Specific Assay of Negative Strand Template to Quantify Intracellular Levels of Rhinovirus Double-Stranded RNA.

Human rhinovirus infections are a major trigger for acute exacerbations of lower airway diseases, including asthma and chronic obstructive pulmonary disease. Disease exacerbation is thought to be regulated via double-stranded RNA (dsRNA)-mediated signaling of proinflammatory and host defense responses in airway epithelial cells. Despite the central role of dsRNA in regulating host cell responses, no method for the quantitative assessment of dsRNA levels during HRV infections has been developed. Conventional RT-PCR for the negative strand template is not effective as self-priming results in apparent signals, even in the absence of primer during reverse transcription. To avoid these issues, we developed a selective assay for the negative strand template that uses a chimeric primer containing a 5' non-viral sequence for reverse transcription and a primer using the non-viral sequence during subsequent PCR. We established that this assay avoided issues of self-priming and is strand specific, as it is unaffected even in the presence of a 1000-fold excess of positive strand. Assays in primary human airway epithelial cells showed that negative strand was detectable within 6 h of virus exposure and peaked at 18 h after virus exposure. The temporal pattern of negative strand induction mirrored that of genomic RNA but was always 1000-fold lower than positive strand, indicating that the negative strand levels regulate levels of dsRNA formation. This assay will permit relative quantification of dsRNA during studies of HRV regulation of epithelial cell function.

Prostanoid Receptors of the EP4-Subtype Mediate Gene Expression Changes in Human Airway Epithelial Cells with Potential Anti-Inflammatory Activity.

There is a clear, unmet clinical need to identify new drugs to treat individuals with asthma, chronic obstructive pulmonary disease (COPD), and idiopathic pulmonary fibrosis (IPF) in whom current medications are either inactive or suboptimal. In preclinical models, EP4-receptor agonists display efficacy, but their mechanism of action is unclear. In this study, using human bronchial epithelial cells as a therapeutically relevant drug target, we hypothesized that changes in gene expression may play an important role. Several prostanoid receptor mRNAs were detected in BEAS-2B cells, human primary bronchial epithelial cells (HBECs) grown in submersion culture and HBECs grown at an air-liquid interface with PTGER4 predominating. By using the activation of a cAMP response element reporter in BEAS-2B cells as a surrogate of gene expression, Schild analysis determined that PTGER4 mRNAs encoded functional EP4-receptors. Moreover, inhibitors of phosphodiesterase 4 (roflumilast N-oxide [RNO]) and cAMP-dependent protein kinase augmented and attenuated, respectively, reporter activation induced by 2-[3-[(1R,2S,3R)-3-hydroxy-2-[(E,3S)-3-hydroxy-5-[2-(methoxymethyl)phenyl]pent-1-enyl]-5-oxo-cyclopentyl]sulphanylpropylsulphanyl] acetic acid (ONO-AE1-329), a selective EP4-receptor agonist. ONO-AE1-329 also enhanced dexamethasone-induced activation of a glucocorticoid response element reporter in BEAS-2B cells, which was similarly potentiated by RNO. In each airway epithelial cell variant, numerous genes that may impart therapeutic benefit in asthma, COPD, and/or IPF were differentially expressed by ONO-AE1-329, and those changes were often augmented by RNO and/or dexamethasone. We submit that an EP4-receptor agonist, either alone or as a combination therapy, may be beneficial in individuals with chronic lung diseases in whom current treatment options are inadequate. SIGNIFICANCE STATEMENT: Using human bronchial epithelial cells as a therapeutically relevant drug target, we report that EP4-receptor activation promoted gene expression changes that could provide therapeutic benefit in individuals with asthma, COPD, and IPF in whom current treatment options are ineffective or suboptimal.

Rhinovirus Induces Basolateral Release of IL-17C in Highly Differentiated Airway Epithelial Cells.

