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1.  Resolution of airway inflammation and hyperreactivity after in vivo transfer of CD4+CD25+ regulatory T cells is interleukin 10 dependent 
The Journal of experimental medicine  2005;202(11):1539-1547.
Deficient suppression of T cell responses to allergen by CD4+CD25+ regulatory T cells has been observed in patients with allergic disease. Our current experiments used a mouse model of airway inflammation to examine the suppressive activity of allergen-specific CD4+CD25+ T cells in vivo. Transfer of ovalbumin (OVA) peptide-specific CD4+CD25+ T cells to OVA-sensitized mice reduced airway hyperreactivity (AHR), recruitment of eosinophils, and T helper type 2 (Th2) cytokine expression in the lung after allergen challenge. This suppression was dependent on interleukin (IL) 10 because increased lung expression of IL-10 was detected after transfer of CD4+CD25+ T cells, and regulation was reversed by anti-IL-10R antibody. However, suppression of AHR, airway inflammation, and increased expression of IL-10 were still observed when CD4+CD25+ T cells from IL-10 gene-deficient mice were transferred. Intracellular cytokine staining confirmed that transfer of CD4+CD25+ T cells induced IL-10 expression in recipient CD4+ T cells, but no increase in IL-10 expression was detected in airway macrophages, dendritic cells, or B cells. These data suggest that CD4+CD25+ T cells can suppress the Th2 cell-driven response to allergen in vivo by an IL-10-dependent mechanism but that IL-10 production by the regulatory T cells themselves is not required for such suppression.
doi:10.1084/jem.20051166
PMCID: PMC1350743  PMID: 16314435
2.  Requirement for Chemokine Receptor 5 in the Development of Allergen-Induced Airway Hyperresponsiveness and Inflammation 
Chemokine receptor (CCR) 5 is expressed on dendritic cells, macrophages, CD8 cells, memory CD4 T cells, and stromal cells, and is frequently used as a marker of T helper type 1 cells. Interventions that abrogate CCR5 or interfere with its ligand binding have been shown to alter T helper type 2–induced inflammatory responses. The role of CCR5 on allergic airway responses is not defined. CCR5-deficient (CCR5−/−) and wild-type (CCR5+/+) mice were sensitized and challenged with ovalbumin (OVA) and allergic airway responses were monitored 48 hours after the last OVA challenge. Cytokine levels in lung cell culture supernatants were also assessed. CCR5−/− mice showed significantly lower airway hyperresponsiveness (AHR) and lower numbers of total cells, eosinophils, and lymphocytes in bronchoalveolar lavage (BAL) fluid compared with CCR5+/+ mice after sensitization and challenge. The levels of IL-4 and IL-13 in BAL fluid of CCR5−/− mice were lower than in CCR5+/+ mice. Decreased numbers of lung T cells were also detected in CCR5−/− mice after sensitization and challenge. Transfer of OVA-sensitized T cells from CCR5+/+, but not transfer of CCR5−/− cells, into CCR5−/− mice restored AHR and numbers of eosinophils in BAL fluid after OVA challenge. Accordingly, the numbers of airway-infiltrating donor T cells were significantly higher in the recipients of CCR5+/+ T cells. Taken together, these data suggest that CCR5 plays a pivotal role in allergen-induced AHR and airway inflammation, and that CCR5 expression on T cells is essential to the accumulation of these cells in the airways.
doi:10.1165/rcmb.2010-0465OC
PMCID: PMC3262662  PMID: 21757680
rodent; T cells; cytokines; chemokines; lung
3.  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.
doi:10.1165/rcmb.2008-0256OC
PMCID: PMC2742748  PMID: 19151321
dendritic cells; CD8+ T cells; airway hyperresponsiveness
4.  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.
Background
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.
Methods
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.
Results
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.
Conclusion
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.
doi:10.1186/1465-9921-14-8
PMCID: PMC3570429  PMID: 23347423
Neutrophil; Elastase; Airway; Hyperresponsiveness; Asthma
5.  Effects of nebulized ketamine on allergen-induced airway hyperresponsiveness and inflammation in actively sensitized Brown-Norway rats 
Since airway hyperresponsiveness (AHR) and allergic inflammatory changes are regarded as the primary manifestations of asthma, the main goals of asthma treatment are to decrease inflammation and maximize bronchodilation. These goals can be achieved with aerosol therapy. Intravenous administration of the anesthetic, ketamine, has been shown to trigger bronchial smooth muscle relaxation. Furthermore, increasing evidence suggests that the anti-inflammatory properties of ketamine may protect against lung injury. However, ketamine inhalation might yield the same or better results at higher airway and lower ketamine plasma concentrations for the treatment of asthma. Here, we studied the effect of ketamine inhalation on bronchial hyperresponsiveness and airway inflammation in a Brown-Norway rat model of ovalbumin(OVA)-induced allergic asthma. Animals were actively sensitized by subcutaneous injection of OVA and challenged by repeated intermittent (thrice weekly) exposure to aerosolized OVA for two weeks. Before challenge, the sensitizened rats received inhalation of aerosol of phosphate-buffered saline (PBS) or aerosol of ketamine or injection of ketamine respectivity. Airway reactivity to acetylcholine (Ach) was measured in vivo, and various inflammatory markers, including Th2 cytokines in bronchoalveolar lavage fluid (BALF), as well as induciable nitric oxide synthase (iNOS) and nitric oxide (NO) in lungs were examined. Our results revealed that delivery of aerosolized ketamine using an ultrasonic nebulizer markedly suppressed allergen-mediated airway hyperreactivity, airway inflammation and airway inflammatory cell infiltration into the BALF, and significantly decreased the levels of interleukin-4 (IL-4) in the BALF and expression of iNOS and the concentration of NO in the inflamed airways from OVA-treated rats. These findings collectively indicate that nebulized ketamine attenuated many of the central components of inflammatory changes and AHR in OVA-provoked experimental asthma, potentially providing a new therapeutic approach against asthma.
doi:10.1186/1476-9255-4-10
PMCID: PMC1876456  PMID: 17480224
6.  IL-35 Production by ICOS+ Tregs Reverses Established IL-17-dependent Allergic Airways Disease 
Background
Recent evidence suggests that IL-17 contributes to airway hyperresponsiveness (AHR); however, the mechanisms that suppress the production of this cytokine remain poorly defined.
