The exaggerated expression of chitinase-like protein YKL-40, the human homologue of breast regression protein–39 (BRP-39), was reported in a number of diseases, including chronic obstructive pulmonary disease (COPD). However, the in vivo roles of YKL-40 in normal physiology or in the pathogenesis of specific diseases such as COPD remain poorly understood. We hypothesized that BRP-39/YKL-40 plays an important role in the pathogenesis of cigarette smoke (CS)–induced emphysema. To test this hypothesis, 10-week-old wild-type and BRP-39 null mutant mice (BRP-39−/−) were exposed to room air (RA) and CS for up to 10 months. The expression of BRP-39 was significantly induced in macrophages, airway epithelial cells, and alveolar Type II cells in the lungs of CS-exposed mice compared with RA-exposed mice, at least in part via an IL-18 signaling–dependent pathway. The null mutation of BRP-39 significantly reduced CS-induced bronchoalveolar lavage and tissue inflammation. However, CS-induced epithelial cell apoptosis and alveolar destruction were further enhanced in the absence of BRP-39. Consistent with these findings in mice, the tissue expression of YKL-40 was significantly increased in the lungs of current smokers compared with the lungs of ex-smokers or nonsmokers. In addition, serum concentrations of YKL-40 were significantly higher in smokers with COPD than in nonsmokers or smokers without COPD. These studies demonstrate a novel regulatory role of BRP-39/YKL-40 in CS-induced inflammation and emphysematous destruction. These studies also underscore that maintaining physiologic concentrations of YKL-40 in the lung is therapeutically important in preventing excessive inflammatory responses or emphysematous alveolar destruction.
YKL-40/BRP-39; COPD; emphysema; cigarette smoke
BRP-39 and its human homolog YKL-40 have been regarded as a prototype of chitinase-like proteins (CLP) in mammals. Exaggerated levels of YKL-40 protein and/or mRNA have been noted in a number of diseases characterized by inflammation, tissue remodeling, and aberrant cell growth. Asthma is an inflammatory disease characterized by airway hyperresponsiveness and airway remodeling. Recently, the novel regulatory role of BRP-39/YKL-40 in the pathogenesis of asthma has been demonstrated both in human studies and allergic animal models. The levels of YKL-40 are increased in the circulation and lungs from asthmatics where they correlate with disease severity, and CHI3L1 polymorphisms correlate with serum YKL-40 levels, asthma and abnormal lung function. Animal studies using BRP-39 null mutant mice demonstrated that BRP-39 was required for optimal allergen sensitization and Th2 inflammation. These studies suggest the potential use of BRP-39 as a biomarker as well as a therapeutic target for asthma and other allergic diseases. Here, we present an overview of chitin/chitinase biology and summarize recent findings on the role of BRP-39 in the pathogenesis of asthma and allergic responses.
BRP-39; human CHI3L1 protein; asthma; hypersensitivity
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).
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 (RL) and dynamic compliance (Cdyn). 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 RL and the decrease in baseline Cdyn caused by chronic exposure to inhaled HDM.
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.
Mouse breast regression protein 39 (BRP-39; Chi3l1) and its human homologue YKL-40 are chitinase-like proteins that lack chitinase activity. Although YKL-40 is expressed in exaggerated quantities and correlates with disease activity in asthma and many other disorders, the biological properties of BRP-39/YKL-40 have only been rudimentarily defined. We describe the generation and characterization of BRP-39−/− mice, YKL-40 transgenic mice, and mice that lack BRP-39 and produce YKL-40 only in their pulmonary epithelium. Studies of these mice demonstrated that BRP-39−/− animals have markedly diminished antigen-induced Th2 responses and that epithelial YKL-40 rescues the Th2 responses in these animals. The ability of interleukin13 to induce tissue inflammation and fibrosis was also markedly diminished in the absence of BRP-39. Mechanistic investigations demonstrated that BRP-39 and YKL-40 play an essential role in antigen sensitization and immunoglobulin E induction, stimulate dendritic cell accumulation and activation, and induce alternative macrophage activation. These proteins also inhibit inflammatory cell apoptosis/cell death while inhibiting Fas expression, activating protein kinase B/AKT, and inducing Faim 3. These studies establish novel regulatory roles for BRP-39/YKL-40 in the initiation and effector phases of Th2 inflammation and remodeling and suggest that these proteins are therapeutic targets in Th2- and macrophage-mediated disorders.