Human rhinovirus (HRV) is a major trigger of acute exacerbations of both asthma and chronic obstructive pulmonary disease. The airway epithelium is the primary site of HRV infection, and responds by releasing proinflammatory and antimicrobial cytokines. Epithelial cells release IL-17C in response to exposure to bacterial, viral, and fungal pathogens. We previously demonstrated a role for HRV in IL-17C production from undifferentiated epithelial cells, and showed that IL-17C could play a role in neutrophil recruitment. To extend these observations, highly differentiated human bronchial epithelial cells (HBE) were infected apically with HRV to assess the effect of dose, time, viral replication, and strain on the IL-17C response. Cellular lysates, and basolateral and apical secretions were analyzed for IL-17C and CXCL1 protein release following HRV or IL-17C stimulation. Upon HRV infection, IL-17C protein was exclusively released basolaterally in a dose-, time-, and viral replication-dependent manner. Several strains of rhinovirus were capable of inducing IL-17C release. Enriched columnar epithelial cell populations contained significantly higher viral titer, and expressed significantly more IL-17C mRNA than enriched basal cell populations. In addition, the kinetic profile of IL-17C release following HRV treatment closely mimics viral shedding kinetics, further implicating the role of rhinovirus replication in IL-17C production. Basolateral treatment of HBEs with IL-17C resulted in a dose-dependent increase in basolateral CXCL1 production. In summary, replicating rhinovirus drives basolateral IL-17C protein release from both apical and basal epithelial cells, which may then act in an autocrine/paracrine manner to promote basolateral CXCL1 protein release.

Rhinovirus replication and innate immunity in highly differentiated human airway epithelial cells.

BACKGROUND: Human rhinovirus (HRV) infections are the primary cause of the common cold and are a major trigger for exacerbations of lower airway diseases, such as asthma and chronic obstructive pulmonary diseases. Although human bronchial epithelial cells (HBE) are the natural host for HRV infections, much of our understanding of how HRV replicates and induces host antiviral responses is based on studies using non-airway cell lines (e.g. HeLa cells). The current study examines the replication cycle of HRV, and host cell responses, in highly differentiated cultures of HBE. METHODS: Highly differentiated cultures of HBE were exposed to initial infectious doses ranging from 104 to 101 50% tissue culture-infective dose (TCID50) of purified HRV-16, and responses were monitored up to 144 h after infection. Viral genomic RNA and negative strand RNA template levels were monitored, along with levels of type I and II interferons and selected antivirals. RESULTS: Regardless of initial infectious dose, relatively constant levels of both genomic and negative strand RNA are generated during replication, with negative strand copy numbers being10,000-fold lower than those of genomic strands. Infections were limited to a small percentage of ciliated cells and did not result in any overt signs of epithelial death. Importantly, regardless of infectious dose, HRV-16 infections were cleared by HBE in the absence of immune cells. Levels of type I and type III interferons (IFNs) varied with initial infectious dose, implying that factors other than levels of double-stranded RNA regulate IFN induction, but the time-course of HRV-16 clearance HBE was the same regardless of levels of IFNs produced. Patterns of antiviral viperin and ISG15 expression suggest they may be generated in an IFN-independent manner during HRV-16 infections. CONCLUSIONS: These data challenge a number of aspects of dogma generated from studies in HeLa cells and emphasize the importance of appropriate cell context when studying HRV infections.

Rhinovirus and Bacteria Synergistically Induce IL-17C Release from Human Airway Epithelial Cells To Promote Neutrophil Recruitment.

Virus-bacteria coinfections are associated with more severe exacerbations and increased risk of hospital readmission in patients with chronic obstructive pulmonary disease (COPD). The airway epithelium responds to such infections by releasing proinflammatory and antimicrobial cytokines, including IL-17C. However, the regulation and role of IL-17C is not well understood. In this study, we examine the mechanisms regulating IL-17C production and its potential role in COPD exacerbations. Human bronchial epithelial cells (HBE) obtained from normal, nontransplanted lungs or from brushings of nonsmokers, healthy smokers, or COPD patients were exposed to bacteria and/or human rhinovirus (HRV). RNA and protein were collected for analysis, and signaling pathways were assessed with pharmacological agonists, inhibitors, or small interfering RNAs. HBE were also stimulated with IL-17C to assess function. HRV-bacterial coinfections synergistically induced IL-17C expression. This induction was dependent on HRV replication and required NF-κB-mediated signaling. Synergy was lost in the presence of an inhibitor of the p38 MAP kinase pathway. HBE exposed to IL-17C show increased gene expression of CXCL1, CXCL2, NFKBIZ, and TFRC, and release CXCL1 protein, a neutrophil chemoattractant. Knockdown of IL-17C significantly reduced induction of CXCL1 in response to HRV-bacterial coinfection as well as neutrophil chemotaxis. HBE from healthy smokers release less IL-17C than cells from nonsmokers, but cells from COPD patients release significantly more IL-17C compared with either nonsmokers or healthy smokers. These data suggest that IL-17C may contribute to microbial-induced COPD exacerbations by promoting neutrophil recruitment.

Human ß-defensin-2 production upon viral and bacterial co-infection is attenuated in COPD.

