TLR4 Research Articles

Expression of interferon-stimulated gene 20 (ISG20), an antiviral effector protein, in glomerular endothelial cells: possible involvement of ISG20 in lupus nephritis

Background In addition to regulating the antiviral response, increased expression of Toll-like receptor 3 (TLR3) in resident renal cells plays a role in developing some forms of glomerulonephritis. TLR3 activation leads to type I interferon (IFN) production, which induces the expression of IFN-stimulated genes (ISGs). However, the role of ISG20 expression in resident renal cells remains unclear. Methods Cultured normal human glomerular endothelial cells (GECs) were treated with polyinosinic-polycytidylic acid (poly IC), Escherichia coli lipopolysaccharide (LPS), R848, and CpG (TLR3, TLR4, TLR7, and TLR9 agonists, respectively). The mRNA levels of ISG20, CX3CL1/fractalkine, and CXCL10/IP-10 were measured by quantitative reverse transcription-polymerase chain reaction. ISG20 protein expression was assessed by Western blotting. RNA interference was used to knockdown IFN-β and ISG20 expression. CX3CL1 protein levels were assessed by enzyme-linked immunosorbent assay. We performed immunofluorescence to examine endothelial ISG20 expression in biopsy specimens from patients with lupus nephritis (LN). Results In GECs, the expression of ISG20 mRNA and protein was increased by polyIC, not by LPS, R848, or CpG treatment. Moreover, ISG20 knockdown prevented poly IC-induced CX3CL1 expression but had no effect on CXCL10 expression. Intense endothelial ISG20 immunoreactivity was observed in biopsy specimens obtained from patients with proliferative LN. Conclusion In GECs, ISG20 was regulated via TLR3 but not via TLR4, TLR7, or TLR9 signaling. Moreover, ISG20 was involved in regulating CX3CL1 production. In addition to regulating antiviral innate immunity, ISG20 may act as a mediator of CX3CL1 production, thereby inducing glomerular inflammation, particularly in patients with LN.

Extracellular RNAs/TLR3 signaling contributes to acute intestinal injury induced by intestinal ischemia reperfusion in mice

Toll-like receptor 3 (TLR3), one pattern recognition receptor activated by viral and endogenous RNA, has been recently reported to regulate ischemia/reperfusion (I/R) injury in various organs. However, the role of TLR3 in the development of intestinal I/R injury remains unclear. The aim of this study is to evaluate the effects of extracellular RNAs/TLR3 signaling in intestinal I/R injury. An intestinal I/R injury model was established with superior mesenteric artery occlusion both in wild-type and TLR3 knockout (KO, −/−) mice, and MODE-K cells were subjected to hypoxia/reoxygenation (H/R) to mimic the I/R model in vivo. Extracellular RNAs (exRNAs), especially double-stranded RNAs (dsRNAs) co-localized with TLR3, were significantly increased both in vitro and in vivo. Compared with wild-type mice, TLR3 knockout obviously attenuated intestinal I/R injury. Both TLR3/dsRNA complex inhibitor and TLR3 siRNA administration reduced TLR3 expressions and subsequently inhibited intestinal inflammatory cytokine production and apoptosis. In conclusion, exRNAs/TLR3 signaling is a key mechanism that regulates intestinal I/R injury in adult mice, and the TLR3/dsRNA complex inhibitor can be an effective approach for attenuating intestinal I/R-induced inflammatory response and apoptosis.

TL-532, a novel specific Toll-like receptor 3 agonist rationally designed for targeting cancers: discovery process and biological characterization.

