Tumor necrosis factor-alpha (TNF-alpha) regulates Toll-like receptor 2 (TLR2) expression in microglia

Microglia represent one effector arm of CNS innate immunity as evident by their role in pathogen recognition. We previously reported that exposure of microglia to Staphylococcus aureus ( S. aureus), a prevalent CNS pathogen, led to elevated Toll-like receptor 2 (TLR2) expression, a pattern recognition receptor capable of recognizing conserved structural motifs associated with gram-positive bacteria such as S. aureus . In this study, we demonstrate that the proinflammatory cytokine tumor necrosis factor-α (TNF-α) enhances TLR2 expression in microglia, whereas interleukin-1β has no significant effect. To determine the downstream signaling events responsible for elevated microglial TLR2 expression in response to TNF-α, a series of signal transduction inhibitors were employed. Treatment with caffeic acid phenethyl ester, an inhibitor of redox-mediated nuclear factor-kappa B activation, significantly attenuated TNF-α-induced TLR2 expression. Similar results were observed with the IKK-2 and IκB-α inhibitors SC-514 and BAY 11-7082, respectively. In contrast, no significant alterations in TLR2 expression were observed with protein kinase C or p38 mitogen-activated protein kinase inhibitors. A definitive role for TNF-α was demonstrated by the inability of S. aureus to augment TLR2 expression in microglia isolated from TNF-α knockout mice. In addition, TLR2 expression was significantly attenuated in brain abscesses of TNF-α knockout mice. Collectively, these results indicate that in response to S. aureus , TNF-α acts in an autocrine/paracrine manner to enhance TLR2 expression in microglia and that this effect is mediated, in part, by activation of the nuclear factor-kappa B pathway.     

Toll-like receptor 2 (TLR2) mediates astrocyte activation in response to the Gram-positive bacterium Staphylococcus aureus

Astrocytes play an important role in initiating and regulating CNS immune responses through the release of proinflammatory cytokines and chemokines. Here we demonstrate that primary astrocytes are capable of recognizing the Gram-positive bacterium Staphylococcus aureus and its cell wall product peptidoglycan (PGN) and respond by producing numerous proinflammatory mediators including interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-1beta (MIP-1beta), MIP-2, and monocyte chemoattractant protein (MCP-1). Astrocytes have recently been shown to express Toll-like receptor 2 (TLR2), a pattern recognition receptor important for recognizing structural components of various Gram-positive bacteria, fungi, and protozoa. However, the functional significance of TLR2 in mediating astrocyte activation remains unknown. Primary astrocytes from TLR2 knockout mice were used to evaluate the role of TLR2 in astrocyte responses to S. aureus and PGN. The results demonstrate that TLR2 is essential for maximal proinflammatory cytokine and chemokine production, but not phagocytosis, in primary astrocytes following S. aureus and PGN exposure. In addition, both stimuli led to a significant increase in TLR2 mRNA expression in wild-type astrocytes as assessed by real-time quantitative RT-PCR. These findings suggest that astrocytes may play a key role in the initial antibacterial immune response in the CNS through engagement of TLR2.     

Differential expression of Toll-like receptor 4 (TLR4) in healthy and infected canine endometrium

This study provides the first report into immunohistochemical localization of Toll-like receptor (TLR) in the canine reproductive tract. TLR4 was investigated in endometrium during the estrous cycle and in pyometra. Pyometra is the most important pathological condition of the uterus due to bacterial infection in dogs. To protect against invading pathogens, the female reproductive tract has evolved immune mechanisms. TLRs are the cellular components of the afferent arm of the innate immune system. The expression of TLR4 was significantly higher in the endometrial stroma compared to the endometrial surface epithelium and glandular epithelium in proestrus. The glandular epithelium and stroma at the diestrous stage expressed TLR4 significantly higher than surface epithelium. Furthermore, when compared to other healthy groups, the glandular epithelium at diestrus also higher expressed TLR4 than other stages. The expression of TLR4 in the surface epithelium was higher in dogs with pyometra compared with all other groups. And, the surface epithelium of dogs suffering from pyometra also expressed TLR4 more intensely than the glandular epithelium. The innate immunity of infected canine endometrium response to bacterial infection is intensely extremely increased by the expression of TLR4. Furthermore, the different levels of TLR4 expression seems related to physiological changes in distinct cell types of endometrium, leukocytes populations, cytokines and sex hormones.     

