Toby K. McGovern

Time course of airway remodelling after an acute chlorine gas exposure in mice

Accidental chlorine (Cl2) gas inhalation is a common cause of acute airway injury. However, little is known about the kinetics of airway injury and repair after Cl2 exposure. We investigated the time course of airway epithelial damage and repair in mice after a single exposure to a high concentration of Cl2 gas. Mice were exposed to 800 ppm Cl2 gas for 5 minutes and studied from 12 hrs to 10 days post-exposure. The acute injury phase after Cl2 exposure (≤ 24 hrs post-exposure) was characterized by airway epithelial cell apoptosis (increased TUNEL staining) and sloughing, elevated protein in bronchoalveolar lavage fluid, and a modest increase in airway responses to methacholine. The repair phase after Cl2 exposure was characterized by increased airway epithelial cell proliferation, measured by immunoreactive proliferating cell nuclear antigen (PCNA), with maximal proliferation occurring 5 days after Cl2 exposure. At 10 days after Cl2 exposure the airway smooth muscle mass was increased relative to controls, suggestive of airway smooth muscle hyperplasia and there was evidence of airway fibrosis. No increase in goblet cells occurred at any time point. We conclude that a single exposure of mice to Cl2 gas causes acute changes in lung function, including pulmonary responsiveness to methacholine challenge, associated with airway damage, followed by subsequent repair and airway remodelling.

Histamine may induce airway remodeling through release of epidermal growth factor receptor ligands from bronchial epithelial cells.

Asthma is a chronic inflammatory disease that is associated with airway remodeling, including hyperplasia of airway epithelial cells and airway smooth muscle cells, and goblet cell differentiation. We wished to address the potential role of histamine, a key biogenic amine involved in allergic reactions, in airway remodeling through the epidermal growth factor receptor (EGFR) pathway. Here, we demonstrate that histamine releases 2 EGFR ligands, amphiregulin and heparin‐binding epidermal growth factor‐like growth factor (HB‐EGF), from airway epithelial cells. Amphiregulin and HB‐EGF were expressed in airway epithelium of patients with asthma. Histamine up‐regulated their mRNA expression (amphiregulin 3.2‐fold, P< 0.001; HB‐EGF 2.3‐fold, P<0.05) and triggered their release (amphiregulin EC50 0.50 μM, 31.2±2.7 pg/ml with 10 μM histamine, P<0.01; HB‐EGF EC50 0.54 μM, 78.5±1.8 pg/ml with 10 μM histamine, P<0.001) compared to vehicle control (amphiregulin 19.3±0.9 pg/ ml; HB‐EGF 60.2± 1.0 pg/ml), in airway epithelial cells. Histamine increased EGFR phosphorylation (2.1‐fold by Western blot analysis) and induced goblet cell differentiation (CLCA1 up‐regulation by real‐time qPCR) in normal human bronchial epithelial (NHBE) cells. Moreover, amphiregulin and HB‐EGF caused proliferation and migration of both NHBE cells and human airway smooth muscle cells. These results suggest that histamine may induce airway remodeling via the epithelial‐derived EGFR ligands amphiregulin and HB‐EGF.—Hirota, N., Risse, P.‐A., Novali, M., McGovern, T., Al‐Alwan, L., McCuaig, S., Proud, D., Hayden, P., Hamid, Q., Martin, J. G. Histamine may induce airway remodeling through release of epidermal growth factor receptor ligands from bronchial epithelial cells. FASEB J. 26, 1704‐1716 (2012). www.fasebj.org

AEOL10150: a novel therapeutic for rescue treatment after toxic gas lung injury.

