Articles in PresS. Am J Physiol Lung Cell Mol Physiol (January 16, 2015). doi:10.1152/ajplung.00305.2014

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Increased IL-33 expression in chronic obstructive pulmonary disease

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Jie Xia, Junling Zhao, Jin Shang, Miao Li, Zhilin Zeng, Jianping Zhao, Jianmiao

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Wang, Yongjian Xu, Jungang Xie

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Department of Respiratory and Critical Care Medicine, Tongji Hospital, Tongji

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Medical College, Huazhong University of Science and Technology, Wuhan 430030,

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China.

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Running title: IL-33 and COPD

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Corresponding Author: Jungang Xie, MD, PhD

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Department of Pulmonary and Critical Care Medicine, Tongji Hospital, Tongji

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Medical College, Huazhong University of Science and Technology, 1095 Jiefang

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Avenue, Wuhan 430030, China. Tel.: 86-27-83663596; Fax: 86-27-83662898. Email:

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[email protected]

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Contributions of authors: Conception: XJ. Design: XJ, XY. Data collection and

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analysis: XJ, ZJ, SJ, LM, ZZ. Drafting the manuscript for important intellectual

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content: XJ, XJ, ZJ, WJ, XY.

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Copyright © 2015 by the American Physiological Society.

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Abstract

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Chronic obstructive pulmonary disease (COPD) is an inflammatory lung disease

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characterized by inflammatory cell activation and the release of inflammatory

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mediators. Interleukin-33 (IL-33) plays a critical role in various inflammatory and

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immunological pathologies, but evidence for its role in COPD is lacking. This study

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aimed to investigate the expression of IL-33 in COPD and to determine whether IL-33

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participates in the initiation and progression of COPD. Levels of serum IL-33 and its

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receptors were measured by ELISA, and serum levels of IL-33, ST2 and interleukin-1

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receptor accessory protein (IL-1RAcP) were elevated in patients with COPD

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compared to control subjects. Flow cytometry analysis further demonstrated an

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increase in peripheral blood lymphocytes (PBLs) expressing IL-33 in COPD patients.

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Immunofluorescence analysis revealed that the main cellular source of IL-33 in lung

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tissue was bronchial epithelial cells (HBEs). Cigarette smoke extract (CSE) and

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lipopolysaccharide (LPS) could enhance the ability of PBLs and HBE to express

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IL-33. Furthermore, PBLs from patients with COPD showed greater IL-33 release in

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response to the stimulus. Collectively, these findings suggest that IL-33 expression

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levels are increased in COPD and related to airway and systemic inflammation.

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Therefore, IL-33 might contribute to the pathogenesis and progression of this disease.

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Key words: chronic obstructive pulmonary disease; Interleukin-33; human bronchial

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epithelial cells; peripheral blood lymphocytes

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Introduction

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Chronic obstructive pulmonary disease (COPD) is a leading public health problem,

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and the incidence of COPD has recently increased—it is projected to rank third

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worldwide for mortality (37). COPD is typically characterized by irreversible and

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slowly progressive shortness of breath upon exertion, which is caused by airway

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obstruction resulting from airway inflammation. Bronchial biopsies from COPD

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patients show infiltration with increased numbers of neutrophils, T cells and

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macrophages (6, 7, 13), which induce the production of various cytokines in the

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airways,

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neutrophil-associated cytokines (5, 16). These cytokines are associated with different

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patterns of inflammation in the airways, and eventually cause chronic bronchitis and

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emphysema by inducing mucus production, alveolar destruction and remodeling of

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the airways.

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IL-33 is a new member of the IL-1 family that has attracted great attention because of

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its function in immune responses. It is mainly expressed by a variety of cells and

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tissues in healthy and inflamed tissues. Immune cells, macrophages and dendritic cells

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can also produce IL-33 in response to specific stimuli (46). IL-33 is known to be a

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ligand for ST2 and plays an important role in the immune response and inflammatory

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diseases. Apart from Th2-type immune response, recent studies indicate that IL-33 has

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a remarkable effect on Th1-type responses by promoting the secretion of Th1-type

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cytokines by NK and NKT cells (48, 55, 56). IL-1 receptor-related protein ST2 and

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IL-1 receptor accessory protein (IL-1RAcP) are required for IL-33-mediated signal

including

IL-6,

IL-8,

tumor

necrosis

factor-α

(TNF-α)

and

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transduction because these molecules form parts of its receptor complex (14).

