Different flavors of Toll guide olfaction.

Toll-like receptors are historically linked to immunity across animal phyla, but accumulating evidence suggests they play additional roles in neuronal networks and in cell-cell interactions. Ward and colleagues now identify Toll-6 and Toll-7 as instructive guidance cues during Drosophila olfactory development.

Toll receptors instruct axon and dendrite targeting and participate in synaptic partner matching in a Drosophila olfactory circuit.

Our understanding of the mechanisms that establish wiring specificity of complex neural circuits is far from complete. During Drosophila olfactory circuit assembly, axons of 50 olfactory receptor neuron (ORN) classes and dendrites of 50 projection neuron (PN) classes precisely target to 50 discrete glomeruli, forming parallel information-processing pathways. Here we show that Toll-6 and Toll-7, members of the Toll receptor family best known for functions in innate immunity and embryonic patterning, cell autonomously instruct the targeting of specific classes of PN dendrites and ORN axons, respectively. The canonical ligands and downstream partners of Toll receptors in embryonic patterning and innate immunity are not required for the function of Toll-6/Toll-7 in wiring specificity, nor are their cytoplasmic domains. Interestingly, both Toll-6 and Toll-7 participate in synaptic partner matching between ORN axons and PN dendrites. Our investigations reveal that olfactory circuit assembly involves dynamic and long-range interactions between PN dendrites and ORN axons.

A novel inflammatory pathway mediating rapid hepcidin-independent hypoferremia.

Regulation of iron metabolism and innate immunity are tightly interlinked. The acute phase response to infection and inflammation induces alterations in iron homeostasis that reduce iron supplies to pathogens. The iron hormone hepcidin is activated by such stimuli causing degradation of the iron exporter ferroportin and reduced iron release from macrophages, suggesting that hepcidin is the crucial effector of inflammatory hypoferremia. Here, we report the discovery of an acute inflammatory condition that is mediated by Toll-like receptors 2 and 6 (TLR2 and TLR6) and which induces hypoferremia in mice injected with TLR ligands. Stimulation of TLR2/TLR6 triggers profound decreases in ferroportin messenger RNA and protein expression in bone marrow-derived macrophages, liver, and spleen of mice without changing hepcidin expression. Furthermore, C326S ferroportin mutant mice with a disrupted hepcidin/ferroportin regulatory circuitry respond to injection of the TLR2/6 ligands FSL1 or PAM3CSK4 by ferroportin downregulation and a reduction of serum iron levels. Our findings challenge the prevailing role of hepcidin in hypoferremia and suggest that rapid hepcidin-independent ferroportin downregulation in the major sites of iron recycling may represent a first-line response to restrict iron access for numerous pathogens.

[Mycoplasma genitalium lipid-associated membrane proteins induce endocervical epithelial cells to express ß-defensin-2].

OBJECTIVE: To observe the expression of human ß-defensin-2 (hBD-2) induced by Mycoplasma genitalium-derived lipid-associated membrane proteins (LAMPs) and its potential mechanism. METHODS: Human endocervical epithelial End1/E6E7 cells were cultured in vitro and stimulated by different concentrations of LAMPs for 48 hours, and the expressions of hBD-2 mRNA and protein were detected by real-time RT-PCR and Western blotting, respectively. Toll-like receptor 2 (TLR2) and TLR6 neutralizing antibodies for End1/E6E7 cell cultivation, and dominant negative plasmids for cell transfection were used to analyze the roles of TLR2, TLR6 and MyD88 in mediating hBD-2 expression. Nuclear translocation of the nuclear factor κB (NF-κB) p65 subunit, and its DNA-binding activity were detected by Western blotting and electrophoretic mobility shift assay (EMSA), respectively. Pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB, was used to investigate the effect of NF-κB on hBD-2 expression. RESULTS: Mycoplasma genitalium-derived LAMPs induced the expressions of hBD-2 mRNA and protein in End1/E6E7 cells. The expressions could be abrogated by TLR2 and TLR6 neutralizing antibodies, or their dominant negative plasmids. In addition, dominant negative plasmids of MyD88 significantly decreased LAMPs-induced hBD-2 expression. Western blotting showed that p65 was translocated to the nucleus, and the DNA binding activity of NF-κB was enhanced after LAMPs treatment. Furthermore, PDTC treatment decreased LAMPs- induced hBD-2 expression. CONCLUSION: Mycoplasma genitalium-derived LAMPs can induce End1/E6E7 cells to express hBD-2, which may be involved in the TLR2, TLR6/Myd88/NF-κB pathways.

