Absence of leukotriene B4 receptor 1 confers resistance to airway hyperresponsiveness and Th2-type immune responses

Bronchial asthma is an increasingly common disorder that remains poorly understood and difficult to manage. The disease is characterized by airway hyperresponsiveness, chronic inflammation, and mucus overproduction. Based on the finding that leukotriene B4 receptor 1 (BLT1) is expressed highly in Th2 lymphocytes, we analyzed the roles of BLT1 using an OVA-induced bronchial asthma model. BLT1-null mice did not develop airway hyperresponsiveness, eosinophilic inflammation, and hyperplasia of goblet cells. Attenuated symptoms were accompanied by reduced IgE production, and accumulation of IL-5 and IL-13 in bronchoalveolar lavage fluid, suggesting attenuated Th2-type immune response in BLT1-null mice. Peribronchial lymph node cells of sensitized BLT1-null mice showed much attenuated proliferation and production of Th2 cytokines upon re-stimulation with Ag in vitro. Thus, LTB4-BLT1 axis is required for the development of Th2-type immune response, and blockade of LTB4 functions through BLT1 would be novel and useful in the effort to ameliorate bronchial asthma and related Th2-biased immune disorders.     

Cutting edge: the absence of C3 demonstrates a role for complement in Th2 effector functions in a murine model of pulmonary allergy

Asthma is a chronic disease of the lung resulting from airway obstruction. Although the initiating causes are not entirely clear, the airway inflammation in asthma is associated with Th2 lymphocytes and their cytokines, particularly IL-4, which play a prominent role in this disease by regulating airway hyperresponsiveness, eosinophil activation, and IgE synthesis. Historically, complement was not thought to contribute to the pathogenesis of asthma. However, using C3-deficient mice in an allergen-induced model of pulmonary allergy, we demonstrate that complement may impact key features of this disease. When challenged with allergen, mice deficient in C3 exhibit diminished airway hyperresponsiveness and lung eosinophilia. Furthermore, these mice also have dramatically reduced numbers of IL-4-producing cells and attenuated Ag-specific IgE and IgG1 responses. Collectively, these results demonstrate that C3-deficient mice have significantly altered allergic lung responses and indicate a role for the complement system in promoting Th2 effector functions in asthma.     

Reversal of thymic stromal lymphopoietin-induced airway inflammation through inhibition of Th2 responses

Lung-specific thymic stromal lymphopoietin (TSLP) expression is sufficient for the development of an asthma-like chronic airway inflammatory disease. However, the nature of the downstream pathways that regulate disease development are not known. In this study, we used IL-4- and Stat6-deficient mice to establish the role of Th2-type responses downstream of TSLP. IL-4 deficiency greatly reduced, but did not eliminate, TSLP-induced airway hyperresponsiveness, airway inflammation, eosinophilia, and goblet cell metaplasia, while Stat6 deficiency eliminated these asthma-like symptoms. We further demonstrate, using the chronic model of TSLP-mediated airway inflammation, that blockade of both IL-4 and IL-13 responses, through administration of an anti-IL-4R alpha mAb, reversed asthma-like symptoms, when given to mice with established disease. Collectively these data provide insight into the pathways engaged in TSLP-driven airway inflammation and demonstrate that simultaneous blockade of IL-4 and IL-13 can reverse established airway disease, suggesting that this may be an effective approach for the therapy of Th2-mediated inflammatory respiratory disease.     

IL-5 and eosinophils are essential for the development of airway hyperresponsiveness following acute respiratory syncytial virus infection

