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Toll-like receptors.

The ability of a host to sense invasion by pathogenic organisms and to respond appropriately to control infection is paramount to survival. In the case of sepsis and septic shock, however, an exaggerated systemic response may, in fact, contribute to the morbidity and mortality associated with overwhelming infections. The innate immune system has evolved as the first line of defense against invading microorganisms. The Toll-like receptors (TLRs) are a part of this innate immune defense, recognizing conserved patterns on microorganisms. These TLRs and their signaling pathways are represented in such diverse creatures as mammals, fruit flies, and plants. Ten members of the TLR family have been identified in humans, and several of them appear to recognize specific microbial products, including lipopolysaccharide, bacterial lipoproteins, peptidoglycan, and bacterial DNA. Signals initiated by the interaction of TLRs with specific microbial patterns direct the subsequent inflammatory response. Thus, TLR signaling represents a key component of the innate immune response to microbial infection.

Animals↗

New insights into the post-translational modification of Toll-like receptor signaling molecules.

Deregulation of Toll-like receptor (TLR) mediated responses can have devastating effects on the host if left unchecked. It is, therefore, critical that control is exerted at several levels. In this review, we discuss post-translational modification of TLRs and their associated signaling molecules as one such means of control. In particular, we focus on the phosphorylation, ubiquitination and de-ubiquitination of various components of TLR signaling pathways.

Animals↗

Evolution and integration of innate immune recognition systems: the Toll-like receptors.

Toll-like receptors (TLRs) recognize specific components of micro-organisms and trigger the activation of innate immunity. TLR-mediated signaling pathways are now rapidly being elucidated. TLR signaling originates from the cytoplasmic Toll/IL-1 receptor (TIR) domain, which is conserved among all TLRs. Furthermore, TIR domain-containing adaptors, such as MyD88, TRIF, TIRAP and TRAM, play essential roles in TLR signaling. MyD88 is essential for inflammatory cytokine production via all TLRs, whereas TRIF is involved in TLR3- and TLR4-mediated MyD88-independent induction of IFN-beta. Thus, innate immunity represents a skillful system that senses microbial invasion and initiates appropriate immune responses.

Adaptor Proteins, Vesicular Transport↗

Pellino 1 is required for interleukin-1 (IL-1)-mediated signaling through its interaction with the IL-1 receptor-associated kinase 4 (IRAK4)-IRAK-tumor necrosis factor receptor-associated factor 6 (TRAF6) complex.

The signaling pathway downstream of the mammalian interleukin-1 receptor (IL-1R)/Toll-like receptor (TLR) is evolutionally conserved with that mediated by the Drosophila Toll protein. Toll initiates its signal through the adapter molecule Tube and the serine-threonine kinase Pelle. Pelle is highly homologous to members of the IL-1R-associated kinase (IRAK) family in mammals. Recently, a novel Pelle-interacting protein called Pellino was identified in Drosophila. We now report a mammalian counterpart of Pellino, termed Pellino 1, which is required for NF kappa B activation and IL-8 gene expression in response to IL-1, probably through its signal-dependent interaction with IRAK4, IRAK, and the tumor necrosis factor receptor-associated factor 6 (TRAF6). The Pellino 1-IRAK-IRAK4-TRAF6 signaling complex is likely to be intermediate, located between the IL-1 receptor complex and the TAK1 complex in the IL-1 pathway.

Amino Acid Sequence↗

Upregulation of costimulatory molecules induced by lipopolysaccharide and double-stranded RNA occurs by Trif-dependent and Trif-independent pathways.

Both lipopolysaccharide (LPS) and double-stranded RNA (dsRNA) are adjuvants for the adaptive immune response, inducing upregulation of costimulatory molecules (UCM) on antigen-presenting cells. Trif, an adapter protein that transduces signals from Toll-like receptor 4 (TLR4) and TLR3, permits the induction of many cytokines, including interferon-beta, which signals through the type I interferon receptor. We show here that LPS-induced UCM was strictly dependent on the TLR4-->Trif axis, whereas dsRNA-induced UCM was only partly dependent on the TLR3-->Trif axis. But both LPS- and dsRNA-induced UCM were entirely dependent on type I interferon receptor signaling. These findings show that UCM involves an autocrine or paracrine loop, and indicate that an alternative TLR3-independent, Trif-independent pathway contributes to dsRNA-induced UCM.

