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Muscarinic M3 receptors on structural cells regulate cigarette smoke-induced neutrophilic airway inflammation in mice.

ABSTRACT: Anticholinergics, blocking the muscarinic M3 receptor, are effective bronchodilators for patients with chronic obstructive pulmonary disease. Recent evidence from M(3) receptor-deficient mice (M(3)R(-/-)) indicates that M3 receptors also regulate neutrophilic inflammation in response to cigarette smoke (CS). M(3) receptors are present on almost all cell types, and in this study we investigated the relative contribution of M(3) receptors on structural cells vs. inflammatory cells to CS-induced inflammation using bone marrow chimeric mice. Bone marrow chimeras (C56Bl/6 mice) were generated, and engraftment was confirmed after 10 wk. Thereafter, irradiated and nonirradiated control animals were exposed to CS or fresh air for four consecutive days. CS induced a significant increase in neutrophil numbers in nonirradiated and irradiated control animals (4- to 35-fold). Interestingly, wild-type animals receiving M(3)R(-/-) bone marrow showed a similar increase in neutrophil number (15-fold). In contrast, no increase in the number of neutrophils was observed in M3R(-/-) animals receiving wild-type bone marrow. The increase in keratinocyte-derived chemokine (KC) levels was similar in all smoke-exposed groups (2.5- to 5.0-fold). Microarray analysis revealed that fibrinogen-? and CD177, both involved in neutrophil migration, were downregulated in CS-exposed M(3)R(-/-) animals receiving wild-type bone marrow compared with CS-exposed wild-type animals, which was confirmed by RT-qPCR (1.6-2.5 fold). These findings indicate that the M(3) receptor on structural cells plays a proinflammatory role in CS-induced neutrophilic inflammation, whereas the M(3) receptor on inflammatory cells does not. This effect is probably not mediated via KC release, but may involve altered adhesion and transmigration of neutrophils via fibrinogen-? and CD177.

SUBMITTER: Kistemaker LE

PROVIDER: S-EPMC4315453 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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Muscarinic M3 receptors on structural cells regulate cigarette smoke-induced neutrophilic airway inflammation in mice.

Kistemaker Loes E M LE van Os Ronald P RP Dethmers-Ausema Albertina A Bos I Sophie T IS Hylkema Machteld N MN van den Berge Maarten M Hiemstra Pieter S PS Wess Jürgen J Meurs Herman H Kerstjens Huib A M HA Gosens Reinoud R

American journal of physiology. Lung cellular and molecular physiology 20141107 1

Anticholinergics, blocking the muscarinic M3 receptor, are effective bronchodilators for patients with chronic obstructive pulmonary disease. Recent evidence from M(3) receptor-deficient mice (M(3)R(-/-)) indicates that M3 receptors also regulate neutrophilic inflammation in response to cigarette smoke (CS). M(3) receptors are present on almost all cell types, and in this study we investigated the relative contribution of M(3) receptors on structural cells vs. inflammatory cells to CS-induced in ...[more]

PMID: 25381025

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Project description:Cigarette smoke (CS)-induced airway inflammation is an important pathologic feature of chronic obstructive pulmonary disease (COPD). Recent studies suggest a potential role of JunD in the regulation of inflammation, but its role in CS-induced airway inflammation has not been reported. This study aimed to determine its role in CS-induced airway inflammation through bioinformatics analysis and in vitro and in vivo experiments. Data from the Gene Expression Omnibus (GEO) database (GSE37147) were analyzed using weighted gene co-expression network analysis (WGCNA) and key driver analysis (KDA), and these analyses were validated using the GSE47460 dataset. The effect of CS on JunD expression was examined in lung tissues of COPD patients and CS-exposed mice and in CS extract (CSE)-exposed BEAS-2B cells. The effects of CSE on airway epithelial inflammatory injury after JunD knockdown or overexpression were also investigated. mRNA-seq and chromatin immunoprecipitation (ChIP)-seq were used to explore the mechanism of JunD-mediated CS-induced airway inflammation. Mice were injected with adeno-associated virus serotype 9 (AAV9)-JunD vector or control vector and then exposed to CS for 4 weeks, and lung tissue morphology and airway inflammation were evaluated. KDA of the lung function-related gene modules in GSE37147 revealed a potential role of JunD in COPD, which was validated in GSE47460. JunD was downregulated in lung tissues of COPD patients and CS-exposed mice and in BEAS-2B cells. JunD knockdown aggravated CSE-induced tumor necrosis factor (TNF)-α and interleukin (IL)-1β release by BEAS-2B cells, while JunD overexpression attenuated these effects. mRNA-seq and ChIP-seq identified several JunD-regulated genes, which are involved in the immune response and TNF signaling pathway and are commonly dysregulated in cell models of airway inflammation. In vivo, JunD overexpression attenuated the CS-induced inflammatory cell infiltration and inflammatory cytokine release in mouse lungs. Thus, JunD is involved in CS-induced airway inflammation and JunD-based therapy may be useful in CS-induced airway disorders.

Project description:Transforming Growth Factor Beta1 (TGF-β1) signaling is upregulated in Chronic Obstructive Pulmonary disease (COPD), smokers, and people living with HIV. Cigarette smoking and HIV are also independent risk factors for COPD. Chronic inflammation is a hallmark of COPD. However, the underlying mechanisms remain unknown. Previous research has suggested that TGF-β1 alters the airway epithelial microRNAome and transcriptome, potentially contributing to lung inflammation. The Lactoperoxidase (LPO) system is an integral component of innate immunity within the airway. LPO plays a crucial role in host defense by catalyzing the oxidation of thiocyanate to hypothiocyanite in the presence of hydrogen peroxide (H2O2), generating a potent antibacterial and antiviral agent. Additionally, the LPO system potentially aids in maintaining cellular redox balance by reducing the levels of H2O2, thus mitigating oxidative stress within the airway epithelium. LPO dysfunction can impair immune responses and exacerbate inflammatory processes in respiratory diseases.In this study, primary bronchial epithelial cells and bronchial cell lines were treated with TGF-β1 and exposed to cigarette smoke to characterize the effect of these factors on LPO and their downstream effects. RT-qPCR and Western Blot were applied to quantify mRNA and proteins' expression. The levels of H2O2 were detected using the Amplex Red Assay. Magnetofection and transfection were applied to probe the effect of miR-449b-5p. Staining procedures using the MitoTracker Green and C12FDG dyes were used to establish mitochondria mass and senescence. The levels of pro-inflammatory cytokines were measured via Luminex assays.We found that TGF-β1 and cigarette smoke suppressed airway LPO expression, increasing H2O2 levels. This increase in H2O2 had downstream effects on mitochondrial homeostasis, epithelial cellular senescence, and the pro-inflammatory cytokine response. We demonstrate for the first time that airway LPO is regulated by TGF-β1-induced miRNA-mediated post-transcriptional silencing through miR-449b-5p in the lungs. Further, we identify and validate miR-449-5p as the candidate miRNA upregulated by TGF-β1, which is involved in LPO suppression. This paper demonstrates a new mechanism by which TGF-β1 can lead to altered redox status in the airway.

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Muscarinic M3 receptors on structural cells regulate cigarette smoke-induced neutrophilic airway inflammation in mice.

Publications

Muscarinic M3 receptors on structural cells regulate cigarette smoke-induced neutrophilic airway inflammation in mice.

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