Respiratory syncytial virus up-regulates TLR4 and sensitizes airway epithelial cells to endotoxin

Airway epithelial cells are unresponsive to endotoxin (lipopolysaccharide (LPS)) exposure under normal conditions. This study demonstrates that respiratory syncytial virus (RSV) infection results in increased sensitivity to this environmental exposure. Infection with RSV results in increased expression of Toll-like receptor (TLR) 4 mRNA, protein, and increased TLR4 membrane localization. This permits significantly enhanced LPS binding to the epithelial monolayer that is blocked by disruption of the Golgi. The increased TLR4 results in an LPS-induced inflammatory response as demonstrated by increased mitogen-activated protein (MAP) kinase activity, IL-8 production, and tumor necrosis factor alpha production. RSV infection also allowed for tumor necrosis factor alpha production subsequent to TLR4 cross-linking with an immobilized antibody. These data suggest that RSV infection sensitizes airway epithelium to a subsequent environmental exposure (LPS) by altered expression and membrane localization of TLR4. The increased interaction between airway epithelial cells and LPS has the potential to profoundly alter airway inflammation.     

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.     

Central role of double-stranded RNA-activated protein kinase in microbial induction of nitric oxide synthase

NO synthase 2 (NOS2) is induced in airway epithelium by influenza virus infection. NOS2 induction late in the course of viral infection may occur in response to IFN-gamma, but early in infection gene expression may be induced by the viral replicative intermediate dsRNA through the dsRNA-activated protein kinase (PKR). Since PKR activates signaling pathways important in NOS2 gene induction, we determined whether PKR is a component in the signal transduction pathway leading to NOS2 gene expression after viral infection of airway epithelium. We show that NOS2 gene expression in human airway epithelial cells occurs in response to influenza A virus or synthetic dsRNA. Furthermore, dsRNA leads to rapid activation of PKR, followed by activation of signaling components including NF-kappaB and IFN regulatory factor 1. NOS2 expression is markedly diminished and IFN regulatory factor 1 and NF-kappaB activation are substantially impaired in PKR null cells. Strikingly, NOS2 induction in response to LPS is abolished in PKR null cells, confirming a central role for PKR in the general signaling pathway to NOS2.     

MDA5 and TLR3 initiate pro-inflammatory signaling pathways leading to rhinovirus-induced airways inflammation and hyperresponsiveness

Rhinovirus (RV), a single-stranded RNA picornavirus, is the most frequent cause of asthma exacerbations. We previously demonstrated in human bronchial epithelial cells that melanoma differentiation-associated gene (MDA)-5 and the adaptor protein for Toll-like receptor (TLR)-3 are each required for maximal RV1B-induced interferon (IFN) responses. However, in vivo, the overall airway response to viral infection likely represents a coordinated response integrating both antiviral and pro-inflammatory pathways. We examined the airway responses of MDA5- and TLR3-deficient mice to infection with RV1B, a minor group virus which replicates in mouse lungs. MDA5 null mice showed a delayed type I IFN and attenuated type III IFN response to RV1B infection, leading to a transient increase in viral titer. TLR3 null mice showed normal IFN responses and unchanged viral titers. Further, RV-infected MDA5 and TLR3 null mice showed reduced lung inflammatory responses and reduced airways responsiveness. Finally, RV-infected MDA5 null mice with allergic airways disease showed lower viral titers despite deficient IFN responses, and allergic MDA5 and TLR3 null mice each showed decreased RV-induced airway inflammatory and contractile responses. These results suggest that, in the context of RV infection, binding of viral dsRNA to MDA5 and TLR3 initiates pro-inflammatory signaling pathways leading to airways inflammation and hyperresponsiveness.     

Respiratory syncytial virus decreases p53 protein to prolong survival of airway epithelial cells

Respiratory syncytial virus (RSV) is a clinically important pathogen. It preferentially infects airway epithelial cells causing bronchiolitis in infants, exacerbations in patients with obstructive lung disease, and life-threatening pneumonia in the immunosuppressed. The p53 protein is a tumor suppressor protein that promotes apoptosis and is tightly regulated for optimal cell growth and survival. A critical negative regulator of p53 is murine double minute 2 (Mdm2), an E3 ubiquitin ligase that targets p53 for proteasome degradation. Mdm2 is activated by phospho-Akt, and we previously showed that RSV activates Akt and delays apoptosis in primary human airway epithelial cells. In this study, we explore further the mechanism by which RSV regulates p53 to delay apoptosis but paradoxically enhance inflammation. We found that RSV activates Mdm2 1-6 h after infection resulting in a decrease in p53 6-24 h after infection. The p53 down-regulation correlates with increased airway epithelial cell longevity. Importantly, inhibition of the PI3K/Akt pathway blocks the activation of Mdm2 by RSV and preserves the p53 response. The effects of RSV infection are antagonized by Nutlin-3, a specific chemical inhibitor that prevents the Mdm2/p53 association. Nutlin-3 treatment increases endogenous p53 expression in RSV infected cells, causing earlier cell death. This same increase in p53 enhances viral replication and limits the inflammatory response as measured by IL-6 protein. These findings reveal that RSV decreases p53 by enhancing Akt/Mdm2-mediated p53 degradation, thereby delaying apoptosis and prolonging survival of airway epithelial cells.     

