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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.

Background

Existing asthma models develop tolerance when chronically exposed to the same allergen.

Objective

To establish a chronic model that sustains features of asthma long after discontinuation of allergen exposure.

Methods

We immunized and exposed mice to a combination of single, double or triple allergens (dust-mite, ragweed, and Aspergillus) intranasally for 8 weeks. Airway hyperreactivity and morphological features of asthma were studied 3 weeks after the allergen exposure. Signaling effects of the allergens were studied on dendritic cells.

Results

Sensitization and repeated exposure to a single allergen induced tolerance. Sensitization to double, and especially triple allergens broke through tolerance and established AHR, eosinophilic inflammation, mast cell and smooth muscle hyperplasia, mucus production and airway remodeling that persisted at least 3 weeks after allergen exposure. Mucosal exposure to triple allergens in the absence of an adjuvant was sufficient to induce chronic airway inflammation. Anti-IL5 and -IL13 antibodies inhibited inflammation and AHR in the acute asthma model but not in the chronic triple allergen model. Multiple allergens produce a synergy in p38 MAPK signaling and maturation of dendritic cells, which provides a heightened T cell co-stimulation at a level that cannot be achieved with a single allergen.

Conclusions

Sensitivity to multiple allergens leads to chronic asthma in mice. Multiple allergens synergize in dendritic cell signaling and T cell stimulation that allows escape from the single allergen-associated tolerance development.

Clinical Implications

We have developed a model of chronic asthma that allows for the study and treatment of long-lasting features of asthma obviating the need for acute de novo allergen challenges.

Rationale: Although there have been numerous studies on the development of allergen-induced inflammation, the mechanisms leading to resolution of inflammation remain poorly understood. This represents an important consideration because failure to resolve allergen driven inflammation potentially leads to irreversible airway remodeling, characteristic of chronic asthma.

Objectives: We investigated the resolution of allergic inflammation and identified the factors responsible.

Methods: BALB/c and C57BL/6 mice were sensitized to ovalbumin and challenged through the airways to induce allergic inflammation. Mice were analyzed at 24 hours and 7 days after the final challenge.

Measurements and Main Results: Airway hyperreactivity (AHR) and increased mucus production were present 7 days after the cessation of allergen challenge in BALB/c mice. Persisting AHR correlated with the continued presence of Th2 cells but not eosinophils in the lungs. The role of Th2 cells in maintaining AHR was confirmed using blocking antibodies against T1/ST2, IL-4, and IL-13 during the resolution period. Moreover, AHR in the “Th1 type” C57BL/6 mouse strain was resolved 1 week after allergen challenge, concomitant with clearance of Th2 cells from the lung. Expression of the T1/ST2 ligand, IL-33, also correlated with maintenance of AHR.

Conclusions: We have used blockade of Th2 function and strain differences to show for the first time that resolution of allergic inflammation and AHR may be dependent on the T1/ST2-IL-33 pathway and the presence of Th2 cells, suggesting they are necessary not only for the development of an allergic response but also for its maintenance.

Atopic asthma is a chronic disease of the airways that has taken on epidemic proportions in the industrialized world. The increase in asthma rates has been linked epidemiologically to the rapid disappearance of Helicobacter pylori, a bacterial pathogen that persistently colonizes the human stomach, from Western societies. In this study, we have utilized mouse models of allergic airway disease induced by ovalbumin or house dust mite allergen to experimentally examine a possible inverse correlation between H. pylori and asthma. H. pylori infection efficiently protected mice from airway hyperresponsiveness, tissue inflammation, and goblet cell metaplasia, which are hallmarks of asthma, and prevented allergen-induced pulmonary and bronchoalveolar infiltration with eosinophils, Th2 cells, and Th17 cells. Protection against asthma was most robust in mice infected neonatally and was abrogated by antibiotic eradication of H. pylori. Asthma protection was further associated with impaired maturation of lung-infiltrating dendritic cells and the accumulation of highly suppressive Tregs in the lungs. Systemic Treg depletion abolished asthma protection; conversely, the adoptive transfer of purified Treg populations was sufficient to transfer protection from infected donor mice to uninfected recipients. Our results thus provide experimental evidence for a beneficial effect of H. pylori colonization on the development of allergen-induced asthma.

