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Allergic asthma is a chronic inflammatory disorder that affects ∼20% of the population worldwide. Microarray analyses of nasal epithelial cells from acute asthmatic patients detected a 50% decrease in expression of Stard7, an intracellular phosphatidylcholine transport protein. To determine whether loss of Stard7 expression promotes allergic responses, mice were generated in which one allele of the Stard7 locus was globally disrupted (Stard7+/− mice). OVA sensitization and challenge of Stard7+/− mice resulted in a significant increase in pulmonary inflammation, mucous cell metaplasia, airway hyperresponsiveness, and OVA-specific IgE compared with OVA-sensitized/challenged wild-type (WT) mice. This exacerbation was largely Th2-mediated with a significant increase in CD4+IL-13+ T cells and IL-4, IL-5, and IL-13 cytokines. The loss of Stard7 was also associated with increased lung epithelial permeability and activation of proinflammatory dendritic cells in sensitized and/or challenged Stard7+/− mice. Notably, OVA-pulsed dendritic cells from Stard7+/− mice were sufficient to confer an exaggerated allergic response in OVA-challenged WT mice, although airway hyperresponsiveness was greater in Stard7+/− recipients compared with WT recipients. Enhanced allergic responses in the lung were accompanied by age-dependent development of spontaneous atopic dermatitis. Overall, these data suggest that Stard7 is an important component of a novel protective pathway in tissues exposed to the extracellular environment.

After birth, the respiratory tract adapts to recurrent exposures to pathogens, allergens, and toxicants by inducing the complex innate and acquired immune systems required for pulmonary homeostasis. In this study, we show that Foxa2, expressed selectively in the respiratory epithelium, plays a critical role in regulating genetic programs influencing Th2 cell-mediated pulmonary inflammation. Deletion of the Foxa2 gene, encoding a winged helix/forkhead box transcription factor that is selectively expressed in respiratory epithelial cells, caused spontaneous pulmonary eosinophilic inflammation and goblet cell metaplasia. Loss of Foxa2 induced the recruitment and activation of myeloid dendritic cells and Th2 cells in the lung, causing increased production of Th2 cytokines and chemokines. Loss of Foxa2-induced expression of genes regulating Th2 cell-mediated inflammation and goblet cell differentiation, including IL-13, IL-4, eotaxins, thymus and activation-regulated chemokine, Il33, Ccl20, and SAM pointed domain-containing Ets transcription factor. Pulmonary inflammation and goblet cell differentiation were abrogated by treatment of neonatal Foxa2∆/∆ mice with mAb against IL-4Rα subunit. The respiratory epithelium plays a central role in the regulation of Th2-mediated inflammation and innate immunity in the developing lung in a process regulated by Foxa2.

The role of complement in the development of maladaptive immunity in experimental allergic asthma is unclear. In this study, we show that C3a receptor (C3aR)-deficient mice are protected from the development of Th2 immunity in a model of house dust mite-induced asthma. C5a receptor (C5aR)-targeting of C3aR-deficient mice during allergen sensitization not only reversed the protective effect but enhanced Th2 cytokine production, airway inflammation, and airway responsiveness, suggesting that the reduced allergic phenotype in C3aR-deficient mice results from protective C5aR signaling. In support of this view, C5aR expression in C3aR-deficient pulmonary dendritic cells (DCs) was increased when compared with wild-type DCs. Moreover, C5aR targeting regulated the frequency of pulmonary plasmacytoid DCs expressing costimulatory molecules B7-H1 and B7-DC. Ex vivo targeting of B7-H1 and B7-DC increased Th2 cytokine production from T cells of wild-type but not of C5aR-targeted mice, suggesting a protective role for C5a through regulation of B7 molecule expression on plasmacytoid DCs.