The mammalian target of rapamycin (mTOR) signaling pathway integrates environmental cues, promotes cell growth / differentiation and regulates immune responses. While inhibition of mTOR with rapamycin has potent immunosuppressive activity, mixed effects have been reported in ovalbumin-induced models of allergic asthma. We investigated the impact of two different rapamycin treatment protocols on the major characteristics of allergic asthma induced by the clinically relevant allergen, house dust mite (HDM). In protocol 1, Balb/c mice were exposed to 10 intranasal HDM doses over a period of 24 days and treated with rapamycin simultaneously during the sensitization/exposure period. In protocol 2, rapamycin was administered after the mice had been sensitized to HDM (I.P. injection) and prior to initiation of two intranasal HDM challenges over 4 days. Airway hyperreactivity (AHR), IgE, inflammatory cells, cytokines, leukotrienes, goblet cells, and activated T cells were assessed. In protocol 1, rapamycin blocked HDM-induced increases in AHR, inflammatory cell counts, IgE, and attenuated goblet cell metaplasia. In protocol 2, rapamycin blocked increases in AHR, IgE, T cell activation, and reduced goblet cell metaplasia, but had no effect on inflammatory cell counts. Increases in IL-13 and leukotrienes were also blocked by rapamycin, although increases in IL-4 were unaffected. These data demonstrate that rapamycin can inhibit cardinal features of allergic asthma including increases in AHR, IgE, and goblet cells most likely due to its ability to reduce the production of two key mediators of asthma, IL-13 and leukotrienes. These findings highlight the importance of the mTOR pathway in allergic airway disease.
Loss-of-function mutations in the bone morphogenetic protein receptor type 2 (BMPR2) gene have been identified in patients with heritable pulmonary arterial hypertension (PAH); however, disease penetrance is low, suggesting additional factors play a role. Inflammation is associated with PAH and vascular remodeling, but whether allergic inflammation triggers vascular remodeling in individuals with BMPR2 mutations is unknown. Our goal was to determine if chronic allergic inflammation would induce more severe vascular remodeling and PAH in mice with reduced BMPR-II signaling. Groups of Bmpr2 hypomorph and wild-type (WT) Balb/c/Byj mice were exposed to house dust mite (HDM) allergen, intranasally for 7 or 20 weeks to generate a model of chronic inflammation. HDM exposure induced similar inflammatory cell counts in all groups compared to controls. Muscularization of pulmonary arterioles and arterial wall thickness were increased after 7 weeks HDM, more severe at 20 weeks, but similar in both groups. Right ventricular systolic pressure (RVSP) was measured by direct cardiac catheterization to assess PAH. RVSP was similarly increased in both HDM exposed groups after 20 weeks compared to controls, but not after 7 weeks. Airway hyperreactivity (AHR) to methacholine was also assessed and interestingly, at 20 weeks, was more severe in HDM exposed Bmpr2 hypomorph mice versus WT. We conclude that chronic allergic inflammation caused PAH and while the severity was mild and similar between WT and Bmpr2 hypomorph mice, AHR was enhanced with reduced BMPR-II signaling. These data suggest that vascular remodeling and PAH resulting from chronic allergic inflammation occurs independently of BMPR-II pathway alterations.
Sensitizations to house dust mites (HDM) trigger strong exacerbated allergen-induced inflammation of the skin and airways mucosa from atopic subjects resulting in atopic dermatitis as well as allergic rhinitis and asthma. Initially, the Th2-biased HDM allergic response was considered to be mediated only by allergen B- and T-cell epitopes to promote allergen-specific IgE production as well as IL-4, IL-5, and IL-13 to recruit inflammatory cells. But this general molecular model of HDM allergenicity must be revisited as a growing literature suggests that stimulations of innate immune activation pathways by HDM allergens offer new answers to the following question: what makes an HDM allergen an allergen? Indeed, HDM is a carrier not only for allergenic proteins but also microbial adjuvant compounds, both of which are able to stimulate innate signaling pathways leading to allergy. This paper will describe the multiple ways used by HDM allergens together with microbial compounds to control the initiation of the allergic response through engagement of innate immunity.
