Shyamala Ganesan

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.

Barrier function of airway tract epithelium

Airway epithelium contributes significantly to the barrier function of airway tract. Mucociliary escalator, intercellular apical junctional complexes which regulate paracellular permeability and antimicrobial peptides secreted by the airway epithelial cells are the three primary components of barrier function of airway tract. These three components act cooperatively to clear inhaled pathogens, allergens and particulate matter without inducing inflammation and maintain tissue homeostasis. Therefore impairment of one or more of these essential components of barrier function may increase susceptibility to infection and promote exaggerated and prolonged innate immune responses to environmental factors including allergens and pathogens resulting in chronic inflammation. Here we review the regulation of components of barrier function with respect to chronic airways diseases.

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.

Rhinovirus-Induced Barrier Dysfunction in Polarized Airway Epithelial Cells Is Mediated by NADPH Oxidase 1

ABSTRACT Previously, we showed that rhinovirus (RV), which is responsible for the majority of common colds, disrupts airway epithelial barrier function, as evidenced by reduced transepithelial resistance (RT), dissociation of zona occludins 1 (ZO-1) from the tight junction complex, and bacterial transmigration across polarized cells. We also showed that RV replication is required for barrier function disruption. However, the underlying biochemical mechanisms are not known. In the present study, we found that a double-stranded RNA (dsRNA) mimetic, poly(I:C), induced tight junction breakdown and facilitated bacterial transmigration across polarized airway epithelial cells, similar to the case with RV. We also found that RV and poly(I:C) each stimulated Rac1 activation, reactive oxygen species (ROS) generation, and Rac1-dependent NADPH oxidase 1 (NOX1) activity. Inhibitors of Rac1 (NSC23766), NOX (diphenylene iodonium), and NOX1 (small interfering RNA [siRNA]) each blocked the disruptive effects of RV and poly(I:C) on RT, as well as the dissociation of ZO-1 and occludin from the tight junction complex. Finally, we found that Toll-like receptor 3 (TLR3) is not required for either poly(I:C)- or RV-induced reductions in RT. Based on these results, we concluded that Rac1-dependent NOX1 activity is required for RV- or poly(I:C)-induced ROS generation, which in turn disrupts the barrier function of polarized airway epithelia. Furthermore, these data suggest that dsRNA generated during RV replication is sufficient to disrupt barrier function.

Quercetin prevents progression of disease in elastase/LPS-exposed mice by negatively regulating MMP expression

BackgroundChronic obstructive pulmonary disease (COPD) is characterized by chronic bronchitis, emphysema and irreversible airflow limitation. These changes are thought to be due to oxidative stress and an imbalance of proteases and antiproteases. Quercetin, a plant flavonoid, is a potent antioxidant and anti-inflammatory agent. We hypothesized that quercetin reduces lung inflammation and improves lung function in elastase/lipopolysaccharide (LPS)-exposed mice which show typical features of COPD, including airways inflammation, goblet cell metaplasia, and emphysema.MethodsMice treated with elastase and LPS once a week for 4 weeks were subsequently administered 0.5 mg of quercetin dihydrate or 50% propylene glycol (vehicle) by gavage for 10 days. Lungs were examined for elastance, oxidative stress, inflammation, and matrix metalloproteinase (MMP) activity. Effects of quercetin on MMP transcription and activity were examined in LPS-exposed murine macrophages.ResultsQuercetin-treated, elastase/LPS-exposed mice showed improved elastic recoil and decreased alveolar chord length compared to vehicle-treated controls. Quercetin-treated mice showed decreased levels of thiobarbituric acid reactive substances, a measure of lipid peroxidation caused by oxidative stress. Quercetin also reduced lung inflammation, goblet cell metaplasia, and mRNA expression of pro-inflammatory cytokines and muc5AC. Quercetin treatment decreased the expression and activity of MMP9 and MMP12 in vivo and in vitro, while increasing expression of the histone deacetylase Sirt-1 and suppressing MMP promoter H4 acetylation. Finally, co-treatment with the Sirt-1 inhibitor sirtinol blocked the effects of quercetin on the lung phenotype.ConclusionsQuercetin prevents progression of emphysema in elastase/LPS-treated mice by reducing oxidative stress, lung inflammation and expression of MMP9 and MMP12.

Pseudomonas aeruginosa Suppresses Interferon Response to Rhinovirus Infection in Cystic Fibrosis but Not in Normal Bronchial Epithelial Cells

ABSTRACT Despite increased morbidity associated with secondary respiratory viral infections in cystic fibrosis (CF) patients with chronic Pseudomonas aeruginosa infection, the underlying mechanisms are not well understood. Here, we investigated the effect of P. aeruginosa infection on the innate immune responses of bronchial epithelial cells to rhinovirus (RV) infection. CF cells sequentially infected with mucoid P. aeruginosa (MPA) and RV showed lower levels of interferons (IFNs) and higher viral loads than those of RV-infected cells. Unlike results for CF cells, normal bronchial epithelial cells coinfected with MPA/RV showed higher IFN expression than RV-infected cells. In both CF and normal cells, the RV-stimulated IFN response requires phosphorylation of Akt and interferon response factor 3 (IRF3). Preinfection with MPA inhibited RV-stimulated Akt phosphorylation and decreased IRF3 phosphorylation in CF cells but not in normal cells. Compared to normal, unstimulated CF cells or normal cells treated with CFTR inhibitor showed increased reactive oxygen species (ROS) production. Treatment of CF cells with antioxidants prior to MPA infection partially reversed the suppressive effect of MPA on the RV-stimulated IFN response. Together, these results suggest that MPA preinfection inhibits viral clearance by suppressing the antiviral response particularly in CF cells but not in normal cells. Further, increased oxidative stress in CF cells appears to modulate the innate immune responses to coinfection.

