Structural analysis of the Toll-like receptor 15 TIR domain.

Toll-like receptors (TLRs) activate innate immunity in response to pathogen-associated molecular patterns (PAMPs). The ectodomain of a TLR directly senses a PAMP and the intracellular TIR domain dimerizes to initiate a signaling cascade. The TIR domains of TLR6 and TLR10, which belong to the TLR1 subfamily, have been structurally characterized in a dimer, whereas those of other subfamilies, including TLR15, have not been explored at the structural or molecular level. TLR15 is a TLR unique to birds and reptiles that responds to virulence-associated fungal and bacterial proteases. To reveal how the TLR15 TIR domain (TLR15TIR) triggers signaling, the crystal structure of TLR15TIR was determined in a dimeric form and a mutational study was performed. TLR15TIR forms a one-domain structure in which a five-stranded ß-sheet is decorated by α-helices, as shown for TLR1 subfamily members. TLR15TIR exhibits substantial structural differences from other TLRs at the BB and DD loops and αC2 helix that are involved in dimerization. As a result, TLR15TIR is likely to form a dimeric structure that is unique in its intersubunit orientation and the contribution of each dimerizing region. Further comparative analysis of TIR structures and sequences provides insights into the recruitment of a signaling adaptor protein by TLR15TIR.

Characterization of toll-like receptor 1 (TLR1) in turbot (Scophthalmus maximus L.).

TLRs are the first and best-characterized pattern recognition receptors conserved across all the species. Different from mammals, the TLRs in teleost fishes are very diversified due to various evolutionary mechanisms. Here, we characterized one TLR1 gene in turbot, with a 2,415 bp open reading frame (ORF), that encoding 804 amino acid residues, and have the highest similarity and identity both to Paralichthys olivaceus with 88.9% and 79.9%. In phylogenetic analysis, it was firstly clustered with P. olivaceus, and then clustered with Takifugu rubripes. TLR1 was widely expressed in all the examined healthy tissues with the highest expression level in spleen, followed by head-kidney. In addition, it was significantly regulated in gill, skin and intestine following Edwardsiella tarda and Vibrio anguillarum challenge with different expression patterns. In in vitro stimulation with pathogen-associated molecular patterns, TLR1 showed significantly strong and elevated responses to LPS, but only responded to LTA and Poly(I:C) at the highest evaluated concentration, while no response was detected using PGN stimulation. Moreover, in subcellular localization analysis, TLR1 was distributed in the cytoplasm, membrane and nucleus. Taken together, TLR1 played vital roles for host immune response to bacterial infection, only with strong binding ability to LPS and involved in the production of inflammatory cytokines. However, the specific ligand for TLR1 and its functional association with other TLRs should be further characterized in fish species.

Gene duplication and adaptive evolution of Toll-like receptor genes in birds.

Toll-like receptors (TLRs) play an important role in innate immune through recognizes pathogens. In order to reveal the evolutionary patterns and adaptive evolution of avian TLRs, we examined 66 representative bird species in 26 orders. Phylogenetic results indicated that TLR1A and TLR1B may have differentiated functionally. Evolutionary analysis showed that the TLR genes in birds under strong Purification selection (0.165-0.4265). A total of 126 common positively selected codons were identified in 10 TLR genes of avian, and most sites were located in the extracellular leucine-rich repeat (LRR) functional domains, and both environment and feeding habits were external factors driving the evolution of avian TLR genes. Environmental pressures had a greater effect on TLR1B, TLR2B, TLR3 and TLR4, while feeding habits were active in affecting TLR2A, TLR2B, TLR15 and TLR21. Our data suggested that TLR genes have been subjected to different selective pressures in the diversification of birds and that these changes enabled them to respond differently to pathogens from diverse sources.

Volatile Anesthetic Sevoflurane Attenuates Toll-Like Receptor 1/2 Activation.

