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

项目总结/摘要 中风相关性肺炎（SAP）是患有急性中风的患者死亡的主要原因。 缺血性中风多中心临床研究表明，预防性抗生素治疗并不能减少 死亡率或SAP的发生率。此外，抗生素耐药性细菌的出现使“黄金” 标准的抗生素治疗无效。因此，需要新的治疗策略来改善 临床结果。利用局灶性脑缺血动物模型的研究表明， 免疫抑制是SAP的原因之一。本提案的总体目标是了解原因 中风诱导的免疫抑制，并确定新的治疗SAP的目标。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，随后是抗原特异性T和B细胞的募集和活化。在 在中风CoBRE的第一阶段，我们使用短暂性大脑中动脉闭塞（tMCAO）小鼠模型， 局灶性脑缺血，分析大脑和肺部的免疫细胞小生境，以及确定 缺血性卒中诱导后肺中炎性细胞因子和趋化因子的表达。我们 初步数据表明：1）缺血性中风增加肺泡巨噬细胞、CD 11b + 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）缺血后脑内单核细胞数量显著增加 中风诱导; 3）缺血性中风减少肺中T细胞、B细胞和NK细胞的数量， 与趋化因子CCL 5和CCL 22的减少相关。这些观察结果表明，缺血 中风事件改变了肺中的免疫细胞小生境，并潜在地损害了它们的功能。 利用tMCAO结合铜绿假单胞菌感染，我们建议进一步检验我们的假设， 是由肺特异性抗菌先天免疫受损引起的。我们的假设将在 三个目标。在目的1中，我们将确定先天性免疫细胞在小鼠体内的吞噬和杀菌活性。 tMCAO和铜绿假单胞菌感染后的肺部。在目标2中，我们将测试我们的假设，单核细胞 缺血性中风后向脑的浸润阻碍了它们向肺的浸润，从而有助于 SAP.我们将通过过继转移来自B6 CD 45.2小鼠的纯化单核细胞到B6 CD 45.2小鼠来检验这一假设。 CD45.1同源株受体小鼠，然后是tMCAO和铜绿假单胞菌感染。在目标3中，我们 通过测定CCL 5和CCL 22在SAP中的保护作用来研究CCL 5和CCL 22在SAP中的保护作用， 这些趋化因子恢复淋巴细胞的可用性并降低SAP的严重性。我们希望这项 研究将确定关键的免疫细胞类型、分子通路和下游介质， 用于缺血性卒中后肺部感染的宿主防御，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.