Toll-like receptor 3 (TLR3) is an innate immune receptor that recognizes double-stranded RNA (dsRNA) and induces inflammation in immune and normal cells to initiate anti-microbial responses. TLR3 acts also as a death receptor only in cancer cells but not in their normal counterparts, making it an attractive target for cancer therapies. To date, all of the TLR3-activating dsRNAs used at preclinical or clinical stages have major drawbacks such as structural heterogeneity, toxicity, and lack of specificity and/or efficacy. We conducted the discovery process of a new family of TLR3 agonists that are chemically manufactured on solid-phase support and perfectly defined in terms of sequence and size. A stepwise discovery process was performed leading to the identification of TL-532, a 70 base pair dsRNA that is potent without transfection reagent and is highly specific for TLR3 without activating other innate nucleic sensors such as RIG-I/MDA5, TLR7, TLR8, and TLR9. TL-532 induces inflammation in murine RAW264.7 myeloid macrophages, in human NCI-H292 lung cancer cells, and it promotes immunogenic apoptosis in tumor cells in vitro and ex vivo without toxicity towards normal primary cells. In conclusion, we identified a novel TLR3 agonist called TL-532 that has promising anticancer properties.

Decreased placental TLR3 is associated with hepatitis B virus vaccine responsiveness in infants born to HBsAg-positive mothers.

Although hepatitis B vaccination has a significant impact on the reduction hepatitis B virus (HBV) infection, babies born to hepatitis B surface antigen (HBsAg) positive mothers bear a high risk of being poor responsive to the vaccine with unilluminated mechanism. Toll-like receptor 3 (TLR3) plays a vital role in placental immunity, which affects the immune response of these babies. This study investigated the role of placental TLR3 in the immune responses of babies born to HBsAg-positive mothers to the HBV vaccine. One hundred pairs of HBsAg-positive mothers and their newborns were recruited. Maternal blood samples were collected before delivery, and placental tissues were collected after delivery. Newborns were administered standard passive and active immunoprophylaxis and followed up until the age of 1. Infant blood samples were collected at 1 year of age. Mothers and infants were tested for HBV serological markers and HBV DNA by electrochemiluminescence immunoassay and fluorescence quantitative polymerase chain reaction. respectively. Placental TLR3 was detected by immunohistochemistry and score in a semi-quantitative fashion, circulating cytokines in infants were detected by enzyme-linked immunosorbent assay. Infants with anti-HBs ≥100 and <100 mIU/mL were classified into the high-responsiveness group and the non- or hypo-responsiveness group. The TLR3 protein was expressed in all placentas. Compared with the high-responsiveness group, the expression of TLR3 in the non- or hypo-responsiveness group was significantly decreased (χ2=10.39, P=0.001). A non-conditional logistic regression model showed that the increased expression of placental TLR3 protein decreased the odds of HBV vaccine non- or hypo-responsiveness in the babies of HBsAg-positive mothers [OR =0.25 (95% CI: 0.11-0.58)], and this association remained significant after accounting for maternal factors, such as HBeAg and HBV DNA, as well as infant cytokines, including IL-6, IL-12, TNF-α, IFN-α, and IFN-γ [OR =0.15 (95% CI: 0.05-0.44)]. Decreased placental TLR3 expression is associated with impaired responsiveness to HBV vaccination in babies born to HBsAg-positive mothers.

Apoptotic Changes, Oxidative Stress and Immunomodulatory Effects in the Liver of Japanese Seabass (Lateolabrax japonicus) Induced by Ammonia-Nitrogen Stress during Keep-Live Transport.

This study investigated the effects of NH3-N on antioxidant responses, histoarchitecture, and immunity of Japanese seabass (Lateolabrax japonicus) during keep-live transport. The findings suggest that NH3-N stress transport alters the transcription of P53, Caspase 9, Bcl2, Caspase 3 and Bax genes, demonstrating that NH3-N stress can trigger the apoptotic pathway of P53-Bax-Bcl2 and Caspase and induce apoptosis. NH3-N stress transport also evoked transcriptional upregulation of inflammatory cytokines (tumor necrosis factor α (TNF-α), Toll-like receptor 3 (TLR-3), nuclear factor kappa β (NF-κB), interleukin 6 (IL-6) and interleukin 1β (IL-1β)) and increased complement C3, C4, lysozyme (LZM) and immunoglobulin (IgM) levels, activating the innate immunological system during keep-live transport. In addition, NH3-N stress transport altered changes in the levels of superoxide dismutase (SOD), catalase (CAT), glutathione-related enzymes, and heat shock proteins 70 and 90 in the liver, indicating that the antioxidant system and Hsp protected the cells from NH3-N-induced oxidative stress. When excess ROS were not removed, they caused the body to respond with immunological and inflammatory responses, as well as apoptosis and tissue damage. This helps towards understanding the effect of NH3-N levels on sea bass during keep-live transport.

