Genetic ablation of glutaredoxin-1 causes enhanced resolution of airways hyperresponsiveness and mucus metaplasia in mice with allergic airways disease

Protein-S-glutathionylation (PSSG) is an oxidative modification of reactive cysteines that has emerged as an important player in pathophysiological processes. Under physiological conditions, the thiol transferase, glutaredoxin-1 (Glrx1) catalyses deglutathionylation. Although we previously demonstrated that Glrx1 expression is increased in mice with allergic inflammation, the impact of Glrx1/PSSG in the development of allergic airways disease remains unknown. In the present study we examined the impact of genetic ablation of Glrx1 in the pathogenesis of allergic inflammation and airway hyperresponsiveness (AHR) in mice. Glrx1(-/-) or WT mice were subjected to the antigen, ovalbumin (OVA), and parameters of allergic airways disease were evaluated 48 h after three challenges, and 48 h or 7 days after six challenges with aerosolized antigen. Although no clear increases in PSSG were observed in WT mice in response to OVA, marked increases were detected in lung tissue of mice lacking Glrx1 48 h following six antigen challenges. Inflammation and expression of proinflammatory mediators were decreased in Glrx1(-/-) mice, dependent on the time of analysis. WT and Glrx1(-/-) mice demonstrated comparable increases in AHR 48 h after three or six challenges with OVA. However, 7 days postcessation of six challenges, parameters of AHR in Glrx1(-/-) mice were resolved to control levels, accompanied by marked decreases in mucus metaplasia and expression of Muc5AC and GOB5. These results demonstrate that the Glrx1/S-glutathionylation redox status in mice is a critical regulator of AHR, suggesting that avenues to increase S-glutathionylation of specific target proteins may be beneficial to attenuate AHR.     

Regulation of mucous cell metaplasia in bronchial asthma

Mucous cell metaplasia (MCM), defined by the appearance of mucous cells in airways where mucous cells were not present, is a consistent pathologic characteristic in the peripheral airways of bronchial asthma. Under mild inflammatory conditions MCM occurs as a result of pre-existing airway epithelial cells (AECs) starting to express mucin genes and differentiating into mucous cells. Under extensive inflammatory responses, AECs proliferate, and the development of MCM involves the differentiation of pre-existing and proliferating cells into mucous cells. Epithelial cell numbers per mm basal lamina are increased by approximately 30%. IL-13 is the central cytokine that is responsible for MCM in asthma through GABA-R- and STAT6-mediated mechanisms involving the calcium-activated chloride channel CLCA. IL-13 is also responsible for the proliferation of AECs by causing cells to produce TGFalpha that acts on the epidermal growth factor (EGF) receptor. Normally, resolution of MCM involves two distinct mechanisms. 1) Some of the metaplastic mucous cells stop the synthesis of mucus and dedifferentiate into Clara or serous cells to reconstitute the epithelium. 2) When proliferation of epithelial cells had occurred, approximately 30% of metaplastic cells are eliminated during the resolution process. Thus, a safe approach to reducing IL-13-induced MCM would involve blocking mucous synthesis and storage, blocking secretion of stored mucus, and eliminating hyperplastic mucous cells. Understanding the molecular mechanisms of each of these processes is necessary for developing effective therapies for reducing mucous hypersecretion in asthma and leading to a repaired epithelium.     

Prostaglandin-generating factor of anaphylaxis induces mucous glycoprotein release and the formation of lipoxygenase products of arachidonate from human airways

The effects of prostaglandin-generating factor of anaphylaxis (PGF-A) upon the lipoxygenaton of arachidonic acid and the promotion of mucous glycoprotein secretion by human airways were analyzed concurrently in order to determine the role that lipoxygenase products play in the secretion of mucus which accompanies immediate hypersensitivity reactions of airways. PGF-A enhanced both mucous glycoprotein relesae and the 5- and 15-lipoxygenation of arachidonic acid as well as the formation of leukotrien B₄ (LTB₄) with similar dose-response relationships. The capacity of PGF-A to stimulate mucous glycoprotein release was inhibited by ETYA but not by indomethacin, suggesting that PGF-A stimulated lipoxygenase products may be involved. Lipoxygenase products of arachidonic acid thus may serve as mediators of the enhancement of mucus secretion from human airways in response to PGF-A.     

The structural basis of airways hyperresponsiveness in asthma.

