Umadevi S. Sajjan

Role of Double-Stranded RNA Pattern Recognition Receptors in Rhinovirus-Induced Airway Epithelial Cell Responses

Rhinovirus (RV), a ssRNA virus of the picornavirus family, is a major cause of the common cold as well as asthma and chronic obstructive pulmonary disease exacerbations. Viral dsRNA produced during replication may be recognized by the host pattern recognition receptors TLR-3, retinoic acid-inducible gene (RIG)-I, and melanoma differentiation-associated gene (MDA)-5. No study has yet identified the receptor required for sensing RV dsRNA. To examine this, BEAS-2B human bronchial epithelial cells were infected with intact RV-1B or replication-deficient UV-irradiated virus, and IFN and IFN-stimulated gene expression was determined by quantitative PCR. The separate requirements of RIG-I, MDA5, and IFN response factor (IRF)-3 were determined using their respective small interfering RNAs (siRNA). The requirement of TLR3 was determined using siRNA against the TLR3 adaptor molecule Toll/IL-1R homologous region-domain-containing adapter-inducing IFN-β (TRIF). Intact RV-1B, but not UV-irradiated RV, induced IRF3 phosphorylation and dimerization, as well as mRNA expression of IFN-β, IFN-λ1, IFN-λ2/3, IRF7, RIG-I, MDA5, 10-kDa IFN-γ-inducible protein/CXCL10, IL-8/CXCL8, and GM-CSF. siRNA against IRF3, MDA5, and TRIF, but not RIG-I, decreased RV-1B-induced expression of IFN-β, IFN-λ1, IFN-λ2/3, IRF7, RIG-I, MDA5, and inflammatory protein-10/CXCL10 but had no effect on IL-8/CXCL8 and GM-CSF. siRNAs against MDA5 and TRIF also reduced IRF3 dimerization. Finally, in primary cells, transfection with MDA5 siRNA significantly reduced IFN expression, as it did in BEAS-2B cells. These results suggest that TLR3 and MDA5, but not RIG-I, are required for maximal sensing of RV dsRNA and that TLR3 and MDA5 signal through a common downstream signaling intermediate, IRF3.

Rhinovirus Disrupts the Barrier Function of Polarized Airway Epithelial Cells

RATIONALE Secondary bacterial infection following rhinovirus (RV) infection has been recognized in chronic obstructive pulmonary disease. OBJECTIVES We sought to understand mechanisms by which RV infection facilitates secondary bacterial infection. METHODS Primary human airway epithelial cells grown at air-liquid interface and human bronchial epithelial (16HBE14o-) cells grown as polarized monolayers were infected apically with RV. Transmigration of bacteria (nontypeable Haemophilus influenzae and others) was assessed by colony counting and transmission electron microscopy. Transepithelial resistance (R(T)) was measured by using a voltmeter. The distribution of zona occludins (ZO)-1 was determined by immunohistochemistry and immunoblotting. MEASUREMENTS AND MAIN RESULTS Epithelial cells infected with RV showed 2-log more bound bacteria than sham-infected cultures, and bacteria were recovered from the basolateral media of RV- but not sham-infected cells. Infection of polarized airway epithelial cell cultures with RV for 24 hours caused a significant decrease in R(T) without causing cell death or apoptosis. Ultraviolet-treated RV did not decrease R(T), suggesting a requirement for viral replication. Reduced R(T) was associated with increased paracellular permeability, as determined by flux of fluorescein isothiocyanate (FITC)-inulin. Neutralizing antibodies to tumor necrosis factor (TNF)-alpha, IFN-gamma and IL-1beta reversed corresponding cytokine-induced reductions in R(T) but not that induced by RV, indicating that the RV effect is independent of these proinflammatory cytokines. Confocal microscopy and immunoblotting revealed the loss of ZO-1 from tight junction complexes in RV-infected cells. Intranasal inoculation of mice with RV1B also caused the loss of ZO-1 from the bronchial epithelium tight junctions in vivo. CONCLUSIONS RV facilitates binding, translocation, and persistence of bacteria by disrupting airway epithelial barrier function.