Viral respiratory infections can cause bronchial hyperresponsiveness and exacerbate asthma. In mice, respiratory syncytial virus (RSV) infection, which induces an immune response dominated by IFN-gamma, results in airway hyperresponsiveness (AHR) and eosinophil influx into the airways, both of which are prevented by pretreatment with anti-IL-5 Ab. To delineate the role of IL-5, IL-4, and IFN-gamma in the development of RSV-induced AHR and lung eosinophilia, we tested the ability of mice deficient in each of these cytokines to develop these symptoms of RSV infection. Mice deficient in either IL-5, IL-4, or IFN-gamma were administered infectious RSV intranasally, and 6 days later, airway responsiveness to inhaled methacholine was assessed by barometric body plethysmography, and numbers of lung eosinophils and production of IFN-gamma, IL-4, and IL-5 by mononuclear cells from peribronchial lymph nodes were monitored. RSV infection resulted in airway eosinophilia and AHR in both IL-4- and IFN-gamma-deficient mice, but not in IL-5-deficient mice. Reconstitution of IL-5-deficient mice with IL-5 restored these responses and enhanced the responses in IL-4-deficient mice. Anti-VLA-4 (very late Ag-4) treatment prevented lung eosinophilia and AHR following RSV infection and IL-5 reconstitution. We conclude that in response to RSV, IL-5 is essential for the influx of eosinophils into the lung and that eosinophils in turn are critical for the development of AHR. IFN-gamma and IL-4 are not essential for these responses to RSV infection.     

Blockade of allergic airway inflammation following systemic treatment with a B7-dendritic cell (PD-L2) cross-linking human antibody

We present a novel immunotherapeutic strategy using a human B7-DC cross-linking Ab that prevents lung inflammation, airway obstruction, and hyperreactivity to allergen in a mouse model of allergic inflammatory airway disease. Dendritic cells (DC) have the ability to skew the immune response toward a Th1 or Th2 polarity. The sHIgM12 Ab functions in vitro by cross-linking the costimulatory family molecule B7-DC (PD-L2) on DC up-regulating IL-12 production, homing to lymph nodes, and T cell-activating potential of these APCs. Using chicken OVA as a model Ag, the administration of sHIgM12 Ab to BALB/c mice blocked lung inflammation, airway pathology, and responsiveness to methacholine, even after animals were presensitized and a Th2-polarized immune response was established. This therapeutic strategy was ineffective in STAT4-deficient animals, indicating that IL-12 production is critical in this system. Moreover, the polarity of the immune response upon in vitro restimulation with Ag is changed in wild-type mice, with a resulting decrease in Th2 cytokines IL-4 and IL-5 and an increase in the immunoregulatory cytokine IL-10. These studies demonstrate that the immune response of hypersensitized responders can be modulated using B7-DC cross-linking Abs, preventing allergic airway disease upon re-exposure to allergen.     

Combined budesonide/formoterol therapy in conjunction with allergen avoidance ameliorates house dust mite-induced airway remodeling and dysfunction

Allergic asthma is characterized by airway inflammation in response to chronic allergen exposure, resulting in remodeling of the airway wall accompanied by dysfunctional airway physiology. However, a link between the immune-inflammatory response to allergen and changes to airway structure and physiology has not yet been fully elucidated. Moreover, the impact of inhaled corticosteroids and beta(2)-agonists, the primary pharmacotherapy for asthma, on this process has not been completely evaluated. In this study, we employed a murine model of chronic exposure to a common environmental aeroallergen, house dust mite, to recapitulate the phenotype of clinical asthma. By examining the therapeutic effects of corticosteroid/beta(2)-agonist combination therapy with budesonide/formoterol (BUD/FORM) in this model of airway disease, we endeavored to determine the impact of BUD/FORM on lung inflammation, structure, and physiology. BUD/FORM was delivered either while allergen exposure was ongoing (concurrent therapy) or following the cessation of allergen exposure (postexposure therapy). Our results show that airway inflammation was substantially reduced in BUD/FORM-treated mice in the concurrent therapy group, whereas in the postexposure therapy group airway inflammation spontaneously resolved. In contrast, BUD/FORM was most effective in resolving several aspects of airway remodeling and bronchial hyperreactivity when delivered in conjunction with allergen withdrawal. This study demonstrates that although both BUD/FORM therapy and allergen avoidance independently reduce airway inflammation, only BUD/FORM therapy in conjunction with allergen avoidance can effectively reverse airway remodeling and bronchial hyperreactivity induced by chronic allergen exposure.     

Mucosal sensitization to German cockroach involves protease-activated receptor-2

Background

Allergic asthma is on the rise in developed countries. A common characteristic of allergens is that they contain intrinsic protease activity, and many have been shown to activate protease-activated receptor (PAR)-2 in vitro. The role for PAR-2 in mediating allergic airway inflammation has not been assessed using a real world allergen.