In vivo function of airway epithelial TLR2 in host defense against bacterial infection

Decreased Toll-like receptor 2 (TLR2) expression has been reported in patients with chronic obstructive pulmonary disease and in a murine asthma model, which may predispose the hosts to bacterial infections, leading to disease exacerbations. Since airway epithelial cells serve as the first line of respiratory mucosal defense, the present study aimed to reveal the role of airway epithelial TLR2 signaling to lung bacterial [i.e., Mycoplasma pneumoniae (Mp)] clearance. In vivo TLR2 gene transfer via intranasal inoculation of adenoviral vector was performed to reconstitute TLR2 expression in airway epithelium of TLR2(-/-) BALB/c mice, with or without ensuing Mp infection. TLR2 and lactotransferrin (LTF) expression in airway epithelial cells and lung Mp load were assessed. Adenovirus-mediated TLR2 gene transfer to airway epithelial cells of TLR2(-/-) mice reconstituted 30-40% TLR2 expression compared with TLR2(+/+) cells. Such airway epithelial TLR2 reconstitution in TLR2(-/-) mice significantly reduced lung Mp load (an appropriate 45% reduction), coupled with elevated LTF expression. LTF expression in mice was shown to be mainly dependent on TLR2 signaling in response to Mp infection. Exogenous human LTF protein dose-dependently decreased lung bacterial load in Mp-infected TLR2(-/-) mice. In addition, human LTF protein directly dose-dependently decreased Mp levels in vitro. These data indicate that reconstitution of airway epithelial TLR2 signaling in TLR2(-/-) mice significantly restores lung defense against bacteria (e.g., Mp) via increased lung antimicrobial protein LTF production. Our findings may offer a deliverable approach to attenuate bacterial infections in airways of asthma or chronic obstructive pulmonary disease patients with impaired TLR2 function.     

TLR3- and Th2 cytokine-dependent production of thymic stromal lymphopoietin in human airway epithelial cells

Thymic stromal lymphopoietin (TSLP) is elevated in asthma and triggers dendritic cell-mediated activation of Th2 inflammatory responses. Although TSLP has been shown to be produced mainly by airway epithelial cells, the regulation of epithelial TSLP expression has not been extensively studied. We investigated the expression of TSLP in cytokine- or TLR ligand-treated normal human bronchial epithelial cells (NHBE). The mRNA for TSLP was significantly up-regulated by stimulation with IL-4 (5.5-fold) and IL-13 (5.3-fold), weakly up-regulated by TNF-alpha, TGF-beta, and IFN-beta, and not affected by IFN-gamma in NHBE. TSLP mRNA was only significantly up-regulated by the TLR3 ligand (dsRNA) among the TLR ligands tested (66.8-fold). TSLP was also induced by in vitro infection with rhinovirus. TSLP protein was detected after stimulation with dsRNA (120 +/- 23 pg/ml). The combination of TNF-alpha and IL-4 produced detectable levels of TSLP protein (40 +/- 13 pg/ml). In addition, TSLP was synergistically enhanced by a combination of IL-4 and dsRNA (mRNA; 207-fold, protein; 325 +/- 75 pg/ml). The induction of TSLP by dsRNA was dependent upon NF-kappaB and IFN regulatory factor 3 (IRF-3) signaling via TLR3 as indicated by a study with small interfering RNA. The potent topical glucocorticoid fluticasone propionate significantly suppressed dsRNA-dependent TSLP production in NHBE. These results suggest that the expression of TSLP is induced in airway epithelial cells by stimulation with the TLR3 ligand and Th2 cytokines and that this response is suppressed by glucocorticoid treatment. This implies that respiratory viral infection and the recruitment of Th2 cytokine producing cells may amplify Th2 inflammation via the induction of TSLP in the asthmatic airway.     