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It has been reported that changes in the serum concentration of IL-33 and ST2 occur

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in allergic and heart diseases (11, 32, 50). Additionally, high expression levels of

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IL-33 have been detected in lung tissues, which play an important role in respiratory

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diseases. IL-33 expression levels are significantly elevated in bronchial asthma and

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bronchial epithelium cells are an important source of IL-33 (36, 41, 42). Although

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elevated sST2 levels have been reported in patients with COPD (17), the relationship

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and function of the IL-33 pathway in the pathogenesis of this disease remain

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unknown.

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In order to investigate the role and source of IL-33 in COPD, we measured the serum

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levels of IL-33, and the receptors ST2 and IL-1RAcP in COPD patients and controls,

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and the intracellular expression levels of IL-33 in peripheral blood and lung tissues.

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IL-33 and its soluble receptors were increased in COPD, and the cellular sources of

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IL-33 in the peripheral blood and lung tissues included peripheral blood lymphocytes

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(PBLs) and human bronchial epithelial (HBE) cells. Moreover, we found that IL-33

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production by these cells could be stimulated by CSE and LPS, which were major risk

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factors for COPD.

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Materials and Methods

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Subjects

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A total of 112 patients with COPD and 115 sex, age and race matched controls were

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chosen for inclusion in our study. The clinical characteristics of these populations are

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described in detail in Table 1. COPD was diagnosed according to the American

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Thoracic Society criteria and the Global Initiative for COPD (GOLD) criteria (57)

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(post-bronchodilator forced expiratory volume in 1 s (FEV1)/forced vital capacity

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ratio 20. Plasma cotinine concentrations were measured by a direct

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ELISA kit (Immunalysis, Pomona, CA) to confirm smoking status. The sensitivity of

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the cotinine assay is 1 ng/ml. Plasma cotinine levels for COPD patients and controls

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were all above the recognized minimum criterion of active smoke exposure

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(9.5 ng/ml)(23). Subjects who suffered from asthma or other obstructive lung diseases,

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or who exhibited disease exacerbation or respiratory infection in the four weeks prior

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to recruitment were excluded from this study. Patients receiving inhaled

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corticosteroids or oral theophylline, anti-inflammatory therapy or oral steroids for

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chronic inflammatory diseases during the previous 4 weeks were also excluded.

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In these COPD and healthy individuals, lung tissues from some patients were

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collected during surgical resection of solitary pulmonary nodules and lung cancer was

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excluded by pathology after surgery. A total of 12 lung specimens were collected,

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including 6 COPD and 6 control subjects. In these subjects, the 12 participants who

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supplied lung tissue specimens also provided venous blood simultaneously, and others

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only provided a blood sample.

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All study subjects were recruited from Tongji Hospital, Tongji Medical College,

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Huazhong University of Science and Technology in Wuhan (Hubei, China) and signed

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a consent form. Our study was approved by the Research Ethics Committee of

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Huazhong University of Science and Technology.

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Cytokine ELISAs

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ELISA was used to measure the protein concentrations of IL-33, ST2 and IL-1RAcP

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in serum or cell culture supernatants. First, an ELISA plate was coated with capture

116

antibody (R&D Systems, Minneapolis, MN, USA). Then, the serum or sample

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supernatants were added to the plate, and horseradish peroxidase (HRP)-conjugated

118

secondary mAb was added. After a washing step, tetramethylbenzidine was added in

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the dark. To stop the reaction, 2 mol/L H2SO4 was added to each well. Absorbance

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was determined at 490 nm and a reference wavelength of 570 nm using a

121

spectrophotometer. Concentrations were calculated based on a standard curve. The

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limits of detection for the IL-33, ST2 and IL-1RAcP ELISA kits were 1.65, 13.5 and

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31.2 pg/mL, respectively.

