UV inhibits allergic airways disease in mice by reducing effector CD4+ T cells

Background In human asthma, and experimental allergic airways disease in mice, antigen‐presenting cells and CD4⁺ effector cells at the airway mucosa orchestrate, and CD4⁺CD25⁺ regulatory T cells attenuate, allergen immunity. UV irradiation of skin before sensitization with ovalbumin (OVA) causes significantly reduced asthma‐like responses in respiratory tissues. Objective To determine whether UV‐induced changes in CD11c⁺ cells, CD4⁺CD25⁺ effector cells or CD4⁺CD25⁺ regulatory cells in the trachea and airway draining lymph nodes (ADLNs) were responsible for reduced allergic airways disease. Methods The phenotype and function of CD11c⁺ cells and CD4⁺CD25⁺ cells in the trachea and ADLNs of UV‐ and non‐irradiated, OVA‐sensitized mice was examined 24 h after a single exposure to aerosolized OVA. Results No changes in the function of CD11c⁺ cells from UV‐irradiated mice were observed. CD4⁺CD25⁺ cells from UV‐irradiated, OVA‐sensitized mice harvested 24 h after OVA aerosol proliferated less in response to OVA in vitro and were unable to suppress the proliferation of OVA‐sensitized responder cells. This result suggested reduced activation of effector T cells in the airway mucosa of UV‐irradiated, OVA‐sensitized mice. To exclude regulatory cells of any type, there was similar proliferation in vivo to aerosolized OVA by CFSE‐loaded, OVA‐TCR‐specific CD4⁺ cells adoptively transferred into UV‐ and non‐irradiated, OVA‐sensitized mice. In addition, there was no difference in the expression of regulatory T cell markers (Foxp3, IL‐10, TGF‐β mRNA). To examine effector T cells, ADLN cells from UV‐irradiated, OVA‐sensitized and ‐challenged mice were cultured with OVA. There was reduced expression of the early activation marker CD69 by CD4⁺CD25⁺ cells, and reduced proliferation in the absence of the regulatory cytokine, IL‐10. Conclusion Reduced allergic airways disease in UV‐irradiated mice is due to fewer effector CD4⁺CD25⁺ cells in the trachea and ADLNs, and not due to UV‐induced regulatory cells. Cite this as: J. P. McGlade, D. H. Strickland, M. J. M. Lambert, S. Gorman, J. A. Thomas, M. A. Judge, J. T. Burchell, G. R. Zosky and P. H. Hart, Clinical & Experimental Allergy, 2010 (40) 772–785.     

Non‐eosinophilic Airway Hyper‐reactivity in Mice,Induced by IFN‐γ Producing CD4+ and CD8+ Lung T cells,is Responsive to Steroid Treatment

Non‐eosinophilic asthma is characterized by infiltration of neutrophils into the lung and variable responsiveness to glucocorticoids. The pathophysiological mechanisms have not been characterized in detail. Here, we present an experimental asthma model in mice associated with non‐eosinophilic airway inflammation and airway hyper‐responsiveness (AHR). For this, BALB/c mice were sensitized by biolistic DNA immunization with a plasmid encoding the model antigen β‐galactosidase (pFascin‐βGal mice). For comparison, eosinophilic airway inflammation was induced by subcutaneous injection of βGal protein (βGal mice). Intranasal challenge of mice in both groups induced AHR to a comparable extent as well as recruitment of inflammatory cells into the airways. In contrast to βGal mice, which exhibited extensive eosinophilic infiltration in the lung, goblet cell hyperplasia and polarization of CD4⁺ T cells into Th2 and Th17 cells, pFascin‐βGal mice showed considerable neutrophilia, but no goblet cell hyperplasia and a predominance of Th1 and Tc1 cells in the airways. Depletion studies in pFascin‐βGal mice revealed that CD4⁺ and CD8⁺ cells cooperated to induce maximum inflammation, but that neutrophilic infiltration was not a prerequisite for AHR induction. Treatment of pFascin‐βGal mice with dexamethasone before intranasal challenge did not affect neutrophilic infiltration, but significantly reduced AHR, infiltration of monocytes and lymphocytes as well as content of IFN‐γ in the bronchoalveolar fluid. Our results suggest that non‐eosinophilic asthma associated predominantly with Th1/Tc1 cells is susceptible to glucocorticoid treatment. pFascin‐βGal mice might represent a mouse model to study pathophysiological mechanisms proceeding in the subgroup of asthmatics with non‐eosinophilic asthma that respond to inhaled steroids.     

