NOTCH3 contributes to rhinovirus-induced goblet cell hyperplasia in COPD airway epithelial cells.

RATIONALE: Goblet cell hyperplasia (GCH) is one of the cardinal features of chronic obstructive pulmonary disease (COPD) and contributes to airways obstruction. Rhinovirus (RV), which causes acute exacerbations in patients with COPD, also causes prolonged airways obstruction. Previously, we showed that RV enhances mucin gene expression and increases goblet cell number in a COPD mouse model. This study examines whether RV causes sustained GCH in relevant models of COPD. METHODS: Mucociliary-differentiated COPD and normal airway epithelial cell cultures and mice with normal or COPD phenotype were infected with RV or sham and examined for GCH by immunofluorescence and/or mucin gene expression. In some experiments, RV-infected COPD cells and mice with COPD phenotype were treated with γ-secretase inhibitor or interleukin-13 neutralising antibody and assessed for GCH. To determine the contribution of NOTCH1/3 in RV-induced GCH, COPD cells transduced with NOTCH1/3 shRNA were used. RESULTS: RV-infected COPD, but not normal cell cultures, showed sustained GCH and increased mucin genes expression. Microarray analysis indicated increased expression of NOTCH1, NOTCH3 and HEY1 only in RV-infected COPD cells. Blocking NOTCH3, but not NOTCH1, attenuated RV-induced GCH in vitro. Inhibition of NOTCH signalling by γ-secretase inhibitor, but not neutralising antibody to IL-13, abrogated RV-induced GCH and mucin gene expression. CONCLUSIONS: RV induces sustained GCH via NOTCH3 particularly in COPD cells or mice with COPD phenotype. This may be one of the mechanisms that may contribute to RV-induced prolonged airways obstruction in COPD.

Impact of community respiratory viral infections in urban children with asthma.

BACKGROUND: Upper respiratory tract viral infections cause asthma exacerbations in children. However, the impact of natural colds on children with asthma in the community, particularly in the high-risk urban environment, is less well defined. OBJECTIVE: We hypothesized that children with high-symptom upper respiratory viral infections have reduced airway function and greater respiratory tract inflammation than children with virus-positive low-symptom illnesses or virus-negative upper respiratory tract symptoms. METHODS: We studied 53 children with asthma from Detroit, Michigan, during scheduled surveillance periods and self-reported respiratory illnesses for 1 year. Symptom score, spirometry, fraction of exhaled nitric oxide (FeNO), and nasal aspirate biomarkers, and viral nucleic acid and rhinovirus (RV) copy number were assessed. RESULTS: Of 658 aspirates collected, 22.9% of surveillance samples and 33.7% of respiratory illnesses were virus-positive. Compared with the virus-negative asymptomatic condition, children with severe colds (symptom score ≥5) showed reduced forced expiratory flow at 25% to 75% of the pulmonary volume (FEF25%-75%), higher nasal messenger RNA expression of C-X-C motif chemokine ligand (CXCL)-10 and melanoma differentiation-associated protein 5, and higher protein abundance of CXCL8, CXCL10 and C-C motif chemokine ligands (CCL)-2, CCL4, CCL20, and CCL24. Children with mild (symptom score, 1-4) and asymptomatic infections showed normal airway function and fewer biomarker elevations. Virus-negative cold-like illnesses demonstrated increased FeNO, minimal biomarker elevation, and normal airflow. The RV copy number was associated with nasal chemokine levels but not symptom score. CONCLUSION: Urban children with asthma with high-symptom respiratory viral infections have reduced FEF25%-75% and more elevations of nasal biomarkers than children with mild or symptomatic infections, or virus-negative illnesses.

Influence of viral infection on the relationships between airway cytokines and lung function in asthmatic children.

