γ-Irradiated cancer cells promote tumor growth by activation of Toll-like receptor 1-mediated inducible nitric oxide synthase in macrophages.

RT is commonly used to treat malignant tumors. However, tumor regrowth is a major limitation to RT as an antitumor treatment. In the present study, we investigated the tumor-promoting effects of high-dose (or ablative) RT treatments on tumor-bearing mice. We focused on the role of macrophages that interact with IR-CCs in the TME, which cause tumor regrowth. We observed that CT26(H-2(d)) tumor growth was enhanced by i.v. injection of IR-CT26 cells compared with NR control CT26 cells. The levels of iNOS gene expression and NO production from RAW264.7 macrophages (H-2(d)) in response to the interaction with IR-CT26 cells were higher than with NR-CT26 cells. When CT26 tumor-bearing mice were treated i.v. with L-NMMA, a NOS inhibitor, the reduction in in vivo tumor growth was higher in the IR-CT26-injected group compared with the NR-CT26-injected control group. In vivo CT26 tumor growth was decreased after transplanting PEM extracted from L-NMMA-treated, tumor-bearing mice. Although iNOS activity was reduced by inhibiting TLR1 expression with TLR1-siRNA, it was enhanced by TLR1 overexpression. Transcriptional activation and protein expression levels of iNOS were also decreased in the presence of TLR1-siRNA but increased as a result of TLR1 overexpression. These results demonstrate that postradiotherapeutic tumor regrowth may be caused by interaction of IR-CCs with macrophages that induce TLR1-mediated iNOS expression and NO production. Our data suggest that iNOS in macrophages could be a useful target to regulate postradiotherapeutic responses in hosts and subsequently limit tumor regrowth.

Toll-like receptor 1 N248S single-nucleotide polymorphism is associated with leprosy risk and regulates immune activation during mycobacterial infection.

Conflicting findings about the association between leprosy and TLR1 variants N248S and I602S have been reported. Here, we performed case-control and family based studies, followed by replication in 2 case-control populations from Brazil, involving 3162 individuals. Results indicated an association between TLR1 248S and leprosy in the case-control study (SS genotype odds ratio [OR], 1.81; P = .004) and the family based study (z = 2.02; P = .05). This association was consistently replicated in other populations (combined OR, 1.51; P < .001), corroborating the finding that 248S is a susceptibility factor for leprosy. Additionally, we demonstrated that peripheral blood mononuclear cells (PBMCs) carrying 248S produce a lower tumor necrosis factor/interleukin-10 ratio when stimulated with Mycobacterium leprae but not with lipopolysaccharide or PAM3cysK4. The same effect was observed after infection of PBMCs with the Moreau strain of bacillus Calmette-Guerin but not after infection with other strains. Finally, molecular dynamics simulations indicated that the Toll-like receptor 1 structure containing 248S amino acid is different from the structure containing 248N. Our results suggest that TLR1 248S is associated with an increased risk for leprosy, consistent with its hypoimmune regulatory function.

Toll-like receptor 1 N248S single-nucleotide polymorphism is associated with leprosy risk and regulates immune activation during mycobacterial infection

Conflicting findings about the association between leprosy and TLR1 variants N248S and I602S have been reported. Here, we performed case-control and family based studies, followed by replication in 2 case-control populations from Brazil, involving 3162 individuals. Results indicated an association between TLR1 248S and leprosy in the case-control study (SS genotype odds ratio [OR], 1.81; P = .004) and the family based study (z = 2.02; P = .05). This association was consistently replicated in other populations (combined OR, 1.51; P < .001), corroborating the finding that 248S is a susceptibility factor for leprosy. Additionally, we demonstrated that peripheral blood mononuclear cells (PBMCs) carrying 248S produce a lower tumor necrosis factor/interleukin-10 ratio when stimulated with Mycobacterium leprae but not with lipopolysaccharide or PAM3cysK4. The same effect was observed after infection of PBMCs with the Moreau strain of bacillus Calmette-Guerin but not after infection with other strains. Finally, molecular dynamics simulations indicated that the Toll-like receptor 1 structure containing 248S amino acid is different from the structure containing 248N. Our results suggest that TLR1 248S is associated with an increased risk for leprosy, consistent with its hypoimmune regulatory function.

