letter nature genetics • volume 25 • june 2000 187 TLR4 mutations are associated with endotoxin hyporesponsiveness in humans Nancy C. Arbour 1 *, Eva Lorenz 1 *, Brian C. Schutte 2 *, Joseph Zabner 1 , Joel N. Kline 1 , Michael Jones 3 , Kathy Frees 1 , Janet L. Watt 1 & David A. Schwartz 1 *These authors contributed equally to this manuscript. Departments of 1 Medicine, 2 Pediatrics and 3 Biostatistics, Department of Veterans Affairs Medical Center, The University of Iowa, Iowa City, Iowa, USA. Correspondence should be addressed to D.A.S. (e-mail: david-schwartz@uiowa.edu or david.schwartz@duke.edu). 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 oth- ers 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 1,2 , underlies the variability in airway responsiveness to inhaled LPS in humans 3 . 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 sig- nalling. 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 evi- dence that common mutations in TLR4 are associated with dif- ferences in LPS responsiveness in humans, and demonstrate that gene-sequence changes can alter the ability of the host to respond to environmental stress. We investigated the genetic basis for the physiologic response to inhaled endotoxin or LPS for several reasons. First, endotoxin is associated with the development and progression of asthma and other forms of airway disease. In the domestic setting, the concen- tration of endotoxin is associated with the clinical severity of asthma 4 , and, among exposed workers, endotoxin is the most sig- nificant component of the bioaerosol that is associated with the development 5 and progression 6 of airway disease. Endotoxin may also have a role in the pathophysiological consequences of air pol- lution 7 . Second, the ability of the host to respond to endotoxin is highly variable. Differences between individuals have been reported in the release and synthesis of cytokines by human monocytes stimulated with LPS in vitro 8 , and a patient with recurrent bacterial infections has been reported to be refractory to the in vivo and in vitro effects of LPS (ref. 9). We have recently found that normal, healthy, non-asthmatic subjects demonstrate a reproducible airway response to an incremental LPS inhalation challenge test; some subjects developed airflow obstruction when challenged with low concentrations of LPS and others were unaf- fected by high concentrations of inhaled LPS (ref. 3). Third, the molecular events leading to cell recognition and response to LPS are becoming more clearly defined. Recent attention has focused on the toll receptor family, specifically TLR4. LPS initiates signal transduction through the TLR4 receptor, and this pathway is enhanced by CD-14 (ref. 10) and MD-2 (ref. 11), and is activated through NF-κB and AP-1 (ref. 12). Mutations in Tlr4 (ref. 1) have been reported in mouse strains that are defective in their response to LPS, and disruption of Tlr4 results in an LPS hyporesponsive phenotype 2 . Based on these observations, we hypothesized that mutations in TLR4 may be associated with diminished airway responsiveness to inhaled LPS in humans. Using SSCP, we screened the entire coding region of TLR4 in all 83 subjects in our study population; 10 (12%) subjects had a band variant detected by SSCP (Fig. 1a). Direct sequencing detected an A→G substitution at nt 896 from the start codon of the TLR4 cDNA (ref. 12). To confirm our findings, we sequenced the 83 unrelated probands in the forward and reverse directions with primers designed to amplify nt 896. We found the same ten indi- viduals to have the A896G substitution and the remaining indi- viduals to have the more common TLR4 allele. One of the ten individuals with the A896G substitution was homozygous for this mutation; the remaining nine had a single, mutant allele. The allelic frequency of the 896 guanine sub- stitution was 6.6% in our study population, 7.9% in a control population from Iowa 13 and 3.3% in the parental chromosomes of the Centre d’Etude du Fig. 1 A common missense mutation in human TLR4. a, The altered electrophoretic mobility of band variants identified by single-stranded conformation polymorphism (SSCP) gel. b, The sequencing results for the samples in lanes 1–4. Lanes 2 and 4 are samples in which a band shift was observed. Upon sequenc- ing, the samples in lanes 1 and 3 were homozygous for alanine at position 896, whereas the sample in lane 2 was identified as heterozygous with both an alanine and a guanine at position 896, and the sample in lane 4 was homozygous for a guanine at position 896. c, The aspartic acid residue at position 299 is conserved between species. The sequence surrounding the amino acid altered by the TLR4 mutation was aligned for human 12 , mouse 1 , rat and hamster (D. Golenbock, pers. comm.). The aspartic acid at position 299 is indicated (arrow). a b c © 2000 Nature America Inc. • http://genetics.nature.com © 2000 Nature America Inc. • http://genetics.nature.com