Compartmental and Temporal Dynamics of Chronic Inflammation and Airway Remodelling in a Chronic Asthma Mouse Model

Background

Allergic asthma is associated with chronic airway inflammation and progressive airway remodelling. However, the dynamics of the development of these features and their spontaneous and pharmacological reversibility are still poorly understood. We have therefore investigated the dynamics of airway remodelling and repair in an experimental asthma model and studied how pharmacological intervention affects these processes.

Methods

Using BALB/c mice, the kinetics of chronic asthma progression and resolution were characterised in absence and presence of inhaled corticosteroid (ICS) treatment. Airway inflammation and remodelling was assessed by the analysis of bronchoalveolar and peribronichal inflammatory cell infiltrate, goblet cell hyperplasia, collagen deposition and smooth muscle thickening.

Results

Chronic allergen exposure resulted in early (goblet cell hyperplasia) and late remodelling (collagen deposition and smooth muscle thickening). After four weeks of allergen cessation eosinophilic inflammation, goblet cell hyperplasia and collagen deposition were resolved, full resolution of lymphocyte inflammation and smooth muscle thickening was only observed after eight weeks. ICS therapy when started before the full establishment of chronic asthma reduced the development of lung inflammation, decreased goblet cell hyperplasia and collagen deposition, but did not affect smooth muscle thickening. These effects of ICS on airway remodelling were maintained for a further four weeks even when therapy was discontinued.

Conclusions

Utilising a chronic model of experimental asthma we have shown that repeated allergen exposure induces reversible airway remodelling and inflammation in mice. Therapeutic intervention with ICS was partially effective in inhibiting the transition from acute to chronic asthma by reducing airway inflammation and remodelling but was ineffective in preventing smooth muscle hypertrophy.     

Bronchial Smooth Muscle Cells of Asthmatics Promote Angiogenesis through Elevated Secretion of CXC-Chemokines (ENA-78, GRO-α, and IL-8)

Background

Airway wall remodelling is a key pathology of asthma. It includes thickening of the airway wall, hypertrophy and hyperplasia of bronchial smooth muscle cells (BSMC), as well as an increased vascularity of the sub-epithelial cell layer. BSMC are known to be the effector cells of bronchoconstriction, but they are increasingly recognized as an important source of inflammatory mediators and angiogenic factors.

Objective

To compare the angiogenic potential of BSMC of asthmatic and non-asthmatic patients and to identify asthma-specific angiogenic factors.

Methods

Primary BSMC were isolated from human airway tissue of asthmatic and non-asthmatic patients. Conditioned medium (CM) collected from BSMC isolates was tested for angiogenic capacity using the endothelial cell (EC)-spheroid in vitro angiogenesis assay. Angiogenic factors in CM were quantified using a human angiogenesis antibody array and enzyme linked immunosorbent assay.

Results

Induction of sprout outgrowth from EC-spheroids by CM of BSMC obtained from asthma patients was increased compared with CM of control BSMC (twofold, p < 0.001). Levels of ENA-78, GRO-α and IL-8 were significantly elevated in CM of BSMC from asthma patients (p < 0.05 vs. non-asthmatic patients). SB 265610, a competitive antagonist of chemokine (CXC-motif) receptor 2 (CXCR2), attenuated the increased sprout outgrowth induced by CM of asthma patient-derived BSMC.

Conclusions

BSMC isolated from asthma patients exhibit increased angiogenic potential. This effect is mediated through the CXCR2 ligands (ENA78, GRO-α and IL-8) produced by BSMC.

Implications

CXCR2 ligands may play a decisive role in directing the neovascularization in the sub-epithelial cell layers of the lungs of asthma patients. Counteracting the CXCR2-mediated neovascularization by pharmaceutical compounds may represent a novel strategy to reduce airway remodelling in asthma.     

Baicalein Reduces Airway Injury in Allergen and IL-13 Induced Airway Inflammation

Background

Baicalein, a bioflavone present in the dry roots of Scutellaria baicalensis Georgi, is known to reduce eotaxin production in human fibroblasts. However, there are no reports of its anti-asthma activity or its effect on airway injury.

