Innate immunity in the human female reproductive tract: antiviral response of uterine epithelial cells to the TLR3 agonist poly(I:C)

The objective of this study was to examine the expression of TLR by human primary uterine epithelial cells (UEC) and to determine whether exposure to the TLR agonist poly(I:C) would induce an antiviral response. The secretion of several cytokines and chemokines was examined as well as the mRNA expression of human beta-defensin-1 and -2 (HBD1 and HBD2), IFN-beta, and the IFN-beta-stimulated genes myxovirus resistance gene 1 and 2',5' oligoadenylate synthetase. The expression of TLR1-9 by UEC was demonstrated by RT-PCR, with only TLR10 not expressed. Stimulation of UEC with the TLR3 agonist poly(I:C) induced the expression of the proinflammatory cytokines TNF-alpha, IL-6, GM-CSF, and G-CSF, as well as the chemokines CXCL8/IL-8, CCL2/MCP-1, and CCL4/MIP-1beta. In addition, poly(I:C) exposure induced the mRNA expression of HBD1 and HBD2 by 6- and 4-fold, respectively. Furthermore, upon exposure to poly(I:C) UEC initiated a potent antiviral response resulting in the induction of IFN-beta mRNA expression 70-fold and myxovirus resistance gene 1 and 2',5' oligoadenylate synthetase mRNA expression (107- and 96-fold), respectively. These results suggest that epithelial cells that line the uterine cavity are sensitive to viral infection and/or exposure to viral dsRNA released from killed epithelial cells. Not only do UEC release proinflammatory cytokines and chemokines that mediate the initiation of an inflammatory response and recruitment of immune cells to the site of infection, but they also express beta-defensins, IFN-beta, and IFN-beta-stimulated genes that can have a direct inhibiting effect on viral replication.     

Protein kinase R mediates intestinal epithelial gene remodeling in response to double-stranded RNA and live rotavirus

As sentinels of host defense, intestinal epithelial cells respond to the viral pathogen rotavirus by activating a gene expression that promotes immune cell recruitment and activation. We hypothesized that epithelial sensing of rotavirus might target dsRNA, which can be detected by TLR3 or protein kinase R (PKR). Accordingly, we observed that synthetic dsRNA, polyinosinic acid:cytidylic acid (poly(I:C)), potently induced gene remodeling in model intestinal epithelia with the specific pattern of expressed genes, including both classic proinflammatory genes (e.g., IL-8), as well as genes that are classically activated in virus-infected cells (e.g., IFN-responsive genes). Poly(I:C)-induced IL-8 was concentration dependent (2-100 mug/ml) and displayed slower kinetics compared with IL-8 induced by bacterial flagellin (ET(50) approximately 24 vs 8 h poly(I:C) vs flagellin, respectively). Although model epithelia expressed detectable TLR3 mRNA, neither TLR3-neutralizing Abs nor chloroquine, which blocks activation of intracellular TLR3, attenuated epithelial responses to poly(I:C). Conversely, poly(I:C)-induced phosphorylation of PKR and inhibitors of PKR, 2-aminopurine and adenine, ablated poly(I:C)-induced gene expression but had no effect on gene expression induced by flagellin, thus suggesting that intestinal epithelial cell detection of dsRNA relies on PKR. Consistent with poly(I:C) detection by an intracellular molecule such as PKR, we observed that both uptake of and responses to poly(I:C) were polarized to the basolateral side. Lastly, we observed that the pattern of pharmacologic inhibition of responses to poly(I:C) was identical to that seen in response to infection by live rotavirus, indicating a potentially important role for PKR in activating intestinal epithelial gene expression in rotavirus infection.     

Poly (I:C), an agonist of toll-like receptor-3, inhibits replication of the Chikungunya virus in BEAS-2B cells

ABSTRACT: BACKGROUND: Double-stranded RNA (dsRNA) and its mimic, polyinosinic acid: polycytidylic acid [Poly (I:C)], are recognized by toll-like receptor 3 (TLR3) and induce interferon (IFN)-beta in many cell types. Poly (I:C) is the most potent IFN inducer. In in vivo mouse studies, intraperitoneal injection of Poly (I:C) elicited IFN-alpha/beta production and natural killer (NK) cells activation. The TLR3 pathway is suggested to contribute to innate immune responses against many viruses, including influenza virus, respiratory syncytial virus, herpes simplex virus 2, and murine cytomegalovirus. In Chikungunya virus (CHIKV) infection, the viruses are cleared within 7-10 days postinfection before adaptive immune responses emerge. The innate immune response is important for CHIKV clearance. RESULTS: The effects of Poly (I:C) on the replication of CHIKV in human bronchial epithelial cells, BEAS-2B, were studied. Poly (I:C) suppressed cytopathic effects (CPE) induced by CHIKV infection in BEAS-2B cells in the presence of Poly (I:C) and inhibited the replication of CHIKV in the cells. The virus titers of Poly (I:C)-treated cells were much lower compared with those of untreated cells. CHIKV infection and Poly (I:C) treatment of BEAS-2B cells induced the production of IFN-beta and increased the expression of anti-viral genes, including IFN-alpha, IFN-beta, MxA, and OAS. Both Poly (I:C) and CHIKV infection upregulate the expression of TLR3 in BEAS-2B cells. CONCLUSIONS: CHIKV is sensitive to innate immune response induced by Poly (I:C). The inhibition of CHIKV replication by Poly (I:C) may be through the induction of TLR3, which triggers the production of IFNs and other anti-viral genes. The innate immune response is important to clear CHIKV in infected cells.     

