Pneumococcal Peptidoglycan-Polysaccharides Regulate Toll-Like Receptor 2 in the Mouse Middle Ear Epithelial Cells

Peptidoglycan-polysaccharides (PGPS) are cell envelope components of Gram staining positive (Gram+) bacteria including Streptococcus pneumoniae, a frequent cause of otitis media (OM) in young children (1) and pneumonia in the elderly (2). Antibiotic application kills S. pneumoniae and leads to release of PGPS, which causes otitis media with effusion (OME), a common condition without active bacterial growth but has a low profile of inflammation in the middle ear cleft on a long-term basis. PGPS is resistant to host enzyme digestion by DNases, RNases, and proteases during purification (3), suggesting durability, and it may serve as a persistent stimulus factor in the middle ear cleft. Consistent with the biochemical property, PGPS is long present in middle ear effusion in humans (3). Other components such as proteins, enzymes, and polysaccharides are present in the middle ear cavity for a limited period of time because they are susceptible to host enzyme digestion. Therefore, PGPS-induced immune and inflammatory responses are thought as persistent events in OME, which causes a low profile of chronic inflammation in the middle ear mucosa. Toll-like receptors (TLRs) are present on the surface of many cell types including epithelial cells, macrophages, monocytes, dendritic cells, lymphocytes, vascular endothelial cells, cardiac myocytes, and adipocytes (4). There are 13 TLR paralogs in mammals that have been found, but TLR2 is the one that recognizes a variety of Gram+ bacterial products such as peptidoglycan, lipoteichoic acid, and lipoarabinomannan, which responds to factors released by Gram staining negative (Gram−) bacteria including nontypeable Hemophilus influenzae (NTHi) (5), coccobacilli, N. meningitidis (6), and the Mycoplasma lipopeptides (7), so-called pathogen-associated molecular patterns (PAMPs). Knockout of TLR2 in mice decreased survival of animals against Gram+ bacterium Staphylococcus aureus (7,8), suggesting that TLR2 plays an important role in the host defense system by activating immune and inflammatory cells. TLR2 is, therefore, recognized as a cell surface receptor for mediating inflammatory responses in the middle ear in the presence of Gram+ metabolites such as cell wall components. It is known that TLR2 mediates immune and inflammatory reactions through NF-κB in host cells (9). However, it is not clear whether it plays a role in the pathogenic continuum of OM through a self-regulatory mechanism. In this study, we demonstrated that pneumococcal PGPS activates NF-κB via the TLR2 receptor in middle ear epithelial cells (MEECs), and then NF-κB, in turn, up-regulated the expression of TLR2 in a positive feedback manner. This perhaps provides an experimental model for OME in the middle ear mucosa in response to bacterial residual challenges.