Background
Chronic obstructive pulmonary disease (COPD) is defined as small airway obstruction that is typically progressive, and is attributed to long-term exposure to toxic gases and particles, principally in relation to cigarette smoking. There is an increased number of alveolar macrophages in lung tissue from COPD patients [
1], which is directly associated with the degree of COPD [
2,
3]. Among the mechanisms relevant to COPD and emphysema development are chronic inflammation, protease-antiprotease imbalance, endoplasmic reticulum stress, accelerated senescence, and oxidative stress.
Alveolar macrophages act to maintain homeostasis of small airways by engulfing apoptotic cells and pathogens, as well as by releasing a variety of cytokines, chemokines, and proteases that regulate airway structure and function [
4‐
8]. Previous studies have provided evidence for defective phagocytic function of alveolar macrophages in COPD [
9‐
12]. Alveolar macrophages from COPD patients are defective in their function to engulf apoptotic bronchial epithelial cells and invading bacteria, despite smoking cessation [
13]. COPD patients have increased susceptibility to pneumococcal pneumonia, at least partly because of defective phagocytosis by alveolar macrophages as a result of smoke exposure [
12]. Thus far, numerous molecules have been identified as possible contributors to this altered alveolar macrophage phagocytosis, but the precise mechanisms remain unknown.
Transient receptor potential cation channel subfamily V member 2 (TRPV) channels are nonselective cation channels that have diverse physiological functions [
14]. Multiple TRPV channel subtypes have been identified in a variety of tissues [
15‐
17]. Recently, TRPV2 expressed in the alveolar macrophage cell membrane was revealed to mediate the earliest steps of macrophage phagocytosis [
18,
19]. Upon exposure to phagocytic substrates, TRPV2 in macrophages is recruited to the nascent phagosome and evokes depolarization of the plasma membrane, which then activates signalling pathways leading to the actin polymerization necessary for phagocytic receptor clustering. TRPV2-deficient macrophages are defective in chemoattractant-elicited motility after pathogen challenge, and TRPV2-deficient mice showed increased mortality after challenge with these pathogens.
In the present study, we aimed to understand the role of TRPV2 in cigarette smoke-induced COPD. We demonstrate that TRPV2 plays a key role during phagocytosis in alveolar macrophages. Cigarette smoke exposure significantly decreased TRPV2 expression in alveolar macrophages, both in vitro and in vivo. Furthermore, our data suggest that TRPV2 knockout mice exhibit increased susceptibility to cigarette smoke-induced COPD.
Methods
Reagents
The following reagents were used: F4/80 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and TRPV2 (LifeSpan BioSciences, Seattle, WA, USA) for Immunofluorescence and Western blot. Research cigarettes were purchased from the University of Kentucky. 4′6′-diamidino-2-phenylondole dihydrochloride (DAPI) was obtained from Vector Laboratories (Burlingame, California, USA).
Mice
TRPV2KO mice were generated as described previously [
20]. All primer sequences for genotyping are shown in Additional file
1 All animal experiments were approved by the Gunma University Animal Care and Use Committee, and were performed in accordance with its guidelines (Permit Number; 17–049). Mice were purchased from SLC Japan (Shizuoka). The smoke-exposed emphysema model was induced in 2-month-old female C57BL/6 mice (weight between 16.5 and 20.7 g) by exposure to the smoke of four unfiltered cigarettes per day (3R4F, University of Kentucky), 6 days per week for 2 and 6 months, by using an experimental-smoking apparatus as described previously [
20,
21]. Mice tolerated cigarette smoke exposure without evidence of toxicity (carboxyhaemoglobin levels at approximately 10%). All mice were euthanized by cutting the inferior vena cava to induce exsanguination and opening of the chest cavity to induce pneumothorax after inhaled sevoflurane before tissue harvest. This method of euthanasia was performed based on a rule of animal experiment of Gunma University.
Bronchoalveolar lavage (BAL)
BAL was performed by using a 20 G intravenous catheter inserted into the trachea. Lungs were lavaged four times with 0.75 ml phosphate-buffered saline (PBS), then centrifuged at 3000 × rpm for 5 min. Cell pellets were resuspended in 1.0 ml PBS, then used to determine total and differential cell counts with a haemocytometer and cytospins via staining with a Diff-Quick stain kit (Sysmex, Kobe, Japan). Macrophages were prepared by plating the BAL cells on culture dishes overnight and then by removing non-adherent cells. About 95% of the BAL cells were adherent cells. We should be aware that some of the adherent cells may not be macrophages but may be fibroblasts or mesenchymal cells.
Lung tissue processing
Right lungs were inflated by instillation of 10% formalin at a constant pressure of 25 cm formalin (for 10 min), then fixed for 24 h before paraffin embedding. Serial sections (4-μm thick) were prepared for histologic analysis.
