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1.  Differential Effects of Dendritic Cell Transfer on Airway Hyperresponsiveness and Inflammation 
Dendritic cells (DCs) are considered to be the most efficient antigen-presenting cells. Intratracheal administration of allergen-pulsed bone marrow–derived dendritic cells (BMDCs) before allergen challenge induces airway hyperresponsiveness (AHR) and inflammation. Ovalbumin (OVA)-pulsed BMDCs from wild-type (WT) mice were transferred into naive WT, CD4−/−, CD8−/−, or IL-13−/− mice. Two days (short protocol) or 10 days (long protocol) after BMDC transfer, mice were challenged with 1% OVA for 3 days and assayed 2 days later. Transfer of OVA-primed BMDCs into BALB/c or C57BL/6 mice elicited AHR in both protocols. Airway eosinophilia, Th2 cytokines, or goblet cell metaplasia were increased in the long but not short protocol. Lung T cells from both protocols produced Th2 cytokines in response to OVA in vitro. Carboxyfluorescein diacetate succinimidylester–labeled BMDCs were observed in bronchoalveolar lavage (BAL) fluid and lung parenchyma at early time points, and were detected in draining lymph nodes 48 hours after transfer. CD8−/− mice developed AHR comparable to WT mice in the short protocol, but decreased levels of AHR, airway eosinophilia, Th2 cytokines in BAL fluid, and goblet cell metaplasia compared with WT mice in the long protocol. CD4−/− or IL-13−/− mice did not develop AHR or airway inflammation in either protocol. These data suggest that allergen-pulsed BMDCs initiate development of AHR that is dependent initially on CD4+ T cells, and at later time periods on CD8+ T cells and IL-13. Thus, the timing of allergen challenge after transfer of allergen-pulsed BMDC affects the development of AHR and airway inflammation.
PMCID: PMC2742748  PMID: 19151321
dendritic cells; CD8+ T cells; airway hyperresponsiveness
2.  Inhibition of Spleen Tyrosine Kinase Prevents Mast Cell Activation and Airway Hyperresponsiveness 
Rationale: Spleen tyrosine kinase (Syk) is important for Fc and B-cell receptor–mediated signaling.
Objective: To determine the activity of a specific Syk inhibitor (R406) on mast cell activation in vitro and on the development of allergen-induced airway hyperresponsiveness (AHR) and inflammation in vivo.
Methods: AHR and inflammation were induced after 10 d of allergen (ovalbumin [OVA]) exposure exclusively via the airways and in the absence of adjuvant. This approach was previously established to be IgE, FcɛRI, and mast cell dependent. Alternatively, mice were passively sensitized with OVA-specific IgE, followed by limited airway challenge. In vitro, the inhibitor was added to cultures of IgE-sensitized bone marrow–derived mast cells (BMMCs) before cross-linking with allergen.
Results: The inhibitor prevented OVA-induced degranulation of passively IgE-sensitized murine BMMCs and inhibited the production of interleukin (IL)-13, tumor necrosis factor α, IL-2, and IL-6 in these sensitized BMMCs. When administered in vivo, R406 inhibited AHR, which developed in BALB/c mice exposed to aerosolized 1% OVA for 10 consecutive d (20 min/d), as well as pulmonary eosinophilia and goblet cell metaplasia. A similar inhibition of AHR was demonstrated in mice passively sensitized with OVA-specific IgE and exposed to limited airway challenge.
Conclusion: This study delineates a functional role for Syk in the development of mast cell– and IgE-mediated AHR and airway inflammation, and these results indicate that inhibition of Syk may be a target in the treatment of allergic asthma.
PMCID: PMC2662982  PMID: 16192454
airway hyperresponsiveness; eosinophils; goblet cell metaplasia; mast cells; spleen tyrosine kinase
3.  Toll-Like Receptor-9 Agonist Inhibits Airway Inflammation, Remodeling and Hyperreactivity in Mice Exposed to Chronic Environmental Tobacco Smoke and Allergen 
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).
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.
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.
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.
PMCID: PMC2853586  PMID: 19851071
Toll-like receptor-9; Airway hyperreactivity; Airway inflammation; Airway remodeling; Eosinophils
4.  Syk Activation in Dendritic Cells Is Essential for Airway Hyperresponsiveness and Inflammation 
We evaluated the role of Syk, using an inhibitor, on allergen-induced airway hyperresponsiveness (AHR) and airway inflammation in a system shown to be B cell– and mast cell–independent. Sensitization of BALB/c mice with ovalbumin (OVA) and alum after three consecutive OVA challenges resulted in AHR to inhaled methacholine and airway inflammation. The Syk inhibitor R406 (30 mg/kg, administered orally, twice daily) prevented the development of AHR, increases in eosinophils and lymphocytes and IL-13 levels in bronchoalveolar lavage (BAL) fluid, and goblet cell metaplasia when administered after sensitization and before challenge with OVA. Levels of IL-4, IL-5, and IFN-γ in BAL fluid and allergen-specific antibody levels in serum were not affected by treatment. Because many of these responses may be influenced by dendritic cell function, we investigated the effect of R406 on bone marrow–derived dendritic cell (BMDC) function. Co-culture of BMDC with immune complexes of OVA and IgG anti-OVA together with OVA-sensitized spleen mononuclear cells resulted in increases in IL-13 production. IL-13 production was inhibited if the BMDCs were pretreated with the Syk inhibitor. Intratracheal transfer of immune complex-pulsed BMDCs (but not nonpulsed BMDCs) to naive mice before airway allergen challenge induced the development of AHR and increases in BAL eosinophils and lymphocytes. All of these responses were inhibited if the transferred BMDCs were pretreated with R406. These results demonstrate that Syk inhibition prevents allergen-induced AHR and airway inflammation after systemic sensitization and challenge, at least in part through alteration of DC function.