Methodology/Principal Findings

In a standard experimental asthma model, male Balb/c mice that were sensitized with ovalbumin (OVA), treated with baicalein (10 mg/kg, ip) or a vehicle control, either during (preventive use) or after OVA challenge (therapeutic use). In an alternate model, baicalein was administered to male Balb/c mice which were given either IL-4 or IL-13 intranasally. Features of asthma were determined by estimating airway hyperresponsiveness (AHR), histopathological changes and biochemical assays of key inflammatory molecules. Airway injury was determined with apoptotic assays, transmission electron microscopy and assessing key mitochondrial functions. Baicalein treatment reduced AHR and inflammation in both experimental models. TGF-β₁, sub-epithelial fibrosis and goblet cell metaplasia, were also reduced. Furthermore, baicalein treatment significantly reduced 12/15-LOX activity, features of mitochondrial dysfunctions, and apoptosis of bronchial epithelia.

Conclusion/Significance

Our findings demonstrate that baicalein can attenuate important features of asthma, possibly through the reduction of airway injury and restoration of mitochondrial function.     

Neutralisation of Interleukin-13 in Mice Prevents Airway Pathology Caused by Chronic Exposure to House Dust Mite

Background

Repeated exposure to inhaled allergen can cause airway inflammation, remodeling and dysfunction that manifests as the symptoms of allergic asthma. We have investigated the role of the cytokine interleukin-13 (IL-13) in the generation and persistence of airway cellular inflammation, bronchial remodeling and deterioration in airway function in a model of allergic asthma caused by chronic exposure to the aeroallergen House Dust Mite (HDM).

Methodology/Principal Findings

Mice were exposed to HDM via the intranasal route for 4 consecutive days per week for up to 8 consecutive weeks. Mice were treated either prophylactically or therapeutically with a potent neutralising anti-IL-13 monoclonal antibody (mAb) administered subcutaneously (s.c.). Airway cellular inflammation was assessed by flow cytometry, peribronchial collagen deposition by histocytochemistry and airway hyperreactivity (AHR) by invasive measurement of lung resistance (R_L) and dynamic compliance (C_dyn). Both prophylactic and therapeutic treatment with an anti-IL-13 mAb significantly inhibited (P<0.05) the generation and maintenance of chronic HDM-induced airway cellular inflammation, peribronchial collagen deposition, epithelial goblet cell upregulation. AHR to inhaled methacholine was reversed by prophylactic but not therapeutic treatment with anti-IL-13 mAb. Both prophylactic and therapeutic treatment with anti-IL-13 mAb significantly reversed (P<0.05) the increase in baseline R_L and the decrease in baseline C_dyn caused by chronic exposure to inhaled HDM.

Conclusions/Significance

These data demonstrate that in a model of allergic lung disease driven by chronic exposure to a clinically relevant aeroallergen, IL-13 plays a significant role in the generation and persistence of airway inflammation, remodeling and dysfunction.     

Aldose Reductase Inhibition Prevents Allergic Airway Remodeling through PI3K/AKT/GSK3β Pathway in Mice

Background

Long-term and unresolved airway inflammation and airway remodeling, characteristic features of chronic asthma, if not treated could lead to permanent structural changes in the airways. Aldose reductase (AR), an aldo-sugar and lipid aldehyde metabolizing enzyme, mediates allergen-induced airway inflammation in mice, but its role in the airway remodeling is not known. In the present study, we have examined the role of AR on airway remodeling using ovalbumin (OVA)-induced chronic asthma mouse model and cultured human primary airway epithelial cells (SAECs) and mouse lung fibroblasts (mLFs).

Methods

Airway remodeling in chronic asthma model was established in mice sensitized and challenged twice a week with OVA for 6 weeks. AR inhibitor, fidarestat, was administered orally in drinking water after first challenge. Inflammatory cells infiltration in the lungs and goblet cell metaplasia, airway thickening, collagen deposition and airway hyper-responsiveness (AHR) in response to increasing doses of methacholine were assessed. The TGFβ1-induced epithelial-mesenchymal transition (EMT) in SAECs and changes in mLFs were examined to investigate AR-mediated molecular mechanism(s) of airway remodeling.

Results

In the OVA-exposed mice for 6 wks inflammatory cells infiltration, levels of inflammatory cytokines and chemokines, goblet cell metaplasia, collagen deposition and AHR were significantly decreased by treatment with AR inhibitor, fidarestat. Further, inhibition of AR prevented TGFβ1-induced altered expression of E-cadherin, Vimentin, Occludin, and MMP-2 in SAECs, and alpha-smooth muscle actin and fibronectin in mLFs. Further, in SAECs, AR inhibition prevented TGFβ1- induced activation of PI3K/AKT/GSK3β pathway but not the phosphorylation of Smad2/3.

