Airway Remodeling In Mice Research Articles

Toll-Like Receptor-9 Agonist Inhibits Airway Inflammation, Remodeling and Hyperreactivity in Mice Exposed to Chronic Environmental Tobacco Smoke and Allergen

Background: As passive environmental tobacco smoke (ETS) exposure in nonsmokers can increase both asthma symptoms and the frequency of asthma exacerbations, we utilized a mouse model, in which ovalbumin (OVA) + ETS induce significantly increased levels of eosinophilic airway inflammation and remodeling compared to either stimulus alone, to determine whether a Toll-like receptor-9 (TLR-9) agonist could reduce levels of airway inflammation, airway remodeling and airway hyperreactivity (AHR). Methods: Mice treated with or without a TLR-9 agonist were sensitized to OVA and challenged with OVA + ETS for 1 month. AHR to methacholine was assessed in intubated and ventilated mice. Lung Th2 cytokines and TGF-β₁ were measured by ELISA. Lungs were processed for histology and immunohistology to quantify eosinophils, mucus, peribronchial fibrosis and smooth muscle changes using image analysis. Results: Administration of a TLR-9 agonist to mice coexposed to chronic ETS and chronic OVA allergen significantly reduced levels of eosinophilic airway inflammation, mucus production, peribronchial fibrosis, the thickness of the peribronchial smooth muscle layer, and AHR. The reduced airway remodeling in mice treated with the TLR-9 agonist was associated with significantly reduced numbers of peribronchial MBP+ and peribronchial TGF-β₁+ cells, and with significantly reduced levels of lung Th2 cytokines [interleukin-5 and interleukin-13] and TGF-β₁. Conclusion: These studies demonstrate that TLR-9-based therapies inhibit airway inflammation, remodeling and AHR in mice coexposed to ETS and allergen who exhibit enhanced airway inflammation and remodeling.

Effects of Pingchuan Recipe on airway remodeling in mice with bronchial asthma

To study the effects of Pingchuan Recipe, a compound traditional Chinese herbal medicine for treating bronchial asthma, on macrophage inflammatory protein-1alpha (MIP-1alpha) and immunoglobulin E (IgE) contents and CD86 expression in a mouse model of bronchial asthma, and to investigate the mechanism of Pingchuan Recipe in regulating airway remodeling in mice with bronchial asthma. Fifty BALB/c mice were randomly divided into normal control group, untreated group, dexamethasone group, low-dose Pingchuan Recipe group and normal-dose Pingchuan Recipe group. Bronchial asthma in mice was induced by intra-abdominal injection of mixed ovalbumin and stimulation by inhaling 5% ovalbumin. Mice in the normal control group and the untreated group did not receive treatment, but mice in the other groups were administered intragastrically with 50 g/ (kg * d) Pingchuan Recipe, 25 g/ (kg * d) Pingchuan Recipe and 0.75 mg/ (kg * d) dexamethasone, respectively. After consecutive treatment for 8 weeks, the MIP-1alpha contents in peripheral blood and bronchoalveolar lavage fluid (BALF) were measured by an avidin biotin complex-enzyme-linked immunosorbent assay (ABC-ELISA) system, and the IgE contents in serum and BALF were measured by ELISA method. Direct immunofluorescence-flow cytometry was used to detect the percentages of CD86(+) and CD3(-) CD86(+) cells in peripheral blood and BALF. General histological changes of the lung tissue were observed by HE staining and collagen fiber staining 12 weeks after activation. Compared with the normal control group, the contents of MIP-1alpha, the percentages of CD86(+) cells and CD3(-) CD86(+) cells in peripheral blood and BALF, and the contents of IgE in serum and BALF were raised significantly (P < 0.01). The contents of MIP-1alpha, the percentages of CD86(+) cells and CD3(-) CD86(+) cells in peripheral blood and BALF, and the contents of IgE in serum and BALF in the treated groups were lower than those in the untreated group (P < 0.01). The contents of MIP-1alpha in peripheral blood and BALF and the contents of IgE in serum and BALF had no significant differences among the treated groups (P > 0.05). The percentage of CD3(-) CD86(+) cells in peripheral blood in low-dose Pingchuan Recipe group was lower than that in the dexamethasone group. Compared with the untreated group, the collagen deposition in the wall of bronchiole in the treated groups was decreased. Pingchuan Recipe can decrease the contents of MIP-1alpha, IgE and the expressions of CD86 in peripheral blood and BALF, which may be one of its mechanisms in improving chronic airway inflammation and inhibiting airway remodeling.