Protein kinase R regulates double-stranded RNA induction of TNF-alpha but not IL-1 beta mRNA in human epithelial cells

Epithelial cells represent the initial site of respiratory viral entry and the first line of defense against such infections. This early antiviral response is characterized by an increase in the production of proinflammatory cytokines such as TNF-alpha and IL-1 beta. dsRNA, which is a common factor present during the life cycle of both DNA and RNA viruses, is known to induce TNF-alpha and IL-1 beta in a variety of cells. In this work we provide data showing that dsRNA treatment induces TNF-alpha and IL-1 beta in human lung epithelial cells via two different mechanisms. Our data show that dsRNA activation of dsRNA-activated protein kinase (PKR) is associated with induction of TNF-alpha but not IL-1 beta expression. An inhibitor of PKR activation blocked the dsRNA-induced elevations in TNF-alpha but not IL-1 beta mRNA in epithelial cells. Data obtained from infection of epithelial cells with a vaccinia virus lacking the PKR inhibitory polypeptide, E3L, revealed that PKR activation was essential for TNF-alpha but not for IL-1 beta expression. In this report, we provide experimental support for the differential regulation of proinflammatory cytokine expression by dsRNA and viral infections in human airway epithelial cells.     

Deletion of TLR3 alters the pulmonary immune environment and mucus production during respiratory syncytial virus infection

The detection of a viral infection by pattern recognition receptors (PAMPs) is an integral part of antiviral immunity. In these studies we have investigated the role of TLR3, which recognizes dsRNA, in Respiratory Syncytial virus (RSV) infection using B6 background mice with a TLR3 deletion. Although we observed no changes in viral growth, we did find that TLR3-/- mice demonstrated significant increases in mucus production in the airways of RSV-infected mice. The qualitative assessment was observed by examining differentially stained lungs, followed by immunohistochemical staining for gob5, a mucus-associated protein. The histopathologic observations were verified using quantitative gene expression analyses examining gob5 gene expression. Changes in pulmonary mucus production were accompanied by an increase in pulmonary IL-13 as well as IL-5 expression and eosinophils in the airways of TLR3-/- mice. Examining leukocytes in the airway indicated an accumulation of eosinophils in TLR3-/- mice, but not wild-type mice, after RSV infection. Isolated lung draining lymph node cells from TLR3-/- mice produced significant increases in Th2-type cytokines, IL-5, and IL-13, compared with wild-type TLR3+/+ mice only after RSV infection. To demonstrate a causative link, we depleted TLR3-/- mice of IL-13 during RSV infection and found that mucus and gob5 expression in the lungs was attenuated. Together, these studies highlight that although TLR3 may not be required for viral clearance, it is necessary to maintain the proper immune environment in the lung to avoid developing pathologic symptoms of disease.     

Rhinovirus increases human beta-defensin-2 and -3 mRNA expression in cultured bronchial epithelial cells

Human beta-defensins (hBDs) are antimicrobial peptides that play important roles in host defense against infection, inflammation and immunity. Previous studies showed that micro-organisms and proinflammatory mediators regulate the expression of these peptides in airway epithelial cells. The aim of the present study was to investigate the modulation of expression of hBDs in cultured primary bronchial epithelial cells (PBEC) by rhinovirus-16 (RV16), a respiratory virus responsible for the common cold and associated with asthma exacerbations. RV16 was found to induce expression of hBD-2 and -3 mRNA in PBEC, but did not affect hBD-1 mRNA. Viral replication appeared essential for rhinovirus-induced beta-defensin mRNA expression, since UV-inactivated rhinovirus did not increase expression of hBD-2 and hBD-3 mRNA. Exposure to synthetic double-stranded RNA (dsRNA) molecule polyinosinic:polycytidylic acid had a similar effect as RV16 on mRNA expression of these peptides in PBEC. In line with this, PBEC were found to express TLR3, a Toll-like receptor involved in recognition of dsRNA. This study shows that rhinovirus infection of PBEC leads to increased hBD-2 and hBD-3 mRNA expression, which may play a role in both the uncomplicated common cold and in virus-associated exacerbations of asthma.     

