Fate and function of border-associated macrophages in pneumococcal meningitis

肺炎球菌脑膜炎中边界相关巨噬细胞的命运和功能

• 批准号: 432002731
• 负责人: Professor Dr. Uwe Ködel
• 金额: --
• 依托单位: Neurologische Klinik und Poliklinik (Campus Großhadern)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2023-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/432002731?language=en
• 关键词: Fate; function; border; associated; macrophages

Pneumococcal meningitis (PM) is one of the most serious infectious diseases of the central nervous system (CNS). Pneumococcal CNS infection generates massive neutrophilic inflammation which causes brain damage and thus contributes to unfavorable disease outcome. PM primarily affects the membranes surrounding the CNS. The CNS boundaries are populated with different subsets of immune cell populations ranging from so-called border-associated macrophages (BAM) over mast cells and dendritic cells to monocytes, neutrophils and lymphocytes, with BAM being the dominant subset under steady-state conditions. Due to their strategic positioning and their diverse repertoires of pattern recognition and opsonic receptors, BAM seem to be central to the orchestration of the inflammatory response to pneumococcal infection. However, current studies on the impact of BAM depletion on meningitis have revealed ambiguous findings, likely due to different study designs and technical issues. Previous work in a zebrafish model of PM and preliminary work of our group suggest that BAM may decline during the disease course, probably due to infection-associated lytic cell death, which may also contribute to the ambiguous findings of previous BAM depletion studies. These (mixed) findings motivate us to characterize the fate and function role of BAM in PM in more detail. The investigations will be done using a well-established mouse model of PM which closely mimics the clinical features of the human disease. For fate-tracking experiments, we will use CX3CR1GFP/+xCCR2RFP/+-transgenic reporter mice, in vivo dye labeling of BAM, post mortem immunostaining using macrophage markers, and combinations of these techniques. The (average) BAM activation state shall be assessed by staining for signature enzymes (namely iNOS and arginase) and real time PCR analysis on FACS-isolated dye-labeled BAM at three characteristic disease stages. To identify BAM cell death, we plan to perform immunhistochemical and/or immunoblot profiling of cell death pathways, focusing on pyroptosis. Next, we intend to evaluate the impact of BAM depletion by intrathecal clodronate liposome application as well as by intrathecal administration of diphtheria toxin (DT) to CD11b-DTR mice on the disease phenotype in early and advanced meningitis. Finally, we want to assess the role of lytic BAM cell death (as well as possible underlying mechanisms) in PM, by using a triple strategy, namely [a] by intrathecal administration of different inhibitors such as the cell death inhibitors VX-765, GSK-782 or NSA, [b] by BAM depletion and reconstitution (using ASC-, GSDMD, and MLKL-deficient macrophages) experiments, and [c] by generating BM chimeric mice with different (e.g., wild type versus ASC-deficient) BAM populations. In our opinion, this research project will markedly improve our knowledge about mechanisms of immunoregulation within the CNS.

肺炎球菌性脑膜炎（PM）是中枢神经系统（CNS）最严重的传染病之一。肺炎球菌性中枢神经系统感染产生大量中性粒细胞炎症，引起脑损伤，从而导致不利的疾病结局。PM主要影响中枢神经系统周围的膜。中枢神经系统边界上分布着不同的免疫细胞群亚群，从所谓的边界相关巨噬细胞（BAM）到肥大细胞和树突状细胞，再到单核细胞、中性粒细胞和淋巴细胞，在稳态条件下，BAM是主要的亚群。由于它们的战略定位和它们的模式识别和听觉受体的多样性，BAM似乎是肺炎球菌感染炎症反应的核心。然而，目前关于BAM耗竭对脑膜炎影响的研究显示了模棱两可的结果，可能是由于不同的研究设计和技术问题。先前在斑马鱼PM模型中的工作和我们小组的初步工作表明，BAM可能在疾病过程中下降，可能是由于感染相关的溶解细胞死亡，这也可能导致先前BAM消耗研究的模糊结果。这些（混合的）发现促使我们更详细地描述BAM在项目管理中的命运和功能角色。研究将使用一种完善的小鼠模型进行，该模型密切模仿人类疾病的临床特征。对于命运追踪实验，我们将使用CX3CR1GFP/+xCCR2RFP/+-转基因报告小鼠，BAM的体内染料标记，巨噬细胞标记的死后免疫染色，以及这些技术的组合。通过标记酶（即iNOS和精氨酸酶）的染色和facs分离的染料标记BAM在三个特征性疾病阶段的实时PCR分析来评估（平均）BAM的激活状态。为了确定BAM细胞死亡，我们计划对细胞死亡途径进行免疫组织化学和/或免疫印迹分析，重点是焦亡。接下来，我们打算评估通过鞘内应用氯膦酸脂质体以及鞘内给药白喉毒素（DT）对CD11b-DTR小鼠的BAM消耗对早期和晚期脑膜炎疾病表型的影响。最后，我们希望通过使用三种策略来评估溶解性BAM细胞死亡在PM中的作用（以及可能的潜在机制），即[a]通过鞘内给药不同的抑制剂，如细胞死亡抑制剂VX-765、GSK-782或NSA， [b]通过BAM消耗和重构（使用ASC-、GSDMD和mlkl缺陷巨噬细胞）实验，以及[c]通过产生具有不同（例如野生型与ASC-缺陷）BAM群体的BM嵌合小鼠。我们认为，该研究项目将显著提高我们对中枢神经系统免疫调节机制的认识。