Osteopontin Induces Airway Remodeling and Lung Fibroblast Activation in a Murine Model of Asthma

Airway remodeling is a central feature of asthma; however, the mechanisms underlying its development have not been fully elucidated. We have demonstrated that osteopontin, an inflammatory cytokine and an extracellular matrix glycoprotein with profibrotic properties, is up-regulated in a murine model of allergen-induced airway remodeling. In the present study, we determined whether osteopontin plays a functional role in airway remodeling. Osteopontin (OPN)-deficient (OPN(-/-)) and wild-type mice were sensitized and exposed to inhaled ovalbumin (OVA) or saline for 5 weeks. Collagen production, peribronchial smooth muscle area, mucus-producing cell number, and bronchoalveolar cell counts were assessed. The functional behavior and phenotype of lung fibroblasts from OVA-treated OPN(-/-) and from wild-type mice were studied using ex vivo cultures. OVA-treated OPN(-/-) mice exhibited reduced lung collagen content, smooth muscle area, mucus-producing cells, and inflammatory cell accumulation as compared with wild-type mice. Reduced matrix metalloproteinase-2 activity and expression of transforming growth factor-beta1 and vascular endothelial growth factor were observed in OVA-treated OPN(-/-) mice. Lung fibroblasts from OVA-treated OPN(-/-) mice showed reduced proliferation, migration, collagen deposition, and alpha-smooth muscle actin expression in comparison with OVA-treated wild-type lung fibroblasts. Thus, OPN is key for the development of allergen-induced airway remodeling in mice. In response to allergen, OPN induces the switching of lung fibroblasts to a pro-fibrogenic myofibroblast phenotype.

Repeated instillations of Dermatophagoides farinae into the airways can induce Th2-dependent airway hyperresponsiveness, eosinophilia and remodeling in mice : Effect of intratracheal treatment of fluticasone propionate

Dermatophagoides farinae are known to be a common environmental allergen causing allergic asthma; however, little is known about their pathophysiological effect via the allergenicities in vivo. Therefore, we first established a mouse model of asthma induced by repeated instillations of D. farinae. Second, to investigate whether the asthmatic responses are Th2-dependent, we examined the effect of the deficiency of interleukin-4 (IL-4) receptor α chain gene. Finally, we examined the effect of fluticasone propionate on this model. Mice were instilled with D. farinae without additional adjuvants into the trachea 8 times. After the final allergen instillation, the airway responsiveness to acetylcholine was measured, and bronchoalveolar lavage and histological examination were carried out. The instillation of the allergen-induced airway hyperresponsiveness, the accumulation of inflammatory cells and increases in the levels of Th2 cytokines and transforming growth factor-β 1 production in the bronchoalveolar lavage fluid dose dependently. The number of goblet cells in the epithelium and the extent of the fibrotic area beneath the basement membrane were also increased in the morphometric study. In contrast, the defect of IL-4/IL-13 signaling through IL-4 receptor α chain completely abrogated all these responses. Furthermore, the simultaneous instillation of fluticasone propionate with the allergen showed significant inhibition or an inhibitory tendency of these changes. These findings demonstrate that the repetitive intratracheal instillations of D. farinae can induce airway remodeling through Th2-type inflammation, and that fluticasone propionate inhibits D. farinae-induced airway remodeling in mice, and this model would be useful for studying mechanisms involved in the development of allergic asthma.