Differences in gene regulation by TLR3 and IPS-1 signaling in murine corneal epithelial cells

Toll-like receptor 3 (TLR3) and interferon-beta promoter stimulator-1 (IPS-1) are associated with antiviral responses to double-stranded RNA viruses and contribute to innate immunity. We previously reported that conjunctival epithelial cell (CEC) TLR3 and IPS-1 pathways respond to the common ligand polyinosinic:polycytidylic acid (polyI:C) to regulate different gene expression patterns as well as CD11c + cell migration in murine-model corneas. However, the differences in the functions and the roles of TLR3 and IPS-1 remain unclear. In this study, we investigated the differences of TLR3 or IPS-1-induced gene expression in corneal epithelial cells (CECs) in response to polyI:C stimulation using cultured murine primary CECs (mPCECs) derived from TLR3 and IPS-1 knockout mice via comprehensive analysis. The genes associated with viral responses were upregulated in the wild-type mice mPCECs after polyI:C stimulation. Among these genes, Neurl3, Irg1, and LIPG were dominantly regulated by TLR3, while interleukin (IL)-6 and IL-15 were dominantly regulated by IPS-1. CCL5, CXCL10, OAS2, Slfn4, TRIM30α, and Gbp9 were complementarily regulated by both TLR3 and IPS-1. Our findings suggest that CECs may contribute to immune responses and that TLR3 and IPS-1 possibly have different functions in the corneal innate immune response.

How Can We Predict Disease Severity in Viral Infections?

How Can We Predict Disease Severity in Viral Infections?

Innate immune pathway modulator screen identifies STING pathway activation as a strategy to inhibit multiple families of arbo and respiratory viruses

RNA viruses continue to remain a threat for potential pandemics due to their rapid evolution. Potentiating host antiviral pathways to prevent or limit viral infections is a promising strategy. Thus, by testing a library of innate immune agonists targeting pathogen recognition receptors, we observe that Toll-like receptor 3 (TLR3), stimulator of interferon genes (STING), TLR8, and Dectin-1 ligands inhibit arboviruses, Chikungunya virus (CHIKV), West Nile virus, and Zika virus to varying degrees. STING agonists (cAIMP, diABZI, and 2',3'-cGAMP) and Dectin-1 agonist scleroglucan demonstrate the most potent, broad-spectrum antiviral function. Furthermore, STING agonists inhibit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and enterovirus-D68 (EV-D68) infection in cardiomyocytes. Transcriptome analysis reveals that cAIMP treatment rescue cells from CHIKV-induced dysregulation of cell repair, immune, and metabolic pathways. In addition, cAIMP provides protection against CHIKV in a chronic CHIKV-arthritis mouse model. Our study describes innate immune signaling circuits crucial for RNA virus replication and identifies broad-spectrum antivirals effective against multiple families of pandemic potential RNA viruses.

Amanita muscaria extract potentiates production of proinflammatory cytokines by dsRNA-activated human microglia.

