CD40 and CD80/86 signaling in cDC1s mediate effective neoantigen vaccination and generation of antigen-specific CX3CR1+ CD8+ T cells.

The use of tumor mutation-derived neoantigen represents a promising approach for cancer vaccines. Preclinical and early phase human clinical studies have shown the successful induction of tumor neoepitope-directed responses; however, overall clinical efficacy of neoantigen vaccines has been limited. One major obstacle of this strategy is the prevailing lack of sufficient understanding of the mechanism underlying the generation of neoantigen-specific CD8+ T cells. Here, we report a correlation between antitumor efficacy of neoantigen/toll-like receptor 3 (TLR3)/CD40 agonists vaccination and an increased frequency of circulating antigen-specific CD8+ T cells expressing CX3C chemokine receptor 1 (CX3CR1) in a preclinical model. Mechanistic studies using mixed bone marrow chimeras identified that CD40 and CD80/86, but not CD70 signaling in Batf3-dependent conventional type 1 dendritic cells (cDC1s) is required for the antitumor efficacy of neoantigen vaccine and generation of neoantigen-specific CX3CR1+ CD8+ T cells. Although CX3CR1+ CD8+ T cells exhibited robust in vitro effector function, in vivo depletion of this subset did not alter the antitumor efficacy of neoantigen/TLR3/CD40 agonists vaccination. These findings indicate that the vaccine-primed CX3CR1+ subset is dispensable for antitumor CD8+ T cell responses, but can be used as a blood-based T-cell biomarker for effective priming of CD8+ T cells as post-differentiated T cells. Taken together, our results reveal a critical role of CD40 and CD80/86 signaling in cDC1s in antitumor efficacy of neoantigen-based therapeutic vaccines, and implicate the potential utility of CX3CR1 as a circulating predictive T-cell biomarker in vaccine therapy.

Conjunctival Expression of Toll-Like Receptor 3 Plays a Pathogenic Role in the Formation of Ultraviolet Light-Induced Pterygium.

Purpose: Toll-like receptor 3 (TLR3), as a damage-associated molecular pattern sensor, can detect self-RNA released from necrotic cells induced by ultraviolet B (UVB) radiation exposure. Pterygium formation is believed to be a tumorigenesis-like process induced by UVB exposure. In this study, we aimed to investigate the expression pattern of TLR3 in pterygium specimens and cultured pterygial epithelial cells (PECs). Methods: Human pterygium and ipsilateral pterygium-free conjunctiva from the same patients were used in this study. The expression of TLR3 and nuclear factor-kappa B (NF-κB) was investigated in these specimens. PECs were exposed to UVB radiation to determine the effect of UVB on the expression of TLR3 and the activation of NF-κB. Results: The immunofluorescence study showed stronger TLR3 expression in superficial epithelial cells in the pterygial epithelium in comparison with the normal conjunctival epithelium. The expression of TLR3 decreased in intensity from the superficial epithelium toward the basal cell layer, implying a correlation between UVB exposure and TLR3 expression. Differential TLR3 expression patterns in pterygial and conjunctival tissues were also found in quantitative PCR analyses. PECs after UVB irradiation had higher protein levels of TLR3 and phospho-NF-κB than those of the PECs without irradiation. Immunofluorescence studies showed that UVB irradiation induced the nuclear translocation of NF-κB in the PECs. In PECs with the targeted TLR3 gene silencing, the expression of phospho-NF-κB was not induced by UVB irradiation. Conclusions: Our results indicate that UVB exposure, TLR3 expression, and NF-κB activation may be a critical sequence that leads to the formation of pterygium.

TLR3 expression by maternal and fetal cells at the maternal-fetal interface in normal and preeclamptic pregnancies.

