Immunotherapeutic role of cabazitaxel treatment in the activation of TLR3 signalling in metastatic castration-resistant prostate cancer in vitro.

BACKGROUND: The activation of toll like receptors (TLR) potentially affect the inflammatory tumor microenvironment and thus is associated with tumor growth or inhibition. Cabazitaxel (CAB) has been effectively used for the treatment of metastatic castration-resistant prostate cancer (mCRPC). However, the immune regulatory role of CAB in the tumor microenvironment is not clear. In this context, we for the first time assessed the immunotherapeutic role of CAB in the TLR3 signalling following activation of Poly I:C in mCRPC cells. METHODS AND RESULTS: The cytotoxic and apoptotic effects of CAB with the induction of Poly I:C were determined by WST-1, Annexin V, acridine orange, RT-PCR analysis, ELISA assay and immunofluorescence staining in DU-145 mCRPC and HUVEC control cells. Our findings showed that CAB treatment with Poly I:C significantly suppressed the proliferation of DU-145 cells through the induction of apoptosis and caspase activation. Additionally, higher concentration of CAB mediated the activation of TLR3 via increased cytoplasmic and nuclear expression of TLR3, TICAM-1 and IRF-3 in mCRPC cells. CONCLUSIONS: Co-treatment of CAB and Poly I:C was more effective in mCRPC cells with less toxicity in control cells. However, further investigations are required to elucidate the molecular mechanisms of TLRs signalling upon CAB treatment at the molecular level to further validate the immunotherapeutic efficacy of CAB in mCRPC.

ω-3 fatty acid alleviates virus-induced myocardial injury by regulating TLR4 and TLR3 expression.

The specific pathogenesis of viral-induced myocardial injury is unclear. TLR regulation plays an important role in virus-induced myocardial injury. The therapeutic effect and possible mechanism of omega-3 fatty acids in patients with viral-induced myocardial injury must be investigated. The study population was randomly divided into three groups: a healthy control group (n = 50); general treatment group (n = 40); and general treatment with ω-3 polyunsaturated fatty acid group (n = 36). We detected the mRNA levels of TLR3 and TLR4, downstream signal pathway proteins, inflammatory factors, oxidative stress markers, and myocardial enzymes in patients and healthy controls. ω-3 fatty acid therapy in patients with virus-induced myocardial injury significantly regulates the expression of TLR3 and TLR4 and their downstream signal protein, increases antioxidant expression, reduces the secretion of inflammatory factors, alleviates myocardial injury, and improves cardiac function. This provides a new strategy to treat virus-induced myocardial injury.

In situ self-assembly of Au-antimiR-155 nanocomplexes mediates TLR3-dependent apoptosis in hepatocellular carcinoma cells.

MicroRNA 155 (miRNA-155) is frequently dysregulated in hepatocellular carcinoma (HCC) and other cancer types. Toll-like receptor 3 (TLR3), a putative miR-155 target, plays a key role in liver pathophysiology, and its downregulation in HCC cells is associated with apoptosis evasion and poor outcomes. Herein, we examined the ability of in situ self-assembled Au-antimiR-155 nanocomplexes (Au-antimiRNA NCs) to activate TLR3 signaling in HCC cells. Gene expression analysis confirmed an inverse relationship between miR-155 and TLR3 expression in HCC samples, and marked upregulation of miR-155 was observed in HCC cells but not in normal L02 hepatocytes. RNA immunoprecipitation confirmed physical interaction between miR-155 and TLR3, while negative regulation of TLR3 expression by miR-155 was demonstrated by luciferase reporter assays. Au-antimiR-155 NCs were self-assembled within HepG2 HCC cells, but not within control L02 cells. They efficiently silenced miR-155, thereby inhibiting proliferation and migration and inducing apoptosis in HepG2 cells. Molecular analyses suggested these effects are secondary to TLR3 signaling mediating NF-κB transcription, caspase-8 activation, and interleukin-1ß (IL-1ß) release. Our results provide a basis for future studies examining the in vivo applicability of this novel Au-antimiRNA NCs delivery system to halt HCC progression by activating pro-apoptotic TLR3 signaling.