Cell culture and stimulation
Primary alveolar macrophages were collected from C57BL/6 mice by the BAL method. MH-S cells (derived from mouse alveolar macrophages) were obtained from ATCC. Cells were cultured in RPMI 1640 medium, supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (Gibco BRL, Gaithersburg, MD, USA) at 37 °C in a 5% CO2 atmosphere.
Morphometry
After fixation, lung sections were stained with haematoxylin and eosin (H&E). Mean linear intercept (Lm; average distance between alveolar walls) was measured by light microscopy, as previously described [
20,
21]. Lm is the average distance between alveolar walls and proportional to the amount of pulmonary emphysema. 20 randomly selected representative images were captured from each slide using a motorized OLYMPUS microscope per lung specimen for each mouse. Lm was manually counted from images taken using a winROOF2013. All measurements were performed by a single blinded investigator. The lengths of all the portions on all lines were summed and divided by the total number of alveolar airspace. Airway and vascular structures were excluded from the analysis.
Immunofluorescence
Immunofluorescence labelling of lung sections was performed by using an antibody against F4/80 (1:100) and TRPV2 (1:250), as previously described [
18,
22]. The secondary antibody used for F4/80 and TRPV2 staining was Alexa Fluor 488-conjugated mouse anti-rat IgG (1:100; Invitrogen Life Technologies Corp., Carlsbad, CA, USA). Sections were then washed and mounted using DAPI-containing media to label cell nuclei.
Phagocytosis assay
Phagocytic function of alveolar macrophages was evaluated by using a FITC-dextran internalization assay. Alveolar macrophages (either MH-S cells or primary alveolar macrophages prepared from mouse BAL fluid) were incubated with 0.35 mg/ml FITC-dextran (Mr = 2,000,000; Sigma-Aldrich, St. Louis, MO, USA) at 37 °C for 6 h. Then, cells were washed three times with PBS. Uptake of FITC-dextran by macrophages was assessed by dissolution in RPMI 1640, and the absorbance of the samples at 485–535 nm was determined by using a Denley plate reader.
Western blot analysis
Cells were washed twice with PBS, harvested, and then lysed in RIPA buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS)) containing complete mini and phosSTOP solution (Roche). After sonication and removal of debris by centrifugation, 20 μg protein from each sample was resolved by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were immunoblotted with the indicated antibodies: cleaved TRPV2 (1:250) and β-actin (1:500). Antigens were revealed by Immobilon Western HRP Substrate (Millipore, Billerica, MA, USA) after incubation with horseradish peroxidase-conjugated anti-rabbit IgG and visualized by enhanced chemiluminescence (Amersham Biosciences, Buckinghamshire, UK). Band densities were quantified by using Image J software. For in vivo analysis, alveolar macrophages collected by BAL from five mice were pooled because the yield of alveolar macrophages by BAL is very few.
RNA isolation and qPCR
Total RNA was extracted from the mouse lung tissue and MH-S cells using ISOGEN reagent (Takara Bio, Kyoto, Japan) according to the manufacturer’s protocol. One microgram of RNA was used for reverse transcription with the RNA PCR Kit (Takara Bio) and qPCR analysis was performed using THUNDERBIRD SYBR qPCR Mix (TOYOBO, Osaka, Japan) according to the manufacturers’ protocol. qPCR analysis was carried out using a MX3000P quantitative system (Stratagene, La Jolla, CA). All primer sequences are shown in Additional file
2.
Statistical analysis
Data are expressed as the mean ± standard deviation. Significant differences were determined by using Student’s t-test and analysis of variance with Tukey–Kramer multiple comparison tests. A P-value of < 0.05 was considered significant.
Discussion
In the present study, we provided several lines of possibility indicating that the development of cigarette smoke-induced COPD is partly attributed to reduced phagocytosis in alveolar macrophages, mediated by cation channel TRPV2. First, CSE reduced TRPV2 expression and phagocytic function, as assessed by FITC-dextran internalization assays in cultured MH-S cells. Second, TRPV2 knockdown experiments with siRNA showed reduction of phagocytic function. Third, 2 months of cigarette smoke reduced TRPV2 expression in mice. Lastly, alveolar macrophages prepared by BAL from cigarette smoked-exposed mice exhibit reduced phagocytosis, compared with macrophages from non-smoke-exposed mice. These results suggest that TRPV2 in alveolar macrophages serves as a protective channel against cigarette smoke-induced COPD by participating in phagocytosis.