Recent interest in mushrooms and their components as potential therapies for mental health, along with recent government and health authority approvals, has necessitated a more comprehensive understanding of their effects on the cellular microenvironment of the brain. Amanita muscaria has been ingested as a treatment for a variety of ailments for centuries, most notably those affecting the central nervous system and conditions associated with neuroinflammation. However, the effects of these extracts on neuroinflammatory cells, such as microglia, are unknown. The effect of commercially-sourced A. muscaria extract (AME-1) on human microglial cell line (HMC3) expression of surface receptors such as CD86, CXCR4, CD45, CD125 and TLR4 was determined by flow cytometry. AME-1 upregulated expression of all of these receptors. The effect of AME-1 on HMC3 production of IL-8 and IL-6 was determined and compared to tumor necrosis factor (TNF), polyinosinic-polycytidylic acid [poly(I:C)], substance P and lipopolysaccharide (LPS), all known activators of HMC-3 and primary microglia. HMC3 produced both IL-8 and IL-6 when activated with LPS, TNF and poly(I:C) but not when they were activated with substance P. Although AME-1 at higher concentrations increased IL-8 production of HMC3 on its own, AME-1 notably potentiated HMC3 production of IL-8 in response to poly(I:C). AME-1 altered expression of toll-like receptor 3 (TLR3) mRNA but not surface protein by HMC3. AME-1 also did not significantly alter expression of retinoic acid-inducible gene I (RIG-I) or melanoma differentiation-associated protein 5 (MDA5), both cytosolic sensors of dsRNA. Metabolomics analysis showed that AME-1 contained several metabolites, including the autophagy inducer, trehalose. Like AME-1, trehalose also potentiated HMC3 poly(I:C) mediated production of IL-8. This study suggests that A. muscaria extracts can modify HMC3 inflammatory responses, possibly due to their trehalose content.

High expression of TLR3 in triple-negative breast cancer predicts better prognosis—data from the Fudan University Shanghai Cancer Center cohort and tissue microarrays

IntroductionWe have previously reported that Toll-like receptor 3 (TLR3) acts as a suppressor gene for breast cancer initiation and progression. In this study, we evaluated the role of TLR3 in breast cancer using our original Fudan University Shanghai Cancer Center (FUSCC) datasets and breast cancer tissue microarrays.MethodsUsing FUSCC multiomics datasets on triple- negative breast cancer (TNBC), we compared the mRNA expression of TLR3 in TNBC tissue and the adjacent normal tissue. A Kaplan–Meier plotter was performed to investigate the expression of TLR3 on prognosis in the FUSCC TNBC cohort. We performed immunohistochemical staining to analyze TLR3 protein expression in the TNBC tissue microarrays. Furthermore, bioinformatics analysis was performed using the Cancer Genome Atlas (TCGA) data to verify the results of our FUSCC study. The relationship between TLR3 and clinicopathological features was analyzed with logistic regression and the Wilcoxon signed-rank test. The association between clinical characteristics and overall survival in TCGA patients was assessed using the Kaplan–Meier method and Cox regression analysis. Gene set enrichment analysis (GSEA) was performed to identify signaling pathways that are differentially activated in breast cancer.ResultsThe mRNA expression of TLR3 was lower in TNBC tissue than in the adjacent normal tissue in the FUSCC datasets. The TLR3 had high expression in immunomodulatory (IM) and mesenchymal-like (MES) subtypes and low expression in luminal androgen receptor (LAR) and basal-like immune-suppressed (BLIS) subtypes. High expression of TLR3 in TNBC predicted better prognosis in the FUSCC TNBC cohort. Immunohistochemical staining of the tissue microarrays showed that TLR3 had lower expression in breast cancer tissues than in the adject normal tissues. Furthermore, the TLR3 expression was positively associated with B cell, CD4 + T cells, CD8 + T cells, neutrophils, macrophages, and myeloid dendritic cells. Bioinformatic analysis using high-throughput RNA-sequencing data from the TCGA demonstrated that the reduced expression of TLR3 in breast cancer was associated with advanced clinicopathological characteristics, survival time, and poor prognosis.ConclusionsTLR3 has low expression in TNBC tissue. High expression of TLR3 in triple-negative breast cancer predicts better prognosis. TLR3 expression may be a potential prognostic molecular marker of poor survival in breast cancer.