Toll-like receptor 6 and connective tissue growth factor are significantly upregulated in mitomycin-C-treated urothelial carcinoma cells under hydrostatic pressure stimulation.

BACKGROUND: Urothelial carcinoma (UC) is the most common histologic subtype of bladder cancer. The administration of mitomycin C (MMC) into the bladder after transurethral resection of the bladder tumor (TURBT) is a common treatment strategy for preventing recurrence after surgery. We previously applied hydrostatic pressure combined with MMC in UC cells and found that hydrostatic pressure synergistically enhanced MMC-induced UC cell apoptosis through the Fas/FasL pathways. To understand the alteration of gene expressions in UC cells caused by hydrostatic pressure and MMC, oligonucleotide microarray was used to explore all the differentially expressed genes. RESULTS: After bioinformatics analysis and gene annotation, Toll-like receptor 6 (TLR6) and connective tissue growth factor (CTGF) showed significant upregulation among altered genes, and their gene and protein expressions with each treatment of UC cells were validated by quantitative real-time PCR and immunoblotting. CONCLUSION: Under treatment with MMC and hydrostatic pressure, UC cells showed increasing apoptosis using extrinsic pathways through upregulation of TLR6 and CTGF.

Local activation of uterine Toll-like receptor 2 and 2/6 decreases embryo implantation and affects uterine receptivity in mice.

Embryo implantation is a complex interaction between maternal endometrium and embryonic structures. Failure to implant is highly recurrent and impossible to diagnose. Inflammation and infections in the female reproductive tract are common causes of infertility, embryo loss, and preterm labor. The current work describes how the activation of endometrial Toll-like receptor (TLR) 2 and 2/6 reduces embryo implantation chances. We developed a morphometric index to evaluate the effects of the TLR 2/6 activation along the uterine horn (UH). TLR 2/6 ligation reduced the endometrial myometrial and glandular indexes and increased the luminal index. Furthermore, TLR 2/6 activation increased the proinflammatory cytokines such as interleukin (IL)-1beta and monocyte chemotactic protein (MCP)-1 in UH lavages in the preimplantation day and IL-1 receptor antagonist in the implantation day. The engagement of TLR 2/6 with its ligand in the UH during embryo transfer severely affected the rate of embryonic implantation (45.00% ± 6.49% vs. 16.69% ± 5.01%, P < 0.05, control vs. test, respectively). Furthermore, this interference with the embryo implantation process was verified using an in vitro model of human embryo implantation where trophoblast spheroids failed to adhere to a monolayer of TLR 2- and TLR 2/6-activated endometrial cells. The inhibition of TLR receptors 2 and 6 in the presence of their specific ligands restored the ability of the spheroids to bind to the endometrial cells. In conclusion, the activation of the innate immune system in the uterus at the time of implantation interfered with the endometrial receptivity and reduced the chances of implantation success.

Toll-like receptor 6 stimulation promotes T-helper 1 and 17 responses in gastrointestinal-associated lymphoid tissue and modulates murine experimental colitis.