Viral-bacterial co-infections are associated with severe exacerbations of COPD. Epithelial antimicrobial peptides, including human ß-defensin-2 (HBD-2), are integral to innate host defenses. In this study, we examined how co-infection of airway epithelial cells with rhinovirus and Pseudomonas aeruginosa modulates HBD-2 expression, and whether these responses are attenuated by cigarette smoke and in epithelial cells obtained by bronchial brushings from smokers with normal lung function or from COPD patients. When human airway epithelial cells from normal lungs were infected with rhinovirus, Pseudomonas aeruginosa, or the combination, co-infection with rhinovirus and bacteria resulted in synergistic induction of HBD-2 (p<0.05). The combination of virus and flagellin replicated this synergistic increase (p<0.05), and synergy was not seen using a flagella-deficient mutant Pseudomonas (p<0.05). The effects of Pseudomonas aeruginosa were mediated via interactions of flagellin with TLR5. The effects of HRV-16 depended upon viral replication but did not appear to be mediated via the intracellular RNA helicases, retinoic acid-inducible gene-I or melanoma differentiation-associated gene-5. Cigarette smoke extract significantly decreased HBD-2 production in response to co-infection. Attenuated production was also observed following co-infection of cells obtained from healthy smokers or COPD patients compared to healthy controls (p<0.05). We conclude that co-exposure to HRV-16 and Pseudomonas aeruginosa induces synergistic production of HBD-2 from epithelial cells and that this synergistic induction of HBD-2 is reduced in COPD patients. This may contribute to the more severe exacerbations these patients experience in response to viral-bacterial co-infections.

Human Rhinovirus Infection of Epithelial Cells Modulates Airway Smooth Muscle Migration.

Airway remodeling, a characteristic feature of asthma, begins in early life. Recurrent human rhinovirus (HRV) infections are a potential inciting stimulus for remodeling. One component of airway remodeling is an increase in airway smooth muscle cell (ASMC) mass with a greater proximity of the ASMCs to the airway epithelium. We asked whether human bronchial epithelial cells infected with HRV produced mediators that are chemotactic for ASMCs. ASMC migration was investigated using the modified Boyden Chamber and the xCELLigence Real-Time Cell Analyzer (ACEA Biosciences Inc., San Diego, CA). Multiplex bead analysis was used to measure HRV-induced epithelial chemokine release. The chemotactic effects of CCL5, CXCL8, and CXCL10 were also examined. Supernatants from HRV-infected epithelial cells caused ASMC chemotaxis. Pretreatment of ASMCs with pertussis toxin abrogated chemotaxis, as did treatment with formoterol, forskolin, or 8-bromo-cAMP. CCL5, CXCL8, and CXCL10 were the most up-regulated chemokines produced by HRV-infected airway epithelial cells. When recombinant CCL5, CXCL8, and CXCL10 were used at levels found in epithelial supernatants, they induced ASMC chemotaxis similar to that seen with epithelial cell supernatants. When examined individually, CCL5 was the most effective chemokine in causing ASMC migration, and treatment of supernatant from HRV-infected epithelial cells with anti-CCL5 antibodies significantly attenuated ASMC migration. These findings suggest that HRV-induced CCL5 can induce ASMC chemotaxis and thus may contribute to the pathogenesis of airway remodeling in patients with asthma.

Rhinovirus-bacteria coexposure synergistically induces CCL20 production from human bronchial epithelial cells.

Exacerbations of chronic obstructive pulmonary disease are triggered by viral or bacterial pathogens, with human rhinovirus (HRV) and nontypeable Hemophilus influenzae (NTHI) among the most commonly detected pathogens. Patients who suffer from concomitant viral and bacterial infection have more severe exacerbations. The airway epithelial cell is the initial site of viral and bacterial interactions, and CCL20 is an epithelial chemokine that attracts immature dendritic cells to the airways and can act as an antimicrobial. As such, it contributes to innate and adaptive immune responses to infection. We used primary cultures of human bronchial epithelial cells and the BEAS-2B cell line to examine the effects of bacterial-viral coexposure, as well as each stimulus alone, on epithelial expression of CXCL8 and, in particular, CCL20. HRV-bacterial coexposure induced synergistic production of CXCL8 and CCL20 compared with the sum of each stimulus alone. Synergistic induction of CCL20 did not require viral replication and occurred with two different HRV serotypes that use different viral receptors. Synergy was also seen with either NTHI or Pseudomonas aeruginosa Synergistic induction of CCL20 was transcriptionally regulated. Although NF-κB was required for transcription, it did not regulate synergy, but NF-IL-6 did appear to contribute. Among MAPK inhibitors studied, neither SB203580 nor PD98059 had any effect on synergy, whereas U0126 prevented synergistic induction of CCL20 by HRV and bacteria, apparently via "off-target" effects. Thus bacterial-viral coexposure synergistically increases innate immune responses compared with individual infections. We speculate that this increased inflammatory response leads to worse clinical outcomes.

