Functional TLR4 Research Articles

TLR2 and Adenosine Receptor A2a Induce ERK1/2 and c-FOS in Oral Squamous Carcinoma Cells

Abstract Squamous cell carcinoma (SCC) is a tenacious cancer prevalent at mucosal and skin surfaces. The level of oral (O)SCC differentiation does not correlate with prognosis, while tumor colonization by microorganisms and chronic inflammation may well contribute to OSCC pathogenesis. Microbial pattern recognition toll-like receptors (TLR) in immune cells induce inflammatory mechanisms, as they activate NF-kB and MAPK ERK1/2 pathways. Activated immune cells downregulate adenosine receptors (AR) A1 and A3, but upregulate inhibitory AR A2a and A2b, which also engage ERK1/2 when stimulated by adenosine. Malignant squamous cells express functional TLR2 and TLR4 as well, but their roles in OSCC cells are poorly understood. To address this gap, we evaluated TLR and AR expression and function in OSCC cells and keratinocytes. Five out of six OSCC lines expressed more TLR2 than TLR1, 4 or 6. TLR2, but not TLR4 stimuli induced AR A2a upregulation, while A2b did not increase. AR A1 and A3 mRNA was not detected in any oral squamous cells. TLR2 and A2a were also expressed in SCC cells and in dysplastic epithelial cells of all 17 archival oral specimens evaluated (use of specimens was approved by AU IRB). TLR2/1, TLR2/6 and AR stimuli induced ERK1/2 phosphorylation and accumulation of c-FOS in TLR2-responsive OSCC, which was similar to the effect of epidermal growth factor (EGF) on these cells. TLR2 and AR stimuli in responsive OSCC also stimulated the expression of the proliferation marker Ki-67. We conclude that products specific for TLR2 and A2a, such as bacterial lipopeptides and adenosine/inosine present in the tumor microenvironment, may contribute to OSCC progression via direct effects on malignant cells.

Cell type-specific regulatory effects of glucocorticoids on cutaneous TLR2 expression and signalling

Glucocorticoids (GCs) induce Toll-like receptor (TLR) 2 expression and synergistically upregulate TLR2 with pro-inflammatory cytokines or bacteria. These paradoxical effects have drawn attention to the inflammatory initiating or promoting effects of GCs, as GC treatment can provoke inflammatory skin diseases. Here, we aimed to investigate the regulatory effects of GCs in human skin cells of different epidermal and dermal layers. We found that Dex induced TLR2 expression mainly in undifferentiated and less in calcium-induced differentiated keratinocytes but not in HaCaT cells or fibroblasts, however, Dex reduced TLR1/6 expression. Stimulation with Dex under inflammatory conditions further increased TLR2 but not TLR1 or TLR6 levels in keratinocytes. Increased ligand-induced interaction of TLR2 with MyD88 and expression of the adaptor protein TRAF6 indicated enhanced TLR2 signalling, whereas TLR2/1 or TLR2/6 signalling was not increased in Dex-pretreated keratinocytes. GC-increased TLR2 expression was negatively regulated by JNK MAPK signalling when stimulated with Propionibacterium acnes. Our results provide novel insights into the molecular mechanisms of glucocorticoid-mediated expression and function of TLR2 in human skin cells and the understanding of the mechanisms of corticosteroid side effects.

Tu1903 - GP96 Deficiency Partially Affects TLR4 Functionality due to an Impaired ERK and P38 Phosphorylation

Tu1903 - GP96 Deficiency Partially Affects TLR4 Functionality due to an Impaired ERK and P38 Phosphorylation

Microbiota, Immune Subversion, and Chronic Inflammation.