Methods

Mice (wild type or PAR-2-deficient) were sensitized to German cockroach (GC) feces (frass) or protease-depleted GC frass by either mucosal exposure or intraperitoneal injection and measurements of airway inflammation (IL-5, IL-13, IL-17A, and IFNγ levels in the lung, serum IgE levels, cellular infiltration, mucin production) and airway hyperresponsiveness were performed.

Results

Following systemic sensitization, GC frass increased airway hyperresponsiveness, Th2 cytokine release, serum IgE levels, cellular infiltration and mucin production in wild type mice. Interestingly, PAR-2-deficient mice had similar responses as wild type mice. Since these data were in direct contrast to our finding that mucosal sensitization with GC frass proteases regulated airway hyperresponsiveness and mucin production in BALB/c mice (Page et. al. 2007 Resp Res 8:91), we backcrossed the PAR-2-deficient mice into the BALB/c strain. Sensitization to GC frass could now occur via the more physiologically relevant method of intratracheal inhalation. PAR-2-deficient mice had significantly reduced airway hyperresponsiveness, Th2 and Th17 cytokine release, serum IgE levels, and cellular infiltration compared to wild type mice when sensitization to GC frass occurred through the mucosa. To confirm the importance of mucosal exposure, mice were systemically sensitized to GC frass or protease-depleted GC frass via intraperitoneal injection. We found that removal of proteases from GC frass had no effect on airway inflammation when administered systemically.

Conclusions

We showed for the first time that allergen-derived proteases in GC frass elicit allergic airway inflammation via PAR-2, but only when allergen was administered through the mucosa. Importantly, our data suggest the importance of resident airway cells in the initiation of allergic airway disease, and could make allergen-derived proteases attractive therapeutic targets.     

Modulation of airway remodeling and airway inflammation by peroxisome proliferator-activated receptor gamma in a murine model of toluene diisocyanate-induced asthma

Toluene diisocyanate (TDI) is a leading cause of occupational asthma. Although considerable controversy remains regarding its pathogenesis, TDI-induced asthma is an inflammatory disease of the airways characterized by airway remodeling. Peroxisome proliferator-activated receptor gamma (PPARgamma) has been shown to play a critical role in the control of airway inflammatory responses. However, no data are available on the role of PPARgamma in TDI-induced asthma. We have used a mouse model for TDI-induced asthma to determine the effect of PPARgamma agonist, rosiglitazone, or pioglitazone, and PPARgamma on TDI-induced bronchial inflammation and airway remodeling. This study with the TDI-induced model of asthma revealed the following typical pathophysiological features: increased numbers of inflammatory cells of the airways, airway hyperresponsiveness, increased levels of Th2 cytokines (IL-4, IL-5, and IL-13), adhesion molecules (ICAM-1 and VCAM-1), chemokines (RANTES and eotaxin), TGF-beta1, and NF-kappaB in nuclear protein extracts. In addition, the mice exposed to TDI developed features of airway remodeling, including thickening of the peribronchial smooth muscle layer, subepithelial collagen deposition, and increased airway mucus production. Administration of PPARgamma agonists or adenovirus carrying PPARgamma2 cDNA reduced the pathophysiological symptoms of asthma and decreased the increased levels of Th2 cytokines, adhesion molecules, chemokines, TGF-beta1, and NF-kappaB in nuclear protein extracts after TDI inhalation. In addition, inhibition of NF-kappaB activation decreased the increased levels of Th2 cytokines, adhesion molecules, chemokines, and TGF-beta1 after TDI inhalation. These findings demonstrate a protective role of PPARgamma in the pathogenesis of the TDI-induced asthma phenotype.     