Objectives
We sought to understand the cellular and molecular basis for suppression of established, IL-17-dependent allergic airways disease.
Methods
Mice were sensitized by airway instillations of ovalbumin (OVA) together with low levels of lipopolysaccharide. Leukocyte recruitment to the lung and AHR were assessed following daily challenges with aerosolized OVA. Flow cytometry and gene targeted mice were used to identify naturally-arising subsets of regulatory T cells (Tregs) and their cytokines required for the suppression of established allergic airway disease.
Results
Allergic sensitization through the airway primed both effector and regulatory responses. Effector responses were initially dominant and led to airway inflammation and IL-17-dependent AHR. However, after multiple daily allergen challenges, IL-17 production and AHR declined, even though pulmonary levels of Th17 cells remained high. This loss of AHR was reversible and required the expansion of a Treg subset expressing both Foxp3 and inducible co-stimulator (ICOS). These Tregs also expressed the regulatory cytokines, IL-10, TGF-beta and IL-35. Whereas IL-10 and TGF-beta were dispensable for suppression of airway hyperresponsiveness, IL-35 was required. Analysis of human ICOS+ Tregs revealed that they also selectively expressed IL-35.
Conclusion
IL-35 production by ICOS+ Tregs can suppress IL-17 production and thereby reverse established, IL-17-dependent AHR in mice. The production of IL-35 by human ICOS+ Tregs suggests that targeting this pathway might be of therapeutic value for treating allergic asthma in humans.
doi:10.1016/j.jaci.2011.08.009
PMCID: PMC3269135  PMID: 21906794
Asthma; airway hyperresponsiveness; AHR; IL-17; Th17; Th2; IL-35; ICOS; ovalbumin
7.  Antigen-sensitized CD4+CD62Llow memory/effector T helper 2 cells can induce airway hyperresponsiveness in an antigen free setting 
Respiratory Research  2005;6(1):46.
Background
Airway hyperresponsiveness (AHR) is one of the most prominent features of asthma, however, precise mechanisms for its induction have not been fully elucidated. We previously reported that systemic antigen sensitization alone directly induces AHR before development of eosinophilic airway inflammation in a mouse model of allergic airway inflammation, which suggests a critical role of antigen-specific systemic immune response itself in the induction of AHR. In the present study, we examined this possibility by cell transfer experiment, and then analyzed which cell source was essential for this process.
Methods
BALB/c mice were immunized with ovalbumin (OVA) twice. Spleen cells were obtained from the mice and were transferred in naive mice. Four days later, AHR was assessed. We carried out bronchoalveolar lavage (BAL) to analyze inflammation and cytokine production in the lung. Fluorescence and immunohistochemical studies were performed to identify T cells recruiting and proliferating in the lung or in the gut of the recipient. To determine the essential phenotype, spleen cells were column purified by antibody-coated microbeads with negative or positive selection, and transferred. Then, AHR was assessed.
Results
Transfer of spleen cells obtained from OVA-sensitized mice induced a moderate, but significant, AHR without airway antigen challenge in naive mice without airway eosinophilia. Immunization with T helper (Th) 1 elicited antigen (OVA with complete Freund's adjuvant) did not induce the AHR. Transferred cells distributed among organs, and the cells proliferated in an antigen free setting for at least three days in the lung. This transfer-induced AHR persisted for one week. Interleukin-4 and 5 in the BAL fluid increased in the transferred mice. Immunoglobulin E was not involved in this transfer-induced AHR. Transfer of in vitro polarized CD4+ Th2 cells, but not Th1 cells, induced AHR. We finally clarified that CD4+CD62Llow memory/effector T cells recruited in the lung and proliferated, thus induced AHR.
Conclusion
These results suggest that antigen-sensitized memory/effector Th2 cells themselves play an important role for induction of basal AHR in an antigen free, eosinophil-independent setting. Therefore, regulation of CD4+ T cell-mediated immune response itself could be a critical therapeutic target for allergic asthma.
doi:10.1186/1465-9921-6-46
PMCID: PMC1180472  PMID: 15921525
8.  Toll-Like Receptor-9 Agonist Inhibits Airway Inflammation, Remodeling and Hyperreactivity in Mice Exposed to Chronic Environmental Tobacco Smoke and Allergen 
Background
As passive environmental tobacco smoke (ETS) exposure in nonsmokers can increase both asthma symptoms and the frequency of asthma exacerbations, we utilized a mouse model, in which ovalbumin (OVA) + ETS induce significantly increased levels of eosinophilic airway inflammation and remodeling compared to either stimulus alone, to determine whether a Toll-like receptor-9 (TLR-9) agonist could reduce levels of airway inflammation, airway remodeling and airway hyperreactivity (AHR).
Methods
Mice treated with or without a TLR-9 agonist were sensitized to OVA and challenged with OVA + ETS for 1 month. AHR to methacholine was assessed in intubated and ventilated mice. Lung Th2 cytokines and TGF-β1 were measured by ELISA. Lungs were processed for histology and immunohistology to quantify eosinophils, mucus, peribronchial fibrosis and smooth muscle changes using image analysis.