Although animal models with ovalbumin have been used to study chronic asthma, there are difficulties in inducing recurrence as well as in maintaining chronic inflammation in this system. Using a murine model of house dust mite (HDM)-induced bronchial asthma, we examined the airway remodeling process in response to the chronic exposure to HDM. During the seventh and twelfth weeks of study, HDM were inhaled through the nose for three consecutive days and airway responsiveness was measured. Twenty-four hours later, bronchoalveolar lavage and histological examination were performed. The degree of overproduction of mucus, subepithelial fibrosis, and the thickness of the peribronchial smooth muscle in the experimental group was clearly increased compared to the control group. In addition, HDM-exposed mice demonstrated severe airway hyperreactivity to methacholine. In the bronchoalveolar lavage fluid, the number of total cells and eosinophils was increased; during the twelfth week, the number of neutrophils increased in the experimental group. With regard to changes in cytokines, the concentrations of IL-4, IL-13, and transforming growth factor-beta (TGF-β) were increased in the experimental group. The data suggest that eosinophils, IL-4, IL-13, and TGF-β might play an important role in the airway remodeling process and that neutrophils may be involved with increased exposure time.
Asthma; Fibrosis; Dermatophagoides farinae; Models
Rationale: Prolonged exposure to 100% O2 causes hyperoxic acute lung injury (HALI), characterized by alveolar epithelial cell injury and death. We previously demonstrated that the murine chitinase-like protein, breast regression protein (BRP)–39 and its human homolog, YKL-40, inhibit cellular apoptosis. However, the regulation and roles of these molecules in hyperoxia have not been addressed.
Objectives: We hypothesized that BRP-39 and YKL-40 (also called chitinase-3–like 1) play important roles in the pathogenesis of HALI.
Methods: We characterized the regulation of BRP-39 during HALI and the responses induced by hyperoxia in wild-type mice, BRP-39–null (−/−) mice, and BRP-39−/− mice in which YKL-40 was overexpressed in respiratory epithelium. We also compared the levels of tracheal aspirate YKL-40 in premature newborns with respiratory failure.
Measurements and Main Results: These studies demonstrate that hyperoxia inhibits BRP-39 in vivo in the murine lung and in vitro in epithelial cells. They also demonstrate that BRP-39−/− mice have exaggerated permeability, protein leak, oxidation, inflammatory, chemokine, and epithelial apoptosis responses, and experience premature death in 100% O2. Lastly, they demonstrate that YKL-40 ameliorates HALI, prolongs survival in 100% O2, and rescues the exaggerated injury response in BRP-39−/− animals. In accord with these findings, the levels of tracheal aspirate YKL-40 were lower in premature infants treated with hyperoxia for respiratory failure who subsequently experienced bronchopulmonary dysplasia or death compared with those that did not experience these complications.
Conclusions: These studies demonstrate that hyperoxia inhibits BRP-39/YKL-40, and that BRP-39 and YKL-40 are critical regulators of oxidant injury, inflammation, and epithelial apoptosis in the murine and human lung.
BRP-39; YKL-40; hyperoxygen; BPD; HALI
Chronic allergic asthma is characterized by Th2-typed inflammation, and contributes to airway remodeling and the deterioration of lung function. However, the initiating factor that links airway inflammation to remodeling is unknown. Thymic stromal lymphopoietin (TSLP), an epithelium-derived cytokine, can strongly activate lung dendritic cells (DCs) through the TSLP-TSLPR and OX40L-OX40 signaling pathways to promote Th2 differentiation. To determine whether TSLP is the underlying trigger of airway remodeling in chronic allergen-induced asthma, we induced allergic airway inflammation in mice by intranasal administration of house dust mite (HDM) extracts for up to 5 consecutive weeks. We showed that repeated respiratory exposure to HDM caused significant airway eosinophilic inflammation, peribronchial collagen deposition, goblet cell hyperplasia, and airway hyperreactivity (AHR) to methacholine. These effects were accompanied with a salient Th2 response that was characterized by the upregulation of Th2-typed cytokines, such as IL-4 and IL-13, as well as the transcription factor GATA-3. Moreover, the levels of TSLP and transforming growth factor beta 1 (TGF-β1) were also increased in the airway. We further demonstrated, using the chronic HDM-induced asthma model, that the inhibition of Th2 responses via neutralization of TSLP with an anti-TSLP mAb reversed airway inflammation, prevented structural alterations, and decreased AHR to methacholine and TGF-β1 level. These results suggest that TSLP plays a pivotal role in the initiation and persistence of airway inflammation and remodeling in the context of chronic allergic asthma.
