dsRNA Analog Research Articles

Down modulation of human TLR3 function by a monoclonal antibody

Toll-like receptors are a family of pattern-recognition receptors that contribute to the innate immune response. Toll-like receptor 3 (TLR3) signals in response to foreign, endogenous and synthetic ligands including viral dsRNA, bacterial RNA, mitochondrial RNA, endogenous necrotic cell mRNA and the synthetic dsRNA analog, poly(I:C). We have generated a monoclonal antibody (mAb CNTO2424) that recognizes the extracellular domain (ECD) of human TLR3 in a conformation-dependent manner. CNTO2424 down-regulates poly(I:C)-induced production of IL-6, IL-8, MCP-1, RANTES, and IP-10 in human lung epithelial cells. In addition, mAb CNTO2424 was able to interfere with the known TLR3-dependent signaling pathways, namely NF-κB, IRF-3/ISRE, and p38 MAPK. The generation of this neutralizing anti-TLR3 mAb provides a unique tool to better understand TLR3 signaling and potential cross-talk between TLR3 and other molecules.

Double-stranded RNA increases kinin B1 receptor expression and function in human airway epithelial cells

Increased levels of kinins have been detected within the airways during upper respiratory viral infections (URIs). Rhinovirus, the major URI associated with acute exacerbations of asthma, is an ssRNA virus that primarily infects the airway epithelium and produces dsRNA during replication. We asked whether dsRNA could increase the expression of kinin receptors in airway epithelial cells, thereby potentiating the inflammatory consequences of kinin generation. Human airway epithelial cell line BEAS-2B was stimulated with the dsRNA analog Poly I:C and kinin receptor expression detected by quantitative RT-PCR as well as radioligand binding. Poly I:C induced an increase in B1 and B2 receptor mRNA levels in BEAS-2B and primary human normal bronchial epithelial cells. At the cell surface, only B1 receptor expression was increased by Poly I:C. Furthermore, pretreatment of BEAS-2B cells with Poly I:C enhanced the induction of phospho-ERK following B1 receptor ligand stimulation. To investigate whether these finding had potential in vivo relevance, we assessed B1 receptor expression in nasal tissue obtained from 8 normal human subjects with URIs and 3 control subjects. Five of the URI subjects demonstrated increased B1 receptor mRNA compared to the 3 control subjects. We suggest that increased expression of B1 receptor in the human airway following a URI could increase the risk of an exacerbation of asthma by contributing to increased inflammation in the airway.

TLR3 Is Essential for the Induction of Protective Immunity against Punta Toro Virus Infection by the Double-Stranded RNA (dsRNA), Poly(I:C12U), but not Poly(I:C): Differential Recognition of Synthetic dsRNA Molecules

In the wake of RNA virus infections, dsRNA intermediates are often generated. These viral pathogen-associated molecular patterns can be sensed by a growing number of host cell cytosolic proteins and TLR3, which contribute to the induction of antiviral defenses. Recent evidence indicates that melanoma differentiation-associated gene-5 is the prominent host component mediating IFN production after exposure to the dsRNA analog, poly(I:C). We have previously reported that Punta Toro virus (PTV) infection in mice is exquisitely sensitive to treatment with poly(I:C(12)U), a dsRNA analog that has a superior safety profile while maintaining the beneficial activity of the parental poly(I:C) in the induction of innate immune responses. The precise host factor(s) mediating protective immunity following its administration remain to be elucidated. To assess the role of TLR3 in this process, mice lacking the receptor were used to investigate the induction of protective immunity, type I IFNs, and IL-6 following treatment. Unlike wild-type mice, those lacking TLR3 were not protected against PTV infection following poly(I:C(12)U) therapy and failed to produce IFN-alpha, IFN-beta, and IL-6. In contrast, poly(I:C) treatment significantly protected TLR3(-/-) mice from lethal challenge despite some deficiencies in cytokine induction. There was no indication that the lack of protection was due to the fact that TLR3-deficient mice had a reduced capacity to fight infection because they were not found to be more susceptible to PTV. We conclude that TLR3 is essential to the induction of antiviral activity elicited by poly(I:C(12)U), which does not appear to be recognized by the cytosolic sensor of poly(I:C), melanoma differentiation-associated gene-5.