We hypothesized that structural airway remodeling contributes to airways hyperresponsiveness (AHR) in asthma. Small, medium, and large airways were analyzed by computed tomography in 21 asthmatic volunteers under baseline conditions (FEV1 = 64% predicted) and after maximum response to albuterol (FEV1 = 76% predicted). The difference in pulmonary function between baseline and albuterol was an estimate of AHR to the baseline smooth muscle tone (BSMT). BSMT caused an increase in residual volume (RV) that was threefold greater than the decrease in forced vital capacity (FVC) because of a simultaneous increase in total lung capacity (TLC). The decrease in FVC with BSMT was the major determinant of the baseline FEV1 (P < 0.0001). The increase in RV correlated inversely with the relaxed luminal diameter of the medium airways (P = 0.009) and directly with the wall thickness of the large airways (P = 0.001). The effect of BSMT on functional residual capacity (FRC) controlled the change in TLC relative to the change in RV. When the FRC increased with RV, TLC increased and FVC was preserved. When the relaxed large airways were critically narrowed, FRC and TLC did not increase and FVC fell. With critical large airways narrowing, the FRC was already elevated from dynamic hyperinflation before BSMT and did not increase further with BSMT. FEV1/FVC in the absence of BSMT correlated directly with large airway luminal diameter and inversely with the fall in FVC with BSMT. These findings suggest that dynamic hyperinflation caused by narrowing of large airways is a major determinant of AHR in asthma.     

NF-kappa B activation in airways modulates allergic inflammation but not hyperresponsiveness

Airways display robust NF-kappaB activation and represent targets for anti-inflammatory asthma therapies, but the functional importance of NF-kappaB activation in airway epithelium remains enigmatic. Therefore, transgenic mice were created in which NF-kappaB activation is repressed specifically in airways (CC10-IkappaBalpha(SR) mice). In response to inhaled Ag, transgenic mice demonstrated significantly ameliorated inflammation, reduced levels of chemokines, T cell cytokines, mucus cell metaplasia, and circulating IgE compared with littermate controls. Despite these findings, Ag-driven airways hyperresponsiveness was not attenuated in CC10-IkappaBalpha(SR) mice. This study clearly demonstrates that airway epithelial NF-kappaB activation orchestrates Ag-induced inflammation and subsequent adaptive immune responses, but does not contribute to airways hyperresponsiveness, the cardinal feature that underlies asthma.     

Chlamydophila pneumoniae induces a sustained airway hyperresponsiveness and inflammation in mice

Background

It has been reported that Chlamydophila (C.) pneumoniae is involved in the initiation and promotion of asthma and chronic obstructive pulmonary diseases (COPD). Surprisingly, the effect of C. pneumoniae on airway function has never been investigated.

Methods

In this study, mice were inoculated intranasally with C. pneumoniae (strain AR39) on day 0 and experiments were performed on day 2, 7, 14 and 21.

Results

We found that from day 7, C. pneumoniae infection causes both a sustained airway hyperresponsiveness and an inflammation. Interferon-γ (IFN-γ) and macrophage inflammatory chemokine-2 (MIP-2) levels in bronchoalveolar lavage (BAL)-fluid were increased on all experimental days with exception of day 7 where MIP-2 concentrations dropped to control levels. In contrast, tumor necrosis factor-α (TNF-α) levels were only increased on day 7. From day 7 to 21 epithelial damage and secretory cell hypertrophy was observed. It is suggested that, the inflammatory cells/mediators, the epithelial damage and secretory cell hypertrophy contribute to initiation of airway hyperresponsiveness.

Conclusion

Our study demonstrates for the first time that C. pneumoniae infection can modify bronchial responsiveness. This has clinical implications, since additional changes in airway responsiveness and inflammation-status induced by this bacterium may worsen and/or provoke breathlessness in asthma and COPD.     

Vitamin-A-induced mucous metaplasia. An in vitro system for modulating tight and gap junction differentiation

Stratified squamous epithelia from 14-day chick embryo shank skin contain rare tight-junctional strands and only small gap junctions. Exposure of this tissue to retinoic acid (vitamin-A) (20 U/ml) in organ culture, however, induces mucous metaplasia, accompanied by tight-junction formation and gap-junction growth; untreated specimens continue to keratinize. To investigate sequential stages of junctional assembly and growth, we examined thin sections and freeze-fracture replicas at daily intervals for 3 days. During the metaplastic process, tight junctions assemble in midepidermal and upper regions, beginning on day 1 and becoming maximal on day 3. Two tight-junctional patterns could be tentatively identified as contributing to the emergence of fully formed zonulae occludentes: (a) the formation of individual ridges along the margins of gap junctions; (b) de novo generation of continuous ramifying strands by fusion of short strand segments and linear particulate aggregates near cellular apices. Gap junction enlargement, already maximal at day 1, occurs primarily three to four cell layers deep. Growth appears to occur by annexation of islands of 20-40 8.5-nm particles into larger lattices of islands separated by particle-free aisles. Eventually, a single gap junction may occupy much of the exposed membrane face in freeze-fractured tissue, but during apical migration of the cells such junctions disappear. The vitamin- A chick-skin system is presented as a responsive model for the controlled study of junction assembly.     

