Allergy

ORIGINAL ARTICLE

EXPERIMENTAL ALLERGY AND IMMUNOLOGY

Chemerin suppresses murine allergic asthma by inhibiting CCL2 production and subsequent airway recruitment of inflammatory dendritic cells L. Zhao1, W. Yang1, X. Yang1, Y. Lin1, J. Lv1, X. Dou2, Q. Luo3, J. Dong3, Z. Chen4, Y. Chu1,5 & R. He1,5 1

Key Laboratory of Medical Molecular Virology of Ministries of Education and Health, Department of Immunology, School of Basic Medical Sciences, Fudan University; 2Department of Dermatology, Huashan Hospital; 3Institute of Chinese Integrative Medicine, Huashan Hospital; 4 Department of Pulmonary Disease, Zhongshan Hospital; 5Biotherapy Research Center, Fudan University, Shanghai, China

To cite this article: Zhao L, Yang W, Yang X, Lin Y, Lv J, Dou X, Luo Q, Dong J, Chen Z, Chu Y, He R. Chemerin suppresses murine allergic asthma by inhibiting CCL2 production and subsequent airway recruitment of inflammatory dendritic cells. Allergy 2014; 69: 763–774.

Keywords allergic asthma; CCL2; chemerin; inflammatory dendritic cells; lung epithelial cells. Correspondence Rui He, Department of Immunology, Shanghai Medical School, 138# Yixueyuan Road, Shanghai 200032, China. Tel.: +86-21-54237419 Fax: +86-21-54237324 E-mail: [email protected] Accepted for publication 4 March 2014 DOI:10.1111/all.12408 Edited by: Angela Haczku

Abstract Background: Chemerin has been implicated to play opposing roles, either proinflammatory or anti-inflammatory, in various tissue inflammation processes primarily through the regulation of tissue recruitment of immune cells. However, the effect of chemerin in allergic asthma has not yet been explored. We sought to investigate the role of chemerin in the murine model of allergic asthma and explore the underlying mechanism. Methods: We examined the effect of intranasal (i.n.) administration of chemerin during antigen challenge in murine models of asthma. Moreover, we examined whether administration of CCL2 or bone marrow-derived dendritic cells (BMDCs) transfer reversed the effects of chemerin on ovalbumin-induced asthma. We finally examined the effect of chemerin on CCL2 expression in activated lung epithelial cells in vitro. Results: The administration of chemerin attenuated allergic airway inflammation and airway hyperreactivity during antigen challenge. Chemerin treatment caused significant decreases in BALF CD4+ T-cell accumulation and mRNA expression of Th2-attracting chemokines, CCL17 and CCL22, which was accompanied by significantly decreased BALF CD11c+CD11b+ inflammatory DC accumulation and CCL2 production. Furthermore, airway administration of exogenous CCL2 or adoptive transfer of CD11c+CD11b+ BMDCs abrogated the suppressive effects of chemerin on allergic asthma. Finally, in vitro study showed that chemerin inhibited CCL2 secretion by low-dose LPS-stimulated lung epithelial cells, which led to decreased chemotaxis of BMDCs. Conclusions: Our study demonstrates that chemerin plays a protective role in allergic asthma by suppressing airway recruitment of inflammatory CD11c+CD11b+ DCs through the inhibition of CCL2 secretion by active lung epithelial cells.

Asthma has increased dramatically in prevalence over the last three decades, affecting 300 million people worldwide Abbreviations AHR, airway hyperreactivity; BALF, bronchoalveolar lavage fluid; BMDC, bone marrow-derived DC; DC, dendritic cell; H&E, hematoxylin and eosin; OVA, ovalbumin; PAS, periodic acid–Schiff; pDC, plasmacytoid DC.