Increased Cytokine Response of Rhinovirus-infected Airway Epithelial Cells in Chronic Obstructive Pulmonary Disease

RATIONALE Airway inflammation is a central feature of chronic obstructive pulmonary disease (COPD). COPD exacerbations are often triggered by rhinovirus (RV) infection. OBJECTIVES We hypothesized that airway epithelial cells from patients with COPD maintain a proinflammatory phenotype compared with control subjects, leading to greater RV responses. METHODS Cells were isolated from tracheobronchial tissues of 12 patients with COPD and 10 transplant donors. Eight patients with COPD had severe emphysema, three had mild to moderate emphysema, and one had no emphysema. All had moderate to severe airflow obstruction, and six met criteria for chronic bronchitis or had at least one exacerbation the previous year. Cells were grown at air-liquid interface and infected with RV serotype 39. Cytokine and IFN expression was measured by ELISA. Selected genes involved in inflammation, oxidative stress, and proteolysis were assessed by focused gene array and real-time polymerase chain reaction. MEASUREMENTS AND MAIN RESULTS Compared with control subjects, cells from patients with COPD demonstrated increased mRNA expression of genes involved in oxidative stress and the response to viral infection, including NOX1, DUOXA2, MMP12, ICAM1, DDX58/RIG-I, STAT1, and STAT2. COPD cells showed elevated baseline and RV-stimulated protein levels of IL-6, IL-8/CXCL8, and growth-related oncogene-alpha/CXCL1. COPD cells demonstrated increased viral titer and copy number after RV infection, despite increased IL-29/IFN-lambda1, IL-28A/IFN-lambda2, and IFN-inducible protein-10/CXCL10 protein levels. Finally, RV-infected COPD cultures showed increased mRNA expression of IL28A/IFNlambda2, IL29/IFNlambda1, IFIH1/MDA5, DDX58/RIG-I, DUOX1, DUOX2, IRF7, STAT1, and STAT2. CONCLUSIONS Airway epithelial cells from patients with COPD show higher baseline levels of cytokine expression and increased susceptibility to RV infection, despite an increased IFN response.

H. influenzae potentiates airway epithelial cell responses to rhinovirus by increasing ICAM-1 and TLR3 expression

Rhinovirus (RV) is an important trigger of chronic obstructive pulmonary disease (COPD) exacerbations. In addition, respiratory viruses are more likely to be isolated in patients with a history of frequent exacerbations, suggesting that these patients are more susceptible to viral infection. To examine potential mechanisms for cooperative effects between bacterial and viral infection in COPD, we studied the responses of cultured human airway epithelial cells to nontypeable Hemophilus influenzae and RV. In both 16HBE14o‐ and primary mucociliary‐differentiated cells, preincubation with H. influenzae enhanced RV serotype 39‐induced protein expression of interleukin (IL)‐8, epithelial‐derived neutrophil attractant‐78, and growth‐related oncogene‐α. H. influenzae infection also increased the binding of RV39 to cultured cells, as well as expression of intercellular adhesion molecule (ICAM)‐1 and Toll‐like receptor (TLR)‐3, receptors for RV and dsRNA, respectively. Neutralizing antibody against tumor necrosis factor‐ inhibited IL‐8 expression induced by H. influenzae and RV39. Finally, siRNA against TLR3 attenuated RV‐induced IL‐8 expression. We conclude that H. influenzae infection increases airway epithelial cell ICAM‐1 and TLR3 expression, leading to enhanced binding of RV and a potentiation of RV‐induced chemokine release. These data provide a cellular mechanism by which H. influenzae infection may increase the susceptibility of COPD patients to RV‐induced exacerbations.—Sajjan, U. S., Jia, Y., Newcomb, D. C., Bentley, J. K., Lukacs, N. W., LiPuma, J. J., Hershenson, M. B. H. influenzae potentiates airway epithelial cell responses to rhinovirus by increasing ICAM‐1 and TLR3 expression FASEB J. 20, E1419 –E1429 (2006)

Human Rhinovirus 1B Exposure Induces Phosphatidylinositol 3-Kinase-dependent Airway Inflammation in Mice

