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

细菌性脑膜炎是全球感染相关死亡的前十大原因之一。欧洲最常见的病原体是肺炎链球菌。蛛网膜下腔肺炎球菌感染会产生医学上已知的最强大的炎症反应之一。这种反应背后的分子机制仅被部分阐明。我们实验室在小鼠身上的研究表明，[i]免疫反应依赖于MyD88，这是一种几乎所有Toll样受体(TLRs，除了TLR3)都使用的适配蛋白，[ii]TLR2和TLR4(但不是TLR9)有助于但不仅仅是MyD88激活。在旨在第一次深入了解额外TLRs在脑膜炎中的作用的试点实验中，我们使用了TLR2-TLR4双缺陷、UNC93B1突变小鼠(3D和TLR2/4-/-小鼠)。编码uc93B1的基因的错义突变通过阻止内体TLRs转位到内溶酶体而取消了内体TLRs的功能。主要的发现是，3D&TLR2/4-/-小鼠的免疫反应与MyD88缺陷小鼠一样受到损害，这表明TLR2、TLR4和内体TLRs的联合作用导致了MyD88的激活。然而，参与肺炎球菌检测的个体内体TLR(S)需要澄清(S)，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TLRs单一或联合缺陷的小鼠品系的表型，包括[i]unc93B1突变小鼠，[ii]TLR3-，TLR7-，TLR9-三缺陷小鼠，[III]TLR2-，TLR3-，TLR4-，TLR7，和TLR-9五重缺陷小鼠[iv]TLR13缺乏小鼠，[v]TLR2-，TLR13双缺乏小鼠，以及[v]TLR2-，TLR4，TLR13三缺乏小鼠，[v]TLR13-，TLR13双缺乏小鼠并与3D&TLR2/4-/-小鼠进行疾病表型比较。下一步，我们计划研究人THP-1来源的巨噬细胞和单核细胞来源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来源的巨噬细胞进行比较。因此，巨噬细胞将在类似蛛网膜下腔的条件下进行培养和挑战。这种方法的理论基础是，[i]人类免疫系统的功能与小鼠的不完全相同，[ii]巨噬细胞具有非凡的可塑性，使它们能够对环境信号做出反应并改变其表型。计划在肺炎球菌攻击前后评估巨噬细胞的表型(例如，细胞表面标志物的表达、细胞因子的产生、细胞活力)。此外，将阐明内体TLRs在肺炎球菌诱导的巨噬细胞激活中的作用(例如，通过使用药物拮抗剂)。在我们看来，这一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了解。

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.