Inhibition of arginase I activity by RNA interference attenuates IL-13-induced airways hyperresponsiveness

Increased arginase I activity is associated with allergic disorders such as asthma. How arginase I contributes to and is regulated by allergic inflammatory processes remains unknown. CD4+ Th2 lymphocytes (Th2 cells) and IL-13 are two crucial immune regulators that use STAT6-dependent pathways to induce allergic airways inflammation and enhanced airways responsiveness to spasmogens (airways hyperresponsiveness (AHR)). This pathway is also used to activate arginase I in isolated cells and in hepatic infection with helminths. In the present study, we show that arginase I expression is also regulated in the lung in a STAT6-dependent manner by Th2-induced allergic inflammation or by IL-13 alone. IL-13-induced expression of arginase I correlated directly with increased synthesis of urea and with reduced synthesis of NO. Expression of arginase I, but not eosinophilia or mucus hypersecretion, temporally correlated with the development, persistence, and resolution of IL-13-induced AHR. Pharmacological supplementation with l-arginine or with NO donors amplified or attenuated IL-13-induced AHR, respectively. Moreover, inducing loss of function of arginase I specifically in the lung by using RNA interference abrogated the development of IL-13-induced AHR. These data suggest an important role for metabolism of l-arginine by arginase I in the modulation of IL-13-induced AHR and identify a potential pathway distal to cytokine receptor interactions for the control of IL-13-mediated bronchoconstriction in asthma.     

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.     

Recombinant VP4 of human rhinovirus induces permeability in model membranes

In common with all nonenveloped viruses, the mechanism of picornavirus membrane penetration during cell entry is poorly understood. The small, myristylated capsid protein VP4 has been implicated in this process. Here we show that recombinant VP4 of human rhinovirus 16 has the ability to associate with and induce membrane permeability in otherwise intact liposomes. This provides further evidence that VP4 plays a key role in picornavirus cell entry.     

NAD(P)H quinone oxidoreductase 1 regulates neutrophil elastase-induced mucous cell metaplasia

Mucous cell metaplasia (MCM) and neutrophil-predominant airway inflammation are pathological features of chronic inflammatory airway diseases. A signature feature of MCM is increased expression of a major respiratory tract mucin, MUC5AC. Neutrophil elastase (NE) upregulates MUC5AC in primary airway epithelial cells by generating reactive oxygen species, and this response is due in part to upregulation of NADPH quinone oxidoreductase 1 (NQO1) activity. Delivery of NE directly to the airway triggers inflammation and MCM and increases synthesis and secretion of MUC5AC protein from airway epithelial cells. We hypothesized that NE-induced MCM is mediated in vivo by NQO1. Male wild-type and Nqo1-null mice (C57BL/6 background) were exposed to human NE (50 μg) or vehicle via oropharyngeal aspiration on days 1, 4, and 7. On days 8 and 11, lung tissues and bronchoalveolar lavage (BAL) samples were obtained and evaluated for MCM, inflammation, and oxidative stress. MCM, inflammation, and production of specific cytokines, granulocyte-macrophage colony-stimulating factor, macrophage inflammatory protein-2, interleukin-4, and interleukin-5 were diminished in NE-treated Nqo1-null mice compared with NE-treated wild-type mice. However, in contrast to the role of NQO1 in vitro, we demonstrate that NE-treated Nqo1-null mice had greater levels of BAL and lung tissue lipid carbonyls and greater BAL iron on day 11, all consistent with increased oxidative stress. NQO1 is required for NE-induced inflammation and MCM. This model system demonstrates that NE-induced MCM directly correlates with inflammation, but not with oxidative stress.     

Neonatal tolerance and patent filarial infection

Lymphatic filariasis occurs in endemic pockets. Patent infections with long-term, high-grade microfilaremia do not develop in nonendemic individuals. It is tempting to speculate that individuals with intact immune responses to filarial antigens are capable of dealing with filarial exposure without developing persistent infection. There are published data that support the idea that only those individuals who are impaired in their immune defense against these parasites owing to neonatal tolerization become productively infected with the filarial parasites. If the model is correct, there are profound implications for global eradication.     

Neutrophil adhesion molecules in experimental rhinovirus infection in COPD

Background

COPD exacerbations are associated with neutrophilic airway inflammation. Adhesion molecules on the surface of neutrophils may play a key role in their movement from blood to the airways. We analysed adhesion molecule expression on blood and sputum neutrophils from COPD subjects and non-obstructed smokers during experimental rhinovirus infections.

