Alexander Poltorak

Phylogenetic variation and polymorphism at the Toll-like receptor 4 locus (TLR4)

BackgroundDifferences in responses to bacterial surface lipopolysaccharides (LPSs) are apparent between and within mammalian species. It has been shown in mice that resistance to LPS is caused by defects in the Toll-like receptor 4 gene (Tlr4), the product of which is thought to bind LPS and mediate LPS signal transduction in immune system cells.ResultsWe have sequenced the Toll-like receptor 4 gene of humans (TLR4; 19.0 kilobases, kb) and mice (Tlr4; 91.7 kb), as well as the coding region and splice junctions of Tlr4 from 35 mouse (Mus musculus) strains, from the chimpanzee and from the baboon. No other discernible genes or regions of interspecies conservation lies close to Tlr4 and, in both humans and mice, flanking sequences and introns are rich in repeats of retroviral origin. Interstrain analyses reveal that Tlr4 is a polymorphic protein and that the extracellular domain is far more variable than the cytoplasmic domain, both among strains and among species. The cytoplasmic domain of the Tlr4 protein is highly variable at the carboxy-terminal end.ConclusionsWe suggest that selective evolutionary pressure exerted by microbes expressing structurally distinguishable LPS molecules has produced the high level of variability in the Tlr4 extracellular domain. The highly variable carboxy-terminal region of the cytoplasmic domain is likely to determine the magnitude of the response to LPS within a species.

Identification of Toll-like receptor 4 (Tlr4) as the sole conduit for LPS signal transduction: genetic and evolutionary studies

One of the most interesting questions confronting the LPS signal transduction field during the 1990s concerned the identity of a postulated co-receptor for LPS. In 1990, LPS was shown to bind to CD14 on the surface of mononuclear phagocytic cells,1 and subsequent gene knockout work proved that the interaction was an important one in LPS signal transduction.2 However, since CD14 was known to be a glycosylphosphoinositol-tethered protein, bereft of a cytoplasmic domain, it had no obvious means of evoking a transmembrane signal. How, then, could the LPS signal be transduced into the cytoplasm? At one time or another, workers in the LPS field held that LPS might signal ‘non-specifically’; that is, without the aid of a protein intermediary within the membrane. Rather, LPS itself might insert into the membrane, and perturb membrane structure in such a way as to initiate a signal. Another school of thought held that a proteolytic enzyme within the membrane or within the cytoplasm might be activated in response to LPS. Among other protein targets of LPS, virtually every type of signaling molecule known has been mentioned, alongside structural proteins (e.g., those involved in vesicle trafficking,3 or microtubular assemblies4). Conventional tools of gene identification, including expression cDNA cloning, subtraction cDNA cloning, the use of the yeast two-hybrid system, and physical approaches to the detection of interactions between proteins all failed to disclose a co-receptor that would bind LPS alongside CD14 and initiate signal transduction. The co-receptor, now known to be the Toll-like receptor 4 (Tlr4) was finally identified as a result of positional cloning work involving the C3H/HeJ and C57BL/10ScCr mouse strains, of which a longer account has been written separately.5 Each of these strains has been known for many years to be profoundly resistant to LPS.6,7 The reason for their resistance has emerged with the discovery that both strains bear mutations affecting the Tlr4 locus. This finding was the outcome of 5 years of intensive genetic and physical mapping work, which culminated in the determination that: (i) Tlr4 is the sole gene in the Lps critical region;8 (ii) a point mutation modifies the Tlr4 cytoplasmic domain in C3H/HeJ mice;9 and (iii) a deletion removes the entire Tlr4 coding region in C57BL/10ScCr mice.9,10 The essential role of Tlr4 in LPS signaling was later confirmed by analysis of mice with a Tlr4 knockout mutation.11

The role of Toll‐like receptor 4 versus interleukin‐12 in immunity to respiratory syncytial virus

Toll‐like receptors (TLR) and IL‐12 represent key elements of innate immunity. Using C57BL/10 ScCr mice it was shown that TLR4 is important for control of infection with respiratory syncytial virus (RSV). Since these mice have an additional defect in the IL‐12R, we reinvestigated immunity to RSV in several C57BL/10 and BALB/c mouse strains lacking a functional TLR4, a functional IL‐12–IL‐12R interaction or both. In the absence of a functional IL‐12 axis, early virus control was impaired in C57BL/10 mice, but not in BALB/c mice. By contrast, TLR4 had no impact on RSV elimination. Pulmonary NK cell recruitment was impaired in IL‐12 deficient BALB/c mice and NK cytotoxicity was reduced in IL‐12/IL‐12R‐deficient mice of both genetic backgrounds. Absence of TLR4 had no impact on NK cell recruitment or NK activity nor on recruitment of other pulmonary inflammatory cells. Activation of RSV‐specific T cell immunity, including T cell mediated immunopathology, was normal in all mutant strains. These findings clearly argue against a significant role for TLR4 and define a limited role for IL‐12 in primary murine RSV infection.