Results
Administration of a TLR-9 agonist to mice coexposed to chronic ETS and chronic OVA allergen significantly reduced levels of eosinophilic airway inflammation, mucus production, peribronchial fibrosis, the thickness of the peribronchial smooth muscle layer, and AHR. The reduced airway remodeling in mice treated with the TLR-9 agonist was associated with significantly reduced numbers of peribronchial MBP+ and peribronchial TGF-β1+ cells, and with significantly reduced levels of lung Th2 cytokines [interleukin-5 and interleukin-13] and TGF-β1.
Conclusion
These studies demonstrate that TLR-9-based therapies inhibit airway inflammation, remodeling and AHR in mice coexposed to ETS and allergen who exhibit enhanced airway inflammation and remodeling.
doi:10.1159/000250437
PMCID: PMC2853586  PMID: 19851071
Toll-like receptor-9; Airway hyperreactivity; Airway inflammation; Airway remodeling; Eosinophils
9.  Aeroallergen-induced eosinophilic inflammation, lung damage, and airways hyperreactivity in mice can occur independently of IL-4 and allergen-specific immunoglobulins. 
Journal of Clinical Investigation  1997;99(6):1329-1339.
In this investigation we have used a mouse model containing certain phenotypic characteristics consistent with asthma and IL-4- and CD40-deficient mice to establish the role of this cytokine and allergen-specific immunoglobulins in the initiation of airways hyperreactivity and morphological changes to the airways in responses to aeroallergen challenge. Sensitization and aerosol challenge of mice with ovalbumin resulted in a severe airways inflammatory response which directly correlated with the induction of extensive airways damage and airways hyperreactivity to beta-methacholine. Inflammatory infiltrates were primarily characterized by the presence of CD4+ T cells and eosinophils. In IL-4-deficient mice, the recruitment of airways eosinophils was impaired, but not abolished in response to aeroallergen. Moreover, the characteristic airways damage and hyperreactivity normally resulting from allergen inhalation were not attenuated. Induction of these structural and functional changes to the airways occurred in the absence of ovalbumin-specific IgE and IgG1, but IgG2a and IgG3 were detected in the sera of IL-4-deficient mice. CD4+ T cells isolated from both wild-type and IL-4-deficient mice given ovalbumin produced significant levels of IL-5 after in vitro stimulation. Treatment of IL-4-deficient mice with anti-IL-5 mAb before aeroallergen challenge abolished blood and airways eosinophilia, lung damage, and airways hyperreactivity. These results indicate that IL-4 is not essential for the development of IL-5-producing CD4+ T cells or for the induction of eosinophilic inflammation and airways damage and hyperreactivity. In response to sensitization and aerosol challenge, CD40-deficient mice did not produce ovalbumin-specific IgE, IgG isotypes, or IgA, and airways inflammation and hyperreactivity were not attenuated. Our results suggest that allergic airways disease can occur via pathways which operate independently of IL-4 and allergen-specific immunoglobulins. Activation of these pathways is intimately associated with IL-5 and eosinophilic inflammation. Such pathways may play a substantive role in the etiology of asthma.
PMCID: PMC507949  PMID: 9077543
10.  IL-12-STAT4-IFN-γ axis is a key downstream pathway in the development of IL-13-mediated asthma phenotypes in a Th2 type asthma model 
Experimental & Molecular Medicine  2010;42(8):533-546.
IL-4 and IL-13 are closely related cytokines that are produced by Th2 cells. However, IL-4 and IL-13 have different effects on the development of asthma phenotypes. Here, we evaluated downstream molecular mechanisms involved in the development of Th2 type asthma phenotypes. A murine model of Th2 asthma was used that involved intraperitoneal sensitization with an allergen (ovalbumin) plus alum and then challenge with ovalbumin alone. Asthma phenotypes, including airway-hyperresponsiveness (AHR), lung inflammation, and immunologic parameters were evaluated after allergen challenge in mice deficient in candidate genes. The present study showed that methacholine AHR and lung inflammation developed in allergen-challenged IL-4-deficient mice but not in allergen-challenged IL-13-deficient mice. In addition, the production of OVA-specific IgG2a and IFN-γ-inducible protein (IP)-10 was also impaired in the absence of IL-13, but not of IL-4. Lung-targeted IFN-γ over-expression in the airways enhanced methacholine AHR and non-eosinophilic inflammation; in addition, these asthma phenotypes were impaired in allergen-challenged IFN-γ-deficient mice. Moreover, AHR, non-eosinophilic inflammation, and IFN-γ expression were impaired in allergen-challenged IL-12Rβ2- and STAT4-deficient mice; however, AHR and non-eosinophilic inflammation were not impaired in allergen-challenged IL-4Rα-deficient mice, and these phenomena were accompanied by the enhanced expression of IL-12 and IFN-γ. The present data suggest that IL-13-mediated asthma phenotypes, such as AHR and non-eosinophilic inflammation, in the Th2 type asthma are dependent on the IL-12-STAT4-IFN-γ axis, and that these asthma phenotypes are independent of IL-4Ralpha-mediated signaling.
doi:10.3858/emm.2010.42.8.054
PMCID: PMC2928926  PMID: 20592486
asthma; interferon-γ; interleukin-12; interleukin-13; respiratory hypersensitivity; Th2 cells
11.  Importance of Myeloid Dendritic Cells in Persistent Airway Disease after Repeated Allergen Exposure 
Rationale: There is conflicting information about the development and resolution of airway inflammation and airway hyperresponsiveness (AHR) after repeated airway exposure to allergen in sensitized mice.
