Jeremy A. Hirota

A GABAergic system in airway epithelium is essential for mucus overproduction in asthma

γ-Aminobutyric acid (GABA) is an important neurotransmitter that, through the subtype A GABA receptor (GABAAR), induces inhibition in the adult brain. Here we show that an excitatory, rather than inhibitory, GABAergic system exists in airway epithelial cells. Both GABAARs and the GABA synthetic enzyme glutamic acid decarboxylase (GAD) are expressed in pulmonary epithelial cells. Activation of GABAARs depolarized these cells. The expression of GAD in the cytosol and GABAARs in the apical membranes of airway epithelial cells increased markedly when mice were sensitized and then challenged with ovalbumin, an approach for inducing allergic asthmatic reactions. Similarly, GAD and GABAARs in airway epithelial cells of humans with asthma increased after allergen inhalation challenge. Intranasal application of selective GABAAR inhibitors suppressed the hyperplasia of goblet cells and the overproduction of mucus induced by ovalbumin or interleukin-13 in mice. These findings show that a previously unknown epithelial GABAergic system has an essential role in asthma.

Group 2 innate lymphoid cells facilitate sensitization to local, but not systemic, TH2-inducing allergen exposures

BACKGROUND Allergic inflammation involves the sensitization of naive CD4(+) T cells to allergens, resulting in a TH2-skewed inflammatory response. Although antigen presentation by dendritic cells to T cells in the lymph node is crucial for TH2 cell development, the innate signals that initiate adaptive type 2 inflammation and the role of group 2 innate lymphoid cells (ILC2s) are poorly understood. OBJECTIVE We sought to investigate the influence of ILC2s and the route of priming on the development of an adaptive type 2 immune response to lung allergens. METHODS Wild-type and ILC2-deficient mice were exposed intranasally or systemically to the TH2-inducing antigens house dust mite or ovalbumin in a model of allergic airway inflammation or the TH17-inducing bacterial antigen Saccharopolyspora rectivirgula in a model of hypersensitivity pneumonitis. The formation of an adaptive immune response was evaluated based on serum antibody titers and production of T cell-derived cytokines (IL-4, IL-5, IL-13 and IL-17A). RESULTS We find that lung ILC2s play a critical role in priming the adaptive type 2 immune response to inhaled allergens, including the recruitment of eosinophils, TH2 cytokine production and serum IgE levels. Surprisingly, systemic priming with ovalbumin, with or without adjuvants, circumvents the requirement for ILC2s in inducing TH2-driven lung inflammation. ILC2s were also found to be dispensable for the sensitization to TH1- or TH17-inducing antigens. CONCLUSION These data highlight a critical role for ILC2s in the development of adaptive type 2 responses to local, but not systemic, antigen exposure.

The airway epithelium nucleotide-binding domain and leucine-rich repeat protein 3 inflammasome is activated by urban particulate matter

BACKGROUND The airway epithelium is the first line of defense against inhaled insults and therefore must be capable of coordinating appropriate inflammatory and immune responses. OBJECTIVE We sought to test the hypothesis that the nucleotide-binding domain and leucine-rich repeat protein 3 (NLRP3) inflammasome, an intracellular danger-sensing complex, plays a critical role in airway epithelium-mediated immune responses to urban particulate matter (PM) exposure. METHODS In this study we (1) identified NLRP3 and caspase-1 expression in human airway epithelium bronchus and primary cells, (2) characterized NLRP3 inflammasome-mediated IL-1β production from human airway epithelium in response to PM, and (3) performed in vivo PM exposure experiments with wild-type and Nlrp3(-/-) mice. RESULTS Our results demonstrate that human airway epithelium contains a functional NLRP3 inflammasome that responds to PM exposure with caspase-1 cleavage and production of IL-1β. Exposure of Nlrp3(-/-) and wild-type mice to PM in vivo demonstrates NLRP3-dependent production of IL-1β in the lung, airway neutrophilia, and increases in CD11c(+hi)/MHC class II(+hi) cell numbers in intrathoracic lymph nodes. CONCLUSION Our study is the first to characterize airway epithelial NLRP3 inflammasome-mediated immune responses to PM exposure, which might have implications in patients with asthma and other lung diseases.

Airway smooth muscle in asthma: phenotype plasticity and function.

Clinical asthma is characterized by reversible airway obstruction which is commonly due to an exaggerated airway narrowing referred to as airway hyperresponsiveness (AHR). Although debate exists on the complex etiology of AHR, it is clear that airway smooth muscle (ASM) mediated airway narrowing is a major contributor to airway dysfunction. More importantly, it is now appreciated that smooth muscle is far from being a simple cell with only contractile ability properties. Rather, it is more versatile with the capacity to exhibit numerous cellular functions as it adapts to the microenvironment to which it is exposed. The emerging ability of individual smooth muscle cells to undergo changes in their phenotype (phenotype plasticity) and function (functional plasticity) in response to physiological and pathological cues is an important and active area of research. This article provides a brief review of the current knowledge and emerging concepts in the field of ASM phenotype and function both under healthy and asthmatic conditions.