Respiratory Syncytial Virus Can Infect Basal Cells and Alter Human Airway Epithelial Differentiation

Respiratory syncytial virus (RSV) is a major cause of morbidity and mortality worldwide, causing severe respiratory illness in infants and immune compromised patients. The ciliated cells of the human airway epithelium have been considered to be the exclusive target of RSV, although recent data have suggested that basal cells, the progenitors for the conducting airway epithelium, may also become infected in vivo. Using either mechanical or chemical injury models, we have demonstrated a robust RSV infection of p63⁺ basal cells in air-liquid interface (ALI) cultures of human bronchial epithelial cells. In addition, proliferating basal cells in 2D culture were also susceptible to RSV infection. We therefore tested the hypothesis that RSV infection of this progenitor cell would influence the differentiation status of the airway epithelium. RSV infection of basal cells on the day of seeding (MOI≤0.0001), resulted in the formation of an epithelium that showed a profound loss of ciliated cells and gain of secretory cells as assessed by acetylated α-tubulin and MUC5AC/MUC5B immunostaining, respectively. The mechanism driving the switch in epithelial phenotype is in part driven by the induced type I and type III interferon response that we demonstrate is triggered early following RSV infection. Neutralization of this response attenuates the RSV-induced loss of ciliated cells. Together, these data show that through infection of proliferating airway basal cells, RSV has the potential to influence the cellular composition of the airway epithelium. The resulting phenotype might be expected to contribute towards both the severity of acute infection, as well as to the longer-term consequences of viral exacerbations in patients with pre-existing respiratory diseases.     

Activation of Ca2+-dependent signaling by TLR2

Upon contact with airway epithelial cells, bacterial products activate Ca(2+) fluxes that are required for induction of NF-kappaB-dependent gene expression. TLR2 is apically displayed on airway cells, making it a likely transducer linking bacterial stimuli and kinases that affect Ca(2+) release. Using biochemical and genetic approaches, we demonstrate that TLR2 ligands stimulate release of Ca(2+) from intracellular stores by activating TLR2 phosphorylation by c-Src, and recruiting PI3K and phospholipase Cgamma to affect Ca(2+) release through inositol (1,4,5) trisphosphate receptors. In the absence of TLR2, murine macrophages as well as airway cells do not generate Ca(2+) fluxes or induce proinflammatory signaling. Thus, Ca(2+) participates as a second messenger in TLR2-dependent signaling and provides another target to modulate proinflammatory responses to bacterial infection.     

Respiratory viruses augment the adhesion of bacterial pathogens to respiratory epithelium in a viral species- and cell type-dependent manner

Secondary bacterial infections often complicate respiratory viral infections, but the mechanisms whereby viruses predispose to bacterial disease are not completely understood. We determined the effects of infection with respiratory syncytial virus (RSV), human parainfluenza virus 3 (HPIV-3), and influenza virus on the abilities of nontypeable Haemophilus influenzae and Streptococcus pneumoniae to adhere to respiratory epithelial cells and how these viruses alter the expression of known receptors for these bacteria. All viruses enhanced bacterial adhesion to primary and immortalized cell lines. RSV and HPIV-3 infection increased the expression of several known receptors for pathogenic bacteria by primary bronchial epithelial cells and A549 cells but not by primary small airway epithelial cells. Influenza virus infection did not alter receptor expression. Paramyxoviruses augmented bacterial adherence to primary bronchial epithelial cells and immortalized cell lines by up-regulating eukaryotic cell receptors for these pathogens, whereas this mechanism was less significant in primary small airway epithelial cells and in influenza virus infections. Respiratory viruses promote bacterial adhesion to respiratory epithelial cells, a process that may increase bacterial colonization and contribute to disease. These studies highlight the distinct responses of different cell types to viral infection and the need to consider this variation when interpreting studies of the interactions between respiratory cells and viral pathogens.     

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.     

Protein kinase R (PKR) interacts with and activates mitogen-activated protein kinase kinase 6 (MKK6) in response to double-stranded RNA stimulation

The double-stranded RNA (dsRNA)-activated protein kinase R (PKR) has been invoked in different signaling pathways. In cells pre-exposed to the PKR inhibitor 2-aminopurine or in PKR-null cells, the activation of p38 mitogen-activated protein kinase (MAPK) following dsRNA stimulation is attenuated. We found that the p38 MAPK activator MKK6, but not its close relatives MKK3 or MKK4, exhibited an increased affinity for PKR following the exposure of cells to poly(rI:rC), a dsRNA analog. In vitro kinase assays revealed that MKK6 was efficiently phosphorylated by PKR, and this could be inhibited by 2-aminopurine. Expression of kinase-inactive PKR (K296R) in cells inhibited the poly(IC)-induced phosphorylation of MKK3/6 detected by phosphospecific antiserum but did not affect the poly(IC)-induced gel migration retardation of MKK3. This suggests that poly(IC)-mediated in vivo activation of MKK6, but not MKK3, is through PKR. Consistent with this observation, PKR was capable of activating MKK6 as assessed in a coupled kinase assay containing the components of the p38 MAPK pathway. Our results indicate that the interaction of MKK6 and PKR provides a mechanism for regulating p38 MAPK activation in response to dsRNA stimulation.