New therapeutics designed as rescue treatments after toxic gas injury such as from chlorine (Cl(2)) are an emerging area of interest. We tested the effects of the metalloporphyrin catalytic antioxidant AEOL10150, a compound that scavenges peroxynitrite, inhibits lipid peroxidation, and has SOD and catalase-like activities, on Cl(2)-induced airway injury. Balb/C mice received 100ppm Cl(2) gas for 5 min. Four groups were studied: Cl(2) only, Cl(2) followed by AEOL10150 1 and 9 h after exposure, AEOL10150 only, and control. Twenty-four hours after Cl(2) gas exposure airway responsiveness to aerosolized methacholine (6.25-50mg/ml) was measured using a small-animal ventilator. Bronchoalveolar lavage (BAL) was performed to assess airway inflammation and protein. Whole lung tissue was assayed for 4-hydroxynonenal. In separate groups, lungs were collected at 72 h after Cl(2) injury to evaluate epithelial cell proliferation. Mice exposed to Cl(2) showed a significantly higher airway resistance compared to control, Cl(2)/AEOL10150, or AEOL10150-only treated animals in response to methacholine challenge. Eosinophils, neutrophils, and macrophages were elevated in BAL of Cl(2)-exposed mice. AEOL10150 attenuated the increases in neutrophils and macrophages. AEOL10150 prevented Cl(2)-induced increase in BAL fluid protein. Chlorine induced an increase in the number of proliferating airway epithelial cells, an effect AEOL10150 attenuated. 4-Hydroxynonenal levels in the lung were increased after Cl(2) and this effect was prevented with AEOL10150. AEOL10150 is an effective rescue treatment for Cl(2)-induced airway hyperresponsiveness, airway inflammation, injury-induced airway epithelial cell regeneration, and oxidative stress.

Dimethylthiourea protects against chlorine induced changes in airway function in a murine model of irritant induced asthma.

BackgroundExposure to chlorine (Cl2) causes airway injury, characterized by oxidative damage, an influx of inflammatory cells and airway hyperresponsiveness. We hypothesized that Cl2-induced airway injury may be attenuated by antioxidant treatment, even after the initial injury.MethodsBalb/C mice were exposed to Cl2 gas (100 ppm) for 5 mins, an exposure that was established to alter airway function with minimal histological disruption of the epithelium. Twenty-four hours after exposure to Cl2, airway responsiveness to aerosolized methacholine (MCh) was measured. Bronchoalveolar lavage (BAL) was performed to determine inflammatory cell profiles, total protein, and glutathione levels. Dimethylthiourea (DMTU;100 mg/kg) was administered one hour before or one hour following Cl2 exposure.ResultsMice exposed to Cl2 had airway hyperresponsiveness to MCh compared to control animals pre-treated and post-treated with DMTU. Total cell counts in BAL fluid were elevated by Cl2 exposure and were not affected by DMTU treatment. However, DMTU-treated mice had lower protein levels in the BAL than the Cl2-only treated animals. 4-Hydroxynonenal analysis showed that DMTU given pre- or post-Cl2 prevented lipid peroxidation in the lung. Following Cl2 exposure glutathione (GSH) was elevated immediately following exposure both in BAL cells and in fluid and this change was prevented by DMTU. GSSG was depleted in Cl2 exposed mice at later time points. However, the GSH/GSSG ratio remained high in chlorine exposed mice, an effect attenuated by DMTU.ConclusionOur data show that the anti-oxidant DMTU is effective in attenuating Cl2 induced increase in airway responsiveness, inflammation and biomarkers of oxidative stress.

LTD4 induces HB-EGF-dependent CXCL8 release through EGFR activation in human bronchial epithelial cells

Airway epithelial cells release proinflammatory mediators that may contribute to airway remodeling and leukocyte recruitment. We explored the hypothesis that leukotriene D₄ (LTD₄) may trigger the release of proremodeling factors through activation of the EGF receptor (EGFR). We particularly focused on the effects of LTD₄ on release of heparin-binding EGF-like factor (HB-EGF) and IL-8 (CXCL8), a potent neutrophil chemoattractant that may be released downstream of EGFR activation. To address this hypothesis, both primary (NHBE) and transformed bronchial human epithelial cells (BEAS-2B) were grown on an air-liquid interface and stimulated with LTD₄. HB-EGF and CXCL8 were evaluated by ELISA in cell culture supernatants. To explore the EGFR signaling pathway, we used a broad-spectrum matrix metalloproteinase (MMP) inhibitor, GM-6001, two selective EGFR tyrosine kinase inhibitors, AG-1478 and PD-153035, an HB-EGF neutralizing antibody, and a specific small interfering RNA (siRNA) against the EGFR. Expression of the CysLT₁ cysteinyl leukotriene receptor was demonstrated by RT-PCR and immunocytochemistry in both BEAS-2B and NHBE cells. Four hours after stimulation with LTD₄, HB-EGF and CXCL8 were significantly increased in cell culture supernatant. GM-6001 and montelukast, a specific CysLT₁ receptor antagonist, blocked the LTD₄-induced increase in HB-EGF. All inhibitors/antagonists decreased LTD₄-induced CXCL8 release. siRNA against EGFR abrogated CXCL8 release following stimulation with LTD₄ and exogenous HB-EGF. These findings suggest LTD₄ induced EGFR transactivation through the release of HB-EGF in human bronchial epithelial cells with downstream release of CXCL8. These effects may contribute to epithelial-mediated airway remodeling in asthma and other conditions associated with cysteinyl leukotriene release.