Anti-inflammatory properties of antiangiogenic fucoidan in retinal pigment epithelium cells

Age-related macular degeneration (AMD) is a multifactorial disease in which angiogenesis, oxidative stress and inflammation are important contributing factors. In this study, we investigated the anti-inflammatory effects of a fucoidan from the brown algae Fucus vesiculosus (FV) in primary porcine RPE cells. Inflammation was induced by lipopolysaccharide (LPS), polyinosinic:polycytidylic acid (Poly I:C), Pam2CSK4 (Pam), or tumor necrosis factor alpha (TNF-α). Cell viability was tested with thiazolyl blue tetrazolium bromide (MTT) test, barrier function by measuring transepithelial electric resistance (TEER), interleukin 6 (IL-6) and interleukin 8 (IL-8) secretion in ELISA, retinal pigment epithelium-specific 65 kDa protein (RPE65) and protectin (CD59) expression in Western blot, gene expression with quantitative polymerase chain reaction (qPCR) (IL6, IL8, MERTK, PIK3CA), and phagocytotic activity in a microscopic assay. FV fucoidan did not influence RPE cell viability. FV fucoidan reduced the Poly I:C proinflammatory cytokine secretion of IL-6 and IL-8. In addition, it decreased the expression of IL-6 and IL-8 in RT-PCR. LPS and TNF-α reduced the expression of CD59 in Western blot, this reduction was lost under FV fucoidan treatment. Also, LPS and TNF-α reduced the expression of visual cycle protein RPE65, this reduction was again lost under FV fucoidan treatment. Furthermore, the significant reduction of barrier function after Poly I:C stimulation is ameliorated by FV fucoidan. Concerning phagocytosis, however, the inflammation-induced reduction was not improved by FV fucoidan. FV and proinflammatory milieu did not relevantly influence phagocytosis relevant gene expression either. In conclusion, we show that fucoidan from FV can reduce proinflammatory stimulation in RPE induced by toll-like receptor 3 (TLR-3) activation and is of high interest as a potential compound for early AMD treatment.

Chicken Glycogen Synthase Kinase 3β Suppresses Innate Immune Responses and Enhances Avian Leukosis Virus Replication in DF-1 Cells.

Glycogen synthase kinase 3β (GSK3β) is a widely distributed multifunctional serine/threonine kinase. In mammals, GSK3β regulates important life activities such as proinflammatory response, anti-inflammatory response, immunity, and cancer development. However, the biological functions of chicken GSK3β (chGSK3β) are still unknown. In the present study, the full-length cDNA of chGSK3β was first cloned and analyzed. Absolute quantification of chicken chGSK3β in 1-day-old specific-pathogen-free birds has shown that it is widely expressed in all tissues, with the highest level in brain and the lowest level in pancreas. Overexpression of chGSK3β in DF-1 cells significantly decreased the gene expression levels of interferon beta (IFN-β), IFN regulatory factor 7 (IRF7), Toll-like receptor 3 (TLR3), melanoma differentiation-associated protein 5 (MDA5), MX-1, protein kinase R (PKR), and oligoadenylate synthase-like (OASL), while promoting the replication of avian leukosis virus subgroup J (ALV-J). Conversely, levels of most of the genes detected in this study were increased when chGSK3β expression was knocked down using small interfering RNA (siRNA), which also inhibited the replication of ALV-J. These results suggest that chGSK3β plays an important role in the antiviral innate immune response in DF-1 cells, and it will be valuable to carry out further studies on the biological functions of chGSK3β. IMPORTANCE GSK3β regulates many life activities in mammals. Recent studies revealed that chGSK3β was involved in regulating antiviral innate immunity in DF-1 cells and also could positively regulate ALV-J replication. These results provide new insights into the biofunction of chGSK3β and the virus-host interactions of ALV-J. In addition, this study provides a basis for further research on the function of GSK3 in poultry.

