Reduced transient receptor potential vanilloid 2 expression in alveolar macrophages causes COPD in mice through impaired phagocytic activity

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).

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.

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.

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.

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 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.

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 (M_r = 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.

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.

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.

To examine the effects of cigarette smoke extract (CSE) on TRPV2 expression in mouse alveolar macrophages in vitro, we cultured MH-S cells, mouse alveolar macrophages, and performed immunocytochemistry and Western blot analyses on cultured MH-S cells. Immunocytochemistry revealed that the number of the TRPV2-positive cells was reduced after exposure to 10% CSE for 24 h. While 57.5 ± 2.9% of F4/80-positive cells were TRPV2+ in vehicle group, 37.2 ± 3.4% of F4/80-positive cells were TRPV2+ in 10% CSE group (Fig. 1). Protein expression levels of TRPV2 in MH-S cells were evaluated by Western blotting. TRPV2 protein levels, relative to β-actin levels, were decreased in a time-dependent manner and were significantly reduced to ~ 20% of control at 24 h exposure to 10% CSE (Fig. 2a, b). The mRNA and protein expression of TRPV2 in MH-S cells was evaluated by qPCR, and Western blot analysis, respectively. The mRNA levels for TRPV2 were decreased by ~ 60% after exposure to 10% CSE at 24 h (Fig. 2c), while TRPV2 protein expression relative to β-actin was decreased by ~ 80% at 24 h (Fig. 2b).

We examined the effects of CSE on phagocytic function of MH-S cells by FITC-dextran internalization assay. MH-S cells were incubated with or without 10% CSE for 6, 12, and 24 h; cells were labelled with FITC-dextran for 6 h by adding 0.35 mg/ml FITC-dextran in the culture medium. Internalized FITC-dextran levels in MH-S cells were determined by measuring the absorbance of cell lysates at 485–535 nm; the luminescence intensity of cell lysates prepared from MH-S cells incubated with FITC-dextran was 10-fold higher than that from the cells incubated with vehicle alone. The luminescence intensity of cell lysates prepared from MH-S cells exposed to 10% CSE for 24 h prior to an incubation with FITC-dextran was 3.5-fold higher than those from the cells incubated with vehicle (Additional file 3: Figure S2). Because MH-S cells gave autofluorescence in the absence of FITC-dextran, we expressed the fold-induction of fluorescence intensity of the cells cultured with versus without FITC-dextran (Fig. 3). Importantly, the luminescence intensity was ~ 3.5-fold higher when MH-S cells were exposed to 10% CSE for 6–24 h prior to an incubation with FITC-dextran or vehicle (Fig. 3a). These results suggest that CSE stimulation reduced the phagocytic function of MH-S cells.

We next examined the effects of silenced TRPV2 expression on phagocytosis. TRPV2 protein levels in MH-S cells transfected with TRPV2 siRNA decreased (Additional file 4: Figure S1A, B). Fold induction of luminescence intensity from internalized FITC-dextran was lower in TRPV2 siRNA-transfected MH-S cells than in siGFP-transfected MH-S cells (6.0-fold vs 10.6-fold, p < 0.05) (Fig. 3b). These results suggest that TRPV2 participates in phagocytosis in MH-S cells.

We examined the expression of TRPV2 in alveolar macrophage collected by bronchoalveolar lavage (BAL) derived from cigarette smoke-exposed mice (Fig. 4a–d). The expression of TRPV2 was markedly decreased after 2 months smoke exposure in mice (Fig. 4a, c). Interestingly, the expression of TRPV2 was increased after six months smoke exposure when COPD is further progressed compared with 2 months (Fig. 4b, d).

We next examined the phagocytosis function of alveolar macrophages from either non-smoke- or cigarette smoke-exposed mice and TRPV2 KO mice at 4 months of age. As shown in Fig. 5 A, alveolar macrophages from non-smoke mice cultured in the presence of FITC-dextran have 6.2-fold higher luminescence luminous intensity relative to those cultured with vehicle; however, this value was reduced to 1.9-fold when alveolar macrophages were prepared from mice exposed to cigarette smoke for 2 months. Consistent with these data, alveolar macrophages derived from mice exposed to cigarette smoke for 6 months showed merely 2-fold greater phagocytosis function, relative to control alveolar macrophages. These results showed that the phagocytic function of alveolar macrophages was reduced by smoking for 2 months, and remained low after 6 months of cigarette smoke exposure. As shown in Fig. 5b, fold induction of luminescence intensity from internalized FITC-dextran was lower in TRPV2KO-derived alveolar macrophages than WT-derived alveolar macrophages (5.9 + 1.3 vs 4.4 + 1.0, p = 0.052), suggesting that alveolar macrophages from TRPV2KO mice exhibit weaker phagocytosis than alveolar macrophages from WT mice.

We examined lung morphological change in TRPV2KO mice and WT mice. An alveolar space gradually enlarges with advancing age in TRPV2KO mice (Fig. 6a), and mean linear intercept was significantly increased in 8-month-old TRPV2KO mice compared to that of 2-month-old TRPV2KO mice (Fig. 6c). Moreover, the emphysematous change was noted in TRPV2 KO mice by 2 months smoke exposure although it was not obvious in WT mice after 2 months-smoke exposure (Fig. 6b). An increase in mean linear intercept in TRPV2 KO mice was more intense than that in WT mice (Fig. 6d). This result shows that alveolar destruction was induced earlier in TRPV2 KO mice than WT mice and suggests that TRPV2KO mice are more susceptible for cigarette smoke-induced emphysema than WT mice.

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.

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.

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.