Methods

Blood and sputum were collected from 9 COPD subjects and 10 smoking and age-matched control subjects at baseline, and neutrophil expression of the adhesion molecules and activation markers measured using flow cytometry. The markers examined were CD62L and CD162 (mediating initial steps of neutrophil rolling and capture), CD11a and CD11b (required for firm neutrophil adhesion), CD31 and CD54 (involved in neutrophil transmigration through the endothelial monolayer) and CD63 and CD66b (neutrophil activation markers). Subjects were then experimentally infected with rhinovirus-16 and repeat samples collected for neutrophil analysis at post-infection time points.

Results

At baseline there were no differences in adhesion molecule expression between the COPD and non-COPD subjects. Expression of CD11a, CD31, CD62L and CD162 was reduced on sputum neutrophils compared to blood neutrophils. Following rhinovirus infection expression of CD11a expression on blood neutrophils was significantly reduced in both subject groups. CD11b, CD62L and CD162 expression was significantly reduced only in the COPD subjects. Blood neutrophil CD11b expression correlated inversely with inflammatory markers and symptom scores in COPD subjects.

Conclusion

Following rhinovirus infection neutrophils with higher surface expression of adhesion molecules are likely preferentially recruited to the lungs. CD11b may be a key molecule involved in neutrophil trafficking in COPD exacerbations.     

Agarose plug instillation causes goblet cell metaplasia by activating EGF receptors in rat airways

We hypothesized that foreign bodies in airways cause inflammation leading to goblet cell metaplasia. Instilled agarose plugs lodged in the bronchi of pathogen-free rats caused a time-dependent increase in Alcian blue-periodic acid-Schiff staining that was detected within 24 h and markedly increased at 72 h. Control bronchi contained no pregoblet or goblet cells, but plugged bronchi contained many pregoblet and goblet cells and a decrease in nongranulated secretory cells. In situ hybridization showed no expression of MUC5AC in control airways, but plugged airways showed a marked expression. Control bronchi showed sparse staining for epidermal growth factor receptor (EGFR) protein, but plugged bronchi showed intense EGFR staining in the epithelium. Pretreatment with an EGFR tyrosine kinase inhibitor (BIBX1522) prevented Alcian blue-periodic acid-Schiff staining and MUC5AC gene expression in plugged bronchi. Pretreatment with tumor necrosis factor-alpha neutralizing antibody or pretreatment with cyclophosphamide abolished plug-induced EGFR protein expression and goblet cell metaplasia. Thus instillation of agarose plugs induces profound goblet cell metaplasia by causing EGFR expression and activation.     

Resistance of differentiated human airway epithelium to infection by rhinovirus

Virtually all in vitro studies of the effects of rhinovirus on human airway epithelium have used cells grown under conditions known to produce low levels of differentiation. The relevance of the results to native epithelium is questionable. Here we grew primary cultures of human tracheal or nasal epithelium under three conditions. One condition produced pseudostratified, mucociliary cells virtually indistinguishable from native epithelium. The other two conditions produced undifferentiated squamous cells lacking cilia. Cells were infected for 6 h with rhinovirus-16. After a 24-h incubation period, we determined levels of viral RNA in the cells, numbers of infectious viral particles released in the mucosal medium, expression of a variety of epithelial cytokines and other proteins, release of IL-6 and IL-8, and transepithelial electrical resistance and voltage. After infection, levels of viral RNA in the poorly differentiated cells were 30 or 130 times those in the differentiated. Furthermore, expression of mRNA for inflammatory cytokines, release of infectious particles, and release of IL-6 and IL-8 were closely correlated with the degree of viral infection. Thus well-differentiated cells are much more resistant to viral infection and its functional consequences than are poorly differentiated cells from the same source.     

Role of interleukin-1 and MyD88-dependent signaling in rhinovirus infection

Rhinoviral infection is an important trigger of acute inflammatory exacerbations in patients with underlying airway disease. We have previously established that interleukin-1β (IL-1β) is central in the communication between epithelial cells and monocytes during the initiation of inflammation. In this study we explored the roles of IL-1β and its signaling pathways in the responses of airway cells to rhinovirus-1B (RV-1B) and further determined how responses to RV-1B were modified in a model of bacterial coinfection. Our results revealed that IL-1β dramatically potentiated RV-1B-induced proinflammatory responses, and while monocytes did not directly amplify responses to RV-1B alone, they played an important role in the responses observed with our coinfection model. MyD88 is the essential signaling adapter for IL-1β and most Toll-like receptors. To examine the role of MyD88 in more detail, we created stable MyD88 knockdown epithelial cells using short hairpin RNA (shRNA) targeted to MyD88. We determined that IL-1β/MyD88 plays a role in regulating RV-1B replication and the inflammatory response to viral infection of airway cells. These results identify central roles for IL-1β and its signaling pathways in the production of CXCL8, a potent neutrophil chemoattractant, in viral infection. Thus, IL-1β is a viable target for controlling the neutrophilia that is often found in inflammatory airway disease and is exacerbated by viral infection of the airways.