High throughput and comprehensive approach to develop multiepitope vaccine against minacious COVID-19.

The ongoing enigmatic COVID-19 outbreak, first reported from Wuhan, China, on last day of the year 2019, which has spread to 213 countries, territories/areas till 28th April 2020, threatens hundreds of thousands human souls. This devastating viral infection has stimulated the urgent development of viable vaccine against COVID-19 across the research institutes around the globe. The World Health Organization (WHO) has also confirmed that the recent pandemic is causing Public Health Emergency of International apprehension. Moreover, the earlier two pathogenic SARS-CoV and MERS-CoV and many others yet to be identified pose a universal menace. Here, in this piece of work, we have utilized an in silico structural biology and advanced immunoinformatic strategies to devise a multi-epitope subunit vaccine against ongoing COVID-19 infection. The engineered vaccine sequence is adjuvanted with ß-3 defensin and comprised of B-cell epitopes, HTL epitopes and CTL epitopes. This is very likely that the vaccine will be able to elicit the strong immune response. Further, specific binding of the engineered vaccine and immune cell receptor TLR3 was estimated by molecular interaction studies. Strong interaction in the binding groove as well as good docking scores affirmed the stringency of engineered vaccine. The interaction is stable with minimal deviation in root-mean square deviation and root-mean-square fluctuation was confirmed by the molecular dynamics simulation experiment. The immune-simulation by C-ImmSim server, which mimics the natural immune environment, yielded more potent immune response data of B-cells, Th cells, Tc cells and IgG for vaccine. The encouraging data obtained from the various in-silico works indicated this vaccine as an effective therapeutic against COVID-19.

MyD88-dependent and -independent signalling via TLR3 and TLR4 are differentially modulated by Δ9-tetrahydrocannabinol and cannabidiol in human macrophages.

Toll-like receptors (TLRs) are sensors of pathogen-associated molecules that trigger inflammatory signalling in innate immune cells including macrophages. All TLRs, with the exception of TLR3, promote intracellular signalling via recruitment of the myeloid differentiation factor 88 (MyD88) adaptor, while TLR3 signals via Toll-Interleukin-1 Receptor (TIR)-domain-containing adaptor-inducing interferon (IFN)-ß (TRIF) adaptor to induce MyD88-independent signalling. Furthermore, TLR4 can activate both MyD88-dependent and -independent signalling (via TRIF). The study aim was to decipher the impact of the highly purified plant-derived (phyto) cannabinoids Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD), when delivered in isolation and in combination (1:1), on MyD88-dependent and -independent signalling in macrophages. We employed the use of the viral dsRNA mimetic poly(I:C) and endotoxin lipopolysaccharide (LPS), to induce viral TLR3 and bacterial TLR4 signalling in human Tamm-Horsfall protein-1 (THP-1)-derived macrophages, respectively. TLR3/TLR4 stimulation promoted the activation of interferon (IFN) regulatory factor 3 (IRF3) and TLR4 promoted the activation of nuclear factor (NF)-κB signalling, with downstream production of the type I IFN-ß, the chemokines CXCL10 and CXCL8, and cytokine TNF-α. THC and CBD (both at 10 µM) attenuated TLR3/4-induced IRF3 activation and induction of CXCL10/IFN-ß, while both phytocannabinoids failed to impact TLR4-induced IκB-α degradation and TNF-α/CXCL8 expression. The role of CB1, CB2 and PPARγ receptors in mediating the effect of THC and CBD on MyD88-independent signalling was investigated. TLRs are attractive therapeutic targets given their role in inflammation and initiation of adaptive immunity, and data herein indicate that both CBD and THC preferentially modulate TLR3 and TLR4 signalling via MyD88-independent mechanisms in macrophages. This offers mechanistic insight into the role of phytocannabinoids in modulating cellular inflammation.

Pre-treatment with the viral Toll-like receptor 3 agonist poly(I:C) modulates innate immunity and protects neutropenic mice infected intracerebrally with Escherichia coli.