Several host-adapted pathogens and commensals have evolved mechanisms to evade the host innate immune system inducing a state of low-grade inflammation. Epidemiological studies have also documented the association of a subset of these microorganisms with chronic inflammatory disorders. In this review, we summarize recent studies demonstrating the role of the microbiota in chronic inflammatory diseases and discuss how specific microorganisms subvert or inhibit protective signaling normally induced by toll-like receptors (TLRs). We highlight our work on the oral pathogen Porphyromonas gingivalis and discuss the role of microbial modulation of lipid A structures in evasion of TLR4 signaling and resulting systemic immunopathology associated with atherosclerosis. P. gingivalis intrinsically expresses underacylated lipid A moieties and can modify the phosphorylation of lipid A, leading to altered TLR4 signaling. Using P. gingivalis mutant strains expressing distinct lipid A moieties, we demonstrated that expression of antagonist lipid A was associated with P. gingivalis-mediated systemic inflammation and immunopathology, whereas strains expressing agonist lipid A exhibited modest systemic inflammation. Likewise, mice deficient in TLR4 were more susceptible to vascular inflammation after oral infection with P. gingivalis wild-type strain compared to mice possessing functional TLR4. Collectively, our studies support a role for P. gingivalis-mediated dysregulation of innate and adaptive responses resulting in immunopathology and systemic inflammation. We propose that anti-TLR4 interventions must be designed with caution, given the balance between the protective and destructive roles of TLR signaling in response to microbiota and associated immunopathologies.

Combinatorial treatment with polyI:C and anti-IL6 enhances apoptosis and suppresses metastasis of lung cancer cells.

Activation of TLR3 stimulates cancer cell apoptosis and triggers secretion of inflammatory cytokines. PolyI:C, a TLR3 agonist, activates immune cells and regresses metastatic lung cancer in vivo. Although polyI:C reportedly kills lung carcinomas, the mechanism remains elusive. Here, we demonstrated that polyI:C suppressed the proliferation and survival of metastatic (NCI-H358 and NCI-H292) and non-metastatic (A549) lung cancer cells. Notably, A549, NCI-H292 and NCI-H358 which are inducible by polyI:C, expressed low-to-medium level of TLR3 protein, and were susceptible to polyI:C treatment. By contrast, NCI-H1299, which endogenously expresses high level of TLR3 protein, was insensitive to polyI:C. We showed that polyI:C stimulated pro-inflammatory cytokines associated with survival and metastasis in a cell type-specific manner. While A549 and NCI-H292 released high levels of IL6, IL8 and GRO, the NCI-H358 cells endogenously secretes abundant levels of these cytokines, and was not further induced by polyI:C. Thus, NCI-H358 was resistant to the inhibition of cytokine-dependent metastasis. NCI-H1299, which was unresponsive to polyI:C, did not produce any of the pro-inflammatory cytokines. Treatment of A549 with a combination of polyI:C and anti-IL6 antibody significantly decreased IL6 production, and enhanced polyI:C-mediated killing and suppression of oncogenicity and metastasis. While polyI:C stimulated the phosphorylation of STAT3 and JAK2, blockade of these proteins enhanced polyI:C-mediated suppression of survival and metastasis. Taken together, polyI:C alone provoked apoptosis of lung cancer cells that express low-to-medium levels of functional TLR3 protein. The combinatorial treatment with polyI:C and anti-IL6 enhanced polyI:C-mediated anticancer activities through IL6/JAK2/STAT3 signalling, and apoptosis via TLR3-mediated caspase 3/8 pathway.

Increased Expression Of Toll-Like Receptor 2 Mrna Following Permanent Middle Cerebral Artery Occlusion In Rat: Role Of TRPV1 Receptors

