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A new approach for the study of lung smooth muscle phenotypes and its application in a murine model of allergic airway inflammation.

ABSTRACT: Phenotypes of lung smooth muscle cells in health and disease are poorly characterized. This is due, in part, to a lack of methodologies that allow for the independent and direct isolation of bronchial smooth muscle cells (BSMCs) and vascular smooth muscle cells (VSMCs) from the lung. In this paper, we describe the development of a bi-fluorescent mouse that permits purification of these two cell populations by cell sorting. By subjecting this mouse to an acute allergen based-model of airway inflammation that exhibits many features of asthma, we utilized this tool to characterize the phenotype of so-called asthmatic BSMCs. First, we examined the biophysical properties of single BSMCs from allergen sensitized mice and found increases in basal tone and cell size that were sustained ex vivo. We then generated for the first time, a comprehensive characterization of the global gene expression changes in BSMCs isolated from the bi-fluorescent mice with allergic airway inflammation. Using statistical methods and pathway analysis, we identified a number of differentially expressed mRNAs in BSMCs from allergen sensitized mice that code for key candidate proteins underlying changes in matrix formation, contractility, and immune responses. Ultimately, this tool will provide direction and guidance for the logical development of new markers and approaches for studying human lung smooth muscle.

SUBMITTER: Paez-Cortez J

PROVIDER: S-EPMC3767675 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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A new approach for the study of lung smooth muscle phenotypes and its application in a murine model of allergic airway inflammation.

Paez-Cortez Jesus J Krishnan Ramaswamy R Arno Anneliese A Aven Linh L Ram-Mohan Sumati S Patel Kruti R KR Lu Jining J King Oliver D OD Ai Xingbin X Fine Alan A

PloS one 20130909 9

Phenotypes of lung smooth muscle cells in health and disease are poorly characterized. This is due, in part, to a lack of methodologies that allow for the independent and direct isolation of bronchial smooth muscle cells (BSMCs) and vascular smooth muscle cells (VSMCs) from the lung. In this paper, we describe the development of a bi-fluorescent mouse that permits purification of these two cell populations by cell sorting. By subjecting this mouse to an acute allergen based-model of airway infla ...[more]

PMID: 24040256

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Project description:Asthma is a heterogeneous disease of the airways characterized by chronic inflammation associated with bronchial and smooth muscle hyperresponsiveness. Currently, different murine models for the study of asthma show poor bronchial hyperresponsiveness due to a scarcity of smooth muscle and large airways, resulting in a failure to reproduce smooth muscle hyperreactivity. Thus, we aimed to standardize a guinea pig model of chronic allergic lung inflammation mimicking airway smooth muscle hyperreactivity observed in asthmatics (Asth). Animals were randomly divided into a control group (Ctrl), which received saline (0.9% NaCl), and the Asth group, subjected to in vivo sensitization with ovalbumin (OVA) nebulization. Morphological analysis was performed by hematoxylin-eosin staining. Bronchial hyperresponsiveness was evaluated by nebulization time in the fifth, sixth, and seventh inhalations (NT5-7) and tracheal isometric contractions were assessed by force transducer. Total antioxidant capacity was measured by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method and protein expression by Western blot. Histologically, the Asth group developed peribronchial cellular infiltrate, epithelial hyperplasia and smooth muscle thickening. After the fourth nebulization, the Asth group developed bronchial hyperreactivity. The trachea from the Asth group contracted after in vitro stimulation with OVA, differing from the Ctrl group, which showed no response. Additionally, airway smooth muscle hyperreactivity to carbachol and histamine was observed in the Asth group only in intact epithelium preparations, but not to KCl, and this effect was associated with an augmented production of reactive oxygen species. Moreover, lung inflammation impaired the relaxant potency of isoproterenol only in intact epithelium preparations, without interfering with nifedipine, and it was found to be produced by transforming growth factor-β negative modulation of β adrenergic receptors and, furthermore, big-conductance Ca2+-sensitive K+ channels. These effects were also associated with increased levels of phosphatidylinositol 3-kinases but not extracellular signal-regulated kinases 1/2 or phosphorylation, and augmented α-actin content as well, explaining the increased smooth muscle mass. Furthermore, pulmonary antioxidant capacity was impaired in the Asth group. Therefore, we developed a standardized and easy-to-use, reproducible guinea pig model of lung inflammation that mimics airway smooth muscle hypercontractility, facilitating the investigation of the mechanisms of bronchial hyperresponsiveness in asthma and new therapeutic alternatives.