Conclusion

Our results demonstrate that allergen-induced airway remodeling is mediated by AR and its inhibition blocks the progression of remodeling via inhibiting TGFβ1-induced Smad-independent and PI3K/AKT/GSK3β-dependent pathway.     

Defective alterations in the collagen network to prostacyclin in COPD lung fibroblasts

Background

Prostacyclin analogs are potent vasodilators and possess anti-inflammatory properties. However, the effect of prostacyclin on extracellular matrix (ECM) in COPD is not well known. Collagen fibrils and proteoglycans are essential ECM components in the lung and fibroblasts are key players in regulating the homeostasis of ECM proteins. The aim was to study the synthesis of prostacyclin and its effect on fibroblast activity and ECM production, and in particular collagen I and the collagen-associated proteoglycans biglycan and decorin.

Methods

Parenchymal lung fibroblasts were isolated from lungs from COPD patients (GOLD stage IV) and from lungs and transbronchial biopsies from control subjects. The prostacyclin analog iloprost was used to study the effect of prostacyclin on ECM protein synthesis, migration, proliferation and contractile capacity of fibroblasts.

Results

TGF-β₁ stimulation significantly increased prostacyclin synthesis in fibroblasts from COPD patients (p < 0.01), but showed no effect on fibroblasts from control subjects. Collagen I synthesis was decreased by iloprost in both control and COPD fibroblasts (p < 0.05). Conversely, iloprost significantly altered biglycan and decorin synthesis in control fibroblasts, but iloprost displayed no effect on these proteoglycans in COPD fibroblasts. Proliferation rate was reduced (p < 0.05) and contractile capacity was increased in COPD fibroblasts (p < 0.05) compared to control fibroblasts. Iloprost decreased proliferative rate in control fibroblasts (p < 0.05), whereas iloprost attenuated contraction capacity in both COPD (p < 0.01) and control fibroblasts (p < 0.05).

Conclusions

Iloprost reduced collagen I synthesis and fibroblast contractility but did not affect the collagen-associated proteoglycans or proliferation rate in fibroblasts from COPD patients. Enhanced prostacyclin production could lead to improper collagen network fibrillogenesis and a more emphysematous lung structure in severe COPD patients.     

Basement membrane and vascular remodelling in smokers and chronic obstructive pulmonary disease: a cross-sectional study

Background

Little is known about airway remodelling in bronchial biopsies (BB) in smokers and chronic obstructive pulmonary disease (COPD). We conducted an initial pilot study comparing BB from COPD patients with nonsmoking controls. This pilot study suggested the presence of reticular basement membrane (Rbm) fragmentation and altered vessel distribution in COPD.

Methods

To determine whether Rbm fragmentation and altered vessel distribution in BB were specific for COPD we designed a cross-sectional study and stained BB from 19 current smokers and 14 ex-smokers with mild to moderate COPD and compared these to 15 current smokers with normal lung function and 17 healthy and nonsmoking subjects.

Results

Thickness of the Rbm was not significantly different between groups; although in COPD this parameter was quite variable. The Rbm showed fragmentation and splitting in both current smoking groups and ex-smoker COPD compared with healthy nonsmokers (p < 0.02); smoking and COPD seemed to have additive effects. Rbm fragmentation correlated with smoking history in COPD but not with age. There were more vessels in the Rbm and fewer vessels in the lamina propria in current smokers compared to healthy nonsmokers (p < 0.05). The number of vessels staining for vascular endothelial growth factor (VEGF) in the Rbm was higher in both current smoker groups and ex-smoker COPD compared to healthy nonsmokers (p < 0.004). In current smoker COPD VEGF vessel staining correlated with FEV1% predicted (r = 0.61, p < 0.02).

Conclusions

Airway remodelling in smokers and mild to moderate COPD is associated with fragmentation of the Rbm and altered distribution of vessels in the airway wall. Rbm fragmentation was also present to as great an extent in ex-smokers with COPD. These characteristics may have potential physiological consequences.     

Impaired macrophage phagocytosis of bacteria in severe asthma

Background

Bacteria are frequently cultured from sputum samples of severe asthma patients suggesting a defect in bacterial clearance from the airway. We measured the capacity of macrophages from patients with asthma to phagocytose bacteria.

Methods

Phagocytosis of fluorescently-labelled polystyrene beads, Haemophilus influenzae or Staphylococcus aureus by broncholaveolar lavage alveolar macrophages (AM) and by monocyte-derived macrophages (MDM) from non-asthmatics, mild-moderate and severe asthmatic patients was assessed using fluorimetry.