BACKGROUND: Although immunomodulatory effects of anesthetics have been increasingly recognized, their underlying molecular mechanisms are not completely understood. Toll-like receptors (TLRs) are one of the major receptors to recognize invading pathogens and danger signals from damaged host tissues to initiate immune responses. Among the TLR family, TLR2 and TLR4 recognize a wide range of ligands and are considered to be important players in perioperative pathophysiology. Based on our recent finding that volatile anesthetics modulate TLR4 function, we tested our hypothesis that they would also modulate TLR2 function. METHODS: The effect of anesthetics isoflurane, sevoflurane, propofol, and dexmedetomidine on TLR2 activation was examined by reporter assays. An anesthetic that affected the activation was subjected to in silico rigid docking simulation on TLR2. To test our prediction that sevoflurane and a TLR1/TLR2 ligand Pam3CSK4 would compete for the same pocket of TLR2, we performed Pam3CSK4 competitive binding assay to TLR2 using HEK cells stably transfected with TLR2 (HEK-TLR2) with or without sevoflurane. We examined the effect of different anesthetics on the functions of human neutrophils stimulated with TLR2 ligands. Kruskal-Wallis test and Mann-Whitney U test were used for statistical analysis. RESULTS: We observed that the attenuation of TLR1/TLR2 activation was seen on sevoflurane exposure but not on isoflurane, propofol, or dexmedetomidine exposure. The attenuation of TLR2/TLR6 activation was not seen in any of the anesthetics tested. The rigid docking simulation predicted that sevoflurane and Pam3CSK4 bound to the same pocket of TLR1/TLR2 complex. The binding of Pam3CSK4 to HEK-TLR2 cells was impaired in the presence of sevoflurane, indicating that sevoflurane and Pam3CSK4 competed for the pocket, as predicted in silico. The stimulation of neutrophils with Pam3CSK4 induced L-selection shedding but did not affect phagocytosis and reactive oxygen species production. L-selectin shedding from neutrophils was attenuated only by sevoflurane, consistent with the result of our reporter assays. CONCLUSIONS: We found that TLR1/TLR2 activation was attenuated by sevoflurane, but we found no evidence for attenuation by isoflurane, propofol, or dexmedetomidine at clinically relevant concentrations. Our structural analysis and competition assay supported that sevoflurane directly bound to TLR2 at the interphase of the TLR1/TLR2 complex. Sevoflurane attenuated neutrophil L-selectin shedding, an important step for neutrophil migration.

The novel small-molecule antagonist MMG-11 preferentially inhibits TLR2/1 signaling.

Toll-like receptor 2 (TLR2) forms heterodimers with either TLR1 or TLR6 to induce protective early inflammatory responses to pathogen- and damage-associated molecular patterns. However, excessive activation is associated with inflammatory and metabolic diseases. Several TLR2 antagonists have been described but pharmacological characterization is still at an early stage. Previously, we identified the potent and selective TLR2 antagonist MMG-11 by computational modelling and experimental validation. Here, we characterized the TLR2 antagonists MMG-11 and CU-CPT22 as well as the TIR-domain binding TLR2 antagonist C29 in TLR-overexpressing promoter cells as well as human and mouse macrophages. In line with our recent studies, MMG-11 abrogated pro-inflammatory cytokine secretion and NF-κB activation induced by different bacterial TLR2 agonists. MMG-11 preferentially inhibited TLR2/1 signaling in promoter cells stably expressing TLR2 heterodimers and mouse macrophages. Furthermore, the TLR2 antagonist blocked ligand-induced interaction of TLR2 with MyD88 and reduced MAP kinase and NF-κB activation. MMG-11 and CU-CPT22 but not C29 displaced Pam3CSK4 in an indirect binding assay confirming the competitive mode of action of MMG-11 and CU-CPT22. Isobologram analysis revealed additive and synergistic effects when the non-competitive antagonist C29 was combined with the competitive antagonist MMG-11 or CU-CPT22, respectively. In conclusion, we provide evidence that MMG-11 acts as a competitive antagonist with a predominance for the TLR2/1 heterodimer in human and mouse cells. Our results also indicate that MMG-11 is a model compound for studying TLR2 signaling.

Regulation of Immune Activation by Optical Control of TLR1/2 Heterodimerization.