IgE-dependent enhancement of Th2 cell-mediated allergic inflammation in the airways

T helper 2 (Th2) cell-derived cytokines, including interleukin (IL)-4, IL-5 and IL-13, play important roles in causing allergic airway inflammation. In contrast to Th2 cells, however, the role of IgE and mast cells in inducing allergic airway inflammation is not understood fully. In the present study, we addressed this point using transgenic mice expressing trinitrophenyl (TNP)-specific IgE (TNP-IgE mice), which enable us to investigate the role of IgE without the influence of antigen-specific T cell activation and other immunoglobulins. When the corresponding antigen, TNP-BSA, was administered intranasally to TNP-IgE mice, a large number of CD4+ T cells were recruited into the airways. In contrast, TNP-BSA administration did not induce eosinophil recruitment into the airways or airway hyperreactivity. Furthermore, when ovalbumin (OVA)-specific Th2 cells were transferred to TNP-IgE mice and the mice were challenged with inhaled OVA, TNP-BSA administration increased OVA-specific T cell recruitment and then enhanced Th2 cell-mediated eosinophil recruitment into the airways. These results indicate that IgE-induced mast cell activation principally induces CD4+ T cell recruitment into the airways and thus plays an important role in enhancing Th2 cell-mediated eosinophilic airway inflammation by recruiting Th2 cells into the site of allergic inflammation.     

Adenoviral infection inhibits allergic airways inflammation in mice

BACKGROUND: Recent epidemiological studies have suggested that exposure to certain viruses and bacteria influences the development of allergy and allergic diseases, such as asthma. However, there is a paucity of experimental evidence examining the consequences of concurrent exposure to allergen and infectious agents, and the potential mechanisms by which allergic disease might be averted as a result. OBJECTIVE: To model this situation experimentally, we investigated whether a virally induced immune response, elicited by a replication-deficient human type 5 adenovirus (RDA) administered at a site distant from the airways, could inhibit ovalbumin (OVA)-induced airways eosinophilic inflammation. METHODS: C57BL/6 mice were infected intramuscularly with RDA 16h prior to intraperitoneal OVA sensitization. Cellular and cytokine responses in the lung/airways were examined after an OVA aerosol challenge. RESULTS: RDA infection significantly inhibited the inflammatory response in the lung tissue after antigen challenge. In the bronchoalveolar lavage (BAL), total cell number, eosinophils and lymphocytes were decreased by 70, 85 and 65%, respectively, after antigen challenge in RDA-treated, compared with untreated, mice. RDA infection had no effect on IgE synthesis. The levels of IL-5, IL-4 and IFNgamma in the BAL after antigen challenge were significantly lower in RDA-treated mice. In vitro production of cytokines by splenocytes in response to OVA restimulation revealed a shift from IL-4 in sensitized, PBS-treated mice, to IFNgamma in sensitized mice treated with RDA. Flow cytometric analysis revealed that RDA infection increased the proportion of CD8 T cells in the BAL; this change in T-cell subsets was accompanied by an increase in both CD4 and CD8 T cells positive for intracellular IFNgamma. Inhibition of antigen-induced airways inflammation was IFNgamma-dependent but did not require IL-12, as RDA-treatment inhibited airways inflammation in IL-12 but not IFNgamma knock-out mice. CONCLUSION: This study demonstrates that an immune response against a replication-deficient adenovirus during the initial exposure to OVA inhibits the development of airways inflammation after antigen aerosol challenge.     

Effect of ozone exposure on allergic sensitization and airway inflammation induced by dendritic cells