An oral Salmonella vaccine promotes the down-regulation of cell surface Toll-like receptor 4 (TLR4) and TLR2 expression in mice

A single oral immunization with the Lon-protease-deficient Salmonella enterica serovar Typhimurium (strain CS2022) induced protective immunity in mice against a subcutaneous challenge with virulent Listeria monocytogenes as well as virulent Salmonella serovar Typhimurium. The populations of cell surface Toll-like receptor 4 (TLR4) and TLR2 on peritoneal macrophages decreased at week 6 after immunization. This population decrease was not reversed after a challenge with either Salmonella or Listeria. These results suggest that oral immunization with CS2022 induced immune tolerance correlated with the down-regulation of cell surface TLR expression. This down-regulation may in part account for the development of cross-protection against a Listeria challenge by immunization with CS2022.     

Biased distribution of single nucleotide polymorphisms (SNPs) in porcine Toll-like receptor 1 (TLR1), TLR2, TLR4, TLR5, and TLR6 genes

Toll-like receptors (TLRs) recognize various microbial components and induce immune responses. Polymorphisms in TLRs may influence their recognition of pathogen-derived molecules; swine TLRs are predicted to be associated with responses to infectious diseases such as pneumonia. In this study, we searched for single nucleotide polymorphisms (SNPs) in the coding sequences of porcine TLR1, TLR2, TLR4, TLR5, and TLR6 genes in 96 pigs from 11 breeds and elucidated 21, 11, 7, 13, and 11 SNPs, respectively, which caused amino acid substitutions in the respective TLRs. Distribution of these nonsynonymous SNPs was biased; many were located in the leucine-rich repeats, particularly in TLR1. These data demonstrated that the heterogeneity of TLR genes was preserved in various porcine breeds despite intensive breeding that was carried out for livestock improvement. It suggests that the heterogeneity in TLR genes is advantageous in increasing the possibility of survival in porcine populations.Electronic SupplementaryMaterial Supplementary material is available for this article at     

Toll-like receptor-4 (TLR4) mediates human β-defensin-2 (HBD-2) induction in response to Chlamydia pneumoniae in mononuclear cells

Monocytes are pivotal effector cells of the innate immune system that are vital for recognizing and eliminating invasive microbial pathogens. When microbial products bind to pathogen-recognition receptors, monocytes are activated and release a broad array of cytokines and defensins that orchestrate the host innate and adaptive immune responses. The aim of the present study is to investigate whether Toll-like receptor-4 (TLR4) mediates human β-defensin-2 (HBD-2) induction in response to Chlamydia pneumoniae in mononuclear cells. We showed that TLR4 is expressed in U937 cells and monocytes infected with viable microorganisms in a time-dependent fashion, while heat-inactivated microorganisms induced a lesser expression, albeit still significant, of TLR4 compared with viable organisms; flow cytometric analysis, in particular, revealed a higher level of TLR4 expression at 48 and 72 h postinfection. In addition, U937 cells and monocytes responded to C. pneumoniae in a TLR4-dependent manner with induction of mRNA and protein of the antimicrobial peptide HBD-2. The treatment of cells with TLR4-neutralizing antibody resulted in a decrease in C. pneumoniae- induced HBD-2 production. This study reveals that TLRs not only recognize ligands but also the types of effector molecules induced, namely, antimicrobial peptides. An understanding of the importance of the TLR-mediated antimicrobial mechanisms may provide new avenues for the development of therapeutic regimens aimed at activating the body's own defenses by stimulating TLR-dependent pathways.     