Absence of the complement anaphylatoxin C3a receptor suppresses Th2 effector functions in a murine model of pulmonary allergy

Asthma is a chronic inflammatory disease of the lung resulting in airway obstruction. The airway inflammation of asthma is strongly linked to Th2 lymphocytes and their cytokines, particularly IL-4, IL-5, and IL-13, which regulate airway hyperresponsiveness, eosinophil activation, mucus production, and IgE secretion. Historically, complement was not thought to contribute to the pathogenesis of asthma. However, our previous reports have demonstrated that complement contributes to bronchial hyperreactivity, recruitment of airway eosinophils, IL-4 production, and IgE responses in a mouse model of pulmonary allergy. To define the complement activation fragments that mediate these effects, we assessed the role of the complement anaphylatoxin C3a in a mouse model of pulmonary allergy by challenging C3aR-deficient mice intranasally with a mixed Ag preparation of Aspergillus fumigatus cell culture filtrate and OVA. Analysis by plethysmography after challenge revealed an attenuation in airway hyperresponsiveness in C3aR-deficient mice relative to wild-type mice. C3aR-deficient mice also had an 88% decrease in airway eosinophils and a 59% reduction in lung IL-4-producing cells. Consistent with the reduced numbers of IL-4-producing cells, C3aR-deficient mice had diminished bronchoalveolar lavage levels of the Th2 cytokines, IL-5 and IL-13. C3aR knockout mice also exhibited decreases in IgE titers as well as reduced mucus production. Collectively, these data highlight the importance of complement activation, the C3a anaphylatoxin, and its receptor during Th2 development in this experimental model and implicate these molecules as possible therapeutic targets in diseases such as asthma.     

CD8+ T cell-mediated airway hyperresponsiveness and inflammation is dependent on CD4+IL-4+ T cells

CD4+ T cells, particularly Th2 cells, play a pivotal role in allergic airway inflammation. However, the requirements for interactions between CD4+ and CD8+ T cells in airway allergic inflammation have not been delineated. Sensitized and challenged OT-1 mice in which CD8+ T cells expressing the transgene for the OVA(257-264) peptide (SIINFEKL) failed to develop airway hyperresponsiveness (AHR), airway eosinophilia, Th2 cytokine elevation, or goblet cell metaplasia. OT-1 mice that received naive CD4+IL-4+ T cells but not CD4+IL-4- T cells before sensitization developed all of these responses to the same degree as wild-type mice. Moreover, recipients of CD4+IL-4+ T cells developed significant increases in the number of CD8+IL-13+ T cells in the lung, whereas sensitized OT-1 mice that received primed CD4+ T cells just before challenge failed to develop these responses. Sensitized CD8-deficient mice that received CD8+ T cells from OT-1 mice that received naive CD4+ T cells before sensitization increased AHR and eosinophil numbers in bronchoalveolar lavage fluid when challenged with allergen. In contrast, sensitized CD8-deficient mice receiving CD8+ T cells from OT-1 mice without CD4+ T cells developed reduced AHR and eosinophil numbers in bronchoalveolar lavage fluid when challenged. These data suggest that interactions between CD4+ and CD8+ T cells, in part through IL-4 during the sensitization phase, are essential to the development of CD8+IL-13+ T cell-dependent AHR and airway allergic inflammation.     

Anti-IL-13 monoclonal antibody inhibits airway hyperresponsiveness, inflammation and airway remodeling

Asthma is a chronic inflammatory disease characterized by reversible bronchial constriction, pulmonary inflammation and airway remodeling. Current standard therapies for asthma provide symptomatic control but fail to target the underlying disease pathology. Furthermore, no therapeutic agent is effective in preventing airway remodeling. Interleukin 13 (IL-13) is a pleiotropic cytokine produced mainly by T cells. A substantial amount of evidence suggests that IL-13 plays a critical role in the pathogenesis of asthma. Therefore, a neutralizing anti-IL-13 monoclonal antibody could provide therapeutic benefits to asthmatic patients. To test the concept we have generated a neutralizing rat anti-mouse IL-13 monoclonal antibody, and evaluated its effects in a chronic mouse model of asthma. Chronic asthma-like response was induced in ovalbumin (OVA) sensitized mice by repeated intranasal OVA challenges. After weeks of challenge, mice developed airway hyperresponsiveness (AHR) to methacholine stimulation, severe airway inflammation, hyper mucus production, and subepithelial fibrosis. When given at the time of each intranasal OVA challenge, anti-IL-13 antibody significantly suppressed AHR, eosinophil infiltration, proinflammatory cytokine/chemokine production, serum IgE, and most interestingly, airway remodeling. Taken together, these results strongly suggest that a neutralizing anti-human IL-13 monoclonal antibody could be an effective therapeutic agent for asthma.     