Results

There were no differences in phagocytosis of polystyrene beads by AMs or MDMs from any of the subject groups. There was reduced phagocytosis of Haemophilus influenzae and Staphylococcus aureus in MDMs from patients with severe asthma compared to non-severe asthma (p < 0.05 and p < 0.01, respectively) and healthy subjects (p < 0.01and p < 0.001, respectively). Phagocytosis of Haemophilus influenzae and Staphylococcus aureus by AM was also reduced in severe asthma compared to normal subjects (p < 0.05). Dexamethasone and formoterol did not suppress phagocytosis of bacteria by MDMs from any of the groups.

Conclusions

Persistence of bacteria in the lower airways may result partly from a reduced phagocytic capacity of macrophages for bacteria. This may contribute to increased exacerbations, airway colonization and persistence of inflammation.     

Elevation of IL-6 in the allergic asthmatic airway is independent of inflammation but associates with loss of central airway function

Background

Asthma is a chronic inflammatory disease of the airway that is characterized by a Th2-type of immune response with increasing evidence for involvement of Th17 cells. The role of IL-6 in promoting effector T cell subsets suggest that IL-6 may play a functional role in asthma. Classically IL-6 has been viewed as an inflammatory marker, along with TNFα and IL-1β, rather than as regulatory cytokine.

Objective

To investigate the potential relationship between IL-6 and other proinflammatory cytokines, Th2/Th17 cytokines and lung function in allergic asthma, and thus evaluate the potential role of IL-6 in this disease.

Methods

Cytokine levels in induced sputum and lung function were measured in 16 healthy control and 18 mild-moderate allergic asthmatic subjects.

Results

The levels of the proinflammatory biomarkers TNFα and IL-1β were not different between the control and asthmatic group. In contrast, IL-6 levels were specifically elevated in asthmatic subjects compared with healthy controls (p < 0.01). Hierarchical regression analysis in the total study cohort indicates that the relationship between asthma and lung function could be mediated by IL-6. Among Th2 cytokines only IL-13 (p < 0.05) was also elevated in the asthmatic group, and positively correlated with IL-6 levels (r_S = 0.53, p < 0.05).

Conclusions

In mild-moderate asthma, IL-6 dissociates from other proinflammatory biomarkers, but correlates with IL-13 levels. Furthermore, IL-6 may contribute to impaired lung function in allergic asthma.     

Ag85B DNA vaccine suppresses airway inflammation in a murine model of asthma

Background

In allergic asthma, Th2 lymphocytes are believed to play important roles in orchestrating airway eosinophilia and inflammation. Resetting the Th1/Th2 imbalance may have a therapeutic role in asthma. The mycobacterium tuberculosis 30-kilodalton major secretory protein (antigen 85B, Ag85B) can protect animals from M. tuberculosis infection by inducing a Th1-dominant response.

Methods

In this study, the Ag85B gene was cloned into pMG plasmids to yield the pMG-Ag85B plasmid. The expression of Ag85B gene in murine bronchial epithelia cells was detected by Western blotting and immunohistochemical staining after intranasal immunization with reconstructed pMG-Ag85B plasmids. The protective effect of pMG-Ag85B plasmids immunization in airway inflammation was evaluated by histological examination and bronchoalveolar lavage (BAL). IL-4 and IFN-γ levels in the BAL and supernatant from splenocyte culture were determined using ELISA kits.

Results

The Ag85B gene was successfully expressed in murine bronchial epithelia cells by intranasal immunization with reconstructed pMG-Ag85B plasmids. Using a murine model of asthma induced by ovalbumin (OVA), pMG-Ag85B immunization significantly inhibited cellular infiltration across the airway epithelium with a 37% decrease in the total number of cells (9.6 ± 2.6 × 10⁵/ml vs. 15.2 ± 3.0 × 10⁵/ml, p < 0.05) and a 74% decrease in the number of eosinophils (1.4 ± 0.2 × 10⁵/ml vs. 5.4 ± 1.1 × 10⁵/ml, p < 0.01) compared with the OVA-sensitized control group. There was no difference in the number of neutrophils in BAL fluid between the pMG-Ag85B group, the OVA-sensitized control group and the empty pMG group. IL-4 production was significantly decreased in the BAL fluid (32.0 ± 7.6 pg/ml vs. 130.8 ± 32.6 pg/ml, p < 0.01) and in the splenocyte supernatant (5.1 ± 1.6 pg/ml vs. 10.1 ± 2.3 pg/ml, p < 0.05) in the pMG-Ag85B group compared with the OVA-sensitized control group, while IFN-γ production was increased in the BAL fluid (137.9 ± 25.6 pg/ml vs. 68.4 ± 15.3 pg/ml, p < 0.05) and in the splenocyte supernatant (20.1 ± 5.4 pg/ml vs. 11.3 ± 3.2 pg/ml, p < 0.05).