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 (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.

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.

Summary

Background

The mechanisms underlying exacerbation of asthma induced by respiratory syncytial virus (RSV) infection have been extensively studied in human and animal models. However, most of these studies focused on acute inflammation and little is known of its long-term consequences on remodelling of the airway tissue.

Objective

The aim of the study was to use a murine model of prolonged allergen-induced airway inflammation to investigate the effect of RSV infection on allergic airway inflammation and tissue remodelling.

Methods

We subjected mice to RSV infection before or during the chronic phase of airway challenges with OVA and compared parameters of airway inflammation and remodelling at the end-point of the prolonged allergen-induced airway inflammation protocol.

Results

RSV infection did not affect the severity of airway inflammation in any of the groups studied. However, RSV infection provoked airway remodelling in non-sensitized, allergen-challenged mice that did not otherwise develop any of the features of allergic airways disease. Increased collagen synthesis in the lung and thickening of the bronchial basal membrane was observed in non-sensitized allergen-challenged mice only after prior RSV infection. In addition, fibroblast growth factor (FGF)-2 but not TGF-β1 was increased in this group following RSV infection.

Conclusion

Our data show for the first time that RSV infection can prime the lung of mice that are not previously systemically sensitized, to develop airway remodelling in response to allergen upon sole exposure via the airways. Moreover, our results implicate RSV-induced FGF-2 in the remodelling process in vivo.

Macrophage migration inhibitory factor (MIF) has recently been forwarded as a critical regulator of inflammatory conditions, and it has been hypothesized that MIF may have a role in the pathogenesis of asthma and chronic obstructive pulmonary disease (COPD). Hence, we examined effects of MIF immunoneutralization on the development of allergen-induced eosinophilic inflammation as well as on lipopolysaccharide (LPS)-induced neutrophilic inflammation in lungs of mice. Anti-MIF serum validated with respect to MIF neutralizing capacity or normal rabbit serum (NRS) was administered i.p. repeatedly during allergen aerosol exposure of ovalbumin (OVA)-immunized mice in an established model of allergic asthma, or once before instillation of a minimal dose of LPS into the airways of mice, a tentative model of COPD. Anti-MIF treatment did not affect the induced lung tissue eosinophilia or the cellular composition of bronchoalveolar lavage fluid (BALF) in the asthma model. Likewise, anti-MIF treatment did not affect the LPS-induced neutrophilia in lung tissue, BALF, or blood, nor did it reduce BALF levels of tumor necrosis factor-alpha (TNF-alpha) and macrophage inflammatory protein-1alpha (MIP-1alpha). The present data suggest that MIF is not critically important for allergen-induced eosinophilic, and LPS-induced neutrophilic responses in lungs of mice. These findings do not support a role of MIF inhibition in the treatment of inflammatory respiratory diseases.

Reactive oxygen species (ROS) play a crucial role in the pathogenesis of acute and chronic respiratory diseases. Antioxidants have been found to ameliorate airway inflammation and hyperresponsiveness in animal models employing short-term exposure to allergen. However, little data are available on the effect of antioxidants on airway remodeling and signaling pathways in chronic asthma. In the present study, we used a long-term exposure murine model of allergic airway disease to evaluate the effects of an antioxidant, L-2-oxothiazolidine-4-carboxylic acid (OTC) or α-lipoic acid (LA) on airway remodeling, focusing on the ROS-related hypoxia-inducible signaling. Long-term challenge of ovalbumin (OVA) increased ROS production, airway inflammation, and airway hyperresponsiveness, and developed features of airway remodeling such as excessive mucus secretion, subepithelial fibrosis, and thickening of the peribronchial smooth muscle layer. Administration of OTC or LA reduced these features of asthma, including airway remodeling, which was accompanied by suppression of transforming growth factor-β1, vascular endothelial growth factor, and T-helper 2 cytokines. In addition, OVA-induced activation of nuclear factor-κB (NF-κB), nuclear factor erythroid 2p45-related factor-2 (Nrf2), hypoxia-inducible factor (HIF)-1α, and HIF-2α was reduced by OTC or LA. Our results also showed that OTC or LA down-regulated phosphoinositide 3-kinase activity and decreased phosphorylation of p38 mitogen-activated protein kinase but not extracellular signal-regulated kinase 1/2 or c-Jun N-terminal kinase. These findings demonstrate that OTC and LA can inhibit activation of NF-κB, Nrf2, and HIF, leading to attenuate allergen-induced airway remodeling.