Th type 1-stimulating activity of lung macrophages inhibits Th2-mediated allergic airway inflammation by an IFN-gamma-dependent mechanism

In the mucosal immune system, resident dendritic cells are specialized for priming Th2-polarized immunity, whereas the Ag-presenting activity of macrophages has been linked with the development of Th1 phenotype. As an immune switch toward Th1 can protect against Th2-mediated allergic response, this study investigated the capacity of lung macrophages to stimulate Th1 responses during the secondary exposure to inhaled allergen, thereby suppressing Th2-mediated allergic airway inflammation in a murine model of allergic asthma. Following airway macrophage depletion in OVA-sensitized mice, lung T cells defaulted to a phenotype that produced less Th1 (IFN-gamma) and more Th2 (IL-4 and IL-5) cytokines, leading to more severe airway hyperreactivity and inflammation after intranasal Ag challenge. After OVA pulsing and adoptive transfer, lung macrophages selectively promoted a Th1 response in Ag-sensitized recipients and did not induce pulmonary eosinophilia. By contrast, OVA pulsing and adoptive transfer of a lung cell preparation, consisting of dendritic cells, B cells, and macrophages, promoted a Th2 response with an associated inflammatory response that was suppressed when macrophages were present and pretreated with IFN-gamma, but exacerbated when macrophages were depleted before IFN-gamma treatment. In addition, Th1-promoting activity of lung macrophages was not related to the autocrine production of IL-12p40. These results suggest that the Th1-promoting APC activity may be an inherent property of the lung macrophage population, and may play an important role, upon stimulation by IFN-gamma, in antagonizing an ongoing Th2 immunity and Th2-dependent allergic responses.     

Global gene expression profiles during acute pathogen-induced pulmonary inflammation reveal divergent roles for Th1 and Th2 responses in tissue repair

T helper 1 responses are typically proinflammatory, while Th2 responses have been considered regulatory. Interestingly, Th2 responses characterize a number of pulmonary diseases, many of which terminate in tissue remodeling and fibrosis. We developed a mouse model using Schistosoma mansoni eggs and cytokine-deficient mice to induce highly polarized Th1- or Th2-type inflammation in the lung. In this study, we examined the pathology and cytokine profiles in Th1- and Th2-polarized environments and used oligonucleotide microarray analysis to decipher the genes responsible for these effects. We further elaborated on the results using IL-10- and IL-13-deficient mice because these cytokines are believed to be the central regulators of Th2-associated pathology. We found that the Th1-polarized mice developed small granulomas with less fibrosis while expressing genes characteristic of tissue damage. Th2-polarized mice, in contrast, formed large granulomas with massive collagen deposition and up-regulated genes associated with wound healing, specifically, arginase, collagens, matrix metalloproteinases (MMPs), and tissue inhibitors of MMP. In addition, several members of the chitinase-like family were up-regulated in the lung following egg challenge. We also developed a method of defining the net collagen deposition using the expression profiles of several collagen, MMP, and tissue inhibitors of MMP genes. We found that Th1-polarized mice did not elaborate collagens or MMPs and therefore did not have a significant capacity for repair in this model. Thus, Th1-mediated inflammation is characterized by tissue damage, while Th2 directs wound healing and fibrosis.     