Allergic diseases, which have escalated in prevalence in recent years, arise as a result of maladaptive immune responses to ubiquitous environmental proteins. Arthropods, such as mites, cockroaches, and crustaceans, are a major source of allergens, yet the majority of us live with or consume these invertebrates with no ill effects. Recent discoveries have raised the possibility that altered innate immune recognition of allergens, through pattern recognition receptors (PRR), may underlie disease susceptibility. Here, we identify the PRR, dectin-1 (CLEC7A), as a key receptor in mediating protection against dust mite-driven type 2 immune responses through the inhibition of epithelial cell IL-33 production, and the downstream recruitment of IL-13-producing innate lymphoid 2 cells (ILC2). Surprisingly, we observed that this effect was not mediated by the prototypical dectin-1 ligand, β-glucan, within allergen extracts. We discovered a novel protein ligand conserved across the arthropod phyla, sensing of which confers protection against aberrant type 2 responses that drive respiratory allergy and food anaphylaxis in mice. While this homeostatic pathway is functional in respiratory epithelial cells from healthy humans, dectin-1 is repressed in asthmatics and patients with chronic-rhinosinusitis. Importantly, we report a novel SNP in CLEC7A in the SAGE and GALAII GWAS studies, associated with lung function. Collectively, our findings reveal a previously unrecognized negative regulatory pathway in which ligation of dectin-1 results in reduction of type 2 immune responses, while disruption of this critical regulatory circuit is associated with susceptibility to a spectrum of allergic disorders.

Allergic asthma is thought to be mediated by CD4+ T lymphocytes producing the Th2-associated cytokines, IL-4, and IL-5. Recently, the costimulatory molecules B7-1 and B7-2, which are expressed on the surface of APC, have been suggested to influence the development of Th1 vs Th2 immune responses. We examined the in vivo role of these costimulatory molecules in the pathogenesis of Th2-mediated allergen-induced airway hyperresponsiveness in a murine model of asthma. In this model, OVA-sensitized A/J mice develop significant increases in airway responsiveness, pulmonary eosinophilia, and pulmonary Th2 cytokine expression following aspiration challenge with OVA as compared with PBS-control animals. Strikingly, administration of anti-B7-2 mAb to OVA-treated mice abolished allergen-induced airway hyperresponsiveness, pulmonary eosinophilia, and elevations in serum IgG1 and IgE levels. Anti-B7-2 treatment of OVA-treated mice reduced both total lung IL-4 and IL-5 mRNA and bronchoalveolar lavage fluid IL-4 and IL-5 protein levels, with no significant changes in IFN-γ message or protein levels. In contrast, treatment with anti-B7-1 mAbs had no effect on allergen-induced airway hyperresponsiveness, IgE production, or cytokine production, however, it significantly suppressed pulmonary eosinophilia. We conclude that B7-2 provides the necessary costimulatory signal required for the development of in vivo allergic responses to inhaled allergen exposure.

Asthma is a chronic inflammatory disease of the airways, driven by a Th2 immune response to allergens like house dust mite (HDM). While Th2 immune responses (IL-13 in particular) alone can induce allergic asthma, Th17 cytokines (IL-17) can exacerbate the disease. Although, the components of HDM that drive asthma remain unclear, HDM is a complex mixture including immunogenic structural moieties like carbohydrates. Such carbohydrate moieties can be recognized by pattern recognition receptors on dendritic cells (DCs) like C-type lectin receptors (CLRs). Recently, CLRs like Dectin-1 & Dectin-2 have been implicated in driving Th2 & Th17 responses against various infections. However, the role of carbohydrate recognition in modulation of HDM-induced allergic asthma remains unclear. Therefore we hypothesized that Dectin-1 & Dectin-2 induce Th2 & Th17 responses following HDM exposure. Surprisingly, Dectin-1 KO mice had exacerbated Th2 immune responses leading to enhance airway resistance. In contrast, mice in which Dectin-2 was blocked (using monoclonal antibody) had diminished HDM-induced Th2 immune responses and airway reactivity. Interestingly, neither Dectin-1 nor Dectin-2 modulated Th17 responses. Overall, our data suggests that while Dectin-1 protects against allergic asthma by negatively regulating Th2 immune responses, Dectin-2 promotes asthma by enhancing Th2 immune responses.