Double‐stranded RNA induces iNOS gene expression in Schwann cells, sensory neuronal death, and peripheral nerve demyelination

Inflammation in the peripheral nervous system (PNS) is one of the characteristics of virus-induced peripheral neuropathy. In this inflammatory response, Schwann cells are actively involved. Previously, toll-like receptor 3 (TLR3) was reported as a receptor for double-stranded RNA (dsRNA) that induces antiviral and inflammatory responses in cells of the innate immune system. In this study, we investigated the expression and putative role of TLR3 in Schwann cells. TLR3 was constitutively expressed in Schwann cells. Stimulation with polyinosinic-polycytidylic acid, a synthetic dsRNA analogue, induced the expression of inducible nitric oxide synthase (iNOS) gene in Schwann cells. Studies on the intracellular signal transduction pathways using iSC, an immortalized Schwann cell line, revealed that dsRNA induces the activation of NF-kappaB, p38, and c-Jun N-terminal kinase (JNK). The activation of NF-kappaB, p38, JNK, and dsRNA-dependent protein kinase is required for dsRNA-mediated iNOS gene expression. However, the activation of PI3 kinase and GSK-3beta inhibited iNOS gene induction, a process mediated by their inhibitory effects on NF-kappaB and p38 activation. dsRNA-induced NO production caused neuronal cell death in cultured dorsal root ganglion. Finally, the introduction of dsRNA into the rat sciatic nerve induced iNOS gene expression and peripheral nerve demyelination in vivo. Taken together, these data suggest that viral RNA may induce inflammatory Schwann cell activation via TLR3 and peripheral nerve damage in the PNS.

Expression of functional Toll-like receptors by salivary gland epithelial cells: increased mRNA expression in cells derived from patients with primary Sjögren's syndrome

Toll-like receptors (TLR) play an essential role in the activation of both innate and adaptive immune responses. Salivary gland epithelial cells (SGEC) may participate in the development of glandular inflammatory reactions that characterize primary Sjögren's syndrome (pSS). In this study we sought to assess the expression and function of several TLR molecules in cultured non-neoplastic SGEC obtained from pSS patients and disease controls. Long-term cultured non-neoplastic SGEC derived from pSS patients (SS-SGEC) and disease controls (control-SGEC), as well as the monocytic cell line THP-1 (positive control cell line), were examined by reverse transcription-polymerase chain reaction (RT-PCR) analysis and quantitative real-time PCR for mRNA expression of TLR1, -2, -3 and -4 molecules. TLR function was assessed by the induction of the expression (flow cytometry) of the immunoregulatory molecules CD54/intercellular adhesion molecule-1 (ICAM-1), CD40, CD86/B7 x 2, major histocompatibility complex (MHC) class I and MHC class II following treatment with the TLR ligands: Staphylococcus aureus peptidoglycan (TLR2), the synthetic dsRNA analogue polyinosinic:cytidylic acid (TLR3) and Escherichia coli lipopolysaccharide (TLR4). SGEC were found to express functional TLR2, -3 and -4 molecules, as attested by dose-dependent up-regulation of surface ICAM-1, CD40 and MHC-I expression (as well as of reciprocal TLR mRNA) following treatment with the respective TLR-ligands. SS-SGEC lines displayed significantly higher constitutive expression of TLR1 (P=0 x 0027), TLR2 (P=0 x 01) and TLR4 (P=0 x 03) mRNA compared to control-SGEC. This study demonstrates that cultured SGEC express functional TLR molecules; the high constitutive TLR expression by SS-SGEC is probably suggestive of the intrinsic activation of epithelial cells in pSS and further supports the role of this type of tissue in pathogenesis of the disorder.