T-helper 1 and 17 (Th1/Th17) responses are important in inflammatory bowel disease (IBD), and research indicates that Toll-like receptor 6 (TLR6) stimulation leads to Th17 cell development within the lung. The gastrointestinal tract, like the lung, is a mucosal surface that is exposed to bacterially derived TLR6 ligands. Thus, we looked at the effects of TLR6 stimulation on the expression of Th17-, Th1-, and regulatory T-cell-associated transcription factors; RORγt, T-bet, and Foxp3, respectively; in CD4+ T cells within gut-associated lymphoid tissue (GALT) in vitro and in vivo. Cells from GALT and spleen were stimulated with anti-CD3 and TLR ligands for TLR1/2 and TLR2/6 (Pam3CSK4 and FSL-1, respectively). FSL-1 was more effective than Pam3CSK4 at inducing Th1 and Th17 responses in the GALT while Pam3CSK4 rivaled FSL-1 in the spleen. TLR6 was further explored in vivo using experimental colitis. Tlr6-/- mice were resistant to colitis, and oral FSL-1 led to more severe colitis in wild-type mice. Similar pro-inflammatory reactions were seen in human peripheral blood mononuclear cells, and TLR6 expression was directly correlated with RORC mRNA levels in inflamed intestines of IBD patients. These results demonstrate that TLR6 supports Th1- and Th17-skewed responses in the GALT and might be an important target for the development of new medical interventions in IBD.

Toll-like receptor 6 plays an important role in host innate resistance to Brucella abortus infection in mice.

Brucella abortus is recognized by several Toll-like receptor (TLR)-associated pathways triggering proinflammatory responses that affect both the nature and intensity of the immune response. Previously, we demonstrated that B. abortus-mediated dendritic cell (DC) maturation and control of infection are dependent on the adaptor molecule MyD88. However, the involvement of all TLRs in response to B. abortus infection is not completely understood. Therefore, we decided to evaluate the requirement for TLR6 in host resistance to B. abortus. Here, we demonstrated that TLR6 is an important component for triggering an innate immune response against B. abortus. An in vitro luciferase assay indicated that TLR6 cooperates with TLR2 to sense Brucella and further activates NF-κB signaling. However, in vivo analysis showed that TLR6, not TLR2, is required for the efficient control of B. abortus infection. Additionally, B. abortus-infected dendritic cells require TLR6 to induce tumor necrosis factor alpha (TNF-α) and interleukin-12 (IL-12). Furthermore, our findings demonstrated that the mitogen-activated protein kinase (MAPK) signaling pathway is impaired in TLR2, TLR6, and TLR2/6 knockout (KO) DCs when infected with B. abortus, which may account for the lower proinflammatory cytokine production observed in TLR6 KO mouse dendritic cells. In summary, the results presented here indicate that TLR6 is required to trigger innate immune responses against B. abortus in vivo and is required for the full activation of DCs to induce robust proinflammatory cytokine production.

The protective role of TLR6 in a mouse model of asthma is mediated by IL-23 and IL-17A.

TLRs are a family of receptors that mediate immune system pathogen recognition. In the respiratory system, TLR activation has both beneficial and deleterious effects in asthma. For example, clinical data indicate that TLR6 activation exerts protective effects in asthma. Here, we explored the mechanism or mechanisms through which TLR6 mediates this effect using mouse models of Aspergillus fumigatus-induced and house dust mite antigen-induced (HDM antigen-induced) chronic asthma. Tlr6-/- mice with fungal- or HDM antigen-induced asthma exhibited substantially increased airway hyperresponsiveness, inflammation, and remodeling compared with WT asthmatic groups. Surprisingly, whole-lung levels of IL-23 and IL-17 were markedly lower in Tlr6-/- versus WT asthmatic mice. Tlr6-/- DCs generated less IL-23 upon activation with lipopolysaccharide, zymosan, or curdlan. Impaired IL-23 generation in Tlr6-/- mice also corresponded with lower levels of expression of the pathogen-recognition receptor dectin-1 and expansion of Th17 cells both in vivo and in vitro. Exogenous IL-23 treatment of asthmatic Tlr6-/- mice restored IL-17A production and substantially reduced airway hyperresponsiveness, inflammation, and lung fungal burden compared with that in untreated asthmatic Tlr6-/- mice. Together, our data demonstrate that TLR6 activation is critical for IL-23 production and Th17 responses, which both regulate the allergic inflammatory response in chronic fungal-induced asthma. Thus, therapeutics targeting TLR6 activity might prove efficacious in the treatment of clinical asthma.