Toll-like Receptor 3 c.1377C/T and -7C/A Polymorphisms Associated with COVID-19 and COVID-19 Severity

Chinese officials have reported the novel coronavirus to the world health organization, which is called the SARS-CoV-2. Toll-like receptor 3 (TLR3) induces antiviral immune responses via the production of type I interferons and inflammatory cytokines. In this study, we aimed to examine TLR3 c.1377C/T and -7C/A polymorphisms in COVID-19 and the association between some clinical parameters. We investigated the frequencies of TLR3 (c.1377C/T and -7C/A) polymorphisms in 150 patients with COVID-19 and 171 healthy individuals as controls. We performed polymerase chain reaction (PCR) based on restriction fragment length polymorphism (RFLP). We also investigated whether TLR3 c.1377C/T and-7C/A were associated with the severity of COVID-19. In addition, CHAID tree-based classification algorithm was created to investigate the severity of the patients in our study. TLR3 c.1377C/T TT genotype frequencies were statistically significant between cases and controls (p= 0.02). For TLR3 -7C/A polymorphism, the findings showed a statistically significant difference in A allele frequencies (p= 0.03). There was a statistically significant difference in the distribution of TLR3 -7C/A CA genotype frequency (p= 0.04). Our findings suggest that TLR3 c.1377C/T and -7C/A polymorphisms may be important on susceptibility or clinical course of COVID-19.

Type I interferon signaling in malignant blasts contributes to treatment efficacy in AML patients

While type I interferon (IFN) is best known for its key role against viral infection, accumulating preclinical and clinical data indicate that robust type I IFN production in the tumor microenvironment promotes cancer immunosurveillance and contributes to the efficacy of various antineoplastic agents, notably immunogenic cell death inducers. Here, we report that malignant blasts from patients with acute myeloid leukemia (AML) release type I IFN via a Toll-like receptor 3 (TLR3)-dependent mechanism that is not driven by treatment. While in these patients the ability of type I IFN to stimulate anticancer immune responses was abolished by immunosuppressive mechanisms elicited by malignant blasts, type I IFN turned out to exert direct cytostatic, cytotoxic and chemosensitizing activity in primary AML blasts, leukemic stem cells from AML patients and AML xenograft models. Finally, a genetic signature of type I IFN signaling was found to have independent prognostic value on relapse-free survival and overall survival in a cohort of 132 AML patients. These findings delineate a clinically relevant, therapeutically actionable and prognostically informative mechanism through which type I IFN mediates beneficial effects in patients with AML.

Pattern-recognition receptors in endometriosis: A narrative review.

Endometriosis is closely associated with ectopic focal inflammation and immunosuppressive microenvironment. Multiple types of pattern recognition receptors (PRRs) are present in the innate immune system, which are able to detect pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs) in both intracellular and external environments. However, the exact role of PRRs in endometriosis and the underlying molecular mechanism are unclear. PRRs are necessary for the innate immune system to identify and destroy invasive foreign infectious agents. Mammals mainly have two types of microbial recognition systems. The first one consists of the membrane-bound receptors, such as toll-like receptors (TLRs), which recognize extracellular microorganisms and activate intracellular signals to stimulate immune responses. The second one consists of the intracellular PRRs, including nod-like receptors (NLRs) and antiviral proteins retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA-5) with helix enzyme domain. In this review, we mainly focus on the key role of PRRs in the pathological processes associated with endometriosis. PRRs recognize PAMPs and can distinguish pathogenic microorganisms from self, triggering receptor ligand reaction followed by the stimulation of host immune response. Activated immune response promotes the transmission of microbial infection signals to the cells. As endometriosis is characterized by dysregulated inflammation and immune response, PRRs may potentially be involved in the activation of endometriosis-associated inflammation and immune disorders. Toll-like receptor 2 (TLR2), toll-like receptor 3 (TLR3), toll-like receptor 4 (TLR4), nod-like receptor family caspase activation and recruitment domain (CARD) domain containing 5 (NLRC5), nod-like receptor family pyrin domain containing 3 (NLRP3), and c-type lectin receptors (CLRs) play essential roles in endometriosis development by regulating immune and inflammatory responses. Absent in melanoma 2 (AIM2)-like receptors (ALRs) and retinoic acid-inducible gene I-like receptors (RLRs) may be involved in the activation of endometriosis-associated immune and inflammation disorders. PRRs, especially TLRs, may serve as potential therapeutic targets for alleviating pain in endometriosis patients. PRRs and their ligands interact with the innate immune system to enhance inflammation in the stromal cells during endometriosis. Thus, targeting PRRs and their new synthetic ligands may provide new therapeutic options for treating endometriosis.