Interferon regulatory factor 7 regulates airway epithelial cell responses to human rhinovirus infection.

BACKGROUND: Human rhinoviruses (HRV) cause the majority of colds and trigger exacerbations of chronic lower airway diseases. Airway epithelial cells are the primary site for HRV infection and replication, and the initiation of host inflammatory responses. At present, the molecular mechanisms that underpin HRV responses in airway epithelial cells are incompletely understood. The aim of this study was to employ microarray profiling, upstream regulator analysis, and siRNA mediated gene silencing to further our understanding of the role of interferon regulatory factor 7 (IRF7) in this response. METHODS: Primary human bronchial epithelial cells (HBE) where transfected with siRNA that targets IRF7 or a non-silencing control (all-star control) using Lipofectamine. The cells were allowed to recover, and then cultured in the presence or absence of HRV-16 for 24 h. Global patterns of gene expression were profiled on microarrays. A subset of genes identified in the microarray study were validated at the mRNA and/or protein level using real time RT-qPCR, ELISA, and western blots. RESULTS: Hundreds of genes were upregulated in HBE during HRV infection. Pathways analysis demonstrated that these genes were mainly involved in type I and II interferon signaling, RIG-I/MDA5 signaling, antigen processing and presentation, and apoptosis. Upstream regulator analysis of these data suggested that IRF7 was a major molecular driver of this response. Knockdown of IRF7 reduced the HRV-driven upregulation of genes involved in antiviral responses (interferon signaling, Toll-like receptor signaling, NOD-like receptor signaling, RIG-I/MDA5 signaling), and increased the expression of genes that promote inflammation (e.g. CXCL5, IL-33, IL1RL1) and the response to oxidative stress. However, the majority of genes that were perturbed by HRV in HBE cells including those that are known to be regulated by IRF7 were insensitive to IRF7 knockdown. Upstream regulator analysis of the part of the response that was insensitive to IRF7 knockdown suggested it was driven by NF-κB, STAT1, STAT3, and IRF1. CONCLUSIONS: Our findings demonstrate that IRF7 regulates the expression of genes involved in antiviral immunity, inflammation, and the response to oxidative stress during HRV infections in HBE cells, and also suggests that other transcription factors play a major role in this response.

Cigarette smoke modulates expression of human rhinovirus-induced airway epithelial host defense genes.

Human rhinovirus (HRV) infections trigger acute exacerbations of chronic obstructive pulmonary disease (COPD) and asthma. The human airway epithelial cell is the primary site of HRV infection and responds to infection with altered expression of multiple genes, the products of which could regulate the outcome to infection. Cigarette smoking aggravates asthma symptoms, and is also the predominant risk factor for the development and progression of COPD. We, therefore, examined whether cigarette smoke extract (CSE) modulates viral responses by altering HRV-induced epithelial gene expression. Primary cultures of human bronchial epithelial cells were exposed to medium alone, CSE alone, purified HRV-16 alone or to HRV-16+ CSE. After 24 h, supernatants were collected and total cellular RNA was isolated. Gene array analysis was performed to examine mRNA expression. Additional experiments, using real-time RT-PCR, ELISA and/or western blotting, validated altered expression of selected gene products. CSE and HRV-16 each induced groups of genes that were largely independent of each other. When compared to gene expression in response to CSE alone, cells treated with HRV+CSE showed no obvious differences in CSE-induced gene expression. By contrast, compared to gene induction in response to HRV-16 alone, cells exposed to HRV+CSE showed marked suppression of expression of a number of HRV-induced genes associated with various functions, including antiviral defenses, inflammation, viral signaling and airway remodeling. These changes were not associated with altered expression of type I or type III interferons. Thus, CSE alters epithelial responses to HRV infection in a manner that may negatively impact antiviral and host defense outcomes.

Human rhinovirus infection up-regulates MMP-9 production in airway epithelial cells via NF-{kappa}B.