The activation of toll-like receptors (TLRs) plays important roles in the immune response. The ability to control the activities of TLRs could be usable as a switch for immune response. Here we have rationally designed and synthesized a photoswitchable Pam3 CSK4 derivative-P10-to control the activation of TLR1/2. The ground-state trans-P10 was able to stimulate and activate antigen-presenting cells (APCs) by promoting TLR1/2 heterodimerization. However, cis-P10, derived from UV irradiation of trans-P10, reduced the activities of APCs by impeding the TLR1/2 heterodimerization. In the absence of UV radiation, the cis-P10 slowly returned to its ground trans state, restoring the activities of the APCs stimulation. Our results indicated that optical control of TLR1/2 heterodimerization mediated by the photoswitchable P10 offers the potential to regulate immune activation and inflammation.

Decoy peptides derived from the extracellular domain of toll-like receptor 2 (TLR2) show anti-inflammatory properties.

Toll-like receptor 2 (TLR2) recognizes bacterial derived- and synthetic-lipopeptides after dimerization with TLR1 or TLR6. Hyper-activation of TLR2 has been described in several inflammatory diseases and the discovery of inhibitors of its pro-inflammatory activity represent potential starting points to develop therapeutics in such pathologies. We designed peptides derived from the TLR2 sequence comprising amino acid residues involved in ligand binding (Pam3CSK4) or heterodimerization (TLR2/TLR1) as pointed out by structural data.2 We identified several peptides (P13, P13(LL), P16, P16(LL)) which inhibited TLR2/1 signaling in HEK293-TLR2 cells (MAPK activation and NF-kB activity). Moreover, P13L and P16L decreased TNFα release in human primary PBMCs and mouse macrophages. The peptides were selective for TLR2/1 as they did not inhibit the activity of other TLRs tested. P13L and P16L inhibited the internalization of Pam3CSK4 fluorescently labeled in macrophages and the heterodimerization of TLR2 with TLR1 as demonstrated by immunoprecipitation studies. Our data demonstrate that peptides derived from the region comprising the leucine-rich repeats (LRR) 11 and 13 in the extracellular domain of TLR2 are good starting points to develop more potent anti-inflammatory peptides with TLR2 inhibitory activity.

Phloretin as a Potent Natural TLR2/1 Inhibitor Suppresses TLR2-Induced Inflammation.

Toll-like receptor 2 (TLR2) responses are involved in various inflammatory immune disorders. Phloretin is a naturally occurring dietary flavonoid that is abundant in fruit. Here, we investigated whether the anti-inflammatory activity of phloretin is mediated through TLR2 pathways, and whether phloretin acts as an inhibitor of TLR2/1 heterodimerization using the TLR2/1 agonist Pam3CSK4. We tested the effects of phloretin on tumor necrosis factor (TNF)-α production induced by various TLRs using known TLR-specific agonists. Phloretin significantly inhibited Pam3CSK4-induced TRL2/1 signaling in Raw264.7 cells compared to TLR signaling induced by the other agonists tested. Therefore, we further tested the effects of phloretin in human embryonic kidney (HEK) 293-hTLR2 cells induced by Pam3CSK4, and confirmed that phloretin has comparable inhibition of TLR2/1 heterodimerization to that induced by the known TLR2 inhibitor CU-CPT22. Moreover, phloretin reduced the secretion of the inflammatory cytokines TNF-α and interleukin (IL)-8 in Pam3CSK4-induced HEK293-hTLR2 cells, whereas it did not significantly reduce these cytokines under Pam2CSK4-induced activation. Western blot results showed that phloretin significantly suppressed Pam3CSK4-induced TLR2 and NF-κB p65 expression. The molecular interactions between phloretin and TLR2 were investigated using bio-layer interferometry and in silico docking. Phloretin bound to TLR2 with micromolar binding affinity, and we proposed a binding model of phloretin at the TLR2⁻TLR1 interface. Overall, we confirmed that phloretin inhibits the heterodimerization of TLR2/1, highlighting TLR2 signaling as a therapeutic target for treating TLR2-mediated inflammatory immune diseases.

