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Here we have identified 'triple D' (3d), a recessive N-ethyl-N-nitrosourea-induced mutation and phenotype in which no signaling occurs via the intracellular Toll-like receptors 3, 7 and 9 (sensors for double-stranded RNA, single-stranded RNA and unmethylated DNA, respectively). The 3d mutation also prevented cross-presentation and diminished major histocompatibility complex class II presentation of exogenous antigen; it also caused hypersusceptibility to infection by mouse cytomegalovirus and other microbes. By positional identification, we found 3d to be a missense allele of Unc93b1, which encodes the 12-membrane-spanning protein UNC-93B, a highly conserved molecule found in the endoplasmic reticulum with multiple paralogs in mammals. Innate responses to nucleic acids and exogenous antigen presentation, which both initiate in endosomes, thus seem to depend on an endoplasmic reticulum–resident protein, which suggests communication between these organellar systems.

Type I interferons (IFN) play a critical role in the Toll-like receptor (TLR)-mediated expression of B7 costimulatory family members. For example, LPS-induced up-regulation of CD80 (B7.1) and CD86 (B7.2) is abrogated in antigen-presenting cells (APC) deficient in TRIF or TRAM, two adaptors that are responsible for TLR4-mediated production of Type I IFN. In this report, we demonstrate that LPS-induced up-regulation of B7-related protein 1 (B7RP-1), a ligand for ICOS, is dependent primarily upon the MyD88-dependent signaling pathway. Signaling via the TRIF pathway sharply limits MyD88-dependent B7RP-1 up-regulation. Hence, LPS induces significantly higher B7RP-1 expression on TRIF- or TRAM-deficient mouse peritoneal macrophages and on TRIF-deficient mouse splenic B cells as compared to wild-type cells. Further studies reveal that Type I IFN are general suppressors of TLR-mediated up-regulation of B7RP-1. These data indicate that Type I IFN play a dual role in the TLR-mediated expression of B7 costimulatory family members and suggest that they may act to limit B7RP-1 expression and thus limit signals derived from B7RP-1-ICOS interaction.

Several subsets of dendritic cells have been shown to produce type I IFN in response to viral infections, thereby assisting the natural killer cell-dependent response that eliminates the pathogen. Type I IFN production can be induced both by unmethylated CpG-oligodeoxynucleotide and by double-stranded RNA. Here, we describe a codominant CpG-ODN unresponsive phenotype that results from anN-ethyl-N-nitrosourea-induced missense mutation in theTlr9gene (Tlr9CpG1). Mice homozygous for theTlr9CpG1allele are highly susceptible to mouse cytomegalovirus infection and show impaired infection-induced secretion of IFN-α/β and natural killer cell activation. We also demonstrate that both the Toll-like receptor (TLR) 9 → MyD88 and TLR3 → Trif signaling pathways are activatedin vivoon viral inoculation, and that each pathway contributes to innate defense against systemic viral infection. Whereas both pathways lead to type I IFN production, neither pathway offers full protection against mouse cytomegalovirus infection in the absence of the other. TheTlr9CpG1mutation alters a leucine-rich repeat motif and lies within a receptor domain that is conserved within the evolutionary cluster encompassing TLRs 7, 8, and 9. In other TLRs, including three mouse-specific TLRs described in this paper, the affected region is not represented. The phenotypic effect of theTlr9CpG1allele thus points to a critical role for TLR9 in viral sensing and identifies a vulnerable amino acid within the ectodomain of three TLR proteins, essential for a ligand response.

Lipopolysaccharide (endotoxin, LPS) is a major recognition marker for the detection of gram-negative bacteria by the host and a powerful initiator of the inflammatory response to infection. Using S- and R-form LPS from wild-type and R-mutants of Salmonella and E. coli, we show that R-form LPS readily activates mouse cells expressing the signaling receptor Toll-like receptor 4/myeloid differentiation protein 2 (TLR4/MD-2), while the S-form requires further the help of the LPS-binding proteins CD14 and LBP, which limits its activating capacity. Therefore, the R-form LPS under physiological conditions recruits a larger spectrum of cells in endotoxic reactions than S-form LPS. We also show that soluble CD14 at high concentrations enables CD14-negative cells to respond to S-form LPS. The presented in vitro data are corroborated by an in vivo study measuring TNF-alpha levels in response to injection of R- and S-form LPS in mice. Since the R-form LPS constitutes ubiquitously part of the total LPS present in all wild-type bacteria its contribution to the innate immune response and pathophysiology of infection is much higher than anticipated during the last half century.