Human rhinovirus (HRV) infections up-regulate proinflammatory mediators and growth factors that are associated with exacerbations of inflammatory airway diseases, such as asthma and chronic obstructive pulmonary disease (COPD). Matrix metalloproteinase (MMP)-9 was shown to be increased in the airways of patients with asthma and COPD. We sought to determine whether HRV infection modulated the expression of MMP-9 and its highest-affinity inhibitor, the tissue inhibitor of metalloproteinase (TIMP)-1, and we explored the mechanism by which this modulation occurs. In vitro studies, using RT-PCR, ELISA, zymography, and a fluorescent activity assay, demonstrated that MMP-9 mRNA, protein, and activity were increased upon infection with HRV, whereas TIMP-1 mRNA and protein remained unchanged. These results were verified in vivo, using nasal lavage samples obtained from subjects with confirmed rhinovirus infections. Human rhinovirus infections were shown to up-regulate NF-kappaB, and NF-kappaB has also been reported to play a role in the expression of MMP-9. We therefore investigated the role of NF-kappaB in HRV-induced MMP-9 expression. Using two inhibitors of IkappaBalpha kinase beta, we observed a concentration-dependent decrease in HRV-induced MMP-9 expression. The role of NF-kappaB in HRV-induced MMP-9 expression was further confirmed using MMP-9 promoter luciferase constructs, which demonstrated that an NF-kappaB site at -620/-607 base pairs was necessary for HRV-induced MMP-9 expression. Electrophoretic mobility shift assays and supershift assays confirmed the nuclear translocation and binding of p50/p65 NF-kappaB subunits to an MMP-9-specific NF-kappaB oligonucleotide. This increase in MMP-9 may be a mechanism by which rhinovirus infections contribute to airway inflammation and, potentially, to airway remodeling.

Selective transcriptional down-regulation of human rhinovirus-induced production of CXCL10 from airway epithelial cells via the MEK1 pathway.

Human rhinovirus (HRV) infections can trigger exacerbations of lower airway diseases. Infection of airway epithelial cells induces production of a number of proinflammatory chemokines that may exacerbate airway inflammation, including CXCL10, a chemoattractant for type 1 lymphocytes and NK cells. Primary human bronchial epithelial cells and the BEAS-2B human bronchial epithelial cell line were used to examine the role of MAPK pathways in HRV-16-induced production of CXCL10. Surprisingly, PD98059 and U0126, two inhibitors of the MEK1/2-ERK MAPK pathway, significantly enhanced HRV-16-induced CXCL10 mRNA and protein. This enhancement was not seen with IFN-beta-induced production of CXCL10. Studies using small interfering RNA revealed that knockdown of MEK1, but not MEK2, was associated with enhanced HRV-induced CXCL10 production. Promoter construct studies revealed that PD98059 and U0126 enhanced HRV-16-induced transcriptional activation of CXCL10. HRV-16-induced promoter activation was regulated by two NF-kappaB binding sites, kappaB1 and kappaB2, and by an IFN-stimulated response element. Inhibitors of the MEK1/2-ERK pathway did not alter HRV-16-induced activation of tandem repeat kappaB1 or kappaB2 constructs, nor did they alter HRV-16-induced nuclear translocation/binding of NF-kappaB to either kappaB1 or kappaB2 recognition sequences. Furthermore, PD98059 and U0126 did not alter phosphorylation or degradation of IkappaBalpha. In contrast, inhibitors of the MEK1/2-ERK pathway, and small interfering RNA knockdown of MEK1, enhanced nuclear translocation/binding of IFN regulatory factor (IRF)-1 to the IFN-stimulated response element recognition sequence in HRV-16 infected cells. We conclude that activation of MEK1 selectively down-regulates HRV-16-induced expression of CXCL10 via modulation of IRF-1 interactions with the gene promoter in human airway epithelial cells.

Nitric oxide inhibits human rhinovirus-induced transcriptional activation of CXCL10 in airway epithelial cells.