BACKGROUND: Epidemiological studies suggest that ozone exposure is related to increased asthma symptoms. Dendritic cells (DCs) are the principal antigen-presenting cells in the airways. OBJECTIVE: We have examined whether ambient doses of ozone (100 ppb for 2 h) enhance allergic sensitization and/or airway inflammation in a mouse model. METHODS: C57BL/6 mice were sensitized to inhaled ovalbumin (OVA) by intratracheal instillation of OVA-pulsed DCs on day 0. Daily exposure to OVA aerosol on days 14-20 resulted in an eosinophilic airway inflammation, as reflected in bronchoalveolar lavage fluid and lung histology. In a first experiment, mice were exposed to ozone or room air immediately prior to and following sensitization. Subsequently, we tested the effect of ozone exposure during antigen challenge in DC-sensitized mice. RESULTS: Exposure to ozone during sensitization did not influence airway inflammation after subsequent allergen challenge. In contrast, in sensitized mice, challenge with OVA together with ozone (days 14-20) resulted in enhanced airway eosinophilia and lymphocytosis, as compared with mice exposed to OVA and room air (1.91 x 106 +/- 0.46 x 106 vs. 0.16 x 106 +/- 0.06 x 106 eosinophils/mL lavage fluid; P = 0.015; 0.49 x 106 +/- 0.11 x 106 vs. 0.08 x 106 +/- 0.03 x 106 lymphocytes/mL lavage fluid; P = 0.004). Ozone exposure without subsequent OVA exposure did not cause airway inflammation. CONCLUSION: Ozone exposure does not increase allergic sensitization but enhances antigen-induced airway inflammation in mice that are sensitized via the airways.     

Interleukin-13-dependent bronchial hyper-responsiveness following isolated upper-airway allergen challenge in a murine model of allergic rhinitis and asthma

BACKGROUND: We have previously shown that isolated allergic sensitization and challenge of the upper airway results in lower-airway inflammation, which supports the concept of the united airways. OBJECTIVE: This study investigates the hypothesis that isolated upper-airway allergic sensitization is sufficient to induce bronchial hyper-responsiveness (BHR), characteristic of asthma, and that IL-13 is an essential mediator in both the upper and lower airways. METHODS: BALB/c mice were sensitized and challenged by intranasal instillation of allergen ovalbumin (OVA) using our standard protocol. BHR to methacholine was determined and inflammation in nares and lung was assessed. RESULTS: Isolated intranasal application of allergen in awake animals resulted in almost exclusive deposition in the upper airways while in anaesthetized mice there was almost equal distribution in the upper and lower airways. We have demonstrated significant BHR to methacholine challenge in animals receiving OVA only in the upper airway. Also noted was concomitant increase in eosinophilic infiltrates in lung and nares as well as increased granulocytes and IL-13 levels in bronchoalveolar lavage (BAL) fluid. Using a polyclonal anti-IL-13 antibody we have shown inhibition of airways inflammation, both in nares and in lung with significant reduction of granulocytes in BAL from anti-IL-13 treated mice (P<0.0001). Anti-IL-13 treatment also abrogates allergen-induced BHR (P<0.01). CONCLUSION: These data suggest that isolated upper-airway allergen deposition initiates allergic responses along the entire airway. IL-13 mediates both airway inflammation and BHR and may play a role in the communication between the upper and lower airways.     

Site-specific sensitization in a murine model of allergic rhinitis: role of the upper airway in lower airways disease

BACKGROUND: Allergic rhinitis (AR) is the most common atopic disease with strong links to asthma. We have developed a murine model of AR to study nasal, bronchial, and systemic immune response to local allergen stimulation. OBJECTIVES: The purpose of this study was to develop and characterize a murine model of AR. METHODS: Six- to 8-week-old BALB/c mice were sensitized by means of intranasal (local) application of ovalbumin (OVA) or systemic intraperitoneal injection. They were then challenged with intranasal OVA, and allergic response was assessed. RESULTS: Intranasal particle deposition was found to be exclusively in the nares. All sensitized animals showed increased levels of OVA-specific serum IgE and IgG after challenge, although the timing to maximal response varied with the route and dose of allergen used. Histology of the upper and lower airways showed marked eosinophilic infiltration, and analysis of bronchoalveolar lavage fluid showed increased IL-5 and PMN infiltrates after challenge. CONCLUSION: Using exclusive local sensitization and challenge of mouse nares, we were able to demonstrate inflammatory changes in both the upper and lower airways, even though distribution of allergen particles appeared to be only in the nares of these animals. This provides further evidence for the importance of the upper airway in lower airways disease. We have shown that the route of administration greatly affects the characteristics of the subsequent immune responses.     

