Differential Involvement of Toll-Like Receptors 2 and 4 in the Host Response to Acute Respiratory Infections with Wild-Type and Mutant Haemophilus influenzae Strains

Nontypeable Haemophilus influenzae (NTHI) is a commensal and opportunistic pathogen. It can cause serious complications, from otitis media in children (26) to exacerbation of chronic obstructive pulmonary disease in adults (21). Since NTHI expresses lipooligosaccharide (LOS), Toll-like receptor 4 (TLR4) was thought to be the major mammalian receptor involved in H. influenzae-dependent signaling. Previous evidence has implicated TLR4 function in H. influenzae clearance in the mouse lung (30). Additional in vitro evidence in human cell lines has also shown TLR2, another member of the toll-like receptor family, to mediate H. influenzae signaling (22, 23), indicating that multiple host receptors mediate the immune response to acute H. influenzae infections. Modifications of the LOS and lipopolysaccharide (LPS) subunits have been described in a large number of bacteria (3, 8, 9, 11). Often, these modifications are thought to improve the survival of the bacteria in the host. To define how modifications in the LOS subunit of H. influenzae affect the induction of the host response, we used two H. influenzae strains to induce acute respiratory tract infections in a mouse model. The LOS subunits expressed by both H. influenzae isolates are well characterized. While strain H. influenzae 2019 expresses a wild-type LOS with hexaacylation (11), the H. influenzae htrB strain expresses LOS isoforms with pentaacylated and tetraacylated lipid A moieties (24, 27). In human airway xenograft studies, these changes in the acylation state did not affect adherence (27), suggesting that the effect of the htrB mutation was limited to the endotoxin moiety, specifically the acylation of lipid A. Since lipid A is known to be the major immunogenic component of bacterial endotoxins (18) and in vitro studies have shown that lipid A exerts its immunogenic effect through efficient activation of TLRs, specifically TLR4 (13, 20), it was not surprising that the H. influenzae htrB strain showed reduced virulence in human xenograft studies (27). TLR4 signaling has recently been shown to induce TLR2 expression in endothelial cells (4), providing evidence for cross talk between several TLRs. In addition to the regulation by TLR4, TLR2, the receptor for lipoproteins, can form complexes with TLR1 and TLR6 (17, 28, 29). The results indicating transcriptional regulation between TLR2 and TLR4 support a close interaction between TLR2 and TLR4 in the initiation of the host response. Since both TLRs were shown to signal in response to acute H. influenzae infections, we used a mouse model of acute H. influenzae lung infections to determine which aspects of the host response were regulated by TLR2 and TLR4. We used a mouse model of acute respiratory infection and measured functional endpoints, such as neutrophil infiltration and cytokine secretion. We determined that TLR4 function was required for cytokine secretion and neutrophil chemotaxis, following an acute infection with wild-type H influenzae. By contrast, H. influenzae htrB strain infections also required TLR2 expression for clearance and neutrophil influx, while cytokine secretion was still largely dependent on TLR4 function. This comparison of the host response induced by wild-type and mutant H. influenzae strains indicated a significant increase in TLR2 expression following an acute infection with the H. influenzae htrB mutant. The change in TLR2 expression suggests that some mutations in LOS may lead to an increased involvement of receptors other than TLR4 as a means of clearing invading pathogens from the lung.