Aerobic Exercise Decreases Chronic Allergic Lung Inflammation and Airway Remodeling in Mice

Aerobic conditioning improves exercise capacity and decreases symptoms in patients with asthma. However, its benefits in the context of allergic airway inflammation are poorly understood. To evaluate the effects of two intensities of aerobic exercise on airway inflammation and remodeling in a model of chronic allergic lung inflammation. Mice were subjected to chronic ovalbumin (OVA) sensitization and to 4 weeks of low (OVA+Low) or moderate (OVA+Mod) exercise training in a treadmill. Airway inflammation and remodeling and expression of helper T-cell type 1 and 2 cytokines were evaluated. OVA-induced allergic airway inflammation and remodeling were characterized by an increase in collagen (288%), elastic fiber (56%), smooth muscle (380%), and epithelial (402%) contents (P < 0.001) when compared with the control group. OVA+Low and OVA+Mod groups presented a decrease in bronchoalveolar lavage fluid eosinophils (respectively, 84 and 75%; P < 0.01) and airway walls (respectively, 94 and 58%; P < 0.001) when compared with the OVA group. OVA+Low and OVA+Mod groups also presented a reduction in the number of peribronchial inflammatory cells expressing IL-4 (respectively, 85 and 75%; P < 0.01) and IL-5 (respectively, 88 and 89%; P < 0.01) when compared with the OVA group. Aerobic conditioning did not change the expression of either IFN-gamma or IL-2 by inflammatory cells or plasma levels of IgE or IgG1. OVA+Low and OVA+Mod groups presented an increase in the expression of IL-10 (P < 0.001). Low and moderate aerobic conditioning also reduced airway remodeling in OVA-sensitized mice when compared with the OVA group. We concluded that low and moderate aerobic exercise decreases airway inflammation and remodeling in a murine model of asthma.

Creatine Supplementation Exacerbates Allergic Lung Inflammation and Airway Remodeling in Mice

Creatine supplement is the most popular nutritional supplement, and has various metabolic functions and sports medicine applications. Creatine supplementation increases muscle mass and can decrease muscular inflammation. Some studies have also suggested a beneficial role of creatine supplementation on chronic pulmonary diseases such as chronic obstructive pulmonary disease and cystic fibrosis. Among athletes, the prevalence of asthma is high, and many of these individuals may be taking creatine. However, the effects of creatine supplementation on chronic pulmonary diseases of allergic origin have not been investigated. In the present study, we analyzed the effects of creatine supplementation on a model of chronic allergic lung inflammation. Thirty-one Balb/c mice were divided into four groups: control, creatine (Cr), ovalbumin (OVA), and OVA+Cr. OVA and OVA+Cr groups were sensitized with intraperitoneal injections of OVA on Days 0, 14, 28, and 42. OVA challenge (OVA 1%) and Cr treatment (0.5 g/kg/d) were initiated on Day 21 and lasted until Day 53. We determined the index of hyperresponsiveness, the serum levels of OVA-specific immunoglobulin (Ig)E and IgG(1), and the total and differential cell counts in bronchoalveolar lavage fluid. We also quantified airway inflammation, and the airway density of IL-4+, IL-5+, IL-2+, IFN-gamma+, and insulin-like growth factor (IGF)-1+ cells, collagen and elastic fibers, and airway smooth muscle thickness. Our results showed that creatine in OVA-sensitized mice increased hyperresponsiveness; eosinophilic inflammation; airway density of IL-4+, IL-5+, and IGF-1 inflammatory cells; airway collagen and elastin content; and smooth muscle thickness. The results show that creatine supplementation exacerbates the lung allergic response to OVA through a T helper cell type 2 pathway and increased IGF-1 expression.

Coexposure to Environmental Tobacco Smoke Increases Levels of Allergen-Induced Airway Remodeling in Mice

