TLR4 Polymorphisms mediate impaired responses to respiratory Syncytial virus and lipopolysaccharide

Severe bronchiolitis following respiratory syncytial virus (RSV) infection occurs in only a small subset of infected infants and the basis for variations in disease severity is not understood. Innate immune responses to RSV are mediated by TLR-4, and the 299Gly and 399Ile alleles of the TLR4 gene have been linked epidemiologically with increased severity of RSV disease in children. We hypothesized that cellular immune responses to RSV mediated by these variant forms of the receptor are defective relative to responses mediated via the common form of the receptor. Human bronchial epithelial cells were transfected with TLR4 constructs encoding the common TLR4 gene sequence (299Asp/399Thr), or the 299Gly or 399Ile alleles, and cytokine responses to in vitro RSV challenge were analyzed in the different transfected cells. Follow-up studies compared RSV-induced responses in PBMC from children expressing these same TLR4 genotypes. Human bronchial epithelial expressing 299Gly or 399Ile displayed normal levels of intracellular TLR4 but failed to efficiently translocate the receptor to the cell surface. This was associated with reduced NF-κB signaling post-TLR4 engagement, reduced production of IFNs, IL-8, IL-10, IL-12p35, IL-18, and CCL8, and the absence of acute-phase TNF-α. These findings were mirrored by blunted PBMC responses to RSV in children expressing the same TLR4 variants. Compromised first-line defense against RSV at the airway-epithelial surface of children expressing these TLR4 variants may thus confer increased susceptibility to severe infections with this virus. The TLR4 gene which encodes the receptor recognizing bacterial LPS is highly polymorphic. Two cosegregating missense polymorphisms have been identified in the TLR4 gene at minor allele frequencies between 8 and 10% in Caucasian populations (1), which result, respectively, in aspartic acid to glycine substitution at position 299 (Asp299Gly) and threonine to isoleucine substitution at position 399 (Thr399Ile) in the receptor protein. These polymorphisms have been linked with blunted airway (2) and systemic inflammatory responses (3) to inhaled LPS in adults and attenuated LPS-induced responses in primary airway epithelial cells (2). Moreover, they have also been associated with increased risk for severe respiratory syncytial virus (RSV)3 bronchiolitis (4, 5, 6) in previously healthy infants. In this regard, studies in mice have shown that TLR4 recognizes not only bacterial but also viral motifs, including the F protein of RSV (7) and, moreover, TLR4-deficient mice infected with RSV show impaired pulmonary cellular responses and delayed viral clearance (7, 8). The primary target for RSV in humans is airway respiratory epithelial cells (9) and the first link of defense against the virus involves epithelial production of a plethora of cytokines, chemokines, and immunomodulatory mediators (9, 10). Although RSV infects >50% of infants during their first year of life, only a small percentage (1–3%) of RSV-infected infants develop infections of sufficient severity to require hospitalization (11). It has been reported (12) that the ability of individual infants to increase TLR4 expression on blood monocytes during acute RSV bronchiolitis is inversely proportional to the degree of ensuing hypoxia, suggesting that the ability to up-regulate TLR4 is closely linked to disease severity. In addition, RSV has also been shown to up-regulate TLR4 in airway epithelial cells in vitro (13) and we have previously reported similar TLR4 up-regulation in the upper airways of infected infants (14). It has also recently been reported that these common TLR4 mutations are associated with an increased risk of severe RSV bronchiolitis (6) and increased risk for hospitalization (4) in previously healthy infants. However, the mechanisms by which TLR4 polymorphisms might impair host defense against RSV are currently unknown. In this present study, we have addressed this issue by comparing the in vitro response to RSV and LPS in bronchial epithelial cells transfected with constructs containing each allele of the TLR4 Asp299Gly (299Asp or 299Gly) and Thr399Ile (399Thr or 399Ile) polymorphisms, measuring markers of cellular immunity. We further extended the studies to investigate the effects of these TLR4 polymorphisms on responses of PBMC to these stimuli