Background: Stroke is a major cause of mortality and long term disability in adults. TRPV1 has a pivotal role in neuroinflammation. Among TLRs, TLR2 significantly participate in induction of inflammation in brain. In this study, the effect of TRPV1 receptor agonist and antagonist on outcome and gene expression of TLR2 in a rat model of permanent middle cerebral artery occlusion (MCAO) was investigated.Methods: Forty male rats were assigned to the following groups: sham, vehicle )stroke), AMG9810 (selective TRPV1 antagonist, 0.5 mg/kg; 3 h after stroke), and capsaicin (1 mg/kg; 3 h after stroke). Stroke was induced by permanent middle cerebral artery occlusion and behavioral functions were assessed 1, 3, and 7 days after stroke. Infarct volume, brain edema and mRNA expression of TLR2 were also evaluated at the end of the study.Results: While stroke animals showed infarctions and behavioral functions, we did not observe any cerebral infarction and behavioral functions in sham-operated animals. AMG9810 decreased neurological deficits 7 days after cerebral ischemia (P<0.01). In the ledged beam-walking test, the slip ratio was increased following ischemia (*P < 0.05). AMG9810 improved this index in animals undergone stroke. However, capsaicin enhanced the slip ratio 3 and 7 days after cerebral ischemia (#P<0.05). TLR2) P<0.05(mRNA expression was elevated in ischemic rats. Conclusion: Our data indicate that pharmacological blockade of TRPV1 by AMG9810 attenuates behavioral function and mRNA expression of TLR2. Therefore, it might be useful as a potential target for the treatment of ischemic stroke.

LPS Sensing Mechanism of Human Astrocytes: Evidence of Functional TLR4 Expression and Requirement of Soluble CD14

LPS Sensing Mechanism of Human Astrocytes: Evidence of Functional TLR4 Expression and Requirement of Soluble CD14

TLRs, future potential therapeutic targets for RA.

Toll like receptors (TLR)s have a central role in regulating innate immunity and in the last decade studies have begun to reveal their significance in potentiating autoimmune diseases such as rheumatoid arthritis (RA). Earlier investigations have highlighted the importance of TLR2 and TLR4 function in RA pathogenesis. In this review, we discuss the newer data that indicate roles for TLR5 and TLR7 in RA and its preclinical models. We evaluate the pathogenicity of TLRs in RA myeloid cells, synovial tissue fibroblasts, T cells, osteoclast progenitor cells and endothelial cells. These observations establish that ligation of TLRs can transform RA myeloid cells into M1 macrophages and that the inflammatory factors secreted from M1 and RA synovial tissue fibroblasts participate in TH-17 cell development. From the investigations conducted in RA preclinical models, we conclude that TLR-mediated inflammation can result in osteoclastic bone erosion by interconnecting the myeloid and TH-17 cell response to joint vascularization. In light of emerging unique aspects of TLR function, we summarize the novel approaches that are being tested to impair TLR activation in RA patients.

Enhanced expression of TREM-1 in splenic cDCs in lupus prone mice and it was modulated by miRNA-150.

Over activation of conventional dendritic cells (cDCs) contributes to the development of systemic lupus erythematosus (SLE). Triggering receptor expressed on myeloid cells 1 (TREM-1) is emerging as a potent amplifier of the inflammatory responses. We sought to determine the expression level of TREM-1 on cDCs in a mice model of SLE and to identify miRNA which could modulate TREM-1 expression. In the present study, TREM-1 expression in splenocytes and on cDCs was strongly up-regulated in vivo, and was enhanced with LPS stimulation in vitro. Blockade of TREM-1 signal impaired the TLR4-induced cytokines production. These indicated that TREM-1 potently amplified the function of TLR4 which enhanced the inflammation responses. A common set of dysregulated miRNAs (miRNA-98, -150 and -494) were identified in splenocytes of mice. Moreover, the results of bioinformatics and the immunoblotting, demonstrated that miRNA-150 inhibited the expression of TREM-1. Together, these data suggested that TREM-1 signaling pathway may be a therapeutic target to prevent the effects of the inflammatory cDCs in SLE and miRNA-150 serves as the important regulator.

Association between TLR2/TLR4 gene polymorphisms and COPD phenotype in aGreek cohort.