Insights into asthmatic airway remodelling through murine models

Asthma is a chronic disorder of the airways associated in many instances with structural changes of the airways, termed airway remodelling. Irritant and allergen‐induced murine models have been used to further understand the mechanisms of airway remodelling. The infiltration of the airways by inflammatory cells, such as T lymphocytes, mast cells, eosinophils, neutrophils and macrophages after repeated allergen challenges may be important effectors in the initiation and perpetuation of airway remodelling through the release of inflammatory mediators and growth factors. Interleukins‐4 and ‐13 have been widely studied in experimental models, and have been shown to play a significant role in airway remodelling. Recently, a role for Th17 cells has been established. Other mediators involved in this process are ligands of the epidermal growth factor receptor, matrix metalloproteases and cysteinyl leukotrienes. A better understanding of the mechanisms leading to airway remodelling in allergic diseases may lead to the identification of novel therapeutic strategies but validation in human subjects is required for potential targets.

Evaluation of respiratory system mechanics in mice using the forced oscillation technique.

The forced oscillation technique (FOT) is a powerful, integrative and translational tool permitting the experimental assessment of lung function in mice in a comprehensive, detailed, precise and reproducible manner. It provides measurements of respiratory system mechanics through the analysis of pressure and volume signals acquired in reaction to predefined, small amplitude, oscillatory airflow waveforms, which are typically applied at the subjects airway opening. The present protocol details the steps required to adequately execute forced oscillation measurements in mice using a computer-controlled piston ventilator (flexiVent; SCIREQ Inc, Montreal, Qc, Canada). The description is divided into four parts: preparatory steps, mechanical ventilation, lung function measurements, and data analysis. It also includes details of how to assess airway responsiveness to inhaled methacholine in anesthetized mice, a common application of this technique which also extends to other outcomes and various lung pathologies. Measurements obtained in naïve mice as well as from an oxidative-stress driven model of airway damage are presented to illustrate how this tool can contribute to a better characterization and understanding of studied physiological changes or disease models as well as to applications in new research areas.

Neutrophils Mediate Airway Hyperresponsiveness after Chlorine-Induced Airway Injury in the Mouse

Chlorine gas (Cl2) inhalation causes oxidative stress, airway epithelial damage, airway hyperresponsiveness (AHR), and neutrophilia. We evaluated the effect of neutrophil depletion on Cl2-induced AHR and its effect on the endogenous antioxidant response, and if eosinophils or macrophages influence Cl2-induced AHR. We exposed male Balb/C mice to 100 ppm Cl2 for 5 minutes. We quantified inflammatory cell populations in bronchoalveolar lavage (BAL), the antioxidant response in lung tissue by quantitative PCR, and nuclear factor (erythroid-derived 2)-like 2 (NRF2) nuclear translocation by immunofluorescence. In vitro, NRF2 nuclear translocation in response to exogenous hypochlorite was assessed using a luciferase assay. Anti-granulocyte receptor-1 antibody or anti-Ly6G was used to deplete neutrophils. The effects of neutrophil depletion on IL-13 and IL-17 were measured by ELISA. Eosinophils and macrophages were depleted using TRFK5 or clodronate-loaded liposomes, respectively. AHR was evaluated with the constant-phase model in response to inhaled aerosolized methacholine. Our results show that Cl2 exposure induced neutrophilia and increased expression of NRF2 mRNA, superoxide dismutase-1, and heme-oxygenase 1. Neutrophil depletion abolished Cl2-induced AHR in large conducting airways and prevented increases in antioxidant gene expression and NRF2 nuclear translocation. Exogenous hypochlorite administration resulted in increased NRF2 nuclear translocation in vitro. After Cl2 exposure, neutrophils occupied 22 ± 7% of the luminal space in large airways. IL-17 in BAL was increased after Cl2, although this effect was not prevented by neutrophil depletion. Neither depletion of eosinophils nor macrophages prevented Cl2-induced AHR. Our data suggest the ability of neutrophils to promote Cl2-induced AHR is dependent on increases in oxidative stress and occupation of luminal space in large airways.