PMCID: PMC2644204  PMID: 16339999
AHR; dendritic cells; eosinophils; mice; Syk
5.  Inhibition of neutrophil elastase attenuates airway hyperresponsiveness and inflammation in a mouse model of secondary allergen challenge: neutrophil elastase inhibition attenuates allergic airway responses 
Respiratory Research  2013;14(1):8.
Chronic asthma is often associated with neutrophilic infiltration in the airways. Neutrophils contain elastase, a potent secretagogue in the airways, nonetheless the role for neutrophil elastase as well as neutrophilic inflammation in allergen-induced airway responses is not well defined. In this study, we have investigated the impact of neutrophil elastase inhibition on the development of allergic airway inflammation and airway hyperresponsiveness (AHR) in previously sensitized and challenged mice.
BALB/c mice were sensitized and challenged (primary) with ovalbumin (OVA). Six weeks later, a single OVA aerosol (secondary challenge) was delivered and airway inflammation and airway responses were monitored 6 and 48 hrs later. An inhibitor of neutrophil elastase was administered prior to secondary challenge.
Mice developed a two-phase airway inflammatory response after secondary allergen challenge, one neutrophilic at 6 hr and the other eosinophilic, at 48 hr. PAR-2 expression in the lung tissues was enhanced following secondary challenge, and that PAR-2 intracellular expression on peribronchial lymph node (PBLN) T cells was also increased following allergen challenge of sensitized mice. Inhibition of neutrophil elastase significantly attenuated AHR, goblet cell metaplasia, and inflammatory cell accumulation in the airways following secondary OVA challenge. Levels of IL-4, IL-5 and IL-13, and eotaxin in BAL fluid 6 hr after secondary allergen challenge were significantly suppressed by the treatment. At 48 hr, treatment with the neutrophil elastase inhibitor significantly reduced the levels of IL-13 and TGF-β1 in the BAL fluid. In parallel, in vitro IL-13 production was significantly inhibited in spleen cells from sensitized mice.
These data indicate that neutrophil elastase plays an important role in the development of allergic airway inflammation and hyperresponsiveness, and would suggest that the neutrophil elastase inhibitor reduced AHR to inhaled methacholine indicating the potential for its use as a modulator of the immune/inflammatory response in both the neutrophil- and eosinophil-dominant phases of the response to secondary allergen challenge.
PMCID: PMC3570429  PMID: 23347423
Neutrophil; Elastase; Airway; Hyperresponsiveness; Asthma
6.  Proton-Sensing Ovarian Cancer G Protein-Coupled Receptor 1 on Dendritic Cells Is Required for Airway Responses in a Murine Asthma Model 
PLoS ONE  2013;8(11):e79985.
Ovarian cancer G protein-coupled receptor 1 (OGR1) stimulation by extracellular protons causes the activation of G proteins and subsequent cellular functions. However, the physiological and pathophysiological roles of OGR1 in airway responses remain largely unknown. In the present study, we show that OGR1-deficient mice are resistant to the cardinal features of asthma, including airway eosinophilia, airway hyperresponsiveness (AHR), and goblet cell metaplasia, in association with a remarkable inhibition of Th2 cytokine and IgE production, in an ovalbumin (OVA)-induced asthma model. Intratracheal transfer to wild-type mice of OVA-primed bone marrow-derived dendritic cells (DCs) from OGR1-deficient mice developed lower AHR and eosinophilia after OVA inhalation compared with the transfer of those from wild-type mice. Migration of OVA-pulsed DCs to peribronchial lymph nodes was also inhibited by OGR1 deficiency in the adoption experiments. The presence of functional OGR1 in DCs was confirmed by the expression of OGR1 mRNA and the OGR1-sensitive Ca2+ response. OVA-induced expression of CCR7, a mature DC chemokine receptor, and migration response to CCR7 ligands in an in vitro Transwell assay were attenuated by OGR1 deficiency. We conclude that OGR1 on DCs is critical for migration to draining lymph nodes, which, in turn, stimulates Th2 phenotype change and subsequent induction of airway inflammation and AHR.
PMCID: PMC3823589  PMID: 24244587
7.  Reversal of Allergen-induced Airway Remodeling by CysLT1 Receptor Blockade 
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.
