Toll-like receptors (TLRs) have been shown to participate in the recognition of pathogens by the innate immune system, but it is not clear how a restricted family of receptors has the capacity to recognize the wide spectrum of TLR stimuli known to exist. We report here that two members of the TLR family, TLR2 and TLR6, together coordinate macrophage activation by Gram-positive bacteria and the yeast cellwall particle, zymosan. TLR6 and TLR2 both are recruited to the macrophage phagosome, where they recognize peptidoglycan, a Gram-positive pathogen component. By contrast, TLR2 recognizes another component, bacterial lipopeptide, without TLR6. The requirement for TLR cooperation is supported by the finding that TLR2 needs a partner to activate tumor necrosis factor-␣ production in macrophages. Dimerization of the cytoplasmic domain of TLR2 does not induce tumor necrosis factor-␣ production in macrophages, whereas similar dimerization of the TLR4 cytoplasmic domain does. We show that the cytoplasmic domain of TLR2 can form functional pairs with TLR6 or TLR1, and this interaction leads to cytokine induction. Thus, the cytoplasmic tails of TLRs are not functionally equivalent, with certain TLRs requiring assembly into heteromeric complexes, whereas others are active as homomeric complexes. Finally, we show that TLR6, TLR2, and TLR1 are recruited to macrophage phagosomes that contain IgG-coated erythrocytes that do not display microbial components. The data suggest that TLRs sample the contents of the phagosome independent of the nature of the contents, and can establish a combinatorial repertoire to discriminate among the large number of pathogen-associated molecular patterns found in nature.

The innate host response to lipopolysaccharide (LPS) obtained from Porphyromonas gingivalis is unusual in that different studies have reported that it can be an agonist for Toll-like receptor 2 (TLR2) as well as an antagonist or agonist for TLR4. In this report it is shown that P. gingivalis LPS is highly heterogeneous, containing more lipid A species than previously described. In addition, purification of LPS can preferentially fractionate these lipid A species. It is shown that an LPS preparation enriched for lipid A species at m/z 1,435 and 1,450 activates human and mouse TLR2, TLR2 plus TLR1, and TLR4 in transiently transfected HEK 293 cells coexpressing membrane-associated CD14. The HEK cell experiments further demonstrated that cofactor MD-2 was required for functional engagement of TLR4 but not of TLR2 nor TLR2 plus TLR1. In addition, serum-soluble CD14 effectively transferred P. gingivalis LPS to TLR2 plus TLR1, but poorly to TLR4. Importantly, bone marrow cells obtained from TLR2؊/؊ and TLR4 ؊/؊ mice also responded to P. gingivalis LPS in a manor consistent with the HEK results, demonstrating that P. gingivalis LPS can utilize both TLR2 and TLR4. No response was observed from bone marrow cells obtained from TLR2 and TLR4 double-knockout mice, demonstrating that P. gingivalis LPS activation occurred exclusively through either TLR2 or TLR4. Although the biological significance of the different lipid A species found in P. gingivalis LPS preparations is not currently understood, it is proposed that the presence of multiple lipid A species contributes to cell activation through both TLR2 and TLR4.

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 lipidIVA 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.

The human neonate and infant are unduly susceptible to infection with a wide variety of microbes. This susceptibility is thought to reflect differences from adults in innate and adaptive immunity, but the nature of these differences is incompletely characterized. The innate immune response directs the subsequent adaptive immune response after integrating information from Toll-like receptors (TLRs) and other environmental sensors. We set out to provide a comprehensive analysis defining differences in response to TLR ligation between human neonates and adults. In response to most TLR ligands, neonatal innate immune cells, including monocytes, conventional and plasmacytoid dendritic cells (cDCs and pDCs, respectively), produced less IL-12p70 and IFN-α (and consequently induced less IFN-γ), moderately less TNF-α, but as much or even more IL-1β, IL-6, IL-23, and IL-10 than adult cells. At the single-cell level, neonatal innate cells generally were less capable of producing multiple cytokines simultaneously, i.e., were less polyfunctional. Overall, our data suggest a robust if not enhanced capacity of the neonate vs. the adult white blood cell TLR-mediated response to support Th17- and Th2-type immunity, which promotes defense against extracellular pathogens, but a reduced capacity to support Th1-type responses, which promote defense against intracellular pathogens.