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Flow cytometry

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Flow cytometry was used to investigate the cellular source of IL-33 in peripheral

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blood. An aliquot of 100 µL whole blood was added to a 6-well plate containing 800

128

µL RPMI-1640 Medium (GIBCO Laboratories, Grand Island, NY). Biolegend

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Brefeldin A Solution (Becton Dickinson, Franklin Lakes, NJ, USA) was added to the

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mixed cell cultures, which were incubated for 4 h. After red blood cells were lysed

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using lysis solution (Becton Dickinson), cytofix/cytoperm solution (BectonDickinson)

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was added to the tube for incubation for 20 min. Then, the cell suspension was

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divided into 2 tubes. In the dark, monoclonal anti-human IL-33- phycoerythrin

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(IL-33-PE; R&D Systems) and IgG2B isotype control-PE (R&D Systems) were added

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to the cells separately. After one final wash, cells were resuspended in 2%

136

paraformaldehyde and assessed using a Becton Dickinson LSR flow cytometer. Data

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were analyzed using CellQuest software.

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Immunofluorescence

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Formalin-fixed and paraffin-embedded lung tissue was obtained from 6 COPD

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patients and 6 healthy control subjects. Then, 5 μm sections mounted onto slides were

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used for immunofluorescence detection of IL-33. After deparaffinazition in methanol

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and rehydration in a graded alcohol series, the sections were washed in TBS and

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boiled in sodium citrate buffer for 20 min in a microwave oven for epitope retrieval.

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Slides were incubated with 10% donkey serum for 1 h at room temperature, and then

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were incubated overnight with mouse anti-human IL-33 antibody (1:100, Alexis

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Biochemicals, Lausen, Switzerland), mouse anti-human ST2 antibody (1:100, Alexis

148

Biochemicals) rabbit anti-human IL-1RAcP antibody (1:200, GeneTex Inc, San

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Antonio, TX, USA) or matched isotype controls (Becton Dickinson) at 4°C. Sections

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were incubated with Alexa Fluor 488-donkey anti-mouse, Alexa Fluor 594-goat

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anti-mouse Alexa Fluor 488- donkey anti-rabbit secondary antibody. Finally, slides

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were visualized using laser scanning confocal microscopy (Zeiss, Oberkochen,

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Germany).

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Total RNA extraction, reverse transcription (RT) and quantitative real-time PCR

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Total RNA from cells and tissue was extracted using TRIzol reagent (Invitrogen,

157

Carlsbad, CA). The cDNA was prepared from 500 ng total RNA using Prime Script

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RT Master Mix (Takara, Dalian, China) following the manufacturer’s instructions.

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Real-time PCR was performed to quantify mRNA levels using an ABI Prism 7500

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sequence detection system with SYBR Premix Ex Taq (Takara, Dalian, China). The

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PCR parameters were 95ºC for 30 s, followed by 40 cycles of 95ºC for 5 s and 60ºC

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for 34 s. Results were expressed as 2–ΔΔCT, normalized to levels of

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glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in untreated cells or control

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subjects. Specific primers used in this study were designed using the program

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BatchPrimer3 v1.0 and were as follows: IL33, GAAGAACACAGCAAGCAAAGC

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and TACCAAAGGCAAAGCACTCC; GADPH, ACCACAGTCCATGCCATCAC

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and TCCACCACCCTGTTGCTGTA.