BACKGROUND: Few longitudinal studies examine inflammation and lung function in asthma. We sought to determine the cytokines that reduce airflow, and the influence of respiratory viral infections on these relationships. METHODS: Children underwent home collections of nasal lavage during scheduled surveillance periods and self-reported respiratory illnesses. We studied 53 children for one year, analyzing 392 surveillance samples and 203 samples from 85 respiratory illnesses. Generalized estimated equations were used to evaluate associations between nasal lavage biomarkers (7 mRNAs, 10 proteins), lung function and viral infection. RESULTS: As anticipated, viral infection was associated with increased cytokines and reduced FVC and FEV1. However, we found frequent and strong interactions between biomarkers and virus on lung function. For example, in the absence of viral infection, CXCL10 mRNA, MDA5 mRNA, CXCL10, IL-4, IL-13, CCL4, CCL5, CCL20 and CCL24 were negatively associated with FVC. In contrast, during infection, the opposite relationship was frequently found, with IL-4, IL-13, CCL5, CCL20 and CCL24 levels associated with less severe reductions in both FVC and FEV1. CONCLUSIONS: In asthmatic children, airflow obstruction is driven by specific pro-inflammatory cytokines. In the absence of viral infection, higher cytokine levels are associated with decreasing lung function. However, with infection, there is a reversal in this relationship, with cytokine abundance associated with reduced lung function decline. While nasal samples may not reflect lower airway responses, these data suggest that some aspects of the inflammatory response may be protective against viral infection. This study may have ramifications for the treatment of viral-induced asthma exacerbations.

TLR2 Activation Limits Rhinovirus-Stimulated CXCL-10 by Attenuating IRAK-1-Dependent IL-33 Receptor Signaling in Human Bronchial Epithelial Cells.

Airway epithelial cells are the major target for rhinovirus (RV) infection and express proinflammatory chemokines and antiviral cytokines that play a role in innate immunity. Previously, we demonstrated that RV interaction with TLR2 causes ILR-associated kinase-1 (IRAK-1) depletion in both airway epithelial cells and macrophages. Further, IRAK-1 degradation caused by TLR2 activation was shown to inhibit ssRNA-induced IFN expression in dendritic cells. Therefore, in this study, we examined the role of TLR2 and IRAK-1 in RV-induced IFN-ß, IFN-λ1, and CXCL-10, which require signaling by viral RNA. In airway epithelial cells, blocking TLR2 enhanced RV-induced expression of IFNs and CXCL-10. By contrast, IRAK-1 inhibition abrogated RV-induced expression of CXCL-10, but not IFNs in these cells. Neutralization of IL-33 or its receptor, ST2, which requires IRAK-1 for signaling, inhibited RV-stimulated CXCL-10 expression. In addition, RV induced expression of both ST2 and IL-33 in airway epithelial cells. In macrophages, however, RV-stimulated CXCL-10 expression was primarily dependent on TLR2/IL-1R. Interestingly, in a mouse model of RV infection, blocking ST2 not only attenuated RV-induced CXCL-10, but also lung inflammation. Finally, influenza- and respiratory syncytial virus-induced CXCL-10 was also found to be partially dependent on IL-33/ST2/IRAK-1 signaling in airway epithelial cells. Together, our results indicate that RV stimulates CXCL-10 expression via the IL-33/ST2 signaling axis, and that TLR2 signaling limits RV-induced CXCL-10 via IRAK-1 depletion at least in airway epithelial cells. To our knowledge, this is the first report to demonstrate the role of respiratory virus-induced IL-33 in the induction of CXCL-10 in airway epithelial cells.

Rhinovirus Delays Cell Repolarization in a Model of Injured/Regenerating Human Airway Epithelium.

Rhinovirus (RV), which causes exacerbation in patients with chronic airway diseases, readily infects injured airway epithelium and has been reported to delay wound closure. In this study, we examined the effects of RV on cell repolarization and differentiation in a model of injured/regenerating airway epithelium (polarized, undifferentiated cells). RV causes only a transient barrier disruption in a model of normal (mucociliary-differentiated) airway epithelium. However, in the injury/regeneration model, RV prolongs barrier dysfunction and alters the differentiation of cells. The prolonged barrier dysfunction caused by RV was not a result of excessive cell death but was instead associated with epithelial-to-mesenchymal transition (EMT)-like features, such as reduced expression of the apicolateral junction and polarity complex proteins, E-cadherin, occludin, ZO-1, claudins 1 and 4, and Crumbs3 and increased expression of vimentin, a mesenchymal cell marker. The expression of Snail, a transcriptional repressor of tight and adherence junctions, was also up-regulated in RV-infected injured/regenerating airway epithelium, and inhibition of Snail reversed RV-induced EMT-like features. In addition, compared with sham-infected cells, the RV-infected injured/regenerating airway epithelium showed more goblet cells and fewer ciliated cells. Inhibition of epithelial growth factor receptor promoted repolarization of cells by inhibiting Snail and enhancing expression of E-cadherin, occludin, and Crumbs3 proteins, reduced the number of goblet cells, and increased the number of ciliated cells. Together, these results suggest that RV not only disrupts barrier function, but also interferes with normal renewal of injured/regenerating airway epithelium by inducing EMT-like features and subsequent goblet cell hyperplasia.