Increased Th2 cytokine secretion, eosinophilic airway inflammation, and airway hyperresponsiveness in neurturin-deficient mice

Neurotrophins such as nerve growth factor and brain-derived neurotrophic factor have been described to be involved in the pathogenesis of asthma. Neurturin (NTN), another neurotrophin from the glial cell line-derived neurotrophic factor family, was shown to be produced by human immune cells: monocytes, B cells, and T cells. Furthermore, it was previously described that the secretion of inflammatory cytokines was dramatically stimulated in NTN knockout (NTN(-/-)) mice. NTN is structurally similar to TGF-β, a protective cytokine in airway inflammation. This study investigates the implication of NTN in a model of allergic airway inflammation using NTN(-/-) mice. The bronchial inflammatory response of OVA-sensitized NTN(-/-) mice was compared with wild-type mice. Airway inflammation, Th2 cytokines, and airway hyperresponsiveness (AHR) were examined. NTN(-/-) mice showed an increase of OVA-specific serum IgE and a pronounced worsening of inflammatory features. Eosinophil number and IL-4 and IL-5 concentration in the bronchoalveolar lavage fluid and lung tissue were increased. In parallel, Th2 cytokine secretion of lung draining lymph node cells was also augmented when stimulated by OVA in vitro. Furthermore, AHR was markedly enhanced in NTN(-/-) mice after sensitization and challenge when compared with wild-type mice. Administration of NTN before challenge with OVA partially rescues the phenotype of NTN(-/-) mice. These findings provide evidence for a dampening role of NTN on allergic inflammation and AHR in a murine model of asthma.     

Effects of thioredoxin on established airway remodeling in a chronic antigen exposure asthma model

The development and treatment of asthma remains a subject of considerable interest in the medical community. Previous studies implicate an important role of cytokines in the pathology of asthma. In this current study, we examined whether redox-active protein thioredoxin 1 (TRX1) could prevent airway remodeling in an ovalbumin (OVA)-driven mouse chronic antigen exposure asthma model. Balb/c mice were sensitized and then challenged nine times with OVA (days 19-45). In this protocol, airway remodeling was established by day 34. Administration of recombinant human TRX1 during antigen challenge (days 18-32) significantly inhibited airway remodeling, eosinophilic pulmonary inflammation, airway hyperresponsiveness and resulted in decreased lung expression of eotaxin, macrophage inflammatory protein-1alpha and IL-13. Airway remodeling and eosinophilic pulmonary inflammation was also prevented in chronic OVA-exposed Balb/c human TRX1 transgenic mice. Importantly, TRX1-administration, after the establishment of airway remodeling (days 35-45), resulted in improved airway pathology. Our results suggest TRX1 prevents the development of airway remodeling, and also improves established airway remodeling by inhibiting production of chemokines and Th2 cytokines in the lungs.     

Requirements for allergen-induced airway hyperreactivity in T and B cell-deficient mice.

BACKGROUND: The pathogenesis of asthma is believed to reflect antigen-induced airway inflammation leading to the recruitment of eosinophils and activation of mast cells through cell-associated IgE. Controversies persist however, regarding the relative importance of different pathogenic cells and effector molecules. MATERIALS AND METHODS: A variety of gene-targeted mice were examined for the induction of cholinergic airway hyperresponsiveness (AH), allergic airway inflammation, mucus production, and serum IgE reactivity following intratracheal challenge with a potent allergen. AH was determined using whole-body plethysmography following acetylcholine challenge. Where possible, results were confirmed using neutralizing antibodies and cell-specific reconstitution of immune deficient mice. RESULTS: T and B cell-deficient, recombinase-activating-gene-deficient mice (RAG -/-) failed to develop significant allergic inflammation and AH following allergen challenge. Reconstitution of RAG -/- mice with CD4+ T cells alone was sufficient to restore allergen-induced AH, allergic inflammation, and goblet cell hyperplasia, but not IgE reactivity. Sensitized B cell-deficient mice also developed airway hyperreactivity and lung inflammation comparable to that of wild-type animals, confirming that antibodies were dispensable. Treatment with neutralizing anti-IL-4 antibody or sensitization of IL-4-deficient mice resulted in loss of airway hyperreactivity, whereas treatment with anti-IL-5 antibody or sensitization of IL-5-deficient mice had no effect. CONCLUSIONS: In mice, CD4+ T cells are alone sufficient to mediate many of the pathognomonic changes that occur in human asthma by a mechanism dependent upon IL-4, but independent of IL-5, IgE, or both. Clarification of the role played by CD4+ T cells is likely to stimulate important therapeutic advances in treatment of asthma.