Antitumor NK activation induced by the Toll-like receptor 3-TICAM-1 (TRIF) pathway in myeloid dendritic cells

Myeloid dendritic cells (mDCs) recognize and respond to polyI:C, an analog of dsRNA, by endosomal Toll-like receptor (TLR) 3 and cytoplasmic receptors. Natural killer (NK) cells are activated in vivo by the administration of polyI:C to mice and in vivo are reciprocally activated by mDCs, although the molecular mechanisms are as yet undetermined. Here, we show that the TLR adaptor TICAM-1 (TRIF) participates in mDC-derived antitumor NK activation. In a syngeneic mouse tumor implant model (C57BL/6 vs. B16 melanoma with low H-2 expresser), i.p. administration of polyI:C led to the retardation of tumor growth, an effect relied on by NK activation. This NK-dependent tumor regression did not occur in TICAM-1(-/-) or IFNAR(-/-) mice, whereas a normal NK antitumor response was induced in PKR(-/-), MyD88(-/-), IFN-beta(-/-), and wild-type mice. IFNAR was a prerequisite for the induction of IFN-alpha/beta and TLR3. The lack of TICAM-1 did not affect IFN production but resulted in unresponsiveness to IL-12 production, mDC maturation, and polyI:C-mediated NK-antitumor activity. This NK activation required NK-mDC contact but not IL-12 function in in vivo transwell analysis. Implanted tumor growth in IFNAR(-/-) mice was retarded by adoptively transferring polyI:C-treated TICACM-1-positive mDCs but not TICAM-1(-/-) mDCs. Thus, TICAM-1 in mDCs critically facilitated mDC-NK contact and activation of antitumor NK, resulting in the regression of low MHC-expressing tumors.

Innate immune response to double-stranded RNA in biliary epithelial cells is associated with the pathogenesis of biliary atresia

Infections of Reoviridae consisting of a double-stranded RNA (dsRNA) genome are a possible cause of biliary atresia (BA). The aim of the present study is to clarify the pathophysiological function of dsRNA viruses in the pathogenesis of BA. The expression of dsRNA pattern-recognizing receptors, Toll-like receptor 3 (TLR3), retinoic acid inducible gene I (RIG-I), melanoma differentiation-associated gene-5 (MDA-5), and dsRNA-activated protein kinase R (PKR) was constitutively detected in cultured human biliary epithelial cells (BECs). Stimulation with polyinosinic-polycytidylic acid [poly(I:C), a synthetic analog of viral dsRNA] induced the activation of transcription factors [nuclear factor (NF)-kappaB and interferon regulatory factor 3 (IRF3)] and the production of interferon-beta1 (IFN-beta1) and MxA as potent antiviral responses. Moreover, poly(I:C) up-regulated the expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), and both poly(I:C) and TRAIL reduced the viability of cultured human BECs by enhancing apoptosis. Experiments in vivo using tissue sections of extrahepatic bile ducts from patients with BA and controls (choledochal cysts and nonbiliary diseases) showed that the activation of NF-kappaB, interferon regulatory factor-3 (IRF-3), and PKR, and the enhancement of TRAIL and single-stranded DNA (ssDNA)-positive apoptosis were significant in BA, although extrahepatic bile ducts diffusely and constantly expressed TLR3 in all diseases. dsRNA viruses could directly induce the expression of TRAIL and apoptosis in human biliary epithelial cells as a result of the biliary innate immune response, supporting the notion that Reoviridae infections are directly associated with the pathogenesis of cholangiopathies in cases of BA.

Distinct Functions between Toll-Like Receptors 3 and 9 in Retinal Pigment Epithelial Cells