Inflammation and oxidative stress at the maternal-fetal interface characterize the placental dysfunction that underlies the pregnancy disorder preeclampsia. Specialized fetal trophoblasts directly interact with leukocytes at both sites of the maternal-fetal interface; the uterine wall decidua; and the placenta. TLR3 has been implicated in the harmful inflammation at the maternal-fetal interface in preeclampsia, but the cellular involvement in the decidua and placenta has not been determined. This study aimed to characterize and quantify cell-specific TLR3 expression and function at the maternal-fetal interface in normal and preeclamptic pregnancies. TLR3 expression was assessed by immunohistochemistry and quantified by a novel image-based and cell-specific quantitation method. TLR3 was expressed at the maternal-fetal interface by all decidual and placental trophoblast types and by maternal and fetal leukocytes. Placental, but not decidual, TLR3 expression was significantly higher in preeclampsia compared to normal pregnancies. This increase was attributed to placental intravillous tissue and associated with both moderate and severe placental dysfunction. TLR3 pathway functionality in the decidua and placenta was confirmed by TLR3 ligand-induced cytokine response, but the TLR3 expression levels did not correlate between the two sites. In conclusion, functional TLR3 was broadly expressed by maternal and fetal cells at both sites of the maternal-fetal interface and the placental intravillous expression was increased in preeclampsia. This suggests TLR3-mediated inflammatory involvement with local regulation at both sites of the maternal-fetal interface in normal and preeclamptic pregnancies.

The intervention mechanism of emodin on TLR3 pathway in the process of central nervous system injury caused by herpes virus infection.

Background and purpose: To investigate the effect of Emodin on the inflammatory response of brain tissue and the expression of the TLR3 pathway in mice with herpes virus encephalitis.Method: Twenty male BALB/c mice were randomly divided into the NS group, HSV-1 group, HSV-1 + Emodin group and HSV-1 + ACV group. The histopathological features and the effect of TLR3 expression were observed by staining and immunohistochemistry (IHC) respectively. The gene expression of TLR3, trif, TRADD, TRAF6, traf3, p38, Nemo and IRF3 was detected by polymerase chain reaction (PCR). The protein production of TLR3 and its downstream molecules was detected by Western blot. The expression of IL-6, TNF-α and IFN-ß in the brain tissues was detected by ELISA.Result: Compared to the HSV-1 group, the pathological changes (inflammatory cell infiltration, necrotic temporal lobe and massive hemorrhage) were not as obvious as those in the HSV-1+emodin and HSV-1+ACV groups. The TLR3 staining increased significantly in the HSV-1 groups and decreased in the HSV-1 + emodin group. Compared with the NS group, the mRNA expression of TLR3, TRIF, TRADD, TRAF6, traf3, p38, NEMO and IRF3 decreased by 20%-60% in the HSV-1 + emodin group and 30% in the HSV-1 + ACV group, respectively. The expression of IL-6, TNF-α and IFN-ß decreased by 30%-50% in the HSV-1 + emodin group and showed no significant change in the HSV-1 + ACV group, respectively.Conclusion: Emodin could inhibit the inflammatory response in the brain of mice with herpes virus encephalitis. The inhibition of TLR3 expression may play an important role in this process.

Pattern of expression of Toll like receptor (TLR)-3 and -4 genes in drug-naïve and antipsychotic treated patients diagnosed with schizophrenia.

Toll like receptors (TLRs), a class of conserved immune molecules are crucially involved in initiating innate immune response to infection. TLR activation and subsequent inflammation are linked to pathogenesis of many brain disorders. Preliminary studies indicate a possible role of TLR-driven immuno-inflammatory responses in schizophrenia. However, gene expression data of TLRs in drug-naïve as well as antipsychotic treated patients diagnosed with schizophrenia are albeit limited. In this study, expression profile of TLR3 and TLR4 genes in peripheral blood mononuclear cells (PBMCs) was compared between drug-naïve patients diagnosed with schizophrenia (N = 31) and healthy controls (N = 30). In addition, the pattern of expression of TLR3 and TLR4 genes were also examined after three months of antipsychotic medication in patients. Compared to healthy controls, gene expression levels of only TLR4 (F = 3.87, p = 0.05, ηp2 = 0.06), not TLR3 (F = 0.17, p = 0.71, ηp2 = 0.003) was significantly up-regulated in drug-naïve patients. The changes in the levels of gene expression of TLR3 (t = 0.09, p = 0.93, d = 0.02) and TLR4 (t = 0.29, p = 0.77, d = 0.06) before and after antipsychotic medication were not found to be statistically significant. This finding suggests possible contribution of TLR4 in immunopathogenetic pathway of schizophrenia.

IL-22Ra1 is induced during influenza infection by direct and indirect TLR3 induction of STAT1.