The development of chronic allergic dermatitis in early life has been associated with increased onset and severity of allergic asthma later in life. However, the mechanisms linking these two diseases are poorly understood. Here, we report that the development of oxazolone-induced chronic allergic dermatitis, in a mouse model, caused enhanced ovalbumin-induced allergic asthma after resolution of the former disease. Our findings show that oxazolone-induced dermatitis caused a marked increase in tissue mast cells, which persisted long after the resolution of this disease. Subsequent ovalbumin sensitization and airway challenge of mice that had recovered from dermatitis resulted in increased allergic airway hyperreactivity. The findings demonstrate that the accumulation of mast cells during dermatitis has the detrimental effect of increasing allergic airway hypersensitivity. Importantly, our findings also show that exposure to a given allergen can modify the immune response to an unrelated allergen.

Allergic airways disease is a consequence of a Th2 response to an allergen leading to a series of manifestations such as production of allergen-specific IgE, inflammatory infiltrates in the airways, and airway hyper-reactivity (AHR). Several strategies have been reported for tolerance induction to allergens leading to protection from allergic airways disease. We now show that CD4 blockade at the time of house dust mite sensitization induces antigen-specific tolerance in mice. Tolerance induction is robust enough to be effective in pre-sensitized animals, even in those where AHR was pre-established. Tolerant mice are protected from airways eosinophilia, Th2 lung infiltration, and AHR. Furthermore, anti-CD4 treated mice remain immune competent to mount immune responses, including Th2, to unrelated antigens. Our findings, therefore, describe a strategy for tolerance induction potentially applicable to other immunogenic proteins besides allergens.

Background

Increased mast cell (MC) density and changes in their distribution in airway tissues is thought to contribute significantly to the pathophysiology of asthma. However, the time sequence for these changes and how they impact small airway function in asthma is not fully understood. The aim of the current study was to characterise temporal changes in airway MC density and correlate these changes with functional airway responses in sheep chronically challenged with house dust mite (HDM) allergen.

Methodology/Principal Findings

MC density was examined on lung tissue from four spatially separate lung segments of allergic sheep which received weekly challenges with HDM allergen for 0, 8, 16 or 24 weeks. Lung tissue was collected from each segment 7 days following the final challenge. The density of tryptase-positive and chymase-positive MCs (MCT and MCTC respectively) was assessed by morphometric analysis of airway sections immunohistochemically stained with antibodies against MC tryptase and chymase.

MCT and MCTC density was increased in small bronchi following 24 weeks of HDM challenges compared with controls (P<0.05). The MCTC/MCT ratio was significantly increased in HDM challenged sheep compared to controls (P<0.05). MCT and MCTC density was inversely correlated with allergen-induced increases in peripheral airway resistance after 24 weeks of allergen exposure (P<0.05). MCT density was also negatively correlated with airway responsiveness after 24 challenges (P<0.01).

Conclusions

MCT and MCTC density in the small airways correlates with better lung function in this sheep model of chronic asthma. Whether this finding indicates that under some conditions mast cells have protective activities in asthma, or that other explanations are to be considered requires further investigation.

Background

Repeated airway challenges with House Dust Mite (HDM) allergen results in marked remodelling and mast cell hyperplasia in the small airways of allergic sheep. We now examine mast cell activation and its association with small airway function and remodelling in these sheep using a novel segmental allergen challenge approach.

Methods

Eight allergic sheep received weekly intra-lung challenges of HDM to the left caudal lung for 24 weeks. Eight separate sheep were used as controls. Baseline lung function was assessed in the left caudal segments of all sheep throughout the challenge regime using a wedged-bronchoscope technique. Airway tissue was collected from challenged segments from all sheep, 7 days following the final intra-lung challenge. The airway tissues were immunohistochemically labelled for chymase-mast cells and eosinophils. Collagen and airway smooth muscle content were assessed on Masson's Trichrome stained sections.