Toll‐like receptor 4 expression levels determine the degree of LPS‐susceptibility in mice

C57BL/10ScCr (Cr) mice carry a deletion of the Toll‐like receptor 4 (tlr4) gene (i.e. they are tlr40/0) and are thus refractory to LPS effects. Insertion of wild‐type tlr4 transgene into the tlr40/0 Cr germ line endowed LPS susceptibility in the two transgenic lines created, indicating that TLR4 is the only limiting factor for LPS responsiveness in Cr mice. The absolute levels of tlr4 mRNA expressed by the heterozygous transgenic (tlr4Tr/0), wild‐type C57BL/10ScSn (Sn) (tlr4+/+) and heterozygous F1 (Sn × Cr) (tlr4+/0) mice varied markedly. However, the pattern of distribution of expression in the different organs was the same in all strains. In different biological assays (B cell mitogenicity, cytokine induction and lethal toxicity) the degree of LPS response obtained in the different strains of mice correlated with the levels of tlr4 mRNA expression. In macrophages, investigation of the LPS‐induced cytokine (IL‐6) response revealed a linear relationship between the response and the logarithm of TLR4–MD‐2 levels.

A Point Mutation in the IL-12Rβ2 Gene Underlies the IL-12 Unresponsiveness of Lps-Defective C57BL/10ScCr Mice

Lps-defective C57BL/10ScCr (Cr) mice are homozygous for a deletion encompassing Toll-like receptor 4 that makes them refractory to the biological activity of LPS. In addition, these mice exhibit an inherited IL-12 unresponsiveness resulting in impaired IFN-γ responses to different microorganisms. By positional cloning methods, we show here that this second defect of Cr mice is due to a mutation in a single gene located on mouse chromosome 6, in close proximity to the Igκ locus. The gene is IL-12Rβ2. Cr mice carry a point mutation creating a stop codon that is predicted to cause premature termination of the translated IL-12Rβ2 after a lysine residue at position 777. The truncated β2 chain can still form a heterodimeric IL-12R that allows phosphorylation of Janus kinase 2, but, unlike the wild-type IL-12R, can no longer mediate phosphorylation of STAT4. Because the phosphorylation of STAT4 is a prerequisite for the IL-12-mediated induction of IFN-γ, its absence in Cr mice is responsible for their defective IFN-γ response to microorganisms.

IRAK1BP1 inhibits inflammation by promoting nuclear translocation of NF-κB p50

An orchestrated balance of pro- and antiinflammatory cytokine release is critical for an innate immune response sufficient for pathogen defense without excessive detriment to host tissues. By using an unbiased forward genetic approach, we previously reported that IL-1R–associated kinase 1 binding protein 1 (IRAK1BP1) down-modulates Toll-like receptor-mediated transcription of several proinflammatory cytokines. To gain insights into the physiological relevance of the inhibitory role of IRAK1BP1 in inflammation, we generated mutant mice lacking IRAK1BP1. Here we report that IRAK1BP1 does not inhibit signaling pathways generally but rather changes the transcriptional profile of activated cells, leading to an increase in IL-10 production and promoting LPS tolerance. This shift in cytokine transcription correlates with an increased ratio of functional NF-κB subunit dimers comprised of p50/p50 homodimers relative to p50/p65 heterodimers. The increase in nuclear p50/p50 was consistent with the ability of IRAK1BP1 to bind to the p50 precursor molecule and IκB family member p105. We conclude that IRAK1BP1 functions through its effects on NF-κB as a molecular switch to bias innate immune pathways toward the resolution of inflammation.

Forward genetic analysis of Toll-like receptor responses in wild-derived mice reveals a novel antiinflammatory role for IRAK1BP1

Although inflammatory cytokines produced by activation of Toll-like receptors (TLRs) are essential for early host defense against infection, they also mediate a vast array of pathologies, including autoimmune disease, hypersensitivity reactions, and sepsis. Thus, numerous regulatory mechanisms exist in parallel with proinflammatory pathways to prevent excessive release of these potent effector molecules. We report elucidation of a novel regulatory function for interleukin receptor–associated kinase (IRAK)-1 binding protein 1 (IRAK1BP1, also known as SIMPL) through quantitative trait locus mapping of the TLR response in wild-derived mouse strains. This gene emerged as a negative regulator of TLR2-mediated interleukin (IL)-6 production in MOLF/Ei mice, which expressed IRAK1BP1 mRNA in an allele-specific manner when crossed with the C57BL/6J strain. Human peripheral blood mononuclear cells and primary macrophages from two other wild-derived mouse strains also induced IRAK1BP1 mRNA by 4 hours after stimulation with agonists of various TLRs. Examination of its effects on IL-6 and other cytokines demonstrated that IRAK1BP1 regulates transcription of a specific subset of TLR-responsive genes, producing an overall antiinflammatory profile. Our results reveal that IRAK1BP1 is a critical factor in preventing dangerous overproduction of proinflammatory cytokines by the innate immune system and in influencing the specificity of TLR responses. Furthermore, these results show that the genetic diversity of wild-derived mouse strains makes them a valuable model of important human gene functions that have been lost in some laboratory-inbred strains.