Genetic diversity of the toll-like receptor 2 (TLR2) in hare (Lepus capensis) populations from Tunisia

Toll-like receptors (TLRs) are a major group of proteins that recognize molecular components of infectious agents, known as pathogen associated molecular patterns (PAMPs). The structure of these genes is similar and characterized by the presence of an ectodomain, a signal transmembrane segment and a highly conserved cytoplasmic domain. The latter domain is homologous to the human interleukin-1 receptor (IL1R) and human IL-18 receptor (IL-18R) and designated TIR domain. The latter domain of the TLR genes was suggested to be very conservative and its evolution is driven by purifying selection. Variability and evolution of the TIR sequences of TLR2 gene were studied in three hare populations from Tunisia with different ecological characteristics (NT–North Tunisia with Mediterranean, CT–Central Tunisia with semi-arid, and ST–South Tunisia with arid climate). Sequencing of a 372 bp fragment of TIR2 revealed 25 alleles among 110 hares. Twenty variable nucleotide positions were detected, of which 7 were non-synonymous. The highest variability was observed in CT, with 16 polymorphic positions. In ST, only 4 polymorphic nucleotide positions were detected with all diversity values lower than those recorded for the other two populations. By using several approaches, no positive selection was detected. However, evidence of purifying selection was found at two positions. The logistic models of the most common TIR2 protein variant that we run to examine whether its occurrence was affected by climatic variation independent of the geographic sample location suggested only a longitudinal effect. Finally, the mapping of the non-synonymous mutations to the inferred tertiary protein structure showed that they were all localized in the different loop regions. Among all non-synonymous substitutions, three were suggested to be deleterious as evidenced by PROVEAN analysis. The observed patterns of variability characterized by low genetic diversity in ST might suggest that the TIR region was more affected, than other markers, by genetic drift or/and that these patterns were shaped by different selective pressures under different ecological conditions. Notably, this low diversity was not detected by other (putatively neutral) microsatellite markers analysed in the course of other studies. But low diversity was also found for two MHC class II adaptive immune genes. As expected from functionally important regions, the evolution of the TIR2 domain is mainly driven by purifying selection. However, the occurrence of deleterious non-synonymous substitutions might highlight the flexible evolution of the TIR genes and/or their interactions with other proteins.     

Toll-like receptor (TLR)2 and TLR3 sensing is required for dendritic cell activation, but dispensable to control Schistosoma mansoni infection and pathology

Toll-like receptors (TLRs) play an important role in the innate recognition of pathogens by dendritic cells (DCs) and in the induction of immune responses. However, relatively little is known about their functions in innate/acquired responses to complex eukaryotic microorganisms, including helminth parasites. That Schistosoma mansoni eggs activate myeloid DCs through TLR2 and TLR3 has been shown by us and others, but the consequences of this combined activation are still unknown. We show that the engagement of both TLR2 and TLR3 by schistosome eggs is important for the production of inflammatory cytokines and interferon-stimulated genes, such as some chemokines, by DCs. Strikingly, DCs sensitized with ovalbumin in the presence of parasite eggs dramatically reduce the release of Th2-type cytokines by ovalbumin-specific T lymphocytes, an effect that fully depends on TLR3. Finally, although TLR2 and TLR3 have no role in host resistance and in egg-induced granuloma formation in S. mansoni-infected mice, they individually and additionally increase the Th1/Th2 balance of the immune response. Thus, TLR2 and TLR3 sensing is required to shape the immune response during murine schistosomiasis, but is dispensable to control infection and pathology.     

Therapeutic targeting of innate immunity with Toll-like receptor 4 (TLR4) antagonists

Early recognition of invading bacteria by the innate immune system has a crucial function in antibacterial defense by triggering inflammatory responses that prevent the spread of infection and suppress bacterial growth. Toll-like receptor 4 (TLR4), the innate immunity receptor of bacterial endotoxins, plays a pivotal role in the induction of inflammatory responses. TLR4 activation by bacterial lipopolysaccharide (LPS) is achieved by the coordinate and sequential action of three other proteins, LBP, CD14 and MD-2 receptors, that bind lipopolysaccharide (LPS) and present it to TLR4 by forming the activated (TLR4-MD-2-LPS)(2) complex. Small molecules active in modulating the TLR4 activation process have great pharmacological interest as vaccine adjuvants, immunotherapeutics or antisepsis and anti-inflammatory agents. In this review we present natural and synthetic molecules active in inhibiting TLR4-mediated LPS signalling in humans and their therapeutic potential. New pharmacological applications of TLR4 antagonists will be also presented related to the recently discovered role of TLR4 in the insurgence and progression of neuropathic pain and sterile inflammations.     