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Cell culture and treatments

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HBEs were purchased from ATCC (Manassas, VA, USA) and PBLs were isolated

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from human peripheral blood samples obtained from study subjects. The cell culture

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medium consisted of DMEM (GIBCO) or RPMI 1640 Medium, 10% fetal bovine

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serum (GIBCO), 100 U/ was recently found in ASMCs bundles penicillin and 100

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μg/ml streptomycin (KeyGEN, Nanjing, China). When cells reached 80% confluence,

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HBEs were digested with 0.25% trypsin and the cell concentration was adjusted to

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106/ml. Meanwhile, PBLs were also suspended in culture medium at a concentration

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of 106/ml. Then, cells and medium with or without irritant (as a control) were added to

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the

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CSE (Murty Pharmaceuticals, Inc., Lexington, KY, USA) and LPS (Sigma–Aldrich,

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USA) were 10 μg/ml and 100 ng/ml respectively. After incubation in a humidified

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incubator (95% air, 5% CO2, 37°C) for 24 h, supernatants were stored at –20ºC for

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ELISA analysis and cells were collected and stored at –80ºC in TRIzol for mRNA

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extraction. All experiments on HBEs were performed in triplicate and were repeated

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at least 3 times.

6-well

tissue

culture

plate.

The

final

concentrations

of

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Cell apoptosis assay

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Cell apoptosis was quantified using Annexin V/PI Apoptosis Detection Kit (KeyGen,

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Nanjing, China). After treatment, HBEs and PBLs were collected and resuspended in

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500μl binding buffer and then incubated with a mixture of 5μl FITC-labeled

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Annexin-V and 5μl propidium iodide (PI) for 15 min at room temperature in the dark.

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Samples were analysed by flow cytometry using a Becton Dickinson LSR flow

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cytometer. The data were analyzed with CellQuest software. Annexin V labeled the

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apoptotic cells, whereas PI labeled the late apoptotic and necrotic cells with

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membrane damage. Undamaged cells remained negative for both parameters.

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DNA extraction and genotyping

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Genomic DNA was extracted from 5 mL whole peripheral blood using a DNA

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extraction kit (Qiagen; Hilden, Germany) according to the standard protocol. PCR

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primers were designed using Primer 3 Input (version 0.4.0) and were as follows:

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rs4986790 and rs49867901, TGTATTCAAGGTCTGGCTGGT and TGTTTCAAAT

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TGGAATGCTGGA; rs11536889, CTGGGATCCCTCCCCTGTA and TTTCCCTG

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ATGACATCCTGA. PCR was performed using the mix of 100 ng genomic DNA, 1.5

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mM MgCl2, 0.2 mM dNTPs, and 1× PCR buffer, 0.5 ul Taq polymerase, and 1 ul of

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the forward and reverse primers in a total of 50 μL. The PCR conditions were as

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follows: 94°C for 1 min, 58°C for 50 sec, 72°C for 90 sec for 40 cycles, 72°C for 10

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min, then followed by 4°C until the reaction mixtures were removed from the cycler.

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PCR products were sequenced using an ABI3730xl sequencer (Applied Biosystems,

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Foster City, Calif).

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Statistical analyses

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Data are presented as means ± SD. Statistical analyses were performed using SPSS

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19.0 (SPSS, Chicago, IL, USA) and Prism 5 software (GraphPad Software Inc., San

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Diego, CA, USA). Student’s t-test, Mann-Whitney test, one-way ANOVA test,

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Kruskal-Wallis test or Friedman’s test followed by Dunn’s test and χ2 test was used as

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appropriate. All statistical tests were two-tailed and P < 0.05 was considered to

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indicate a statistically significant difference.

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Results

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Levels of IL-33 and its receptors are elevated in serum from COPD patients

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Protein levels of IL-33, sST2 and IL-1RAcP in the serum were measured in COPD

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patients and controls. Significantly higher levels of IL-33 in serum were detected in

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COPD patients (319.24±77.47 pg/ml) than control subjects (222.60±26.11 pg/ml,

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Figure 1A). ST2 and IL-1RAcP are receptors for IL-33. Because of different ST2

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mRNA splice variants, there are two ST2 protein isoforms (10). The form that we

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detected in serum was sST2, which has been widely studied in allergic and heart

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disease as an inflammatory marker (11, 32, 50). A significant difference in ST2 levels

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was observed between COPD patients (3.13±0.95 ng/ml) and control subjects

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(0.93±0.66 ng/ml, Figure 1B). Additionally, we found that the soluble IL-1RAcP

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levels in serum from subjects with COPD (2.85±0.10 ng/ml) was higher than that in

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healthy subjects (0.98±0.05 ng/ml, Figure 1C). Thus, our data indicated that COPD

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patients showed increased levels of IL-33 and its soluble receptors in serum compared

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to healthy subjects.