RATIONALE Infection with rhinovirus (RV) triggers exacerbations of asthma and chronic obstructive lung disease. OBJECTIVES We sought to develop a mouse model of RV employing RV1B, a minor group serotype that binds to the low-density lipoprotein receptor. METHODS C57BL/6 mice were inoculated intranasally with RV1B, replication-deficient ultraviolet (UV)-irradiated RV1B, or RV39, a major group virus. MEASUREMENTS AND MAIN RESULTS Viral RNA was present in the lungs of RV1B-treated mice, but not in those exposed to UV-irradiated RV1B or RV39. Lung homogenates of RV-treated mice contained infectious RV 4 days after inoculation. RV1B exposure induced neutrophilic and lymphocytic airway inflammation, as well as increased lung expression of KC, macrophage-inflammatory protein-2, and IFN-alpha and IFN-beta. RV1B-exposed mice showed airway hyperresponsiveness 1 and 4 days after inoculation. UV-irradiated RV1B induced modest neutrophilic airway inflammation and hyperresponsiveness 1 day after exposure. Both RV1B and UV-irradiated RV1B, but not RV39, increased lung phosphorylation of Akt. Confocal immunofluorescence showed colocalization of RV1B and phospho-Akt in the airway epithelium. Finally, pretreatment with the phosphatidylinositol 3-kinase inhibitor LY294002 attenuated chemokine production and neutrophil infiltration. CONCLUSIONS We conclude that RV1B induces airway inflammation in vivo. Evidence is presented that viral replication occurs in vivo and is required for maximal responses. On the other hand, viral replication was not required for a subset of RV-induced responses, including neutrophilic inflammation, airway hyperresponsiveness, and Akt phosphorylation. Finally, phosphatidylinositol 3-kinase/Akt signaling is required for maximal RV1B-induced airway neutrophilic inflammation, likely via its essential role in virus internalization.

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.

Rhinovirus Infection of Allergen-Sensitized and -Challenged Mice Induces Eotaxin Release from Functionally Polarized Macrophages

Human rhinovirus is responsible for the majority of virus-induced asthma exacerbations. To determine the immunologic mechanisms underlying rhinovirus (RV)-induced asthma exacerbations, we combined mouse models of allergic airways disease and human rhinovirus infection. We inoculated OVA-sensitized and challenged BALB/c mice with rhinovirus serotype 1B, a minor group strain capable of infecting mouse cells. Compared with sham-infected, OVA-treated mice, virus-infected mice showed increased lung infiltration with neutrophils, eosinophils and macrophages, airway cholinergic hyperresponsiveness, and increased lung expression of cytokines including eotaxin-1/CCL11, IL-4, IL-13, and IFN-γ. Administration of anti–eotaxin-1 attenuated rhinovirus-induced airway eosinophilia and responsiveness. Immunohistochemical analysis showed eotaxin-1 in the lung macrophages of virus-infected, OVA-treated mice, and confocal fluorescence microscopy revealed colocalization of rhinovirus, eotaxin-1, and IL-4 in CD68-positive cells. RV inoculation of lung macrophages from OVA-treated, but not PBS-treated, mice induced expression of eotaxin-1, IL-4, and IL-13 ex vivo. Macrophages from OVA-treated mice showed increased expression of arginase-1, Ym-1, Mgl-2, and IL-10, indicating a shift in macrophage activation status. Depletion of macrophages from OVA-sensitized and -challenged mice reduced eosinophilic inflammation and airways responsiveness following RV infection. We conclude that augmented airway eosinophilic inflammation and hyperresponsiveness in RV-infected mice with allergic airways disease is directed in part by eotaxin-1. Airway macrophages from mice with allergic airways disease demonstrate a change in activation state characterized in part by altered eotaxin and IL-4 production in response to RV infection. These data provide a new paradigm to explain RV-induced asthma exacerbations.