Soluble Toll-like receptor 1 family members in the amniotic fluid of women with preterm prelabor rupture of the membranes.

OBJECTIVE: To determine soluble Toll-like receptor (sTLR) 1, sTLR2 and sTLR6 concentrations in amniotic fluid (AF) of women with preterm prelabor rupture of membranes (PPROM) and if there is an association with microbial invasion of the amniotic cavity and histological chorioamnionitis (HCA). METHODS: Cross-sectional study was performed. Forty-two women with singleton PPROM pregnancies at a gestational age between 24 + 0 and 36 + 6 weeks were included in the study (twenty-two women with presence of both microbial invasion of the amniotic cavity and HCA, and 20 women without microbial invasion of the amniotic cavity and HCA). Amniocenteses were performed, and the concentrations of sTLRs were determined by sandwich enzyme-linked immunosorbent assays. RESULTS: Women with microbial invasions of the amniotic cavity and HCA (n = 22) had significantly higher median sTLR1, sTLR2 and sTLR6 levels than those without (n = 20). (20.4 ng/mL vs. 0.44 ng/mL; p < 0.0001, 577.6 ng/mL vs. 60.7 ng/mL; p < 0.0001 and 0.44 ng/mL vs. 0.26 ng/mL; p = 0.02, respectively). CONCLUSIONS: Women with microbial invasion of the amniotic cavity and HCA had higher AF sTLR1, 2 and 6 levels.

MyD88-dependent TLR1/2 signals educate dendritic cells with gut-specific imprinting properties.

Gut-associated dendritic cells (DC) synthesize all-trans retinoic acid, which is required for inducing gut-tropic lymphocytes. Gut-associated DC from MyD88(-/-) mice, which lack most TLR signals, expressed low levels of retinal dehydrogenases (critical enzymes for all-trans retinoic acid biosynthesis) and were significantly impaired in their ability to induce gut-homing T cells. Pretreatment of extraintestinal DC with a TLR1/2 agonist was sufficient to induce retinal dehydrogenases and to confer these DC with the capacity to induce gut-homing lymphocytes via a mechanism dependent on MyD88 and JNK/MAPK. Moreover, gut-associated DC from TLR2(-/-) mice, or from mice in which JNK was pharmacologically blocked, were impaired in their education to imprint gut-homing T cells, which correlated with a decreased induction of gut-tropic T cells in TLR2(-/-) mice upon immunization. Thus, MyD88-dependent TLR2 signals are necessary and sufficient to educate DC with gut-specific imprinting properties and contribute in vivo to the generation of gut-tropic T cells.

TLR1/TLR2 agonist induces tumor regression by reciprocal modulation of effector and regulatory T cells.

Using TLR agonists in cancer treatment can have either beneficial or detrimental effects. Therefore, it is important to determine their effect on the tumor growth and understand the underlying mechanisms in animal tumor models. In this study, we report a general immunotherapeutic activity of a synthetic bacterial lipoprotein (BLP), a TLR1/TLR2 agonist, on established lung carcinoma, leukemia, and melanoma in mice. Systemic treatment of 3LL tumor-bearing mice with BLP, but not LPS, led to a dose-dependent tumor regression and a long-lasting protective response against tumor rechallenge. The BLP-mediated tumor remission was neither mediated by a direct tumoricidal activity nor by innate immune cells, because it lacked therapeutic effect in immunodeficient SCID mice. Instead, BLP treatment reduced the suppressive function of Foxp3(+) regulatory T cells (Tregs) and enhanced the cytotoxicity of tumor-specific CTL in vitro and in vivo. Furthermore, adoptive cotransfer of BLP-pretreated but not untreated CTL and Tregs from wild-type but not from TLR2(-/-) mice was sufficient to restore antitumor immunity in SCID mice by reciprocally modulating Treg and CTL function. These results demonstrate that the TLR1/TLR2 agonist BLP may have a general tumor therapeutic property involving reciprocal downregulation of Treg and upregulation of CTL function. This property may play an important role in the development of novel antitumor strategies.

Human airway epithelial cell responses to Neisseria lactamica and purified porin via Toll-like receptor 2-dependent signaling.