Adaptor Proteins, Signal Transducing↗

Toll-like receptor activation in the pathogenesis of systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is characterized by the production of pathogenic autoantibodies to nucleoproteins and DNA. The level of anti-DNA antibodies correlates with disease severity, and the deposition of these immune complexes (ICs) in the kidneys is thought to contribute to disease pathogenesis. Recent evidence suggests that the DNA component of ICs purified from SLE patients (SLE DNA-ICs) contributes to the development of SLE pathology. SLE DNA-ICs induce proliferation of self-reactive B cells and cytokine production by plasmacytoid dendritic cells (PDCs) in a TLR9-dependent manner. One of the cytokines induced by DNA-containing ICs is interferon alpha (IFN-alpha). Elevated serum levels of IFN-alpha and overexpression of interferon-induced genes have been observed in SLE patient blood and shown to correlate with disease severity. We have recently found that the mechanism of IFN-alpha production by PDCs depends on TLR9 and FcgammaRIIa (CD32), and CD32 delivers SLE DNA-ICs to intracellular lysosomes containing TLR9. This CD32-TLR9 pathway, which is operative in PDCs, is distinct from the BCR/TLR9 pathway in B cells and may prove to be a novel target for future SLE therapies. In this article, the role of toll-like receptors, cytokines, and Fc receptors expressed by PDCs in the pathogenesis of SLE is summarized.

Animals↗

A DAP12-mediated pathway regulates expression of CC chemokine receptor 7 and maturation of human dendritic cells.

Gene targeting of the adaptor molecule DAP12 in mice caused abnormal distribution and impaired antigen presentation capacity of dendritic cells (DCs). However, the DAP12-associated receptors expressed on DCs and their functions have not been identified yet. Here we show that the triggering receptor expressed on myeloid cells-2 (TREM-2) is a cell surface receptor on human monocyte-derived DCs, which is associated with DAP12. TREM-2/DAP12 promotes upregulation of CC chemokine receptor 7, partial DC maturation, and DC survival through activation of protein tyrosine kinases and extracellular signal-regulated kinase. In contrast to Toll-like receptor-mediated signaling, TREM2/DAP12 stimulation is independent of nuclear factor-kappaB and p38 stress-activated protein kinase. This novel DC activation pathway may regulate DC homeostasis and amplify DC responses to pathogens, explaining the phenotype observed in DAP12-deficient mice.

Adaptor Proteins, Signal Transducing↗

Lack of genetic association of the Toll-like receptor 4 (TLR4) Asp299Gly and Thr399Ile polymorphisms with spondylarthropathies in a Hungarian population.

OBJECTIVES: Bacteria have long been suggested as aetiological factors in the genetically susceptible host in spondylarthropathies, including ankylosing spondylitis (AS) and reactive arthritis (ReA). Variability of the Toll-like receptor 4 (TLR4) gene has been shown to play a role in the inflammatory response to certain bacterial infections. We investigated whether TLR4 Asp299Gly and Thr399Ile polymorphisms contribute to the genetic background of spondylarthropathies in a cohort of Hungarian patients with AS and ReA. METHODS: DNA was obtained from patients with AS (n=138), ReA (n=91) and ethnically matched healthy controls (n=140). Genotyping was carried out by polymerase chain reaction-restriction fragment length polymorphism analysis and the results were confirmed by direct sequencing. RESULTS: No significant differences in allele or genotype frequencies were observed between controls and either the AS patients or the ReA patients. Clinical characteristics of these groups were unrelated to the presence of any of these polymorphisms. CONCLUSIONS: Toll-like receptor 4 Asp299Gly and Thr399Ile polymorphisms do not contribute to disease susceptibility in either AS or ReA. Functional abnormalities of the TLR4 signalling pathway suggested in spondylarthropathies seem not to be genetically determined by these two common polymorphisms.

Adult↗

Toll-like receptor-mediated responses of primary intestinal epithelial cells during the development of colitis.