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IL-33 expression in peripheral blood cells, including PBLs and neutrophils

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Significantly higher levels of serum IL-33 protein could be observed in COPD

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patients. To corroborate the ELISA findings and characterize the cellular sources of

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IL-33 in peripheral blood, we also measured IL-33 expression in PBLs and

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neutrophils from 20 COPD patients and 20 controls by flow cytometry. We found that

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IL-33-positive cells were detectable in the lymphocyte group and the frequency of

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IL-33-positive cells was higher in COPD patients than in control patients (11.62 ±

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8.848 vs. 2.238 ± 4.490, respectively, Figure2). A low frequency of IL-33-positive

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cells was detected in the neutrophil group and there was no significant difference

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between COPD and control patients.

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IL-33 is highly expressed in the lungs of COPD patients

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We next investigated whether IL-33 expression was increased in the lungs of COPD

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patients. Immunofluorescence staining showed expression of IL-33 in HBEs, but there

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was little signal detected in airway smooth muscle cells (ASMCs; Figure 3A–B). In

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agreement with previous reports that IL-33 expression could be observed in nuclei of

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endothelial cells (2, 36, 51), IL-33 was detected in the nuclei of epithelial cells.

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Additionally, the IL-33 staining intensity of COPD was obviously higher than that of

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the control groups (Figure 3C). Finally, we discovered that ST2 and IL1-RAcP were

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expressed in the cytoplasm of HBEs, and IL-1RAcP was also expressed in ASMCs

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(Figure 3D). However, the expression levels of ST2 and IL-1RAcP were not

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significantly different between COPD and control patients. The isotype controls were

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negative in the epithelial cells.

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IL-33 expression is up-regulated by CSE and LPS in HBEs and PBLs

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Based on our observations that HBEs and PBLs were the cellular sources of IL-33, we

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further characterized the regulatory factors involved in controlling IL-33 expression,

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including CSE and LPS, which are risk factors for the development of COPD. The

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protein levels of IL-33 in supernatants of HBEs cultured with or without stimulation

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were measured by ELISA. IL-33 secretion was markedly increased after CSE

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stimulation compared to untreated cells (Figure 4A). We further assessed the effect of

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LPS treatment on IL-33 expression by HBEs and found that there was also a

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significant difference between cells treated with or without LPS. A stronger response

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was induced in cultured HBEs when those cells were treated with a mixture of LPS

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and CSE. This finding indicated that that a mixture of LPS and CSE might

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synergistically stimulate IL-33 expression in HBEs.

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Expression of IL-33 in cultured PBLs was examined in cells from 20 COPD patients

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and 20 healthy controls with or without stimulation. After treatment with CSE, LPS

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alone or together, the expression level of IL-33 in PBLs from COPD patients was

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significantly increased compared to that of untreated cells (Figure 4B). Notably, a

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stronger response was induced in cultured PBLs from COPD patients when those cells

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were stimulated with the mixture of CSE and LPS. These data were similar to those

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obtained using CSE and LPS-treated HBEs. However, there were no statistical

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differences in IL33 gene expression levels between treated and untreated PBLs from

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healthy controls (P = 0.174, Friedman’s test). Importantly, after stimulation with the

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mixture, IL33 gene expression levels in PBLs from COPD patients were significantly

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higher than those from healthy controls. These findings indicated that the responses of

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cells from COPD patients and healthy controls to these stimuli were different.

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In addition, when cells were double stained with Annexin V and PI, no apparent

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differences were observed in the cell death (apoptosis and necrosis) rates between the

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cells incubated with CSE and LPS and those incubated with media alone (Figure 4C),

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which suggested that IL-33 was actively secreted from HBEs and PBLs without

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apparent cell damage.