Toll-like receptor (TLR) 2-9 agonists-induced cytokines and chemokines: I. Comparison with T cell receptor-induced responses

The cells of innate and adaptive immunity, although activated by different ligands, engage in cross talk to ensure a successful immune outcome. To better understand this interaction, we examined the demographic picture of individual TLR (TLRs 2-9) -driven profiles of eleven cytokines (IFN-alpha/beta, IFN-gamma, IL-12p40/IL-12p70, IL-4, 1L-13, TNF-alpha, IL-1beta, IL-2, IL-10) and four chemokines (MCP-1, MIP1beta, IL-8, and RANTES), and compared them with direct T-cell receptor triggered responses in an assay platform using human PBMCs. We find that T-cell activation by a combination of anti-CD3/anti-CD28/PHA induced a dominant IL-2, IL-13, and Type-II interferon (IFN-gamma) response without major IL-12 and little Type-I interferon (IFN-alphabeta) release. In contrast, TLR7 and TLR9 agonists induced high levels of Type-I interferons. The highest IFN-gamma levels were displayed by TLR8 and TLR7/8 agonists, which also induced the highest levels of pro-inflammatory cytokines IL-12, TNF-alpha, and IL-1beta. Amongst endosomal TLRs, TLR7 displayed a unique profile producing weak IL-12, IFN-gamma, TNF-alpha, IL-1beta, and IL-8. TLR7 and TLR9 resembled each other in their cytokine profile but differed in MIP-1beta and MCP1 chemokine profiles. Gram positive (TLR2, TLR2/6) and gram negative (TLR4) pathogen-derived TLR agonists displayed significant similarities in profile, but not in potency. TLR5 and TLR2/6 agonists paralleled TLR2 and TLR4 in generating pro-inflammatory chemokines MCP-1, MIP-1beta, RANTES, and IL-8 but yielded weak TNF-alpha and IL-1 responses. Taken together, the data show that diverse TLR agonists, despite their operation through common pathways induce distinct cytokine/chemokine profiles that in turn have little or no overlap with TCR-mediated response.     

Evolution of an intronic microsatellite polymorphism in Toll-like receptor 2 among primates

Nonhuman primates express varying responses to Mycobacterium tuberculosis: New World monkeys appear to be resistant to tuberculosis (TB) while Old World monkeys seem to be particularly susceptible. The aim of this study was to elucidate the presence of the regulatory guanine–thymine (GT) repeat polymorphisms in intron 2 of Toll-like receptor 2 (TLR2) associated with the development of TB in humans and to determine any variations in these microsatellite polymorphisms in primates. We sequenced the region encompassing the regulatory GT repeat microsatellites in intron 2 of TLR2 in 12 different nonhuman primates using polymerase chain reaction amplification, TA cloning, and automatic sequencing. The nonhuman primates included for this study were as follows: chimpanzee (Pan troglodytes), bonobo (Pan paniscus), gorilla (Gorilla gorilla), orangutan (Pongo pygmaeus), Celebes ape (Macaca nigra), rhesus monkey (Macaca mulatta), pigtail macaque (Macaca nemestrina), patas monkey (Erythrocebus patas), spider monkey (Ateles geoffroyi), Woolly monkey (Lagothrix lagotricha), tamarin (Saguinus labiatus), and ring-tailed lemur (Lemur catta). Nucleotide sequences encompassing the regulatory GT repeat region are similar across species and are completely conserved in great apes. However, Old World monkeys lack GT repeats altogether, while New World monkeys and ring-tailed lemurs have much more complex structures around the position of the repeats. In conclusion, the genetic structures encompassing the regulatory GT repeats in intron 2 of human TLR2 are similar among nonhuman primates. The sequence is most conserved in New World monkeys and less in Old World monkeys.     