The modulation of large airway smooth muscle phenotype and effects of epidermal growth factor receptor inhibition in the repeatedly allergen-challenged rat

Allergen challenges induce airway hyperresponsiveness (AHR) and increased airway smooth muscle (ASM) mass in the sensitized rat. Whether the remodeled ASM changes its phenotype is uncertain. We examined, in sensitized Brown Norway rats, the effects of multiple ovalbumin (Ova) challenges on ASM remodeling and phenotype and the role of the epidermal growth factor receptor (EGFR) in these processes. Rats were sensitized with Ova and challenged three times at 5-day intervals with phosphate-buffered saline or Ova and pretreated with the EGFR inhibitor AG-1478 (5 mg/kg) or its vehicle dimethyl sulfoxide. Ova challenges increased ASM mass in all-sized airways and in large airway mRNA expression of smooth muscle myosin heavy chain (sm-MHC), assessed by laser capture. Myosin light chain kinase and the fast myosin isoform SM-B mRNA expressions were not affected. Ova induced AHR to methacholine, and, based on the constant-phase model, this was largely attributable to the small airways and lung derecruitment at 48 h that recovered by 1 wk. The EGFR ligands amphiregulin and heparin-binding epidermal growth factor (HB-EGF) were increased in bronchoalveolar lavage fluid at 48 h after Ova exposure. AG-1478 inhibited AHR and prevented ASM growth. Epithelial gene expression of EGFR, HB-EGF, matrix metalloproteinase (MMP)-9, Gro-α, and transforming growth factor-β was unaffected by Ova challenges. We conclude that EGFR drives remodeling of ASM, which results from repeated Ova challenge. Furthermore, the latter results in excessive small airway and, to a lesser degree, large airway narrowing to methacholine, and large airway gene expression of contractile protein is conserved.

Evolution of the Immune Response to Chronic Airway Colonization with Aspergillus fumigatus Hyphae

ABSTRACT Airway colonization by the mold Aspergillus fumigatus is common in patients with underlying lung disease and is associated with chronic airway inflammation. Studies probing the inflammatory response to colonization with A. fumigatus hyphae have been hampered by the lack of a model of chronic colonization in immunocompetent mice. By infecting mice intratracheally with conidia embedded in agar beads (Af beads), we have established an in vivo model to study the natural history of airway colonization with live A. fumigatus hyphae. Histopathological examination and galactomannan assay of lung homogenates demonstrated that hyphae exited beads and persisted in the lungs of mice up to 28 days postinfection without invasive disease. Fungal lesions within the airways were surrounded by a robust neutrophilic inflammatory reaction and peribronchial infiltration of lymphocytes. Whole-lung cytokine analysis from Af bead-infected mice revealed an increase in proinflammatory cytokines and chemokines early in infection. Evidence of a Th2 type response was observed only early in the course of colonization, including increased levels of interleukin-4 (IL-4), elevated IgE levels in serum, and a mild increase in airway responsiveness. Pulmonary T cell subset analysis during infection mirrored these results with an initial transient increase in IL-4-producing CD4+ T cells, followed by a rise in IL-17 and Foxp3+ cells by day 14. These results provide the first report of the evolution of the immune response to A. fumigatus hyphal colonization.