Environmental tobacco smoke (ETS) can increase asthma symptoms and the frequency of asthma attacks. However, the contribution of ETS to airway remodeling in asthma is at present unknown. In this study, we have used a mouse model of allergen-induced airway remodeling to determine whether the combination of chronic exposure to ETS and chronic exposure to OVA allergen induces greater levels of airway remodeling than exposure to either chronic ETS or chronic OVA allergen alone. Mice exposed to chronic ETS alone did not develop significant eosinophilic airway inflammation, airway remodeling, or increased airway hyperreactivity to methacholine. In contrast, mice exposed to chronic OVA allergen had significantly increased levels of peribronchial fibrosis, increased thickening of the smooth muscle layer, increased mucus, and increased airway hyperreactivity which was significantly enhanced by coexposure to the combination of chronic ETS and chronic OVA allergen. Mice coexposed to chronic ETS and chronic OVA allergen had significantly increased levels of eotaxin-1 expression in airway epithelium which was associated with increased numbers of peribronchial eosinophils, as well as increased numbers of peribronchial cells expressing TGF-beta1. These studies suggest that chronic coexposure to ETS significantly increases levels of allergen-induced airway remodeling (in particular smooth muscle thickness) and airway responsiveness by up-regulating expression of chemokines such as eotaxin-1 in airway epithelium with resultant recruitment of cells expressing TGF-beta1 to the airway and enhanced airway remodeling.

Remodeling associated expression of matrix metalloproteinase 9 but not tissue inhibitor of metalloproteinase 1 in airway epithelium: Modulation by immunostimulatory DNA

Matrix metalloproteinase 9 (MMP-9) and its tissue inhibitor of metalloproteinase 1 (TIMP-1) are hypothesized to play a role in the pathogenesis of airway remodeling in asthma. We have used a mouse model of airway remodeling to determine the pattern of expression of MMP-9 and TIMP-1 in airway epithelium and peribronchial cells, and assess whether TIMP-1, an inhibitor of MMP-9, is expressed at the same sites in the airway. In addition, we have investigated whether immunostimulatory sequences (ISSs) of DNA modulate levels of expression of MMP-9, TIMP-1, and peribronchial fibrosis. Levels of lung MMP-9 and TIMP-1 were assessed by zymography, ELISA, and immunohistochemistry. Repetitive ovalbumin challenge induced a significant increase in levels of MMP-9, TIMP-1, and peribronchial collagen deposition. The pattern of expression of MMP-9 and TIMP-1 in the remodeled airway was significantly different. MMP-9 but not TIMP-1 was expressed in airway epithelium, whereas both MMP-9 and TIMP-1 were expressed in peribronchial inflammatory cells. ISS significantly reduced expression of MMP-9 in airway epithelium (which immunostained positive for Toll receptor 9), as well as in peribronchial inflammatory cells. In vitro studies demonstrated that ISS inhibited bone marrow macrophage generation of MMP-9. Allergen-induced peribronchial fibrosis is associated with expression of MMP-9 and TIMP-1 at different anatomical sites in the remodeled airway. The ability of ISS to inhibit the expression of MMP-9 in airway epithelium (a site where its inhibitor TIMP-1 is not induced by allergen challenge) may be important in determining whether ISS contributes to reductions in airway remodeling by reducing levels of MMP-9. Immunostimulatory sequences of DNA, which are being investigated as novel therapeutics in asthma, inhibit airway remodeling in mice as well as epithelial expression of MMP-9, an enzyme that degrades the extracellular matrix proteins surrounding the airway.

Combination of corticosteroid therapy and allergen avoidance reverses allergen-induced airway remodeling in mice

Allergen avoidance and anti-inflammatory therapy are standard therapeutic approaches guidelines advocate to control asthma symptoms. Currently, it is not known whether such strategies reduce airway remodeling. We have therefore used a mouse model of allergen-induced airway remodeling to determine whether allergen avoidance combined with corticosteroid therapy can reverse established airway remodeling. Mice were sensitized to ovalbumin and then repetitively challenged with intranasal ovalbumin for 3 months to develop structural features of airway remodeling including peribronchial fibrosis and increased thickness of the peribronchial smooth muscle layer. At this time point, mice were treated with allergen avoidance, allergen avoidance and corticosteroids, or corticosteroids for 1 month to determine whether either strategy could reverse established airway remodeling. Mice repetitively challenged with ovalbumin developed peribronchial fibrosis (increased total lung collagen and increased peribronchial trichrome staining) as well as increased thickness of the peribronchial smooth muscle layer. Allergen avoidance significantly reduced airway inflammation and mucus expression, slightly reduced peribronchial fibrosis, and had no effect on the thickness of the peribronchial smooth muscle layer. Addition of corticosteroids to allergen avoidance significantly reduced levels of peribronchial fibrosis as well as the thickness of the peribronchial smooth muscle layer. Allergen avoidance reduces airway inflammation and mucus expression but has more limited immediate effects on reducing structural features of established airway remodeling. The combination of allergen avoidance and corticosteroid therapy is effective in reversing established features of airway remodeling including peribronchial fibrosis and the increased thickness of the smooth muscle layer.