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TLR4 Polymorphisms Analysis in COPD Patients and Controls

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rs4986790, rs4986791 and rs11536889 polymorphisms of Toll-like receptor 4 (TLR4)

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gene have been detected in 20 COPD patients and 20 controls. The genotype

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distribution of TLR4 SNPs in these subjects was summarized in Table 2. In the 40

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samples, no heterozygous or homozygous variant genotypes of the rs4986790 and

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rs49867901 polymorphism were detected. Our data showed that there were no

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significant differences in the rs11536889 genotype distribution between COPD

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patients and controls (P >0.05).

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Discussion

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There is increasing evidence implicating IL-33 in various inflammatory diseases, such

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as rheumatoid arthritis (33), atopic dermatitis (21), ulcerative colitis (39), and asthma

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(30, 42, 46); however, no study to date has investigated the role of IL-33 in COPD.

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Herein, we show that levels of IL-33 expression are elevated in COPD, and confirm

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that the HBEs in the airway and PBLs in the peripheral blood were the cellular

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sources of IL-33. IL-33 could be induced by CSE and LPS, which are risk factors for

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COPD, and we found that the sensitivity of PBLs from COPD subjects to stimuli was

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much higher than that of healthy subjects.

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Our study is the first to provide evidence that the expression of IL-33 and its receptors

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are increased in the serum of COPD patients. A recent study showed that the

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concentrations of serum IL-33 in patients with COPD during acute episodes were

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significantly lower than those in individuals with healthy controls and patients at the

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stable phase of COPD (53). However, as an alarmin, it has been demonstrated that

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IL-33 could be released when cells sense inflammatory signals or undergo necrosis

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(35). Increased serum levels of IL-33 was found to be associated with numerous

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inflammatory diseases, including systemic lupus erythematosus (34), systemic

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sclerosis (59), inflammatory skin disorders (59) and Crohn's disease (9). Moreover,

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previous study in animal model showed that the serum level of IL-33 was elevated in

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COPD (43), which is consistent with the detection of increased serum IL-33 levels in

316

COPD patients in our study. Additionally, immunofluorescence staining showed that

317

the expression of IL-33 was also increased in lung tissues of COPD patients,

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indicating that IL-33-upregulation was probably related to systemic and airway

319

inflammation in COPD. Furthermore, both serum sST2 and IL-1RAcp levels showed

320

a striking increase in patients with COPD compared to those in healthy subjects.

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Because a beneficial effect of the high levels of soluble IL-1RAcp and ST2 is to

322

antagonize the pro-inflammatory function of IL-33 (58), the enhanced levels of these

323

molecules demonstrated increased IL-33 expression and release in COPD.

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Recent studies have implicated IL-33 in the pathogenesis of asthma. In asthma, IL-33

325

can significantly enhance inflammation and cause pathological changes in the airways

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by recruiting inflammatory cells and inducing the release of inflammatory cytokines

327

(40, 49, 52). Interestingly, a similar pattern of inflammation also exists in COPD. In

328

IL-33-treated mice, the most obvious histopathological changes in the lungs were

329

epithelial lining hypertrophy, goblet cell hypertrophy and mucus hypersecretion (46),

330

which are the prominent pathophysiological features of COPD patients. By activating

331

macrophages to secrete cytokines or by direct stimulation, IL-33 can promote

332

neutrophil maturation (56). The activation and recruitment of neutrophils to the

333

airway has been strongly linked to the inflammatory response in COPD. In IL-33

334

transgenic mice, increased IL-8 was detected in the BALF (61), which is a potent

335

chemotactic cytokine for neutrophils and is thought to play a central role in COPD (8).

336

Combined with the finding of higher IL-33 expression in peripheral blood and

337

airways from COPD patients in our study, we speculate that IL-33 may play an

338

important role in the pathogenesis of COPD.

