nature immunology • volume 3 no 4 • april 2002 • http://immunol.nature.com A RTICLES 354 Adeline M. Hajjar 1, *, Robert K. Ernst 2, *, Jeff H.Tsai 1 , Christopher B.Wilson 1,3† and Samuel I. Miller 2,4† Published online: 25 March 2002, DOI: 10.1038/ni777 Lipopolysaccharide (LPS) is the principal proinflammatory component of the Gram-negative bacterial envelope and is recognized by the Toll-like receptor 4 (TLR4)–MD-2 receptor complex. Bacteria can alter the acylation state of their LPS in response to environmental changes. One opportunistic bacterium, Pseudomonas aeruginosa, synthesizes more highly acylated (hexa-acylated) LPS structures during adaptation to the cystic fibrosis airway. Here we show that human, but not murine, TLR4–MD-2 recognizes this adaptation and transmits robust proinflammatory signals in response to hexa-acylated but not penta-acylated LPS from P. aeruginosa. Whereas responses to lipid IVA and taxol are dependent on murine MD-2, discrimination of P. aeruginosa LPS structures is mediated by an 82-amino-acid region of human TLR4 that is hypervariable across species. Thus, in contrast to mice, humans use TLR4 to recognize a molecular signature of bacterial-host adaptation to modulate the innate immune response. 1 Department of Immunology, 2 Department of Microbiology, 3 Department of Pediatrics and 4 Department of Medicine, University of Washington, Seattle,WA 98195, USA. *These authors contributed equally to this work. † These authors share senior authorship. Correspondence should be addressed to S. I. M. (millersi@u.washington.edu) or C. B.W. (cbwilson@u.washington.edu). Human Toll-like receptor 4 recognizes host-specific LPS modifications The major Gram-negative bacterial surface component LPS is recog- nized by mammalian cells inducing mediators of inflammation 1 . Much evidence indicates that TLR4 transduces the signals that lead to the production of inflammatory mediators in response to LPS 2–4 . LPS- hyporesponsive strains of mice (C3H/HeJ and C57BL/10ScCr) have mutations in the TLR4 gene that generate a dominant-negative protein (a cytoplasmic proline-to-histidine substitution) or a null mutation, respectively 5,6 . Mice deficient in TLR4 are also hyporesponsive to LPS, whereas mice lacking TLR2, which was once proposed to mediate sig- nals in response to LPS, respond normally 7,8 . TLR4 is part of a trimol- ecular LPS receptor complex 9 that contains two coreceptors: MD-2, which is required for TLR4 function 10–12 ; and CD14, which is a high- affinity glycosyl-phosphatidyl inositol–linked LPS-binding protein 13 . The structure of the proinflammatory component of LPS, lipid A, varies between bacteria of different species 1 . Gram-negative bacteria including Pseudomonas aeruginosa (PA) can modulate the structure of their LPS on invasion of host tissues (or when grown under magne- sium-limiting conditions in vitro) to resist killing by the innate immune system and to maintain outer membrane integrity 14,15 . Such modifica- tions include the addition of ethanolamine, aminoarabinose and palmi- tate to lipid A, which promotes resistance to cationic antimicrobial pep- tides 16–18 . The environmental bacterium PA is an opportunistic pathogen in humans, particularly in people with cystic fibrosis (CF). Most chil- dren with CF develop PA lung infection by 3 years 19 , and in nearly all individuals affected with CF, death results from a chronic progressive lung inflammatory response to unremitting PA infection 20,21 . Lipopolysaccharide from environmental isolates and from laborato- ry-adapted strains of PA grown in conventional bacterial culture media has a penta-acylated structure (LA LPS), which is less acylated than that from enteric bacteria such as Escherichia coli. In contrast, isolates from the airways of CF-affected individuals synthesize hexa-acylated LPS (CF LPS), which contains additional palmitate and aminoarabi- nose 15 . This adaptation is not found in PA LPS from bloodstream iso- lates or urinary tract isolates, or from individuals with the chronic lung disease bronchiectasis (BR). Whereas CF LPS stimulates robust pro- duction of proinflammatory mediators by human umbilical-vein endothelial cells, penta-acylated PA LPS does not 15 , which suggests that the acylation state of LPS may affect LPS-mediated responses 22 . The basis for the differences in proinflammatory activity of CF LPS compared with penta-acylated PA LPS is unknown. Species-specific differences in the recognition of lipidIVA, a tetra-acy- lated biosynthetic precursor of lipid A, and taxol, a plant-derived mol- ecule that is unrelated to LPS in structure 23 , have been attributed to either the TLR4 (refs. 3,4), or both the MD-2 and TLR4 (refs. 24,25) components of the LPS receptor complex. Whether there are similar species-specific differences in the recognition of TLR4 agonists from pathogenic microbes is unknown. Here we show that human cells and human TLR4 (hTLR4), but not murine cells or murine TLR4 (mTLR4), discriminate between lipid A structures of PA LPS. Human TLR4 responded to low concentrations of hexa-acylated CF LPS but required much higher concentrations of penta-acylated LPS. This differential recognition of PA LPS is depen- dent only on a hypervariable 82-amino-acid (aa) region in the extracel- lular domain of hTLR4, and not on any other regions of murine TLR4 or human MD-2. By contrast, differential responsiveness to lipidIVA and taxol are mediated by both MD-2 and TLR4. These data show that an © 2002 Nature Publishing Group http://immunol.nature.com