T cells are necessary for ILC2 activation in house dust mite‐induced allergic airway inflammation in mice

Allergic asthma is a chronic inflammation of the airways mediated by an adaptive type 2 immune response. Upon allergen exposure, group 2 innate lymphoid cells (ILC2s) can be rapidly activated and represent an early innate source of IL‐5 and IL‐13. Here, we used a house dust mite (HDM)‐driven asthma mouse model to study the induction of ILC2s in allergic airway inflammation. In BALF, lungs, and lymph nodes, ILC2 activation is critically dependent on prior sensitization with HDM. Importantly, T cells are required for ILC2 induction, whereby T‐cell activation precedes ILC2 induction. During HDM‐driven allergic airway inflammation the accumulation of ILC2s in BALF is IL‐33 independent, although infiltrating ILC2s produce less cytokines in Il33^?/? mice. Transfer of in vitro polarized OVA‐specific OT‐II Th2 cells alone or in combination with Th17 cells followed by OVA and HDM challenge is not sufficient to induce ILC2, despite significant eosinophilic inflammation and T‐cell activation. In this asthma model, ILC2s are therefore not an early source of Th2 cytokines, but rather contribute to type 2 inflammation in which Th2 cells play a key role. Taken together, ILC2 induction in HDM‐mediated allergic airway inflammation in mice critically depends on activation of T cells.     

Activation of invariant natural killer T cells in regional lymph nodes as new antigen‐specific immunotherapy via induction of interleukin‐21 and interferon‐γ

Invariant natural killer T (iNKT) cells play important immunoregulatory functions in allergen‐induced airway hyperresponsiveness and inflammation. To clarify the role of iNKT cells in allergic rhinitis (AR), we generated bone marrow‐derived dendritic cells (BMDCs), which were pulsed by ovalbumin (OVA) and α‐galactosylceramide (OVA/α‐GalCer‐BMDCs) and administered into the oral submucosa of OVA‐sensitized mice before nasal challenge. Nasal symptoms, level of OVA‐specific immunoglobulin (IgE), and T helper type 2 (Th2) cytokine production in cervical lymph nodes (CLNs) were significantly ameliorated in wild‐type (WT) mice treated with OVA/α‐GalCer‐BMDCs, but not in WT mice treated with OVA‐BMDCs. These anti‐allergic effects were not observed in Jα18^–/– recipients that lack iNKT cells, even after similar treatment with OVA/α‐GalCer‐BMDCs in an adoptive transfer study with CD4⁺ T cells and B cells from OVA‐sensitized WT mice. In WT recipients of OVA/α‐GalCer‐BMDCs, the number of interleukin (IL)‐21‐producing iNKT cells increased significantly and the Th1/Th2 balance shifted towards the Th1 dominant state. Treatment with anti‐IL‐21 and anti‐interferon (IFN)‐γ antibodies abrogated these anti‐allergic effects in mice treated with α‐GalCer/OVA‐BMDCs. These results suggest that activation of iNKT cells in regional lymph nodes induces anti‐allergic effects through production of IL‐21 or IFN‐γ, and that these effects are enhanced by simultaneous stimulation with antigen. Thus, iNKT cells might be a useful target in development of new treatment strategies for AR.     

Interleukin (IL)-5 but not immunoglobulin E reconstitutes airway inflammation and airway hyperresponsiveness in IL-4-deficient mice

We studied the role of interleukin (IL)-4, IL-5, and allergen-specific immunoglobulin (Ig) E in the development of allergen-induced sensitization, airway inflammation, and airway hy-perresponsiveness (AHR). Normal, IL-4-, and IL-5-deficient C57BL/6 mice were sensitized intraperitoneally to ovalbumin (OVA) and repeatedly challenged with OVA via the airways. After allergen sensitization and airway challenge, normal and IL-5-deficient, but not IL-4-deficient, mice developed increased serum levels of total and antigen-specific IgE levels and increased IL-4 production in the lung tissue compared with nonsensitized control mice. Only normal mice showed significantly increased IL-5 production in the lung tissue and an eosinophilic infiltration of the peribronchial regions of the airways, whereas both IL-4- and IL-5-deficient mice had little or no IL-5 production and no significant eosinophilic airway inflammation. Associated with the inflammatory responses in the lung, only normal mice developed increased airway responsiveness to methacholine after sensitization and airway challenge; in both IL-4- and IL-5-deficient mice, airway responsiveness was similar to that in nonsensitized control mice. Reconstitution of sensitized, IL-4-deficient mice before allergen airway challenge with IL-5, but not with allergen-specific IgE, restored eosinophilic airway inflammation and the development of AHR. These data demonstrate the importance of IL-4 for allergen-driven airway sensitization and that IL-5, but not allergen-specific IgE, is required for development of eosinophilic airway inflammation and AHR after this mode of sensitization and challenge.     