339

Furthermore, we characterized the sources of IL-33 in COPD. We made the novel

340

observation that PBLs and HBEs in the airways produced IL-33 cytokine. We

341

detected IL-33 protein immunoreactivity in HBEs from lung tissue sections from

342

COPD and control subjects, and the expression of IL-33 was significantly higher in

343

COPD patients than in controls. However, we failed to observe immunostaining of

344

ASMCs. Furthermore, our flow cytometry results showed that PBLs were the major

345

source of IL-33 in the serum, particularly in COPD patients. By contrast,

346

IL-33-positive neutrophils were scarcely detectable in either patients or healthy

347

controls.

348

Although IL-33 was initially found in the endothelial cells of high endothelial venules

349

(4), previous reports confirmed the expression of IL-33 in various cell types,

350

including epithelial cells, smooth muscle cells, keratinocytes, fibroblasts, dendritic

351

cells and macrophages (28, 36, 46, 54). IL-33 expression has also been detected in

352

venules from chronically inflamed tissues from patients with Crohn’s disease or

353

rheumatoid arthritis. Moreover, IL-33 expression levels were correlated with

354

inflammation and tissue damage, which can lead to apoptosis and the release of IL-33

355

protein. Several studies have demonstrated that IL-33 is highly expressed in epithelial

356

cells from many tissues, including the skin (18), gastrointestinal tract (29) and airway

357

(27), which make contact with allergens, pathogens, tobacco smoke and other

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common environmental agents (36). Recent studies of epithelial cells from asthma

359

patients reported significantly increased expression and secretion levels of IL-33

360

compared to those from controls (42). In our study, IL-33 was mainly derived from

361

airway epithelial cells and PBLs, which may account for the airway and systemic

362

inflammation observed in COPD.

363

Another important finding of our study is that CSE and LPS significantly enhanced

364

IL-33 expression in both PBLs and HBEs. It has been reported that IL-33 and ST2 can

365

be induced in CS-exposed mice (43). However, our study is the first to provide

366

evidence that IL-33 expression in HBEs and PBLs could be induced by CSE and LPS,

367

which are risk factors for the development of COPD. Moreover, low levels of IL-33

368

were detected in the supernatants of cultured ASMCs, irrespective of whether they

369

were stimulated, which was consistent with the negative immunostaining of ASMCs

370

in lung tissue. More importantly, we noted a significantly higher release of IL-33 for

371

COPD patients compared to healthy controls after PBLs were stimulated with the

372

mixture of CSE and LPS.

373

As an alarm signal, IL-33 is released to activate the immune system in response to

374

inflammatory signals or tissue injury caused by factors such as toxins, high free fatty

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acids, inflammation or oxidative stress (4, 31). Increased IL-33 has been reported to

376

be detected in synovial fibroblasts and keratinocytes after activation with IL-1β and

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TNF-α (26, 47). It has been speculated that IL-33 protein could be released when cell

378

damage occurs to alert the immune system to ‘danger’ (60). Cigarette smoking is the

379

key cause of the initiation of COPD, and LPS plays an important role in the

380

progression of COPD. Both of stimuli can activate inflammatory cells and enhance

381

cytokine release (15, 20). Consequently, we surmised that CSE and LPS are related to

382

the high expression of IL-33 in COPD, and they may play an important role in the

383

progression of COPD through modulating the release of IL-33.

384

In order to explore the mechanism of the high expression of IL-33 in COPD after CSE

385

and LPS stimulating, rs4986790, rs4986791 and rs11536889 polymorphisms of TLR4

386

gene were detected. TLR4 is a transmembrane protein that plays an essential role in

387

detecting LPS. After LPS binding to TLR4, the intracellular signaling events could

388

affect the production of proinflammatory cytokines (1). TLR4 SNPs have been

389

described in associating with greater susceptibility to various inflammatory diseases,

390

including rheumatoid arthritis (44), atherosclerosis (24), asthma (12) and COPD (45).

