Animals

Female BALB/c Cr Slc mice (6-weeks) were purchased from Japan-SLC (Hamamatsu, Japan). This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources Commission on Life Sciences National Research Council, USA. The protocol was approved by Institutional Animal Care and Use Committee (08–183) in Yonsei University College of Medicine (Seoul, Korea), which has been fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International.

Mice exposed intranasally to GCE (100 μg/mouse) 3 times per week for 2 or 4 weeks (short- and long-term GCE exposure models, respectively).

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Figure 4. GCE promotes allergic phenotype through the TNF-α pathway.

Lung homogenates were harvested and used for measuring (a) IL-5, (b) IL-13 and (c) IFN-γ. (d) The level of serum IgE in the blood. Lung tissues were stained with (e) PAS and (f) Masson's Trichrome. (g) PAS-positive cells in peri-bronchial regions and (h) total collagen deposition in the lung tissue were quantitatively calculated. * indicates statistical significance between “Sham” and “GCE” (n = 5, p<0.05). ^# indicates statistical significance between “GCE” and “a-TNF” (n = 5, p<0.05). All data are representative of three independent experiments.

https://doi.org/10.1371/journal.pone.0047971.g004

GCE preparation

GCE was prepared as previously described [7]. Fifty grams of frozen German cockroaches were homogenized in liquid nitrogen and defatted in 200 mL of ethyl ether and 200 mL of ethyl acetate. Extractions were preformed with slow stirring at 4°C overnight in PBS containing 6 mmol/L 2-mercaptoethanol and 1 mg/mL 1-phenyl-3-(2-thiazolyl)-2-thiourea to prevent melanization. The extract was then centrifuged at 10,000×g for 30 minutes at 4°C, and the supernatant was finally filtered through 0.2-μm filters. The endotoxin from the supernatant was removed by the Detoxi-Gel^TM Endotoxin Removing Gel (Pierce Biotechnology, Rockford, IL). The endotoxin from the GCE was removed and measured to be below 0.01 EU/mL by the chromogenic Limulus Amebocyte Lysate test (Lonza, Walkersville, MD). The GCE contained 0.11 U/mg Bla g1.

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Figure 5. Serine protease in GCE develops pulmonary inflammation, alveolar macrophage infiltration and TNF-α expression.

(a) Scheme for the inhibition of GCE protease activity of GCE-induced asthma model. (b) AHR and (c) BAL cell count. (d) Alveolar macrophage, interstitial macrophage, and dendritic cell population in the lung tissue. (e) Quantitative analysis of alveolar macrophage from “d” panel. (f) Intracellular expressions of TNF-α in the alveolar macrophages of the GCE-induced asthma experiment. (g) Relative MFI ratio in “f” panel. * indicates statistical significance between “Sham” and “GCE” (n = 5, p<0.05). ^# indicates statistical significance between “GCE” and “GCE+Aprotinin” (n = 5, p<0.05). All data are representative of three independent experiments. AMs, alveolar macrophages; IMs, interstitial macrophages; DCs, dendritic cells.

https://doi.org/10.1371/journal.pone.0047971.g005

Protease activity measurement

Protease activity of GCE was measured using a Protease Fluorescent Detection Kit (Sigma-Aldrich, St. Louis, MO) according to the supplier's recommendations [21]. A fluorescein isothiocyanate (FITC)-casein and GCE were prepared and the fluorescence intensity was recorded by Luminescence Spectrometer (Perkin-Elmer, Oak Brook, IL).

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Figure 6. Alveolar macrophages are the major source of TNF-α in the GCE-induced asthma model.

(a) Scheme for the alveolar macrophage depletion of GCE-induced asthma model. Cl₂MDP, Cl₂MDP-containing liposomes; Liposome, control liposomes. (b) Macrophages from BAL fluid and (c) AHR in alveolar macrophage-depleted animals. (d) Intracellular TNF-α production in lung tissue. (e) Relative MFI ratio in “d” panel. * indicates statistical significance between “Liposome/sham” and “Liposome/GCE” (n = 5, p<0.05). ^# indicates statistical significance between “Liposome/GCE” and “Cl₂MDP/GCE” (n = 5, p<0.05). All data are representative of three independent experiments. Liposome/sham, control liposome-treated group; Liposome/GCE, control liposome-treated GCE-treated group; Cl₂MDP/sham, Cl₂MDP-containing liposome-treated sham group; Cl₂MDP/GCE, Cl₂MDP-containing liposome-treated GCE-treated group.

https://doi.org/10.1371/journal.pone.0047971.g006

Cell culture

MH-S and RAW264.7 cells (ATCC, Manassas, VA) were stimulated with GCE, ENMD-1068 (Enzo Life Sciences, Farmingdale, NY), aprotinin, or polymixin B (PMB; Sigma-Aldrich). The cells were cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum and 100 U/mL penicillin-streptomycin.