Retinal pigment epithelial cells (RPE cells) are key players in the first-line defense against invading organisms such as viruses and bacteria. The interaction between RPE cells and viral or bacterial components is very important for clearance of these organisms. Toll-like receptors are a family of recognition receptors involved in innate immunity. Each TLR acts as a primary sensor of conserved microbial components and drives the induction of specific biological responses. TLR 3 is involved in the recognition of viral components, such as double-stranded RNA (dsRNA) and poly(I:C), while TLR 9 recognizes viral or bacterial DNA without methylation at CpG motifs. In the present study, we investigated the expression and function of TLR 3 and 9 in RPE cells. PCR analysis revealed expression of genes for TLR 3 and 9 in RPE cells. Expression of TLR 3 and 9 protein was detected in RPE cells by flow cytometry. TLR 3 and 9 showed strong intracellular expression. To detect angiogenetic factors produced by RPE cells, culture supernatant was examined with the Human Angiogenesis Antibody Array, which can simultaneously detect 20 different angiogenetic factors including cytokines, chemokines, soluble cytokine receptors, and growth factors. RPE cells showed high production of interleukin-8 (IL-8) and monocyte chemotactic protein-I (MCP-I). Furthermore, stimulation of RPE cells with the dsRNA analogue poly(I:C) enhanced the secretion of IL-8 and MCP-I, as well as enhancing the expression of junctional adhesion molecule-I (Jam-I) and intracellular adhesion molecule-I (ICAM-I), and promoted the adhesion of monocyte to these cells. In contrast, stimulation with the CpG-DNA motif only enhanced the secretion of IL-8. However, CpG-DNA motif enhanced phagocytosis in RPE cells. These results may indicate that TLR 3 and 9 play a distinct role in the inflammatory response that clears viruses from the retina.

Double-Stranded RNA Acts as a Strong Danger Signal in Human Myeloid Leukemia Cells Leading to Increased Immunogenicity.

Leukemic cells exert immunosuppressive effects that interfere with dendritic cell function and hamper effective anti-leukemic immune responses. Recently, Toll-like receptor 3 (TLR3) was characterized in dendritic cells as an intracellular double-stranded (ds)RNA receptor which is triggered by viral infection or incubation with the synthetic dsRNA analogue polyriboinosinic polyribocytidylic acid [poly(I:C)], leading to maturation and activation of dendritic cells. Until now, little was known on the expression of TLR3 in leukemic cells and their responsiveness to dsRNA treatment. We assessed TLR3 expression in primary and transformed acute myeloid leukemia (AML) cells and hypothesized that the immunogenicity of AML cells could be improved by treatment with the synthetic TLR3 agonist poly(I:C), thereby mimicking viral infection of these leukemic cells. In view of this hypothesis, we electroporated or pulsed transformed and primary AML cells with poly(I:C) and analyzed the effect of poly(I:C) loading on TLR3 expression, costimulatory molecules, cytokine production and allogeneic T cell response. We also assessed the uptake of poly(I:C)-loaded leukemic cells by immature dendritic cells and the subsequent effect on dendritic cell activation and maturation status. We observed that primary and transformed AML cells respond to poly(I:C) electroporation by upregulation of TLR3 expression, apoptosis, elevated levels of costimulatory molecules CD80 and CD86 and by production of type I interferons (IFN). Furthermore, poly(I:C)-electroporated AML cells induced interferon-gamma production by allogeneic T cells. Upon phagocytosis of poly(I:C)-electroporated AML cells, dendritic cells showed an increased expression of maturation markers and marked production of proinflammatory cytokines. In contrast, this set of immune effects was absent or suboptimal when AML cells were passively pulsed with poly(I:C), indicating the superiority of transfection over pulsing with poly(I:C). These results demonstrate that poly(I:C) electroporation is a promising novel strategy to increase the immunogenicity of AML cells.

Essential role of mda-5 in type I IFN responses to polyriboinosinic:polyribocytidylic acid and encephalomyocarditis picornavirus

The innate immune system recognizes viral dsRNA through two distinct pathways; the Toll-like receptor 3 (TLR3) pathway detects dsRNA phagocytosed in endosomes; the helicases retinoic acid-induced protein I (RIG-I) and melanoma differentiation-associated gene-5 (mda-5) detect cytoplasmic dsRNA generated during viral replication. Both RIG-I and mda-5 can bind polyriboinosinic:polyribocytidylic acid (polyI:C), the synthetic analog of viral dsRNA, and mediate type I IFN responses to polyI:C and multiple RNA viruses in vitro. We generated mda-5-deficient mice and showed that mda-5 is the dominant receptor mediating type I IFN secretion in response to polyI:C in vitro and in vivo. Moreover, mda-5-/- mice exhibited a selectively impaired antiviral response to encephalomyocarditis picornavirus, indicating functional specialization of mda-5 in vivo.

Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses

The innate immune system senses viral infection by recognizing a variety of viral components (including double-stranded (ds)RNA) and triggers antiviral responses. The cytoplasmic helicase proteins RIG-I (retinoic-acid-inducible protein I, also known as Ddx58) and MDA5 (melanoma-differentiation-associated gene 5, also known as Ifih1 or Helicard) have been implicated in viral dsRNA recognition. In vitro studies suggest that both RIG-I and MDA5 detect RNA viruses and polyinosine-polycytidylic acid (poly(I:C)), a synthetic dsRNA analogue. Although a critical role for RIG-I in the recognition of several RNA viruses has been clarified, the functional role of MDA5 and the relationship between these dsRNA detectors in vivo are yet to be determined. Here we use mice deficient in MDA5 (MDA5-/-) to show that MDA5 and RIG-I recognize different types of dsRNAs: MDA5 recognizes poly(I:C), and RIG-I detects in vitro transcribed dsRNAs. RNA viruses are also differentially recognized by RIG-I and MDA5. We find that RIG-I is essential for the production of interferons in response to RNA viruses including paramyxoviruses, influenza virus and Japanese encephalitis virus, whereas MDA5 is critical for picornavirus detection. Furthermore, RIG-I-/- and MDA5-/- mice are highly susceptible to infection with these respective RNA viruses compared to control mice. Together, our data show that RIG-I and MDA5 distinguish different RNA viruses and are critical for host antiviral responses.

Toll‐like receptor 3 mediated hyperphosphorylation of tau in human SH‐SY5Y neuroblastoma cells

Abstract Neurofibrillary tangles of abnormally phosphorylated tau are one of the characteristic pathological hallmarks of Alzheimer's disease (AD). In addition, immunological and inflammatory changes including complements and activated microglia are also common phenomena in AD. However, these pathological changes are yet to be interlinked in a common explainable background. In this study, the relevant mechanism of phosphorylation of tau protein and an innate immune signal transduction system were investigated. Toll‐like receptor 3 (TLR3) is a receptor working in the innate immune system and its expression in the brain has already been reported. Total RNA was isolated from SH‐SY5Y cells and reverse transcriptase polymerase chain reaction was done to see endogenous expression of TLR3 in SH‐SY5Y cells that was further confirmed at protein level by Western blot analysis. Cells were treated with 50 µg/mL of polyinosinic–polycytidylic acid (pIpC), a synthetic analog of dsRNA and the changes of phosphorylation of tau protein were investigated. Further the level of phosphorylation of tau protein was investigated after the cells had been previously treated with 10 ng/mL of lipopolysaccharide (LPS) for 6 h to induce over‐expression of TLR3. Increased phosphorylation of tau protein at PHF‐1 site (Ser396/404), activation of Jun N‐terminal kinase and p38 MAPK were observed in cells treated with pIpC. These effects were enhanced when cells were pretreated with LPS, a known transducer of TLR3. These data suggest that toll‐like receptor 3, an innate immune molecule, might be a potential link to mediate hyperphosphorylation of tau in neurodegenerative processes of AD.

In vitro‐Generated Viral Double‐Stranded RNA in Contrast to Polyinosinic : Polycytidylic Acid Induces Interferon‐α in Human Plasmacytoid Dendritic Cells

Double-stranded RNA (dsRNA) arises in the cytoplasm during viral replication and was shown to participate in the interferon (IFN)-alpha induction process. Besides the intracellular recognition, released dsRNA from dying, infected cells can function as a pathogen-associated molecular pattern (PAMP) for the innate immune system. In the present study, in vitro-generated dsRNA fragments of genomic sequences of Newcastle disease virus were used to induce IFN-alpha release in human peripheral blood mononuclear cells (PBMC), in immature myeloid dendritic cells (mDC) and in immature plasmacytoid DC (pDC). The extracellular administration of dsRNA fragments but not the application of the corresponding single-stranded RNA (ssRNA) strands led to an IFN-alpha production in PBMC. The synthetic dsRNA analogue polyinosinic acid : polycytidylic acid [Poly(I : C)] could only stimulate IFN-alpha production in enriched mDC but not in pDC. In contrast, dsRNA fragments induced IFN-alpha only in pDC. Complexation of dsRNA fragments with transfection reagents increased the efficiency of IFN-alpha induction and commuted ssRNA molecules into IFN-alpha inducers. However, stimulation of in vitro-generated murine Toll-like receptor 7 (TLR7) knockout DC and human TLR-transfected HEK293 cells with dsRNA fragments gave no evidences for the involvement of pDC-specific TLR7 or TLR9 in the observed IFN-alpha induction.