Activation of Toll-like receptor 3 impairs the dengue virus serotype 2 replication through induction of IFN-β in cultured hepatoma cells

Toll-like receptors (TLRs) play an important role in innate immunity against invading pathogens. Although TLR signaling has been indicated to protect cells from infection of several viruses, the role of TLRs in Dengue virus (DENV) replication is still unclear. In the present study, we examined the replication of DENV serotype 2 (DENV2) by challenging hepatoma cells HepG2 with different TLR ligands. Activation of TLR3 showed an antiviral effect, while pretreatment of other TLR ligands (including TLR1/2, TLR2/6, TLR4, TLR5 or TLR7/8) did not show a significant effect. TLR3 ligand poly(I:C) treatment prior to viral infection or simultaneously, but not post-treatment, significantly down-regulated virus replication. Pretreatment with poly(I:C) reduced viral mRNA expression and viral staining positive cells, accompanying an induction of the type I interferon (IFN-β) and type III IFN (IL-28A/B). Intriguingly, neutralization of IFN-β alone successfully restored the poly(I:C)-inhibited replication of DENV2. The poly(I:C)-mediated effects, including IFN induction and DENV2 suppression, were significantly reversed by IKK inhibitor, further suggesting that IFN-β is the dominant factor involved in the poly(I:C) mediated antiviral effect. Our study presented the first evidence to show that activation of TLR3 is effective in blocking DENV2 replication via IFN-β, providing an experimental clue that poly(I:C) may be a promising immunomodulatory agent against DENV infection and might be applicable for clinical prevention.     

干扰素的信号传导和抗病毒效应机制

干扰素是一种具有抗病毒、抗增殖和免疫调节功能的细胞因子,它在宿主天然免疫防御中起着重要的作用。干扰素诱导的信号通路除了最初的Jak-Stat途径以外,很多新发现的信号途径对于干扰素反应也是必需的。干扰素反应最终导致抗病毒的效应蛋白通路,包括2′,5′-寡聚腺苷酸合成酶、蛋白激酶、ISG15等途径。这些效应蛋白通过抑制病毒转录、降解病毒RNA、抑制翻译和修饰蛋白的功能来控制病毒复制的过程。     

Polyinosinic-polycytidylic acid treatment of Friend retrovirus-infected mice improves functional properties of virus-specific T cells and prevents virus-induced disease

The induction of type I IFN is the most immediate host response to viral infections. Type I IFN has a direct antiviral activity mediated by antiviral enzymes, but it also modulates the function of cells of the adaptive immune system. Many viruses can suppress type I IFN production, and in retroviral infections, the initial type I IFN is weak. Thus, one strategy of immunotherapy in viral infection is the exogenous induction of type I IFN during acute viral infection by TLR ligands. Along these lines, the TLR3/MDA5 ligand polyinosinic-polycytidylic acid [poly(I:C)] has already been used to treat viral infections. However, the immunological mechanisms underlying this successful therapy have not been defined until now. In this study, the Friend retrovirus (FV) mouse model was used to investigate the mode of action of poly(I:C) in antiretroviral immunotherapy. Postexposure, poly(I:C) treatment of FV-infected mice resulted in a significant reduction in viral loads and protection from virus-induced leukemia. This effect was IFN dependent because type I IFN receptor-deficient mice could not be protected by poly(I:C). The poly(I:C)-induced IFN response resulted in the expression of antiviral enzymes, which suppressed FV replication. Also, the virus-specific T cell response was augmented. Interestingly, it did not enhance the number of virus-specific CD4(+) and CD8(+) T cells, but rather the functional properties of these cells, such as cytokine production and cytotoxic activity. The results demonstrate a direct antiviral and immunomodulatory effect of poly(I:C) and, therefore, suggests its potential for clinical treatment of retroviral infections.     