Certain families of allergens contain domains that bear structural and functional similarity to homologs from pathogens enabling them to sabotage host immunity by hijacking crucial innate immune pathways. We focused on members of the Bet v 1-like superfamily that share a remarkably conserved architecture, the so called Bet v 1-fold, with the aim to identify structural mimicry as one possible cause of allergenicity. Here we show that Bet v 1, the major birch pollen (BP) allergen, has functional homology with a bacterial homolog, namely TTHA0849 from Thermus thermophilus. TTHA0849 and Bet v 1 competed for the same uptake mechanism by monocyte-derived DCs (MoDCs) of BP allergic and normal donors as visualized by fluorescence microscopy. Using a pre-designed antibacterial response PCR array, we could reveal striking similarities in gene expression patterns induced by Bet v 1 in MoDCs of both donor groups to those induced by TTHA0849. Importantly, Bet v 1 and its bacterial homolog also triggered the transcription of the Th2 signature cytokines IL-4 and IL-13, as well as EGR-3, a marker of early cell activation, in MoDCs from BP allergic but not normal donors. These findings propose that conserved structural features are present on both Bet v 1 and TTHA0849 targeting the same pathways common to allergic and normal individuals. Altered signal processing and translation in predisposed allergic individuals might be a mechanism underlying the phenomenon of allergic sensitization.

Allergic diseases are increasingly prevalent in Westernized environments. Recent data indicate that the molecular substrates of allergenicity are a function of direct interactions with pathways of innate immune activation that evolved to sense microbial infection. After lipid-binding proteins exemplified by Der p 2, proteases represent the next most common class of allergens. The relevant mechanisms by which proteases lead to innate immune activation remain under-defined. α2-macroglobulin (α2M) is a plasma protein that inhibits extracellular proteases, targeting them for internalization by LDL receptor-related protein 1 (LRP1). In antigen presenting cells (APC), LRP1 directs α2M-associated proteins for antigen presentation. We hypothesized that protease adjuvanticity and allergenicity result from interactions with α2M and LRP1. Thus, we intratracheally challenged mice with papain, an occupational allergen and cysteine protease. In C57Bl/6 mice, this induced eosinophil and lymphocyte recruitment to the lung and increases in total and papain-specific IgE. While α2M-deficient mice exhibited no defects in the generation of allergic responses to papain, deletion of LRP-1 on APCs resulted in increased allergic inflammatory responses after challenge. These data suggest a role for α2M/LRP-1 in the clearance of protease allergens and suppression of excessive immune activation.

Although allergic asthma is a chronic inflammatory disease of the lung mediated by Th2 cells, the pathways leading to this response are not clear. Recent studies have highlighted influence of pulmonary dendritic cells (DCs) on T-cell differentiation. Rapid DC recruitment into the bronchial mucosa has been well documented in animals and humans in response to allergen. Central to early DC recruitment, CCL20 is the only known ligand for CCR6, a receptor preferentially expressed on immature DCs. The potential importance of CCL20/CCR6 in allergic sensitization was demonstrated in a cockroach model of allergic asthma in which Ccr6-/- mice were protected against asthma. At present, the mechanisms by which allergen induces secretion of DC-attractant chemokines are unknown. We previously showed that bronchial epithelial cells release CCL20 relatively rapidly after house dust mite (HDM) (Nathan, 2009). This induction was not prevented by the inhibiting protein synthesis (cycloheximide) suggesting release through a transcriptionally independent mechanism. These data suggested that HDM may induced the release of preformed stores of CCL20

Asthma is a major public health burden worldwide. Excessive mucus production and mucus plugging are key pathologic features of asthma, yet the mechanisms responsible for mucus production remain largely unknown and therapies to effectively target mucus hypersecretion are lacking. Using a murine asthma model, we showed that SerpinB3a, the mouse ortholog of the serine protease inhibitors, SERPINB4 and B3, contribute to house dust mite induced airway hyperresponsiveness (AHR), mucus production and goblet cell hyperplasia and expression of SPDEF, a transcription factor that mediates goblet cell differentiation. Microarray analysis revealed attenuated expression of multiple IL-13 regulated genes that contribute to mucus production in the Serpinb3a null mice and IL-13 treated mice showed attenuated AHR and mucus production. Our data have revealed a novel non-redundant role for SERPINB4 and B3 in mediating mucus production through regulation of SPDEF expression.

