A pilot study to assess the effects of preventing fluid retention in the legs by wearing compression stockings on overnight airway narrowing in mild asthma.

PURPOSE: Nocturnal asthma is a sign of asthma worsening and could be partially due to more fluid drawn into the thorax during sleep by gravitational force and/or pharyngeal collapse in those with obstructive sleep apnea. Wearing compression stockings during the day reduces fluid shift from the legs to the neck overnight. However, the potential effect of wearing compression stockings to reduce fluid accumulation in the leg and to improve nocturnal small airway narrowing in patients with asthma has not been investigated. This study investigates whether reducing leg fluid volume by wearing compression stockings during the day would attenuate small airway narrowing in patients with asthma before and after sleep. METHODS: We enrolled 11 participants with asthma. All participants underwent overnight polysomnography with or without wearing compression stockings for 2 weeks. Before and after sleep, leg fluid volume (LFV) was measured by bioelectrical impedance, and airway narrowing was primarily assessed by respiratory system resistance and reactance at 5 Hz (R5 and X5 respectively) using oscillometry. RESULTS: After 2 weeks of wearing compression stockings, the LFV measured in the evening was reduced (∆ = - 192.6 ± 248.3 ml, p = 0.02), and R5 and X5 improved (∆ = - 0.7 ± 0.9 cmH2O/L/s, p = 0.03 and 0.2 ± 1.4 cmH2O/L/s, p = 0.05 respectively). No changes were observed in the morning. CONCLUSIONS: Preventing fluid retention in the legs by wearing compression stockings for 2 weeks during the day, reduced LFV and airway narrowing in the evening in all participants with asthma, but not in the morning after sleep.

Potentiation of long-acting ß2-agonist and glucocorticoid responses in human airway epithelial cells by modulation of intracellular cAMP.

INTRODUCTION: Over 300 million people in the world live with asthma, resulting in 500,000 annual global deaths with future increases expected. It is estimated that around 50-80% of asthma exacerbations are due to viral infections. Currently, a combination of long-acting beta agonists (LABA) for bronchodilation and glucocorticoids (GCS) to control lung inflammation represent the dominant strategy for the management of asthma, however, it is still sub-optimal in 35-50% of moderate-severe asthmatics resulting in persistent lung inflammation, impairment of lung function, and risk of mortality. Mechanistically, LABA/GCS combination therapy results in synergistic efficacy mediated by intracellular cyclic adenosine monophosphate (cAMP). HYPOTHESIS: Increasing intracellular cAMP during LABA/GCS combination therapy via inhibiting phosphodiesterase 4 (PDE4) and/or blocking the export of cAMP by ATP Binding Cassette Transporter C4 (ABCC4), will potentiate anti-inflammatory responses of mainstay LABA/GCS therapy. METHODS: Expression and localization experiments were performed using in situ hybridization and immunohistochemistry in human lung tissue from healthy subjects, while confirmatory transcript and protein expression analyses were performed in primary human airway epithelial cells and cell lines. Intervention experiments were performed on the human airway epithelial cell line, HBEC-6KT, by pre-treatment with combinations of LABA/GCS with PDE4 and/or ABCC4 inhibitors followed by Poly I:C or imiquimod challenge as a model for viral stimuli. Cytokine readouts for IL-6, IL-8, CXCL10/IP-10, and CCL5/RANTES were quantified by ELISA. RESULTS: Using archived human lung and human airway epithelial cells, ABCC4 gene and protein expression were confirmed in vitro and in situ. LABA/GCS attenuation of Poly I:C or imiquimod-induced IL-6 and IL-8 were potentiated with ABCC4 and PDE4 inhibition, which was greater when ABCC4 and PDE4 inhibition was combined. Modulation of cAMP levels had no impact on LABA/GCS modulation of Poly I:C-induced CXCL10/IP-10 or CCL5/RANTES. CONCLUSION: Modulation of intracellular cAMP levels by PDE4 or ABCC4 inhibition potentiates LABA/GCS efficacy in human airway epithelial cells challenged with viral stimuli. The data suggest further exploration of the value of adding cAMP modulators to mainstay LABA/GCS therapy in asthma for potentiated anti-inflammatory efficacy.

Induction of bystander tolerance and immune deviation after Fel d 1 peptide immunotherapy.