Autophagy negatively regulates keratinocyte inflammatory responses via scaffolding protein p62/SQSTM1.

The scaffolding adaptor protein p62/SQSTM1 (p62) has been shown to be an autophagy receptor that acts as a link between the ubiquitination and autophagy machineries. However, the roles of autophagy and p62 in human keratinocytes are not well understood. In this study, we show that keratinocyte autophagy negatively regulates p62 expression, which is essential for the prevention of excessive inflammation and the induction of cathelicidin in human keratinocytes. Stimulation of TLR2/6 or TLR4 in primary human keratinocytes robustly activated autophagy pathways and up-regulated p62 expression through induction of NADPH oxidases 2 and 4 and the generation of reactive oxygen species. MyD88 and TNFR-associated factor 6, key signaling molecules that mediate TLR activation, played an essential role in the induction of autophagy and p62 expression. Additionally, blockade of autophagy significantly increased the generation of inflammatory cytokines and expression of p62 in primary human keratinocytes. Notably, silencing hp62 through RNA interference resulted in a significant decrease in NF-κB activation, inflammatory cytokine production, cathelicidin expression, and cell proliferation (as well as cyclin D1 expression) in keratinocytes. Epidermal expression of p62 was further found to be significantly higher in psoriatic skin than in skin affected by atopic dermatitis or from healthy controls. Collectively, our data provide new insights into the roles of autophagy and p62 in controlling cutaneous inflammation.

Cyclooxygenase-2 enhances antimicrobial peptide expression and killing of Staphylococcus aureus.

Antimicrobial peptides such as human ß-defensins (hBDs) and cathelicidins are critical for protection against infection and can be induced by activation of TLRs, a pathway that also activates cyclooxygenase(Cox)-2 expression. We hypothesized that Cox-2 is induced by TLR activation and is necessary for optimal AMP production, and that inhibitors of Cox-2 may therefore inhibit antimicrobial action. Normal human keratinocytes (NHEKs) stimulated with a TLR2/6 ligand, macrophage-activating lipopeptide-2, or a TLR3 ligand, polyinosinic-polycytidylic acid, increased Cox-2 mRNA and protein and increased PGE(2), a product of Cox-2. Treatment with a Cox-2 selective inhibitor (SC-58125) or Cox-2 small interfering RNA attenuated hBD2 and hBD3 production in NHEKs when stimulated with macrophage-activating lipopeptide-2, polyinosinic-polycytidylic acid, or UVB (15 mJ/cm(2)), but it did not attenuate vitamin D3-induced cathelicidin. SC-58125 also inhibited TLR-dependent NF-κB activation. Conversely, treatment with Cox-derived prostanoids PGD(2) or 15-deoxy-Δ(12,14)-PGJ(2) induced hBD3 or hBD2 and hBD3, respectively. The functional significance of these observations was seen in NHEKs that showed reduced anti-staphylococcal activity when treated with a Cox-2 inhibitor. These findings demonstrate a critical role for Cox-2 in hBD production and suggest that the use of Cox-2 inhibitors may adversely influence the risk for bacterial infection.

Internalization and coreceptor expression are critical for TLR2-mediated recognition of lipoteichoic acid in human peripheral blood.