Investigating the effect of key mutations on the conformational dynamics of toll-like receptor dimers through molecular dynamics simulations and protein structure networks.

The Toll-like receptors (TLRs) are critical components of the innate immune system due to their ability to detect conserved pathogen-associated molecular patterns, present in bacteria, viruses, and other microorganisms. Ligand detection by TLRs leads to a signaling cascade, mediated by interactions among TIR domains present in the receptors, the bridging adaptors and sorting adaptors. The BB loop is a highly conserved region present in the TIR domain and is crucial for mediating interactions among TIR domain-containing proteins. Mutations in the BB loop of the Toll-like receptors, such as the A795P mutation in TLR3 and the P712H mutation (Lpsd mutation) in TLR4, have been reported to disrupt or alter downstream signaling. While the phenotypic effect of these mutations is known, the underlying effect of these mutations on the structure, dynamics and interactions with other TIR domain-containing proteins is not well understood. Here, we have attempted to investigate the effect of the BB loop mutations on the dimer form of TLRs, using TLR2 and TLR3 as case studies. Our results based on molecular dynamics simulations, protein-protein interaction analyses and protein structure network analyses highlight significant differences between the dimer interfaces of the wild-type and mutant forms and provide a logical reasoning for the effect of these mutations on adaptor binding to TLRs. Furthermore, it also leads us to propose a hypothesis for the differential requirement of signaling and bridging adaptors by TLRs. This could aid in further understanding of the mechanisms governing such signaling pathways.

SNPs in the Toll1 receptor of Litopenaeus vannamei are associated with immune response.

Tolls and Toll-like receptors (TLRs) are important regulators in the innate immune system and their genetic variations usually affect the host's susceptibility/resistance to pathogen infections. In this study, we report on the single nucleotide polymorphisms (SNPs) of Toll1 in Litopenaeus vannamei (LvToll1) and how this is associated with immune response. PCR-DGGE analysis revealed genetic polymorphisms in LvToll1 at both the genomic DNA (gDNA) and cDNA levels. Using high-throughput sequencing, 223 SNPs were identified at the gDNA level, of which 145 were non-synonymous SNP (nsSNP), with 3 nsSNPs having frequency over 1%. On the other hand, 60 SNPs were identified at the cDNA level including 38 nsSNPs and 4 nsSNPs with frequency over 1%. Upon challenging shrimps with Streptococcus iniae, Vibrio parahaemolyticus and white spot syndrome virus (WSSV), LvToll1 was shown to generate 6, 4 and 4 novel bands, respectively when analyzed with PCR-DGGE. Sequencing analysis of these bands showed that they contained 6, 4 and 2 nsSNPs, respectively. Moreover, the nsSNP C1526T was detected in S. iniae-resistant but not in susceptible shrimps. Most significantly, the C1526T mutation could shorten the α-helix of the LRR domain and was predicted to affect the function of LvToll1, indicating that SNP C1526T might be associated with shrimp's resistance to pathogen infections. In sum, our findings here reveal that the genetic polymorphisms of Toll receptor are linked with the immune response to pathogen infections in L. vannamei.

Detection of the Lipopeptide Pam3CSK4 Using a Hybridized Toll-like Receptor Electrochemical Sensor.

Electrochemical detection of Pam3CSK4, a synthetic triacylated lipopeptide that mimics the structural moieties of its natural Gram negative bacterial pathogen-associated molecular pattern (PAMP) counterpart, has been achieved using hybridized toll-like receptors (TLR) combining TLR1 and TLR2 onto a single sensor surface. These sensors represent the first hybridized TLR sensors. The limit of detection for Pam3CSK4 attained was 7.5 µg/mL, which is within the same order of magnitude for that of the more labor-intensive and time-consuming cell-assay technique, 2.0 µg/mL. The results gathered in these electrochemical experiments show that sensors fabricated by immobilizing a mixture of cooperative TLR1 and -2 generate higher responses when exposed to the analyte in comparison to the control sensors fabricated using pure TLR1 or -2 standalone. A PAMP selectivity test was carried out in line with our inspiration from the mammalian innate immune response. TLRs1-5 as standalone biorecognition elements and the hybridized "TLR1 and 2" sensor surface were investigated, understanding the known TLR-PAMP interactions, through the exploitation of this electrochemical sensor fabrication technique. The experimental result is consistent with observations from previously published in vivo and in vitro studies, and it is the first demonstration of the simultaneous evaluation of electrochemical responses from multiple, unique fabricated TLR sensor surfaces against the same analyte.