Results

Resting lung function in the left caudal segment is elevated in 4 out of 8 sheep at the end of the repeated allergen challenge regime. Chymase mast cell density was significantly increased in the small bronchial walls of the HDM-challenged group compared to the control group (52 ± 8 vs 8 ± 4; P < 0.01). There were significant increases in bronchial collagen deposition in HDM-exposed segments compared to control segments (0.17 ± 0.02 vs 0.11 ± 0.02 mm2/BM, P < 0.05). A correlation analysis of individual sheep data showed that there was a trend for a direct association between the increases in bronchial collagen deposition and the density of chymase-labelled mast cells (rs = 0.71, P = 0.088). Eosinophil density in the small bronchial walls of HDM-challenged segments was also significantly increased compared to controls (65 ± 19 vs 11 ± 3 cells/mm2, P < 0.001), but not associated with collagen content. The bronchial smooth muscle content was not different between HDM-challenged and unexposed control segments.

Conclusions

The results show that repeated exposure to allergen results in significant increases in density of chymase-labelled mast cells, together with increased levels of collagen content in the small airways. The segmental challenge protocol allows for a novel approach to characterise the progressive remodelling events occurring in the small airways in chronic asthma.

Mast cell numbers and allergen specific IgE are increased in the lungs of patients with allergic asthma and this can be reproduced in mouse models. The increased number of mast cells is likely due to recruitment of mast cell progenitors that mature in situ. We hypothesized that formation of IgE immune complexes in the lungs of sensitized mice increase the migration of mast cell progenitors to this organ. To study this, a model of allergic airway inflammation where mice were immunized with ovalbumin (OVA) in alum twice followed by three daily intranasal challenges of either OVA coupled to trinitrophenyl (TNP) alone or as immune complexes with IgE-anti-TNP, was used. Mast cell progenitors were quantified by a limiting dilution assay. IgE immune complex challenge of sensitized mice elicited three times more mast cell progenitors per lung than challenge with the same dose of antigen alone. This dose of antigen challenge alone did not increase the levels of mast cell progenitors compared to unchallenged mice. IgE immune complex challenge of sensitized mice also enhanced the frequency of mast cell progenitors per 106 mononuclear cells by 2.1-fold. The enhancement of lung mast cell progenitors by IgE immune complex challenge was lost in FcRγ deficient mice but not in CD23 deficient mice. Our data show that IgE immune complex challenge enhances the number of mast cell progenitors in the lung through activation of an Fc receptor associated with the FcRγ chain. This most likely takes place via activation of FcεRI, although activation via FcγRIV or a combination of the two receptors cannot be excluded. IgE immune complex-mediated enhancement of lung MCp numbers is a new reason to target IgE in therapies against allergic asthma.

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.

Rationale: Airway hyperreactivity and remodeling are characteristic features of asthma. Interactions between the airway epithelium and environmental allergens are believed to be important in driving development of pathology, particularly because altered epithelial gene expression is common in individuals with asthma.

Objectives: To investigate the interactions between a modified airway epithelium and a common aeroallergen in vivo.

Methods: We used an adenoviral vector to generate mice overexpressing the transforming growth factor-β signaling molecule, Smad2, in the airway epithelium and exposed them to house dust mite (HDM) extract intranasally.

Measurements and Main Results: Smad2 overexpression resulted in enhanced airway hyperreactivity after allergen challenge concomitant with changes in airway remodeling. Subepithelial collagen deposition was increased and smooth muscle hyperplasia was evident resulting in thickening of the airway smooth muscle layer. However, there was no increase in airway inflammation in mice given the Smad2 vector compared with the control vector. Enhanced airway hyperreactivity and remodeling did not correlate with elevated levels of Th2 cytokines, such as IL-13 or IL-4. However, mice overexpressing Smad2 in the airway epithelium showed significantly enhanced levels of IL-25 and activin A after HDM exposure. Blocking activin A with a neutralizing antibody prevented the increase in lung IL-25 and inhibited subsequent collagen deposition and also the enhanced airway hyperreactivity observed in the Smad2 overexpressing HDM-exposed mice.

Conclusions: Epithelial overexpression of Smad2 can specifically alter airway hyperreactivity and remodeling in response to an aeroallergen. Moreover, we have identified novel roles for IL-25 and activin A in driving airway hyperreactivity and remodeling.

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.