Lipoteichoic acid (LTA), a ubiquitous cell wall component of Gram-positive bacteria, represents a potent immunostimulatory molecule. Because LTA of a mutant Staphylococcus aureus strain lacking lipoproteins (Deltalgt-LTA) has been described to be immunobiologically inactive despite a lack of ascertained structural differences to wild-type LTA (wt-LTA), we investigated the functional requirements for the recognition of Deltalgt-LTA by human peripheral blood cells. In this study, we demonstrate that Deltalgt-LTA-induced immune activation critically depends on the immobilization of LTA and the presence of human serum components, which, to a lesser degree, was also observed for wt-LTA. Under experimental conditions allowing LTA-mediated stimulation, we found no differences between the immunostimulatory capacity of Deltalgt-LTA and wt-LTA in human blood cells, arguing for a limited contribution of possible lipoprotein contaminants to wt-LTA-mediated immune activation. In contrast to human blood cells, TLR2-transfected human embryonic kidney 293 cells could be activated only by wt-LTA, whereas activation of these cells by Deltalgt-LTA required the additional expression of TLR6 and CD14, suggesting that activation of human embryonic kidney 293 cells expressing solely TLR2 is probably mediated by residual lipoproteins in wt-LTA. Notably, in human peripheral blood, LTA-specific IgG Abs are essential for Deltalgt-LTA-mediated immune activation and appear to induce the phagocytic uptake of Deltalgt-LTA via engagement of FcgammaRII. In this study, we have elucidated a novel mechanism of LTA-induced cytokine induction in human peripheral blood cells that involves uptake of LTA and subsequent intracellular recognition driven by TLR2, TLR6, and CD14.

CD47 and TLR-2 cross-talk regulates neutrophil transmigration.

Neutrophil (PMN) infiltration into tissues is a hallmark of acute inflammation and is crucial for the rapid removal of microbial pathogens. Previous studies have shown that PMN transmigration is regulated by the cell surface protein CD47. However this phenomenon in the context of microbial invasion and subsequent TLR signaling is poorly understood. In this study, we assessed the role of TLR2 and CD47 costimulation in regulating PMN transmigration. Human PMN transmigration across acellular collagen-coated filters toward the bacterial chemoattractant fMLP was more significantly inhibited by MALP-2 (TLR2/6 agonist) than Pam(3)CSK(4) (TLR2/1 agonist). Subsequent experiments demonstrated that treatment with MALP-2 or anti-human CD47 mAbs delayed human PMN transfilter migration, while combined treatment led to further delayed inhibition. Interestingly, stimulation of PMNs with MALP-2 resulted in an increase in surface expression of CD11b, but not CD47. In experiments addressing the role of TLR agonists in regulating CD47-mediated PMN transmigration, incubation with MALP-2 or with anti-mouse CD47 mAbs did not inhibit transfilter migration of TLR2(-/-) or MyD88(-/-)-deficient murine bone marrow-derived PMNs. Similarly, inhibition of MyD88 homodimerization reversed the attenuation of human PMN transmigration induced by MALP-2 or anti-human CD47 mAbs. Separate experiments demonstrated that CD47(-/-) murine bone marrow-derived PMNs exhibited 4-fold decreased sensitivity toward MALP-2. Collectively, these findings suggest that activation of CD47 signaling enhances PMN sensitivity toward TLR2 activation which, in turn, signals their arrival at a site of invasion and may facilitate antimicrobial function.

Engagement of Toll-like receptor 2 in mouse macrophages infected with Mycobacterium avium induces non-oxidative and TNF-independent anti-mycobacterial activity.

Toll-like receptor (TLR) 2 plays an important role in the immune response to mycobacterial infections, being required for optimal immunity against certain virulent Mycobacterium avium strains. Here we analyzed the role of TLR2 in the intra-macrophagic growth of M. avium, using macrophages from TLR2-deficient mice. We found that the engagement of TLR2/TLR6 and/or TLR2/TLR1 receptors induced bacteriostasis of M. avium inside bone marrow-derived macrophages in a MyD88-dependent way. Additionally, lipoproteins from the cell envelope of M. avium with a molecular mass of 20-25 kDa triggered this TLR2 pathway, leading to a decrease in the growth of the mycobacteria. Although TLR2 engagement induced the production of TNF, this cytokine as well as nitric oxide and superoxide molecules were not necessary for TLR2-mediated bacteriostasis. Finally, TLR ligation did not induce the expression of the 47-kDa guanosine triphosphatase (LRG-47) but it promoted an increased maturation of the phagosome with regards to acquisition of LAMP1. Our data show that triggering TLR2 inhibited M. avium growth by an as-yet-unknown mechanism that may involve increased phagosome maturation.

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.

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.

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.