Aberrantly activated EGFR contributes to enhanced IL-8 expression in COPD airways epithelial cells via regulation of nuclear FoxO3A

Background Decreased activity of forkhead transcription factor class O (FoxO)3A, a negative regulator of NF-κB-mediated chemokine expression, is implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). Previously, we showed that quercetin reduces lung inflammation in a murine model of COPD. Here, we examined the mechanisms underlying decreased FoxO3A activation and its modulation by quercetin in COPD human airway epithelial cells and in a COPD mouse model. Methods Primary COPD and normal human airway epithelial cells were treated with quercetin, LY294002 or erlotinib for 2 weeks. IL-8 was measured by ELISA. FoxO3A, Akt, and epidermal growth factor (EGF) receptor (EGFR) phosphorylation and nuclear FoxO3A levels were determined by Western blot analysis. Effects of quercetin on lung chemokine expression, nuclear FoxO3A levels and phosphorylation of EGFR and Akt were determined in COPD mouse model. Results Compared with normal, COPD cells showed significantly increased IL-8, which negatively correlated with nuclear FoxO3A levels. COPD bronchial biopsies also showed reduced nuclear FoxO3A. Decreased FoxO3A in COPD cells was associated with increased phosphorylation of EGFR, Akt and FoxO3A and treatment with quercetin, LY294002 or erlotinib increased nuclear FoxO3A and decreased IL-8 and phosphorylation of Akt, EGFR and FoxO3A, Compared with control, elastase/LPS-exposed mice showed decreased nuclear FoxO3A, increased chemokines and phosphorylation of EGFR and Akt. Treatment with quercetin partially reversed these changes. Conclusions In COPD airways, aberrant EGFR activity increases PI 3-kinase/Akt-mediated phosphorylation of FoxO3A, thereby decreasing nuclear FoxO3A and increasing chemokine expression. Quercetin restores nuclear FoxO3A and reduces chemokine expression partly by modulating EGFR/PI 3-kinase/Akt activity.

Rhinovirus Attenuates Non-typeable Hemophilus influenzae-stimulated IL-8 Responses via TLR2-dependent Degradation of IRAK-1

Bacterial infections following rhinovirus (RV), a common cold virus, are well documented, but pathogenic mechanisms are poorly understood. We developed animal and cell culture models to examine the effects of RV on subsequent infection with non-typeable Hemophilus influenzae (NTHi). We focused on NTHI-induced neutrophil chemoattractants expression that is essential for bacterial clearance. Mice infected with RV1B were superinfected with NTHi and lung bacterial density, chemokines and neutrophil counts determined. Human bronchial epithelial cells (BEAS-2B) or mouse alveolar macrophages (MH-S) were infected with RV and challenged with NHTi, TLR2 or TLR5 agonists. Chemokine levels were measured by ELISA and expression of IRAK-1, a component of MyD88-dependent TLR signaling, assessed by immunoblotting. While sham-infected mice cleared all NTHi from the lungs, RV-infected mice showed bacteria up to 72 h post-infection. However, animals in RV/NTHi cleared bacteria by day 7. Delayed bacterial clearance in RV/NTHi animals was associated with suppressed chemokine levels and neutrophil recruitment. RV-infected BEAS-2B and MH-S cells showed attenuated chemokine production after challenge with either NTHi or TLR agonists. Attenuated chemokine responses were associated with IRAK-1 protein degradation. Inhibition of RV-induced IRAK-1 degradation restored NTHi-stimulated IL-8 expression. Knockdown of TLR2, but not other MyD88-dependent TLRs, also restored IRAK-1, suggesting that TLR2 is required for RV-induced IRAK-1 degradation. In conclusion, we demonstrate for the first time that RV infection delays bacterial clearance in vivo and suppresses NTHi-stimulated chemokine responses via degradation of IRAK-1. Based on these observations, we speculate that modulation of TLR-dependent innate immune responses by RV may predispose the host to secondary bacterial infection, particularly in patients with underlying chronic respiratory disorders.

Quercetin inhibits rhinovirus replication in vitro and in vivo

Abstract Rhinovirus (RV), which is responsible for the majority of common colds, also causes exacerbations in patients with asthma and chronic obstructive pulmonary disease. So far, there are no drugs available for treatment of rhinovirus infection. We examined the effect of quercetin, a plant flavanol on RV infection in vitro and in vivo. Pretreatment of airway epithelial cells with quercetin decreased Akt phosphosphorylation, viral endocytosis and IL-8 responses. Addition of quercetin 6h after RV infection (after viral endocytosis) reduced viral load, IL-8 and IFN responses in airway epithelial cells. This was associated with decreased levels of negative and positive strand viral RNA, and RV capsid protein, abrogation of RV-induced eIF4GI cleavage and increased phosphorylation of eIF2α. In mice infected with RV, quercetin treatment decreased viral replication as well as expression of chemokines and cytokines. Quercetin treatment also attenuated RV-induced airway cholinergic hyperresponsiveness. Together, our results suggest that quercetin inhibits RV endocytosis and replication in airway epithelial cells at multiple stages of the RV life cycle. Quercetin also decreases expression of pro-inflammatory cytokines and improves lung function in RV-infected mice. Based on these observations, further studies examining the potential benefits of quercetin in the prevention and treatment of RV infection are warranted.