BACKGROUND: Individuals with impaired immunity are more susceptible to infections than immunocompetent subjects. No vaccines are currently available to induce protection against E. coli meningoencephalitis. This study evaluated the potential of poly(I:C) pre-treatment to induce trained immunity. Poly(I:C) was administered as a non-specific stimulus of innate immune responses to protect immunocompetent and neutropenic wild-type mice from a subsequent challenge by the intracranial injection of E. coli K1. METHODS: Three days prior to infection, mice received an intraperitoneal injection of poly(I:C) or vehicle. Kaplan-Meier survival curves were analyzed. In short-term experiments, bacterial titers and the inflammatory response were characterized in the blood, cerebellum, and spleen homogenates. NK cell subpopulations in the brain and spleen were analyzed by flow cytometry. Numbers of microglia and activation scores were evaluated by histopathology. RESULTS: Pre-treatment with 200 µg poly(I:C) increased survival time, reduced mortality, and enhanced bacterial clearance in the blood, cerebellum, and spleen at early infection in neutropenic mice. Poly(I:C)-mediated protection correlated with an augmented number of NK cells (CD45+NK1.1+CD3-) and Iba-1+ microglial cells and a higher production of IFN-γ in the brain. In the spleen, levels of CCL5/RANTES and IFN-γ were increased and sustained in surviving poly(I:C)-treated animals for 14 days after infection. In immunocompetent animals, survival time was not significantly prolonged in poly(I:C)-treated animals although poly(I:C) priming reduced brain bacterial concentrations compared with vehicle-injected animals at early infection. CONCLUSIONS: Pre-treatment with the viral TLR3 agonist poly(I:C) modulated innate immune responses and strengthened the resistance of neutropenic mice against E. coli K1 meningoencephalitis.

Transcriptome Modifications in Porcine Adipocytes via Toll-Like Receptors Activation.

Adipocytes are the most important cell type in adipose tissue playing key roles in immunometabolism. We previously reported that nine members of the Toll-like receptor (TLR) family are expressed in an originally established porcine intramuscular pre-adipocyte (PPI) cell line. However, the ability of TLR ligands to modulate immunometabolic transcriptome modifications in porcine adipocytes has not been elucidated. Herein, we characterized the global transcriptome modifications in porcine intramuscular mature adipocytes (pMA), differentiated from PPI, following stimulation with Pam3csk4, Poly(I:C) or LPS which are ligands for TLR2, TLR3, and TLR4, respectively. Analysis of microarray data identified 530 (218 up, 312 down), 520 (245 up, 275 down), and 525 (239 up, 286 down) differentially expressed genes (DEGs) in pMA following the stimulation with Pam3csk4, Poly(I:C), and LPS, respectively. Gene ontology classification revealed that DEGs are involved in several biological processes including those belonging to immune response and lipid metabolism pathways. Functionally annotated genes were organized into two groups for downstream analysis: immune response related genes (cytokines, chemokines, complement factors, adhesion molecules, and signal transduction), and genes involved with metabolic and endocrine functions (hormones and receptors, growth factors, and lipid biosynthesis). Differential expression analysis revealed that EGR1, NOTCH1, NOS2, TNFAIP3, TRAF3IP1, INSR, CXCR4, PPARA, MAPK10, and C3 are the top 10 commonly altered genes of TLRs induced transcriptional modification of pMA. However, the protein-protein interaction network of DEGs identified EPOR, C3, STAR, CCL2, and SAA2 as the major hub genes, which were also exhibited higher centrality estimates in the Gene-Transcription factor interaction network. Our results provide new insights of transcriptome modifications associated with TLRs activation in porcine adipocytes and identified key regulatory genes that could be used as biomarkers for the evaluation of treatments having immunomodularoty and/or metabolic functional beneficial effects in porcine adipocytes.

Chronic ethanol consumption: role of TLR3/TRIF-dependent signaling.