Identification of a Differentially Expressed TIR-NBS-LRR Gene in a Major QTL Associated to Leaf Rust Resistance in Salix.

An earlier identified major quantitative trait locus for resistance towards the willow leaf rust fungus Melampsora larici-epitea in a Salix viminalis x (S. viminalis × S. schwerinii) population was used to identify potential resistance genes to the rust pathogen. Screening a genomic bacterial artificial chromosome library with markers from the peak position of the QTL region revealed one gene with TIR-NBS-LRR (Toll Interleukin1 Receptor-Nucleotide Binding Site-Leucine-Rich Repeat) domain structure indicative of a resistance gene. The resistance gene analog was denoted RGA1 and further analysis revealed a number of non-synonymous single nucleotide polymorphisms in the LRR domain between the resistant and susceptible Salix genotypes. Gene expression levels under controlled conditions showed a significantly lower constitutive expression of RGA1 in the susceptible genotype. In addition, the susceptible genotype showed a significantly reduced expression level of the RGA1 gene at 24 hours post inoculation with M. larici-epitea. This indicates that the pathogen may actively suppress RGA1 gene expression allowing a compatible plant-pathogen interaction and causing infection.

Red squirrels in the British Isles are infected with leprosy bacilli.

Leprosy, caused by infection with Mycobacterium leprae or the recently discovered Mycobacterium lepromatosis, was once endemic in humans in the British Isles. Red squirrels in Great Britain (Sciurus vulgaris) have increasingly been observed with leprosy-like lesions on the head and limbs. Using genomics, histopathology, and serology, we found M. lepromatosis in squirrels from England, Ireland, and Scotland, and M. leprae in squirrels from Brownsea Island, England. Infection was detected in overtly diseased and seemingly healthy animals. Phylogenetic comparisons of British and Irish M. lepromatosis with two Mexican strains from humans show that they diverged from a common ancestor around 27,000 years ago, whereas the M. leprae strain is closest to one that circulated in Medieval England. Red squirrels are thus a reservoir for leprosy in the British Isles.

Comparative genomic evidence for duplication of TLR1 subfamily and miiuy croaker TLR1 perceives LPS stimulation via MyD88 and TIRAP.

Being indispensable pattern recognition receptors in innate immune responses in host protection, Toll-like receptors (TLRs) play an important role in pathogen recognition. Fish TLRs exhibit high variety and distinct features, although little is known about their function on ligand recognition and signaling pathway in fish. This paper reports the evolutionary spectrum of the TLR1 subfamily (referred to as TLR1, TLR6, and TLR10) as determined using the comparative genomic approach. We hypothesized that the TLR1 subfamily underwent two rounds of gene duplication events; the first duplication occurred prior to the divergence of amphibians, and the second one occurred prior to the divergence of eutherians. To further study the function of fish TLR1, we identified miiuy croaker (Miichthys miiuy) TLR1 (mmiTLR1) and determined its potential ability to perceive Vibrio anguillarum and lipopolysaccharide stimulation. Data further suggested that mmiTLR1 is dependent on TIRAP and MyD88 for signal transmission. In addition, immunocytochemistry showed the speculative interaction between MyD88 and mmiTLR1 TIR domain. Overall, we systematically and comprehensively analyzed evolution of TLR1 subfamily and the function of mmiTLR1, which will provide the basis for future scientific research on fish TLRs.

Molecular and functional characterization of Toll-like receptor (Tlr)1 and Tlr2 in common carp (Cyprinus carpio).