Suppression of TRPV2 expression in alveolar macrophages by cigarette smoke in vitro and in vivo
Immunocytochemistry of MH-S cells revealed that 10% CSE stimulation reduced the number of TRPV2-positive cells, while the number of F4/F80-positive cells seemed to be less affected by CSE. These results suggest that the CSE-related reduction in TRPV2-positive cells is not due to cell apoptosis. Consistent with this, qPCR and Western blot analyses revealed significant reduction of TRPV2 mRNA and protein levels, respectively, relative to β-actin levels. These findings deserve special attention because it is well documented that cigarette smoke induces oxidative damage, primarily through reactive oxygen species (ROS); ROS has been implicated in apoptosis induction in a variety of cells, including lung epithelial cells, via induction of caspase-3 activity [
23,
24]. The molecular mechanisms regulating a decrease in TRPV2 expression in the absence of apoptosis remain to be determined, but based on our finding that TRPV2 mRNA levels were robustly reduced by CSE, we suspect that ROS-mediated pathways that suppress TRPV2 gene transcription or TRPV2 mRNA function may regulate TRPV2 protein levels. Understanding the molecular mechanisms governing suppression of TRPV2 gene expression upon CSE treatment should provide novel insights into the mechanisms of CSE-mediated macrophage dysfunction.
It is notable that, while TRPV2 protein levels were strongly suppressed after 2 months of cigarette smoke exposure, they were restored by 6 months of exposure, when airspace enlargement developed further. The precise mechanisms of increased TRPV2 expression at 6 months of cigarette smoke exposure are unclear, but may involve compensatory mechanisms to prevent the destruction of lung architecture; we have previously reported that collagen type IV and surfactant protein-A (SP-A), which protect against development of COPD, are upregulated despite progression of destructive changes in the lung [
25]. Compensatory increases in TRPV2 during COPD progression may underscore the importance of this channel in maintaining alveolar structure.
We prepared macrophages from BAL cells by plating the BAL cells on culture dishes overnight and then by removing the non-adherent cells. We should be aware that this methods may have allowed some non-macrophages to be included in adherent cells. Thus, a decrease in TRPV2 protein in macrophages in BAL cells may be, at least in part, due to a decrease in the number of macrophages in BAL cells rather than the reduced expression of TRPV2 in macrophages.
The role of phagocytosis by alveolar macrophage in cigarette smoke-induced COPD
Increased oxidative stress and a subsequent chronic inflammatory response are responsible for many deleterious effects of cigarette smoke [
26‐
30]. In addition, increased apoptosis and defective alveolar macrophage phagocytosis contribute to cigarette smoke-induced COPD [
13]; several studies have reported a large number of apoptotic cells in the lungs of patients with COPD [
31] [
32]. Therefore, CSE and cigarette smoke exposure may suppress TRPV2-mediated phagocytosis. TRPV2KO mice have increased susceptibility to cigarette smoke-induced COPD, which suggests that impaired phagocytosis is important in the development of COPD.
The role of TRPV2 in cigarette smoke-induced COPD
Our finding that reduction of TRPV2 expression increases susceptibility to cigarette smoke-induced COPD is consistent with results reported by Link et al., who showed that TRPV2 plays a critical role in macrophage phagocytosis [
18]. Their study revealed that TRPV2-deficient peritoneal macrophages were defective in phagocytosis of a wide variety of phagocytic substrates, such as complement-coated latex beads, zymosan particles, and bacteria. TRPV2 is also required for phagocytic receptor clustering. In our study, we did not examine phagocytic substrates of alveolar macrophages in our cigarette smoke exposure mouse model. However, cigarette smoke contains a complex mixture of > 5000 chemicals, including ROS and carbonyl compounds, which directly injure lung epithelial surfaces [
33]. Many apoptotic cells including endothelial cells, alveolar epithelial cells, and inflammatory cells, accumulate in lung tissues of COPD patients [
34]. Therefore, in our cigarette smoke exposure mouse model, apoptotic cells are likely to serve as a major substrate for macrophage phagocytosis, also known as efferocytosis. Further studies are necessary to test whether alveolar macrophages utilize signalling through TRPV2 channels to cluster apoptotic cell recognition receptors and activate subsequent intracellular mechanisms that can change the phenotype and function of alveolar macrophages, given that recognition receptors for apoptotic cells may be distinct from those for microbial particles [
35].
Our finding that TRPV2-mediated phagocytosis is impaired in smoke-exposure mice suggests that TRPV2 agonists may have potential to ameliorate cigarette-induced COPD in humans. It is intriguing to speculate that probenecid, the prototypical uricosuric agent which has been reported to activate TRPV2 [
36], may have therapeutic potential for COPD. This possibility warrants thorough investigation in future studies.
Conclusion
The present study demonstrated that alveolar macrophages, in which TRPV2 is genetically disrupted or silenced by siRNA, exhibit defective phagocytic function. Together, these results suggest that TRPV2 may be a potential target for the treatment of cigarette smoke-induced COPD.
Acknowledgments
We are grateful to Keiko Arai, Miki Matsui and Takko Kobayashi for technical assistance. We thank Ryan Chastain-Gross, Ph.D., from Edanz Group (
www.edanzediting.com/ac) for editing a draft of this manuscript.
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