Chronic ethanol consumption stimulates neuroimmune signaling in the brain, and Toll-like receptor (TLR) activation plays a key role in ethanol-induced inflammation. However, it is unknown which of the TLR signaling pathways, the myeloid differentiation primary response gene 88 (MyD88) dependent or the TIR-domain-containing adapter-inducing interferon-ß (TRIF) dependent, is activated in response to chronic ethanol. We used voluntary (every-other-day) chronic ethanol consumption in adult C57BL/6J mice and measured expression of TLRs and their signaling molecules immediately following consumption and 24 hours after removing alcohol. We focused on the prefrontal cortex where neuroimmune changes are the most robust and also investigated the nucleus accumbens and amygdala. Tlr mRNA and components of the TRIF-dependent pathway (mRNA and protein) were increased in the prefrontal cortex 24 hours after ethanol and Cxcl10 expression increased 0 hour after ethanol. Expression of Tlr3 and TRIF-related components increased in the nucleus accumbens, but slightly decreased in the amygdala. In addition, we demonstrate that the IKKε/TBK1 inhibitor Amlexanox decreases immune activation of TRIF-dependent pathway in the brain and reduces ethanol consumption, suggesting the TRIF-dependent pathway regulates drinking. Our results support the importance of TLR3 and the TRIF-dependent pathway in ethanol-induced neuroimmune signaling and suggest that this pathway could be a target in the treatment of alcohol use disorders.

Tumor necrosis factor-α synergistically enhances polyinosinic-polycytidylic acid-induced toll-like receptor 3 signaling in cultured normal human mesangial cells: possible involvement in the pathogenesis of lupus nephritis.

AIM: It has been reported that tumor necrosis factor (TNF)-α plays dual controversial roles, beneficial or detrimental, in the pathogenesis of murine lupus nephritis (LN). However, its precise role in the development of human LN remains to be determined. METHODS: We examine the effect of pretreatment with TNF-α on the toll-like receptor 3 (TLR3) signaling induced by polyinosinic-polycytidylic acid (poly IC), a synthetic analog of viral dsRNA that makes "pseudoviral" infection in cultured normal human mesangial cells, and analyzed the expression of CC chemokine ligand 5 (CCL5) via TLR3/interferon (IFN)-ß/retinoic acid-inducible gene-I (RIG-I) pathway by reverse transcriptase-polymerase chain reaction, Western blotting and enzyme-linked immunosorbent assay. RESULTS: We found synergistic effect of TNF-α, even at low level, on the expression of CCL5 induced by poly IC in a concentration-dependent manner, in comparison with that by poly IC alone. Knockdown of either IFN-ß or RIG-I decreased CCL5 expression induced by TNF-α followed by poly IC. CONCLUSION: Pretreatment with TNF-α leads marked activation of the TLR3/IFN-ß/RIG-I/CCL5 axis induced by "pseudoviral" infection. Since chronic local activation of proinflammatory cytokines including TNF-α in resident renal cells may exist in patients with active lupus, synergistic effect of TNF-α and "pseudoviral" infection is possibly involved in the development of LN.

Dendritic cell subsets require cis-activation for cytotoxic CD8 T-cell induction.

Dendritic cells (DCs) are required for the induction of cytotoxic T cells (CTL). In most tissues, including the lung, the resident DCs fall into two types expressing the integrin markers CD103 and CD11b. The current supposition is that DC function is predetermined by lineage, designating the CD103(+) DC as the major cross-presenting DC able to induce CTL. Here we show that Poly I:C (TLR3 agonist) or R848 (TLR7 agonist) do not activate all endogenous DCs. CD11b(+) DCs can orchestrate a CTL response in vivo in the presence of a TLR7 agonist but not a TLR3 agonist, whereas CD103(+) DCs require ligation of TLR3 for this purpose. This selectivity does not extend to antigen cross-presentation for T-cell proliferation but is required for induction of cytotoxicity. Thus, we demonstrate that the ability of DCs to induce functional CTLs is specific to the nature of the pathogen-associated molecular pattern (PAMP) encountered by endogenous DC.