The mammalian target of rapamycin (mTOR) modulates immune responses and cellular proliferation. The objective of this study was to assess whether inhibition of mTOR with rapamycin modifies disease severity in two experimental murine models of house dust mite (HDM)-induced asthma. In an induction model, rapamycin was administered to BALB/c mice coincident with nasal HDM challenges for 3 weeks. In a treatment model, nasal HDM challenges were performed for 6 weeks and rapamycin treatment was administered during weeks 4 through 6. In the induction model, rapamycin significantly attenuated airway inflammation, airway hyperreactivity (AHR) and goblet cell hyperplasia. In contrast, treatment of established HDM-induced asthma with rapamycin exacerbated AHR and airway inflammation, whereas goblet cell hyperplasia was not modified. Phosphorylation of the S6 ribosomal protein, which is downstream of mTORC1, was increased after 3 weeks, but not 6 weeks of HDM-challenge. Rapamycin reduced S6 phosphorylation in HDM-challenged mice in both the induction and treatment models. Thus, the paradoxical effects of rapamycin on asthma severity paralleled the activation of mTOR signaling. Lastly, mediastinal lymph node re-stimulation experiments showed that treatment of rapamycin-naive T cells with ex vivo rapamycin decreased antigen-specific Th2 cytokine production, whereas prior exposure to in vivo rapamycin rendered T cells refractory to the suppressive effects of ex vivo rapamycin. We conclude that rapamycin had paradoxical effects on the pathogenesis of experimental HDM-induced asthma. Thus, consistent with the context-dependent effects of rapamycin on inflammation, the timing of mTOR inhibition may be an important determinant of efficacy and toxicity in HDM-induced asthma.
Despite its reported pro-inflammatory activity, cyclooxygenase (COX)-2 has been proposed to play a protective role in asthma. Accordingly, COX-2 might be down-regulated in the airway cells of asthmatics. This, together with results of experiments to assess the impact of COX-2 blockade in ovalbumin (OVA)-sensitized mice in vivo, led us to propose a novel experimental approach using house dust mite (HDM)-sensitized mice in which we mimicked altered regulation of COX-2.
Allergic inflammation was induced in BALBc mice by intranasal exposure to HDM for 10 consecutive days. This model reproduces spontaneous exposure to aeroallergens by asthmatic patients. In order to impair, but not fully block, COX-2 production in the airways, some of the animals received an intranasal antisense oligonucleotide. Lung COX-2 expression and activity were measured along with bronchovascular inflammation, airway reactivity, and prostaglandin production.
We observed impaired COX-2 mRNA and protein expression in the lung tissue of selective oligonucleotide-treated sensitized mice. This was accompanied by diminished production of mPGE synthase and PGE2 in the airways. In sensitized mice, the oligonucleotide induced increased airway hyperreactivity (AHR) to methacholine, but a substantially reduced bronchovascular inflammation. Finally, mRNA levels of hPGD synthase remained unchanged.
Intranasal antisense therapy against COX-2 in vivo mimicked the reported impairment of COX-2 regulation in the airway cells of asthmatic patients. This strategy revealed an unexpected novel dual effect: inflammation was improved but AHR worsened. This approach will provide insights into the differential regulation of inflammation and lung function in asthma, and will help identify pharmacological targets within the COX-2/PG system.
Prostaglandin E2 (PGE2), experimentally administered to asthma patients or assayed in murine models, improves allergen-driven airway inflammation. The mechanisms are unknown, but fluctuations of the endogenous cyclooxygenase (COX)-2/prostaglandin/E prostanoid (EP) receptor pathway activity likely contribute to the clinical outcome. We analyzed the activity of the pathway in mice sensitized to aeroallergens, and then studied its modulation under exogenous PGE2.
Mice were exposed to house dust mite (HDM) aeroallergens, a model that enable us to mimic the development of allergic asthma in humans, and were then treated with either subcutaneous PGE2 or the selective EP1/3 receptor agonist sulprostone. Simultaneously with airway responsiveness and inflammation, lung COX-2 and EP receptor mRNA expression were assessed. Levels of PGE2, PGI2, PGD2 were also determined in bronchoalveolar lavage fluid.