The human airway epithelium is constantly exposed to microbial products from colonizing organisms. Regulation of Toll-like receptor (TLR) expression and specific interactions with bacterial ligands is thought to mitigate exacerbation of inflammatory processes induced by the commensal flora in these cells. The genus Neisseria comprises pathogenic and commensal organisms that colonize the human nasopharynx. Neisseria lactamica is not associated with disease, but N. meningitidis occasionally invades the host, causing meningococcal disease and septicemia. Upon colonization of the airway epithelium, specific host cell receptors interact with numerous Neisseria components, including the PorB porin, at the immediate bacterial-host cell interface. This major outer membrane protein is expressed by all Neisseria strains, regardless of pathogenicity, but its amino acid sequence varies among strains, particularly in the surface-exposed regions. The interaction of Neisseria PorB with TLR2 is essential for driving TLR2/TLR1-dependent cellular responses and is thought to occur via the porin's surface-exposed loop regions. Our studies show that N. lactamica PorB is a TLR2 ligand but its binding specificity for TLR2 is different from that of meningococcal PorB. Furthermore, N. lactamica PorB is a poor inducer of proinflammatory mediators and of TLR2 expression in human airway epithelial cells. These effects are reproduced by whole N. lactamica organisms. Since the responsiveness of human airway epithelial cells to colonizing bacteria is in part regulated via TLR2 expression and signaling, commensal organisms such as N. lactamica would benefit from expressing a product that induces low TLR2-dependent local inflammation, likely delaying or avoiding clearance by the host.

Human TLRs 10 and 1 share common mechanisms of innate immune sensing but not signaling.

TLRs are central receptors of the innate immune system that drive host inflammation and adaptive immune responses in response to invading microbes. Among human TLRs, TLR10 is the only family member without a defined agonist or function. Phylogenetic analysis reveals that TLR10 is most related to TLR1 and TLR6, both of which mediate immune responses to a variety of microbial and fungal components in cooperation with TLR2. The generation and analysis of chimeric receptors containing the extracellular recognition domain of TLR10 and the intracellular signaling domain of TLR1, revealed that TLR10 senses triacylated lipopeptides and a wide variety of other microbial-derived agonists shared by TLR1, but not TLR6. TLR10 requires TLR2 for innate immune recognition, and these receptors colocalize in the phagosome and physically interact in an agonist-dependent fashion. Computational modeling and mutational analysis of TLR10 showed preservation of the essential TLR2 dimer interface and lipopeptide-binding channel found in TLR1. Coimmunoprecipitation experiments indicate that, similar to TLR2/1, TLR2/10 complexes recruit the proximal adaptor MyD88 to the activated receptor complex. However, TLR10, alone or in cooperation with TLR2, fails to activate typical TLR-induced signaling, including NF-kappaB-, IL-8-, or IFN-beta-driven reporters. We conclude that human TLR10 cooperates with TLR2 in the sensing of microbes and fungi but possesses a signaling function distinct from that of other TLR2 subfamily members.

Cot/Tpl2 regulates IL-23 p19 expression in LPS-stimulated macrophages through ERK activation

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We have previously reported that a serine/threonine protein kinase, Cot/Tpl2, is a negative regulatorof Th1-type immunity through inhibiting IL-12 expression in antigen presenting cells (APCs)stimulated by Toll-like receptor (TLR) ligands. We here show that Cot/Tpl2-/- macrophages produce significantly less IL-23, an important regulator of Th17-type response, than the wild-type counterparts in response to lipopolysaccharide (LPS), which is a ligand for TLR4. The decreased IL-23 production in Cot/Tpl2-/- macrophages is, at least partly, regulated at the transcriptional level, as the LPS-mediated IL-23 p19 mRNA induction was significantly less in Cot/Tpl2-/- macrophages. Chemical inhibition of extracellular signal-regulated kinase (ERK) activity similarly inhibited IL-23 expression inLPS-stimulated wild-type macrophages. As Cot/Tpl2 is an essential upstream component of theERK activation pathway of LPS, it is suggested that Cot/Tpl2 positively regulates IL-23 expression through ERK activation. These results indicate that Cot/Tpl2 may be involved in balancing Th1/Th17 differentiation by regulating the expression ratio of IL-12 and IL-23 in APCs (AU)

TLR expression on neutrophils at the pulmonary site of infection: TLR1/TLR2-mediated up-regulation of TLR5 expression in cystic fibrosis lung disease.