Toll like receptor-3 priming alters diesel exhaust particle-induced cytokine responses in human bronchial epithelial cells.

Inflammation is considered central in the pathology of health effects from airborne particulate matter (PM). Preexisting inflammatory disorders, such as asthma, but also pulmonary infections, appear to be a risk factor of adverse health effects from PM exposure. Thus, to assess whether and how preexisting inflammation may sensitize lung cells toward additional proinflammatory effects of PM, human bronchial epithelial cells (BEAS-2B) were primed with the highly proinflammatory Toll-like receptor 3 (TLR3) ligand, Poly I:C, prior to exposure with diesel exhaust particles (DEP). DEP-exposure alone induced increased gene-expression of interleukin-6 (IL-6) and CXCL8 (IL-8) but did not affect expression of CCL5 (RANTES), while TLR3-priming alone induced expression of IL-6, CXCL8 and CCL5. DEP-exposure exacerbated IL-6 and CXCL8 responses in TLR3-primed cells, while TLR3-induced CCL5 was suppressed by DEP. TLR3-priming and DEP-exposure resulted in possible additive effects on p38 phosphorylation and IκB-degradation, while DEP rather suppressed ERK and JNK-activation. However, TLR3-priming elicited a considerable increase in p65-phosphorylation at serine 536 which is known to enhance the transcriptional activity of NF-κB. DEP-exposure was unable to induce p65-phosphorylation. Thus TLR3-priming may affect susceptibility toward DEP by activating both shared and complementing pathways required for optimal expression of proinflammatory genes such as IL-6 and CXCL8. The study underscores that primed "sick" cells may be more susceptible toward effects of particle-exposure and respond both stronger and differently compared to unprimed "healthy" cells.

Cathelicidin antimicrobial peptide LL-37 augments interferon-ß expression and antiviral activity induced by double-stranded RNA in keratinocytes.

BACKGROUND: Cathelicidin antimicrobial peptide LL-37 has the capacity to kill a wide range of microbes and to modify host immunity. Recently, our group observed that the activation of keratinocytes by LL-37 and DNA greatly increases interferon (IFN)-ß through Toll-like receptor (TLR)9. However, the effect of LL-37 on the induction of IFN-ß through TLR3, a sensor of double-stranded (ds) RNA, in keratinocytes is not well known. OBJECTIVES: To investigate whether LL-37 could affect TLR3 signalling and antiviral activity in normal human epidermal keratinocytes (NHEKs). METHODS: We investigated the production of IFN-ß in NHEKs stimulated with a TLR3 ligand, poly (I:C), in the presence of LL-37. To examine the effect of LL-37 and poly (I:C) on antiviral activity, a virus plaque assay using herpes simplex (HS) virus type-1 was carried out. The uptake of poly (I:C) conjugated with fluorescein isothiocyanate (FITC) into the keratinocytes was observed in the presence of LL-37. Immunostaining for TLR3 and LL-37 was performed using skin samples from HS. RESULTS: LL-37 and poly (I:C) synergistically induced the expression of IFN-ß in NHEKs. Furthermore, co-stimulation with LL-37 and poly (I:C) significantly decreased the viral plaque numbers compared with poly (I:C) or LL-37 alone. LL-37 enhanced the uptake of FITC-conjugated poly (I:C) into cells. Immunohistochemical analysis demonstrated that the expression of TLR3 and LL-37 is upregulated in HS lesions. CONCLUSIONS: Our findings suggest that LL-37 augments the antiviral activity induced by dsRNA in keratinocytes, which may contribute to the innate immune response to cutaneous viral infections such as HS.

Inhibition of phosphodiesterase 4 modulates cytokine induction from toll like receptor activated, but not rhinovirus infected, primary human airway smooth muscle.