HDM-induced airway hyperreactivity and inflammation were accompanied by increased COX-2 mRNA production. In parallel, airway PGE2 and PGI2, but not PGD2, were upregulated, and the EP2 receptor showed overexpression. Subcutaneous PGE2 attenuated aeroallergen-driven airway eosinophilic inflammation and reduced endogenous PGE2 and PGI2 production. Sulprostone had neither an effect on airway responsiveness or inflammation nor diminished allergen-induced COX-2 and PGE2 overexpression. Finally, lung EP2 receptor levels remained high in mice treated with PGE2, but not in those treated with sulprostone.
The lung COX-2/PGE2/EP2 receptor pathway is upregulated in HDM-exposed mice, possibly as an effort to attenuate allergen-induced airway inflammation. Exogenous PGE2 downregulates its endogenous counterpart but maintains EP2 overexpression, a phenomenon that might be required for administered PGE2 to exert its protective effect.
Vitamin A may have some influence on the immune system, but the role in allergy modulation is still unclear.
To clarify whether high levels of retinoic acid (RA) affects allergic response in vivo, we used a murine experimental model of airway allergic disease.
Ovalbumin (OVA)-immunization/OVA-challenge (OVA/OVA) and house dust mite (HDM)-immunization/HDM-challenge (HDM/HDM) experimental murine models of allergic airway disease, using C57Bl.10/Q groups of mice (n = 10) treated subcutaneously with different concentrations of all-trans RA (0, 50, 500 and 2,500 ug) every 2-days were used to assess the allergic immune response.
Levels of total and specific-IgE in sera were increased in all groups of RA treated OVA/OVA and HDM/HDM mice. Percentage and total amount of recruited eosinophil in airways by bronchoalveolar lavage fluid (BALF) were significantly enhanced in groups treated with 50, 500 and 2,500 ug of RA compared to non-treated mice. However, the group of mice treated with 2,500 ug had less eosinophil recruitment than the other two groups (50 and 500 ug). In parallel, levels of IL-5 and total IgE in BALF were also significantly diminished in the group treated with 2,500 ug compared to the other 2 groups (50 and 500 ug). Finally, total lung resistance was decreased in group treated with 2,500 ug compared to non-treated mice.
Our results suggest that retinoic acid directly enhances allergic response in vivo, but in higher doses may produce of immune suppression.
Asthma frequently commences in early life during airway and immune development and exposure to new environmental challenges. Endobronchial biopsies from children with asthma are abnormal, and lung function is maximally reduced by 6 years of age. As longitudinal biopsy studies are unethical in children, the relationship between development of pathology and reduced lung function is unknown. We aimed to establish a novel neonatal mouse model of allergic airways disease to investigate the developmental sequence of the pathophysiologic features of asthma. Neonatal Balb/c mice were challenged three times weekly from Day 3 of life using intranasal house dust mite (HDM) or saline for up to 12 weeks. Weekly assessments of airway inflammation and remodeling were made. Airway hyperresponsiveness (AHR) to methacholine was assessed from Week 2 onward. Total and eosinophilic inflammation was significantly increased in the lungs of HDM-exposed neonates from Week 2 onwards, and a peak was seen at 3 weeks. Goblet cells and peribronchiolar reticulin deposition were significantly increased in HDM-exposed neonates from Week 3, and peribronchiolar collagen was significantly greater from Week 4. HDM-exposed neonates had increased AHR from Week 2 onward. Although inflammation and AHR had subsided after 4 weeks without allergen challenge, the increased reticulin and collagen deposition persisted in HDM-exposed mice. Neonatal mice exposed to intranasal HDM developed eosinophilic inflammation, airway remodeling, and AHR as reported in pediatric asthma. Importantly, all abnormalities developed in parallel, not sequentially, between 2 and 3 weeks of age.
pediatric; remodeling; asthma pathophysiology; mouse model; allergic airways disease
House dust mite (HDM) induces allergic asthma in sensitized individuals, although the mechanisms by which HDM is sensed and recognized by the airway mucosa, leading to dendritic cell (DC) recruitment, activation, and subsequent TH2-mediated responses, are unknown.
We sought to define the pathways by which HDM activates respiratory epithelium to induce allergic airway responses.
Using a human airway epithelial cell line (16HBE14o-), we studied secretion of the DC chemokine CCL20 after exposure to HDM or other allergens, investigated components of the HDM responsible for the induction of chemokine release, and examined activation of signaling pathways. Central findings were also confirmed in primary human bronchial cells.