Nod-like receptor X-1 is required for rhinovirus-induced barrier dysfunction in airway epithelial cells.

UNLABELLED: Barrier dysfunction of airway epithelium may increase the risk for acquiring secondary infections or allergen sensitization. Both rhinovirus (RV) and polyinosinic-polycytidilic acid [poly(I·C)], a double-stranded RNA (dsRNA) mimetic, cause airway epithelial barrier dysfunction, which is reactive oxygen species (ROS) dependent, implying that dsRNA generated during RV replication is sufficient for disrupting barrier function. We also demonstrated that RV or poly(I·C)-stimulated NADPH oxidase 1 (NOX-1) partially accounts for RV-induced ROS generation. In this study, we identified a dsRNA receptor(s) contributing to RV-induced maximal ROS generation and thus barrier disruption. We demonstrate that genetic silencing of the newly discovered dsRNA receptor Nod-like receptor X-1 (NLRX-1), but not other previously described dsRNA receptors, abrogated RV-induced ROS generation and reduction of transepithelial resistance (R(T)) in polarized airway epithelial cells. In addition, both RV and poly(I·C) stimulated mitochondrial ROS, the generation of which was dependent on NLRX-1. Treatment with Mito-Tempo, an antioxidant targeted to mitochondria, abolished RV-induced mitochondrial ROS generation, reduction in R(T), and bacterial transmigration. Furthermore, RV infection increased NLRX-1 localization to the mitochondria. Additionally, NLRX-1 interacts with RV RNA and poly(I·C) in polarized airway epithelial cells. Finally, we show that NLRX-1 is also required for RV-stimulated NOX-1 expression. These findings suggest a novel mechanism by which RV stimulates generation of ROS, which is required for disruption of airway epithelial barrier function. IMPORTANCE: Rhinovirus (RV), a virus responsible for a majority of common colds, disrupts the barrier function of the airway epithelium by increasing reactive oxygen species (ROS). Poly(I·C), a double-stranded RNA (dsRNA) mimetic, also causes ROS-dependent barrier disruption, implying that the dsRNA intermediate generated during RV replication is sufficient for this process. Here, we demonstrate that both RV RNA and poly(I·C) interact with NLRX-1 (a newly discovered dsRNA receptor) and stimulate mitochondrial ROS. We show for the first time that NLRX-1 is primarily expressed in the cytoplasm and at the apical surface rather than in the mitochondria and that NLRX-1 translocates to mitochondria following RV infection. Together, our results suggest a novel mechanism for RV-induced barrier disruption involving NLRX-1 and mitochondrial ROS. Although ROS is necessary for optimal viral clearance, if not neutralized efficiently, it may increase susceptibility to secondary infections and alter innate immune responses to subsequently inhaled pathogens, allergens, and other environmental factors.

Enhancement of Inducible Nitric Oxide Synthase Activity by Low Molecular Weight Peptides Derived from Protamine: A Potential Therapy for Chronic Rhinosinusitis.

Nitric oxide (NO) is a key immune defense agent that is produced from l-arginine in the airways by leukocytes and airway epithelial cells, primarily via inducible nitric oxide synthase (iNOS). Deficiencies in nasal NO levels have been associated with diseases such as primary ciliary dyskinesia and chronic rhinosinusitis. Herein, we demonstrate a proof-of-concept regarding a potential new therapeutic approach for such disorders. We show that arginine-rich low molecular weight peptides (LMWPs) derived from the FDA-approved protamine (obtained from salmon sperm) are effective at significantly raising NO production in both RAW 264.7 mouse macrophage and LA4 mouse epithelial cell lines. LMWP is produced using a stable, easily produced immobilized thermolysin gel column followed by size-exclusion purification. Monomeric l-arginine induces concentration-dependent increases in NO production in stimulated RAW 264.7 and LA4 cells, as measured by stable nitrite in the cell media. In stimulated RAW 264.7 cells, LMWP significantly increases iNOS expression and total NO production 12-24 h post-treatment compared to cells given equivalent levels of monomeric l-arginine. For stimulated LA4 cells, LMWPs are effective in significantly increasing NO production compared to equivalent l-arginine monomer concentrations over 24 h but do not substantially enhance iNOS expression. The use of the arginase inhibitor S-boronoethyl-l-cysteine in combination with LMWPs results in even higher NO production by stimulated RAW 264.7 cells and LA4 cells. Increases in NO due to LMWPs, compared to l-arginine, occur only after 4 h, which may be due to iNOS elevation rather than increased substrate availability.