BACKGROUND: Influenza attacks the epithelium of the lung, causing cell death and disruption of the epithelial barrier leading to fluid buildup in the lung and impairment of gas exchange. Limited treatment options for severe influenza pneumonia prioritize the need for the discovery of effective therapies. IL-22 is a cytokine that promotes tissue integrity and has strong promise as a treatment option. While research has been focused on the cytokine itself, there is limited understanding of the regulation of the IL-22 receptor (IL-22Ra1) at the epithelial surface during infection. METHODS: IL-22Ra1 levels were measured by qRT-PCR, western blot and immunofluorescence following H1N1 influenza infection (A/PR/8/34 H1N1) or synthetic TLR3 mimetic, Poly (I:C). Regulation of the receptor was determined using STAT inhibitors (STAT1, STAT3 and PanSTAT inhibitors), TLR3 inhibition, and neutralization of interferon alpha receptor 2 (IFNAR2). Significance was determined by a p-value of greater than 0.05. Significance between two groups was measured using unpaired t-test and significance between more than two groups was measured using one-way ANOVA with Tukey Multiple Comparison Test. RESULTS: Here we show both in vivo and in vitro that IL-22Ra1 was induced as early as 24 h after influenza (H1N1 PR8) infection. This induction was triggered by toll-like receptor 3 (TLR3) as a TLR3 mimetic [Poly (I:C)] also induced IL-22Ra1 and inhibition of endosomal formation required for TLR3 function inhibited this process. This upregulation was dependent upon IFNß signaling through STAT1. Importantly, induction of IL-22Ra1 significantly increased IL-22 signaling as evidenced by pSTAT3 levels following IL-22 treatment. CONCLUSION: Collectively, these data suggest epithelial cells may optimize the beneficial effects of IL-22 through the induction of the IL-22 receptor during viral infection in the lung.

Bronchial mucosal IFN-α/ß and pattern recognition receptor expression in patients with experimental rhinovirus-induced asthma exacerbations.

BACKGROUND: The innate immune system senses viral infection through pattern recognition receptors (PRRs), leading to type I interferon production. The role of type I interferon and PPRs in rhinovirus-induced asthma exacerbations in vivo are uncertain. OBJECTIVES: We sought to compare bronchial mucosal type I interferon and PRR expression at baseline and after rhinovirus infection in atopic asthmatic patients and control subjects. METHODS: Immunohistochemistry was used to detect expression of IFN-α, IFN-ß, and the PRRs: Toll-like receptor 3, melanoma differentiation-associated gene 5, and retinoic acid-inducible protein I in bronchial biopsy specimens from 10 atopic asthmatic patients and 15 nonasthmatic nonatopic control subjects at baseline and on day 4 and 6 weeks after rhinovirus infection. RESULTS: We observed IFN-α/ß deficiency in the bronchial epithelium at 3 time points in asthmatic patients in vivo. Lower epithelial IFN-α/ß expression was related to greater viral load, worse airway symptoms, airway hyperresponsiveness, and reductions in lung function during rhinovirus infection. We found lower frequencies of bronchial subepithelial monocytes/macrophages expressing IFN-α/ß in asthmatic patients during infection. Interferon deficiency at baseline was not accompanied by deficient PRR expression in asthmatic patients. Both epithelial and subepithelial PRR expression were induced during rhinovirus infection. Rhinovirus infection-increased numbers of subepithelial interferon/PRR-expressing inflammatory cells were related to greater viral load, airway hyperresponsiveness, and reductions in lung function. CONCLUSIONS: Bronchial epithelial IFN-α/ß expression and numbers of subepithelial IFN-α/ß-expressing monocytes/macrophages during infection were both deficient in asthmatic patients. Lower epithelial IFN-α/ß expression was associated with adverse clinical outcomes after rhinovirus infection in vivo. Increases in numbers of subepithelial cells expressing interferon/PRRs during infection were also related to greater viral load/illness severity.

The Toll-Like Receptor 3 Agonist Poly(I:C) Induces Rapid and Lasting Changes in Gene Expression Related to Glutamatergic Function and Increases Ethanol Self-Administration in Rats.