Induction of antigen-specific CD8+ cytotoxic T cells by dendritic cells co-electroporated with a dsRNA analogue and tumor antigen mRNA

The maturation state of dendritic cells (DCs) is an important determinant for the initiation and regulation of adaptive immune responses. In this study, we wanted to assess whether functional activation of human monocyte-derived DCs can be achieved by electroporation of an activation signal in the form of double-stranded (ds) RNA and whether simultaneous electroporation of the dsRNA with tumor antigen encoding mRNA can lead to the induction of a cytotoxic T-lymphocyte (CTL) response. Electroporation of immature DCs with poly(I:C(12)U), a dsRNA analogue, resulted in phenotypic as well as functional changes, indicative of DC maturation. Co-electroporation of DCs with both poly(I:C(12)U) and Melan-A/MART-1 encoding mRNA induced strong anti-Melan-A/MART-1 CD8(+) T-cell responses in vitro. Higher numbers of Melan-A/MART-1-specific CTLs were consistently obtained with poly(I:C(12)U)-activated DCs compared to DCs matured in the presence of an inflammatory cytokine cocktail. These results indicate that DC co-electroporation with both dsRNA and tumor antigen encoding mRNA induces fully activated and antigen-loaded DCs that promote antigen-specific CTL responses and may provide the basis for future immunotherapeutic strategies.

Responsiveness to double stranded RNA in human biliary epithelial cells: Involvement of innate immunity in the pathogenesis of cholangiopathy

Viral infections including reovirus and rotavirus have been suspected as a possible etiology of biliary atresia and choledochal cysts, but a role of these double stranded RNA (dsRNA) viruses is still unknown in the pathogenesis of cholangiopathies. Toll-like receptor (TLR) 3 and the recently identified cytosolic RNA helicases RIG-I (retinoic acid inducible gene I) and MDA5 (melanoma differentiation-associated gene 5) recognize dsRNA and activate NF-kappaB and interferon regulatory factor 3 (IRF3) leading to the induction of IFN-beta production. In order to clarify the innate immunity to dsRNA virus in cholangiopathies with reference to extrahepatic biliary atresia and choledochal cysts, we examined the expression of TLR3, RIG-I, and MDA5 and the effects of dsRNA using 3 cultured human biliary epithelial cells (HIBECs). The expression of TLR3, RIG-I, and MDA5 were demonstrated in all of three HIBEC cells by RT-PCR. In addition, HIBEC cells produced IFN-beta after the treatment with poly(I:C) (synthetic analog of dsRNA), but not with bacterial components such as peptidoglycan and lipopolysaccharide. Furthermore, the poly(I:C) treatment upregulated the activation of NF-kappaB. In contrast, IRF3 had already activated under basal conditions and its synergistic activation by poly(I:C) was unclear in HIBEC cells. In conclusion, human biliary epithelial cells can cause an innate immunity response to dsRNA virus, supporting the immune-mediated mechanisms in the pathogenesis of cholangiopathies in biliary atresia and choledochal cysts.

