There is much variability between individuals in the response to inhaled toxins, but it is not known why certain people develop disease when challenged with environmental agents and others remain healthy. To address this, we investigated whether TLR4 (encoding the toll-like receptor-4), which has been shown to affect lipopolysaccharide (LPS) responsiveness in mice, underlies the variability in airway responsiveness to inhaled LPS in humans. Here we show that common, co-segregating missense mutations (Asp299Gly and Thr399Ile) affecting the extracellular domain of the TLR4 receptor are associated with a blunted response to inhaled LPS in humans. Transfection of THP-1 cells demonstrates that the Asp299Gly mutation (but not the Thr399Ile mutation) interrupts TLR4-mediated LPS signalling. Moreover, the wild-type allele of TLR4 rescues the LPS hyporesponsive phenotype in either primary airway epithelial cells or alveolar macrophages obtained from individuals with the TLR4 mutations. Our findings provide the first genetic evidence that common mutations in TLR4 are associated with differences in LPS responsiveness in humans, and demonstrate that gene-sequence changes can alter the ability of the host to respond to environmental stress.

The Asp299Gly TLR4 polymorphism, which attenuates receptor signaling and diminishes the inflammatory response to gram-negative pathogens, is associated with a decreased risk of atherosclerosis. This finding is consistent with the hypothesis that innate immunity may play a part in atherogenesis.

Mutations in the TLR4 receptor may predispose people to develop septic shock with gram-negative microorganisms.

The toll-like receptor 2 (TLR2) has gained importance as a major mammalian receptor for lipoproteins derived from the cell wall of a variety of bacteria, such as Borrelia burgdorferi, Treponema pallidum, and Mycoplasma fermentans. We were interested in identifying mutations in the TLR2 gene that might prove to be associated with altered susceptibility to septic shock. We performed a mutation screen of the TLR2 gene using single-stranded conformational polymorphism in 110 normal, healthy study subjects and detected an Arg753Gln mutation in three individuals. No other missense mutations were detected in the TLR2 open reading frame. Functional studies demonstrate that the Arg753Gln polymorphism, in comparison to the wild-type TLR2 gene, is significantly less responsive to bacterial peptides derived from B. burgdorferi and T. pallidum. In a septic shock population, the Arg753Gln TLR2 polymorphism occurred in 2 out of 91 septic patients. More importantly, both of the subjects with the TLR2 Arg753Gln polymorphism had staphylococcal infections. These findings suggest that a mutation in the TLR2 gene may predispose individuals to lifethreatening bacterial infections.Microbial pathogens are a major health concern worldwide. Invasion of the host by microbial pathogens causes the activation of the innate immune response, which acts as a first line of defense against pathogenic bacteria. The innate immune response triggers a sequence of events that results in the production and secretion of cytokines and chemokines, activation of macrophages and monocytes, and in some cases initiation of adaptive immunity (10).The toll receptors appear to be very important in initiating the innate immune response. C3H/HeJ mice which have a mutation in the toll-like receptor 4 (TLR4) gene (Pro712His) and C57BL6/10ScCr mice which have a deletion of TLR4 are both hyporesponsive to lipopolysaccharide (LPS) (12). The TLR4 mutation in C3H/HeJ mice was the first direct proof for identifying TLR4 as a major LPS receptor in mammals. Subsequently TLR4 Ϫ/Ϫ mice were shown to be LPS hyporesponsive as well (6). Binding of LPS to TLR4, via a complex involving CD14 as well as MD-2, is thought to initiate a signaling cascade that involves several kinases and accessory molecules such as MyD88 and IRAK, leading eventually to a nuclear translocation of transcription factors NF-B and AP-1 (14). The binding of these transcription factors is responsible for initiating cytokine and chemokine expression subsequent to LPS exposure. Recently, mutations in the TLR4 receptor at residues 299 and 399 were shown to be associated with hyporesponsiveness to inhaled endotoxin in humans (2). Furthermore, the Asp299Gly TLR4 mutation may increase the risk to carriers of developing gram-negative septic shock (E. Lorenz, J. Mira, K. Frees, and D. Schwartz, submitted for publication). However, TLR4 does not seem to be involved in mediating the cellular response to gram-positive pathogens. TLR2, which is highly homologous to TLR4, has been shown to mediate a response to LPS, albei...

TLR4 is the signal-transducing receptor for structurally diverse microbial molecules such as bacterial LPS, respiratory syncytial virus fusion (F) protein, and chlamydial heat shock protein 60. Previous studies associated two polymorphic mutations in the extracellular domain of TLR4 (Asp299Gly and Thr399Ile) with decreased LPS responsiveness. To analyze the molecular basis for diminished responsiveness, site-specific mutations (singly or coexpressed) were introduced into untagged and epitope (Flag)-tagged wild-type (WT) TLR4 expression vectors to permit a direct comparison of WT and mutant signal transduction. Coexpression of WT TLR4, CD14, and MD-2 expression vectors in HEK293T cells was first optimized to achieve optimal LPS-induced NF-κB reporter gene expression. Surprisingly, transfection of cells with MD-2 at high input levels often used in the literature suppressed LPS-induced signaling, whereas supraoptimal CD14 levels did not. Under conditions where WT and polymorphic variants were comparably expressed, significant differences in NF-κB activation were observed in response to LPS and two structurally unrelated TLR4 agonists, chlamydial heat shock protein 60 and RSV F protein, with the double, cosegregating mutant TLR4 exhibiting the greatest deficiency. Overexpression of Flag-tagged WT and mutant vectors at input levels resulting in agonist-independent signaling led to equivalent NF-κB signaling, suggesting that these mutations in TLR4 affect appropriate interaction with agonist or coreceptor. These data provide new insights into the importance of stoichiometry among the components of the TLR4/MD-2/CD14 complex. A structural model that accounts for the diminished responsiveness of mutant TLR4 polymorphisms to structurally unrelated TLR4 agonists is proposed.