BACKGROUND: Growing evidence suggests that neuroimmune signaling via Toll-like receptors (TLRs) alters brain circuitry related to alcohol use disorders. Both ethanol (EtOH) exposure and the TLR3 agonist, poly(I:C), increase brain TLR3 expression in neurons and glia. Furthermore, previous studies have shown that cortical TLR3 expression is correlated with lifetime EtOH intake in humans. METHODS: The current experiments investigated the consequences of poly(I:C) treatment on gene expression in 2 brain regions contributing to alcohol reinforcement, the insular cortex (IC) and nucleus accumbens (Acb) and on operant EtOH self-administration, in Long Evans rats. RESULTS: TLR3 activation increased mRNA levels of neuroimmune genes (TLR3, COX2), glutamatergic genes (mGluR2, mGluR3, GLT1), and the trophic factor BDNF in Acb and IC. Furthermore, increases in each of these genes were correlated with increases in TLR3 mRNA, suggesting that TLR3 induction of these genes may impact excitatory transmission in IC and Acb. TLR3 activation also increased EtOH self-administration 18 days postinjection and enhanced the effects of the mGluR2/3 agonist LY379268 to reduce EtOH self-administration following poly(I:C). CONCLUSIONS: Together, these findings suggest lasting consequences of TLR3 activation on gene expression including increases in Group II mGluRs in the Acb. Furthermore, we show an important role for TLR3 signaling in EtOH intake, and a functional involvement of Group II mGluRs.

Interferon-stimulated gene 60 (ISG60) constitutes a negative feedback loop in the downstream of TLR3 signaling in hCMEC/D3 cells.

Brain capillary endothelial cells are the component of blood brain barrier, and the first line of defense against viruses invading into brain. We demonstrate that treatment of hCMEC/D3 cells, a human brain capillary endothelial cell line, with a Toll-like receptor 3 (TLR3) agonist polyinosinic-polycytidylic acid (poly IC) induces the expression of interferon (IFN)-stimulated gene 60 (ISG60), and this reaction was mediated by IFN-ß. Knockdown of ISG60 increased the poly IC-induced expression of IFN-ß and an IFN-ß-inducible chemokine CXCL10. This indicates that ISG60 constitutes a negative feedback loop in the downstream of TLR3/IFN-ß. ISG60 in brain capillary endothelial cells may contribute to prevent excess immune reactions associated with viral infections.

Ontogeny of white matter, toll-like receptor expression, and motor skills in the neonatal ferret.

Inflammation caused by perinatal infection, superimposed with hypoxia and/or hyperoxia, appears to be important in the pathogenesis of preterm neonatal encephalopathy, with white matter particularly vulnerable during the third trimester. The associated inflammatory response is at least partly mediated through Toll-like receptor (TLR)-dependent mechanisms. Immunohistochemistry, gene expression, and behavioral studies were used to characterize white matter development and determine TLR3 and TLR4 expression and accumulation in the neonatal ferret brain. Expression of markers of white matter development increased significantly between postnatal day (P)1 and P10 (NG2, PDGFRα) or P15 (Olig2), and either remained elevated (NG2), or decreased again at P40 (PDGFRα, Olig2). Olig2 immunostaining within the internal capsule was also greatest at P15. Myelin basic protein (MBP) immunostaining and mRNA expression increased markedly from P15 to P40 and into adulthood, which correlated with increasing performance on behavioral tests (negative geotaxis, cliff aversion, righting reflex, and catwalk gait analysis). TLR4 and TLR3 positive staining was low at all ages, but TLR3 and TLR4 mRNA expression both increased significantly from P1 to P40. Following lipopolysaccharide (LPS) and hypoxia/hyperoxia exposure at P10, meningeal and parenchymal inflammation was seen, including an increase in TLR4 positive cells. These data suggest that the neuroinflammation associated with prematurity could be modeled in the newborn ferret.

TLR3 expression status predicts prognosis in patients with advanced thoracic esophageal squamous cell carcinoma after esophagectomy.

BACKGROUND: The relationship between Toll-like receptors (TLRs) and esophageal squamous cell carcinoma (ESCC) is not completely understood. METHODS: RT-qPCR was used to evaluate the mRNA expression of TLR1-10 in 13 ESCC lines. We then used ESCC tissue microarray (TMA) to confirm expression of TLR3 protein in patients with ESCC. RESULTS: All ESCC lines showed 10-60 times higher TLR3 mRNA expression than PBLs. High expression of TLR3 correlated with favorable 5-year overall survival (OS) and disease specific survival (DSS) among patients with ESCC after esophagectomy (p < 0.01). Additionally, In the adjuvant chemotherapy group, TLR3 high patients had significantly better 5-year OS compared to TLR3 low patients (60.2%, 34.4%, respectively) but not in the surgery alone group. CONCLUSION: High TLR3 expression is an independent prognostic factor and has the potential to serve as a clinically useful marker of the need for adjuvant chemotherapy after esophagectomy in patients with advanced thoracic ESCC.