Novel Role of Toll-Like Receptor 3 in Hepatitis C-Associated Glomerulonephritis

Hepatitis C virus (HCV) infection is frequently complicated by glomerulonephritis with immune complexes containing viral RNA. We examined the potential influence of Toll-like receptors (TLRs), specifically TLR3 recognition of viral dsRNA exemplified by polyriboinosinic:polyribocytidylic acid [poly(I:C) RNA]. Normal human kidney stained positive for TLR3 on mesangial cells (MCs), vascular smooth muscle cells, and collecting duct epithelium. Cultured MCs have low TLR3 mRNA levels with predominant intracellular protein localization, which was increased by tumor necrosis factor-alpha, interleukin (IL)-1beta, interferon (IFN)-gamma, and the TLR3 ligand poly(I:C) RNA. Poly(I:C) RNA stimulation of MCs increased mRNA and protein synthesis of IL-6, IL-1beta, M-CSF, IL-8/CXCL8, RANTES/CCL5, MCP-1/CCL2, and ICAM-I; it also increased anti-proliferative and proapoptotic effects, the latter of which was decreased by inhibiting caspase-8. In microdissected glomeruli of normal and non-HCV membranoproliferative glomerulonephritis biopsies, TLR3 mRNA expression was low. In contrast TLR3 mRNA expression was significantly increased in hepatitis C-positive glomerulonephritis and was associated with enhanced mRNA for RANTES/CCL5 and MCP-1/CCL2. We hypothesize that immune complexes containing viral RNA activate mesangial TLR3 during HCV infection, thereby contributing to chemokine/cytokine release and effecting proliferation and apoptosis. Thus, TLR3 expression on renal cells, and especially MCs, may establish a link between viral infections and glomerular diseases.

Characterization of Toll-like receptors in primary lung epithelial cells: strong impact of the TLR3 ligand poly(I:C) on the regulation of Toll-like receptors, adaptor proteins and inflammatory response

BackgroundBacterial and viral exacerbations play a crucial role in a variety of lung diseases including COPD or asthma. Since the lung epithelium is a major source of various inflammatory mediators that affect the immune response, we analyzed the inflammatory reaction of primary lung epithelial cells to different microbial molecules that are recognized by Toll-like receptors (TLR).MethodsThe effects of TLR ligands on primary small airway epithelial cells were analyzed in detail with respect to cytokine, chemokine and matrix metalloproteinase secretion. In addition, the regulation of the expression of TLRs and their adaptor proteins in small airway epithelial cells was investigated.ResultsOur data demonstrate that poly(I:C), a synthetic analog of viral dsRNA, mediated the strongest proinflammatory effects among the tested ligands, including an increased secretion of IL-6, IL-8, TNF-α, GM-CSF, GRO-α, TARC, MCP-1, MIP-3α, RANTES, IFN-β, IP-10 and ITAC as well as an increased release of MMP-1, MMP-8, MMP-9, MMP-10 and MMP-13. Furthermore, our data show that poly(I:C) as well as type-1 and type-2 cytokines have a pronounced effect on the expression of TLRs and molecules involved in TLR signaling in small airway epithelial cells. Poly(I:C) induced an elevated expression of TLR1, TLR2 and TLR3 and increased the gene expression of the general TLR adaptor MyD88 and IRAK-2. Simultaneously, poly(I:C) decreased the expression of TLR5, TLR6 and TOLLIP.ConclusionPoly(I:C), an analog of viral dsRNA and a TLR3 ligand, triggers a strong inflammatory response in small airway epithelial cells that is likely to contribute to viral exacerbations of pulmonary diseases like asthma or COPD. The pronounced effects of poly(I:C) on the expression of Toll-like receptors and molecules involved in TLR signaling is assumed to influence the immune response of the lung epithelium to viral and bacterial infections. Likewise, the regulation of TLR expression by type-1 and type-2 cytokines is important considering the impact of exogenous and endogenous TLR ligands on Th1 or Th2 driven pulmonary inflammations like COPD or asthma, respectively.

Expression of toll‐like receptors by human muscle cells in vitro and in vivo: TLR3 is highly expressed in inflammatory and HIV myopathies, mediates IL‐8 release, and up‐regulation of NKG2D‐ligands