PMCID: PMC2662952  PMID: 16387808
eosinophils; fibrosis; mucus; smooth muscle
8.  Chronic OVA allergen challenged Siglec-F deficient mice have increased mucus, remodeling, and epithelial Siglec-F ligands which are up-regulated by IL-4 and IL-13 
Respiratory Research  2010;11(1):154.
In this study we examined the role of Siglec-F, a receptor highly expressed on eosinophils, in contributing to mucus expression, airway remodeling, and Siglec-F ligand expression utilizing Siglec-F deficient mice exposed to chronic allergen challenge.
Wild type (WT) and Siglec-F deficient mice were sensitized and challenged chronically with OVA for one month. Levels of airway inflammation (eosinophils), Siglec-F ligand expresion and remodeling (mucus, fibrosis, smooth muscle thickness, extracellular matrix protein deposition) were assessed in lung sections by image analysis and immunohistology. Airway hyperreactivity to methacholine was assessed in intubated and ventilated mice.
Siglec-F deficient mice challenged with OVA for one month had significantly increased numbers of BAL and peribronchial eosinophils compared to WT mice which was associated with a significant increase in mucus expression as assessed by the number of periodic acid Schiff positive airway epithelial cells. In addition, OVA challenged Siglec-F deficient mice had significantly increased levels of peribronchial fibrosis (total lung collagen, area of peribronchial trichrome staining), as well as increased numbers of peribronchial TGF-β1+ cells, and increased levels of expression of the extracellular matrix protein fibronectin compared to OVA challenged WT mice. Lung sections immunostained with a Siglec-Fc to detect Siglec-F ligand expression demonstrated higher levels of expression of the Siglec-F ligand in the peribronchial region in OVA challenged Siglec-F deficient mice compared to WT mice. WT and Siglec-F deficient mice challenged intranasally with IL-4 or IL-13 had significantly increased levels of airway epithelial Siglec-F ligand expression, whereas this was not observed in WT or Siglec-F deficient mice challenged with TNF-α. There was a significant increase in the thickness of the peribronchial smooth muscle layer in OVA challenged Siglec-F deficient mice, but this was not associated with significant increased airway hyperreactivity compared to WT mice.
Overall, this study demonstrates an important role for Siglec-F in modulating levels of chronic eosinophilic airway inflammation, peribronchial fibrosis, thickness of the smooth muscle layer, mucus expression, fibronectin, and levels of peribronchial Siglec-F ligands suggesting that Siglec-F may normally function to limit levels of chronic eosinophilic inflammation and remodeling. In addition, IL-4 and IL-13 are important regulators of Siglec-F ligand expression by airway epithelium.
PMCID: PMC2988013  PMID: 21040544
9.  Aldose Reductase Inhibition Prevents Allergic Airway Remodeling through PI3K/AKT/GSK3β Pathway in Mice 
PLoS ONE  2013;8(2):e57442.
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).
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.
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.
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.
PMCID: PMC3584054  PMID: 23460857
10.  Protein tyrosine phosphatase SHP2 regulates TGF-β1 production in airway epithelia and asthmatic airway remodeling in mice 
Allergy  2012;67(12):1547-1556.
Transforming growth factor (TGF)-β1 produced in airway epithelia has been suggested as a contributor to the airway remodeling observed in asthma patients. The protein tyrosine phosphatase SHP2 is a demonstrable modulator of TGF-β1 production and thus a potential regulator of airway remodeling.
To define the signal event by which SHP2 regulates asthmatic responses in airway epithelial cells by using a mouse model of experimental OVA-induced airway remodeling.
The airways of Shp2flox/flox mice were infected with recombinant adenovirus vectors expressing a Cre recombinase–green fluorescence protein (GFP) fusion protein as part of allergen provocation studies using mice sensitized with ovalbumin (OVA) and repeatedly challenged with OVA. Several endpoint pathologies were assessed, including airway hyper-responsiveness (AHR), lung inflammatory score, peribronchial collagen deposition, and α-smooth muscle actin (SMA) hyperplasia. In vitro studies using airway epithelial cells (BEAS-2B) were used to investigate the role of SHP2 in the regulation of pulmonary remodeling events, including the expression of collagen, α-SMA, and TGF-β1.
Chronic OVA challenges in wild-type mice resulted in airway remodeling and lung dysfunction (e.g., increased inflammatory scores, collagen deposition (fibrosis), smooth muscle hyperplasia, and a significant increase in AHR). These endpoint pathology metrics were each significantly attenuated by conditional shp2 gene knockdown in airway epithelia. In vitro studies using BEAS-2B cells also demonstrated that the level of TGF-β1 production by these cells correlated with the extent of shp2 gene expression.
SHP2 activities in airway epithelial cells appear to modulate TGF-β1 production and, in turn, regulate allergic airway remodeling following allergen provocation.
Clinical Implications
Our findings identify SHP2 as a previously underappreciated contributor to the airway remodeling and lung dysfunction associated with allergen challenge. As such, SHP2 represents a potentially novel therapeutic target for the treatment of asthmatics.