Long-chain polyunsaturated fatty acids are consumed during allergic inflammation and affect T helper type 1 (Th1)- and Th2-mediated hypersensitivity differently

Studies have shown that atopic individuals have decreased serum levels of n‐3 fatty acids. Indicating these compounds may have a protective effect against allergic reaction and/or are consumed during inflammation. This study investigated whether fish (n‐3) or sunflower (n‐6) oil supplementation affected T helper type 1 (Th1)‐ and Th2‐mediated hypersensitivity in the skin and airways, respectively, and whether the fatty acid serum profile changed during the inflammatory response. Mice were fed regular chow, chow + 10% fish oil or chow + 10% sunflower oil. Mice were immunized with ovalbumin (OVA) resolved in Th1 or Th2 adjuvant. For Th1 hypersensitivity, mice were challenged with OVA in the footpad. Footpad swelling, OVA‐induced lymphocyte proliferation and cytokine production in the draining lymph node were evaluated. In the airway hypersensitivity model (Th2), mice were challenged intranasally with OVA and the resulting serum immunoglobulin (Ig)E and eosinophilic lung infiltration were measured. In the Th1 model, OVA‐specific T cells proliferated less and produced less interferon (IFN)‐γ, tumour necrosis factor (TNF) and interleukin (IL)‐6 in fish oil‐fed mice versus controls. Footpad swelling was reduced marginally. In contrast, mice fed fish oil in the Th2 model produced more OVA‐specific IgE and had slightly higher proportions of eosinophils in lung infiltrate. A significant fall in serum levels of long‐chain n‐3 fatty acids accompanied challenge and Th2‐mediated inflammation in Th2 model. Fish oil supplementation affects Th1 and Th2 immune responses conversely; significant consumption of n‐3 fatty acids occurs during Th2‐driven inflammation. The latter observation may explain the association between Th2‐mediated inflammation and low serum levels of n‐3 fatty acids.     

Obesity promotes prolonged ovalbumin‐induced airway inflammation modulating T helper type 1 (Th1), Th2 and Th17 immune responses in BALB/c mice

Clinical and epidemiological studies indicate that obesity affects the development and phenotype of asthma by inducing inflammatory mechanisms in addition to eosinophilic inflammation. The aim of this study was to assess the effect of obesity on allergic airway inflammation and T helper type 2 (Th2) immune responses using an experimental model of asthma in BALB/c mice. Mice fed a high‐fat diet (HFD) for 10 weeks were sensitized and challenged with ovalbumin (OVA), and analyses were performed at 24 and 48 h after the last OVA challenge. Obesity induced an increase of inducible nitric oxide synthase (iNOS)‐expressing macrophages and neutrophils which peaked at 48 h after the last OVA challenge, and was associated with higher levels of interleukin (IL)‐4, IL‐9, IL‐17A, leptin and interferon (IFN)‐γ in the lungs. Higher goblet cell hyperplasia was associated with elevated mast cell influx into the lungs and trachea in the obese allergic mice. In contrast, early eosinophil influx and lower levels of IL‐25, thymic stromal lymphopoietin (TSLP), CCL11 and OVA‐specific immunoglobulin (IgE) were observed in the obese allergic mice in comparison to non‐obese allergic mice. Moreover, obese mice showed higher numbers of mast cells regardless of OVA challenge. These results indicate that obesity affects allergic airway inflammation through mechanisms involving mast cell influx and the release of TSLP and IL‐25, which favoured a delayed immune response with an exacerbated Th1, Th2 and Th17 profile. In this scenario, an intense mixed inflammatory granulocyte influx, classically activated macrophage accumulation and intense mucus production may contribute to a refractory therapeutic response and exacerbate asthma severity.     

MMP induced by Gr‐1+ cells are crucial for recruitment of Th cells into the airways