TLR3 promotes MMP-9 production in primary human airway epithelial cells through Wnt/ß-catenin signaling.

BACKGROUND: Airway epithelial cells (AEC) act as the first line of defence in case of lung infections. They constitute a physical barrier against pathogens and they participate in the initiation of the immune response. Yet, the modalities of pathogen recognition by AEC and the consequences on the epithelial barrier remain poorly documented. METHOD: We investigated the response of primary human AEC to viral (polyinosinic-polycytidylic acid, poly(I:C)) and bacterial (lipopolysaccharide, LPS) stimulations in combination with the lung remodeling factor Transforming Growth Factor-ß (TGF-ß). RESULTS: We showed a strong production of pro-inflammatory cytokines (Interleukin (IL)-6, Tumor Necrosis Factor α, TNFα) or chemokines (CCL2, CCL3, CCL4, CXCL10, CXCL11) by AEC stimulated with poly(I:C). Cytokine and chemokine production, except CXCL10, was Toll Like Receptor (TLR)-3 dependent and although they express TLR4, we found no cytokine production after LPS stimulation. Poly(I:C), but not LPS, synergised with TGF-ß for the production of matrix metalloproteinase-9 (MMP-9) and fibronectin. Mechanistic analyses suggest the secretion of Wnt ligands by AEC along with a degradation of the cellular junctions after poly(I:C) exposure, leading to the release of ß-catenin from the cell membrane and stimulation of the Wnt/ß-catenin pathway. CONCLUSION: Our results highlight the cross talk between TGF-ß and TLR signaling in bronchial epithelium and its impact on the remodeling process.

Expression of miR-155 associated with Toll-like receptors 3, 7, and 9 transcription in the olfactory bulbs of cattle naturally infected with BHV5.

Bovine herpesvirus 5 (BHV5) infection of young cattle is frequently associated with fatal neurological disease and, as such, represents an attractive model for studying the pathogenesis of viral-induced meningoencephalitis. Following replication in the nasal mucosa, BHV5 invades the central nervous system (CNS) mainly through the olfactory pathway. The innate immune response triggered by the host face to virus replication through the olfactory route is poorly understood. Recently, an upregulation of conserved pathogen-associated molecular pattern, as Toll-like receptors (TLRs), has been demonstrated in the CNS of BHV5 experimentally infected cows. A new perspective to understand host-pathogen interactions has emerged elucidating microRNAs (miRNAs) network that interact with innate immune response during neurotropic viral infections. In this study, we demonstrated a link between the expression of TLRs 3, 7, and 9 and miR-155 transcription in the olfactory bulbs (OB) of 16 cows suffering from acute BHV5-induced neurological disease. The OBs were analyzed for viral antigens and genome, miR-155 and TLR 3, 7, and 9 expression considering three major regions: olfactory receptor neurons (ORNs), glomerular layer (GL), and mitral cell layer (ML). BHV5 antigens and viral genomes, corresponding to glycol-C gene, were detected in all OBs regions by fluorescent antibody assay (FA) and PCR, respectively. TLR 3, 7, and 9 transcripts were upregulated in ORNs and ML, yet only ORN layers revealed a positive correlation between TLR3 and miR-155 transcription. In ML, miR-155 correlated positively with all TLRs studied. Herein, our results evidence miR-155 transcription in BHV5 infected OB tissue associated to TLRs expression specifically ORNs which may be a new window for further studies.

LncRNA CRNDE triggers inflammation through the TLR3-NF-κB-Cytokine signaling pathway.

Colorectal neoplasia differentially expressed (CRNDE), an oncogene, is highly expressed in many tumor cells and affects cellular proliferation, migration, invasion, and apoptosis. Its function and mechanism of action is a research hotspot. In this study, microarray analysis was performed to discover the differentially expressed genes in CRNDE over-expression cells. RT² Profiler PCR Array was used to study the expression of genes related to the toll-like receptor (TLR) pathway. We found that over-expression of CRNDE in astrocytes increased the expression of key factors in the toll-like receptor signaling pathway, especially toll-like receptor-3-mediated MyD88-independent pathway. Furthermore, it up-regulated expression levels of downstream transcription factor such as nuclear factor kappa B and numerous cytokines. In contrast, CRNDE knockdown in glioma U87MG cell line showed an opposite trend in the expression of the above-mentioned genes. We speculated that CRNDE might trigger inflammation to regulate tumorigenesis and tumor development through the toll-like receptor pathway.