PMCID: PMC3942166  PMID: 23057634
airway epithelia; asthma; mice; protein tyrosine phosphatase SHP2; remodeling
11.  Effect of aging on pulmonary inflammation, airway hyperresponsiveness and T and B cell responses in antigen sensitized and challenged mice 
The effect of aging on several pathologic features of allergic-asthma (pulmonary inflammation, eosinophilia, mucus-hypersecretion), and their relationship with airway hyperresponsiveness (AHR) is not well characterized.
To evaluate lung inflammation, mucus-metaplasia and AHR in relationship to age in murine models of allergic-asthma comparing young and older mice.
Young (6-week) and older (6-, 12- 18-month) BALB/c mice were sensitized and challenged with ovalbumin (OVA). AHR and bronchoalveolar fluid (BALF) total inflammatory cell count and differential were measured. To evaluate mucus-metaplasia, quantitative PCR for the major airway mucin-associated gene, MUC-5AC, from lung tissue was measured, and lung tissue sections stained with periodic acid-Schiff (PAS) for goblet-cell enumeration. Lung tissue cytokine gene expression was determined by qPCR, and systemic cytokine protein levels by ELISA from spleen-cell cultures. Antigen-specific serum IgE was determined by ELISA.
AHR developed in both aged and young OVA-sensitized/challenged mice (OVA-mice), and was more significantly increased in young OVA-mice than in aged OVA-mice. However, BALF eosinophil numbers were significantly higher, and lung histology showed greater inflammation in aged OVA-mice than in young OVA-mice. MUC-5AC expression and numbers of PAS+ staining bronchial epithelial cells were significantly increased in the aged OVA-mice. All aged OVA-mice had increased IL-5 and IFN-γ mRNA expression in the lung and IL-5 and IFN-γ protein levels from spleen cell cultures compared to young OVA-mice. OVA-IgE was elevated to a greater extent in aged OVA-mice.
Although pulmonary inflammation and mucus-metaplasia after antigen sensitization/challenge occurred to a greater degree in older mice, the increase in AHR was significantly less compared with younger OVA-mice. Antigen treatment produced a unique cytokine profile in older mice (elevated IFN-γ and IL-5) compared with young mice (elevated IL-4 and IL-13). Thus, the airway response to inflammation is lessened in aging animals, and may represent age-associated events leading to different phenotypes in response to antigen provocation.
PMCID: PMC2818115  PMID: 17845421
Aging; murine; asthma; airway hyperresponsiveness; eosinophil; inflammation
12.  An inhalation model of airway allergic response to inhalation of environmental Aspergillus fumigatus conidia in sensitized BALB/c mice 
Fungal exposure may elicit a number of pulmonary diseases in man, including allergic asthma. Fungal sensitization is linked to asthma severity, although the basis for this increased pathology remains ambiguous. To recapitulate environmental fungal allergen exposure in a human, a nose-only inhalation delivery of Aspergillus fumigatus conidia was employed in mice previously sensitized to Aspergillus antigen extract. BALB/c mice were immunized with subcutaneous and intraperitoneal injections of soluble A. fumigatus extract in alum, followed by 3 intranasal inoculations of the same fungal antigens dissolved in saline to elicit global sensitization in a manner similar to other published models. The animals were then challenged with a 10-min inhaled dose of live conidia blown directly from the surface of a mature A. fumigatus culture. After a single challenge with inhaled A. fumigatus conidia, allergic pulmonary inflammation and airway hyperresponsiveness were significantly increased above that of either naïve animals or animals that had been sensitized to A. fumigatus antigens but not challenged with conidia. The architecture of the lung was changed by inhalation of conidia with epithelial thickness, goblet cell metaplasia, and peribronchial collagen deposition significantly increased when compared to controls. Additionally, α-smooth muscle actin staining of histological sections showed visual evidence of increased peribronchial smooth muscle mass after fungal challenge. In summary, the inhalation of live A. fumigatus spores to the sensitized airways of BALB/c mice advances the study of the pulmonary response to fungus by providing a more natural route of exposure and, for the first time, demonstrates the consistent development of fibrosis and smooth muscle changes accompanying exposure to inhaled fungal spores in a mouse model.
PMCID: PMC3113699  PMID: 20482452
Asthma; Allergy; Fungi; Aspergillus; Murine model
13.  Anti-Siglec-F Antibody Reduces Allergen-Induced Eosinophilic Inflammation and Airway Remodeling1 
Siglec-F is a sialic acid-binding Ig superfamily receptor that is highly expressed on eosinophils. We have investigated whether administration of an anti-Siglec-F Ab to OVA-challenged wild-type mice would reduce levels of eosinophilic inflammation and levels of airway remodeling. Mice sensitized to OVA and challenged repetitively with OVA for 1 mo who were administered an anti-Siglec-F Ab had significantly reduced levels of peribronchial eosinophilic inflammation and significantly reduced levels of subepithelial fibrosis as assessed by either trichrome staining or lung collagen levels. The anti-Siglec-F Ab reduced the number of bone marrow, blood, and tissue eosinophils, suggesting that the anti-Siglec-F Ab was reducing the production of eosinophils. Administration of a F(ab′)2 fragment of an anti-Siglec-F Ab also significantly reduced levels of eosinophilic inflammation in the lung and blood. FACS analysis demonstrated increased numbers of apoptotic cells (annexin V+/CCR3+ bronchoalveolar lavage and bone marrow cells) in anti-Siglec-F Ab-treated mice challenged with OVA. The anti-Siglec-F Ab significantly reduced the number of peribronchial major basic protein+/TGF-β+ cells, suggesting that reduced levels of eosinophil-derived TGF-β in anti-Siglec-F Ab-treated mice contributed to reduced levels of peribronchial fibrosis. Administration of the anti-Siglec-F Ab modestly reduced levels of periodic acid-Schiff-positive mucus cells and the thickness of the smooth muscle layer. Overall, these studies suggest that administration of an anti-Siglec-F Ab can significantly reduce levels of allergen-induced eosinophilic airway inflammation and features of airway remodeling, in particular subepithelial fibrosis, by reducing the production of eosinophils and increasing the number of apoptotic eosinophils in lung and bone marrow.