Hyperresponsiveness to cholinergic stimulation of increased airway resistance is a defining characteristic of asthma. Both IL-4 and IL-13 activate Stat6 via IL-4Rα to induce airway hyperresponsiveness (AHR) in mouse models of asthma and increase mouse and human smooth muscle contractility in vitro. However, the relatively small size of mouse vs. human airways, differences in airway smooth muscle cell distribution between mouse and human, and observations that selective IL-13 stimulation of Stat6 in airway epithelium is sufficient to induce AHR in mice have raised questions about the importance of direct IL-4/IL-13 effects on smooth muscle in murine models of allergic airway disease and whether these models are appropriate for studying AHR in human asthma. To determine whether direct effects of IL-4/IL-13 on smooth muscle contribute to murine allergic airway disease, we produced mice that express IL-4Rα only on smooth muscle and evaluated the effects of intratracheal IL-4, IL-13 and allergen on their airways. We demonstrate that IL-4 and IL-13 induce AHR in these mice, without stimulating goblet cell hyperplasia or airway eosinophilia. Similar results were observed following airway inoculation of these mice with a potent allergen. Thus, allergen-stimulated IL-4/IL-13 can induce AHR in mice through direct effects on airway smooth muscle.

Infiltration of eosinophils into the lung in experimental asthma is dependent on the adhesion molecule vascular cell adhesion molecule-1 (VCAM-1) on endothelial cells. Ligation of VCAM-1 activates endothelial cell NADPH oxidase which is required for VCAM-1-dependent leukocyte migration in vitro. To examine whether endothelial-derived NADPH oxidase modulates eosinophil recruitment in vivo, mice deficient in NADPH oxidase (CYBB mice) were irradiated and received wild type hematopoietic cells to generate chimeric CYBB mice. In response to OVA challenge, the chimeric CYBB mice had increased numbers of eosinophils bound to the endothelium as well as reduced eosinophilia in the lung tissue and bronchoalveolar lavage. This occurred independent of changes in VCAM-1 expression, cytokine/chemokine levels (IL-5, IL-10, IL-13, IFNã, or eotaxin), or numbers of T cells, neutrophils or mononuclear cells in the lavage fluids or lung tissue of OVA-challenged mice. Importantly, the OVA-challenged chimeric CYBB mice had reduced airway hyperresponsiveness (AHR). The AHR in OVA-challenged chimeric CYBB mice was restored by bypassing the endothelium with intratracheal administration of eosinophils. These data suggest that VCAM-1 induction of NADPH oxidase in the endothelium is necessary for the eosinophil recruitment during allergic inflammation. (supported by NIH HL069428, J.M.C-M.)

IL-13 is the primary inducer of AHR in mouse models of asthma and increases contractility of isolated muscle cells in vitro and the contractility of intestinal smooth muscle in worm-infected mice. Consequently, we evaluated whether smooth muscle responsiveness to IL-13 is sufficient for development of AHR in mice inoculated intratracheally with IL-13. The smooth muscle-specific promoter SMP8 was used to generate mice that express IL-4Rα only on smooth muscle cells (SMonly mice, produced by crossing SMP8-IL-4Rα transgenic mice to IL-4Rα-deficient mice). SMonly mice failed to develop goblet cell hyperplasia or BAL eosinophilia in response to intratracheal IL-13 but developed AHR to inhaled methacholine to the same extent as wild-type mice when tested by barometric plethysmography with a Buxco apparatus and significant, albeit reduced AHR to injected acetylcholine when anesthetized, intubated and evaluated by the airway time-pressure index (APTI) technique. Thus, smooth muscle expression of IL-4Rα is sufficient for IL-13 induction of AHR and this is not dependent on goblet cell hyperplasia or pulmonary eosinophilia. Studies designed to determine whether smooth muscle expression of IL-4Rα is necessary for IL-13 induction of AHR are in progress. Supported by the US Dept of Veterans Affairs and NIH.