BACKGROUND: Virus-induced exacerbations of Chronic Obstructive Pulmonary Disease (COPD) are a significant health burden and occur even in those receiving the best current therapies. Rhinovirus (RV) infections are responsible for half of all COPD exacerbations. The mechanism by which exacerbations occur remains undefined, however it is likely to be due to virus-induced inflammation. Given that phophodiesterase 4 (PDE4) inhibitors have an anti-inflammatory effect in patients with COPD they present a potential therapy prior to, and during, these exacerbations. METHODS: In the present study we investigated whether the PDE4 inhibitor piclamilast (10(-6) M) could alter RV or viral mimetic (5 µg/mL of imiquimod or poly I:C) induced inflammation and RV replication in primary human airway smooth muscle cells (ASMC) and bronchial epithelial cells (HBEC). The mediators IL-6, IL-8, prostaglandin E2 and cAMP production were assayed by ELISA and RV replication was assayed by viral titration. RESULTS: We found that in ASMCs the TLR3 agonist poly I:C induced IL-8 release was reduced while induced IL-6 release by the TLR7/8 agonist imiquimod was further increased by the presence of piclamilast. However, in RV infected ASMCs, virus replication and induced mediator release were unaltered by piclamilast, as was also found in HBECs. The novel findings of this study reveal that although PDE inhibitors may not influence RV-induced cytokine production in ASMCs and replication in either ASMCs or HBECs, they have the capacity to be anti-inflammatory during TLR activation by modulating the induction of these chemotactic cytokines. CONCLUSION: By extrapolating our in vitro findings to exacerbations of COPD in vivo this suggests that PDE4 inhibitors may have beneficial anti-inflammatory properties when patients are infected with bacteria or viruses other than RV.

Cannabinoid 1 receptors are critical for the innate immune response to TLR4 stimulation.

Sickness behaviors are host defense adaptations that arise from integrated autonomic outputs in response to activation of the innate immune system. These behaviors include fever, anorexia, and hyperalgesia intended to promote survival of the host when encountering pathogens. Cannabinoid (CB) receptor activation can induce hypothermia and attenuate LPS-evoked fever. The aim of the present study was to examine the role of CB1 receptors in the LPS-evoked febrile response. CB1 receptor-deficient (CB1(-/-)) mice did not display LPS-evoked fever; likewise, pharmacological blockade of CB1 receptors in wild-type mice blocked LPS-evoked fever. This unresponsiveness is not limited to thermogenesis, as the animals were not hyperalgesic after LPS administration. A Toll-like receptor (TLR)3 agonist and viral mimetic polyinosinic:polycytidylic acid evoked a robust fever in CB1(-/-) mice, suggesting TLR3-mediated responses are functional. LPS-evoked c-Fos activation in areas of the brain associated with the febrile response was evident in wild-type mice but not in CB1(-/-) mice. Liver and spleen TLR4 mRNA were significantly lower in CB1(-/-) mice compared with wild-type mice, and peritoneal macrophages from CB1(-/-) mice did not release proinflammatory cytokines in response to LPS. These data indicate that CB1 receptors play a critical role in LPS-induced febrile responses through inhibiting TLR4-mediated cytokine production.

Mechanisms of chemokine responses by polycyclic aromatic hydrocarbons in bronchial epithelial cells: sensitization through toll-like receptor-3 priming.

We have previously observed that 1-nitropyrene (1-NP) and its amine metabolite 1-aminopyrene (1-AP) induce differential chemokine responses in human bronchial epithelial cells (BEAS-2B) characterized by maximum responses for CXCL8 (IL-8) and CCL5 (RANTES), respectively. In the present study, we further explored the effects of 1-NP and 1-AP on chemokine responses. The results suggest that the differential effect of 1-NP and 1-AP on CXCL8 and CCL5 in BEAS-2B cells was mainly related to effects at higher concentrations, which in the case of 1-NP seemed to be linked to ROS-formation and/or metabolic activation by CYP-enzymes. However, at a low concentration (1 µM) where neither 1-NP, 1-AP nor unsubstituted pyrene had any effect on chemokine responses, we found that all three PAHs potentiated CXCL8 and CCL5 responses induced by the TLR3 ligand polyinosinic:polycytidylic acid (Poly I:C) in BEAS-2B cells. As neither benzo[a]pyrene nor ß-naphthoflavone induced a similar effect in Poly I:C-primed cells, the response seemed independent of aryl hydrocarbon receptor-mediated mechanisms. The results show that priming cells with an inflammogenic stimuli like Poly I:C sensitizes the cells toward additional pro-inflammatory effects of certain PAHs. The study underscores that testing on healthy cells or animals may not be sufficient to fully evaluate chemokine responses and the pro-inflammatory potential of organic chemicals.