Toll-like receptor 3 agonist poly I:C reinforces the potency of cytotoxic chemotherapy via the TLR3-UNC93B1-IFN-β signaling axis in paclitaxel-resistant colon cancer

Type I interferon (IFN) signaling in neoplastic cells has a chemo-sensitizing effect in cancer therapy. Toll-like receptor 3 (TLR3) activation promotes IFN-β production, which induces apoptosis and impairs proliferation in some cancer cells. Herein, we tested whether the TLR3 agonist polyinosinic: polycytidylic acid (poly I:C) can improve chemotherapeutic efficacy in paclitaxel (PTX) resistant cell lines. Human colon cancer cell lines HCT116, SW620, HCT-8 (sensitive to PTX), and HCT-8/PTX (resistant to PTX) were treated with poly I:C and the cell viability was measured. Results showed that poly I:C specifically impaired the cell viability of HCT-8/PTX by simultaneously promoting cell apoptosis and inhibiting cell proliferation. In addition, when TLR3 was overexpressed in HCT-8/PTX cells, we found that TLR3 contributed to the production of IFN-β that reduced cell viability, and poly I:C preferentially activated the TLR3-UNC93B1 signaling pathway to mediate this effect. Moreover, cotreatment of poly I:C and PTX acted synergistically to induce cell apoptosis of HCT-8/PTX via upregulating the expression of TLR3 and its molecular chaperone UNC93B1, assisting in the secretion of IFN-β. Notably, a combination of poly I:C and PTX synergistically inhibited the PTX-resistant tumor growth in vivo without side effects. In conclusion, our studies demonstrate that poly I:C reinforces the potency of cytotoxic chemotherapeutics in PTX-resistant cell line through the TLR3-UNC93B1-IFN-β signaling pathway, which supplies a novel mechanism of poly I:C for the chemotherapy sensitizing effect in a PTX-resistant tumor.     

Diesel exhaust enhances virus- and poly(I:C)-induced Toll-like receptor 3 expression and signaling in respiratory epithelial cells

Prior exposure of respiratory epithelial cells to an aqueous-trapped solution of diesel exhaust (DE(as)) enhances the susceptibility to influenza infections. Here, we examined the effect of DE(as) on the Toll-like receptor 3 (TLR3) pathway, which is responsible for the recognition of and response to viruses and double-stranded RNA. Flow cytometric and confocal microscopy analyses showed that TLR3 is predominantly expressed in the cytoplasm of respiratory epithelial cells. To examine the effect of DE on TLR3 expression and function, differentiated human bronchial or nasal epithelial cells as well as A549 cells were exposed to DE(as) and then infected with influenza A or treated with polyriboinosinic acid-polyribocytidylic acid [poly(I:C)], a synthetic form of double-stranded RNA. Exposure to DE(as) before infection with influenza or stimulation with poly(I:C) significantly upregulated the expression of TLR3. Additionally, preexposure to DE(as) significantly increased the poly(I:C)-induced expression of IL-6. Overexpression of a dominant-negative mutant form of TNF receptor-associated factor 6 reversed the effects of DE(as) on poly(I:C)-induced IL-6 expression, suggesting that the response was TLR3 dependent. Similarly, preexposure to DE(as) significantly increased nuclear levels of interferon regulatory factor 3 and the expression of IFN-beta in response to poly(I:C). Pretreatment with wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase, was able to abate the effect of DE(as) on poly(I:C)-induced IFN-beta expression. Together, these results indicate that exposure of respiratory epithelial cells to DE(as) could potentially alter the response to viral infections by increasing the expression and function of TLR3.     

RIG-I/MDA5/MAVS are required to signal a protective IFN response in rotavirus-infected intestinal epithelium

Rotavirus is a dsRNA virus that infects epithelial cells that line the surface of the small intestine. It causes severe diarrheal illness in children and ～500,000 deaths per year worldwide. We studied the mechanisms by which intestinal epithelial cells (IECs) sense rotavirus infection and signal IFN-β production, and investigated the importance of IFN-β production by IECs for controlling rotavirus production by intestinal epithelium and virus excretion in the feces. In contrast with most RNA viruses, which interact with either retinoic acid-inducible gene I (RIG-I) or melanoma differentiation-associated gene 5 (MDA5) inside cells, rotavirus was sensed by both RIG-I and MDA5, alone and in combination. Rotavirus did not signal IFN-β through either of the dsRNA sensors TLR3 or dsRNA-activated protein kinase (PKR). Silencing RIG-I or MDA5, or their common adaptor protein mitochondrial antiviral signaling protein (MAVS), significantly decreased IFN-β production and increased rotavirus titers in infected IECs. Overexpression of laboratory of genetics and physiology 2, a RIG-I-like receptor that interacts with viral RNA but lacks the caspase activation and recruitment domains required for signaling through MAVS, significantly decreased IFN-β production and increased rotavirus titers in infected IECs. Rotavirus-infected mice lacking MAVS, but not those lacking TLR3, TRIF, or PKR, produced significantly less IFN-β and increased amounts of virus in the intestinal epithelium, and shed increased quantities of virus in the feces. We conclude that RIG-I or MDA5 signaling through MAVS is required for the activation of IFN-β production by rotavirus-infected IECs and has a functionally important role in determining the magnitude of rotavirus replication in the intestinal epithelium.     