Methods: Sensitized BALB/c and C57BL/6 mice were exposed to repeated allergen challenge on 3, 7, or 11 occasions. Airway function in response to inhaled methacholine was monitored; bronchoalveolar lavage fluid inflammatory cells were counted; and goblet cell metaplasia, peribronchial fibrosis, and smooth muscle hypertrophy were quantitated on tissue sections. Bone marrow–derived dendritic cells were generated after differentiation of bone marrow cells in the presence of growth factors.
Results: Sensitization to ovalbumin (OVA) in alum, followed by three airway exposures to OVA, induced lung eosinophilia, goblet cell metaplasia, mild peribronchial fibrosis, and peribronchial smooth muscle hypertrophy; increased levels of interleukin (IL)-4, IL-5, IL-13, granulocyte-macrophage colony–stimulating factor, transforming growth factor-β1, eotaxin-1, RANTES (regulated on activation, normal T-cell expressed and secreted), and OVA-specific IgG1 and IgE; and resulted in AHR. After seven airway challenges, development of AHR was markedly decreased as was the production of IL-4, IL-5, and IL-13. Levels of IL-10 in both strains and the level of IL-12 in BALB/c mice increased. After 11 challenges, airway eosinophilia and peribronchial fibrosis further declined and the cytokine and chemokine profiles continued to change. At this time point, the number of myeloid dendritic cells and expression of CD80 and CD86 in lungs were decreased compared with three challenges. After 11 challenges, intratracheal instillation of bone marrow–derived dendritic cells restored AHR and airway eosinophilia.
Conclusions: These data suggest that repeated allergen exposure leads to progressive decreases in AHR and allergic inflammation, through decreases in myeloid dendritic cell numbers.
doi:10.1164/rccm.200505-783OC
PMCID: PMC2662981  PMID: 16192450
airway hyperresponsiveness; chronic asthma; cytokine; dendritic cells; eosinophil
12.  Modulation of Distinct Asthmatic Phenotypes in Mice by Dose-Dependent Inhalation of Microbial Products 
Background: Humans with asthma display considerable heterogeneity with regard to T helper (Th) 2–associated eosinophilic and Th17-associated neutrophilic inflammation, but the impact of the environment on these different forms of asthma is poorly understood.
Objective: We studied the nature and longevity of asthma-like responses triggered by inhalation of allergen together with environmentally relevant doses of inhaled lipopolysaccharide (LPS).
Methods: Ovalbumin (OVA) was instilled into the airways of mice together with a wide range of LPS doses. Following a single OVA challenge, or multiple challenges, animals were assessed for pulmonary cytokine production, airway inflammation, and airway hyperresponsiveness (AHR).
Results: Mice instilled with OVA together with very low doses (≤ 10–3 μg) of LPS displayed modest amounts of Th2 cytokines, with associated airway eosinophilia and AHR after a single challenge, and these responses were sustained after multiple OVA challenges. When the higher but still environmentally relevant dose of 10–1 μg LPS was used, mice initially displayed similar Th2 responses, as well as Th17-associated neutrophilia. After multiple OVA challenges, however, the 10–1 μg LPS animals also accumulated large numbers of allergen-specific T regulatory (Treg) cells with high levels of inducible co-stimulatory molecule (ICOS). As a result, asthma-like features in these mice were shorter-lived than in mice sensitized using lower doses of LPS.
Conclusions: The nature and longevity of Th2, Th17, and Treg immune responses to inhaled allergen are dependent on the quantity of LPS inhaled at the time of allergic sensitization. These findings might account in part for the heterogeneity of inflammatory infiltrates seen in lungs of asthmatics.
Citation: Whitehead GS, Thomas SY, Cook DN. 2014. Modulation of distinct asthmatic phenotypes in mice by dose-dependent inhalation of microbial products. Environ Health Perspect 122:34–42; http://dx.doi.org/10.1289/ehp.1307280
doi:10.1289/ehp.1307280
PMCID: PMC3888577  PMID: 24168764
13.  Nerve growth factor exacerbates allergic lung inflammation and airway remodeling in a rat model of chronic asthma 
Nerve growth factor (NGF) is critical in the pathogenesis of allergic airway inflammation in vivo and induces proliferation of airway smooth muscle cells and matrix metalloproteinase-9 (MMP-9) expression in vitro. However, the effects of NGF on chronic pulmonary diseases of allergic origin remain unknown. To investigate the effects of NGF on lung inflammation and airway remodeling, 32 Wistar rats were randomly divided into four groups: control, NGF, ovalbumin (OVA) and anti-rat-β-NGF antibody (anti-NGF). Aerosolized OVA was administered to the rats in the NGF, OVA and anti-NGF groups to generate the asthmatic rat model, and NGF or anti-NGF was administered 3 h prior to OVA inhalation every two days. On day 70, bronchial responsiveness tests, bronchoalveolar lavage (BAL) and cell counting were conducted. The levels of serum OVA-specific immunoglobulin E (IgE) and of T-helper cell type-2 (Th2) cytokines [interleukin (IL)-4 and IL-13] in the BAL fluid were measured by enzyme-linked immunosorbent assay. The expression levels of NGF protein and MMP-9 mRNA, and the activity of MMP-9 in the lungs were detected by western blot analysis, quantitative polymerase chain reaction and gelatin zymography analysis, respectively. Our results showed that NGF significantly increased eosinophilic airway inflammation, persistent airway hyperresponsiveness (AHR), the serum levels of OVA-specific IgE and the levels of Th2 cytokines in the BAL fluid, and also increased the expression levels and activity of MMP-9. However, anti-NGF treatment significantly inhibited eosinophilic airway inflammation, persistent AHR and airway remodeling. The results showed that NGF may have exacerbated the development of airway inflammation, AHR and airway remodeling through a Th2 pathway and by increasing the level of MMP-9 expression. Therefore, anti-NGF is potentially beneficial for preventing and treating patients with asthma.
doi:10.3892/etm.2013.1284
PMCID: PMC3820700  PMID: 24223654
nerve growth factor; asthma; airway remodeling; T-helper cell type-2 cytokines; matrix metalloproteinase-9
14.  Haemophilus influenzae Infection Drives IL-17-Mediated Neutrophilic Allergic Airways Disease 
PLoS Pathogens  2011;7(10):e1002244.