Activation of toll-like receptor 3 attenuates alcoholic liver injury by stimulating Kupffer cells and stellate cells to produce interleukin-10 in mice.

BACKGROUND & AIMS: The important function of toll-like receptor (TLR) 4 in Kupffer cells and hepatic stellate cells (HSCs) has been well documented in alcoholic liver injury. However, little is known about the role of TLR3. Thus, we tested whether TLR3 activation in HSCs and Kupffer cells could attenuate alcoholic liver injury in vivo, and investigated its possible mechanism in vitro. METHODS: Alcoholic liver injury was achieved by feeding wild type (WT), TLR3 knockout (TLR3(-/-)) and interleukin (IL)-10(-/-) mice with high-fat diet plus binge ethanol drinking for 2 weeks. To activate TLR3, polyinosinic-polycytidylic acid (poly I:C) was injected into mice. For in vitro studies, HSCs and Kupffer cells were isolated and treated with poly I:C. RESULTS: In WT mice, poly I:C treatment reduced alcoholic liver injury and fat accumulation by suppressing nuclear factor-κB activation and sterol response element-binding protein 1c expression in the liver. In addition, freshly isolated HSCs and Kupffer cells from poly I:C-treated mice showed enhanced expression of IL-10 compared to controls. Infiltrated macrophage numbers and the expression of tumor necrosis factor-α, monocyte chemoattractant protein-1 and IL-6 on these cells were decreased after poly I:C treatment. In vitro, poly I:C treatment enhanced the expression of IL-10 via a TLR3-dependent mechanism in HSCs and Kupffer cells. Finally, the protective effects of poly I:C on alcoholic liver injury were diminished in TLR3(-/-) and IL-10(-/-) mice. CONCLUSIONS: TLR3 activation ameliorates alcoholic liver injury via the stimulation of IL-10 production in HSCs and Kupffer cells. TLR3 could be a novel therapeutic target for the treatment of alcoholic liver injury.

ErbB1 epidermal growth factor receptor is a valid target for reducing the effects of multiple inhibitors of axonal regeneration.

Pharmacological inhibitors of epidermal growth factor receptor (ErbB1) attenuate the ability of CNS myelin to inhibit axonal regeneration. However, it has been claimed that such effects are mediated by off-target interactions. We have tested the role of ErbB1 in axonal regeneration by culturing neurons from ErbB1 knockout mice in the presence of various inhibitors of axonal regeneration: CNS myelin, chondroitin sulfate proteoglycans (CSPG), fibrinogen or polyinosinic:polycytidylic acid (poly I:C). We confirmed that ErbB1 was activated in cultures of cerebellar granule cells exposed to inhibitors of axonal regeneration and that ErbB1 kinase inhibitors promoted neurite outgrowth under these conditions. In the presence of myelin, fibrinogen, CSPG and poly I:C ErbB1 -/- neurons grew longer neurites than neurons expressing ErbB1. Furthermore, inhibitors of ErbB1 kinase did not improve neurite outgrowth from ErbB1 -/- neurons, ruling out an off-target mechanism of action. ErbB1 kinase activity is therefore a valid target for promoting axonal elongation in the presence of many of the molecules believed to contribute to the failure of axonal regeneration in the injured CNS.

Adolescent binge drinking increases expression of the danger signal receptor agonist HMGB1 and Toll-like receptors in the adult prefrontal cortex.