(1). Asthma is a heterogeneous airway disorder, among which allergic asthma is the most common form. Allergic asthma is characterized by Th2-mediated inappropriate immune response to harmless inhaled allergen, which leads to increased IgE production, airway eosinophilia, increased mucus production, and airway hyperreactivity (AHR) (1, 2). The initiation and amplification of allergen-specific Th2 immune response is regulated by many factors, among

Allergy 69 (2014) 763–774 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

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Chemerin suppresses asthma inflammatory DC recruitment

which dendritic cells (DCs) play a crucial role (3). Dendritic cells can be generally divided into two subsets with distinct role in allergic asthma: Myeloid dendritic cells (mDCs) promote the initiation of allergen-specific Th2 cells (4), while plasmacytoid DCs (pDCs) mediate tolerance following allergen exposure (5). Emerging evidence suggests that DCs also influence the effector phase of allergic asthma, as depletion of mDCs from the airways of an OVA-induced asthma model during allergen challenge attenuates the characteristic features of asthma (6), while removal of pDCs enhances airway inflammation (7). Therefore, targeting DCs could be an effective strategy to control Th2 immune response in allergic asthma. Chemerin is a recently identified chemotactic protein acting on pDCs and NK cells, which are dependent on the expression of its functional receptor, ChemR23 (8). Previous studies found increased chemerin levels in various inflammatory sites including skin lesions of acute psoriasis and lupus as well as in the kidneys of patients with severe lupus nephritis, which is associated with the tissue infiltration of pDCs, suggesting a pro-inflammatory role of chemerin (9– 11). However, emerging data suggest that chemerin also plays an anti-inflammatory role, as the administration of exogenous chemerin exerts potential anti-inflammatory activities in experimental peritonitis and acute lung injury (12, 13). Interestingly, two recent studies demonstrated that chemerin is upregulated in the airways of acute viral pneumonia and cigarette-smoke-induced lung inflammation and seems to play opposing roles in these two models (14, 15). CCRL2, another receptor for chemerin, recently was also reported to be involved in the migration of lung DCs to regional lymph nodes and in the induction of Th2-mediated allergic airway inflammation (16). However, the role for chemerin in the development of allergic asthma has not yet been investigated. Here, we aimed to investigate whether the administration of exogenous chemerin affects allergeninduced experimental allergic asthma and, if so, to further determine the underlying mechanism.

Methods

concentrations of IL-4, IL-13, and CCL2 were determined using ELISA kits (eBioscience). Ovalbumin-induced allergic asthma and treatment regimens Mice were sensitized and challenged with ovalbumin (OVA) (Grade V; Sigma-Aldrich, St Louis, MO, USA) as per the protocol shown in Fig. 1A. Some mice were intranasally (i.n.) treated with chemerin (200 ng/mouse) following OVA challenge. Some mice were locally treated with CCL2 (400 ng/mouse) or bone marrow (BM)-derived CD11c+CD11b+ dendritic cells (DCs) (1 9 106/mouse) following OVA challenge and chemerin administration. Analysis of bronchoalveolar lavage fluid and lung tissues Bronchoalveolar lavage fluid (BALF) was obtained 24 h after the last OVA challenge. Differential cell counts were determined from stained cytospins by Wright-Giemsa. The percentages of CD4+ T cells and CD11c+CD11b+ DCs were determined by flow cytometry. Lung sections were stained with hematoxylin and eosin (H&E) or periodic acid–Schiff (PAS). Immunohistochemical staining of CCL2 was performed on paraffin-embedded lung tissue sections. After rehydration, sections were heated in the microwave for antigen retrieval and then sequentially incubated with 3% H2O2 for blocking endogenous peroxidase activity, with 3% bovine serum albumin (Sigma-Aldrich) for blocking nonspecific staining, with a rabbit polyclonal anti-CCL2 antibody (1 : 200 dilution; Abcam, Cambridge, MA, USA) at 4°C overnight in a humidified chamber. A ready-to-use peroxidase-conjugated anti-rabbit second antibody (GeneTech, Shanghai, China) was used. Measurement of airway hyperreactivity Mice were anesthetized for the measurement of pulmonary mechanics (Buxco Electronics, Wilmington, NC) 24 h after the last OVA challenge as previously described (17). Quantitative real-time PCR

Mice Six-week-old female BALB/c mice (Chinese Academy of Science, Shanghai, China) were housed under pathogen-free conditions. All the animal experiments were approved by the Animal Care and Use Committee at Fudan University, Shanghai. Reagents Recombinant BSA-free chemerin was purchased from R&D systems, Minneapolis, MN, USA (chemerin was produced in E. coli with

Chemerin suppresses murine allergic asthma by inhibiting CCL2 production and subsequent airway recruitment of inflammatory dendritic cells.

Chemerin has been implicated to play opposing roles, either pro-inflammatory or anti-inflammatory, in various tissue inflammation processes primarily ...
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