Cystic fibrosis (CF) lung disease is characterized by infection with Pseudomonas aeruginosa and a sustained accumulation of neutrophils. In this study, we analyzed 1) the expression of MyD88-dependent TLRs on circulating and airway neutrophils in P. aeruginosa-infected CF patients, P. aeruginosa-infected non-CF bronchiectasis patients, and noninfected healthy control subjects and 2) studied the regulation of TLR expression and functionality on neutrophils in vitro. TLR2, TLR4, TLR5, and TLR9 expression was increased on airway neutrophils compared with circulating neutrophils in CF and bronchiectasis patients. On airway neutrophils, TLR5 was the only TLR that was significantly higher expressed in CF patients compared with bronchiectasis patients and healthy controls. Studies using confocal microscopy and flow cytometry revealed that TLR5 was stored intracellularly in neutrophils and was mobilized to the cell surface in a protein synthesis-independent manner through protein kinase C activation or after stimulation with TLR ligands and cytokines characteristic of the CF airway microenvironment. The most potent stimulator of TLR5 expression was the bacterial lipoprotein Pam(3)CSK(4). Ab-blocking experiments revealed that the effect of Pam(3)CSK(4) was mediated through cooperation of TLR1 and TLR2 signaling. TLR5 activation enhanced the phagocytic capacity and the respiratory burst activity of neutrophils, which was mediated, at least partially, via a stimulation of IL-8 production and CXCR1 signaling. This study demonstrates a novel mechanism of TLR regulation in neutrophils and suggests a critical role for TLR5 in neutrophil-P. aeruginosa interactions in CF lung disease.

Human TLR1 deficiency is associated with impaired mycobacterial signaling and protection from leprosy reversal reaction.

Toll-like receptors (TLRs) are important regulators of the innate immune response to pathogens, including Mycobacterium leprae, which is recognized by TLR1/2 heterodimers. We previously identified a transmembrane domain polymorphism, TLR1_T1805G, that encodes an isoleucine to serine substitution and is associated with impaired signaling. We hypothesized that this TLR1 SNP regulates the innate immune response and susceptibility to leprosy. In HEK293 cells transfected with the 1805T or 1805G variant and stimulated with extracts of M. leprae, NF-kappaB activity was impaired in cells with the 1805G polymorphism. We next stimulated PBMCs from individuals with different genotypes for this SNP and found that 1805GG individuals had significantly reduced cytokine responses to both whole irradiated M. leprae and cell wall extracts. To investigate whether TLR1 variation is associated with clinical presentations of leprosy or leprosy immune reactions, we examined 933 Nepalese leprosy patients, including 238 with reversal reaction (RR), an immune reaction characterized by a Th1 T cell cytokine response. We found that the 1805G allele was associated with protection from RR with an odds ratio (OR) of 0.51 (95% CI 0.29-0.87, p = 0.01). Individuals with 1805 genotypes GG or TG also had a reduced risk of RR in comparison to genotype TT with an OR of 0.55 (95% CI 0.31-0.97, p = 0.04). To our knowledge, this is the first association of TLR1 with a Th1-mediated immune response. Our findings suggest that TLR1 deficiency influences adaptive immunity during leprosy infection to affect clinical manifestations such as nerve damage and disability.

Toll-like receptor 2, 3, 4, 5 ligands and interleukin-4 synergistically induce TARC production in nasal polyp fibroblasts.