BACKGROUND: Human rhinovirus (HRV) infections trigger exacerbations of asthma and chronic obstructive pulmonary disease. Nitric oxide (NO) inhibits HRV replication in human airway epithelial cells and suppresses HRV-induced epithelial production of several cytokines and chemokines. OBJECTIVE: We sought to delineate the mechanisms by which NO inhibits HRV-induced epithelial production of CXCL10, a chemoattractant for type 1 T cells and natural killer cells. METHODS: Primary human bronchial epithelial cells or cells of the BEAS-2B human bronchial epithelial cell line were exposed to HRV-16 in the presence or absence of the NO donor 3-(2-hydroxy-2-nitroso-1-propylhydrazino)-1-propanamine (PAPA NONOate). A cGMP analogue and an inhibitor of soluble guanylyl cyclase were used to examine the role of the cyclic guanosine monophosphate (cGMP) pathway in the actions of NO. BEAS-2B cells were transfected with CXCL10 promoter-luciferase constructs and the effects of PAPA NONOate were examined to study mechanisms of transcriptional regulation. Electrophoretic mobility shift assays were also used. RESULTS: PAPA NONOate inhibited HRV-16-induced increases in CXCL10 mRNA and protein. Inhibition of CXCL10 production occurred through a cGMP-independent pathway. PAPA NONOate inhibited HRV-16-induced CXCL10 transcription by blocking nuclear translocation, binding, or both of both nuclear factor kappaB and IFN response factors (IRFs) to their respective recognition elements in the CXCL10 promoter. CONCLUSIONS: NO inhibits HRV-16-induced production of CXCL10 by inhibiting viral activation of nuclear factor kappaB and of IRFs, including IRF-1, through a cGMP-independent pathway. The broad-ranging inhibition of HRV-induced epithelial cytokine and chemokine production by NO suggests a potential therapeutic utility of NO donors in viral exacerbations of asthma and chronic obstructive pulmonary disease.

Gene expression profiles during in vivo human rhinovirus infection: insights into the host response.

RATIONALE: Human rhinovirus infections cause colds and trigger exacerbations of lower airway diseases. OBJECTIVES: To define changes in gene expression profiles during in vivo rhinovirus infections. METHODS: Nasal epithelial scrapings were obtained before and during experimental rhinovirus infection, and gene expression was evaluated by microarray. Naturally acquired rhinovirus infections, cultured human epithelial cells, and short interfering RNA knockdown were used to further evaluate the role of viperin in rhinovirus infections. MEASUREMENTS AND MAIN RESULTS: Symptom scores and viral titers were measured in subjects inoculated with rhinovirus or sham control, and changes in gene expression were assessed 8 and 48 hours after inoculation. Real-time reverse transcription-polymerase chain reaction for viperin and rhinoviruses was used in naturally acquired infections, and viperin mRNA levels and viral titers were measured in cultured cells. Rhinovirus-induced changes in gene expression were not observed 8 hours after viral infection, but 11,887 gene transcripts were significantly altered in scrapings obtained 2 days postinoculation. Major groups of up-regulated genes included chemokines, signaling molecules, interferon-responsive genes, and antivirals. Viperin expression was further examined and also was increased in naturally acquired rhinovirus infections, as well as in cultured human epithelial cells infected with intact, but not replication-deficient, rhinovirus. Knockdown of viperin with short interfering RNA increased rhinovirus replication in infected epithelial cells. CONCLUSIONS: Rhinovirus infection significantly alters the expression of many genes associated with the immune response, including chemokines and antivirals. The data obtained provide insights into the host response to rhinovirus infection and identify potential novel targets for further evaluation.

Human rhinovirus infection enhances airway epithelial cell production of growth factors involved in airway remodeling.

BACKGROUND: Childhood human rhinovirus (HRV) infections are associated with an increased risk of asthma. We reasoned that HRV infections might be important in the pathogenesis of airway remodeling, thereby providing a mechanism by which these children are at risk of asthma. OBJECTIVE: We sought to determine whether HRV infection of airway epithelial cells regulates production of growth factors associated with airway remodeling and to determine whether vascular endothelial growth factor (VEGF) was upregulated in airways during HRV-induced natural colds. METHODS: Cultured human airway epithelial cells were infected with HRV. Amphiregulin, activin A, and VEGF protein levels were assayed by means of ELISA, and VEGF mRNA was quantified by using real-time RT-PCR. Pharmacologic inhibitors were used to assess the role of mitogen-activated protein kinase and nuclear factor kappaB pathways. Nasal lavage samples from subjects with confirmed natural HRV infections were assayed for VEGF protein and compared with baseline levels and with control levels. RESULTS: HRV infection upregulated amphiregulin, activin A, and VEGF protein levels compared with control media (P < .05). VEGF gene expression was maximally induced 3 hours after infection. HRV-induced generation of VEGF was regulated by p38 mitogen-activated protein kinase and extracellular signal-regulated kinase 1/2 pathways but did not depend on nuclear factor kappaB activation. In subjects with HRV infections, VEGF levels during peak cold symptoms were significantly higher than at baseline (P = .005) or in control subjects (P < .01). CONCLUSION: HRV-16 infection upregulates amphiregulin, activin A, and VEGF in airway epithelial cells, and HRV infections in vivo upregulate airway VEGF production.

