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Pathogen recognition within the subarachnoid space: Investigations in a mouse model of pneumococcal meningitis

蛛网膜下腔内的病原体识别：肺炎球菌脑膜炎小鼠模型的研究

基本信息

批准号：
315106912
负责人：
Professor Dr. Carsten Jürgen Kirschning
金额：
--
依托单位：
Institut für Medizinische Mikrobiologie
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2016
资助国家：
德国
起止时间：
2015-12-31 至 2018-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/315106912?language=en
关键词：
Pathogen recognition within subarachnoid space

项目摘要

Bacterial meningitis is among the top 10 causes of infection-related deaths worldwide. The most frequent etiologic agent in Europe is Streptococcus pneumoniae. Pneumococcal infection of the subarachnoid space generates one of the most powerful inflammatory responses known in medicine. The molecular mechanisms underlying this response have been elucidated only in part. Studies from our laboratory in mice have demonstrated that [i] the immune response depends on Myd88, an adapter protein used by almost all Toll-like receptors (TLRs, except TLR 3), and [ii] TLR2 and TLR4 (but not TLR9) contribute to but not solely account for Myd88 activation. In pilot experiments aimed at getting first insight into the role of additional TLRs in meningitis, we used TLR2-TLR4-double-deficient, UNC93B1-mutant mice (3d&TLR2/4-/- mice). A missense mutation in the gene coding for UNC93B1 abolishes the function of endosomal TLRs by preventing their translocation to endolysosomes. The major finding was that 3d&TLR2/4-/- mice were equally impaired in their immune response than Myd88-deficient mice, suggesting that the combined action of TLR2, TLR4, and endosomal TLRs is responsible for Myd88 activation. The individual endosomal TLR(s) involved in pneumococcal sensing, however, need(s) to be clarified, which is the major goal of this project. In the first place, we plan to evaluate the phenotype of mouse strains with single or combined deficiencies in TLRs in our meningitis model, including [i] UNC93B1-mutant mice, [ii] TLR3-, TLR7-, TLR9-triple-deficient mice, [iii] TLR2-, TLR3-, TLR4-, TLR7-, and TLR-9-quintuple deficient mice [iv] TLR13-deficient mice, [v] TLR2-, TLR13-double-deficient mice, as well as [v] TLR2-, TLR4-, TLR13-triple-deficient mice, and to compare their disease phenotype with that of 3d&TLR2/4-/- mice. In the next step, we plan to investigate the reactivity of human THP-1-derived and monocyte-derived macrophages to pneumococcal challenge and compare their response with that of murine bone-marrow-derived macrophages. Thereby, macrophages will be cultured and challenged under conditions resembling those of the subarachnoid space. The rationale for this approach is that [i] the human immune system does not function in precisely the same way as that of mice, and [ii] macrophages have a remarkable plasticity that allows them to respond to environmental signals and change their phenotype. The macrophages are planned to be assessed for their phenotype (e.g., cell surface marker expression, cytokine production, cell viability) before and after pneumococcal challenge. Moreover, the involvement of endosomal TLRs in pneumococci-induced macrophage activation will be clarified (e.g., by using pharmacologic antagonists). In our opinion, this research project will markedly improve our knowledge about mechanisms of immune regulation within the central nervous system.

细菌性脑膜炎是全世界感染相关死亡的十大原因之一。在欧洲最常见的病原体是肺炎链球菌。蛛网膜下腔的肺炎球菌感染产生医学上已知的最强有力的炎症反应之一。这种反应的分子机制只得到部分阐明。我们实验室在小鼠中的研究表明，[i]免疫应答依赖于Myd 88，这是一种几乎所有Toll样受体（TLR，除了TLR 3）使用的衔接蛋白，[ii] TLR 2和TLR 4（但不是TLR 9）有助于但不仅仅是Myd 88激活。在旨在首次了解额外TLR在脑膜炎中的作用的初步实验中，我们使用了TLR 2-TLR 4双缺陷的UNC 93 B1突变小鼠（3d&TLR2/4-/-小鼠）。编码UNC 93 B1的基因中的错义突变通过阻止内体TLR易位至内溶酶体而消除了内体TLR的功能。主要发现是3d&TLR2/4-/-小鼠的免疫应答与Myd 88缺陷小鼠的免疫应答同样受损，这表明TLR 2、TLR 4和内体TLR的联合作用是Myd 88活化的原因。然而，参与肺炎球菌传感的单个内体TLR需要澄清，这是本项目的主要目标。首先，我们计划在我们的脑膜炎模型中评估具有TLR的单一或组合缺陷的小鼠品系的表型，包括[i] UNC 93 B1突变小鼠，[ii] TLR 3-、TLR 7-、TLR 9-三重缺陷小鼠，[iii] TLR 2-、TLR 3-、TLR 4-、TLR 7-和TLR-9-五重缺陷小鼠，[iv] TLR 13-缺陷小鼠，[v] TLR 2-、TLR 3-、TLR 4-、TLR 7-和TLR-9-五重缺陷小鼠，[v] TLR 13-缺陷小鼠，[v] TLR 13 TLR 13-双缺陷小鼠，以及[v] TLR 2-、TLR 4-、TLR 13-三缺陷小鼠，并将它们的疾病表型与3d&TLR2/4-/-小鼠的疾病表型进行比较。在下一步中，我们计划研究人THP-1衍生的和单核细胞衍生的巨噬细胞对肺炎球菌攻击的反应性，并将它们的反应与小鼠骨髓衍生的巨噬细胞的反应进行比较。因此，将在类似于蛛网膜下腔的条件下培养和激发巨噬细胞。这种方法的基本原理是，[i]人类免疫系统的功能与小鼠的功能不完全相同，[ii]巨噬细胞具有显着的可塑性，使它们能够对环境信号做出反应并改变其表型。计划评估巨噬细胞的表型（例如，细胞表面标志物表达、细胞因子产生、细胞活力）。此外，将阐明内体TLR参与肺炎球菌诱导的巨噬细胞活化（例如，通过使用药理学拮抗剂）。我们认为，这项研究项目将显著提高我们对中枢神经系统内免疫调节机制的认识。