Liposome-encapsulated Cl₂MDP

Liposomes encapsulated with dichloromethylenediphosphonic acid disodium salt (Cl₂MDP; Sigma-Aldrich) were prepared as previously described [22]. Briefly, 86 mg phosphatidylcholine and 8 mg cholesterol were dissolved in 10 mL chloroform or 4 mL PBS, and a lipid film was manufactured by low vacuum rotary evaporation. The suspension, which was dissolved in either 10 mL Cl₂MDP or 4 mL PBS, was kept at room temperature for 2 hours and then sonicated for 5 minutes. The suspensions were then centrifuged at 100,000×g for 30 minutes to remove free Cl₂MDP. The Cl₂MDP-containing liposomes and the control liposomes were resuspended in 2 mL PBS.

AHR measurement

AHR was measured as previously described [23]. Mice were anesthetized (pentobarbital sodium, intraperitoneally), ventilated (flexiVent 5.1®; SCIREQ, Montreal, Canada) and challenged with a saline aerosol followed by increasing concentrations of methacholine (MCh; Sigma-Aldrich). Aerosols were generated with an ultrasonic nebulizer (Omron Healthcare, Kyoto, Japan) and delivered to the inspiratory line of the flexiVent using a bias flow of medical air.

BAL fluid

Bronchoalveolar lavage (BAL) fluid was obtained as previously described [23]. To collect BAL fluid, the lungs were lavaged with 1 mL Hank's balanced salt solution (HBSS) via the tracheostomy tube. Total cell numbers were counted with a hemocytometer. After the procedure, BAL fluid was centrifuged at 1,500×g for 3 minutes at 4°C, and then smears of BAL cells were prepared by cytocentrifugation (Cytospin3, Thermo, Billerica, MA) at 1,000 rpm for 3 minutes. BAL cells were stained with Hemacolor Staining Kit (Merck, Darmstadt, Germany) counted, and classified as neutrophils, eosinophils, lymphocytes, or macrophages.

Lung homogenate

For assessment of cytokine levels, lung tissues were homogenized in 20 mL/g tissue protein extraction reagent (Thermo Fisher Scientific Inc., Rockford, IL) using a tissue homogenizer (Biospec Products, Bartlesville, OK). Homogenates were incubated at 4°C for 30 min and then centrifuged at 1,000×g for 10 min. Supernatants were collected, passed through a 0.45-micron filter (Gelman Sciences, Ann Arbor, MI), and then stored at −70°C for assessment of cytokine levels.

Immunocytohistochemistry

Immunofluorescence staining of PAR-2 and TNF-α in Raw 264.7 cells and lung tissues was examined by confocal laser scanning microscopy (LSM700, Carl Zeiss, Jena, Germany). Cytospin-fixed RAW 264.7 cells or formalin-fixed, paraffin-embedded lung tissues were stained with FITC anti-mouse PAR-2 (SAM11, Santa Cruz Biotechnology, Santa Cruz, CA), phycoerythrin (PE) anti-mouse CD11b, and allophycocyanin (APC) anti-mouse TNF-α (BD Pharmingen^TM, San Jose, CA) at 4°C for 30 minutes. After staining, the samples were washed and observed under confocal laser scanning microscopy with excitation wavelengths of 493, 565, and 645 nm and emission wavelengths of 525, 575, and 660 nm, respectively.

Intracellular cytokine staining

Intracellular cytokine staining was performed using a Cytofix/Cytoperm kit (BD Biosciences, San Diego, CA) according to the supplier's recommendations. The cells were stained with PE/Cy7-anti-mouse CD4, PE-anti-mouse CD11b, PerCP/Cy5.5-anti-mouse CD11c and PE/Cy7-anti-mouse F4/80, permeabilized, and stained intracellularly with FITC-anti-mouse IFN-γ, PE-anti-mouse IL-5, APC-anti mouse IL-17, APC-anti-mouse TNF-α (BD Pharmingen), FITC- and PE-anti-mouse PAR-2 (Santa-Cruz Biotechnology).

Flow cytometric analysis

We performed multicolor-flow cytometric analysis (LSRII; BD Biosciences). The data were analyzed using FACSDiva (BD Biosciences) or FlowJo ver.7.6.2 (Three Star, Ashland, OR) and expressed as a percentage value or mean fluorescence intensity (MFI). The relative-MFI ratio was calculated relative to the control group.

ELISA

TNF-α, IFN-γ, IL-5 and IL-13 were detected by enzyme-linked immunosorbent assay (ELISA) with a DuoSet® ELISA (R&D Systems, Minneapolis, MN). Immunoglobulin (Ig)E was detected with an IgE ELISA set (BD Biosciences).

Pulmonary pathology analysis

Periodic Acid-Schiff (PAS) and Masson's Trichrome staining were performed in the formalin-fixed/paraffin-embedded lung tissues. Tissue sections were examined with an Olympus BX40 microscope in conjunction with an Olympus U-TV0.63XC digital camera (Olympus Corp., Melvile, NY). Images were acquired using DP Controller and Manager software (Olympus Corp.). PAS⁺cells per millimeter of bronchial basement membrane (mmBM) and Trichrome⁺pixels per total area (%) were measured by MetaMorph 4.6 (Universal Imaging, Downingtown, PA).