Genetic Control of Severe Egg-Induced Immunopathology and IL-17 Production in Murine Schistosomiasis

Infection with the trematode parasite Schistosoma mansoni results in a distinct heterogeneity of disease severity, both in humans and in an experimental mouse model. Severe disease is characterized by pronounced hepatic egg-induced granulomatous inflammation in a proinflammatory cytokine environment, whereas mild disease corresponds with reduced hepatic inflammation in a Th2 skewed cytokine environment. This marked heterogeneity indicates that genetic differences play a significant role in disease development, yet little is known about the genetic basis of dissimilar immunopathology. To investigate the role of genetic susceptibility in murine schistosomiasis, quantitative trait loci analysis was performed on F2 progeny derived from SJL/J and C57BL/6 mice, which develop severe and mild pathology, respectively. In this study, we show that severe liver pathology in F2 mice 7 wk after infection significantly correlated with an increase in the production of the proinflammatory cytokines IL-17, IFN-γ, and TNF-α by schistosome egg Ag-stimulated mesenteric lymph node cells. Quantitative trait loci analysis identified several genetic intervals controlling immunopathology as well as IL-17 and IFN-γ production. Egg granuloma size exhibited significant linkage to two loci, D4Mit203 and D17Mit82, both of which were inherited in a BL/6 dominant manner. Furthermore, a significant reduction of hepatic granulomatous inflammation and IL-17 production in interval-specific congenic mice demonstrated that the two identified genetic loci have a decisive effect on the development of immunopathology in murine schistosomiasis.

Toll-like Receptor-mediated Down-regulation of the Deubiquitinase Cylindromatosis (CYLD) Protects Macrophages from Necroptosis in Wild-derived Mice

Background: Necroptosis is a regulated signaling pathway leading to necrotic cell death. Results: Genetic mapping identified that down-regulation of the deubiquitinase CYLD confers resistance to necroptosis in a wild-derived mouse strain. Conclusion: Different strains of inbred mice regulate cell death pathways using distinct mechanisms. Significance: Genetic diversity of wild-derived mice underlies phenotypic diversity, which can identify novel mechanisms of regulation in cell death signaling. Pathogen recognition by the innate immune system initiates the production of proinflammatory cytokines but can also lead to programmed host cell death. Necroptosis, a caspase-independent cell death pathway, can contribute to the host defense against pathogens or cause damage to host tissues. Receptor-interacting protein (RIP1) is a serine/threonine kinase that integrates inflammatory and necroptotic responses. To investigate the mechanisms of RIP1-mediated activation of immune cells, we established a genetic screen on the basis of RIP1-mediated necroptosis in wild-derived MOLF/EiJ mice, which diverged from classical laboratory mice over a million years ago. When compared with C57BL/6, MOLF/EiJ macrophages were resistant to RIP1-mediated necroptosis induced by Toll-like receptors. Using a forward genetic approach in a backcross panel of mice, we identified cylindromatosis (CYLD), a deubiquitinase known to act directly on RIP1 and promote necroptosis in TNF receptor signaling, as the gene conferring the trait. We demonstrate that CYLD is required for Toll-like receptor-induced necroptosis and describe a novel mechanism by which CYLD is down-regulated at the transcriptional level in MOLF/EiJ macrophages to confer protection from necroptosis.

Mannose receptor 1 mediates cellular uptake and endosomal delivery of CpG-motif containing oligodeoxynucleotides.

Recognition of microbial components is critical for activation of TLRs, subsequent innate immune signaling, and directing adaptive immune responses. The DNA sensor TLR9 traffics from the endoplasmic reticulum to endolysosomal compartments where it is cleaved by resident proteases to generate a competent receptor. Activation of TLR9 by CpG-motif containing oligodeoxynucleotides (CpG ODNs) is preceded by agonist endocytosis and delivery into the endolysosomes. The events that dictate this process remain largely unknown; furthermore, it is unclear whether the receptors involved in mediating uptake of exogenous DNA are conserved for both naturally derived pathogenic DNA and synthetic ODNs. In this study, we report that peritoneal macrophages from a wild-derived inbred mouse strain, MOLF/Ei, are hyporesponsive to CpG ODN but are fully responsive to bacterial DNA, thus implying that microbial recognition is not fully recapitulated by a synthetic analog. To identify the gene responsible for the CpG ODN defect, we have performed genome-wide linkage analysis. Using N2 backcross mice, we mapped the trait with high resolution to a single locus containing Mrc1 as the gene conferring the trait. We show that mannose receptor 1 (MRC1; CD206) is involved in CpG ODN uptake and trafficking in wild-derived MOLF/Ei peritoneal macrophages. Furthermore, we show that other strains of wild-derived mice also require MRC1 for CpG-induced cytokine responses. These findings reveal novel functions for MRC1 and demonstrate that wild-derived mice are important and indispensable model for understanding naturally occurring regulators of inflammatory responses in innate immune pathways.