Syk associates with clathrin and mediates phosphatidylinositol 3-kinase activation during human rhinovirus internalization.

Human rhinovirus (HRV) causes the common cold. The most common acute infection in humans, HRV is a leading cause of exacerbations of asthma and chronic obstruction pulmonary disease because of its ability to exacerbate airway inflammation by altering epithelial cell biology upon binding to its receptor, ICAM-1. ICAM-1 regulates not only viral entry and replication but also signaling pathways that lead to inflammatory mediator production. We recently demonstrated the Syk tyrosine kinase to be an important mediator of HRV-ICAM-1 signaling: Syk regulates replication-independent p38 MAPK activation and IL-8 expression. In leukocytes, Syk regulates receptor-mediated internalization via PI3K. Although PI3K has been shown to regulate HRV-induced IL-8 expression and clathrin-mediated endocytosis of HRV, the role of airway epithelial Syk in this signaling pathway is not known. We postulated that Syk regulates PI3K activation and HRV endocytosis in the airway epithelium. Using confocal microscopy and immunoprecipitation, we demonstrated recruitment of the normally cytosolic Syk to the plasma membrane upon HRV16-ICAM-1 binding, along with Syk-clathrin coassociation. Subsequent incubation at 37 degrees C to permit internalization revealed redistribution of Syk to punctate structures resembling endosomes and colocalization with HRV16. Internalized HRV was not detected in cells overexpressing the kinase inactive Syk(K396R) mutant, indicating that kinase activity was necessary for endocytosis. HRV-induced PI3K activation was dependent on Syk; Syk knockdown by small interfering RNA significantly decreased phosphorylation of the PI3K substrate Akt. Together, these data reveal Syk to be an important mediator of HRV endocytosis and HRV-induced PI3K activation.

Interleukin-17A modulates human airway epithelial responses to human rhinovirus infection.

Human rhinovirus (HRV) infections are associated with exacerbations of asthma and chronic obstructive pulmonary disease that are characterized by a selective neutrophil infiltration. IL-17A, a cytokine derived primarily from activated T cells, has been linked to neutrophilic inflammation of the airways. We hypothesized that IL-17A alters the response of HRV-infected epithelial cells to modulate airway inflammatory cell populations. IL-17A synergistically enhanced HRV-16-induced epithelial production of the neutrophil chemoattractant, IL-8, as well as human beta-defensin-2 (HBD-2), a chemoattractant for immature dendritic cells and memory T cells, but suppressed viral production of the eosinophil chemoattractant, RANTES. These effects were not due to alterations of viral uptake or replication by IL-17A. The synergy between HRV-16 and IL-17A for IL-8 protein production was both dose- and time-dependent. IL-8 induction by IL-17A or HRV-16, alone and in combination, was reduced by inhibitors of the p38 and p44/42 MAPK pathways. By contrast, induction of HBD-2 depended on the activation of the p38 and JNK pathways. The ability of IL-17A to synergistically enhance HRV-induced IL-8 is mediated posttranscriptionally, since IL-8 promoter activation by the combination of the two stimuli was merely additive, whereas the combination of IL-17A and HRV-16 led to stabilization of IL-8 mRNA. Similarly, stimulation of HBD-2 promoter constructs by the combination of IL-17A and HRV-16 was no more than the sum of the individual responses. Further studies are needed to examine HBD-2 mRNA stability. Taken together, these data represent the first demonstration that IL-17A can modify epithelial responses to HRV in a manner that would be expected to favor the recruitment of neutrophils, immature dendritic cells, and memory T cells to the airways.

Syk is downstream of intercellular adhesion molecule-1 and mediates human rhinovirus activation of p38 MAPK in airway epithelial cells.