We demonstrate that exposure of airway epithelium to HDM results in specific and rapid secretion of CCL20, a chemokine attractant for immature DCs. The induction of CCL20 secretion is dose and time dependent and quite specific to HDM because other allergens, such as ragweed pollen and cockroach antigen, fail to significantly induce CCL20 secretion. Induction of CCL20 secretion is not protease or Toll-like receptor 2/4 dependent but, interestingly, relies on β-glucan moieties within the HDM extract, as evidenced by the ability of other β-glucans to competitively inhibit its secretion and by the fact that disruption of these structures by treatment of HDM with β-glucanase significantly reduces subsequent chemokine secretion.
Taken together, our results describe a novel mechanism for specific pattern recognition of HDM-derived β-glucan moieties, which initiates allergic airway inflammation and, through recruitment of DCs, might link innate pattern recognition at the airway surface with adaptive immune responses.
Asthma; allergy; house dust mite; epithelium; dendritic cell; chemokine; pattern recognition; innate immunity
House dust mites (HDM; Dermatophagoides sp.) are one of the commonest aeroallergens worldwide and up to 85% of asthmatics are typically HDM allergic. Allergenicity is associated both with the mites themselves and with ligands derived from mite-associated bacterial and fungal products. Murine models of allergic airways disease for asthma research have recently switched from the use of surrogate allergen ovalbumin together with adjuvant to use of the HDM extract. This has accelerated understanding of how adaptive and innate immunity generate downstream pathology. We review the myriad ways in which HDM allergic responses are orchestrated. Understanding the molecular pathways that elicit HDM-associated pathology is likely to reveal novel targets for therapeutic intervention.
The mammalian target of rapamycin (mTOR) plays an important role in cell growth/differentiation, integrating environmental cues, and regulating immune responses. Our lab previously demonstrated that inhibition of mTOR with rapamycin prevented house dust mite (HDM)-induced allergic asthma in mice. Here, we utilized two treatment protocols to investigate whether rapamycin, compared to the steroid, dexamethasone, could inhibit allergic responses during the later stages of the disease process, namely allergen re-exposure and/or during progression of chronic allergic disease. In protocol 1, BALB/c mice were sensitized to HDM (three i.p. injections) and administered two intranasal HDM exposures. After 6 weeks of rest/recovery, mice were re-exposed to HDM while being treated with rapamycin or dexamethasone. In protocol 2, mice were exposed to HDM for 3 or 6 weeks and treated with rapamycin or dexamethasone during weeks 4–6. Characteristic features of allergic asthma, including IgE, goblet cells, airway hyperreactivity (AHR), inflammatory cells, cytokines/chemokines, and T cell responses were assessed. In protocol 1, both rapamycin and dexamethasone suppressed goblet cells and total CD4+ T cells including activated, effector, and regulatory T cells in the lung tissue, with no effect on AHR or total inflammatory cell numbers in the bronchoalveolar lavage fluid. Rapamycin also suppressed IgE, although IL-4 and eotaxin 1 levels were augmented. In protocol 2, both drugs suppressed total CD4+ T cells, including activated, effector, and regulatory T cells and IgE levels. IL-4, eotaxin, and inflammatory cell numbers were increased after rapamycin and no effect on AHR was observed. Dexamethasone suppressed inflammatory cell numbers, especially eosinophils, but had limited effects on AHR. We conclude that while mTOR signaling is critical during the early phases of allergic asthma, its role is much more limited once disease is established.
Chronic allergic asthma is characterized by Th2-polarized inflammation and leads to airway remodeling and fibrosis but the mechanisms involved are not clear. To determine whether epithelial-mesenchymal transition contributes to airway remodeling in asthma, we induced allergic airway inflammation in mice by intranasal administration of house dust mite (HDM) extract for up to 15 consecutive weeks. We report that respiratory exposure to HDM led to significant airway inflammation and thickening of the smooth muscle layer in the wall of the large airways. Transforming growth factor beta-1 (TGF-β1) levels increased in mouse airways while epithelial cells lost expression of E-cadherin and occludin and gained expression of the mesenchymal proteins vimentin, alpha-smooth muscle actin (α-SMA) and pro-collagen I. We also observed increased expression and nuclear translocation of Snail1, a transcriptional repressor of E-cadherin and a potent inducer of EMT, in the airway epithelial cells of HDM-exposed mice. Furthermore, fate-mapping studies revealed migration of airway epithelial cells into the sub-epithelial regions of the airway wall. These results show the contribution of EMT to airway remodeling in chronic asthma-like inflammation and suggest that Th2-polarized airway inflammation can trigger invasion of epithelial cells into the subepithelial regions of the airway wall where they contribute to fibrosis, demonstrating a previously unknown plasticity of the airway epithelium in allergic airway disease.