Alteration of Toll-like receptor 4 activation by 4-hydroxy-2-nonenal mediated by the suppression of receptor homodimerization

Toll-like receptors (TLRs) detect invading microbial pathogens and initiate immune responses as part of host defense mechanisms. They also respond to host-derived substances released from injured cells and tissues to ensure wound healing and tissue homeostasis. Dysregulation of TLRs increases the risk of chronic inflammatory diseases and immune disorders. Inflammatory events are often accompanied by oxidative stress, which generates lipid peroxidation products such as 4-hydroxy-2-nonenal (4-HNE). Therefore, we investigated if 4-HNE affects TLR activation. We found that 4-HNE blocked LPS (a TLR4 agonist)-induced activation of NFκB and IRF3 as well as expression of IFNβ, IP-10, RANTES, and TNFα. To investigate the mechanism of inhibition by 4-HNE, we examined its effects on TLR4 dimerization, one of the initial steps in TLR4 activation. 4-HNE suppressed both ligand-induced and ligand-independent receptor dimerization. The thiol donors, DTT and NAC, prevented the inhibitory effects of 4-HNE on TLR4 dimerization, and LC–MS/MS analysis showed that 4-HNE formed adducts with cysteine residues of synthetic peptides derived from TLR4. These observations suggest that the reactivity of 4-HNE with sulfhydryl moieties is implicated in the inhibition of TLR4 activation. Furthermore, inhibition of TLR4 activation by 4-HNE resulted in down-regulation of the phagocytic activity of macrophages. Collectively, these results demonstrate that 4-HNE blocks TLR4-mediated macrophage activation, gene expression, and phagocytic functions, at least partly by suppressing receptor dimerization. They further suggest that 4-HNE influences innate immune responses at sites of infection and inflammation by inhibiting TLR4 activation.     

人 Toll-like receptor 2 配基模拟肽的初步研究

TLR-2 (Toll-like receptor 2) 是介导天然免疫的重要模式识别分子,可参与识别多种病原体及其产物 . 为探索被 TLR-2 所识别配基的结构共性,以真核细胞表达的人 TLR-2 胞外段蛋白 (A26~T588) 为钓饵筛选噬菌体 12 肽库,获得一高度保守的阳性噬菌体克隆 P12-1 ,实验发现 P12-1 可与不同形式的 TLR-2 胞外段结合,并且可刺激细胞分泌 TNFα,提示 P12-1 可能模拟 TLR-2 配基的结构与生物学活性 .     

Divergent Toll-like receptors in catfish (Ictalurus punctatus): TLR5S, TLR20, TLR21

Toll-like receptors (TLR) mediate pathogen recognition in vertebrate species through detection of conserved microbial ligands. Families of TLR molecules have been described from the genomes of the teleost fish model species zebrafish and Takifugu, but much research remains to characterize the full length sequences and pathogen specificities of individual TLR members in fish. While the majority of these pathogen receptors are conserved among vertebrate species with clear orthologues present in fish for most mammalian TLRs, several interesting differences are present in the TLR repertoire of teleost fish when compared to that of mammals. A soluble form of TLR5 has been reported from salmonid fish and Takifugu rubripes which is not present in mammals, and a large group of TLRs (arbitrarily numbered 19-23) was identified from teleost genomes with no easily discernible orthologues in mammals. To better understand these teleost adaptations to the TLR family, we have isolated, sequenced, and characterized the full-length cDNA and gene sequences of TLR5S, TLR20, and TLR21 from catfish as well as studied their expression pattern in tissues. We also mapped these genes to bacterial artificial chromosome (BAC) clones for genome analysis. While TLR5S appeared to be common in teleost fish, and TLR21 is common to birds, amphibians and fish, TLR20 has only been identified in zebrafish and catfish. Phylogenetic analysis of catfish TLR20 indicated that it is closely related to murine TLR11 and TLR12, two divergent TLRs about which little is known. All three genes appear to exist in catfish as single copy genes.     

Impaired host defense to infection and Toll-like receptor 2-independent killing of Borrelia burgdorferi clinical isolates in TLR2-deficient C3H/HeJ mice

To investigate the role of Toll-like receptor 2 (TLR2)-mediated signaling in host innate defense and development of Lyme disease, the pathogenicity of Borrelia burgdorferi sensu stricto clinical isolates representing two distinct genotypes (RST1 and RST3A) was assessed in TLR2(-/-) C3H/HeJ mice. All TLR2(-/-) mice infected with a B. burgdorferi RST1 isolate developed severe arthritis. The numbers of spirochetes in heart, joint and ear biopsy specimens were significantly higher in TLR2(-/-) mice than in wild-type mice similarly infected as determined by real-time quantitative polymerase chain reaction. Interestingly, despite the higher spirochete levels in heart tissues, milder carditis was observed in TLR2(-/-) than in wild-type mice infected with this RST1 isolate (P=0.02). By contrast, no positive cultures were obtained from any of the blood and tissue specimens from TLR2(-/-) mice inoculated with two RST3A clinical isolates. The data suggest that there is impaired host innate defense against infection and TLR2-independent killing of B. burgdorferi clinical isolates in TLR2-deficient C3H/HeJ mice.     