Conclusion

In a murine model of asthma induced by OVA, intranasal immunization with pMG-Ag85B significantly reduced allergic airway inflammation with less eosinophil infiltration. This protective effect was associated with decreased IL-4 and increased IFN-γ production in the BAL fluid and in the supernatant of cultured splenocytes.     

Surfactant protein D attenuates sub-epithelial fibrosis in allergic airways disease through TGF-β

Background

Surfactant protein D (SP-D) can regulate both innate and adaptive immunity. Recently, SP-D has been shown to contribute to the pathogenesis of airway allergic inflammation and bleomycin-induced pulmonary fibrosis. However, in allergic airways disease, the role of SP-D in airway remodeling remains unknown. The objective of this study was to determine the contribution of functional SP-D in regulating sub-epithelial fibrosis in a mouse chronic house dust mite model of allergic airways disease.

Methods

C57BL/6 wild-type (WT) and SP-D−/− mice (C57BL/6 background) were chronically challenged with house dust mite antigen (Dermatophagoides pteronyssinus, Dp). Studies with SP-D rescue and neutralization of TGF-β were conducted. Lung histopathology and the concentrations of collagen, growth factors, and cytokines present in the airspace and lung tissue were determined. Cultured eosinophils were stimulated by Dp in presence or absence of SP-D.

Results

Dp-challenged SP-D−/− mice demonstrate increased sub-epithelial fibrosis, collagen production, eosinophil infiltration, TGF-β1, and IL-13 production, when compared to Dp-challenged WT mice. By immunohistology, we detected an increase in TGF-β1 and IL-13 positive eosinophils in SP-D−/− mice. Purified eosinophils stimulated with Dp produced TGF-β1 and IL-13, which was prevented by co-incubation with SP-D. Additionally, treatment of Dp challenged SP-D−/− mice with exogenous SP-D was able to rescue the phenotypes observed in SP-D−/− mice and neutralization of TGF-β1 reduced sub-epithelial fibrosis in Dp-challenged SP-D−/− mice.

Conclusion

These data support a protective role for SP-D in the pathogenesis of sub-epithelial fibrosis in a mouse model of allergic inflammation through regulation of eosinophil-derived TGF-β.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0143-9) contains supplementary material, which is available to authorized users.     

Obstructive Sleep Apnoea Modulates Airway Inflammation and Remodelling in Severe Asthma

Background

Obstructive sleep apnoea (OSA) is frequently observed in severe asthma but the causal link between the 2 diseases remains hypothetical. The role of OSA-related systemic and airway neutrophilic inflammation in asthma bronchial inflammation or remodelling has been rarely investigated. The aim of this study was to compare hallmarks of inflammation in induced sputum and features of airway remodelling in bronchial biopsies from adult patients with severe asthma with and without OSA.

Materials and Methods

An overnight polygraphy was performed in 55 patients referred for difficult-to-treat asthma, who complained of nocturnal respiratory symptoms, poor sleep quality or fatigue. We compared sputum analysis, reticular basement membrane (RBM) thickness, smooth muscle area, vascular density and inflammatory cell infiltration in bronchial biopsies.

Results

In total, 27/55 patients (49%) had OSA diagnosed by overnight polygraphy. Despite a moderate increase in apnoea-hypopnoea index (AHI; 14.2±1.6 event/h [5–35]), the proportion of sputum neutrophils was higher and that of macrophages lower in OSA than non-OSA patients, with higher levels of interleukin 8 and matrix metalloproteinase 9. The RBM was significantly thinner in OSA than non-OSA patients (5.8±0.4 vs. 7.8±0.4 μm, p<0.05). RBM thickness and OSA severity assessed by the AHI were negatively correlated (rho = -0.65, p<0.05). OSA and non-OSA patients did not differ in age, sex, BMI, lung function, asthma control findings or treatment.

Conclusion

Mild OSA in patients with severe asthma is associated with increased proportion of neutrophils in sputum and changes in airway remodelling.     