The airway epithelium is the primary target of inhaled pathogens such as human rhinovirus (HRV). Airway epithelial cells express ICAM-1, the major receptor for HRV. HRV binding to ICAM-1 mediates not only viral entry and replication but also a signaling cascade that leads to enhanced inflammatory mediator production. The specific signaling molecules and pathways activated by HRV-ICAM-1 interactions are not well characterized, although studies in human airway epithelia implicate a role for the p38 MAPK in HRV-induced cytokine production. In the current study, we report that Syk, an important immunoregulatory protein tyrosine kinase, is highly expressed by primary and cultured human airway epithelial cells and is activated in response to infection with HRV16. Biochemical studies revealed that ICAM-1 engagement by HRV and cross-linking Abs enhanced the coassociation of Syk with ICAM-1 and ezrin, a cytoskeletal linker protein. In polarized airway epithelial cells, Syk is diffusely distributed in the cytosol under basal conditions but, following engagement of ICAM-1 by cross-linking Abs, is recruited to the plasma membrane. The enhanced Syk-ICAM-1 association following HRV exposure is accompanied by Syk phosphorylation. ICAM-1 engagement by HRV and cross-linking Abs also induced phosphorylation of p38 in a Syk-dependent manner, and conversely, knockdown of Syk by short interfering (si)RNA substantially diminished p38 activation and IL-8 gene expression. Taken together, these observations identify Syk as an important mediator of the airway epithelial cell inflammatory response by modulating p38 phosphorylation and IL-8 gene expression following ICAM-1 engagement by HRV.

Chronic arthritis down-regulates peripheral mu-opioid receptor expression with concomitant loss of endomorphin 1 antinociception.

OBJECTIVE: To determine whether peripheral administration of the endogenous mu-opioid peptide endomorphin 1 could reduce knee joint pain, using animal models of acute and chronic arthritis. METHODS: Extracellular electrophysiologic recordings were made of rat knee joint primary afferent nerve activity in response to noxious hyperrotation of the joint. Neuronal activity was assessed before and following local injection of endomorphin 1. Comparisons were made between normal knees and knees with adjuvant-induced monarthritis, tested at 48 hours and 1 week posttreatment. Expression of mu-opioid receptors in the dorsal root ganglia ipsilateral to the chronically inflamed joints was determined by real-time polymerase chain reaction (PCR) and immunohistochemical analysis. RESULTS: In normal knees, endomorphin 1 caused up to a 75% reduction in joint afferent nerve activity, which was blocked by the mu-opioid receptor antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-amide. The inhibitory effect of endomorphin 1 was sustained in acutely inflamed knees. Conversely, in chronically inflamed joints, endomorphin 1 had no observable effect on the primary afferent nerve firing rate elicited by a noxious mechanical stimulus and, as such, was significantly different from the rate in normal joints. Immunohistochemical and real-time PCR analysis of the L3-L5 dorsal root ganglia ipsilateral to the chronic arthritis lesion revealed a reduction in mu-opioid receptor protein and gene expression compared with that in normal control animals. CONCLUSION: Taken together, these results provide the first electrophysiologic evidence that selective activation of peripheral mu-opioid receptors reduces normal knee joint mechanosensitivity to a noxious stimulus. Furthermore, the analgesic effect of endomorphin 1 is lost during chronic inflammation due to down-regulation of mu-opioid receptor expression in afferent nerve cell bodies. These findings begin to explain the ambiguous efficacy of peripherally administered mu-opioid drugs in controlling chronic inflammatory joint pain.

Human airway epithelial cells produce IP-10 (CXCL10) in vitro and in vivo upon rhinovirus infection.

Human rhinovirus (HRV) infections trigger exacerbations of asthma and chronic obstructive pulmonary disease (COPD) and are associated with lymphocytic infiltration of the airways. We demonstrate that infection of primary cultures of human airway epithelial cells, or of the BEAS-2B human bronchial epithelial cell line, with human rhinovirus type 16 (HRV-16) induces expression of CXCL10 [IFN-gamma-inducible protein 10 (IP-10)], a ligand for the CXCR3 receptor found on activated type 1 T lymphocytes and natural killer cells. IP-10 mRNA reached maximal levels 24 h after HRV-16 infection then declined, whereas protein levels peaked 48 h after infection with no subsequent new synthesis. Cytosolic levels of AU-rich factor 1, a protein associated with mRNA destabilization, increased beginning 24 h after HRV-16 infection. Generation of IP-10 required virus capable of replication but was not dependent on prior induction of type 1 interferons. Transfection of synthetic double-stranded RNA into epithelial cells induced robust production of IP-10, whereas transfection of single-stranded RNA had no effect. Induction of IP-10 gene expression by HRV-16 depended upon activation of NF-kappaB, as well as other transcription factor recognition sequences further upstream in the IP-10 promoter. In vivo infection of human volunteers with HRV-16 strikingly increased IP-10 protein in nasal lavages during symptomatic colds. Levels of IP-10 correlated with symptom severity, viral titer, and numbers of lymphocytes in airway secretions. Thus IP-10 may play a role in the pathogenesis of HRV-induced colds and in HRV-induced exacerbations of COPD and asthma.