Th2 lymphocytes deliver essential signals for induction of asthmatic airway inflammation. We previously found that airway antigen challenge induces recruitment of Gr‐1⁺ neutrophils prior to the recruitment of Th2 cells. We examined, therefore, whether Gr‐1⁺ cells contribute to the development of Th2‐dependent airway inflammation. Systemic depletion of Gr‐1⁺ cells using the RB6‐8C5 monoclonal antibody reduced Th2 cell recruitment following i.n. antigen challenge. The levels of both MMP‐9 and the tissue inhibitor of matrix metalloproteinases‐1 mRNA were up‐regulated in the lungs of mice 12 h after i.n. antigen challenge. Up‐regulation of tissue inhibitor of matrix metalloproteinases‐1 was independent of Gr‐1⁺ cells, whereas up‐regulation of MMP‐9 RNA and total gelatinolytic activity was dramatically reduced in mice depleted of Gr‐1⁺ cells. At 24 h after challenge, total lung collagenolytic activity was also up‐regulated, in a Gr‐1⁺ cell‐dependent fashion. Systemic inhibition of MMP‐8 and MMP‐9 reduced the airway recruitment of Th cells, resulting in significantly reduced eosinophilic inflammation. These data suggest that antigen challenge via the airway activates Gr‐1⁺ cells and consequently MMP to facilitate the recruitment of Th cells in the airway inflammatory response.     

Allergic sensitization is enhanced in early life through toll‐like receptor 7 activation

Background Prospective cohort studies suggest that children hospitalized in early life with severe infections are significantly more likely to develop recurrent wheezing and asthma. Objective Using an inhalational mouse model of allergic airways inflammation, we sought to determine the effect of viral and bacterial‐associated molecular patterns on the magnitude of the allergic inflammatory response and whether this effect was age dependent. Methods BALB/c mice were sensitized by intranasal administration of endotoxin^low ovalbumin (OVA) in the absence or presence of viral single‐stranded (ss)RNA, lipoteichoic acid or flagellin as neonates (within the first 24 h of life) or as weanlings (4 weeks of age). Mice were challenged four times with OVA at 6 weeks of age and end‐points (bronchoalveolar lavage cytology, histology, antigen‐specific T and B cell responses) determined at 7 weeks of age. Results Inhalational sensitization (<24 h or 4 weeks of age) and challenge with OVA induced a mild allergic inflammatory response in the airways as indicated by increased numbers of eosinophils and mucus cells, elevated serum OVA‐specific IgG1, and production of T helper 2 (Th2) cytokines. Mice sensitized to endotoxin^low OVA at birth in the presence of ssRNA or lipoteichoic acid, but not flagellin, showed an increase in the numbers of airway and tissue eosinophils, mucus producing cells and antigen‐specific production of IL‐13 as compared with mice exposed only to endotoxin^low OVA. By contrast, all three TLR ligands failed to increase the magnitude of OVA‐induced allergic inflammation in mice sensitized as weanlings. Conclusions Recognition of distinct microbial‐associated patterns in early life may preferentially promote the de novo differentiation of bystander, antigen‐specific CD4⁺ T cells toward a Th2 phenotype, and promote an asthma‐like phenotype upon cognate antigen exposure in later life.     

Muc-5/5ac mucin messenger RNA and protein expression is a marker of goblet cell metaplasia in murine airways

Airway inflammation, hyperreactivity, increased number of goblet cells, and mucus overproduction characterize asthma. Respiratory challenge with ovalbumin (OVA) of sensitized mice has been shown by several laboratories to cause pulmonary pathology similar to that observed in human allergic asthma. Recently, interleukin (IL)-13 has been shown to be a central mediator in this process. Because the airways of healthy mice have few, if any, mucus-producing cells, an increase in the number of these cells likely reflects induction of mucin-gene expression. The purpose of this study was to identify mucin genes induced as a result of airway goblet-cell metaplasia (GCM) in mice sensitized and challenged with OVA or in mice treated with IL-13 alone. BALB/c mice were sensitized by intraperitoneal injection (Days 0, 4, 7, 11, and 14) and intranasal instillation (Day 14) of 100 microg of OVA in saline, and then challenged by intranasal instillation (Days 25, 26, and 27) of the same. IL-13-treated mice received 5 microg of IL-13 by intranasal instillation on three consecutive days. Control mice were given saline alone. All mice were studied 24 h after the last challenge. Histologic analysis of the lungs revealed both a striking peribronchial and perivascular lymphocytic and eosinophilic inflammation and airway GCM in OVA-treated mice, and also airway GCM without inflammation in IL-13-treated mice. Northern blot analysis of lung RNA demonstrated (1) expression of Muc-5/5ac messenger RNA (mRNA) in OVA-treated and IL-13-treated mice, but not in control mice; (2) expression of Muc-1 mRNA at comparable levels in all mice regardless of treatment; and (3) no expression of Muc-2 or Muc-3 mRNA in control or treated mice. Western blot analysis demonstrated the expression of Muc-5/5ac protein (both apomucin and glycosylated mucin) in lung lysates of OVA-treated (but not control) mice, and also the expression of Muc-5/5ac mucins in the bronchoalveolar lavage fluid of OVA-treated and IL-13-treated mice. These findings demonstrate that airway GCM is associated with the induction of pulmonary expression of Muc-5/5ac mRNA and mucin in murine models of allergic asthma.     