The recessive mutation 'Heedless' (hdl) was detected in third-generation N-ethyl-N-nitrosourea-mutated mice that showed defective responses to microbial inducers. Macrophages from Heedless homozygotes signaled by the MyD88-dependent pathway in response to rough lipopolysaccharide (LPS) and lipid A, but not in response to smooth LPS. In addition, the Heedless mutation prevented TRAM-TRIF-dependent signaling in response to all LPS chemotypes. Heedless also abolished macrophage responses to vesicular stomatitis virus and substantially inhibited responses to specific ligands for the Toll-like receptor 2 (TLR2)-TLR6 heterodimer. The Heedless phenotype was positionally ascribed to a premature stop codon in Cd14. Our data suggest that the TLR4-MD-2 complex distinguishes LPS chemotypes, but CD14 nullifies this distinction. Thus, the TLR4-MD-2 complex receptor can function in two separate modes: one in which full signaling occurs and one limited to MyD88-dependent signaling.

Toll-like receptor 2 (TLR2) is required for the recognition of numerous molecular components of bacteria, fungi and protozoa. The breadth of the ligand repertoire seems unusual, even if one considers that TLR2 may form heteromers with TLRs 1 and 6 (ref. 12), and it is likely that additional proteins serve as adapters for TLR2 activation. Here we show that an N-ethyl-N-nitrosourea-induced nonsense mutation of Cd36 (oblivious) causes a recessive immunodeficiency phenotype in which macrophages are insensitive to the R-enantiomer of MALP-2 (a diacylated bacterial lipopeptide) and to lipoteichoic acid. Homozygous mice are hypersusceptible to Staphylococcus aureus infection. Cd36(obl) macrophages readily detect S-MALP-2, PAM(2)CSK(4), PAM(3)CSK(4) and zymosan, revealing that some--but not all--TLR2 ligands are dependent on CD36. Already known as a receptor for endogenous molecules, CD36 is also a selective and nonredundant sensor of microbial diacylglycerides that signal via the TLR2/6 heterodimer.

The innate immune system senses pathogens largely through signals initiated by proteins known as 'Toll-like receptors' (TLRs), of which ten representatives are known to be encoded in the human genome. The understanding of the biochemical circuitry that maintains the innate capacity for immune recognition and response has loomed as a major hurdle in immunology. A total of five adapter proteins with cytoplasmic domain homology to the TLRs are known to exist in mammals. These proteins show preferential association with individual TLR family members, giving a particular character to the signals that distinct microorganisms initiate, and also initiate the adaptive immune response. The adaptive immune response is dependent upon upregulation of costimulatory molecules (UCM) such as CD80 and CD86. Forward genetic analysis has revealed that this upregulation depends upon an adapter encoded by a locus known as Lps2, and upon type I interferon receptor signaling.