PMCID: PMC2788790  PMID: 19783675
14.  Chronic OVA allergen challenged TNF p55/p75 Receptor deficient mice have reduced airway remodeling 
International immunopharmacology  2011;11(8):1038-1044.
The role of Tumor necrosis factor-α (TNF-α) in contributing to allergen induced airway remodeling in asthma is unknown. In this study we have utilized a mouse model of chronic OVA allergen induced airway remodeling to determine whether TNF p55/p75 receptor deficient mice (abbreviated TNF-R KO) had reduced levels of airway remodeling. Chronic OVA challenged WT mice had significantly increased levels of lung eosinophilic inflammation as well as features of airway remodeling including increased peribronchial fibrosis, thickness of the peribronchial smooth muscle layer, mucus expression, and deposition of extracellular matrix proteins. In contrast, TNF-R KO mice had significantly reduced levels of major basic protein positive peribronchial eosinophils and significantly reduced peribronchial fibrosis assessed by quantitating the area of peribronchial trichrome staining and total lung collagen. In addition, TNF-R KO mice had significantly reduced thickness of the peribronchial smooth muscle layer, area of peribronchial α-smooth muscle actin immunostaining, and levels of the extracellular matrix protein fibronectin. There was a non-significant trend for reduced mucus expression in TNF-R KO mice. Levels of peribronchial cells immunostaining positive for TGF-β1 were significantly reduced in TNF-R KO mice suggesting that reduced levels of TGF-β1 expression in TNF-R KO mice may contribute to reduced airway remodeling. Overall, this study suggests an important role for TNF-α in contributing to many features of allergen induced airway remodeling including changes in levels of peribronchial smooth muscle, subepithelial fibrosis, and deposition of extracellular matrix.
PMCID: PMC3121895  PMID: 21382533
eosinophil; fibronectin; smooth muscle
15.  Prolonged allergen challenge in mice leads to persistent airway remodelling 
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.
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.
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).
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.
In conclusion, we have demonstrated that prolonged allergen challenge results in persistent airway wall remodelling.
PMCID: PMC3428844  PMID: 15005746
airway remodelling; allergic airway inflammation; asthma; eosinophils; Th2 cytokines
16.  Modulation of the Effects of Lung Immune Response on Bone Marrow by Oral Antigen Exposure 
BioMed Research International  2013;2013:474132.
Allergic airway inflammation is attenuated by oral tolerization (oral exposure to allergen, followed by conventional sensitization and challenge with homologous antigen), which decreases airway allergen challenge-induced eosinophilic infiltration of the lungs and bone marrow eosinophilia. We examined its effects on bone marrow eosinophil and neutrophil production. Mice of wild type (BP-2, BALB/c, and C57BL/6) and mutant strains (lacking iNOS or CD95L) were given ovalbumin (OVA) or water (vehicle) orally and subsequently sensitized and challenged with OVA (OVA/OVA/OVA and H2O/OVA/OVA groups, resp.). Anti-OVA IgG and IgE, bone marrow eosinophil and neutrophil numbers, and eosinophil and neutrophil production ex vivo were evaluated. T lymphocytes from OVA/OVA/OVA or control H2O/OVA/OVA donors were transferred into naïve syngeneic recipients, which were subsequently sensitized/challenged with OVA. Alternatively, T lymphocytes were cocultured with bone marrow eosinophil precursors from histocompatible sensitized/challenged mice. OVA/OVA/OVA mice of the BP-2 and BALB/c strains showed, relative to H2O/OVA/OVA controls, significantly decreased bone marrow eosinophil counts and ex vivo eosinopoiesis/neutropoiesis. Full effectiveness in vivo required sequential oral/subcutaneous/intranasal exposures to the same allergen. Transfer of splenic T lymphocytes from OVA/OVA/OVA donors to naive recipients prevented bone marrow eosinophilia and eosinopoiesis in response to recipient sensitization/challenge and supressed eosinopoiesis upon coculture with syngeneic bone marrow precursors from sensitized/challenged donors.
PMCID: PMC3793322  PMID: 24171165
17.  Time Sequence of Airway Remodeling in a Mouse Model of Chronic Asthma: the Relation with Airway Hyperresponsiveness 
Journal of Korean Medical Science  2007;22(2):183-191.