Diesel exhaust augments allergen-induced lower airway inflammation in allergic individuals: a controlled human exposure study

Rationale Traffic-related air pollution has been shown to augment allergy and airway disease. However, the enhancement of allergenic effects by diesel exhaust in particular is unproven in vivo in the human lung, and underlying details of this apparent synergy are poorly understood. Objective To test the hypothesis that a 2 h inhalation of diesel exhaust augments lower airway inflammation and immune cell activation following segmental allergen challenge in atopic subjects. Methods 18 blinded atopic volunteers were exposed to filtered air or 300 µg PM2.5/m3 of diesel exhaust in random fashion. 1 h post-exposure, diluent-controlled segmental allergen challenge was performed; 2 days later, samples from the challenged segments were obtained by bronchoscopic lavage. Samples were analysed for markers and modifiers of allergic inflammation (eosinophils, Th2 cytokines) and adaptive immune cell activation. Mixed effects models with ordinal contrasts compared effects of single and combined exposures on these end points. Results Diesel exhaust augmented the allergen-induced increase in airway eosinophils, interleukin 5 (IL-5) and eosinophil cationic protein (ECP) and the GSTT1 null genotype was significantly associated with the augmented IL-5 response. Diesel exhaust alone also augmented markers of non-allergic inflammation and monocyte chemotactic protein (MCP)-1 and suppressed activity of macrophages and myeloid dendritic cells. Conclusion Inhalation of diesel exhaust at environmentally relevant concentrations augments allergen-induced allergic inflammation in the lower airways of atopic individuals and the GSTT1 genotype enhances this response. Allergic individuals are a susceptible population to the deleterious airway effects of diesel exhaust. Trial registration number NCT01792232.

Regional differences in the pattern of airway remodeling following chronic allergen exposure in mice

BackgroundAirway remodeling present in the large airways in asthma or asthma models has been associated with airway dysfunction in humans and mice. It is not clear if airways distal to the large conducting airways have similar degrees of airway remodeling following chronic allergen exposure in mice. Our objective was to test the hypothesis that airway remodeling is heterogeneous by optimizing a morphometric technique for distal airways and applying this to mice following chronic exposure to allergen or saline.MethodsIn this study, BALB/c mice were chronically exposed to intranasal allergen or saline. Lung sections were stained for smooth muscle, collagen, and fibronectin content. Airway morphometric analysis of small (0–50000 μm2), medium (50000 μm2–175000 μm2) and large (>175000 μm2) airways was based on quantifying the area of positive stain in several defined sub-epithelial regions of interest. Optimization of this technique was based on calculating sample sizes required to detect differences between allergen and saline exposed animals.ResultsFollowing chronic allergen exposure BALB/c mice demonstrate sustained airway hyperresponsiveness. BALB/c mice demonstrate an allergen-induced increase in smooth muscle content throughout all generations of airways, whereas changes in subepithelial collagen and fibronectin content are absent from distal airways.ConclusionWe demonstrate for the first time, a systematic objective analysis of allergen induced airway remodeling throughout the tracheobronchial tree in mice. Following chronic allergen exposure, at the time of sustained airway dysfunction, BALB/c mice demonstrate regional differences in the pattern of remodeling. Therefore results obtained from limited regions of lung should not be considered representative of the entire airway tree.

The nucleotide-binding domain, leucine-rich repeat protein 3 inflammasome/IL-1 receptor I axis mediates innate, but not adaptive, immune responses after exposure to particulate matter under 10 μm.

Exposure to particulate matter (PM), a major component of air pollution, contributes to increased morbidity and mortality worldwide. Inhaled PM induces innate immune responses by airway epithelial cells that may lead to the exacerbation or de novo development of airway disease. We have previously shown that 10-μm PM (PM10) activates the nucleotide-binding domain, leucine-rich repeat protein (NLRP) 3 inflammasome in human airway epithelial cells. Our objective was to determine the innate and adaptive immune responses mediated by the airway epithelium NLRP3 inflammasome in response to PM10 exposure. Using in vitro cultures of human airway epithelial cells and in vivo studies with wild-type and Nlrp3(-/-) mice, we investigated the downstream consequences of PM10-induced NLPR3 inflammasome activation on cytokine production, cellular inflammation, dendritic cell activation, and PM10-facilitated allergic sensitization. PM10 activates an NLRP3 inflammasome/IL-1 receptor I (IL-1RI) axis in airway epithelial cells, resulting in IL-1β, CC chemokine ligand-20, and granulocyte/macrophage colony-stimulating factor production, which is associated with dendritic cell activation and lung neutrophilia. Despite these profound innate immune responses in the airway epithelium, the NLRP3 inflammasome/IL-1RI axis is dispensable for PM10-facilitated allergic sensitization. We demonstrate the importance of the lung NLRP3 inflammasome in mediating PM10 exposure-associated innate, but not adaptive, immune responses. Our study highlights a mechanism by which PM10 exposure can contribute to the exacerbation of airway disease, but not PM10-facilitated allergic sensitization.

