Fate and function of border-associated macrophages in pneumococcal meningitis

肺炎球菌性脑膜炎（PM）是一种严重的中枢神经系统（CNS）感染性疾病。肺炎球菌中枢神经系统感染产生大量嗜酸性炎症，导致脑损伤，从而导致不利的疾病结局。PM主要影响CNS周围的膜。CNS边界填充有免疫细胞群体的不同子集，范围从所谓的边界相关巨噬细胞（BAM）超过肥大细胞和树突细胞到单核细胞、嗜中性粒细胞和淋巴细胞，其中BAM是稳态条件下的主要子集。由于它们的战略定位及其模式识别和调理素受体的多样性，BAM似乎是肺炎球菌感染的炎症反应的协调的中心。然而，目前关于BAM耗竭对脑膜炎影响的研究显示了模糊的结果，可能是由于不同的研究设计和技术问题。先前在PM的斑马鱼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性细胞死亡，这也可能导致先前BAM耗竭研究的模糊结果。这些（混合）的发现促使我们更详细地描述BAM在PM中的命运和功能作用。研究将使用完善的PM小鼠模型进行，该模型密切模拟人类疾病的临床特征。对于命运追踪实验，我们将使用CX 3CR 1GFP/+ xCCR 2 RFP/+转基因报告小鼠，BAM的体内染料标记，使用巨噬细胞标记物的死后免疫染色，以及这些技术的组合。在三个特征性疾病阶段，通过对特征酶（即iNOS和iNOS酶）染色和对FACS分离的染料标记的BAM进行真实的实时PCR分析来评估（平均）BAM活化状态。为了鉴定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，我们打算评估鞘内注射氯膦酸盐脂质体以及鞘内注射白喉毒素（DT）对CD 11b-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，我们想评估溶解性BAM细胞死亡的作用，（以及可能的潜在机制），即[a]通过鞘内给予不同的抑制剂，如细胞死亡抑制剂VX-765、GSK-782或NSA，[B]通过BAM消耗和重建（使用ASC-、GSDMD和MLKL-缺陷型巨噬细胞）实验，和[c]通过产生具有不同（例如，野生型与ASC缺陷型）BAM群体。在我们看来，这项研究项目将显着提高我们对中枢神经系统内免疫调节机制的认识。

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.