Asthma is etiologically and clinically heterogeneous, making the genomic basis of asthma difficult to identify. We exploited the strain-dependence of a murine model of allergic airway disease to identify different genomic responses in the lung. BALB/cJ and C57BL/6J mice were sensitized with the immunodominant allergen from the Dermatophagoides pteronyssinus species of house dust mite (Der p 1), without exogenous adjuvant, and the mice then underwent a single challenge with Der p 1. Allergic inflammation, serum antibody titers, mucous metaplasia, and airway hyperresponsiveness were evaluated 72 hours after airway challenge. Whole-lung gene expression analyses were conducted to identify genomic responses to allergen challenge. Der p 1–challenged BALB/cJ mice produced all the key features of allergic airway disease. In comparison, C57BL/6J mice produced exaggerated Th2-biased responses and inflammation, but exhibited an unexpected decrease in airway hyperresponsiveness compared with control mice. Lung gene expression analysis revealed genes that were shared by both strains and a set of down-regulated genes unique to C57BL/6J mice, including several G-protein–coupled receptors involved in airway smooth muscle contraction, most notably the M2 muscarinic receptor, which we show is expressed in airway smooth muscle and was decreased at the protein level after challenge with Der p 1. Murine strain–dependent genomic responses in the lung offer insights into the different biological pathways that develop after allergen challenge. This study of two different murine strains demonstrates that inflammation and airway hyperresponsiveness can be decoupled, and suggests that the down-modulation of expression of G-protein–coupled receptors involved in regulating airway smooth muscle contraction may contribute to this dissociation.

Asthma is a leading cause of morbidity in children. Risk factors include chronic exposure to allergens and air pollution. While chronically activated mast cells contribute to the pathophysiology of asthma in part through their proteases such as chymase and tryptase, previous studies of airway mast cell abundance and distribution in asthmatics have been inconsistent. To determine whether repeated episodic exposures to environmental pollutants during postnatal lung development alter airway mast cell abundance and distribution, we exposed infant rhesus monkeys to a known human allergen, house dust mite antigen (HDMA), and/or a known environmental pollutant, ozone (O3), and quantitatively compared the abundance of tryptase- or chymase-positive mast cells in three airway levels. Mast cells are resident in multiple compartments of the airway wall in infant rhesus monkeys raised from birth in filtered air. Tryptase- and chymase-positive cells were most abundant in trachea and least in terminal bronchioles. The majority of tryptase-positive and almost all chymase-positive cells were in extracellular matrix and smooth muscle bundles. Chronic exposure to HDMA elevated the abundance of both tryptase- and chymase-positive cells in the trachea and intrapulmonary bronchi. Neither exposure to O3 nor HDMA + O3 increased mast cell accumulations in the airway wall. We conclude that during postnatal airway development (1) mast cells are a resident airway cell population even in the absence of toxic air contaminants; (2) aeroallergen exposure alters large airway mast cell distribution and abundance, increasing chymase-positive mast cells; and (3) this response is attenuated by exposure to oxidant air pollutants.

Allergic inflammation and severe allergic reactions (anaphylaxis) are important in allergen induced diseases. Bacterial products such as lipopolysaccharide (LPS) are ubiquitous and can facilitate allergen induced Th2 immune responses. Phosphatase SHP-1 is critical in regulating immunological homeostasis and in allergen induced Th2 immune responses in the lung. However, the mechanisms underlying the initiation of allergic inflammation and allergen induced anaphylaxis are still not completely elucidated and it is unclear whether SHP-1 plays any role in LPS-induced airway inflammation and in allergen-induced anaphylaxis. In this study we tested the hypothesis that phosphatase SHP-1 plays an important role in allergic inflammation and anaphylaxis and determined whether its effects are through regulation of mast cell functions. SHP-1 deficient (mev/+ and mev/mev) and mast cell deficient (KitW-sh) mice were examined in their responses to LPS airway stimulation and to ovalbumin (OVA) allergen induced systemic anaphylaxis. Compared to wild type mice, mev/+ mice had significantly enhanced LPS induced airway inflammation and OVA induced anaphylactic responses, including hypothermia and clinical symptoms. These changes were mast cell dependent as KitW-sh mice had reduced responses whereas adoptive transfer of mast cells restored the responses. However, T and B cells were not involved and macrophages did not play a significant role in LPS induced airway inflammation. Interestingly, basophil differentiation from SHP-1 deficient bone marrow cells was significantly reduced. These findings provided evidence that through regulation of mast cell functions SHP-1 plays a critical role as a negative regulator in allergic inflammation and in allergen induced anaphylaxis. In addition, SHP-1 seems to be required for normal basophil development.

