*4.3.2 Roles of TLR4 in pneumococcal infection*

On inoculation with a non-lethal dose of *S. pneumoniae*, TLR4 mutant mice exhibited decreased survival rates, accompanied by increased bacterial growth, monocyte and lymphocyte infiltration, and interstitial inflammation in the lungs [69]. Notably, a recent study demonstrated that although mice lacking TLR4 also displayed lower viability and augmented colonization in the lung after intranasal *S. pneumoniae* inoculation compared with that of wild-type mice, this exacerbation of infection was accompanied by an attenuated pro-inflammatory profile, reduced live alveolar macrophages, diminished infiltration of neutrophils and monocytes, and inhibition of monocyte differentiation into macrophages [70]. In addition, MyD88 deletion was not able to completely reproduce these phenotypes, implying that pro-inflammatory responses via both MyD88- and TRIF-dependent TLR4 signaling are necessary for the mobilization and activation of phagocytes [70]. Therefore, TLR4 signaling could have led to the sufficient elimination of bacteria and subsequent protection of alveolar macrophages from pneumococcal cytotoxicity.

#### *4.3.3 Roles of TLR9 in pneumococcal infection*

Both TLR9-knockout and wild-type mice developed pulmonary inflammation during *S. pneumoniae* infection, but TLR9-knockout mice exhibited worse survival and more bacterial invasion from the bronchoalveolar fluids into the lung tissue and blood stream, with abrogated upregulation of phagocytic activity in alveolar macrophages [71]. This early finding indicates that the activation of TLR9 signaling is indispensable for maximizing phagocytosis in alveolar macrophages during pneumococcal infection. The priming effects of TLR agonists have also been investigated. A prior inhalational challenge with the TLR9 agonist ODN2395 in combination with the TLR2 agonist Pam2CSK4 protected mice from death due to *S. pneumoniae* infection, although administration of agonists of any individual TLR had no protective effect [72]. However, ODN2395/Pam2CSK4 stimulation enhanced intracellular bacterial death in isolated tracheal epithelial cells, but not in alveolar macrophages. Taken together, maintaining basal levels of TLR9 expression and signaling in alveolar macrophages is likely to be critical for defensing the host from pneumococcal infection.