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.

Combined exposure to cigarette smoke and nontypeable Haemophilus influenzae drives development of a COPD phenotype in mice.

BACKGROUND: Cigarette smoke (CS) is the major etiologic factor of chronic obstructive pulmonary disease (COPD). CS-exposed mice develop emphysema and mild pulmonary inflammation but no airway obstruction, which is also a prominent feature of COPD. Therefore, CS may interact with other factors, particularly respiratory infections, in the pathogenesis of airway remodeling in COPD. METHODS: C57BL/6 mice were exposed to CS for 2 h a day, 5 days a week for 8 weeks. Mice were also exposed to heat-killed non-typeable H. influenzae (HK-NTHi) on days 7 and 21. One day after the last exposure to CS, mice were sacrificed and lung inflammation and mechanics, emphysematous changes, and goblet cell metaplasia were assessed. Mice exposed to CS or HK-NTHi alone or room air served as controls. To determine the susceptibility to viral infections, we also challenged these mice with rhinovirus (RV). RESULTS: Unlike mice exposed to CS or HK-NTHi alone, animals exposed to CS/HK-NTHi developed emphysema, lung inflammation and goblet cell metaplasia in both large and small airways. CS/HK-NTHi-exposed mice also expressed increased levels of mucin genes and cytokines compared to mice in other groups. CS/HK-NTHi-exposed mice infected with RV demonstrated increased viral persistence, sustained neutrophilia, and further increments in mucin gene and chemokine expression compared to other groups. CONCLUSIONS: These findings indicate that in addition to CS, bacteria may also contribute to development of COPD, particularly changes in airways. Mice exposed to CS/HK-NTHi are also more susceptible to subsequent viral infection than mice exposed to either CS or HK-NTHi alone.

Macrophage activation state determines the response to rhinovirus infection in a mouse model of allergic asthma.

BACKGROUND: The mechanisms by which viruses cause asthma exacerbations are not precisely known. Previously, we showed that, in ovalbumin (OVA)-sensitized and -challenged mice with allergic airway inflammation, rhinovirus (RV) infection increases type 2 cytokine production from alternatively-activated (M2) airway macrophages, enhancing eosinophilic inflammation and airways hyperresponsiveness. In this paper, we tested the hypothesis that IL-4 signaling determines the state of macrophage activation and pattern of RV-induced exacerbation in mice with allergic airways disease. METHODS: Eight week-old wild type or IL-4 receptor knockout (IL-4R KO) mice were sensitized and challenged with OVA and inoculated with RV1B or sham HeLa cell lysate. RESULTS: In contrast to OVA-treated wild-type mice with both neutrophilic and eosinophilic airway inflammation, OVA-treated IL-4R KO mice showed increased neutrophilic inflammation with few eosinophils in the airways. Like wild-type mice, IL-4R KO mice showed OVA-induced airway hyperreactivity which was further exacerbated by RV. There was a shift in lung cytokines from a type 2-predominant response to a type 1 response, including production of IL-12p40 and TNF-α. IL-17A was also increased. RV infection of OVA-treated IL-4R KO mice further increased neutrophilic inflammation. Bronchoalveolar macrophages showed an M1 polarization pattern and ex vivo RV infection increased macrophage production of TNF-α, IFN-γ and IL-12p40. Finally, lung cells from OVA-treated IL-4R KO mice showed reduced CD206+ CD301+ M2 macrophages, decreased IL-13 and increased TNF-α and IL-17A production by F4/80+, CD11b+ macrophages. CONCLUSIONS: OVA-treated IL-4R KO mice show neutrophilic airway inflammation constituting a model of allergic, type 1 cytokine-driven neutrophilic asthma. In the absence of IL-4/IL-13 signaling, RV infection of OVA-treated mice increased type 1 cytokine and IL-17A production from conventionally-activated macrophages, augmenting neutrophilic rather than eosinophilic inflammation. In mice with allergic airways inflammation, IL-4R signaling determines macrophage activation state and the response to subsequent RV infection.

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.

Elastase/LPS-exposed mice exhibit impaired innate immune responses to bacterial challenge: role of scavenger receptor A.