Plasma antibodies against heat shock protein 70 correlate with the incidence and severity of asthma in a Chinese population

Background

The heat shock proteins (Hsps) are induced by stresses such as allergic factors and inflammatory responses in bronchi epithelial cells and therefore may be detectable in patients with asthma. However, the etiologic link between anti-Hsps and asthma (its severity and related inflammatory responses such as interleukin-4 and immunoglobulin E) has not been established. We determined whether antibodies against Hsp60 and Hsp70 were present in patients with asthma and evaluated their associations with risk and severity of asthma.

Methods

We determined the levels of anti-Hsp60 and anti-Hsp70 by immunoblot and their associations with risk and symptom severity of asthma in 95 patients with asthma and 99 matched non-symptomatic controls using multivariate logistic regression analysis.

Results

Compared to the controls, asthma patients were more likely to have detectable anti-Hsp60 (17.2% vs 5.1%) and anti-Hsp70 (33.7% vs 8.1%) (p ≤ 0.001). In particular, the presence of anti-Hsp70 was associated with a greater than 2 fold risk for asthma (adjusted OR = 2.21; 95% CI = 1.35~3.59). Furthermore, both anti-Hsp60 and anti-Hsp70 levels were positively correlated with symptom severity (p < 0.05) as well as interleukin-4 and immunoglobulin E (p < 0.05). Individuals with antibodies against anti-Hsp60 and anti-Hsp70 were more likely to have a family history of asthma (p < 0.001) and higher plasma concentrations of total immunoglobulin E (p = 0.001) and interleukin-4 (p < 0.05) than those without antibodies.

Conclusions

These data suggest that anti-Hsp60 and especially anti-Hsp70 correlate with the attacks and severity of asthma. The underlying molecular mechanisms linking antibodies to heat shock proteins and asthma remain to be investigated.     

Inhaled salmeterol and/or fluticasone alters structure/function in a murine model of allergic airways disease

Background

The relationship between airway structural changes (remodeling) and airways hyperresponsiveness (AHR) is unclear. Asthma guidelines suggest treating persistent asthma with inhaled corticosteroids and long acting β-agonists (LABA). We examined the link between physiological function and structural changes following treatment fluticasone and salmeterol separately or in combination in a mouse model of allergic asthma.

Methods

BALB/c mice were sensitized to intraperitoneal ovalbumin (OVA) followed by six daily inhalation exposures. Treatments included 9 daily nebulized administrations of fluticasone alone (6 mg/ml), salmeterol (3 mg/ml), or the combination fluticasone and salmeterol. Lung impedance was measured following methacholine inhalation challenge. Airway inflammation, epithelial injury, mucus containing cells, and collagen content were assessed 48 hours after OVA challenge. Lungs were imaged using micro-CT.

Results and Discussion

Treatment of allergic airways disease with fluticasone alone or in combination with salmeterol reduced AHR to approximately naüve levels while salmeterol alone increased elastance by 39% compared to control. Fluticasone alone and fluticasone in combination with salmeterol both reduced inflammation to near naive levels. Mucin containing cells were also reduced with fluticasone and fluticasone in combination with salmeterol.

Conclusions

Fluticasone alone and in combination with salmeterol reduces airway inflammation and remodeling, but salmeterol alone worsens AHR: and these functional changes are consistent with the concomitant changes in mucus metaplasia.     

Early-life viral infection and allergen exposure interact to induce an asthmatic phenotype in mice

Background

Early-life respiratory viral infections, notably with respiratory syncytial virus (RSV), increase the risk of subsequent development of childhood asthma. The purpose of this study was to assess whether early-life infection with a species-specific model of RSV and subsequent allergen exposure predisposed to the development of features of asthma.

Methods

We employed a unique combination of animal models in which BALB/c mice were neonatally infected with pneumonia virus of mice (PVM, which replicates severe RSV disease in human infants) and following recovery, were intranasally sensitised with ovalbumin. Animals received low-level challenge with aerosolised antigen for 4 weeks to elicit changes of chronic asthma, followed by a single moderate-level challenge to induce an exacerbation of inflammation. We then assessed airway inflammation, epithelial changes characteristic of remodelling, airway hyperresponsiveness (AHR) and host immunological responses.