Diesel exhaust particle (DEP) exposure enhances allergic inflammation and has been linked to the incidence of asthma. Oxidative stress on the thiol molecules cysteine (Cys) and glutathione (GSH) can promote inflammatory host responses. The effect of DEP on the thiol oxidation/reduction (redox) state in the asthmatic lung is unknown.
To determine if DEP exposure alters the Cys or GSH redox state in the asthmatic airway.
Bronchoalveolar lavage fluid was obtained from a house dust mite (HDM) induced murine asthma model exposed to DEP. GSH, glutathione disulfide (GSSG), Cys, cystine (CySS), and s-glutathionylated cysteine (CySSG) were determined by high pressure liquid chromatography.
DEP co-administered with HDM, but not DEP or HDM alone, decreased total Cys, increased CySS, and increased CySSG without significantly altering GSH or GSSG.
DEP exposure promotes oxidation and S-glutathionylation of cysteine amino acids in the asthmatic airway, suggesting a novel mechanism by which DEP may enhance allergic inflammatory responses.
Antigen-specific CD4+ T cells play an important role in the allergic immune response to house dust mite (HDM) allergens in humans. The group 1 allergen of Dermatophagoides spp. is a major target antigen in both B and T cell recognition of HDM. In vitro studies have shown that the presentation of peptides to human T cells under appropriate conditions may lead to a state of specific nonresponsiveness. Therefore, to determine if peptides are able to modulate the function of allergen- reactive T cells in vivo, we have used a murine model of T cell recognition of the HDM allergen Der p 1. The results demonstrate that inhalation of low concentrations of peptide containing the major T cell epitope of Der p 1 (residues 111-139), induces tolerance in naive C57BL/6J mice such that they become profoundly unresponsive to an immunogenic challenge with the intact allergen. When restimulated in vitro with antigen, lymph node T cells isolated from tolerant mice secrete very low levels of interleukin 2, proliferative poorly, and are unable to provide cognate help to stimulate specific antibody production. Furthermore, intranasal peptide therapy was able to inhibit an ongoing immune response to the allergen in mice and this has potential implications in the development of allergen-based immunotherapy.
Allergic asthma is a complex process arising out of the interaction between the immune system and aeroallergens. Yet, the relationship between aeroallergen exposure, allergic sensitization and disease remains unclear. This knowledge is essential to gain further insight into the origin and evolution of allergic diseases. The objective of this research is to develop a computational view of the interaction between aeroallergens and the host by investigating the impact of dose and length of aeroallergen exposure on allergic sensitization and allergic disease outcomes, mainly airway inflammation and to a lesser extent lung dysfunction and airway remodeling.
Methods and Principal Findings
BALB/C mice were exposed intranasally to a range of concentrations of the most pervasive aeroallergen worldwide, house dust mite (HDM), for up to a quarter of their lifespan (20 weeks). Actual biological data delineating the kinetics, nature and extent of responses for local (airway inflammation) and systemic (HDM-specific immunoglobulins) events were obtained. Mathematical equations for each outcome were developed, evaluated, refined through several iterations involving in vivo experimentation, and validated. The models accurately predicted the original biological data and simulated an extensive array of previously unknown responses, eliciting two- and three-dimensional models. Our data demonstrate the non-linearity of the relationship between aeroallergen exposure and either allergic sensitization or airway inflammation, identify thresholds, behaviours and maximal responsiveness for each outcome, and examine inter-variable relationships.
This research provides a novel way to visualize allergic responses in vivo and establishes a basic experimental platform upon which additional variables and perturbations can be incorporated into the system.
Barrier epithelial cells and airway dendritic cells (DC) make up the first line of defence against inhaled substances like house dust mite (HDM) allergen and endotoxin. We hypothesized that these cells need to communicate to cause allergic disease. Using irradiated chimeric mice, we demonstrate that TLR4 expression on radioresistant lung structural cells is required and sufficient for DC activation in the lung and for priming of effector T helper responses to HDM. TLR4 triggering on structural cells caused production of the innate proallergic cytokines thymic stromal lymphopoietin, granulocyte-macrophage colony stimulating factor, interleukin-25 and IL-33. The absence of TLR4 on structural cells, but not on hematopoietic cells, abolished HDM driven allergic airway inflammation. Finally, inhalation of a TLR4 antagonist to target exposed epithelial cells suppressed the salient features of asthma including bronchial hyperreactivity. Our data identify an innate immune function of airway epithelial cells that drives allergic inflammation via activation of mucosal DCs.