A subset of patients with stable asthma has prominent neutrophilic and reduced eosinophilic inflammation, which is associated with attenuated airways hyper-responsiveness (AHR). Haemophilus influenzae has been isolated from the airways of neutrophilic asthmatics; however, the nature of the association between infection and the development of neutrophilic asthma is not understood. Our aim was to investigate the effects of H. influenzae respiratory infection on the development of hallmark features of asthma in a mouse model of allergic airways disease (AAD). BALB/c mice were intraperitoneally sensitized to ovalbumin (OVA) and intranasally challenged with OVA 12–15 days later to induce AAD. Mice were infected with non-typeable H. influenzae during or 10 days after sensitization, and the effects of infection on the development of key features of AAD were assessed on day 16. T-helper 17 cells were enumerated by fluorescent-activated cell sorting and depleted with anti-IL-17 neutralizing antibody. We show that infection in AAD significantly reduced eosinophilic inflammation, OVA-induced IL-5, IL-13 and IFN-γ responses and AHR; however, infection increased airway neutrophil influx in response to OVA challenge. Augmented neutrophilic inflammation correlated with increased IL-17 responses and IL-17 expressing macrophages and neutrophils (early, innate) and T lymphocytes (late, adaptive) in the lung. Significantly, depletion of IL-17 completely abrogated infection-induced neutrophilic inflammation during AAD. In conclusion, H. influenzae infection synergizes with AAD to induce Th17 immune responses that drive the development of neutrophilic and suppress eosinophilic inflammation during AAD. This results in a phenotype that is similar to neutrophilic asthma. Infection-induced neutrophilic inflammation in AAD is mediated by IL-17 responses.
Author Summary
Approximately 50% of asthmatics have non-eosinophilic inflammation, and 20% of these patients have severe neutrophilic inflammation and increased IL-8 levels. These so-called neutrophilic asthmatics have persistent airway colonization with bacteria, and Haemophilus influenzae is one of the bacteria most commonly isolated. However, how H. influenzae is associated with the pathogenesis of neutrophilic asthma is unknown. In this study we used mouse models to investigate the relationship between H. influenzae infection and allergic airways disease (AAD). We showed that infection promoted the development of hallmark features of neutrophilic asthma. Infection suppressed Th2 cytokines, eosinophilic inflammation, and AHR in AAD, while increasing neutrophilic inflammation and IL-17 responses. Importantly, inhibition of IL-17 during AAD reduced airway neutrophils and neutrophil chemokines, suggesting that infection drives the development of neutrophilic inflammation through an IL-17-mediated mechanism. This provides novel insights into the mechanisms that may underpin infection-induced neutrophilic asthma. These data also suggest that treatments targeting infection may lead to improved management of neutrophilic asthma.
doi:10.1371/journal.ppat.1002244
PMCID: PMC3188527  PMID: 21998577
15.  Low-Dose Lipopolysaccharide Affects Lung Allergic Responses by Regulating Jagged1 Expression on Antigen-Pulsed Dendritic Cells 
Background
Notch signaling pathways govern immune function and the regulation of Th1 and Th2 differentiation. We previously demonstrated essential interactions between Notch on CD4+ T cells and Jagged1 on antigen-presenting cells in Th2 differentiation for the full development of allergen-induced airway hyperresponsiveness (AHR) and allergic airway inflammation.
Methods
Bone marrow-derived dendritic cells (BMDCs) were differentiated and incubated with different preparations of ovalbumin (OVA), including lipopolysaccharide (LPS)-depleted and LPS-spiked preparations. In some experiments recipient mice also received soluble Jagged1-Fc in addition to allergen-pulsed BMDCs. Ten days following transfer of BMDCs, mice were exposed to three airway challenges with OVA, and airway responsiveness to inhaled methacholine, airway inflammation and cytokine production were monitored 48 h later. Notch ligand expression was assessed by real-time PCR.
Results
Induction of Jagged1 expression on antigen-pulsed BMDCs was dependent on low-dose endotoxin. In vivo, transfer of endotoxin-free, antigen-pulsed BMDCs failed to induce AHR or airway eosinophilia on allergen challenge. However, administration of exogenous Jagged1-Fc together with endotoxin-free, allergen-pulsed BMDCs fully restored the responses to allergen challenge.
Conclusions
These data demonstrate that LPS regulates the expression of Jagged1 on BMDCs, which is essential for the full development of lung allergic responses.
doi:10.1159/000324836
PMCID: PMC3180653  PMID: 21912175
Asthma; Dendritic cells; Endotoxin; Notch ligands
16.  Attenuated expression of tenascin-c in ovalbumin-challenged STAT4-/- mice 
Respiratory Research  2011;12(1):2.
Background
Asthma leads to structural changes in the airways, including the modification of extracellular matrix proteins such as tenascin-C. The role of tenascin-C is unclear, but it might act as an early initiator of airway wall remodelling, as its expression is increased in the mouse and human airways during allergic inflammation. In this study, we examined whether Th1 or Th2 cells are important regulators of tenascin-C in experimental allergic asthma utilizing mice with impaired Th1 (STAT4-/-) or Th2 (STAT6-/-) immunity.