Toll-like receptors (TLRs) are key sensors of the innate immune system, and individual TLRs respond to specific molecules derived from microbes. MyD88 is a Toll/Interleukin-1/Resistance (TIR) domain-containing adaptor protein required for signaling by all TLRs except TLR3. While the structural basis of association between MyD88 and TIR-domain receptors is obscure, MyD88-deficient mice show no responses to bacterial flagellin, peptidoglycan (PGN), lipoteichoic acid (LTA), bacterial lipopeptides such as PAM2CSK4, PAM3CSK4 and R- or S-MALP-2, DNA bearing unmethylated CpG dinucleotides (CpG DNA), or Resiquimod (RSQ). Using germline ENU mutagenesis, we have produced a large number of phenotypic variants that have abnormal TLR signaling. We now report the identification of a new mutation called Pococurante (Poc), originally detected in screening because macrophages from this mouse showed no response to the tri-acylated lipopeptide PAM3CSK4, the di-acylated lipopeptide S-MALP-2, LTA, CpG DNA, RSQ, and a markedly reduced response to LPS: the ligands for TLRs 2/1, 2/6, 9, 7 and 4 respectively. They also had no response to interleukin-1, a cytokine that signals by way of a MyD88-dependent TIR domain receptor. However, Poc mice showed a normal response to PGN, as well as R-MALP-2 and PAM2CSK4 lipopeptides. The latter three ligands are sensed in a TLR2-dependent, MyD88-dependent fashion. The Poc phenotype was ascribed to a point mutation of MyD88 affecting a surface residue (I179N). Because the mutation is discriminatory, permitting MyD88 to carry a signal from some TIR domain receptors but not others, we infer that it resides at the receptor:adaptor signaling interface. A new model of TIR receptor:adaptor interaction is proposed on the basis of docking studies that take account of the Poc phenotype, made using the protein-protein docking program SURFDOCK. We note that S-MALP-2 is dependent upon TLR2/6 heterodimers, while PAM3CSK4 sensing depends upon TLR2/1 heterodimers. Since the Poc mutation forbids detection of both these ligands while it allows detection of PAM2CSK4 and R-MALP-2, it may be inferred that TLR2 signal transduction entails greater structural diversity than was previously supposed. The involvement of TLR2 homodimers, or the incorporation of subunits yet unknown into the receptor complex, cannot be excluded.

The mammalian Toll-like receptors (TLRs) comprise a key interface between cells of the host and all classes of microbial pathogen. By sensing nucleic acids, the endosomal TLRs 3, 7, and 9 play a particularly important role in the detection of viral infection, and permit the host to mount an immediate and efficacious anti-viral response. In an effort to identify novel components of the TLR signaling apparatus, we have pursued a program of germline saturation mutagenesis with N-ethyl-N-nitrosourea (ENU) in mice. The germline mutants are produced on the C57BL/6 background, and peritoneal macrophages from individual animals are screened for their competence to respond to TLR-dependent microbial inducers. A strong phenodeviant called 3d was identified in the F3 generation as a non-responder to nucleoside-based molecules such as unmethylated CpG oligodeoxynucleotides, Resiquimod (a drug of the imidazoquinoline class) and Poly I:C. TNFa production induced by TLRs 3, 7, and 9 was completely prevented in 3d homozygotes. However, heterozygotes were unaffected, and the ability to produce TNFa in response to other bacterial compounds, sensed by TLRs 1, 2, 4, and 6, was intact. 3d homozygotes show extreme susceptibility to infection by mouse cytomegalovirus (MCMV) infection in vivo. After IP inoculation with MCMV (104 PFU), 3d mutants showed impaired production of type I and type II interferons, TNF, and IL-12 in serum (p

The mammalian Toll-like receptors (TLRs) activate cells of the innate immune system when stimulated by diverse ligands of microbial origin. In some instances, these ligands are directly engaged by the TLRs; however, this is not necessarily true in all cases. TLR2 recognizes multiple, structurally disparate microbial ligands, consistent with a requirement for co-receptors in ligand binding. Using N-ethyl-N-nitrosourea, we generated the recessive immunodeficiency phenotype oblivious, in which macrophages show diminished awareness of the S-enantiomer of the di-acylated bacterial lipopeptide MALP-2 and lipoteichoic acid, together with spontaneous ocular colonization by Gram-positive organisms and hypersusceptibility to Staphylococcus aureus infection. Oblivious macrophages readily detect the tri-acylated bacterial lipopeptide PAM3CSK4 as well as zymosan, revealing that some TLR2 ligands are activated via an Oblivious-independent pathway. The gene responsible for the oblivious phenotype has been positionally cloned. In its ability to carry the lipoteichoic acid and MALP-2 signal to the transmembrane signaling receptors TLR2 and TLR6, Oblivious serves a function analogous to CD14, which concentrates the lipopolysacchardide signal for transduction by TLR4. Besides microbial molecules, oblivious also serves as a receptor for endogenous molecules and may mediate (some) of the inflammatory events involved in the development of atherosclerosis.