Results

Allergic airway inflammation, including recruitment of eosinophils, was prominent only in animals that had recovered from neonatal infection with PVM and then been sensitised and chronically challenged with antigen. Furthermore, only these mice exhibited an augmented Th2-biased immune response, including elevated serum levels of anti-ovalbumin IgE and IgG₁ as well as increased relative expression of Th2-associated cytokines IL-4, IL-5 and IL-13. By comparison, development of AHR and mucous cell change were associated with recovery from PVM infection, regardless of subsequent allergen challenge. Increased expression of IL-25, which could contribute to induction of a Th2 response, was demonstrable in the lung following PVM infection. Signalling via the IL-4 receptor α chain was crucial to the development of allergic inflammation, mucous cell change and AHR, because all of these were absent in receptor-deficient mice. In contrast, changes of remodelling were evident in mice that received chronic allergen challenge, regardless of neonatal PVM infection, and were not dependent on signalling via the IL-4 receptor.

Conclusion

In this mouse model, interaction between early-life viral infection and allergen sensitisation/challenge is essential for development of the characteristic features of childhood asthma, including allergic inflammation and a Th2-biased immune response.     

Increase of nitrosative stress in patients with eosinophilic pneumonia

Background

Exhaled nitric oxide (NO) production is increased in asthma and reflects the degree of airway inflammation. The alveolar NO concentration (Calv) in interstitial pneumonia is reported to be increased. However, it remains unknown whether NO production is increased and nitrosative stress occurs in eosinophilic pneumonia (EP). We hypothesized that nitrosative stress markers including Calv, inducible type of NO synthase (iNOS), and 3-nitrotyrosine (3-NT), are upregulated in EP.

Methods

Exhaled NO including fractional exhaled NO (FE_NO) and Calv was measured in ten healthy subjects, 13 patients with idiopathic pulmonary fibrosis (IPF), and 13 patients with EP. iNOS expression and 3-NT formation were assessed by immunocytochemistory in BALf cells. The exhaled NO, lung function, and systemic inflammatory markers of the EP patients were investigated after corticosteroid treatment for 4 weeks.

Results

The Calv levels in the EP group (14.4 ± 2.0 ppb) were significantly higher than those in the healthy subjects (5.1 ± 0.6 ppb, p < 0.01) and the IPF groups (6.3 ± 0.6 ppb, p < 0.01) as well as the FE_NO and the corrected Calv levels (all p < 0.01). More iNOS and 3-NT positive cells were observed in the EP group compared to the healthy subject and IPF patient. The Calv levels had significant positive correlations with both iNOS (r = 0.858, p < 0.05) and 3-NT positive cells (r = 0.924, p < 0.01). Corticosteroid treatment significantly reduced both the FE_NO (p < 0.05) and the Calv levels (p < 0.01). The magnitude of reduction in the Calv levels had a significant positive correlation with the peripheral blood eosinophil counts (r = 0.802, p < 0.05).

Conclusions

These results suggested that excessive nitrosative stress occurred in EP and that Calv could be a marker of the disease activity.     

The clinical features of the overlap between COPD and asthma

Background

The coexistence of COPD and asthma is widely recognized but has not been well described. This study characterizes clinical features, spirometry, and chest CT scans of smoking subjects with both COPD and asthma.

Methods

We performed a cross-sectional study comparing subjects with COPD and asthma to subjects with COPD alone in the COPDGene Study.

Results

119 (13%) of 915 subjects with COPD reported a history of physician-diagnosed asthma. These subjects were younger (61.3 vs 64.7 years old, p = 0.0001) with lower lifetime smoking intensity (43.7 vs 55.1 pack years, p = 0.0001). More African-Americans reported a history of asthma (33.6% vs 15.6%, p < 0.0001). Subjects with COPD and asthma demonstrated worse disease-related quality of life, were more likely to have had a severe COPD exacerbation in the past year, and were more likely to experience frequent exacerbations (OR 3.55 [2.19, 5.75], p < 0.0001). Subjects with COPD and asthma demonstrated greater gas-trapping on chest CT. There were no differences in spirometry or CT measurements of emphysema or airway wall thickness.

Conclusion

Subjects with COPD and asthma represent a relevant clinical population, with worse health-related quality of life. They experience more frequent and severe respiratory exacerbations despite younger age and reduced lifetime smoking history.

Trial registration

ClinicalTrials.gov: {"type":"clinical-trial","attrs":{"text":"NCT00608764","term_id":"NCT00608764"}}NCT00608764     

MMP-9 gene variants increase the risk for non-atopic asthma in children

Background

Atopic and non-atopic wheezing may be caused by different etiologies: while eosinophils are more important in atopic asthmatic wheezers, neutrophils are predominantly found in BAL samples of young children with wheezing. Both neutrophils as well as eosinophils may secrete matrix metalloproteinase 9 (MMP-9). Considering that MMP-9 plays an important role in airway wall thickening and airway inflammation, it may influence the development of obstructive airway phenotypes in children. In the present study we investigated whether genetic variations in MMP-9 influence the development of different forms of childhood asthma.

