Restoring immune-vascular axis integrity to alleviate acute lung injury in sepsis

项目摘要/摘要 脓毒症引起的多器官功能障碍综合征，主要影响肺和肾脏，是一种主要的 世界范围内致病和死亡的原因。急性肺损伤(ALI)或其最严重的形式，急性呼吸系统损伤 窘迫综合征(ARDS)是脓毒症最常见的并发症，目前尚无有效的治疗方法 除了可以得到支持性的护理。因此，减轻脓毒症引起的ALI的新的治疗策略是 迫切需要，在当前的SARS-CoV-2大流行面前更是如此。 脓毒症是一种威胁生命的器官功能障碍，由宿主对感染的反应失调引起， 其中免疫细胞和内皮细胞都扮演着关键角色。病原体诱导的炎症级联反应和 内皮细胞通过自分泌或旁分泌环路相互影响，以增加血管通透性和 导致免疫失调，破坏免疫血管的动态平衡。因此，治疗的目标是 需要恢复免疫-血管动态平衡来减轻脓毒症引起的并发症，如ALI/ARDS。 与血管通透性和炎症有关的神经粘连蛋白-1(Nrp1)可能被证明是一种可行的 通过恢复免疫血管的完整性来治疗败血症所致的ALI的目标。因此，核心假设是 我们认为Nrp1在脓毒症所致ALI的免疫血管功能障碍中起关键作用。 靶向Nrp1以恢复免疫血管动态平衡是治疗这种疾病的一个可行的选择。 为了验证我们的假设，我们提出了三个具体目标。在目标1中，我们将分析ALI的影响- 内皮特异性Nrp1基因敲除小鼠肺内皮细胞相关细胞因子的体外研究 老鼠。我们还将通过大剂量脂多糖(LPS)治疗或盲肠治疗在这些小鼠中发展ALI。 结扎穿孔(CLP)评估内皮Nrp1在脓毒症所致ALI严重程度中的作用 活体血管通透性、白细胞滚动、渗出。目标2将调查共培养的效果 从髓系特异性和CD4T细胞特异性Nrp1-1分离的外周血单核细胞(PBMC) 用野生型小鼠肺内皮细胞敲除小鼠。此外，我们将在这些Nrp1中发展ALI- 基因敲除小鼠模型研究Nrp1在这两种免疫细胞群中的多效性作用 脓毒症诱导的ALI。AIM 3将评估一种内皮细胞靶向脂质体的治疗效果 一种小分子Nrp1抑制剂(EG00229)的配方，以改善脓毒症诱导的ALI的严重程度。 我们预计，我们的提案将提供对Nrp1在免疫中的作用的更深入的见解- 脓毒症所致ALI中血管功能障碍的研究进展 以Nrp1为目标，克服其严重性。我们建议的最终目标是使大量 患有败血症所致肺部并发症的患者，目前尚无有效的治疗方法。

PROJECT SUMMARY/ABSTRACT Sepsis-induced multi organ dysfunction syndrome, which primarily affects the lungs and kidneys, is a major cause of morbidity and mortality worldwide. Acute lung injury (ALI) or its most severe form, acute respiratory distress syndrome (ARDS), are the most common complications of sepsis, for which no effective treatment except supportive care is available. Hence, novel therapeutic strategies for mitigating sepsis-induced ALI are desperately needed, more so in the face of the current SARS-CoV-2 pandemic. Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to an infection, where both immune cells and endothelial cells play critical roles. Pathogen-induced inflammatory cascade and the endothelium impact each other via autocrine or paracrine loops to increase vascular permeability and cause immune dysregulation to disrupt immune-vascular homeostasis. Hence, therapies aimed towards reinstating immune-vascular homeostasis are needed to alleviate sepsis-induced complications like ALI/ARDS. Neuropilin-1 (NRP1), implicated in vascular permeability and inflammation, may prove to be a viable target for treating sepsis-induced ALI by restoring immune-vascular integrity. Hence, the central hypothesis of our proposal is that NRP1 plays a critical role in the immune-vascular dysfunction in sepsis-induced ALI and targeting NRP1 for restoring the immune-vascular homeostasis is a viable therapeutic option for this disease. To validate our hypothesis, we propose three specific aims. In Aim 1, we will analyze the effect of ALI- relevant cytokines in vitro in mouse lung endothelial cells isolated from endothelial-specific NRP1 knockout mice. We will also develop ALI in these mice by high dose lipopolysaccharides (LPS) treatment or by cecal ligation and puncture (CLP) to evaluate the role of endothelial NRP1 in the severity of sepsis-induced ALI, in vivo vascular permeability, leukocyte rolling and extravasation. Aim 2 will investigate the effect of coculturing peripheral blood mononuclear cells (PBMCs) isolated from myeloid-specific and CD4+T-cell specific NRP1- knockout mice with wild-type mouse lung endothelial cells. Moreover, we will develop ALI in these NRP1- knockout mice models to investigate the pleotropic role of NRP1 in these two immune cell populations during sepsis-induced ALI. Aim 3 will evaluate the therapeutic efficacy of an endothelial cell (EC)-targeting liposomal formulation of a small-molecule NRP1 inhibitor (EG00229), in ameliorating the severity of sepsis-induced ALI. We anticipate that our proposal will provide a deeper insight into the role of NRP1 in the immune- vascular dysfunction in sepsis-induced ALI and lead to the development of a viable therapeutic strategy of targeting NRP1 to overcome its severity. The ultimate goal of our proposal is to benefit a large number of patients suffering from sepsis-induced lung complications for which there is no effective therapy till date.