Toll-like receptors (TLRs) are fundamental components of innate immunity that play significant roles in the defence against pathogen invasion. In this study, we present the molecular characterization of the full-length coding sequence of tlr1, tlr2a and tlr2b from common carp (Cyprinus carpio). Each is encoded within a single exon and contains a conserved number of leucine-rich repeats, a transmembrane region and an intracellular TIR domain for signalling. Indeed, sequence, phylogenetic and synteny analysis of carp tlr1, tlr2a and tlr2b support that these genes are orthologues of mammalian TLR1 and TLR2. The tlr genes are expressed in various immune organs and cell types. Furthermore, the carp sequences exhibited a good three-dimensional fit with the heterodimer structure of human TLR1-TLR2, including the potential to bind to the ligand Pam3CSK4. This supports the possible formation of carp Tlr1-Tlr2 heterodimers. However, we were unable to demonstrate Tlr1/Tlr2-mediated ligand binding in transfected cell lines through NF-κB activation, despite showing the expression and co-localization of Tlr1 and Tlr2. We discuss possible limitations when studying ligand-specific activation of NF-κB after expression of Tlr1 and/or Tlr2 in human but also fish cell lines and we propose alternative future strategies for studying ligand-binding properties of fish Tlrs.

Structural characterisation of Toll-like receptor 1 (TLR1) and Toll-like receptor 6 (TLR6) in elephant and harbor seals.

Pinnipeds are a diverse clade of semi-aquatic mammals, which act as key indicators of ecosystem health. Their transition from land to marine environments provides a complex microbial milieu, making them vulnerable to both aquatic and terrestrial pathogens, thereby contributing to pinniped population decline. Indeed, viral pathogens such as influenza A virus and phocine distemper virus (PDV) have been identified as the cause of several of these mass mortality events. Furthermore, bacterial infection with mammalian Brucella sp. and methicillin-resistant Staphylococcus aureus strains have also been observed in marine mammals, posing further risk to both co-habiting endangered species and public health. During these disease outbreaks, mortality rates have varied amongst different pinniped species. Analyses of innate immune receptors at the host-pathogen interface have previously identified variants which may drive these species-specific responses. Through a combination of both sequence- and structure-based methods, this study characterises members of the Toll-like receptor (TLR) 1 superfamily from both harbour and elephant seals, identifying variations which will help us to understand these species-specific innate immune responses, potentially aiding the development of specific vaccine-adjuvants for these species.

Three novel Toll genes (PtToll1-3) identified from a marine crab, Portunus trituberculatus: Different tissue expression and response to pathogens.

The Toll signaling pathway is one of the most important regulators of the immune response in both vertebrates and invertebrates. Herein, three novel Toll (PtToll1-3) cDNA sequences were cloned from the swimming crab, Portunus trituberculatus. PtToll1 has 1003 amino acid residues and consists of an extracellular domain containing 15 leucine-rich repeats (LRRs) and a cytoplasmic Toll/interleukin-1 receptor (TIR) domain of 139 residues. PtToll2 encodes 1196 peptides, with an extracellular domain containing 28 LRRs and a cytoplasmic TIR domain. PtToll3 is 1229 residues long and contains 26 LRRs and a cytoplasmic TIR domain. Based on sequence and phylogenetic analyses, PtToll1 distinctly clustered with almost all crustacean Tolls, except Litopenaeus vannamei Toll3. However, PtToll2 and PtToll3 were separated from most reported crustacean Tolls, which mostly clustered with Drosophila melanogaster (Dm) Toll8, L. vannamei Toll3, and DmToll6. Reverse transcription PCR and real-time quantitative PCR analyses showed that PtToll1 and PtToll3 were constitutively expressed in all tissues tested, but PtToll2 mRNA was only highly enriched in gills. Upon challenges with Vibrio alginolyticus, Candida lusitaniae, or white spot syndrome virus (WSSV), the three Tolls exhibited different responses: the PtToll1 transcript was up-regulated in response to C. lusitaniae or V. alginolyticus challenge, but did not respond to WSSV challenge; both PtToll2 and PtToll3 mRNAs were down-regulated 12 h after C. lusitaniae or V. alginolyticus infection. However, WSSV elicited the expression of PtToll2 at 6 h post-infection, but suppressed transcription of PtToll3 at 24 h post-infection. The study provides valuable data for understanding the role of Toll pathways in the host defense against microbial pathogens, which will facilitate future studies on host-pathogen interactions in crabs.