TLR3 and MDA5 Knockout DF-1 cells Enhance Replication of Avian Orthoavulavirus 1.

Despite the essential role of innate immunity in defining the outcome of viral infections, the roles played by different components of the avian innate immune system are poorly delineated. Here, we investigated the potential implication of avian toll-like receptor (TLR) 3 (TLR3) and melanoma differentiation-associated (MDA) gene 5 (MDA5) receptors of double-stranded RNA (dsRNA) in induction of the interferon pathway and avian orthoavulavirus 1 (AOAV-1) replication in chicken-origin DF-1 fibroblast cells. TLR3 and MDA5 knockout (KO) DF-1 cells were generated using our avian-specific CRISPR/Cas9 system and stimulated with a synthetic dsRNA ligand polyinosinic:polycytidylic acid [poly(I:C)] or infected with AOAV-1 (previously known as Newcastle disease virus). Poly(I:C) treatment in cell culture media resulted in significant upregulation of interferon (IFN)α, IFNβ, and Mx1 gene expression in wild type (WT) DF-1 cells but not in TLR3-MDA5 double KO cells. Interestingly, poly(I:C) treatment induced rapid cell degeneration in WT and MDA5 KO cells, but not in TLR3 knockout or TRL3-MDA5 double knockout (DKO) cells, directly linking poly(I:C)-induced cell degeneration to TLR3-mediated host response. The double knockout cells supported significantly higher replication of AOAV-1 virus than did the WT cells. However, no correlation between the level of virus replication and type I IFN response was observed. Our study suggests that innate immune response is host- and pathogen specific, and further investigation is needed to understand the relevance of dsRNA receptor-mediated immune responses in viral replication and pathogenesis in avian species.

Pestiviruses infection: Interferon-virus mutual regulation.

Pestiviruses are a class of viruses that in some cases can cause persistent infection of the host, thus posing a threat to the livestock industry. Interferons (IFNs) are a group of secreted proteins that play a crucial role in antiviral defense. In this review, on the one hand, we elaborate on how pestiviruses are recognized by the host retinoic acid-inducible gene-I (RIG-I), melanoma-differentiation-associated protein 5 (MDA5), and Toll-like receptor 3 (TLR3) proteins to induce the synthesis of IFNs. On the other hand, we focus on reviewing how pestiviruses antagonize the production of IFNs utilizing various strategies mediated by self-encoded proteins, such as the structural envelope protein (Erns) and non-structural protein (Npro). Hence, the IFN signal transduction pathway induced by pestiviruses infection and the process of pestiviruses blockade on the production of IFNs intertwines into an intricate regulatory network. By reviewing the interaction between IFN and pestiviruses (based on studies on BVDV and CSFV), we expect to provide a theoretical basis and reference for a better understanding of the mechanisms of induction and evasion of the innate immune response during infection with these viruses.

Early activation of Toll-like receptor-3 reduces the pathological progression of Alzheimer’s disease in APP/PS1 mouse