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

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

Basophils as a primary inducer of the T helper type 2 immunity in ovalbumin‐induced allergic airway inflammation

Antigen‐induced allergic airway inflammation is mediated by T helper type 2 (Th2) cells and their cytokines, but the mechanism that initiates the Th2 immunity is not fully understood. Recent studies show that basophils play important roles in initiating Th2 immunity in some inflammatory models. Here we explored the role of basophils in ovalbumin (OVA) ‐induced airway allergic inflammation in BALB/c mice. We found that OVA sensitization and challenge resulted in a significant increase in the amount of basophils in blood and lung, along with the up‐regulation of activation marker of CD200R. However, depletion of basophils with MAR‐1 or Ba103 antibody attenuated airway inflammation, represented by the significantly decreased amount of the Th2 subset in spleen and draining lymph nodes, interlukin‐4 level in lung and OVA‐special immunoglobulin E (sIgE) levels in serum. On the other hand, adoptive transfer of basophils from OVA‐challenged lung tissue to naive BALB/c mice provoked the Th2 immune response. In addition, pulmonary basophils from OVA‐challenged mice were able to uptake DQ‐OVA and express MHC class II molecules and CD40 in vivo, as well as to release interleukin‐4 following stimulation by IgE–antigen complexes and promote Th2 polarization in vitro. These findings demonstrate that basophils may participate in Th2 immune responses in antigen‐induced allergic airway inflammation and that they do so through facilitating antigen presentation and providing interleukin‐4.     

Anti‐Asthma Simplified Herbal Medicine Intervention‐induced long‐lasting tolerance to allergen exposure in an asthma model is interferon‐γ, but not transforming growth factor‐β dependent

Background Chronic allergic asthma is the result of a T‐helper type 2 (Th2)‐biased immune status. Current asthma therapies control symptoms in some patients, but a long‐lasting therapy has not been established. Anti‐Asthma Simplified Herbal Medicine Intervention (ASHMI^?), a Chinese herbal formula improved symptoms and lung function, and reduced Th2 responses in a controlled trial of patients with persistent moderate to severe asthma. Objective We evaluated the persistence of ASHMI^? beneficial effects following therapy in a murine model of chronic asthma and the immunological mechanisms underlying such effects. Methods BALB/c mice sensitized intraperitoneally with ovalbumin (OVA) received 3 weekly intratracheal OVA challenges to induce airway hyper‐reactivity (AHR) and inflammation (OVA mice). Additionally, OVA mice were treated with ASHMI^? (OVA/ASHMI^?) or water (OVA/sham) for 4 weeks, and then challenged immediately and 8 weeks post‐therapy. In other experiments, OVA mice received ASHMI^? treatment with concomitant neutralization of IFN‐γ or TGF‐β. Effects on airway responses, cytokine‐ and OVA‐specific IgE levels were determined 8 weeks post‐therapy. Results Before treatment, OVA mice exhibited AHR and pulmonary eosinophilic inflammation following OVA challenge, which was almost completely resolved immediately after completing treatment with ASHMI^? and did not re‐occur following OVA re‐challenge up to 8 weeks post‐therapy. Decreased allergen‐specific IgE and Th2 cytokine levels, and increased IFN‐γ levels also persisted at least 8 weeks post‐therapy. ASHMI^? effects were eliminated by the neutralization of IFN‐γ, but not TGF‐β, during therapy. Conclusion ASHMI^? induced long‐lasting post‐therapy tolerance to antigen‐induced inflammation and AHR. IFN‐γ is a critical factor in ASHMI^? effects. Cite this as: K. Srivastava, T. Zhang, N. Yang, H. Sampson and X. M. Li, Clinical & Experimental Allergy, 2010 (40) 1678–1688.     