We previously reported that Fms-like tyrosine kinase 3 ligand (Flt3-L) reversed airway hyperresponsiveness (AHR) and airway inflammation, and increased the number of regulatory CD11chighCD8αhighCD11blow dendritic cells and CD4+CD25+ICOS+Foxp3+IL-10+ T-regulatory cells in the lung of allergen-sensitized and -challenged mice. In this study, we evaluated the effect of Flt3-L on Th17 cells and expression of suppressors of cytokine signaling (SOCS) proteins in the lungs of house dust mite (HDM)–sensitized and –challenged mice. BALB/c mice were sensitized and challenged with HDM, and AHR to methacholine was established. Mice were treated with Flt3-L (5 μg, intraperitoneal) daily for 10 days. Levels of IL-4, -5, -6, -8, and -13, and transforming growth factor (TGF)–β in the bronchoalveolar lavage fluid (BALF) were examined by ELISA. Flt3-L treatment reversed existing AHR to methacholine and substantially decreased eosinophils, neutrophils, IL-5, -6, -8, and IL-13, and TGF-β levels in the BALF. HDM-sensitized and -challenged mice showed a significant increase in lung CD4+IL-17+IL-23R+CD25− T cells with high expression of retinoic acid–related orphan receptor (ROR)–γt transcripts. However, administration of Flt3-L substantially decreased the number of lung CD4+IL-17+IL-23R+CD25− T cells, with significantly decreased expression of ROR-γt mRNA in these cells. HDM sensitization caused a significant increase in the expression of SOCS-1, -3, and -5 in the lung. Flt3-L treatment abolished the increase in SOCS-1 and SOCS-3 proteins, whereas SOCS-5 expression was significantly reduced. These data suggest that the therapeutic effect of Flt3-L in reversing the hallmarks of allergic asthma in a mouse model is mediated by decreasing IL-6 and TGF-β levels in the BALF, which, in turn, decrease CD4+IL-17+IL-23R+ROR-γt+CD25− T cells and the expression of SOCS-1 and SOCS-3 in the lung of HDM-sensitized and -challenged mice.
airway hyperresponsiveness; house dust mite; retinoic acid–related orphan receptor–γt; suppressors of cytokine signaling; T helper cell type 17
New treatments are needed for severe asthmatics to improve disease control and avoid severe toxicities associated with oral corticosteroids. We have used a murine model of house dust mite (HDM)-induced asthma to identify steroid-unresponsive genes that might represent targets for new therapeutic approaches for severe asthma. This strategy identified apolipoprotein E as a steroid-unresponsive gene with increased mRNA expression in the lungs of HDM-challenged mice. Furthermore, apolipoprotein E functioned as an endogenous negative regulator of airway hyperreactivity and goblet cell hyperplasia in experimental HDM-induced asthma. The ability of apolipoprotein E, which is expressed by lung macrophages, to attenuate AHR, and goblet cell hyperplasia is mediated by low density lipoprotein (LDL) receptors expressed by airway epithelial cells. Consistent with this, administration of an apolipoprotein E mimetic peptide, corresponding to amino acids 130–149 of the LDL receptor-binding domain of the holo-apoE protein, significantly reduced AHR and goblet cell hyperplasia in HDM-challenged apoE−/− mice. These findings identified the apolipoprotein E – LDL receptor pathway as a new druggable target for asthma that can be activated by administration of apoE-mimetic peptides. Similarly, apolipoprotein A-I may have therapeutic potential in asthma based upon its anti-inflammatory, anti-oxidative, and anti-fibrotic properties. Furthermore, administration of apolipoprotein A-I mimetic peptides has attenuated airway inflammation, airway remodeling, and airway hyperreactivity in murine models of experimental asthma. Thus, site-directed delivery of inhaled apolipoprotein E or apolipoprotein A-I mimetic peptides may represent novel treatment approaches that can be developed for asthma, including severe disease.
asthma; apolipoprotein E; apolipoprotein A-I; apolipoprotein E mimetic peptide; apolipoprotein A-I mimetic peptide