Background

Reactive oxygen species (ROS) play a crucial role in the pathogenesis of acute and chronic respiratory diseases. Antioxidants have been found to ameliorate airway inflammation and hyperresponsiveness in animal models employing short-term exposure to allergen. However, little data are available on the effect of antioxidants on airway remodeling and signaling pathways in chronic asthma.

Methods

In the present study, we used a long-term exposure murine model of allergic airway disease to evaluate the influence of an antioxidant, L-2-oxothiazolidine-4-carboxylic acid (OTC) or α-lipoic acid (LA) on airway remodeling and to explore possible transcription factors and kinases involved in this effect.

Results

Long-term challenge of ovalbumin (OVA) increased ROS production, airway inflammation, and airway hyperresponsiveness, and developed features of airway remodeling such as excessive mucus secretion, subepithelial fibrosis, and thickening of the peribronchial smooth muscle layer. Administration of OTC or LA reduced these features of asthma including airway remodeling, which was accompanied by suppression of transforming growth factor-β1, vascular endothelial growth factor, and T-helper 2 cytokines. In addition, OVA-induced activation of nuclear factor-κB (NF-κB), nuclear factor erythroid 2p45-related factor-2 (Nrf2), hypoxia-inducible factor (HIF)-1α, and HIF-2α was reduced by OTC or LA. Our results also showed that OTC or LA down-regulated phosphoinositide 3-kinase activity and decreased phosphorylation of p38 mitogen-activated protein kinase but not extracellular signal-regulated kinase 1/2 or c-Jun N-terminal kinase.

Conclusions

These findings demonstrate that OTC and LA can inhibit activation of NF-κB, Nrf2, and HIF and thus attenuate allergen-induced airway remodeling, suggesting that antioxidants may provide therapeutic benefit in chronic asthma and other airway disorders.

Summary

Background

Inflammatory infiltrates, airway hyper-responsiveness, goblet cell hyperplasia and subepithelial thickening are characteristic of chronic asthma. Current animal models of allergen-induced airway inflammation generally concentrate on the acute inflammation following allergen exposure and fail to mimic all of these features.

Objective

The aim of this study was to use a murine model of prolonged allergen-induced airway inflammation in order to characterize the cells and molecules involved in the ensuing airway remodelling. Moreover, we investigated whether remodelling persists in the absence of continued allergen challenge.

Methods

Acute pulmonary eosinophilia and airways hyper-reactivity were induced after six serial allergen challenges in sensitized mice (acute phase). Mice were subsequently challenged three times a week with ovalbumin (OVA) (chronic phase) up to day 55. To investigate the persistence of pathology, one group of mice were left for another 4 weeks without further allergen challenge (day 80).

Results

The extended OVA challenge protocol caused significant airway remodelling, which was absent in the acute phase. Specifically, remodelling was characterized by deposition of collagen as well as airway smooth muscle and goblet cell hyperplasia. Importantly, these airway changes, together with tissue eosinophilia were sustained in the absence of further allergen challenge. Examination of cytokines revealed a dramatic up-regulation of IL-4 and tumour growth factor-β1 during the chronic phase. Interestingly, while IL-4 levels were significantly increased during the chronic phase, levels of IL-13 fell. Levels of the Th1-associated cytokine IFN-γ also increased during the chronic phase.

Conclusion

In conclusion, we have demonstrated that prolonged allergen challenge results in persistent airway wall remodelling.