Bacillus‐derived poly‐γ‐glutamic acid attenuates allergic airway inflammation through a Toll‐like receptor‐4‐dependent pathway in a murine model of asthma

Background Asthma is an inflammatory disease of the airways that is mediated by Th2 responses. Poly‐γ‐glutamic acid (γ‐PGA) is an extracellular polymeric compound that is synthesized by Bacillus cells. Previously, we found that γ‐PGA promoted Th1 cell development in a manner dependent on antigen‐presenting cells, but inhibited Th2 cell development. Objective To investigate the effect of γ‐PGA on dendritic cells (DCs), and its potential for treating Th2‐mediated allergic asthma. Methods Wild‐type, Toll‐like receptor (TLR)‐2 deficient, and TLR‐4‐defective mice were used. DCs derived from the bone marrow and extracted from the lung were stimulated with γ‐PGA and assayed for the expression of signalling molecules, costimulatory molecules, and cytokines. Mice were sensitized and challenged with ovalbumin (OVA) to induce asthma. They were repeatedly injected intranasally with γ‐PGA before and during the challenge period, and inflammation and structural remodelling of the airways were examined. Results γ‐PGA selectively signalled conventional DCs to activate NF‐κB and mitogen‐activated protein kinase, leading to the up‐regulation of CD86, CD40, and IL‐12, but not IL‐10 and IL‐6. These effects of γ‐PGA were dependent on TLR‐4 and independent of TLR‐2. Importantly, the intranasal administration of γ‐PGA to OVA‐sensitized/challenged mice reduced the airway hyperresponsiveness and allergic inflammation such as leucocyte influx, goblet cell hyperplasia, eosinophilia, and Th2 cytokine production. In addition to lowered IgE titres, the treatment of mice with γ‐PGA significantly reduced the multiplication and Th2 polarization of mediastinal lymph node T cells upon allergen‐specific restimulation. These anti‐asthmatic effects of γ‐PGA were also abolished in TLR‐4‐defective mice. Conclusions and Clinical Relevance Our data indicate that γ‐PGA activates DCs to favour Th1 cell induction through a TLR‐4‐dependent pathway and alleviates pathologic symptoms in a Th2‐biased asthmatic model. These findings highlight the potential of γ‐PGA for the treatment of asthma and other allergic disease in which Th2 polarization plays an important role. Cite this as: K. Lee, S.‐H. Kim, H. J. Yoon, D. J. Paik, J. M. Kim and J. Youn, Clinical & Experimental Allergy, 2011 (41) 1143–1156.     

Endotoxins prevent murine IgE production,T(H)2 immune responses,and development of airway eosinophilia but not airway hyperreactivity

BACKGROUND: Contact with immunomodulatory factors, such as LPS, in early infancy is associated with decreased allergen sensitization. OBJECTIVE: We sought to study the effects of systemic or airway exposure with LPS on the development of allergen sensitization, eosinophilic airway inflammation, and increased in vivo airway reactivity (AR) in a mouse model. METHODS: BALB/c mice were systemically sensitized with ovalbumin (OVA) plus adjuvant on days 1 and 14 and challenged through the airways with allergen on days 34 to 36. We performed measurement of OVA-specific IgE serum levels, in vitro T(H)2 cytokine production, differential cell counts in bronchoalveolar lavage fluids, and assessment of in vivo AR to inhaled methacholine by means of barometric whole-body plethysmography. RESULTS: Systemic LPS administration before OVA sensitization reduced OVA-specific IgE serum levels (426 +/- 76 vs 880 +/- 104 U/mL, P <.01), T(H)2 cytokine production by splenic mononuclear cells (IL-4: 0.08 +/- 0.01 vs 0.17 +/- 0.01 ng/mL; IL-5: 1.98 +/- 0.52 vs 4.11 +/- 0.54 ng/mL; P <.01), and extent of airway eosinophilia (total cell counts: 93 vs 376 x 10(3)/mL; eosinophils: 23% vs 51%; P <.01) compared with that in OVA-sensitized mice. Local LPS administration to sensitized mice before airway allergen challenges particularly induced IFN-gamma production by peribronchial lymph node cells in vitro (1718 +/- 315 vs 483 +/- 103 ng/mL, P <.01) associated with reduced airway eosinophilia compared with that seen in OVA-sensitized mice. Development of increased AR was not affected by systemic or local LPS exposure. Inhibitory effects of LPS on allergen sensitization and eosinophilic airway inflammation were inhibited by administration of anti-IL-12 antibodies before LPS exposure. CONCLUSION: These data indicate that local and systemic application of LPS modulates systemic and local T(H)1/T(H)2 immune responses in a distinct but similarly IL-12-dependent mode.