The interleukin-2-deficient (IL-2(-/-)) mouse model of ulcerative colitis was used to test the hypothesis that colonic epithelial cells (CEC) directly respond to bacterial antigens and that alterations in Toll-like receptor (TLR)-mediated signaling may occur during the development of colitis. TLR expression and activation of TLR-mediated signaling pathways in primary CEC of healthy animals was compared with CEC in IL-2(-/-) mice during the development of colitis. In healthy animals, CEC expressed functional TLR, and in response to the TLR4 ligand LPS, proliferated and secreted the cytokines IL-6 and monocyte chemoattractant protein-1 (MCP-1). However, the TLR-responsiveness of CEC in IL-2(-/-) mice was different with decreased TLR4 responsiveness and augmented TLR2 responses that result in IL-6 and MCP-1 secretion. TLR signaling in CEC did not involve NF-kappaB (p65) activation with the inhibitory p50 form of NF-kappaB predominating in CEC in both the healthy and inflamed colon. Development of colitis was, however, associated with the activation of MAPK family members and upregulation of MyD88-independent signaling pathways characterized by increased caspase-1 activity and IL-18 production. These findings identify changes in TLR expression and signaling during the development of colitis that may contribute to changes in the host response to bacterial antigens seen in colitis.

Adaptor Proteins, Signal Transducing↗

MyD88 but not TRIF is essential for osteoclastogenesis induced by lipopolysaccharide, diacyl lipopeptide, and IL-1alpha.

Myeloid differentiation factor 88 (MyD88) plays essential roles in the signaling of the Toll/interleukin (IL)-1 receptor family. Toll-IL-1 receptor domain-containing adaptor inducing interferon-beta (TRIF)-mediated signals are involved in lipopolysaccharide (LPS)-induced MyD88-independent pathways. Using MyD88-deficient (MyD88-/-) mice and TRIF-deficient (TRIF-/-) mice, we examined roles of MyD88 and TRIF in osteoclast differentiation and function. LPS, diacyl lipopeptide, and IL-1alpha stimulated osteoclastogenesis in cocultures of osteoblasts and hemopoietic cells obtained from TRIF-/- mice, but not MyD88-/- mice. These factors stimulated receptor activator of nuclear factor-kappaB ligand mRNA expression in TRIF-/- osteoblasts, but not MyD88-/- osteoblasts. LPS stimulated IL-6 production in TRIF-/- osteoblasts, but not TRIF-/- macrophages. LPS and IL-1alpha enhanced the survival of TRIF-/- osteoclasts, but not MyD88-/- osteoclasts. Diacyl lipopeptide did not support the survival of osteoclasts because of the lack of Toll-like receptor (TLR)6 in osteoclasts. Macrophages expressed both TRIF and TRIF-related adaptor molecule (TRAM) mRNA, whereas osteoblasts and osteoclasts expressed only TRIF mRNA. Bone histomorphometry showed that MyD88-/- mice exhibited osteopenia with reduced bone resorption and formation. These results suggest that the MyD88-mediated signal is essential for the osteoclastogenesis and function induced by IL-1 and TLR ligands, and that MyD88 is physiologically involved in bone turnover.

Adaptor Proteins, Signal Transducing↗

Essential roles for NF-kappa B and a Toll/IL-1 receptor domain-specific signal(s) in the induction of I kappa B-zeta.

I kappa B-zeta, a new negative-regulator of nuclear factor-kappa B (NF-kappa B), is strongly induced by lipopolysaccharide or interleukin-1 beta stimulation, but not by tumor necrosis factor-alpha. Here, we analyzed the mechanisms for transcriptional induction of I kappa B-zeta. I kappa B-zeta mRNA was induced by overexpression of MyD88 or TRAF6, but not TRAF2. Stimulation of macrophages with peptidoglycan or CpG DNA, which activated Toll-like receptor 2 or 9, respectively, also resulted in I kappa B-zeta induction. Thus, activation of the MyD88-dependent signaling pathway, commonly found downstream of different Toll/interleukin-1 receptor (TIR) domains, is sufficient for I kappa B-zeta induction. The induction was inhibited by treatment with various inhibitors of NF-kappa B activation or by overexpressing I kappa B-alpha or beta, indicating essential roles for NF-kappa B in I kappa B-zeta induction. However, overexpression of the NF-kappa B subunits induced I kappa B-alpha, but not I kappa B-zeta. These results indicate the existence of another signal essential for I kappa B-zeta induction, which is specifically mediated by the TIR domain-mediated signaling pathway.

Adaptor Proteins, Signal Transducing↗

Interferon gene regulation: not all roads lead to Tolls.

Many infectious agents elicit a type I interferon response but, until recently, the molecular details that coordinate interferon (IFN)-alpha and -beta expression during infection were unknown. Innate immune pattern recognition receptors, including Toll-like receptors and cytoplasmic RNA helicases such as retinoic acid-inducible gene, that sense viral nucleic acids have been discovered. Using distinct mechanisms, these receptors trigger cellular signaling pathways, culminating in the activation of interferon regulatory factors that transcriptionally induce IFN-alpha and IFN-beta genes.