Macrophages orchestrate innate immunity by phagocytosing pathogens and coordinating inflammatory responses. Effective defence requires the host to discriminate between different pathogens. The specificity of innate immune recognition in Drosophila is mediated by the Toll family of receptors; Toll mediates anti-fungal responses, whereas 18-wheeler mediates anti-bacterial defence. A large number of Toll homologues have been identified in mammals, and Toll-like receptor 4 is critical in responses to Gram-negative bacteria. Here we show that Toll-like receptor 2 is recruited specifically to macrophage phagosomes containing yeast, and that a point mutation in the receptor abrogates inflammatory responses to yeast and Gram-positive bacteria, but not to Gram-negative bacteria. Thus, during the phagocytosis of pathogens, two classes of innate immune receptors cooperate to mediate host defence: phagocytic receptors, such as the mannose receptor, signal particle internalization, and the Toll-like receptors sample the contents of the vacuole and trigger an inflammatory response appropriate to defence against the specific organism.

Toll-like receptor (TLR) 2 and TLR4 play important roles in the early, innate immune response to microbial challenge. TLR2 is preferentially involved in the inflammatory response to lipoteichoic acid, lipopeptides, and glycans from a variety of microbes, whereas TLR4 is essential for a complete response to LPSs. We report here that TLR2 transduces the response to phenol-soluble modulin, a factor secreted by Staphylococcus epidermidis. The TLR2-mediated response to this modulin was enhanced by TLR6 but inhibited by TLR1, indicating a functional interaction between these receptors. We also demonstrate that a response to phenol-soluble modulin mediated by TLR2 and TLR6 was more refractory to inhibition by TLR1 than one mediated by TLR2 alone.

Rationale: Polymorphisms affecting Toll-like receptor (TLR)-mediated responses could predispose to excessive inflammation during an infection and contribute to an increased risk for poor outcomes in patients with sepsis. Objectives: To identify hypermorphic polymorphisms causing elevated TLR-mediated innate immune cytokine and chemokine responses and to test whether these polymorphisms are associated with increased susceptibility to death, organ dysfunction, and infections in patients with sepsis. Methods: We screened single-nucleotide polymorphisms (SNPs) in 43 TLR-related genes to identify variants affecting TLR-mediated inflammatory responses in blood from healthy volunteers ex vivo. The SNP associated most strongly with hypermorphic responses was tested for associations with death, organ dysfunction, and type of infection in two studies: a nested case-control study in a cohort of intensive care unit patients with sepsis, and a case-control study using patients with sepsis, patients with sepsis-related acute lung injury, and healthy control subjects. Measurements and Main Results: The SNP demonstrating the most hypermorphic effect was the G allele of TLR1 27202A/G (rs5743551), which associated with elevated TLR1-mediated cytokine production (P , 2 3 10 220 ). TLR1 27202G marked a coding SNP that causes higher TLR1-induced NF-kB activation and higher cell surface TLR1 expression. In the cohort of patients with sepsis TLR1 27202G predicted worse organ dysfunction and death (odds ratio, 1.82; 95% confidence interval, 1.07-3.09). In the case-control study TLR1 27202G was associated with sepsis-related acute lung injury (odds ratio, 3.40; 95% confidence interval, 1.59-7.27). TLR1 27202G also associated with a higher prevalence of gram-positive cultures in both clinical studies. Conclusions: Hypermorphic genetic variation in TLR1 is associated with increased susceptibility to organ dysfunction, death, and grampositive infection in sepsis.

The glycosylphosphatidylinositol (GPI) anchors of Plasmodium falciparum have been proposed to be the major factors that contribute to malaria pathogenesis through their ability to induce proinflammatory responses. In this study we identified the receptors for P. falciparum GPI-induced cell signaling that leads to proinflammatory responses and studied the GPI structure-activity relationship. The data show that GPI signaling is mediated mainly through recognition by TLR2 and to a lesser extent by TLR4. The activity of sn-2-lysoGPIs is comparable with that of the intact GPIs, whereas the activity of Man 3 -GPIs is about 80% that of the intact GPIs. The GPIs with three (intact GPIs and Man 3 -GPIs) and two fatty acids (sn-2-lyso-GPIs) appear to differ considerably in the requirement of the auxiliary receptor, TLR1 or TLR6, for recognition by TLR2. The former are preferentially recognized by TLR2/TLR1, whereas the latter are favored by TLR2/TLR6. However, the signaling pathways initiated by all three GPI types are similar, involving the MyD88-dependent activation of extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 and NF-B-signaling pathways. The signaling molecules of these pathways differentially contribute to the production of various cytokines and nitric oxide (Zhu, J., Krishnegowda, G., and Gowda, D. C. (2004) J. Biol. Chem. 280, 8617-8627). Our data also show that GPIs are degraded by the macrophage surface phospholipases predominantly into inactive species, indicating that the host can regulate GPI activity at least in part by this mechanism. These results imply that macrophage surface phospholipases play important roles in the GPI-induced innate immune responses and malaria pathogenesis.