OBJECTIVE: Although type 2 T helper (Th2) cytokines such as IL-4 and IL-5 play a crucial role in the pathogenesis of chronic sinusitis with allergy, the mechanism underlying the predominance of Th2 cytokines has yet to be clarified. Thymus and activation-regulated chemokine (TARC) has been known to facilitate the recruitment of Th2 polarized cells, resulting in high levels of Th2 cytokines in the sinus mucosa as well as nasal polyps. The nasal and sinus cavities are ideal sites for studying the interplay between microbial Toll-like receptor (TLR) ligands and chemokines. We investigated whether nasal polyp fibroblasts produce TARC when stimulated with the breakdown products of microorganisms (TLR ligands) and a Th2 cytokine (IL-4). METHODS: Fibroblast lines were established from nasal polyp tissues. The expression of TARC mRNA was evaluated by real-time RT-PCR. The amount of TARC in the supernatants was measured by ELISA. RESULTS: Combined stimulation with TLR 2, 3, 4, 5 ligands and IL-4 induced TARC gene expression and protein production in the cultured nasal polyp fibroblasts. This response was time-dependent. CONCLUSIONS: These results suggest that nasal polyp fibroblasts contribute to innate immunity and may play an important role in the recruitment of Th2 cells into nasal polyps through the production of TARC.

The type 1 diabetes locus Idd6 controls TLR1 expression.

The Idd6 locus on mouse chromosome 6, which controls the development of type 1 diabetes in the NOD mouse, affects proliferation rates of T cells and the activity of regulatory CD4+CD25+ T cells. Using a transcriptional profiling approach, we show that splenocytes and thymocytes from diabetes-resistant Idd6 NOD.C3H-congenic mouse strains exhibit a constitutive and specific down-regulation of Toll-like receptor 1 (Tlr1) gene expression compared with diabetes prone NOD mice. This phenotype correlates with a diminished proliferation capacity of both CD4+CD25- effector and CD4+CD25+ regulatory T cells upon in vitro stimulation of the TLR1/TLR2 pathway by the ligand palmitoyl-3-cysteine-serine-lysine 4, and with the constitutive down-regulation of Tnf-alpha and IL-6 in macrophages of Idd6- congenic mice. These data suggest that TLR1 is involved in the regulation of mechanisms that impinge on diabetes development in the NOD mouse.

Toll-like receptors 1 and 6 are involved in TLR2-mediated macrophage activation by hepatitis C virus core and NS3 proteins.

Hepatitis C virus (HCV) is a leading cause of end-stage liver disease through sustained inflammation of the liver produced by the host's immune system. The mechanism for HCV evasion or activation of the immune system is not clear. TLRs are cellular activators of the innate immune system. We recently reported that TLR2-mediated innate immune signaling pathways are activated by HCV core and NS3 proteins. TLR2 activation requires homo- or heterodimerization with TLR1 or TLR6. Here, we aimed to determine whether TLR2 coreceptors participated in cellular activation by HCV core or NS3 proteins. By designing small interfering RNAs targeted to TLR2, TLR1, and TLR6, we showed that knockdown of each of these receptors impairs pro- and anti-inflammatory cytokine activation by TLR-specific ligands as well as by HCV core and NS3 proteins in human embryonic kidney-TLR2 cells and in primary human macrophages. We found that HCV core and NS3 proteins induced TNF-alpha and IL-10 production in human monocyte-derived macrophages, which was impaired by TLR2, TLR1, and TLR6 knockdown. Contrary to human data, results from TLR2, TLR1, or TLR6 knockout mice indicated that the absence of TLR2 and its coreceptor TLR6, but not TLR1, prevented the HCV core and NS3 protein-induced peritoneal macrophage activation. In conclusion, TLR2 may use TLR1 and TLR6 coreceptors for HCV core- and NS3-mediated activation of macrophages and innate immunity in humans. These results imply that multiple pattern recognition receptors could participate in cellular activation by HCV proteins.

The central leucine-rich repeat region of chicken TLR16 dictates unique ligand specificity and species-specific interaction with TLR2.