BackgroundToll-like receptor 3 (TLR3) plays an important role in the immune/inflammatory response in the nervous system and is a main pathological feature of Alzheimer’s disease (AD). This study investigates the role of early activation of TLR3 in the pathophysiological process of AD.MethodsIn the experiment, the agonist of TLR3, Poly(I:C), was intraperitoneally injected into the APP/PS1 mouse model of AD and wild-type control mice starting from the age of 4 to 9 months. At the age of 14 months, behavioral tests were conducted. Western blot and immunohistochemistry staining were used to evaluate the level of amyloid β-protein (Aβ), the activation of inflammatory cells, and neuron loss. In addition, the levels of inflammatory cytokines were measured using a quantitative polymerase chain reaction.ResultsThe results demonstrated that the early activation of TLR3 attenuated neuronal loss and neurobehavioral dysfunction. Moreover, the early activation of TLR3 reduced Aβ deposition, inhibited the activation of microglia and astrocytes, and decreased the transcription of pro-inflammatory factors in the hippocampus.ConclusionsThe results indicated that the activation of TLR3 by Poly (I:C) in the early stage of development of AD in a mouse model attenuated neuron loss and improved neurobehavioral functions. The underlying mechanisms could be attributed to its role in Aβ clearance, the inhibition of glial cells, and the regulation of neuroinflammation in the hippocampus.

The Impact of Variation in the Toll-like Receptor 3 Gene on Epizootic Hemorrhagic Disease in Illinois Wild White-Tailed Deer (Odocoileus virginianus).

Epizootic hemorrhagic disease (EHD) leads to high mortality in white-tailed deer (Odocoileus virginianus) and is caused by a double-stranded RNA (dsRNA) virus. Toll-like receptor 3 (TLR3) plays a role in host immune detection and response to dsRNA viruses. We, therefore, examined the role of genetic variation within the TLR3 gene in EHD among 84 Illinois wild white-tailed deer (26 EHD-positive deer and 58 EHD-negative controls). The entire coding region of the TLR3 gene was sequenced: 2715 base pairs encoding 904 amino acids. We identified 85 haplotypes with 77 single nucleotide polymorphisms (SNPs), of which 45 were synonymous mutations and 32 were non-synonymous. Two non-synonymous SNPs differed significantly in frequency between EHD-positive and EHD-negative deer. In the EHD-positive deer, phenylalanine was relatively less likely to be encoded at codon positions 59 and 116, whereas leucine and serine (respectively) were detected less frequently in EHD-negative deer. Both amino acid substitutions were predicted to impact protein structure or function. Understanding associations between TLR3 polymorphisms and EHD provides insights into the role of host genetics in outbreaks of EHD in deer, which may allow wildlife agencies to better understand the severity of outbreaks.

Toll-like receptor-3 contributes to the development of aortic valve stenosis

Aortic valve stenosis (AS) development is driven by distinct molecular and cellular mechanisms which include inflammatory pathways. Toll-like-receptor-3 (TLR3) is a lysosomal pattern-recognition receptor that binds double-stranded RNA and promotes pro-inflammatory cellular responses. In recent years, TLR3 has emerged as a major regulator of vascular inflammation. The exact role of TLR3 in the development of AS has not been investigated. Isolated human valvular interstitial cells (VICs) were stimulated with the TLR3-agonist polyIC and the resulting pro-inflammatory and pro-osteogenic response measured. Severe AS was induced in wildtype- and TLR3−/− mice via mechanical injury of the aortic valve with a coronary springwire. TLR3 activation was achieved by polyIC injection every 24 h after wire injury, while TLR3 inhibition was realized using Compound 4a (C4a) every 48 h after surgery. Endothelial mesenchymal transition (EndoMT) of human valvular endothelial cells (VECs) was assessed after polyIC stimulation. Stimulation of human VICs with polyIC promoted a strong inflammatory and pro-osteogenic reaction. Similarly, injection of polyIC marginally increased AS development in mice after wire injury. AS induction was significantly decreased in TLR3−/− mice, confirming the role of endogenous TLR3 ligands in AS pathology. Pharmacological inhibition of TLR3 with C4a not only prevented the upregulation of inflammatory cytokines and osteogenic markers in VICs, and EndoMT in VECs, but also significantly abolished the development of AS in vivo. Endogenous TLR3 activation significantly contributes to AS development in mice. Pharmacological inhibition of TLR3 with C4a prevented AS formation. Therefore, targeting TLR3 may be a viable treatment option.