BACKGROUND: Treatment of patients with cat allergy with peptides derived from Fel d 1 (the major cat allergen) ameliorated symptoms of cat allergy in phase 2 clinical trials. OBJECTIVE: We sought to demonstrate that the tolerance induced by Fel d 1 peptide immunotherapy can be exploited to reduce allergic responses to a second allergen, ovalbumin (OVA), in mice sensitized dually to OVA and Fel d 1. METHODS: Induction of tolerance to OVA was achieved through simultaneous exposure to both allergens after peptide treatment. Functional tolerance to each allergen was assessed in a model of allergic airways disease in which treated mice were protected from eosinophilia, goblet cell hyperplasia, and TH2 cell infiltration. RESULTS: Suppression of allergic responses to cat allergen challenge was associated with significant increases in numbers of CD4+CD25+Foxp3+ T cells, IL-10+ cells, and CD19+IL-10+ B cells, whereas the response to OVA was associated with a marked reduction in numbers of TH2 cytokine-secreting T cells and less prominent changes in outcomes associated with immune regulation. CONCLUSIONS: These observations suggest that immune tolerance induced by peptide immunotherapy can be used experimentally to treat an allergic response to another allergen and that the molecular mechanisms underlying induction of tolerance to a treatment-specific allergen and a bystander allergen might be different.

GRP78 and CHOP modulate macrophage apoptosis and the development of bleomycin-induced pulmonary fibrosis.

Endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) have been associated with fibrotic lung disease, although exactly how they modulate this process remains unclear. Here we investigated the role of GRP78, the main UPR regulator, in an experimental model of lung injury and fibrosis. Grp78(+/-) , Chop(-/-) and wild type C57BL6/J mice were exposed to bleomycin by oropharyngeal intubation and lungs were examined at days 7 and 21. We demonstrate here that Grp78(+/-) mice were strongly protected from bleomycin-induced fibrosis, as shown by immunohistochemical analysis, collagen content and lung function measurements. In the inflammatory phase of this model, a reduced number of lung macrophages associated with an increased number of TUNEL-positive cells were observed in Grp78(+/-) mice. Dual immunohistochemical and in situ hybridization experiments showed that the macrophage population from the protected Grp78(+/-) mice was also strongly positive for cleaved caspase-3 and Chop mRNA, respectively. In contrast, the administration of bleomycin to Chop(-/-) mice resulted in increased quasi-static elastance and extracellular matrix deposition associated with an increased number of parenchymal arginase-1-positive macrophages that were negative for cleaved caspase-3. The data presented indicate that the UPR is activated in fibrotic lung tissue and strongly localized to macrophages. GRP78- and CHOP-mediated macrophage apoptosis was found to protect against bleomycin-induced fibrosis. Overall, we demonstrate here that the fibrotic response to bleomycin is dependent on GRP78-mediated events and provides evidence that macrophage polarization and apoptosis may play a role in this process. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

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

Gamma-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.

A new short-term mouse model of chronic obstructive pulmonary disease identifies a role for mast cell tryptase in pathogenesis.

BACKGROUND: Cigarette smoke-induced chronic obstructive pulmonary disease (COPD) is a life-threatening inflammatory disorder of the lung. The development of effective therapies for COPD has been hampered by the lack of an animal model that mimics the human disease in a short timeframe. OBJECTIVES: We sought to create an early-onset mouse model of cigarette smoke-induced COPD that develops the hallmark features of the human condition in a short time-frame. We also sought to use this model to better understand pathogenesis and the roles of macrophages and mast cells (MCs) in patients with COPD. METHODS: Tightly controlled amounts of cigarette smoke were delivered to the airways of mice, and the development of the pathologic features of COPD was assessed. The roles of macrophages and MC tryptase in pathogenesis were evaluated by using depletion and in vitro studies and MC protease 6-deficient mice. RESULTS: After just 8 weeks of smoke exposure, wild-type mice had chronic inflammation, mucus hypersecretion, airway remodeling, emphysema, and reduced lung function. These characteristic features of COPD were glucocorticoid resistant and did not spontaneously resolve. Systemic effects on skeletal muscle and the heart and increased susceptibility to respiratory tract infections also were observed. Macrophages and tryptase-expressing MCs were required for the development of COPD. Recombinant MC tryptase induced proinflammatory responses from cultured macrophages. CONCLUSION: A short-term mouse model of cigarette smoke-induced COPD was developed in which the characteristic features of the disease were induced more rapidly than in existing models. The model can be used to better understand COPD pathogenesis, and we show a requirement for macrophages and tryptase-expressing MCs.

Increased ornithine-derived polyamines cause airway hyperresponsiveness in a mouse model of asthma.