Animals↗

Cloning and characterization of two Toll/Interleukin-1 receptor-like genes TIL3 and TIL4: evidence for a multi-gene receptor family in humans.

Remarkable structural and functional similarities exist between the Drosophila Toll/Cactus/Dorsal signaling pathway and the mammalian cytokine-mediated interleukin-1 receptor (IL-1R)/I-kappaB/NF-kappaB activation cascade. In addition to a role regulating dorsal-ventral polarity in the developing Drosophila embryo, signaling through Drosophila Toll (dToll) activates the nonclonal, or innate, immune response in the adult fly. Recent evidence indicates that a human homologue of the dToll protein participates in the regulation of both innate and adaptive human immunity through the activation of NF-kappaB and the expression of the NF-kappaB-controlled genes IL-1, IL-6, and IL-8, thus affirming the evolutionary conservation of this host defense pathway. We report here the cloning of two novel human genes, TIL3 and TIL4 (Toll/IL-1R-like-3, -4) that exhibit homology to both the leucine-rich repeat extracellular domains and the IL-1R-like intracellular domains of human and Drosophila Toll. Northern analysis showed distinctly different tissue distribution patterns with TIL3 expressed predominantly in ovary, peripheral blood leukocytes, and prostate, and TIL4 expressed primarily in peripheral blood leukocytes and spleen. Chromosomal mapping by fluorescence in situ hybridization localized the TIL3 gene to chromosome 1q41-42 and TIL4 to chromosome 4q31.3-32. Functional studies showed that both TIL3 and TIL4 are able to activate NF-kappaB, though in a cell type-dependent fashion. Together with human Toll, TIL3 and TIL4 encode a family of genes with conserved structural and functional features involved in immune modulation.

Amino Acid Sequence↗

Negative regulation of toll-like receptor-mediated immune responses.

Toll-like receptors (TLRs) are involved in host defence against invading pathogens, functioning as primary sensors of microbial products and activating signalling pathways that induce the expression of immune and pro-inflammatory genes. However, TLRs have also been implicated in several immune-mediated and inflammatory diseases. As the immune system needs to constantly strike a balance between activation and inhibition to avoid detrimental and inappropriate inflammatory responses, TLR signalling must be tightly regulated. Here, we discuss the various negative regulatory mechanisms that have evolved to attenuate TLR signalling to maintain this immunological balance.

Animals↗

Differential induction of endotoxin tolerance by lipopolysaccharides derived from Porphyromonas gingivalis and Escherichia coli.

Exposure of mononuclear phagocytes to enterobacterial LPS induces a state of transient hyporesponsiveness to subsequent LPS exposure, termed endotoxin tolerance. In the present study, LPS derived from the oral periodontal pathogen, Porphyromonas gingivalis, was compared with that derived from the enterobacterium, Escherichia coli, for the ability to induce endotoxin tolerance. Pretreatment of the human macrophage cell line, THP-1, with E. coli LPS resulted in a severe reduction in the levels of IL-1beta, IL-6, and TNF-alpha upon secondary stimulation. In contrast, pretreatment of THP-1 cells with P. gingivalis LPS resulted in a mitigation of IL-1beta, but not IL-6 and TNF-alpha production upon subsequent exposure to P. gingivalis LPS: primary or secondary stimulation with < or =100 ng/ml P. gingivalis LPS resulted in comparable levels of IL-6 and TNF-alpha, while stimulation of THP-1 cells with > or =1 microg/ml P. gingivalis LPS induced a significant enhancement in IL-6 and TNF-alpha levels upon secondary exposure. To identify possible mechanisms for these differences, changes in the expression of molecules involved in the LPS-signaling pathway were assessed. Pretreatment of THP-1 cells with E. coli LPS resulted in a significant reduction in surface Toll-like receptor 4 (TLR4) expression and an inability to degrade I-kappaB-alpha or I-kappaB-beta proteins upon secondary stimulation. In contrast, pretreatment of THP-1 cells with P. gingivalis LPS resulted in a significant enhancement of both CD14 and TLR2, while maintaining the ability to degrade I-kappaB-beta only upon secondary stimulation. Thus, E. coli and P. gingivalis LPS differentially affect CD14 and TLR expression as well as secondary LPS-associated responses.