Methods
Balb/c wildtype (WT), STAT4-/- and STAT6-/- mice were sensitized with intraperitoneally injected ovalbumin (OVA) followed by OVA or PBS airway challenge. Airway hyperreactivity (AHR) was measured and samples were collected. Real time PCR and immunohistochemistry were used to study cytokines and differences in the expression of tenascin-C. Tenascin-C expression was measured in human fibroblasts after treatment with TNF-α and IFN-γ in vitro.
Results
OVA-challenged WT mice showed allergic inflammation and AHR in the airways along with increased expression of TNF-α, IFN-γ, IL-4 and tenascin-C in the lungs. OVA-challenged STAT4-/- mice exhibited elevated AHR and pulmonary eosinophilia. The mRNA expression of TNF-α and IFN-γ was low, but the expression of IL-4 was significantly elevated in these mice. OVA-challenged STAT6-/- mice had neither AHR nor pulmonary eosinophilia, but had increased expression of mRNA for TNF-α, IFN-γ and IL-4. The expression of tenascin-C in the lungs of OVA-challenged STAT4-/- mice was weaker than in those of OVA-challenged WT and STAT6-/- mice suggesting that TNF-α and IFN-γ may regulate tenascin-C expression in vivo. The stimulation of human fibroblasts with TNF-α and IFN-γ induced the expression of tenascin-C confirming our in vivo findings.
Conclusions
Expression of tenascin-C is significantly attenuated in the airways of STAT4-/- mice, which may be due to the impaired secretion of TNF-α and IFN-γ in these mice.
doi:10.1186/1465-9921-12-2
PMCID: PMC3024219  PMID: 21205293
17.  Resolution of Allergic Inflammation and Airway Hyperreactivity Is Dependent upon Disruption of the T1/ST2–IL-33 Pathway 
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.
doi:10.1164/rccm.200805-666OC
PMCID: PMC2675564  PMID: 19179489
Th2 cells; IL-13; IL-4
18.  TLR-2 Activation Induces Regulatory T Cells and Long-Term Suppression of Asthma Manifestations in Mice 
PLoS ONE  2013;8(2):e55307.
Asthma is a chronic inflammatory disease of the airways characterized by variable airway obstruction and airway hyperresponsiveness (AHR). The T regulatory (Treg) cell subset is critically important for the regulation of immune responses. Adoptive transfer of Treg cells has been shown to be sufficient for the suppression of airway inflammation in experimental allergic asthma. Intervention strategies aimed at expanding the Treg cell population locally in the airways of sensitized individuals are therefore of high interest as a potential therapeutic treatment for allergic airway disease. Here, we aim to test whether long-term suppression of asthma manifestations can be achieved by locally expanding the Treg cell subset via intranasal administration of a TLR-2 agonist. To model therapeutic intervention aimed at expanding the endogenous Treg population in a sensitized host, we challenged OVA-sensitized mice by OVA inhalation with concomitant intranasal instillation of the TLR-2 agonist Pam3Cys, followed by an additional series of OVA challenges. Pam3Cys treatment induced an acute but transient aggravation of asthma manifestations, followed by a reduction or loss of AHR to methacholine, depending on the time between Pam3Cys treatment and OVA challenges. In addition, Pam3Cys-treatment induced significant reductions of eosinophils and increased numbers of Treg cells in the lung infiltrates. Our data show that, despite having adverse acute effects, TLR2 agonist treatment as a therapeutic intervention induces an expansion of the Treg cell population in the lungs and results in long-term protection against manifestation of allergic asthma upon subsequent allergen provocation. Our data indicate that local expansion of Tregs in allergic airway disease is an interesting therapeutic approach that warrants further investigation.
doi:10.1371/journal.pone.0055307
PMCID: PMC3564817  PMID: 23393567
19.  Effects of Corni fructus on ovalbumin-induced airway inflammation and airway hyper-responsiveness in a mouse model of allergic asthma 
Background
Allergic asthma is a chronic inflammatory lung disease that is characterized by airway hyperresponsiveness (AHR) to allergens, airway oedema, increased mucus secretion, excess production of T helper-2 (Th2) cytokines, and eosinophil accumulation in the lungs. Corni fructus (CF) is a fruit of Cornus officinalis Sieb. Et. Zucc. (Cornaceae) and has been used in traditional Korean medicine as an anti-inflammatory, analgesic, and diuretic agent. To investigate the anti-asthmatic effects of CF and their underlying mechanism, we examined the influence of CF on the development of pulmonary eosinophilic inflammation and airway hyperresponsiveness in a mouse model of allergic asthma.
Methods
In this study, BALB/c mice were systemically sensitized to ovalbumin (OVA) by intraperitoneal (i.p.), intratracheal (i.t.) injections and intranasal (i.n.) inhalation of OVA. We investigated the effect of CF on airway hyperresponsiveness, pulmonary eosinophilic infiltration, various immune cell phenotypes, Th2 cytokine production, and OVA-specific immunoglobulin E (IgE) production.
Results
The CF-treated groups showed suppressed eosinophil infiltration, allergic airway inflammation, and AHR via reduced production of interleuin (IL) -5, IL-13, and OVA-specific IgE.
Conclusions
Our data suggest that the therapeutic effects of CF in asthma are mediated by reduced production of Th2 cytokines (IL-5), eotaxin, and OVA-specific IgE and reduced eosinophil infiltration.
doi:10.1186/1476-9255-9-9
PMCID: PMC3328291  PMID: 22439901
Corni fructus; Asthma; Eosinophil; IL-5; CCR3
20.  Exaggerated IL-17 response to epicutaneous sensitization mediates airway inflammation in the absence of IL-4 and IL-13 
Background
Atopic dermatitis (AD) is characterized by local and systemic Th2 responses to cutaneously introduced allergens and is a risk factor for asthma. Blockade of Th2 cytokines has been suggested as therapy for AD.