Role of interleukin-5 and eosinophils in allergen-induced airway remodeling in mice.

Asthma is a chronic inflammatory disease characterized by variable bronchial obstruction, hyperresponsiveness, and by tissue damage known as airway remodeling. In the present study we demonstrate that interleukin (IL)-5 plays an obligatory role in the airway remodeling observed in experimental asthma. BALB/c mice sensitized by intraperitoneal injections of ovalbumin and exposed daily to aerosol of ovalbumin for up to 3 wk, develop eosinophilic infiltration of the bronchi and subepithelial and peribronchial fibrosis. The lesions are associated with increased amounts of hydroxyproline in the lungs and elevated levels of eosinophils and transforming growth factor (TGF)-beta1 in the bronchoalveolar lavage fluid. After 1 wk of allergen challenge, TGF-beta is mainly produced by eosinophils accumulated in the peribronchial and perivascular lesions. At a later stage of the disease, the main source of TGF-beta is myofibroblasts, identified by alpha-smooth muscle actin mAb. We show that all these lesions, including fibrosis, are abolished in sensitized and allergen-exposed IL-5 receptor-null mice, whereas they are markedly accentuated in IL-5 transgenic animals. More importantly, treatment of wild-type mice with neutralizing anti-IL-5 antibody, administered before each allergen challenge, almost completely prevented subepithelial and peribronchial fibrosis. These findings demonstrated that eosinophils are involved in allergen-induced subepithelial and peribronchial fibrosis probably by producing a fibrogenic factor, TGF-beta1.

Immunostimulatory DNA inhibits transforming growth factor-beta expression and airway remodeling.

Immunostimulatory sequences of DNA (ISS) inhibit eosinophilic airway inflammation, Th2 responses, and airway hyperreactivity (AHR) in mouse models of acute ovalbumin (OVA)-induced airway inflammation. To determine whether ISS inhibits airway remodeling, we developed a mouse model of airway remodeling in which OVA-sensitized mice were repeatedly exposed to intranasal OVA administration for 1-6 mo. Mice chronically exposed to OVA developed sustained eosinophilic airway inflammation and sustained AHR to methacholine compared with control mice. In addition, the mice chronically exposed to OVA developed features of airway remodeling, including thickening of the peribronchial smooth muscle layer, peribronchial myofibroblast accumulation, expression of the profibrotic growth factor transforming growth factor-beta, and subepithelial collagen deposition (assessed by quantitation of the area of peribronchial trichrome staining using image analysis, and immunostaining with anti-collagen V antibodies). Administration of ISS systemically every other week significantly inhibited the development of AHR, eosinophilic inflammation, airway mucus production, and importantly, airway remodeling in mice chronically exposed to OVA for 3-6 mo. In addition, ISS significantly reduced bronchoalveolar lavage and lung levels of the profibrotic cytokine transforming growth factor-beta. These studies demonstrate that ISS prevents not only Th2-mediated airway inflammation in response to acute allergen challenge, but also airway remodeling associated with chronic allergen challenge.