Methods

Genotyping of four HapMap derived tagging SNPs in the MMP-9 gene was performed using MALDI-TOF MS in three cross sectional study populations of German children (age 9-11; N = 4,264) phenotyped for asthma and atopic diseases according to ISAAC standard procedures. Effects of single SNPs and haplotypes were studied using SAS 9.1.3 and Haploview.

Results

SNP rs2664538 significantly increased the risk for non-atopic wheezing (OR 2.12, 95%CI 1.40-3.21, p < 0.001) and non-atopic asthma (OR 1.66, 95%CI 1.12-2.46, p = 0.011). Furthermore, the minor allele of rs3918241 may be associated with decreased expiratory flow measurements in non-atopic children. No significant effects on the development of atopy or total serum IgE levels were observed.

Conclusions

Our results have shown that homozygocity for MMP-9 variants increase the risk to develop non-atopic forms of asthma and wheezing, which may be explained by a functional role of MMP-9 in airway remodeling. These results suggest that different wheezing disorders in childhood are affected differently by genetic alterations.     

Comparison of exhaled breath condensate pH using two commercially available devices in healthy controls,asthma and COPD patients

Background

Analysis of exhaled breath condensate (EBC) is a non-invasive method for studying the acidity (pH) of airway secretions in patients with inflammatory lung diseases.

Aim

To assess the reproducibility of EBC pH for two commercially available devices (portable RTube and non-portable ECoScreen) in healthy controls, patients with asthma or COPD, and subjects suffering from an acute cold with lower-airway symptoms. In addition, we assessed the repeatability in healthy controls.

Methods

EBC was collected from 40 subjects (n = 10 in each of the above groups) using RTube and ECoScreen. EBC was collected from controls on two separate occasions within 5 days. pH in EBC was assessed after degasification with argon for 20 min.

Results

In controls, pH-measurements in EBC collected by RTube or ECoScreen showed no significant difference between devices (p = 0.754) or between days (repeatability coefficient RTube: 0.47; ECoScreen: 0.42) of collection. A comparison between EBC pH collected by the two devices in asthma, COPD and cold patients also showed good reproducibility. No differences in pH values were observed between controls (mean pH 8.27; RTube) and patients with COPD (pH 7.97) or asthma (pH 8.20), but lower values were found using both devices in patients with a cold (pH 7.56; RTube, p < 0.01; ECoScreen, p < 0.05).

Conclusion

We conclude that pH measurements in EBC collected by RTube and ECoScreen are repeatable and reproducible in healthy controls, and are reproducible and comparable in healthy controls, COPD and asthma patients, and subjects with a common cold.     

Corticosteroid suppression of lipoxin A4 and leukotriene B4from alveolar macrophages in severe asthma

Background

An imbalance in the generation of pro-inflammatory leukotrienes, and counter-regulatory lipoxins is present in severe asthma. We measured leukotriene B₄ (LTB₄), and lipoxin A₄ (LXA₄) production by alveolar macrophages (AMs) and studied the impact of corticosteroids.

Methods

AMs obtained by fiberoptic bronchoscopy from 14 non-asthmatics, 12 non-severe and 11 severe asthmatics were stimulated with lipopolysaccharide (LPS,10 μg/ml) with or without dexamethasone (10^-6M). LTB₄ and LXA₄ were measured by enzyme immunoassay.

Results

LXA₄ biosynthesis was decreased from severe asthma AMs compared to non-severe (p < 0.05) and normal subjects (p < 0.001). LXA₄ induced by LPS was highest in normal subjects and lowest in severe asthmatics (p < 0.01). Basal levels of LTB₄ were decreased in severe asthmatics compared to normal subjects (p < 0.05), but not to non-severe asthma. LPS-induced LTB₄ was increased in severe asthma compared to non-severe asthma (p < 0.05). Dexamethasone inhibited LPS-induced LTB₄ and LXA₄, with lesser suppression of LTB₄ in severe asthma patients (p < 0.05). There was a significant correlation between LPS-induced LXA₄ and FEV₁ (% predicted) (r_s = 0.60; p < 0.01).

Conclusions

Decreased LXA₄ and increased LTB₄ generation plus impaired corticosteroid sensitivity of LPS-induced LTB₄ but not of LXA₄ support a role for AMs in establishing a pro-inflammatory balance in severe asthma.