以及最先进的技术，例如空间活体成像和单细胞/空间转录组学。我们 将使用互补的遗传和药理学技术来进行因果实验来证明我们的 假说，同时创建丰富的人类缺血再灌注损伤分子图谱。这些研究将有助于 我们的研究结果快速转化为临床实践。

项目成果

Fracture of dual lumen cannula leading to cerebrovascular accident in a patient supported with ECMO.

• DOI: 10.1007/s10047-021-01306-z
• 发表时间: 2022-09
• 期刊: Journal of artificial organs : the official journal of the Japanese Society for Artificial Organs
• 作者: Argaw ST;Devlin PJ;Clark JA;Garza-Castillon R;Kurihara C;Bharat A
• 通讯作者: Bharat A