The ligand specificity of human TLR (hTLR) 2 is determined through the formation of functional heterodimers with either hTLR1 or hTLR6. The chicken carries two TLR (chTLR) 2 isoforms, type 1 and type 2 (chTLR2t1 and chTLR2t2), and one putative TLR1/6/10 homologue (chTLR16) of unknown function. In this study, we report that transfection of HeLa cells with the various chicken receptors yields potent NF-kappaB activation for the receptor combination of chTLR2t2 and chTLR16 only. The sensitivity of this complex was strongly enhanced by human CD14. The functional chTLR16/chTLR2t2 complex responded toward both the hTLR2/6-specific diacylated peptide S-(2,3-bispalmitoyloxypropyl)-Cys-Gly-Asp-Pro-Lys-His-Pro-Lys-Ser-Phe (FSL-1) and the hTLR2/1 specific triacylated peptide tripalmitoyl-S-(bis(palmitoyloxy)propyl)-Cys-Ser-(Lys)(3)-Lys (Pam(3)CSK(4)), indicating that chTLR16 covers the functions of both mammalian TLR1 and TLR6. Dissection of the species specificity of TLR2 and its coreceptors showed functional chTLR16 complex formation with chTLR2t2 but not hTLR2. Conversely, chTLR2t2 did not function in combination with hTLR1 or hTLR6. The use of constructed chimeric receptors in which the defined domains of chTLR16 and hTLR1 or hTLR6 had been exchanged revealed that the transfer of leucine-rich repeats (LRR) 6-16 of chTLR16 into hTLR6 was sufficient to confer dual ligand specificity to the human receptor and to establish species-specific interaction with chTLR2t2. Collectively, our data indicate that diversification of the central LRR region of the TLR2 coreceptors during evolution has put constraints on both their ligand specificity and their ability to form functional complexes with TLR2.

Ganglioside GD1a is an essential coreceptor for Toll-like receptor 2 signaling in response to the B subunit of type IIb enterotoxin.

Innate recognition and signaling by Toll-like receptors (TLRs) is facilitated by functionally associated coreceptors, although the cooperativity mechanisms involved are poorly understood. As a model we investigated TLR2 interactions with the GD1a ganglioside binding subunit of type IIb Escherichia coli enterotoxin (LT-IIb-B(5)). Both LT-IIb-B(5) and a GD1a binding-defective mutant (LT-IIb-B(5)(T13I)) could modestly bind to TLR2, but only the wild-type molecule displayed a dramatic increase in TLR2 binding activity in the presence of GD1a (although not in the presence of irrelevant gangliosides). Moreover, fluorescence resonance energy transfer experiments indicated that LT-IIb-B(5) induces lipid raft recruitment of TLR2 and TLR1 and their clustering with GD1a, in contrast to the GD1a binding-defective mutant, which moreover fails to activate TLR2 signaling. LT-IIb-B(5)-induced cell activation was critically dependent upon the Toll/IL-1 receptor domain-containing adaptor protein, which was induced to colocalize with TLR2 and GD1a, as shown by confocal imaging. Therefore, GD1a provides TLR2 coreceptor function by enabling the ligand to recruit, bind, and activate TLR2. These findings establish a model of TLR2 coreceptor function and, moreover, suggest novel mechanisms of adjuvanticity by non-toxic derivatives of type II enterotoxins dependent upon GD1a/TLR2 cooperative activity.

Toll-like receptor 9 acts at an early stage in host defence against pneumococcal infection.

Toll-like receptor 9 (TLR9) induces an inflammatory response by recognition of unmethylated CpG dinucleotides, mainly present in prokaryotic DNA. So far, TLR9-deficient mice have been shown to be more sensitive than wild-type mice to viral, but not to bacterial infections. Here, we show that mice deficient in TLR9 but not in TLR1, TLR2, TLR4 and TLR6 or IL-1R/IL-18R are more susceptible to a respiratory tract bacterial infection caused by Streptococcus pneumoniae. Intranasal challenge studies revealed that TLR9 plays a protective role in the lungs at an early stage of infection prior to the entry of circulating inflammatory cells. Alveolar as well as bone marrow-derived macrophages deficient in either TLR9 or the myeloid adaptor differentiation protein MyD88 were impaired in pneumococcal uptake and in pneumococcal killing. Our data suggest that in the airways, pneumococcal infection triggers a TLR9 and MyD88-dependent activation of phagocytic activity from resident macrophages leading to an early clearance of bacteria from the lower respiratory tract.

Membrane sorting of toll-like receptor (TLR)-2/6 and TLR2/1 heterodimers at the cell surface determines heterotypic associations with CD36 and intracellular targeting.