Vibrio cholerae porin OmpU mediates M1-polarization of macrophages/monocytes via TLR1/TLR2 activation.

Polarization of the monocytes and macrophages toward the M1 and M2 states is important for hosts' defense against the pathogens. Moreover, it plays a crucial role to resolve the overwhelming inflammatory responses that can be harmful to the host. Polarization of macrophages/monocytes can be induced by pathogen-associated molecular patterns (PAMPs). PAMP-mediated monocyte/macrophage polarization is important during the infection, as pathogen can suppress host immune system by altering the polarization status of the macrophages/monocytes. OmpU, an outer membrane porin protein of Vibrio cholerae, possesses the ability to induce pro-inflammatory responses in monocytes/macrophages. It is also able to down-regulate the LPS-mediated activation of the monocytes/macrophages. Such observation leads us to believe that OmpU may induce a state that can be called as M1/M2-intermediate state. In the present study, we evaluated a set of M1 and M2 markers in RAW 264.7 murine macrophage cell line, and THP-1 human monocytic cell line, in response to the purified OmpU protein. We observed that OmpU, as a PAMP, induced M1-polarization by activating the Toll-like receptor (TLR) signaling pathway. OmpU induced formation of TLR1/TLR2-heterodimers. OmpU-mediated TLR-activation led to the MyD88 recruitment to the TLR1/TLR2 complex. MyD88, in turn, recruited IRAK1. Ultimately, OmpU-mediated signaling led to the activation and subsequent nuclear translocation of the NFκB p65 subunit. We also observed that blocking of the TLR1, TLR2, IRAK1, and NFκB affected OmpU-mediated production of M1-associated pro-inflammatory cytokines such as TNFα and IL-6.

N-tetradecylcarbamyl lipopeptides as novel agonists for Toll-like receptor 2.

New analogues (UPam) of triacylated lipopeptide Pam3CysSK4, a popular agonist of Toll-like receptor 2 (TLR2), were designed making use of the cocrystal structure of a TLR2 heterodimer (with TLR1) with Pam3CysSK4. Twenty-two UPam derivatives that feature an N-tetradecylcarbamyl chain to mimic the native N-palmitoyl moiety and various small amino acids residues at the penultimate N-terminal position were prepared via solid-phase synthesis. In vitro evaluation of immunostimulatory properties revealed new potent TLR2 ligands.

Lipopeptides are signaled by Toll-like receptor 1, 2 and 6 in endolysosomes.

Toll-like receptors (TLRs) recognize a variety of microbial products and induce defense responses. Pathogen sensing by TLRs occurs either on the cell surface or in endolysosomes. TLR-dependent responses are greatly influenced by the site of pathogen sensing. TLR heterodimers TLR1/TLR2 and TLR2/TLR6 recognize tri- or diacylated microbial lipopeptides, respectively. Although TLR1, 2 and 6 are believed to localize on the cell surface of immune cells, little is known about where lipopeptides are signaled. In this study, we established mAbs to TLR1, 2 and 6. TLR1, 2 and 6 were expressed on the surface of B cells, monocytes and dendritic cells in a manner dependent on a TLR-specific chaperone PRAT4A (protein associated with TLR4 A). Cell surface localization of TLR1 or TLR6 was not necessarily required for TLR2 response. Furthermore, a dynamin inhibitor 'Dynasore' abolished the lipopeptide responses by preventing lipopeptide internalization into LAMP-1 and LAMP-2 positive compartments. Our findings suggest that lipopeptides elicit TLR1/2 and TLR2/6 signaling in the endolysosomes, but not on the cell surface.