Mechanistic Study of Stroke-induced Immune Suppression and Identification of Immune Modulatory Targets for Stroke-associated Pneumonia

项目摘要/摘要 卒中相关性肺炎(SAP)是急性肺炎患者死亡的主要原因 缺血性中风。多中心临床研究表明预防性抗生素治疗不会减少 死亡率或SAP的发生率。此外，抗药性细菌的出现为人类带来了 标准的“抗生素治疗无效。因此，需要新的治疗策略来改进。 临床结果。利用局灶性脑缺血动物模型的研究表明，中风诱导的 免疫抑制是SAP发病的原因之一。这项建议的总体目标是了解原因 对中风诱导的免疫抑制进行研究，并确定SAP的新治疗靶点。先天免疫细胞 包括中性粒细胞、单核细胞、巨噬细胞和树突状细胞(DC)是肺免疫的第一线 防御，紧随其后的是抗原特异性T和B细胞的招募和激活。在……里面 卒中模型的第一阶段，我们使用了一过性大脑中动脉闭塞(TMCAO)的小鼠模型 以分析大脑和肺中的免疫细胞生态位，以及确定 炎性细胞因子和趋化因子在缺血性卒中诱导后肺组织中的表达我们的 初步数据表明：1)缺血性卒中肺泡巨噬细胞CD11b+ DC和肺中的中性粒细胞，但单核细胞数量保持不变，尽管表达增加 2)脑缺血后单核细胞数量明显增加 3)缺血性中风降低肺中T细胞、B细胞和NK细胞的数量， 与趋化因子CCL5和CCL22减少相关。这些观察结果表明，脑缺血 中风事件会改变肺部的免疫细胞位置，并可能损害其功能。 使用tMCAO和铜绿假单胞菌感染，我们建议进一步检验我们的假设，SAP 是由于肺部特异性、抗菌性先天免疫功能受损所致。我们的假设将在 三个目标。在目标1中，我们将测定先天免疫细胞的吞噬和杀菌活性。 TMCAO和铜绿假单胞菌感染后的肺部。在目标2中，我们将检验我们的假设单核细胞 缺血性卒中后脑的渗透阻碍了它们对肺的渗透，从而促进了 萨普。我们将通过过继将纯化的单核细胞从B6CD45.2小鼠转移到B6小鼠来验证这一假设 CD45.1同源品系受体小鼠，其次为tMCAO和铜绿假单胞菌感染。在《目标3》中，我们将 研究CCL5和CCL22在SAP中的保护作用 这些趋化因子可恢复淋巴细胞的可获得性并减轻SAP的严重程度。我们希望这一点 研究将确定关键的免疫细胞类型、分子途径和关键的下游介质 用于宿主防御缺血性中风后的肺部感染，并最终改善临床结果。

Project Summary/Abstract Stroke-associated pneumonia (SAP) is the major cause of mortality in patients who have suffered from acute ischemic stroke. Multi-center clinical studies suggested that prophylactic antibiotic treatments do not reduce the incidence of mortality or SAP. Moreover, the emergence of antibiotic resistant bacteria renders the “gold standard” antibiotic treatments ineffective. Therefore, novel therapeutic strategies are needed to improve clinical outcomes. Studies utilizing animal models of focal brain ischemia demonstrated that stroke-induced immune suppression is one of the causes of SAP. The overall goal of this proposal is to understand the cause of stroke-induced immune suppression and identify novel therapeutic targets for SAP. Innate immune cells including neutrophils, monocytes, macrophages, and dendritic cells (DCs) are the first line of lung immune defense, which is followed by subsequent recruitment and activation of the antigen-specific T and B cells. In phase one of the Stroke CoBRE, we used transient middle cerebral artery occlusion (tMCAO), a mouse model of focal brain ischemia, to analyze immune cell niches in the brain and the lungs, as well as determined the expression of inflammatory cytokines and chemokines in the lungs after ischemic stroke induction. Our preliminarily data demonstrate that 1) ischemic stroke increases the number of alveolar macrophages, CD11b+ DCs, and neutrophils in the lungs, but monocyte number remains unchanged despite an increased expression of monocyte chemoattractant CCL2; 2) monocyte number is significantly increased in the brain after ischemic stroke induction; 3) ischemic stroke decreases the number of T cells, B cells, and NK cells in the lungs, correlating with the reduction of chemokines CCL5 and CCL22. These observations suggest that ischemic stroke events alter the immune cell niches in the lungs and potentially impair their functions. Using tMCAO coupled with P. aeruginosa infection, we propose to further test our hypothesis that SAP is caused by the impairment of the lung-specific, anti-bacterial innate immunity. Our hypothesis will be tested in three aims. In Aim 1, we will determine the phagocytic and bactericidal activities of innate immune cells in the lungs following tMCAO and P. aeruginosa infection. In Aim 2, we will test our hypothesis that monocyte infiltration to the brain after ischemic stroke impedes their infiltrating to the lungs, and thereby contributes to SAP. We will test this hypothesis by adoptive transfer of purified monocytes from B6 CD45.2 mice to the B6 CD45.1 congenic strain recipient mice, followed by tMCAO and P. aeruginosa infection. In Aim 3, we will investigate the protective role of CCL5 and CCL22 in SAP by determining if intratracheal administration of these chemokines restores lymphocyte availability and reduces severity of SAP. We are hopeful that this research will identify key immune cell types, molecular pathways, and downstream mediators that are critical for the host defense against lung infections following ischemic stroke, and ultimately improve clinical outcomes.