391

The rs4986790 and rs4986791 polymorphisms of TLR4 gene have been reported to be

392

strongly associated with endotoxin hyporesponsiveness to LPS in Caucasians (3) by

393

affecting the extracellular domain of TLR4. Another study showed that the

394

rs11536889 polymorphisms in the TLR4 gene were likely associated with COPD in

395

Japanese subjects (22). However, in the present study, we observed no rs4986790 and

396

rs4986791 polymorphisms in our samples, which is consistent with previous studies

397

on Chinese Han populations (19), Korean (25) and Japanese (38). Moreover, the

398

analysis of rs11536889 polymorphisms showed that there was no significant

399

difference between COPD patients and controls, which means TLR4 SNPs may not be

400

related to LPS hyporesponsiveness in our study.

401

In conclusion, this study provides compelling evidence for elevated IL-33 expression

402

in COPD, including in the airways and peripheral blood. IL-33 is mainly produced by

403

airway epithelial cells in airway inflammation and PBLs in systemic inflammation.

404

Moreover, CSE and LPS, which are risk factors for COPD, could both promote IL-33

405

secretion. Our study supports the importance of IL-33 signaling in the pathogenesis of

406

COPD, but the target cells and mechanism of action remain to be elucidated.

407

Therefore, more studies should be carried out to achieve a more comprehensive

408

understanding of the role of IL-33 in COPD and to determine the therapeutic value of

409

anti-IL-33 therapy in COPD patients.

410 411

ACKNOWLEDGMENTS

412

Present address of J. Xie: Department of Respiratory and Critical Care Medicine,

413

Tongji Hospital, Tongji Medical College, Huazhong University of Science and

414

Technology, Wuhan, China

415 416

Grants

417

This study was supported by the National Natural Science Foundation of China (No.

418

81370145, 81370156, 81470227 and 81200029), The National Support Programme of

419

the Twelfth five-year plan: clinical translational research on respiratory diseases (No.

420

2012BAI05B01).

421 422

Disclosures

423

No conflicts of interest, financial or otherwise, are declared by the authors.

424 425

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426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455

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the effector function of CD8(+) T cells. European Journal Of Immunology 41: 3351-3360, 2011. 61. Zhiguang X, Wei C, Steven R, Wei D, Wei Z, Rong M, Zhanguo L, and Lianfeng Z. Over-expression of IL-33 leads to spontaneous pulmonary inflammation in mIL-33 transgenic mice. Immunol Lett 131: 159-165, 2010.

650

Table 1. The characteristics of COPD patients and controls. COPD

Control

N

112

115

Age (years)

65.27±13.84

60.63±11.67

Sex (M:F)

95:17

97:18

Smoking (pack year)

34.16±17.96

33.84±15.29

Serum Cotinine (ng/ml)

151.50±138.47

145.38±107.19

FEV1 (L)

1.12±0.47

1.98±0.63

FEV1 % pred

55.71±13.33

106.31±12.69

FEV1/FVC

53.18±7.55

86.21±8.58

651

FEV1, forced expiratory volume in 1 second; FVC, forced vital capacity; FEV1 %

652

pred, FEV1 percent predicted. Data shown as means ± SD.

653 654 655 656 657 658 659 660 661 662 663 664 665

666

Table 2. Distribution of the TLR SNPs in COPD patients and controls. Genotype

COPD, n (%)

Control, n (%)

AA

20(100)

20(100)

AG

0(0)

0(0)

GG

0(0)

0(0)

CC

20(100)

20(100)

CT

0(0)

0(0)

TT

0(0)

0(0)

GG

13

14

CG

7

6

CC

0

0

χ2

P value*

0.114

0.736

rs4986790

rs49867901

rs11536889

667 668 669 670 671 672 673 674 675 676 677 678 679 680

*χ2 test

681

Figure legends

682

Figure 1. The levels of IL-33, sST2 and IL-1RAcP in plasma samples from patients

683

with COPD were significantly higher than those in controls. Each molecule was

684

separately measured by ELISA using plasma from study subjects. A, The levels of

685

IL-33 in plasma from subjects. B, The levels of sST2 in plasma from subjects. C, The

686

levels of IL-1RAcP in plasma from subjects. Data are presented as means ± SD. ****

687

P