Objectives
To examine the effect of the absence of IL-4 and IL-13 on the Th-17 response to epicutaneous (EC) sensitization in a mouse model of allergic skin inflammation with features of AD.
Methods
Wild-type (WT), IL-4KO, IL-13KO and IL-4/13 double KO (DKO) mice were subjected to EC sensitization with ovalbumin (OVA) or saline and airway challenged with OVA. Systemic immune responses to OVA, skin and airway inflammation, and airway hyperresponsiveness (AHR) were examined.
Results
OVA sensitized DKO mice exhibited impaired Th2 driven responses with undetectable OVA specific IgE and severely diminished eosinophil infiltration at sensitized skin sites, but intact dermal infiltration with CD4+ cells. DKO mice mounted an exaggerated IL-17A, but normal IFN-γ and IL-5 systemic responses. Airway challenge of these mice with OVA caused marked upregulation of IL-17 mRNA expression in the lungs, increased neutrophilia in bronchoalveolar lavage fluid (BALF), airway inflammation characterized by mononuclear cell infiltration with no detectable eosinophils, and bronchial hyperresponsiveness to methacholine that were reversed by IL-17 blockade. IL-4, but not IL-13, was identified as the major Th2 cytokine that downregulates the IL-17 response in EC sensitized mice.
Conclusion
EC sensitization in the absence of IL-4/IL-13 induces an exaggerated Th17 response systemically, and in lungs following antigen challenge that results in airway inflammation and AHR.
Clinical implications
Blockade of IL-4 may promote IL-17-mediated airway inflammation in AD.
doi:10.1016/j.jaci.2009.07.040
PMCID: PMC2895457  PMID: 19815118
IL-17; Th2 cytokines; atopic dermatitis; asthma
21.  Transforming Growth Factor-β1 Suppresses Airway Hyperresponsiveness in Allergic Airway Disease 
Rationale: Asthma is characterized by increases in airway resistance, pulmonary remodeling, and lung inflammation. The cytokine transforming growth factor (TGF)-β has been shown to have a central role in asthma pathogenesis and in mouse models of allergic airway disease.
Objectives: To determine the contribution of TGF-β to airway hyperresponsiveness (AHR), we examined the time course, source, and isoform specificity of TGF-β production in an in vivo mouse asthma model. To then elucidate the function of TGF-β in AHR, inflammation, and pulmonary fibrosis, we examined the effects of blocking TGF-β signaling with neutralizing antibody.
Methods: Mice were sensitized and challenged with ovalbumin (OVA) to establish allergic airway disease. TGF-β activity was neutralized by intranasal administration of monoclonal antibody.
Measurements and Main Results: TGF-β1 protein levels were increased in OVA-challenged lungs versus naive controls, and airway epithelial cells were shown to be a likely source of TGF-β1. In addition, TGF-β1 levels were elevated in OVA-exposed IL-5–null mice, which fail to recruit eosinophils into the airways. Neutralization of TGF-β1 with specific antibody had no significant effect on airway inflammation and eosinophilia, although anti–TGF-β1 antibody enhanced OVA-induced AHR and suppressed pulmonary fibrosis.
Conclusions: These data show that TGF-β1 is the main TGF-β isoform produced after OVA challenge, with a likely cellular source being the airway epithelium. The effects of blocking TGF-β1 signaling had differential effects on AHR, fibrosis, and inflammation. While TGF-β neutralization may be beneficial to abrogating airway remodeling, it may be detrimental to lung function by increasing AHR.
doi:10.1164/rccm.200702-334OC
PMCID: PMC2078678  PMID: 17761617
lung; mice; hypersensitivity; cytokines
22.  Allergic Sensitization through the Airway Primes Th17-dependent Neutrophilia and Airway Hyperresponsiveness 
Rationale: In humans, immune responses to inhaled aeroallergens develop in the lung and draining lymph nodes. Many animal models of asthma bypass this route and instead use intraperitoneal injections of allergen using aluminum hydroxide as an adjuvant.
Objectives: We investigated whether allergic sensitization through the airway elicits immune responses qualitatively different than those arising in the peritoneum.
Methods: Mice were sensitized to allergen through the airway using low-dose LPS as an adjuvant, or through the peritoneum using aluminum hydroxide as an adjuvant. After a single allergen challenge, ELISA and flow cytometry were used to measure cytokines and leukocyte subsets. Invasive measurements of airway resistance were used to measure allergen-induced airway hyperreactivity (AHR).
Measurements and Main Results: Sensitization through the peritoneum primed strong Th2 responses and eosinophilia, but not AHR, after a single allergen challenge. By contrast, allergic sensitization through the airway primed only modest Th2 responses, but strong Th17 responses. Th17 cells homed to the lung and released IL-17 into the airway on subsequent encounter with inhaled allergen. As a result, these mice developed IL-17–dependent airway neutrophilia and AHR. This AHR was neutrophil-dependent because it was abrogated in CXCR2-deficient mice and also in wild-type mice receiving a neutrophil-depleting antibody. Individually, neither IL-17 nor ongoing Th2 responses were sufficient to confer AHR, but together they acted synergistically to promote neutrophil recruitment, eosinophil recruitment and AHR.