Morphometric Analysis of Mouse Airways After Chronic Allergen Challenge

Understanding the mechanisms of airway remodeling in chronic allergic conditions such as asthma is increasingly dependent on the use of animal models. Techniques for quantifying structural changes are required that are reproducible and responsive and that can be applied to different staining techniques in both human and animal airway tissues. Here, we characterize a morphometric technique to quantify changes in extracellular matrix and contractile tissue as two indices of airway remodeling in mice. Specific aims were to establish the optimum projection beneath the epithelium to assess remodeling changes and to determine whether such changes are reproducible within different areas of the lung. Finally, based on the variance within measurements, we calculated sample size requirements for research applications of this technique. BALB/c mice were sensitized to ovalbumin and studied after chronic allergen challenge. Lungs were formalin fixed and sectioned were then assayed for extracellular matrix or contractile tissue using morphometric/colorimetric techniques. In this model, the optimum projected distance to measure changes in extracellular matrix or contractile tissue was 20 μm beneath the epithelium; projecting beyond this depth resulted in decreased ability to detect allergen-induced changes (signal) because of increased irrelevant staining of surrounding parenchymal tissue (noise). The technique was responsive, because an allergen-induced signal was detected in all airway sections and all lung regions studied (p < 0.05). The power of this analysis was such that allergen-induced changes can be reliably (>80% power) detected using 8 to 10 mice. This morphometric technique provides a valid and objective method to assess structural changes in the airways of mice after chronic allergen exposure.

Time course study on the development of allergen-induced airway remodeling in mice: the effect of allergen avoidance on established airway remodeling.

We carried out a time course study on the development of allergen-induced airway remodeling in a mouse model of allergic asthma. Moreover, we examined the effect of allergen avoidance on the established airway remodeling. BALB/c mice were sensitized to ovalbumin (OA) with alum, and exposed daily for 3 weeks to aerosolized OA. At each designated point, bronchial responsiveness was measured, and bronchoalveolar lavage and histological examination were carried out. The numbers of inflammatory leukocytes in the airways and the percentage of goblet cells in the epithelium, Th2 cytokine production, IgE production, collagen deposition beneath the basement membrane and bronchial responsiveness to acetylcholine were all markedly increased after repeated antigen challenge for 1-3 weeks. In contrast, after cessation of antigen exposure, goblet cell hyperplasia, inflammatory infiltrates and bronchial hyperresponsiveness were gradually attenuated and had almost resolved 4 weeks after cessation, but subepithelial fibrosis was still observed at this time point. The present findings demonstrated that epithelial changes following repeated allergen challenge are rapidly induced and recover after the cessation of exposure, but subepithelial fibrosis has a late onset and relatively irreversible changes, and subepithelial fibrosis in contrast to goblet cells hyperplasia did not appear to contribute to bronchial hyperresponsiveness, at least, in this mouse model.

Antigen challenge and airway remodeling in mice

Antigen challenge and airway remodeling in mice

Tuberculosis and Bronchiectasis

Although airway remodeling is a central feature of COPD, the mechanisms underlying its development have not been fully elucidated. The goal of this study was to determine whether histone deacetylase (HDAC) 2 protects against cigarette smoke (CS)-induced airway remodeling through IL-17A-dependent mechanisms.Sputum samples and lung tissue specimens were obtained from control subjects and patients with COPD. The relationships between HDAC2, IL-17A, and airway remodeling were investigated. The effect of HDAC2 on IL-17A-mediated airway remodeling was assessed by using in vivo models of COPD induced by CS and in vitro culture of human bronchial epithelial cells and primary human fibroblasts exposed to CS extract, IL-17A, or both.HDAC2 and IL-17A expression in the sputum cells and lung tissue samples of patients with COPD were associated with bronchial wall thickening and collagen deposition. Il-17a deficiency (Il-17a–/–) resulted in attenuation of, whereas Hdac2 deficiency (Hdac2+/–) exacerbated, CS-induced airway remodeling in mice. IL-17A deletion also attenuated airway remodeling in CS-exposed Hdac2+/– mice. HDAC2 regulated IL-17A production partially through modulation of CD4+ T cells during T helper 17 cell differentiation and retinoid-related orphan nuclear receptor γt in airway epithelial cells. In vitro, IL-17A deficiency attenuated CS-induced mouse fibroblast activation from Hdac2+/– mice. IL-17A-induced primary human fibroblast activation was at least partially mediated by autocrine production of transforming growth factor beta 1.These findings suggest that activation of HDAC2 and/or inhibition of IL-17A production could prevent the development of airway remodeling by suppressing airway inflammation and modulating fibroblast activation in COPD.