P-113d, an Antimicrobial Peptide Active against Pseudomonas aeruginosa, Retains Activity in the Presence of Sputum from Cystic Fibrosis Patients

ABSTRACT Antimicrobial peptides are a source of novel agents that could be useful for treatment of the chronic lung infections that afflict cystic fibrosis (CF) patients. Efficacy depends on antimicrobial activity against the major pathogens of CF patients, Pseudomonas aeruginosa, Staphylococcus aureus, and Haemophilus influenzae, in the environment of the CF patients airway. We describe the in vitro efficacies of derivatives of histatins, which are histidine-rich peptides produced by the salivary glands of humans and higher primates. P-113, a peptide containing 12 of the 24 amino acid residues of the parent molecule, histatin 5, retained full antibacterial activity and had a good spectrum of activity in vitro against the prominent pathogens of CF patients. However, P-113 was not active in the presence of purulent sputum from CF patients. In contrast, P-113d, the mirror-image peptide with the amino acid residues in the d configuration, was stable in sputum, was as active as P-113 against pathogens of CF patients in the absence of sputum and retained significant activity in the presence of sputum from CF patients. Recombinant human DNase, which effectively liquefies sputum, enhanced the activity of P-113d in undiluted sputum against both exogenous (added) bacteria and endogenous bacteria. Because of its properties, P-113d shows potential as an inhalant in chronic suppressive therapy for CF patients.

Elastase- and LPS-exposed mice display altered responses to rhinovirus infection

Viral infection is associated with approximately one-half of acute exacerbations of chronic obstructive pulmonary disease (COPD), which in turn, accelerate disease progression. In this study, we infected mice exposed to a combination of elastase and LPS, a constituent of cigarette smoke and a risk factor for development of COPD, with rhinovirus serotype 1B, and examined animals for viral persistence, airway resistance, lung volume, and cytokine responses. Mice exposed to elastase and LPS once a week for 4 wk showed features of COPD such as airway inflammation and obstruction, goblet cell metaplasia, reduced lung elastance, increased total lung volume, and increased alveolar chord length. In general, mice exposed to elastase or LPS alone showed intermediate effects. Compared with rhinovirus (RV)-infected PBS-exposed mice, RV-infected elastase/LPS-exposed mice showed persistence of viral RNA, airway hyperresponsiveness, increased lung volume, and sustained increases in expression of TNFalpha, IL-5, IL-13, and muc5AC (up to 14 days postinfection). Furthermore, virus-induced IFNs, interferon response factor-7, and IL-10 were deficient in elastase/LPS-treated mice. Mice exposed to LPS or elastase alone cleared virus similar to PBS-treated control mice. We conclude that limited exposure of mice to elastase/LPS produces a COPD-like condition including increased persistence of RV, likely due to skewing of the immune response towards a Th2 phenotype. Similar mechanisms may be operative in COPD.

Rhinovirus infection liberates planktonic bacteria from biofilm and increases chemokine responses in cystic fibrosis airway epithelial cells

Background Intermittent viral exacerbations in patients with cystic fibrosis (CF) with chronic Pseudomonas aeruginosa (PA) infection are associated with increased bacterial load. A few clinical studies suggest that rhinoviruses (RV) are associated with the majority of viral-related exacerbations in CF and require prolonged intravenous antibiotic treatment. These observations imply that acute RV infection may increase lower respiratory symptoms by increasing planktonic bacterial load. However, the underlying mechanisms are not known. Methods Primary CF airway epithelial cells differentiated into mucociliary phenotype were infected with mucoid PA (MPA) followed by RV and examined for bacterial density, biofilm mass, levels of chemokines and hydrogen peroxide (H2O2). The need for dual oxidase 2, a component of NADPH oxidase, in RV-induced generation of H2O2 in CF cells was assessed using gene-specific siRNA. Results Superinfection with RV increased chemokine responses in CF mucociliary-differentiated airway epithelial cells with pre-existing MPA infection in the form of biofilm. This was associated with the presence of planktonic bacteria at both the apical and basolateral epithelial cell surfaces. Further, RV-induced generation of H2O2 via dual oxidase 2 in CF cells was sufficient for dispersal of planktonic bacteria from the biofilm. Inhibition of NADPH oxidase reduced bacterial transmigration across mucociliary-differentiated CF cells and the interleukin-8 response in MPA- and RV-infected cells. Conclusion This study shows that acute infection with RV liberates planktonic bacteria from biofilm. Planktonic bacteria, which are more proinflammatory than their biofilm counterparts, stimulate increased chemokine responses in CF airway epithelial cells which, in turn, may contribute to the pathogenesis of CF exacerbations.