Project description:Allergic asthma is characterized by airway eosinophilia, increased mucin production and allergen-specific IgE. Fc gamma receptor IIb (FcgammaRIIb), an inhibitory IgG receptor, has recently emerged as a negative regulator of allergic diseases like anaphylaxis and allergic rhinitis. However, no studies to date have evaluated its role in allergic asthma. Our main objective was to study the role of FcgammaRIIb in allergic lung inflammation. We used a murine model of allergic airway inflammation. Inflammation was quantified by BAL inflammatory cells and airway mucin production. FcgammaRIIb expression was measured by qPCR and flow cytometry and the cytokines were quantified by ELISA. Compared to wild type animals, FcgammaRIIb deficient mice mount a vigorous allergic lung inflammation characterized by increased bronchoalveolar lavage fluid cellularity, eosinophilia and mucin content upon ragweed extract (RWE) challenge. RWE challenge in sensitized mice upregulated FcgammaRIIb in the lungs. Disruption of IFN-gamma gene abrogated this upregulation. Treatment of naïve mice with the Th1-inducing agent CpG DNA increased FcgammaRIIb expression in the lungs. Furthermore, treatment of sensitized mice with CpG DNA prior to RWE challenge induced greater upregulation of FcgammaRIIb than RWE challenge alone. These observations indicated that RWE challenge upregulated FcgammaRIIb in the lungs by IFN-gamma- and Th1-dependent mechanisms. RWE challenge upregulated FcgammaRIIb on pulmonary CD14+/MHC II+ mononuclear cells and CD11c+ cells. FcgammaRIIb deficient mice also exhibited an exaggerated RWE-specific IgE response upon sensitization when compared to wild type mice. We propose that FcgammaRIIb physiologically regulates allergic airway inflammation by two mechanisms: 1) allergen challenge mediates upregulation of FcgammaRIIb on pulmonary CD14+/MHC II+ mononuclear cells and CD11c+ cells by an IFN-gamma dependent mechanism; and 2) by attenuating the allergen specific IgE response during sensitization. Thus, stimulating FcgammaRIIb may be a therapeutic strategy in allergic airway disorders.

Project description:BackgroundIn recent years, particulate matter 2.5 (PM2.5) exposure has been considered a key dangerous factor in chronic obstructive pulmonary disease (COPD). The dysfunction of airway smooth muscle cells (ASMCs) facilitates lung inflammation and fibrosis in COPD. Therefore, we explored whether PM2.5 could promote the inflammatory response and fibrosis in ASMCs in vivo and in vitro via the wingless-related integration site 5a (Wnt5a)/c-Jun N-terminal kinase (JNK)/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway.MethodsWnt5a expression in the bronchoalveolar lavage fluid (BALF) of COPD patients exposed to PM2.5 was measured by enzyme-linked immunosorbent assay (ELISA). Mice were intratracheally injected with PM2.5 and a Wnt5a antagonist (BOX5). ASMCs were transfected with Wnt5a small interfering RNA (siRNA), BOX5 and the JNK inhibitor SP600125 before PM2.5 stimulation. Hematoxylin and eosin (H&E) staining was performed to measure the inflammatory response and airway fibrosis. The production of Wnt5a/JNK/NF-KB pathway factors was analyzed by Western blotting. The secretion of interleukin-6 (IL-6), IL-8 and tumor necrosis factor-α (TNF-α) was measured by ELISA. The expression levels of alpha smooth muscle actin (α-SMA), collagen I and collagen III were assessed by quantitative real time polymerase chain reaction (qRT-PCR) and Western blotting.ResultsWe found that the increase in Wnt5a expression in the BALF of COPD patients was positively correlated with the levels of PM2.5 exposure. The Wnt5a/JNK/NF-κB pathway was activated in the lung samples of PM2.5-induced model mice and PM2.5-exposed ASMCs, which promoted the production of α-SMA, collagen I and collagen III and increased the secretion of IL-6, IL-8 and TNF-α. Furthermore, our results showed that BOX5 could prevent these effects. Wnt5a siRNA blocked the activation of the Wnt5a/JNK/NF-κB pathway and inhibited the effects of PM2.5 on fibrosis and inflammation in ASMCs. SP600125 blocked the phosphorylation of NF-κB and inhibited inflammation and fibrosis in PM2.5-exposed ASMCs.ConclusionsThese findings suggest that PM2.5 stimulation of ASMCs induces pulmonary inflammatory factor expression and collagen deposition during COPD via the Wnt5a/JNK pathway, which indicates that modulating the Wnt5a/JNK pathway could be a promising therapeutic strategy for PM2.5-induced COPD.

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A new approach for the study of lung smooth muscle phenotypes and its application in a murine model of allergic airway inflammation.

Publications

A new approach for the study of lung smooth muscle phenotypes and its application in a murine model of allergic airway inflammation.

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