Molecular mechanisms of HBeAg in persistent HBV infection.

PURPOSE: The aim of this study is to investigate the T-lymphocyte subpopulation and expression of programmed cell death-1 (PD-1), toll-like receptor (TLR)3, TLR4, and interferon (INF)-γ to illustrate the relationship between hepatitis B e antigen (HBeAg) and persistent hepatitis B virus (HBV) infection. METHODS: Blood was taken from normal subjects into anticoagulation tubes to separate peripheral blood mononuclear cells (PBMCs). The PBMCs were divided into four groups and cultured with various concentrations of HBeAg for 72 h. Changes in the T-cell subset were analyzed through cell counting by flow cytometry, and expression of TLR3, TLR4, and PD-1 was assessed by flow cytometry and Western blot. The concentration of IFN-γ was analyzed using enzyme-linked immunospot (ELISPOT) experiments. RESULTS: PBMCs were stimulated with various concentrations of HBeAg for 72 h and assayed by flow cytometry to determine CD4+ and CD8+ cell counts. The relative frequencies of CD4+ and CD8+ subpopulations and the CD4+/CD8+ ratio decreased compared with the control group, and T-cell impairment was significantly associated with higher HBeAg load. TLR3, TLR4, and PD-1 protein expression was assessed using flow cytometry and Western blotting. Expression of TLR3, TLR4, and PD-1 increased with increasing concentration of HBeAg. ELISPOT experiments were used to determine the concentration of IFN-γ. IFN-γ production in treatment groups was lower than in the control group. Comparing IFN-γ production in treatment groups, IFN-γ production in PBMCs stimulated with high dose of HBeAg was lower than for those stimulated with low-dose HBeAg. CONCLUSIONS: HBeAg can inhibit proliferation of lymphocytes, increase TLR3, TLR4, and PD-1 expression, and decrease IFN-γ production. This may be one of the molecular mechanisms of HBV immune tolerance.

Upregulated TLR3 Promotes Neuropathic Pain by Regulating Autophagy in Rat With L5 Spinal Nerve Ligation Model.

Microglia, rapidly activated following peripheral nerve injury (PNI), accumulate within the spinal cord and adopt inflammation that contributes to development and maintenance of neuropathic pain. Microglia express functional Toll-like receptors (TLRs), which play pivotal roles in regulating inflammatory processes. However, little is known about the role of TLR3 in regulating neuropathic pain after PNI. Here TLR3 expression and autophagy activation was assayed in dorsal root ganglions and in microglia following PNI by using realtime PCR, western blot and immunohistochemistry. The role of TLR3/autophagy signaling in regulating tactile allodynia was evaluated by assaying paw mechanical withdrawal threshold and cold allodynia after intrathecal administration of Poly (I:C) and 3-methyladenine (3-MA). We found that L5 spinal nerve ligation (SNL) induces the expression of TLR3 in dorsal root ganglions and in primary rat microglia at the mRNA and protein level. Meanwhile, L5 SNL results in an increased activation of autophagy, which contributes to microglial activation and subsequent inflammatory response. Intrathecal administration of Poly (I:C), a TLR3 agonist, significantly increases the activation of microglial autophagy, whereas TLR3 knockdown markedly inhibits L5 SNL-induced microglial autophagy. Poly (I:C) treatment promotes the expression of proinflammatory mediators, whereas 3-MA (a specific inhibitor of autophagy) suppresses Poly (I:C)-induced secretion of proinflammatory cytokines. Autophagy inhibition further inhibits TLR3-mediated mechanical and cold hypersensitivity following SNL. These results suggest that inhibition of TLR3/autophagy signaling contributes to alleviate neurophathic pain triggered by SNL.

Association of mRNA expression of toll-like receptor 2 and 3 with hepatitis B viral load in chronic hepatitis, cirrhosis, and hepatocellular carcinoma.