During the course of establishing an animal model of chronic asthma, we tried to elucidate the time sequence of airway hyperresponsiveness (AHR), airway inflammation, airway remodeling, and associated cytokines. Seven-week-old female BALB/c mice were studied as a chronic asthma model using ovalbumin (OVA). After sensitization, mice were exposed twice weekly to aerosolized OVA, and were divided into three groups depending on the duration of 4 weeks, 8 weeks, and 12 weeks. At each time point, airway responsiveness, inflammatory cells, cytokines in bronchoalveolar lavage fluids (BALF), serum OVA-specific IgE, IgG1, IgG2a, and histological examination were carried out. AHR to methacholine, increased levels of OVA-specific IgG1 and IgG2a, and goblet cell hyperplasia were continuously sustained at each time point of weeks. In contrast, we observed a time-dependent decrease in serum OVA-specific IgE, BALF eosinophils, BALF cytokines such as IL-13, transforming growth factor-beta1, and a time-dependent increase in BALF promatrix metalloproteinase-9 and peribronchial fibrosis. In this OVA-induced chronic asthma model, we observed airway remodelings as well as various cytokines and inflammatory cells being involved in different time-dependent manners. However, increased airway fibrosis did not directly correlate with a further increase in airway hyperresponsiveness.
PMCID: PMC2693579  PMID: 17449921
Asthma; Disease Models, Animal; Mice; Bronchial Hyperreactivity; Chronic Disease
18.  Combined Sensitization of Mice to Extracts of Dust Mites, Ragweed and Aspergillus Breaks through Tolerance and Establishes Chronic Features of Asthma in Mice 
Existing asthma models develop tolerance when chronically exposed to the same allergen.
To establish a chronic model that sustains features of asthma long after discontinuation of allergen exposure.
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.
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.
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.
PMCID: PMC2683988  PMID: 19348928
chronic asthma; mouse; inflammation; airway hyperreactivity; tolerance; dendritic cells
19.  Adam8 Limits the Development of Allergic Airway Inflammation in Mice 
To determine whether a disintegrin and a metalloproteinase-8 (Adam8) regulates allergic airway inflammation (AAI) and airway hyper-responsiveness (AHR), we compared AAI and AHR in wild type (WT) versus Adam8−/− mice in different genetic backgrounds sensitized and challenged with ovalbumin (OVA) or house dust mite protein extract (HDM). OVA- and HDM-treated Adam8−/− mice had higher lung leukocyte counts, more airway mucus metaplasia, greater lung levels of some TH2 cytokines, and higher methacholine-induced increases in central airway resistance than allergen-treated WT mice. Studies of OVA-treated Adam8 bone marrow chimeric mice confirmed that leukocyte-derived Adam8 predominantly mediated Adam8’s anti-inflammatory activities in murine airways. Airway eosinophils and macrophages both expressed Adam8 in WT mice with AAI. Adam8 limited AAI and AHR in mice by reducing leukocyte survival because: 1) Adam8−/− mice with AAI had fewer apoptotic eosinophils and macrophages in their airways than WT mice with AAI; and 2) Adam8−/− macrophages and eosinophils had reduced rates of apoptosis compared with WT leukocytes when the intrinsic (but not the extrinsic) apoptosis pathway was triggered in the cells in vitro. ADAM8 was robustly expressed by airway granulocytes in lung sections from human asthma patients but, surprisingly, airway macrophages had less ADAM8 staining than airway eosinophils. Thus, ADAM8 has anti-inflammatory activities during AAI in mice by activating the intrinsic apoptosis pathway in myeloid leukocytes. Strategies that increase ADAM8 levels in myeloid leukocytes may have therapeutic efficacy in asthma.
PMCID: PMC3679335  PMID: 23670189
20.  120 The Features of Airway Remodeling Are More Severe in Female Mice 
Epidemiological studies have already shown that females are dominant in terms of the sex ratio of adult asthma prevalence and severe asthma. It has also been reported that female mice are more susceptible to the development of allergic airway inflammation and airway hyperresponsiveness (AHR) than males. However, there have been few reports of studies on sex difference in the pathogenesis of severe asthma, especially airway remodeling in an animal model. In this study, we investigated sex difference in formation of airway remodeling using a long-term antigen challenged asthma model.
Following ovalbumin (OVA)/alum intraperitoneal injection, male or female mice (BALB/c) were challenged with aerosolized 1% OVA on 3 days/week for 5 weeks, and we investigated the sex difference in AHR, airway inflammation, as well as airway remodeling.
In OVA-sensitized and -challenged (OVA/OVA) female mice, AHR, the number of eosinophils and lymphocytes, as well as Th2 cytokines and growth factors in BAL fluid were increased compared with OVA/OVA male mice. On the other hand, there is no significant difference in the level of eotaxin in BAL fluid. The histological features of airway remodeling, including goblet cell hyperplasia, subepithelial fibrosis and myofibroblast hypertrophy, were also increased in OVA/OVA female mice. Moreover, serum total and OVA-specific IgE were significantly elevated in OVA/OVA female mice.
