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）导致的原发性移植物功能障碍（PGD）的发生率极高（>50%）， 肺移植后结局受损的主要原因，导致短期死亡率以及 慢性肺移植排斥PGD是由中性粒细胞募集到同种异体移植物中驱动的， 外渗进入肺泡腔，经历NETosis，导致不可逆的肺损伤。我们和其他人已经 发现消耗中性粒细胞可以改善PGD，但鉴于其在临床上的重要性， 病原体清除因此，在上一个供资期间，我们重点了解了 在移植后驱动中性粒细胞运输到肺。我们的工作帮助发现了一个迷人的 A）供体来源Ly 6ClowCCR 2-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CXCL 12以募集受体中性粒细胞，B）骨- 将储存在脾中的骨髓来源的受体Ly 6ChiglycCR 2+经典单核细胞（CM）动员至 同种异体移植物通过供体NCM依赖性激活供体肺泡巨噬细胞（AM），C）进入后 在同种异体移植物中，CM通过IL-1β依赖性下调IL-1 β的表达而使肺内皮透化。 闭合蛋白-2和紧密连接蛋白Claudin-5的结合，以使受体中性粒细胞外渗。实验 在前一个周期中进行，额外的初步数据进一步表明，CM通过以下方式激活： Toll受体信号传导（TLR）和IL-1β的释放是NLRP 3炎性体依赖性的。令人兴奋的是，我们的数据 揭示了脾在引发骨髓来源的CM以激活NLRP 3炎性体中的新功能。 单细胞转录组学和高分辨率空间活体成像表明，脾脏嗜金属性 巨噬细胞不依赖于CCR 2-CCL 2轴而释放TGF-β，将CM募集到脾脏，而红髓则不依赖于CCR 2-CCL 2轴。 巨噬细胞通过激活NLRP 3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，从单核细胞库转变为单核细胞库。 对损伤作出协调反应所必需的活性免疫器官。总的来说，我们的数据强烈支持 假设肺移植后，脾致敏CM释放的IL-1 β被募集到肺中， 依赖于TLR信号传导和NLRP 3炎性体的活化。我们将用两个例子来验证我们的假设。 目标：目标1。确定CM从脾迁移到移植肺后的激活机制。 目标2.确定脾脏是否同时作为单核细胞启动的储库和位点来介导肺移植物 损伤我们的实验设计利用了我们的脾和肺顺序移植模型， 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，以证明我们的 同时建立了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

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