During Hepatitis B virus infection, the pathogen sensors Toll-like receptors (TLRs) play a role in innate immunity system. The study aimed to investigate mRNA expression levels of TLR2 and TLR3 in Hepatitis B virus (HBV) mediated chronic hepatitis B (CHB), cirrhosis (CIRR), and hepatocellular carcinoma (HCC), and to correlate viral load with severity of these diseases and expression of TLRs. A total of 180 HBV DNA positive samples were selected for the study. HVB-DNA was detected by multiplex PCR. Viral load estimation was done by using the Ampisure HBV Quantitative kit as per manufacture instructions. Expression levels of TLR2 and TLR3 were determined by real time PCR. The viral load was estimated to be 6.64log10 IU/ml in CHB, 4.88log10 IU/ml in CIRR, and 4.86log10 IU/ml in HCC. No significant association of viral load was found with increasing age. Upregulation of TLR2 expression in CHB when individually compared with CIRR and HCC was found to be statistically significant. Downregulation of TLR3 expressions in CIRR when compared to both CHB and HCC individually were found to be statistically significant. No significant effect of viral load on the expression of TLR2 and 3 were found. With severity of the disease from CHB to HCC, the HBV load decreases. The study suggests the possibility of HBV interacting with signalling of both analysed TLR receptors which partially explains the induction of immune tolerance pathways by Hepatitis B virus. J. Med. Virol. 89:1008-1014, 2017. © 2017 Wiley Periodicals, Inc.

Autoimmune Regulator Expression in DC2.4 Cells Regulates the NF-κB Signaling and Cytokine Expression of the Toll-Like Receptor 3 Pathway.

Autoimmune regulator (Aire) mutations result in autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED), which manifests as multi-organ autoimmunity and chronic mucocutaneous candidiasis (CMC). Indendritic cells (DCs), pattern recognition receptors (PRR), such as Toll-like receptors (TLRs), are closely involved in the recognition of various pathogens, activating the intercellular signaling pathway, followed by the activation of transcription factors and the expression of downstream genes, which take part in mediating the immune response and maintaining immune tolerance. In this study, we found that Aire up-regulated TLR3 expression and modulated the downstream cytokine expression and nuclear factor-κB (NF-κB) of the TLR3 signaling pathway.

Down-Regulation of RNA 3'-Terminal Phosphate Cyclase Attenuates Toll-Like Receptor 3-Mediated Axonal Loss in the Retina and Optic Nerve.

PURPOSE: To investigate the role of RNA 3'-terminal phosphate cyclase (Rtca) in Toll-like receptor 3 (TLR3)-mediated loss of retinal ganglion cells (RGCs) and their axons. METHODS: Polyinosinic-polycytidylic acid (Poly[I:C]) or PBS was injected into the vitreous humor of C57BL/6J and Tlr3 knockout mice. C57BL/6J mouse eyes were treated with Rtca silencing RNA or control RNA, with or without PBS or Poly(I:C). At 24, 48, and 72 hours after treatments, RGC loss was determined with the brain-specific homeobox/POU domain protein 3a antibody, and axonal loss was assessed by using the neuronal class III beta-tubulin (Tuj1) antibody. Axonal loss in the optic nerves was determined by anterograde-labeling of Cholera Toxin B. Western blot assays were performed to determine TLR3, Rtca, c-jun N-terminal kinase 3 (JNK3), and phospho-JNK3 (pJNK3) levels, and immunohistochemistry assays were performed to determine the cells that synthesize Rtca. RESULTS: Poly(I:C) significantly up-regulated the protein levels of TLR3, Rtca, JNK3, and pJNK3 in the retina. Rtca levels were increased in RGCs, and an increase in Rtca levels promoted significant loss of RGCs and their axons. In Tlr3 knockout mouse retinas, Poly(I:C) failed to elevate Rtca, JNK3, and pJNK3 protein levels and did not promote significant axonal loss. Also, Rtca silencing RNA down-regulated Rtca, JNK3, and pJNK3 in C57BL/6J mouse retinas, and down-regulation of Rtca attenuated Poly(I:C)-mediated loss of RGCs and their axons. CONCLUSIONS: The results presented in this study show that the activation of TLR3 promotes the loss of RGCs and their axons by elevating Rtca levels in the retina. Also, the results presented in this study show that Rtca regulates JNK3 expression in the retina.