The role played by the β-galactoside-binding lectin galectin-3 (Gal-3) in airway remodeling, a characteristic feature of asthma that leads to airway dysfunction and poor clinical outcome in humans, was investigated in a murine model of chronic allergic airway inflammation. Wild-type (WT) and Gal-3 knock-out (KO) mice were subjected to repetitive allergen challenge with ovalbumin (OVA) up to 12 weeks and bronchoalveolar lavage fluid (BALF) and lung tissue collected after the last challenge were evaluated for cellular features associated with airway remodeling. Compared to WT mice, chronic OVA challenge in Gal-3 KO mice resulted in diminished remodeling of the airways with significantly reduced mucus secretion, sub-epithelial fibrosis, smooth muscle thickness, and peribronchial angiogenesis. The higher degree of airway remodeling in WT mice was associated with higher Gal-3 expression in the BALF as well as lung tissue. Cell counts in BALF and lung immunohistology demonstrated that eosinophil infiltration in OVA-challenged Gal-3 KO mice was significantly reduced compared to WT mice. Evaluation of cellular mediators associated with eosinophil recruitment and airway remodeling revealed that levels of eotaxin-1, IL-5, IL-13, FIZZ1 and TGF-β were substantially lower in Gal-3 KO mice. Finally, leukocytes from Gal-3 KO mice demonstrated decreased trafficking (rolling) on vascular endothelial adhesion molecules compared to WT cells. Overall, these studies demonstrate that Gal-3 is an important lectin that promotes airway remodeling via airway recruitment of inflammatory cells, specifically eosinophils, and the development of a Th2 phenotype as well as increased expression of eosinophil-specific chemokines, pro-fibrogenic and angiogenic mediators.

Rationale: Airway inflammation in asthma is accompanied by structural changes, including goblet cell metaplasia, smooth muscle cell layer thickening, and subepithelial fibrosis. This allergen-induced airway remodeling can be replicated in a mouse asthma model.

Objectives: The study goal was to determine whether established airway remodeling in a mouse asthma model is reversible by administration of the cysteinyl leukotriene (CysLT)1 receptor antagonist montelukast, the corticosteroid dexamethasone, or the combination montelukast + dexamethasone.

Methods: BALB/c mice, sensitized by intraperitoneal ovalbumin (OVA) as allergen, received intranasal OVA periodically Days 14–73 and montelukast or dexamethasone or placebo from Days 73–163.

Measurements and Main Results: Allergen-induced trafficking of eosinophils into the bronchoalveolar lavage fluid and lung interstitium and airway goblet cell metaplasia, smooth muscle cell layer thickening, and subepithelial fibrosis present on Day 73 persisted at Day 163, 3 mo after the last allergen challenge. Airway hyperreactivity to methacholine observed on Day 73 in OVA-treated mice was absent on Day 163. In OVA-treated mice, airway eosinophil infiltration and goblet cell metaplasia were reduced by either montelukast or dexamethasone alone. Montelukast, but not dexamethasone, reversed the established increase in airway smooth muscle mass and subepithelial collagen deposition. By immunocytochemistry, CysLT1 receptor expression was significantly increased in airway smooth muscle cells in allergen-treated mice compared with saline-treated controls and was reduced by montelukast, but not dexamethasone, administration.

Conclusions: These data indicate that established airway smooth muscle cell layer thickening and subepithelial fibrosis, key allergen-induced airway structural changes not modulated by corticosteroids, are reversible by CysLT1 receptor blockade therapy.

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.

Both nature and induced regulatory T (Treg) lymphocytes are potent regulators of autoimmune and allergic disorders. Defects in endogenous Treg cells have been reported in patients with allergic asthma, suggesting that disrupted Treg cell-mediated immunological regulation may play an important role in airway allergic inflammation. In order to determine whether adoptive transfer of induced Treg cells generated in vitro can be used as an effective therapeutic approach to suppress airway allergic inflammation, exogenously induced Treg cells were infused into ovalbumin-sensitized mice prior to or during intranasal ovalbumin challenge. The results showed that adoptive transfer of induced Treg cells prior to allergen challenge markedly reduced airway hyperresponsiveness, eosinophil recruitment, mucus hyper-production, airway remodeling, and IgE levels. This effect was associated with increase of Treg cells (CD4+FoxP3+) and decrease of dendritic cells in the draining lymph nodes, and with reduction of Th1, Th2, and Th17 cell response as compared to the controls. Moreover, adoptive transfer of induced Treg cells during allergen challenge also effectively attenuate airway inflammation and improve airway function, which are comparable to those by natural Treg cell infusion. Therefore, adoptive transfer of in vitro induced Treg cells may be a promising therapeutic approach to prevent and treat severe asthma.