Nontypeable Haemophilus influenzae (NTHi) is an important bacterial pathogen associated with lower respiratory tract colonization and with acute exacerbations and disease progression in chronic obstructive pulmonary disease (COPD). Why the immune system fails to eliminate NTHi and the exact contribution of the organism to COPD progression are not well understood, in part because we lack an animal model that mimics all aspects of COPD. For this study, we used an established murine model that exhibits typical features of COPD. Elastase/LPS-exposed mice infected with NTHi showed persistence of bacteria up to 5 days after infection, whereas mice exposed to elastase, LPS, or PBS cleared all bacteria by 3 days. Elastase/LPS-exposed mice also showed sustained lung neutrophilic inflammation, goblet cell metaplasia, airway hyperresponsiveness, and progression of emphysema at 15 days after infection. Alveolar macrophages isolated from elastase/LPS-exposed mice showed impaired bacterial phagocytosis, reduced expression of MARCO and of mannose receptor, and absent expression of scavenger receptor-A (SR-A). Neutralization of SR-A significantly decreased phagocytosis of NTHi by normal alveolar macrophages. Our results suggest that elastase/LPS-exposed mice show impaired bacterial clearance and sustained lung inflammation. Lack of SR-A expression may, in part, be responsible for impaired phagocytosis of bacteria by alveolar macrophages of elastase/LPS-exposed mice. These data validate the suitability of elastase/LPS model for investigating NTHi pathogenesis and progression of disease in COPD.

Rhinovirus-induced barrier dysfunction in polarized airway epithelial cells is mediated by NADPH oxidase 1.

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 (R(T)), 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 R(T), 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 R(T). 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.

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.

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.

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

BACKGROUND: Chronic 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. METHODS: Mice 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. RESULTS: Quercetin-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. CONCLUSIONS: Quercetin prevents progression of emphysema in elastase/LPS-treated mice by reducing oxidative stress, lung inflammation and expression of MMP9 and MMP12.

Randomised clinical trial to determine the safety of quercetin supplementation in patients with chronic obstructive pulmonary disease.

INTRODUCTION: Quercetin is a plant flavonoid and has potent antioxidant and anti-inflammatory properties. In a preclinical model of chronic obstructive pulmonary disease (COPD), quercetin reduced markers of both oxidative stress and lung inflammation and also reduced rhinovirus-induced progression of lung disease. Although quercetin appears to be an attractive natural alternative to manage COPD, the safety of quercetin supplementation in this population is unknown. METHODS: We recruited COPD patients with mild-to-severe lung disease with FVE1 ranging between >35% and <80% and supplemented with either placebo or quercetin at 500, 1000 or 2000 mg/day in a dose-escalation manner. The duration of quercetin supplementation was 1 week. RESULTS: Patients had no study drug-related severe adverse events based on blood tests, which included both complete blood counts and evaluation of comprehensive metabolic panel. One of the patients reported mild adverse events included gastro-oesophageal reflux disease, which was observed in both placebo and quercetin groups. CONCLUSIONS: Quercetin was safely tolerated up to 2000 mg/day as assessed by lung function, blood profile and COPD assessment test questionnaire. TRIAL REGISTRATION NUMBER: NCT01708278.

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.

Quercetin prevents rhinovirus-induced progression of lung disease in mice with COPD phenotype.

Acute exacerbations are the major cause of morbidity and mortality in patients with chronic obstructive pulmonary disease (COPD). Rhinovirus, which causes acute exacerbations may also accelerate progression of lung disease in these patients. Current therapies reduces the respiratory symptoms and does not treat the root cause of exacerbations effectively. We hypothesized that quercetin, a potent antioxidant and anti-inflammatory agent with antiviral properties may be useful in treating rhinovirus-induced changes in COPD. Mice with COPD phenotype maintained on control or quercetin diet and normal mice were infected with sham or rhinovirus, and after 14 days mice were examined for changes in lung mechanics and lung inflammation. Rhinovirus-infected normal mice showed no changes in lung mechanics or histology. In contrast, rhinovirus-infected mice with COPD phenotype showed reduction in elastic recoiling and increase in lung inflammation, goblet cell metaplasia, and airways cholinergic responsiveness compared to sham-infected mice. Interestingly, rhinovirus-infected mice with COPD phenotype also showed accumulation of neutrophils, CD11b+/CD11c+ macrophages and CD8+ T cells in the lungs. Quercetin supplementation attenuated rhinovirus-induced all the pathologic changes in mice with COPD phenotype. Together these results indicate that quercetin effectively mitigates rhinovirus-induced progression of lung disease in a mouse model of COPD. Therefore, quercetin may be beneficial in the treatment of rhinovirus-associated exacerbations and preventing progression of lung disease in COPD.

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.