These results indicate that female mice are dominant in terms of forming airway remodeling as compared with male mice. The involvement of sex difference for sensitization and growth factor release in lung tissue based on inflammatory cell infiltration is indicated for the mechanism of sex difference of airway remodeling.
PMCID: PMC3513133
21.  Alternaria induces STAT-6 dependent acute airway eosinophila and epithelial FIZZ1 expression that promotes airway fibrosis and epithelial thickness 
The fungal allergen, Alternaria, is specifically associated with severe asthma, including life-threatening exacerbations. To better understand the acute innate airway response to Alternaria, naïve WT mice were challenged once intranasally with Alternaria. Naïve WT mice developed significant BAL eosinophila following Alternaria challenge when analyzed 24 hours later. In contrast to Alternaria, neither Aspergillus nor Candida induced BAL eosinophilia. Gene microarray analysis of airway epithelial cell brushings demonstrated that Alternaria-challenged naïve WT mice had an over 20 fold increase level of expression of “Found in Inflammatory Zone 1” (FIZZ1/Retnla), a resistin-like molecule. Lung immunostaining confirmed strong airway epithelial FIZZ1 expression present as early as 3 hours after a single Alternaria challenge that persisted for at least 5 days and was significantly reduced in STAT6-deficient, but not PAR-2-deficient mice. Bone marrow chimera studies revealed that STAT6 expressed in lung cells was required for epithelial FIZZ1 expression, while in contrast, STAT6 present in bone marrow derived cells contributed to airway eosinophilia. Studies investigating which cells in the non-challenged lung bind FIZZ1 demonstrated that CD45+CD11c+ (macrophages and dendritic cells) as well as collagen-1 producing CD45 negative cells (fibroblasts) can bind to FIZZ1. Importantly, direct administration of recombinant FIZZ1 to naïve WT mice led to airway eosinophilia, peribronchial fibrosis, and increased thickness of the airway epithelium. Thus, Alternaria induces STAT-6 dependent acute airway eosinophila and epithelial FIZZ1 expression that promotes airway fibrosis and epithelial thickness. This may provide some insight into the uniquely pathogenic aspects of Alternaria-associated asthma.
PMCID: PMC3294141  PMID: 22327070
22.  Airway hyperresponsiveness is associated with airway remodeling but not inflammation in aging Cav1-/- mice 
Respiratory Research  2013;14(1):110.
Airway inflammation and airway remodeling are the key contributors to airway hyperresponsiveness (AHR), a characteristic feature of asthma. Both processes are regulated by Transforming Growth Factor (TGF)-β. Caveolin 1 (Cav1) is a membrane bound protein that binds to a variety of receptor and signaling proteins, including the TGF-β receptors. We hypothesized that caveolin-1 deficiency promotes structural alterations of the airways that develop with age will predispose to an increased response to allergen challenge.
AHR was measured in Cav1-deficient and wild-type (WT) mice 1 to 12 months of age to examine the role of Cav1 in AHR and the relative contribution of inflammation and airway remodeling. AHR was then measured in Cav1-/- and WT mice after an ovalbumin-allergen challenge performed at either 2 months of age, when remodeling in Cav1-/- and WT mice was equivalent, and at 6 months of age, when the Cav1-/- mice had established airway remodeling.
Cav1-/- mice developed increased thickness of the subepithelial layer and a correspondingly increased AHR as they aged. In addition, allergen-challenged Cav1-/- mice had an increase in AHR greater than WT mice that was largely independent of inflammation. Cav1-/- mice challenged at 6 months of age have decreased AHR compared to those challenged at 2 months with correspondingly decreased BAL IL-4 and IL-5 levels, inflammatory cell counts and percentage of eosinophils. In addition, in response to OVA challenge, the number of goblet cells and α-SMA positive cells in the airways were reduced with age in response to OVA challenge in contrast to an increased collagen deposition further enhanced in absence of Cav1.
A lack of Cav1 contributed to the thickness of the subepithelial layer in mice as they aged resulting in an increase in AHR independent of inflammation, demonstrating the important contribution of airway structural changes to AHR. In addition, age in the Cav1-/- mice is a contributing factor to airway remodeling in the response to allergen challenge.
PMCID: PMC4015038  PMID: 24138138
23.  Allergen-Induced Coexpression of bFGF and TGF-β1 by Macrophages in a Mouse Model of Airway Remodeling: bFGF Induces Macrophage TGF-β1 Expression in vitro 
Basic fibroblast growth factor (bFGF) is a cytokine that is mitogenic for fibroblasts and smooth muscle and may play a role in airway remodeling in asthma. We have used a mouse model of chronic ovalbumin (OVA) allergen-induced airway remodeling to determine whether bFGF and fibroblast growth factor receptor-1 are expressed and regulated by corticosteroids in the airway, as well as to determine whether bFGF mediates expression of another proremodeling cytokine, transforming growth factor (TGF)-β1.