Akt contributes to activation of the TRIF-dependent signaling pathways of TLRs by interacting with TANK-binding kinase 1

Toll/IL-1R domain-containing adaptor inducing IFN-β (TRIF) is an adaptor molecule that is recruited to TLR3 and -4 upon agonist stimulation and triggers activation of IFN regulatory factor 3 (IRF3) and expression of type 1 IFNs, which are critical for cellular antiviral responses. We show that Akt is a downstream molecule of TRIF/TANK-binding kinase 1 (TBK1) and plays an important role in the activation of IRF3 by TLR3 and -4 agonists. Blockade of Akt by a dominant-negative mutant or by short interfering RNA decreased IRF3 activation and IFN-β expression induced by polyinosinic:polycytidylic acid [poly(I:C)], LPS, TRIF, and TBK1. Association of endogenous TBK1 and Akt was observed in macrophages when stimulated with poly(I:C) and LPS. In vitro kinase assays combined with reversed-phase liquid chromatography mass spectrometry analysis showed that TBK1 enhanced phosphorylation of Akt on Ser(473), whereas knockdown of TBK1 expression by short interfering RNA in macrophages decreased poly(I:C)- and LPS-induced Akt phosphorylation. Embryonic fibroblasts derived from TBK1 knockout mice also showed impaired Akt phosphorylation in response to poly(I:C) and LPS. To our knowledge, our results demonstrate a new regulatory mechanism for Akt activation mediated by TBK1 and a novel role of Akt in TLR-mediated immune responses.     

Distinct poly(I-C) and virus-activated signaling pathways leading to interferon-beta production in hepatocytes

Innate cellular antiviral defenses are likely to influence the outcome of infections by many human viruses, including hepatitis B and C viruses, agents that frequently establish persistent infection leading to chronic hepatitis, cirrhosis, and liver cancer. However, little is known of the pathways by which hepatocytes, the cell type within which these hepatitis agents replicate, sense infection, and initiate protective responses. We show that cultured hepatoma cells, including Huh7 cells, do not activate the interferon (IFN)-beta promoter in response to extracellular poly(I-C). In contrast, the addition of poly(I-C) to culture media activates the IFN-beta promoter and results in robust expression of IFN-stimulated genes (ISG) in PH5CH8 cells, which are derived from non-neoplastic hepatocytes transformed with large T antigen. Small interfering RNA knockdown of TLR3 or its adaptor, Toll-interleukin-1 receptor domain-containing adaptor inducing IFN-beta (TRIF), blocked extracellular poly(I-C) signaling in PH5CH8 cells, whereas poly(I-C) responsiveness could be conferred on Huh7 hepatoma cells by ectopic expression of Toll-like receptor 3 (TLR3). In contrast to poly(I-C), both cell types signal the presence of Sendai virus infection through a TLR3-independent intracellular pathway requiring expression of retinoic acid-inducible gene I (RIG-I), a putative cellular RNA helicase. Silencing of RIG-I expression impaired only the response to Sendai virus and not extracellular poly(I-C). We conclude that hepatocytes contain two distinct antiviral signaling pathways leading to expression of type I IFNs, one dependent upon TLR3 and the other dependent on RIG-I, with little cross-talk between these pathways.     

Toll-like receptor 3 activation induces antiviral immune responses in mouse sertoli cells

Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns and elicit antimicrobial immune responses. In the testis, viruses can induce pathological conditions, such as orchitis, and may participate in the etiology of testicular cancer; however, the molecular mechanisms involved remain under investigation. It has been suggested that because they constitutively express interferon (IFN)-inducible antiviral proteins, Sertoli cells participate in the testicular antiviral defense system. Previously, we demonstrated a key function of mouse Sertoli cells in the bactericidal testicular defense mechanism mediated by a panel of TLRs. To better characterize the potential role of Sertoli cells in the response against testicular viral infections, we investigated the TLR3 expression and function in these cells. Sertoli cells express TLR3, and under stimulation with the synthetic double-stranded RNA analogue poly (I:C), they produce the proinflammatory molecule ICAM1 and secrete functionally active CCL2 chemokine. Using both pharmacological and genetic approaches, we found that these effects are TLR3-dependent. Moreover, using ELISA, we found that IFNA is constitutively produced and not further inducible, whereas IFNB1 is absent and dramatically induced only by transfected poly (I:C), indicating different control mechanisms underlying IFNA and IFNB1 production. To conclude, poly (I:C) elicits both inflammatory and antiviral responses in Sertoli cells.