Adolescence is a critical developmental stage of life during which the prefrontal cortex (PFC) matures, and binge drinking and alcohol abuse are common. Recent studies have found that ethanol increases neuroinflammation via upregulated high-mobility group box 1 (HMGB1) signaling through Toll-like receptors (TLRs). HMGB1/TLR 'danger signaling' induces multiple brain innate immune genes that could alter brain function. To determine whether adolescent binge drinking persistently increases innate immune gene expression in the PFC, rats (P25-P55) were exposed to adolescent intermittent ethanol (AIE [5.0 g/kg, 2-day on/2-day off schedule]). On P56, HMGB1/TLR danger signaling was assessed using immunohistochemistry (i.e., +immunoreactivity [+IR]). In a separate group of subjects, spatial and reversal learning on the Barnes maze was assessed in early adulthood (P64-P75), and HMGB1/TLR danger signaling was measured using immunohistochemistry for +IR and RT-PCR for mRNA in adulthood (P80). Immunohistochemical assessment at P56 and 24 days later at P80 revealed increased frontal cortical HMGB1, TLR4, and TLR3 in the AIE-treated rats. Adolescent intermittent ethanol treatment did not alter adult spatial learning on the Barnes maze, but did cause reversal learning deficits and increased perseverative behavior. Barnes maze deficits correlated with the expression of danger signal receptors in the PFC. Taken together, these findings provide evidence that adolescent binge drinking leads to persistent upregulation of innate immune danger signaling in the adult PFC that correlates with adult neurocognitive dysfunction.

Toll-like receptor 3-initiated antiviral responses in mouse male germ cells in vitro.

The testis is an immunoprivileged site where local cell-initiated innate immunity plays a crucial role in antimicrobial responses. Toll-like receptors (TLRs) mediate innate immune responses in testicular somatic cells. Although several TLRs are expressed in some stages of male germ cells, the potential role of TLRs in triggering antimicrobial responses in the germ cells has yet to be exclusively studied. The current study demonstrates that TLR3 is constitutively expressed in spermatogonia and spermatocytes and can be activated by a synthetic double-strained RNA analog, polyinosinic-polycytidylic acid. TLR3 activation in these male germ cells up-regulates the expression of proinflammatory cytokines, such as interleukin IL1B, IL6, and tumor necrosis factor alpha, through activation of nuclear factor kappa B; it also induces production of type 1 interferons (IFNA and IFNB) through the activation of IFN regulatory factor 3. In addition, TLR3 activation increases the production of two major antiviral proteins, namely, double-stranded RNA-activated protein kinase and MX1 protein, by germ cells. Data in this article describe an antiviral response of male germ cells through the activation of TLR3 in vitro.

Established and novel NF-κB inhibitors lead to downregulation of TLR3 and the proliferation and cytokine secretion in HNSCC.

The transcriptional activation of NF-κB signalling has been identified as a major pathway involved in inflammation and tumor aggressiveness in a number of human cancers. Here we identify the impact of miscellaneous known and so far unknown NF-κB inhibitors originating from different drug classes on the function and proliferation of HNSCC. In detail: HNSCC cell lines were exposed to Acetylsalicylic Acid (ASA), Celecoxib, Dexamethasone, Curcumin and EPs 7630. Our major interest was to detect upstream alterations in cell signalling after applying NF-κB inhibiting substances. The inhibition of NF-κB signalling leads to an upstream regulation of Toll-like-receptor 3 (TLR3), a predominant receptor driving cell expansion. We find a marked downregulation of TLR3 and IKK complex, documenting upstream responses to NF-κB inhibition by every agent tested. In a second step we further analyse proliferation, cytokine production and alterations in the expression of downstream proteins such as cyclin D1 and c-Myc. Our data demonstrate decreased proliferation in response to incubation with aforementioned agents. Modulation of TLR3 and NF-κB expression is accompanied by altered profiles of IL-6 and IL-8 which are relevant cytokines in HNSCC progression. Proto-oncogenes cyclin D1 and c-myc are downregulated by all substances. Apart from the interplay of cytokines and TLR3, we substantiated EPs 7630 as a new and natural NF-κB inhibitor.