Sangbrita Chattoraj

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.

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.

Pseudomonas aeruginosa alginate promotes Burkholderia cenocepacia persistence in cystic fibrosis transmembrane conductance regulator knockout mice.

ABSTRACT Pseudomonas aeruginosa, a major respiratory pathogen in cystic fibrosis (CF) patients, facilitates infection by other opportunistic pathogens. Burkholderia cenocepacia, which normally infects adolescent patients, encounters alginate elaborated by mucoid P. aeruginosa. To determine whether P. aeruginosa alginate facilitates B. cenocepacia infection in mice, cystic fibrosis transmembrane conductance regulator knockout mice were infected with B. cenocepacia strain BC7 suspended in either phosphate-buffered saline (BC7/PBS) or P. aeruginosa alginate (BC7/alginate), and the pulmonary bacterial load and inflammation were monitored. Mice infected with BC7/PBS cleared all of the bacteria within 3 days, and inflammation was resolved by day 5. In contrast, mice infected with BC7/alginate showed persistence of bacteria and increased cytokine levels for up to 7 days. Histological examination of the lungs indicated that there was moderate to severe inflammation and pneumonic consolidation in isolated areas at 5 and 7 days postinfection in the BC7/alginate group. Further, alginate decreased phagocytosis of B. cenocepacia by professional phagocytes both in vivo and in vitro. P. aeruginosa alginate also reduced the proinflammatory responses of CF airway epithelial cells and alveolar macrophages to B. cenocepacia infection. The observed effects are specific to P. aeruginosa alginate, because enzymatically degraded alginate or other polyuronic acids did not facilitate bacterial persistence. These observations suggest that P. aeruginosa alginate may facilitate B. cenocepacia infection by interfering with host innate defense mechanisms.