The particular microenvironment of the skeletal muscle can be the site of complex immune reactions. Toll-like receptors (TLRs) mediate inflammatory stimuli from pathogens and endogenous danger signals and link the innate and adaptive immune system. We investigated innate immune responses in human muscle. Analyzing TLR1-9 mRNA in cultured myoblasts and rhabdomyosarcoma cells, we found constitutive expression of TLR3. The TLR3 ligand Poly (I:C), a synthetic analog of dsRNA, and IFN-gamma increased TLR3 levels. TLR3 was mainly localized intracellularly and regulated at the protein level. Poly (I:C) challenge 1) activated nuclear factor-kappaB (NF-kappaB), 2) increased IL-8 release, and 3) up-regulated NKG2D ligands and NK-cell-mediated lysis of muscle cells. We examined muscle biopsy specimens of 6 HIV patients with inclusion body myositis/polymyositis (IBM/PM), 7 cases of sporadic IBM and 9 nonmyopathic controls for TLR3 expression. TLR3 mRNA levels were elevated in biopsy specimens from patients with IBM and HIV-myopathies. Muscle fibers in inflammatory myopathies expressed TLR3 in close proximity of infiltrating mononuclear cells. Taken together, our study suggests an important role of TLR3 in the immunobiology of muscle, and has substantial implications for the understanding of the pathogenesis of inflammatory myopathies or therapeutic interventions like vaccinations or gene transfer.

Stable expression of EBERs in immortalized nasopharyngeal epithelial cells confers resistance to apoptotic stress

Epstein-Barr virus (EBV) infection is closely associated with the development of nasopharyngeal carcinoma (NPC). The EBV-encoded RNAs (EBERs) are the most abundant EBV transcripts (about 10(7) copies per cell) in EBV infected cells. However, the cellular function of EBER expression, particularly in nasopharyngeal epithelial cells, remains poorly understood. EBERs acquire secondary structures analogous to double-stranded RNA (dsRNA) and may bind to the double-stranded RNA-dependent protein kinase (PKR) and interfere with its function. Activation of PKR involves autophosphorylation resulting in protein synthesis inhibition and cellular apoptosis. Induction of cellular apoptosis by activation of PKR may be an antiviral response adopted by virally infected cells. We have examined the functional properties of EBER expression in an immortalized nasopharyngeal epithelial cell line (NP69). Expression of EBERs was achieved by transfecting the NP69 cells with an EBER-expressing plasmid, pESK10. The EBER-expressing NP69 cells attained a higher growth rate compared to cells transfected with control plasmid (pcDNA3). However, the EBER-expressing NP69 cells did not form colonies in soft agar and were non-tumorigenic in nude mice. To investigate if EBERs may protect the nasopharyngeal epithelial cells from apoptotic insults, we treated the EBER-expressing NP69 cells with a dsRNA analogue, poly(I).poly(C) (pIC), to activate PKR in cells and examined for their responses. Lower level of PKR phosphorylation and elevation of Bcl-2 were observed in EBER-expressing NP69 cells. In addition, other apoptotic markers including the cleaved forms of caspase-3 and poly(ADP)ribose polymerase (PARP) were found to be lower in EBER-expressing NP69 cells after treatment with pIC. Lower phosphorylation levels of p38 MAPK (mitogen-activated protein kinase) and c-jun were also observed in EBER-expressing NP cells. Our results suggest that EBER expression may confer an apoptotic-resistant phenotype in immortalized nasopharyngeal epithelial cells.

Expression of B7-H1 and B7-DC on the airway epithelium is enhanced by double-stranded RNA

Viral infection in the airway provokes various immune responses, including Th1 and Th2 responses, which are partly initiated by double-stranded RNA (dsRNA), a viral product for its replication. B7-H1 (PD-L1) and B7-DC (PD-L2) are B7-family molecules that bind to programmed death-1 (PD-1) on lymphocytes and are implicated in peripheral tolerance. We investigated the effect of dsRNA on the expression of B7-H1 and B7-DC on airway epithelial cell lines. B7-H1 and B7-DC were constitutively expressed on the cells, and their expression was profoundly upregulated by stimulation with an analog of viral dsRNA, polyinosinic–polycytidylic acid. B7-H1 and B7-DC were also upregulated by stimulation with IFN-γ, IL-13, and the supernatant from T cell clones. A relatively high concentration of dexamethasone (1 μM) was required to suppress the upregulation of B7-H1 or B7-DC. These results suggest that epithelial B7-H1 and B7-DC play a role in virus-associated immune responses in the airways.