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

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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.