Considering that the innate immune system plays apivotal role in the pathogenesis of chronic obstructive pulmonary disease (COPD), we hypothesized that functional single-nucleotide polymorphisms (SNPs) of innate immune genes affect the disease phenotype and prognosis. To elucidate the contribution of common functional TLR2 and TLR4 SNPs and genotypic deficiency of the mannose-binding lectin (MBL) protein, both as single parameters and in combination, in Greek COPD patients. In acohort of 114 Greek COPD patients, we confirmed that the presence of TLR4-D299G or TLR4-T399I SNPs was significantly associated with an earlier COPD stage (p= 0.003 and p= 0.009, respectively). In comparison, the absence of any analyzed polymorphism, including those of TLR2-R753Q and genotypic MBL deficiency, was significantly associated with amore severe disease phenotype, characterized by more frequent exacerbations (p= 0.045). Our findings support the notion that the presence of innate immune SNPs, such as functional polymorphisms of TLRs along with MBL deficiency, might exert aprotective effect on the COPD phenotype, similar with other immune-mediated disorders.

High-Density Lipoprotein (HDL) Counter-Regulates Serum Amyloid A (SAA)-Induced sPLA2-IIE and sPLA2-V Expression in Macrophages.

Human serum amyloid A (SAA) has been demonstrated as a chemoattractant and proinflammatory mediator of lethal systemic inflammatory diseases. In the circulation, it can be sequestered by a high-density lipoprotein, HDL, which carries cholesterol, triglycerides, phospholipids and apolipoproteins (Apo-AI). The capture of SAA by HDL results in the displacement of Apo-AI, and the consequent inhibition of SAA’s chemoattractant activities. It was previously unknown whether HDL similarly inhibits SAA-induced sPLA2 expression, as well as the resultant HMGB1 release, nitric oxide (NO) production and autophagy activation. Here we provided compelling evidence that human SAA effectively upregulated the expression and secretion of both sPLA2-IIE and sPLA2-V in murine macrophages, which were attenuated by HDL in a dose-dependent fashion. Similarly, HDL dose-dependently suppressed SAA-induced HMGB1 release, NO production, and autophagy activation. In both RAW 264.7 cells and primary macrophages, HDL inhibited SAA-induced secretion of several cytokines (e.g., IL-6) and chemokines (e.g., MCP-1 and RANTES) that were likely dependent on functional TLR4 signaling. Collectively, these findings suggest that HDL counter-regulates SAA-induced upregulation and secretion of sPLA2-IIE/V in addition to other TLR4-dependent cytokines and chemokines in macrophage cultures.

Molecular cloning and characterization of LrTLR4, analysis of its inductive expression and associated down-stream signaling molecules following lipopolysaccharide stimulation and Gram-negative bacterial infection

Toll-like receptors (TLRs) play key roles in innate immunity from lower to higher vertebrates. Among various TLR types, TLR4 was reported to recognize LPS in higher vertebrates resulting in the activation of down-stream signaling pathway. Except in some teleosts, function of TLR4 in most fish species including rohu (Labeo rohita) a commercially important fish species in the South-East Asian countries remained unknown. To investigate it, full-length cDNA of Labeo rohita TLR4 (LrTLR4) was cloned, and it consisted of 2729 bp, with a single ORF of 2469 bp encoding a polypeptide of 822 aa with a predicted molecular mass of 94.753 kDa. Structurally, LrTLR4 consisted of 25 LRRs (leucine rich repeat regions), one TM (trans-membrane) domain and one TIR (Toll/interleukin-1 receptor) domain, and was similar to higher vertebrate's TLR4. Phylogenetically, LrTLR4 exhibited highest (85%) identity with the common carp TLR4b amino acids sequence, and formed a separate subgroup in the phylogenetic tree. LrTLR4 was widely expressed in all tested organs/tissues, and amidst the tissues highest expression was detected in blood and the lowest in eye. In response to LPS-stimulation, LrTLR4 was induced with the activation of MyD88-dependent and TRIF-dependent signaling pathway resulting in pro-inflammatory cytokines (interleukin 6 and 8) and type I IFN gene expression. Infection of rohu with a Gram-negative fish pathogen (Aeromonas hydrophila), also activated LrTLR4. Together, these findings suggest the important role of TLR4 in LPS sensing and augmentation of innate immunity against Gram-negative bacterial infection in fish.

Pig lacks functional NLRC4 and NAIP genes.