Toll-like receptors (TLRs) are receptors of the innate immune system responsible for recognizing pathogen-associated molecular patterns. TLR2 seems to be the most promiscuous TLR receptor able to recognize the most diverse set of pathogen-associated patterns. Its promiscuity has been attributed to its unique ability to heterodimerize with TLRs 1 and 6 and, most recently, to its association with CD36 in response to diacylated lipoproteins. Thus, it seems that TLR2 forms receptor clusters in response to different microbial ligands. In this study we investigated TLR2 cell surface heterotypic interactions in response to different ligands as well as internalization and intracellular trafficking. Our data show that TLR2 forms heterodimers with TLR1 and TLR6 and that these heterodimer pre-exist and are not induced by the ligand. Upon stimulation by the specific ligand, these heterodimers are recruited within lipid rafts. In contrast, heterotypic associations of TLR2/6 with CD36 are not preformed and are ligand-induced. All TLR2 receptor clusters accumulate in lipid rafts and are targeted to the Golgi apparatus. This localization and targeting is ligand-specific. Activation occurs at the cell surface, and the observed trafficking is independent of signaling.

Structural and functional evidence for the role of the TLR2 DD loop in TLR1/TLR2 heterodimerization and signaling.

The Toll/Interleukin-1 receptor (TIR) domain of the Toll-like receptors (TLRs) plays an important role in innate host defense signaling. The TIR-TIR platform formed by the dimerization of two TLRs promotes homotypic protein-protein interactions with additional cytoplasmic adapter molecules to form an active signaling complex resulting in the expression of pro- and anti-inflammatory cytokine genes. To generate a better understanding of the functional domains of TLR2 we performed a random mutagenesis analysis of the human TLR2 TIR domain and screened for TLR2/1 signaling-deficient mutants. Based upon the random mutagenesis results, we performed an alanine scanning mutagenesis of the TLR2 DD loop and part of the alphaD region. This resulted in the identification of four residues crucial for TLR2/1 signaling: Arg-748, Phe-749, Leu-752, and Arg-753. Computer-assisted energy minimization and docking studies indicated three regions of interaction in the TLR2/1 TIR-docked heterodimer. In Region I, residues Arg-748 and Phe-749 in TLR2 DD loop were involved in close contacts with Gly-676 in the TLR1 BB loop. Because this model suggested that steric hindrance would significantly alter the binding interactions between DD loop of TLR2 and BB loop of TLR1, Gly-676 in TLR1 was rationally mutated to Ala and Leu. As expected, in vitro functional studies involving TLR1 G676A and TLR1 G676L resulted in reduced PAM(3)CSK(4) mediated NF-kappaB activation lending support to the computerized predictions. Additionally, mutation of an amino acid residue (TLR2 Asp-730) in Region II also resulted in decreased activity in agreement with our model, providing new insights into the structure-function relationship of TLR2/1 TIR domains.

Toll-like receptor 2 is required for inflammatory responses to Francisella tularensis LVS.

Francisella tularensis, a gram-negative bacterium, is the etiologic agent of tularemia and has recently been classified as a category A bioterrorism agent. Infections with F. tularensis result in an inflammatory response that plays an important role in the pathogenesis of the disease; however, the cellular mechanisms mediating this response have not been completely elucidated. In the present study, we determined the role of Toll-like receptors (TLRs) in mediating inflammatory responses to F. tularensis LVS, and the role of NF-kappaB in regulating these responses. Stimulation of bone marrow-derived dendritic cells from C57BL/6 wild-type (wt) and TLR4-/- but not TLR2-/- mice, with live F. tularensis LVS elicited a dose-dependent increase in the production of tumor necrosis factor alpha. F. tularensis LVS also induced in a dose-dependent manner an up-regulation in the expression of the costimulatory molecules CD80 and CD86 and of CD40 and the major histocompatibility complex class II molecules on dendritic cells from wt and TLR4-/- but not TLR2-/- mice. TLR6, not TLR1, was shown to be involved in mediating the inflammatory response to F. tularensis LVS, indicating that the functional heterodimer is TLR2/TLR6. Stimulation of dendritic cells with F. tularensis resulted in the activation of NF-kappaB, which resulted in a differential effect on the production of pro- and anti-inflammatory cytokines. Taken together, our results demonstrate the role of TLR2/TLR6 in the host's inflammatory response to F. tularensis LVS in vitro and the regulatory function of NF-kappaB in modulating the inflammatory response.