Animals↗

Mycobacterium tuberculosis inhibits macrophage responses to IFN-gamma through myeloid differentiation factor 88-dependent and -independent mechanisms.

Mycobacterium tuberculosis overcomes macrophage bactericidal activities and persists intracellularly. One mechanism by which M. tuberculosis avoids macrophage killing might be through inhibition of IFN-gamma-mediated signaling. In this study we provide evidence that at least two distinct components of M. tuberculosis, the 19-kDa lipoprotein and cell wall peptidoglycan (contained in the mycolylarabinogalactan peptidoglycan (mAGP) complex), inhibit macrophage responses to IFN-gamma at a transcriptional level. Moreover, these components engage distinct proximal signaling pathways to inhibit responses to IFN-gamma: the 19-kDa lipoprotein inhibits IFN-gamma signaling in a Toll-like receptor (TLR)2-dependent and myeloid differentiation factor 88-dependent fashion whereas mAGP inhibits independently of TLR2, TLR4, and myeloid differentiation factor 88. In addition to inhibiting the induction of specific IFN-gamma responsive genes, the 19-kDa lipoprotein and mAGP inhibit the ability of IFN-gamma to activate murine macrophages to kill virulent M. tuberculosis without inhibiting production of NO. These results imply that inhibition of macrophage responses to IFN-gamma may contribute to the inability of an apparently effective immune response to eradicate M. tuberculosis.

Acylation↗

Peptidoglycan induces nuclear factor-kappaB activation and cyclooxygenase-2 expression via Ras, Raf-1, and ERK in RAW 264.7 macrophages.

In this study, we investigated the signaling pathway involved in cyclooxygenase-2 (COX-2) expression caused by peptidoglycan (PGN), a cell wall component of the Gram-positive bacterium Staphylococcus aureus, in RAW 264.7 macrophages. PGN caused dose- and time-dependent increases in COX-2 expression, which was attenuated by a Ras inhibitor (manumycin A), a Raf-1 inhibitor (GW 5074), and an MEK inhibitor (PD 098059). Treatment of RAW 264.7 macrophages with PGN caused time-dependent activations of Ras, Raf-1, and ERK. The PGN-induced increase in Ras activity was inhibited by manumycin A. Raf-1 phosphorylation at Ser-338 by PGN was inhibited by manumycin A and GW 5074. The PGN-induced increase in ERK activity was inhibited by manumycin A, GW 5074, and PD 098059. Stimulation of cells with PGN activated IkappaB kinase alpha/beta (IKKalpha/beta), IkappaBalpha phosphorylation, IkappaBalpha degradation, and kappaB-luciferase activity. Treatment of macrophages with an NF-kappaB inhibitor (pyrrolidine dithiocarbamate), an IkappaBalpha phosphorylation inhibitor (Bay 117082), and IkappaB protease inhibitors (l-1-tosylamido-2-phenylethyl chloromethyl ketone and calpain inhibitor I) all inhibited PGN-induced COX-2 expression. The PGN-mediated increase in the activities of IKKalpha/beta and kappaB-luciferase were also inhibited by the Ras dominant negative mutant (RasN17), manumycin A, GW 5074, and PD 098059. Further studies revealed that PGN induced the recruitment of p85alpha and Ras to Toll-like receptor 2 in a time-dependent manner. Our data demonstrate for the first time that PGN activates the Ras/Raf-1/ERK pathway, which in turn initiates IKKalpha/beta and NF-kappaB activation, and ultimately induces COX-2 expression in RAW 264.7 macrophages.

Animals↗

Toll-like receptors in innate immunity.

Functional characterization of Toll-like receptors (TLRs) has established that innate immunity is a skillful system that detects invasion of microbial pathogens. Recognition of microbial components by TLRs initiates signal transduction pathways, which triggers expression of genes. These gene products control innate immune responses and further instruct development of antigen-specific acquired immunity. TLR signaling pathways are finely regulated by TIR domain-containing adaptors, such as MyD88, TIRAP/Mal, TRIF and TRAM. Differential utilization of these TIR domain-containing adaptors provides specificity of individual TLR-mediated signaling pathways. Several mechanisms have been elucidated that negatively control TLR signaling pathways, and thereby prevent overactivation of innate immunity leading to fatal immune disorders. The involvement of TLR-mediated pathways in autoimmune and inflammatory diseases has been proposed. Thus, TLR-mediated activation of innate immunity controls not only host defense against pathogens but also immune disorders.

Animals↗