The NLRC4 inflammasome, which recognizes flagellin and components of the type III secretion system, plays an important role in the clearance of intracellular bacteria. Here, we examined the genomic sequences carrying two genes encoding key components of the NLRC4 inflammasome-NLR family, CARD-containing 4 (NLRC4), and NLR apoptosis inhibitory protein (NAIP)-in pigs. Pigs have a single locus encoding NLRC4 and NAIP. Comparison of the sequences thus obtained with the corresponding regions in humans revealed the deletion of intermediate exons in both pig genes. In addition, the genomic sequences of both pig genes lacked valid open reading frames encoding functional NLRC4 or NAIP protein. Additional pigs representing multiple breeds and wild boars also lacked the exons that we failed to find through genome sequencing. Furthermore, neither the NLRC4 nor the NAIP gene was expressed in pigs. These findings indicate that pigs lack the NLRC4 inflammasome, an important factor involved in monitoring bacterial proteins and contributing to the clearance of intracellular pathogens. These results also suggest that genetic polymorphisms affecting the molecular functions of TLR2, TLR4, TLR5, and other pattern recognition receptors associated with the recognition of bacteria have a more profound influence on disease resistance in pigs than in other species.

Bartonella quintana lipopolysaccharide (LPS): structure and characteristics of a potent TLR4 antagonist for in-vitro and in-vivo applications

The pattern recognition receptor TLR4 is well known as a crucial receptor during infection and inflammation. Several TLR4 antagonists have been reported to inhibit the function of TLR4. Both natural occurring antagonists, lipopolysaccharide (LPS) from Gram-negative bacteria as well as synthetic compounds based on the lipid A structure of LPS have been described as potent inhibitors of TLR4. Here, we have examined the characteristics of a natural TLR4 antagonist, isolated from Bartonella quintana bacterium by elucidating its chemical primary structure. We have found that this TLR4 antagonist is actually a lipooligosaccharide (LOS) instead of a LPS, and that it acts very effective, with a high inhibitory activity against triggering by the LPS-TLR4 system in the presence of a potent TLR4 agonist (E. coli LPS). Furthermore, we demonstrate that B. quintana LPS is not inactivated by polymyxin B, a classical cyclic cationic polypeptide antibiotic that bind the lipid A part of LPS, such as E. coli LPS. Using a murine LPS/D-galactosamine endotoxaemia model we showed that treatment with B. quintana LPS could improve the survival rate significantly. Since endogenous TLR4 ligands have been associated with several inflammatory- and immune-diseases, B. quintana LPS might be a novel therapeutic strategy for TLR4-driven pathologies.

TLR3 signaling is downregulated by a MAVS isoform in epithelial cells

Innate immune responses to dsRNA result in signaling through the TLR3 pathway and/or the RIG-I/MDA-5/MAVS pathway which can activate type I IFN, proinflammatory cytokines and apoptosis. It is not clear whether MAVS could play a role in TLR3-dependent responses to extracellular dsRNA. Using a model of epithelial cells that express a functional TLR3 signaling pathway, we found that TLR3-dependent responses to extracellular dsRNA are negatively regulated by MAVS, precisely “miniMAVS”, a recently described 50kDa isoform of MAVS. This regulation of TLR3 by a MAVS isoform constitutes an endogenous regulatory mechanism in epithelial cells that could help prevent a potentially damaging excessive inflammatory response.

Toll-like receptor 4 modulates the cochlear immune response to acoustic injury.