Role for NLRP3 Inflammasome-mediated, IL-1β-dependent Responses in Severe, Steroid-resistant Asthma.

&NA; Rationale: Severe, steroid‐resistant asthma is the major unmet need in asthma therapy. Disease heterogeneity and poor understanding of pathogenic mechanisms hampers the identification of therapeutic targets. Excessive nucleotide‐binding oligomerization domain‐like receptor family, pyrin domain‐containing 3 (NLRP3) inflammasome and concomitant IL‐1&bgr; responses occur in chronic obstructive pulmonary disease, respiratory infections, and neutrophilic asthma. However, the direct contributions to pathogenesis, mechanisms involved, and potential for therapeutic targeting remain poorly understood, and are unknown in severe, steroid‐resistant asthma. Objectives: To investigate the roles and therapeutic targeting of the NLRP3 inflammasome and IL‐1&bgr; in severe, steroid‐resistant asthma. Methods: We developed mouse models of Chlamydia and Haemophilus respiratory infection‐mediated, ovalbumin‐induced severe, steroid‐resistant allergic airway disease. These models share the hallmark features of human disease, including elevated airway neutrophils, and NLRP3 inflammasome and IL‐1&bgr; responses. The roles and potential for targeting of NLRP3 inflammasome, caspase‐1, and IL‐1&bgr; responses in experimental severe, steroid‐resistant asthma were examined using a highly selective NLRP3 inhibitor, MCC950; the specific caspase‐1 inhibitor Ac‐YVAD‐cho; and neutralizing anti‐IL‐1&bgr; antibody. Roles for IL‐1&bgr;‐induced neutrophilic inflammation were examined using IL‐1&bgr; and anti‐Ly6G. Measurements and Main Results: Chlamydia and Haemophilus infections increase NLRP3, caspase‐1, IL‐1&bgr; responses that drive steroid‐resistant neutrophilic inflammation and airway hyperresponsiveness. Neutrophilic airway inflammation, disease severity, and steroid resistance in human asthma correlate with NLRP3 and IL‐1&bgr; expression. Treatment with anti‐IL‐1&bgr;, Ac‐YVAD‐cho, and MCC950 suppressed IL‐1&bgr; responses and the important steroid‐resistant features of disease in mice, whereas IL‐1&bgr; administration recapitulated these features. Neutrophil depletion suppressed IL‐1&bgr;‐induced steroid‐resistant airway hyperresponsiveness. Conclusions: NLRP3 inflammasome responses drive experimental severe, steroid‐resistant asthma and are potential therapeutic targets in this disease.

In Vivo Role of Platelet-Derived Growth Factor–BB in Airway Smooth Muscle Proliferation in Mouse Lung

Airway smooth muscle (ASM) hyperplasia in asthma likely contributes considerably to functional changes. Investigating the mechanisms behind proliferation of these cells may lead to therapeutic benefit. Platelet-derived growth factor (PDGF)-BB is a well known ASM mitogen in vitro but has yet to be directly explored using in vivo mouse models in the context of ASM proliferation and airway responsiveness. To determine the in vivo influence of PDGF-BB on gene transcripts encoding contractile proteins, ASM proliferation, and airway physiology, we used an adenovirus overexpression system and a model of chronic allergen exposure. We used adenovirus technology to selectively overexpress PDGF-BB in the airway epithelium of mice. Outcome measurements, including airway physiology, real time RT-PCR measurements, proliferating cell nuclear antigen staining, and airway smooth muscle quantification, were performed 7 days after exposure. The same outcome measurements were performed 24 hours and 4 weeks after a chronic allergen exposure model. PDGF-BB overexpression resulted in airway hyperresponsiveness, decreased lung compliance, increased airway smooth muscle cell numbers, positive proliferating cell nuclear antigen-stained airway smooth muscle cells, and a reduction in genes encoding contractile proteins. Chronic allergen exposure resulted in elevations in lung lavage PDGF-BB, which were observed in conjunction with changes in gene transcript expression encoding contractile proteins and ASM proliferation. We demonstrate for the first time in vivo that PDGF-BB induces ASM hyperplasia and changes in lung mechanics in mice and that, during periods of allergen exposure changes in lung, PDGF-BB are associated with changes in airway structure and function.

Human airway epithelial cell innate immunity: relevance to asthma.

The innate immunity function of the human airway epithelium is responsible for orchestrating defence against inhaled viruses, bacteria, fungi, allergens, pollution, and other environmental insults. Epithelial cells present a mechanically tight, pseudostratified, multi-cell barrier that secretes mucus, surfactants, and anti-microbial peptides to manage minor insults. Secondary to the mechanical impedances, cell surface and cytoplasmic pattern recognition receptors await detection of more aggressive insults. The differentiation state of the airway epithelium contributes to innate immunity by compartmentalizing receptors and mediator production. Activation of innate immune receptors triggers production of interferons, cytokines, and chemokines, which influence adaptive immune responses. Mounting evidence suggests that these responses are aberrant in asthma and may contribute to disease progression and exacerbations. In this review, we discuss the recent evidence supporting these statements, focusing primarily on data generated from using human samples.