Up-regulation of arginase contributes to airways hyperresponsiveness (AHR) in asthma by reducing L-arginine bioavailability for the nitric oxide (NO) synthase isozymes. The product of arginase activity, L-ornithine, can be metabolized into polyamines by ornithine decarboxylase. We tested the hypothesis that increases in L-ornithine-derived polyamines contribute to AHR in mouse models of allergic airways inflammation. After measuring significantly increased polyamine levels in sputum samples from human subjects with asthma after allergen challenge, we used acute and subacute ovalbumin sensitization and challenge mouse models of allergic airways inflammation and naive mice to investigate the relationship of AHR to methacholine and polyamines in the lung. We found that spermine levels were elevated significantly in lungs from the acute model, which exhibits robust AHR, but not in the subacute murine model of asthma, which does not develop AHR. Intratracheal administration of spermine significantly augmented airways responsiveness to methacholine in both naive mice and mice with subacute airways inflammation, and reduced nitrite/nitrate levels in lung homogenates, suggesting that the AHR developed as a consequence of inhibition of constitutive NO production in the airways. Chronic inhibition of polyamine synthesis using an ornithine decarboxylase inhibitor significantly reduced polyamine levels, restored nitrite/nitrate levels to normal, and abrogated the AHR to methacholine in the acute model of allergic airways inflammation. We demonstrate that spermine increases airways responsiveness to methacholine, likely through inhibition of constitutive NO synthesis. Thus, inhibition of polyamine production may represent a new therapeutic target to treat airway obstruction in allergic asthma.

Mepolizumab for prednisone-dependent asthma with sputum eosinophilia.

BACKGROUND: Eosinophilic inflammation, which may be a consequence of interleukin-5 action, is a characteristic feature of some forms of asthma. However, in three previous clinical trials involving patients with asthma, blockade of this cytokine did not result in a significant improvement in outcomes. We studied the prednisone-sparing effect of mepolizumab, a monoclonal antibody against interleukin-5, in a rare subgroup of patients who have sputum eosinophilia and airway symptoms despite continued treatment with prednisone. Secondary objectives were to examine its effect on the number of eosinophils in sputum and blood, symptoms, and airflow limitation. METHODS: In this randomized, double-blind, parallel-group trial involving patients with persistent sputum eosinophilia and symptoms despite prednisone treatment, we assigned 9 patients to receive mepolizumab (administered in five monthly infusions of 750 mg each) and 11 patients to receive placebo. RESULTS: There were 12 asthma exacerbations in 10 patients who received placebo, 9 of whom had sputum eosinophilia at the time of exacerbation. In comparison, only one patient who received mepolizumab had an asthma exacerbation, and this episode was not associated with sputum eosinophilia (P=0.002). Patients who received mepolizumab were able to reduce their prednisone dose by a mean (+/-SD) of 83.8+/-33.4% of their maximum possible dose, as compared with 47.7+/-40.5% in the placebo group (P=0.04). The use of mepolizumab was associated with a significant decrease in the number of sputum and blood eosinophils. Improvements in eosinophil numbers, asthma control, and forced expiratory volume in 1 second were maintained for 8 weeks after the last infusion. There were no serious adverse events. CONCLUSIONS: Mepolizumab reduced the number of blood and sputum eosinophils and allowed prednisone sparing in patients who had asthma with sputum eosinophilia despite prednisone treatment. (ClinicalTrials.gov number, NCT00292877.)

Modeling asthma in mice: what have we learned about the airway epithelium?

Clinical reports of areas of damaged airway epithelium associated with shed epithelial cells in bronchoalveolar lavage fluid, aberrant epithelial repair processes, and altered cytokine and growth factor release have highlighted some fundamental differences between the airway epithelium in individuals with and without asthma. However, the consequences of these epithelial changes are not clearly defined, and may be difficult to assess in the clinic. In this Review, we answer the two questions. (1) What in vivo models and methods have been used to inform us about airway epithelium damage, repair, and immune responses? Our response focuses on genetic influences as well as allergen exposure, environmental/chemical, and mechanical models. (2) How can we improve on existing mouse models to understand changes in airway epithelium biology in asthma? In answering the second question, we include exciting recent studies that have combined multiple exposure methods and/or epithelium-centric outcome measurements. By addressing these two questions, we propose that future interrogation of epithelial responses of both existing and nascent mouse models may provide greater understanding of the mechanisms underlying airway inflammation and remodeling in asthma with hope of generating novel therapeutic targets.