Acoustic overstimulation traumatizes the cochlea, resulting in auditory dysfunction. As a consequence of acoustic injury, the immune system in the cochlea is activated, leading to the production of inflammatory mediators and the infiltration of immune cells. However, the molecular mechanisms responsible for initiating these immune responses remain unclear. Here, we investigate the functional role of Toll-like receptor 4 (Tlr4), a cellular receptor that activates the innate immune system, in the regulation of cochlear responses to acoustic overstimulation. Using a Tlr4 knockout mouse model, we examined how Tlr4 deficiency affects sensory cell pathogenesis, auditory dysfunction and cochlear immune activity. We demonstrate that Tlr4 knockout does not affect sensory cell viability under physiological conditions, but reduces the level of sensory cell damage and cochlear dysfunction after acoustic injury. Together, these findings suggest that Tlr4 promotes sensory cell degeneration and cochlear dysfunction after acoustic injury. Acoustic injury provokes a site-dependent inflammatory response in both the organ of Corti and the tissues of the lateral wall and basilar membrane. Tlr4 deficiency affects these inflammatory responses in a site-dependent manner. In the organ of Corti, loss of Tlr4 function suppresses the production of interleukin 6 (Il6), a pro-inflammatory molecule, after acoustic injury. By contrast, the production of inflammatory mediators, including Il6, persists in the lateral wall and basilar membrane. In addition to immune molecules, Tlr4 knockout inhibits the expression of major histocompatibility complex class II, an antigen-presenting molecule, in macrophages, suggesting that Tlr4 participates in the antigen-presenting function of macrophages after acoustic trauma. Together, these results suggest that Tlr4 regulates multiple aspects of the immune response in the cochlea and contributes to cochlear pathogenesis after acoustic injury.

Impairment of toll-like receptors 2 and 4 leads to compensatory mechanisms after sciatic nerve axotomy.

BackgroundPeripheral nerve injury results in retrograde cell body-related changes in the spinal motoneurons that will contribute to the regenerative response of their axons. Successful functional recovery also depends on molecular events mediated by innate immune response during Wallerian degeneration in the nerve microenvironment. A previous study in our lab demonstrated that TLR 2 and 4 develop opposite effects on synaptic stability in the spinal cord after peripheral nerve injury. Therefore, we suggested that the better preservation of spinal cord microenvironment would positively influence distal axonal regrowth. In this context, the present work aimed to investigate the influence of TLR2 and TLR4 on regeneration and functional recovery after peripheral nerve injury.MethodsEighty-eight mice were anesthetized and subjected to unilateral sciatic nerve crush (C3H/HeJ, n = 22, C3H/HePas, n = 22; C57Bl6/J, n = 22 and TLR2−/−, n = 22). After the appropriate survival times (3, 7, 14 days, and 5 weeks), all mice were killed and the sciatic nerves and tibialis cranialis muscles were processed for immunohistochemistry and transmission electron microscopy (TEM). Gait analysis, after sciatic nerve crushing, was performed in another set of mice (minimum of n = 8 per group), by using the walking track test (CatWalk system).ResultsTLR4 mutant mice presented greater functional recovery as well as an enhanced p75NTR and neurofilament protein expression as compared to the wild-type strain. Moreover, the better functional recovery in mutant mice was correlated to a greater number of nerve terminal sprouts. Knockout mice for TLR2 exhibited 30 % greater number of degenerated axons in the distal stump of the sciatic nerve and a decreased p75NTR and neurofilament protein expression compared to the wild type. However, the absence of TLR2 receptor did not influence the overall functional recovery. End-point equivalent functional recovery in transgenic mice may be a result of enhanced axonal diameter found at 2 weeks after lesion.ConclusionsAltogether, the present results indicate that the lack of TLR2 or the absence of functional TLR4 does affect the nerve regeneration process; however, such changes are minimized through different compensatory mechanisms, resulting in similar motor function recovery, as compared to wild-type mice. These findings contribute to the concept that innate immune-related molecules influence peripheral nerve regeneration by concurrently participating in processes taking place both at the CNS and PNS.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0579-6) contains supplementary material, which is available to authorized users.

Obesity Takes Its Toll on Visceral Pain: High-Fat Diet Induces Toll-Like Receptor 4-Dependent Visceral Hypersensitivity.