Conclusions: Allergic sensitization through the airway primes modest Th2 responses but strong Th17 responses that promote airway neutrophilia and acute AHR. These findings support a causal role for neutrophils in severe asthma.
doi:10.1164/rccm.200904-0573OC
PMCID: PMC2778149  PMID: 19661246
asthma; lung; immunity
23.  Chitosan IFN-γ-pDNA Nanoparticle (CIN) Therapy for Allergic Asthma 
Background
Allergic subjects produce relatively low amounts of IFN-γ, a pleiotropic Th-1 cytokine that downregulates Th2-associated airway inflammation and hyperresponsiveness (AHR), the hallmarks of allergic asthma. Adenovirus-mediated IFN-γ gene transfer reduces AHR, Th2 cytokine levels and lung inflammation in mice, but its use would be limited by the frequency of gene delivery required; therefore, we tested chitosan/IFN-γ pDNA nanoparticles (CIN) for in situ production of IFN-γ and its in vivo effects.
Methods
CIN were administered to OVA-sensitized mice to investigate the possibility of using gene transfer to modulate ovalbumin (OVA)-induced inflammation and AHR.
Results
Mice treated with CIN exhibit significantly lower AHR to methacholine challenge and less lung histopathology. Production of IFN-γ is increased after CIN treatment while the Th2-cytokines, IL-4 and IL-5, and OVA-specific serum IgE are reduced compared to control mice. AHR and eosinophilia are also significantly reduced by CIN therapy administered therapeutically in mice with established asthma. CIN was found to inhibit epithelial inflammation within 6 hours of delivery by inducing apoptosis of goblet cells. Experiments performed on STAT4-defective mice do not show reduction in AHR with CIN treatment, thus implicating STAT4 signaling in the mechanism of CIN action.
Conclusion
These results demonstrate that mucosal CIN therapy can effectively reduce established allergen-induced airway inflammation and AHR.
doi:10.1186/1479-0556-1-3
PMCID: PMC280670  PMID: 14613519
24.  Antiasthmatic Effects of Hesperidin, a Potential Th2 Cytokine Antagonist, in a Mouse Model of Allergic Asthma 
Mediators of Inflammation  2011;2011:485402.
Background and Objective. The features of asthma are airway inflammation, reversible airflow obstruction, and an increased sensitivity to bronchoconstricting agents, termed airway hyperresponsiveness (AHR), excess production of Th2 cytokines, and eosinophil accumulation in the lungs. To investigate the antiasthmatic potential of hesperidin as well as the underlying mechanism involved, we studied the inhibitory effect and anti-inflammatory effect of hesperidin (HPN) on the production of Th2 cytokines, eotaxin, IL-17, -OVA-specific IgE in vivo asthma model mice. Methods. In this paper, BALB/c mice were systemically sensitized to ovalbumin (OVA) followed intratracheally, intraperitoneally, and by aerosol allergen challenges. We investigated the effect of HPN on airway hyperresponsiveness, pulmonary eosinophilic infiltration, various immune cell phenotypes, Th2 cytokine production and OVA-specific IgE production in a mouse model of asthma. Results. In BALB/c mice, we found that HPN-treated groups had suppressed eosinophil infiltration, allergic airway inflammation, and AHR, and these occurred by suppressing the production of IL-5, IL-17, and OVA-specific IgE. Conclusions. Our data suggest that the therapeutic mechanism by which HPN effectively treats asthma is based on reductions of Th2 cytokines (IL-5), eotaxin, OVA-specific IgE production, and eosinophil infiltration via inhibition of GATA-3 transcription factor.
doi:10.1155/2011/485402
PMCID: PMC3136080  PMID: 21772663
25.  Transfer of in vivo primed transgenic T cells supports allergic lung inflammation and FIZZ1 and Ym1 production in an IL-4Rα and STAT6 dependent manner 
BMC Immunology  2011;12:60.
Background
CD4+ T helper type 2 (TH2) cells, their cytokines IL-4, IL-5 and IL-13 and the transcription factor STAT6 are known to regulate various features of asthma including lung inflammation, mucus production and airway hyperreactivity and also drive alternative activation of macrophages (AAM). However, the precise roles played by the IL-4/IL-13 receptors and STAT6 in inducing AAM protein expression and modulating specific features of airway inflammation are still unclear. Since TH2 differentiation and activation plays a pivotal role in this disease, we explored the possibility of developing an asthma model in mice using T cells that were differentiated in vivo.
Results
In this study, we monitored the activation and proliferation status of adoptively transferred allergen-specific naïve or in vivo primed CD4+ T cells. We found that both the naïve and in vivo primed T cells expressed similar levels of CD44 and IL-4. However, in vivo primed T cells underwent reduced proliferation in a lymphopenic environment when compared to naïve T cells. We then used these in vivo generated effector T cells in an asthma model. Although there was reduced inflammation in mice lacking IL-4Rα or STAT6, significant amounts of eosinophils were still present in the BAL and lung tissue. Moreover, specific AAM proteins YM1 and FIZZ1 were expressed by epithelial cells, while macrophages expressed only YM1 in RAG2-/- mice. We further show that FIZZ1 and YM1 protein expression in the lung was completely dependent on signaling through the IL-4Rα and STAT6. Consistent with the enhanced inflammation and AAM protein expression, there was a significant increase in collagen deposition and smooth muscle thickening in RAG2-/- mice compared to mice deficient in IL-4Rα or STAT6.
Conclusions
These results establish that transfer of in vivo primed CD4+ T cells can induce allergic lung inflammation. Furthermore, while IL-4/IL-13 signaling through IL-4Rα and STAT6 is essential for AAM protein expression, lung inflammation and eosinophilia are only partially dependent on this pathway. Further studies are required to identify other proteins and signaling pathways involved in airway inflammation.
doi:10.1186/1471-2172-12-60
PMCID: PMC3212823  PMID: 22014099

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