The airway levels and localization of bFGF, FGF receptor-1 and TGF-β1 were determined by ELISA, immunohistology and image analysis in the remodeled airways of chronic OVA-challenged mice treated with either corticosteroids or diluent. In vitro cultures of bone narrow-derived macrophages were used to determine whether bFGF induced TGF-β1 expression.
Mice chronically challenged with OVA developed significant airway remodeling that was associated with significantly increased levels of bFGF and TGF-β1. Immunohistochemistry demonstrated significantly increased bFGF and FGF receptor-1 expression by peri- bronchial F4/80+ cells. Double-label immunofluorescence microscopy studies demonstrated that peribronchial macrophages coexpressed bFGF and TGF-β1. In vitro studies demonstrated that incubation of bone marrow-derived macrophages with bFGF induced expression of TGF-β1. Mice treated with corticosteroids and subjected to chronic OVA challenge had significantly reduced levels of bFGF, FGF receptor-1, peribronchial TGF-β1+ cells and airway remodeling.
Overall, this study demonstrates that allergen challenge stimulates peribronchial macrophages to coexpress bFGF and TGF-β1 and that bFGF may potentiate macrophage release of TGF-β1 through autocrine and/or paracrine pathways.
PMCID: PMC2997445  PMID: 21109744
Eosinophils; Allergy; Transforming growth factor-β1; Basic fibroblast growth factor
24.  In Utero Exposure to Environmental Tobacco Smoke Potentiates Adult Responses to Allergen in BALB/c Mice 
Environmental Health Perspectives  2007;115(4):548-555.
Fetal stress has been linked to adult atherosclerosis, obesity, and diabetes. Epidemiology studies have associated fetal exposure to maternal smoking and postnatal exposure to environmental tobacco smoke (ETS) with increased asthma risk.
We tested the hypothesis, in a mouse model of asthma, that in utero ETS exposure alters airway function and respiratory immune responses in adults.
Pregnant Balb/c mice were exposed daily to ETS or HEPA-filtered air (AIR). Offspring inhaled aerosolized ovalbumin (OVA) or saline in weeks 7–8. Regardless of whether they inhaled OVA or saline, mice were sensitized by OVA injections in weeks 11 and 13 followed by OVA aerosol challenge in weeks 14–15. At three time points, we assessed OVA-specific serum immunoglobins, bronchoalveolar lavage cells and cytokines, lung and nasal histopathology, and airway hyperresponsiveness (AHR).
At 6 weeks, we found no significant differences between in utero ETS and AIR mice. At 10 weeks, following OVA aerosol, ETS mice displayed greater AHR than AIR mice (α = 0.05), unaccompanied by changes in histopathology, cytokine profile, or antibody levels. At 15 weeks, mice that had inhaled saline in weeks 7–8 developed airway inflammation: eosinophilia (α = 0.05), interleukin-5 (α = 0.05), and AHR (α = 0.05) were greater in ETS mice than in AIR mice. Mice that had inhaled OVA in weeks 7–8 demonstrated no airway inflammation after sensitization and challenge.
In utero ETS exposure exacerbates subsequent adult responses to initial allergen exposure.
PMCID: PMC1852677  PMID: 17450223
airway hyperresponsiveness; allergens; asthma; environmental tobacco smoke (ETS); in utero; tolerance
25.  A protective role for periostin and TGF-β in IgE-mediated allergy and airway hyperresponsiveness 
The pathophysiology of asthma involves allergic inflammation and remodelling in the airway and airway hyperresponsiveness (AHR) to cholinergic stimuli, but many details of the specific underlying cellular and molecular mechanisms remain unknown. Periostin is a matricellular protein with roles in tissue repair following injury in both the skin and heart. It has recently been shown to be up-regulated in the airway epithelium of asthmatics and to increase active TGF-β. Though one might expect periostin to play a deleterious role in asthma pathogenesis, to date its biological role in the airway is unknown.
To determine the effect of periostin deficiency on airway responses to inhaled allergen.
In vivo measures of airway responsiveness, inflammation, and remodelling were made in periostin deficient mice and wild-type controls following repeated intranasal challenge with Aspergillus fumigatus antigen. In vitro studies of the effects of epithelial cell-derived periostin on murine T cells were also performed.
Surprisingly, compared with wild-type controls, periostin deficient mice developed increased AHR and serum IgE levels following allergen challenge without differences in two outcomes of airway remodelling (mucus metaplasia and peribronchial fibrosis). These changes were associated with decreased expression of TGF-β1 and Foxp3 in the lungs of periostin deficient mice. Airway epithelial cell-derived periostin-induced conversion of CD4+ CD25− cells into CD25+, Foxp3+ T cells in vitro in a TGF-β dependent manner.
Conclusions and Clinical Relevance
Allergen-induced increases in serum IgE and bronchial hyperresponsiveness are exaggerated in periostin deficient mice challenged with inhaled aeroallergen. The mechanism of periostin’s effect as a brake on allergen-induced responses may involve augmentation of TGF-β-induced T regulatory cell differentiation.
PMCID: PMC3271792  PMID: 22093101
airway hyperresponsiveness; asthma; IgE; mouse model; mucus metaplasia, peribronchial fibrosis; periostin; TGF-β

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