Exposure to high-fat diet induces both, peripheral and central alterations in TLR4 expression. Moreover, functional TLR4 is required for the development of high-fat diet-induced obesity. Recently, central alterations in TLR4 expression have been associated with the modulation of visceral pain. However, it remains unknown whether there is a functional interaction between the role of TLR4 in diet-induced obesity and in visceral pain. In the present study we investigated the impact of long-term exposure to high-fat diet on visceral pain perception and on the levels of TLR4 and Cd11b (a microglial cell marker) protein expression in the prefrontal cortex (PFC) and hippocampus. Peripheral alterations in TLR4 were assessed following the stimulation of spleenocytes with the TLR4-agonist LPS. Finally, we evaluated the effect of blocking TLR4 on visceral nociception, by administering TAK-242, a selective TLR4-antagonist. Our results demonstrated that exposure to high-fat diet induced visceral hypersensitivity. In parallel, enhanced TLR4 expression and microglia activation were found in brain areas related to visceral pain, the PFC and the hippocampus. Likewise, peripheral TLR4 activity was increased following long-term exposure to high-fat diet, resulting in an increased level of pro-inflammatory cytokines. Finally, TLR4 blockage counteracted the hyperalgesic phenotype present in mice fed on high-fat diet. Our data reveal a role for TLR4 in visceral pain modulation in a model of diet-induced obesity, and point to TLR4 as a potential therapeutic target for the development of drugs to treat visceral hypersensitivity present in pathologies associated to fat diet consumption.

TLR2 tolerance in EAE

Abstract TLR2 expression is enhanced in MS and EAE, but its role in these diseases is controversial. In adoptive transfer EAE, exogenous TLR ligands are not administered yet TLR2-deficient mice have been reported to develop attenuated disease. Despite this evidence for a disease-promoting function of TLR2, others have shown the opposite, i.e. that administration of TLR2 ligands can inhibit EAE in WT mice. To understand this paradox we tested the postulate that although TLR2 signaling may be EAE-promoting, repeated TLR2 ligand administration can lead to diminished TLR2 responsiveness (tolerance) and attenuated disease. To establish our approach, we administered Pam2Cys to SJL/J mice for 5 days and induced TLR2 tolerance that persisted for 4–5 days, reflected in decreased serum TNFα in response to a second TLR2 ligand (p<0.0001 day 1; p<0.01 day 3; p=0.069 day 5). Next, we used this approach to induce TLR2 tolerance in the context of EAE. SJL/J mice received EAE-causing, PLP-reactive LNCs on day 0 and tolerance was induced at various time points. Surprisingly, initiating the TLR2 tolerance protocol at day −2 or day +8 showed no effect on disease. In contrast, initiating the protocol between day +4 and +6 lead to significant inhibition of EAE onset and severity that lasted at least 5 days (p=0.025) and was associated with TLR2 tolerance (p<0.0001). In sum, we have used TLR tolerance as a novel probe to identify a narrow kinetic window in which TLR2 is required for EAE. Additionally, we have demonstrated the potential use of TLR tolerance as a new therapeutic approach in EAE and MS. Ongoing studies will characterize the relevant TLR2-expressing cells in EAE and the potential role of the microbiome in inducing homeostatic TLR tolerance that regulates autoimmunity.

Toll-like receptor 2 of tongue sole Cynoglossus semilaevis: Signaling pathway and involvement in bacterial infection.

Toll-like receptor (TLR) 2 is a member of the TLR family that plays a pivotal role in innate immunity. In mammals, TLR2 is known to recognize specific microbial structures and trigger MyD88-dependent signaling to induce various cytokine responses. In this study, we examined the expression and function of the tongue sole Cynoglossus semilaevis TLR2, CsTLR2. CsTLR2 is composed of 898 amino acid residues and shares 25.6%-27.3% overall sequence identities with known teleost TLR2. CsTLR2 is a transmembrane protein with a toll/interleukin-1 receptor domain and eight leucine-rich repeats. Expression of CsTLR2 occurred in multiple tissues and was upregulated during bacterial infection. Stimulation of the CsTLR2 pathway led to enhanced expression of MyD88-dependent signaling molecules. Recombinant CsTLR2 (rCsTLR2) corresponding to the extracellular region was able to bind to a wide range of bacteria. Under both invitro and invivo conditions, rCsTLR2 significantly reduced bacterial infection. These observations add new insights into the signaling and function of teleost TLR2.