Effect of herbal melanin on IL-8: a possible role of Toll-like receptor 4 (TLR4)

The production of IL-8 can be induced by LPS via TLR4 signaling pathway. In this study, we tested the effect of a herbal melanin (HM) extract, from black cumin seeds (Nigella sativa L.), on IL-8 production. We used HM and LPS in parallel to induce IL-8 production by THP-I, PBMCs, and TLR4-transfected HEK293 cells. Both HM and LPS induced IL-8 mRNA expression and protein production in THP-1 and PBMCs. On applying similar treatment to HEK293 cells that express TLR4, MD2, and CD14, both HM and LPS significantly induced IL-8 protein production. We have also demonstrated that HM and LPS had identical effects in terms of IL-8 stimulation by HEK293 transfected with either TLR4 or MD2-CD14. Melanin extracted from N. sativa L. mimics the action of LPS in the induction of IL-8 by PBMC and the other used cell lines. Our results suggest that HM may share a signaling pathway with LPS that involves TLR4.     

Mechanistic insights into high mobility group box-1 (HMGb1)-induced Toll-like receptor 4 (TLR4) dimer formation

Abstract

Supplemental data for this article can be accessed here.High mobility group box-1 (HMGb1), an endogenous danger-associated molecular pattern protein (DAMP) whose extracellular release has been associated with sterile injury and various inflammatory diseases and conditions, has been shown to be a valuable clinical drug target. Elucidation of the specific interactions with the HMGb1 receptor, Toll-like receptor 4 (TLR4) and adaptor protein myeloid differentiation factor-2 (MD-2), will lead to more precisely targeted therapeutics. We sought to examine detailed interactions and dynamics of the HMGb1 A-box and B-box fragments, as well as the intact protein using in silico protein–protein docking (ZDOCK, ZRANK) and molecular dynamics (Schrödinger Desmond, New York, NY). Mutagenesis and SPR-binding studies allowed us to draw further conclusions regarding the details of the HMGb1–TLR4–MD2 interaction and shed light on the reasons for the opposing biological activities of HMGb1 A-box and B-box fragments. From our findings, we hypothesize that disulfide A-box fragment binds as an anchor toward the TLR4–MD-2 but does not facilitate the TLR4 dimer formation, thereby competing with the HMGb1-binding site and preventing HMGb1-induced signaling and downstream inflammation, whereas the pro-inflammatory B-box fragment retains the MD-2 active conformation and binds to both TLR4 proteins in the complex to aid TLR4 dimer formation, which activates the intracellular signaling for downstream inflammatory pathways and cytokine release.

Communicated by Ramaswamy H. Sarma     

Lipopolysaccharides can protect mesenchymal stem cells (MSCs) from oxidative stress-induced apoptosis and enhance proliferation of MSCs via Toll-like receptor(TLR)-4 and PI3K/Akt

Apoptosis of implanted mesenchymal stem cells (MSCs) limits the efficiency of MSC therapy. Recent studies showed the ligands of Toll-like receptors (TLRs) could control the function of these cells. We have investigated the effect of lipopolysaccharides (LPS), a ligand of TLR4, on the survival of MSCs and explored the roles of TLR4 and PI3K/Akt. H₂O₂/serum deprivation(H₂O₂/SD) induced apoptosis of MSCs but LPS-preconditioning (1.0 μg/ml) protected MSCs from H₂O₂/SD-induced apoptosis and promoted their proliferation. Western blotting showed that 1.0 μg/ml LPS enhanced phosphorylation of both Akt at Ser 473 and nuclear factor-kappa B (NF-κB) p65 at Ser 536. However, the protective effects of LPS on survival were not observed in TLR4^lps-del MSCs. The results suggest appropriate treatments with LPS can protect MSCs from oxidative stress-induced apoptosis and improve the survival of MSCs via the TLR4 and PI3K/Akt pathway.