Statistical analysis

The data are expressed as mean±standard error. Statistical analyses were measured using SPSS ver.12.0 (Chicago, IL). Paired groups were compared using the Student's t-test, and all differences were considered significant at p<0.05.

Serine protease activity of GCE activates PAR-2 in macrophages

German cockroaches exhibit protease activity that includes serine- and cysteine-degrading enzymes. We measured the protease activity in GCE on a FITC-casein substrate. Compared to the typical standard curve of trypsin from 0.16 µg/mL to 20 µg/mL on the FITC-casein substrate, one milligram of the GCE contained protease activity that was equivalent to 21.87 μg/mL trypsin (Figure 1A and 1B). To identify the specific protease activity in GCE, GCE was incubated with several protease inhibitors that inhibited serine or cysteine protease classes. When GCE was incubated with aprotinin-a serine protease inhibitor, E-64-a cysteine protease inhibitor, phenylmethanesulfonyl fluoride (PMSF)-a serine/cysteine protease inhibitor, and soybean trypsin inhibitor (STI)-a trypsin inhibitor, each protease activities were decreased. In addition, one of these inhibitors, aprotinin, markedly reduced protease activity in the GCE (Figure 1B).

To determine PAR-2 immunolocalization following GCE stimulation in the mouse macrophages, alveolar macrophage cell-lines (MH-S cells) and peritoneal macrophage cell-lines (RAW264.7 cells) were incubated with either GCE or a combination of GCE and aprotinin. GCE stimulation led to internalization of the PAR-2 in the MH-S cells, while the cells incubated with a combination of GCE and aprotinin revealed similar patterns of control, as visualized by confocal imaging of intracellular staining (Figure 1C) and cell surface staining samples (Figure S1). In the RAW264.7 cells, PAR-2 expression of the cell surface decreased following GCE stimulation, but the cells incubated with a combination of GCE and aprotinin revealed similar to the control (Figure S2)

To confirm a specific interaction between the GCE and the PAR-2, MH-S cells were incubated with an ENMD-1068, a novel selective PAR-2 antagonist [24], [25], [26], and then stimulated with GCE. The intracellular expression of PAR-2 and TNF-α was markedly inhibited by ENMD-1068 (Figure 1D–G).

GCE induces TNF-α production in macrophages

To examine whether PAR-2 activation by serine proteases within GCE induces inflammation via macrophages, we studied TNF-α production and secretion in the MH-S and RAW264.7 cells. Intracellular TNF-α levels were significantly increased in GCE- stimulated MH-S cells, but these levels were not increased when GCE protease activity was inhibited by aprotinin. In GCE+PMB-stimulated condition, the endotoxin level less than 0.1 EU/mL in GCE had no effect in GCE-stimulated cells (Figure 2A and 2B). These results showed that serine protease but not endotoxin in GCE is critical for TNF-α production. The culture supernatants from cells grown under each condition revealed similar patterns of TNF-α production (Figure 2C). The results of RAW264.7 cells revealed similar to the MH-S cells (Figure S3).

To identify the kinetics of PAR-2 and TNF-α expression during GCE stimulation process, GCE was administered intranasally to BALB/c mice 3 times per week for 2 weeks (short-term GCE exposure model; Figure 2D). Intracellular PAR-2 and TNF-α levels of alveolar macrophages (CD11c⁺ and F4/80⁺ cells) were increased continuously for up to 2 weeks (Figure 2E–2G).

According to the immunohistochemistric analysis, TNF-α accumulation of macrophages were increased in the lung tissues of long-term GCE exposure model (3 times per week for 4 weeks; Figure S4).

GCE promotes pulmonary inflammation through the release of TNF-α

To determine whether GCE promotes allergic asthma-like symptoms, we used a long-term GCE exposure model (Figure 3A). In an AHR assay, mice that received GCE developed pulmonary resistance based on the requirement for increasing doses of MCh inhalation (Figure 3B). The mice receiving GCE exhibited inflammatory cell infiltration in the BAL fluid, and the majority of the cells in the BAL fluid were macrophages, lymphocytes, and neutrophils (Figure 3C). Intracellular TNF-α production and secretion of the macrophages were increased in the long-term GCE exposure model (Figure 3D–3F).

To confirm the helper T cell differentiation during GCE-stimulation, intracellular and secreted levels of different cytokines in CD4⁺ T cells were measured. The intracellular levels of IFN-γ, IL-5 and IL-17 were increased in the CD4⁺ T cells of the long-term GCE exposure mice (Figure S5A and S5B).IFN-γ, IL-5, IL-10, IL-13 and IL-17 production in lung, and serum IgE levels were also increased in this model (Figure 4A–4D; Figure S5C). Histologically, goblet cell hyperplasia and collagen deposition in the peri-bronchiolar area were exacerbated in the long-term GCE exposure model as compared to controls (Figure 4E–4H).

To confirm the role of TNF-α in the long-term GCE exposure model, we blocked TNF-α in these animals. We intravenously injected goat anti-mouse TNF-α polyclonal antibody (10 µg/mouse, R&D Systems) or goat IgG (10 µg/mouse, R&D Systems) into the animals intravenously 12 hours before the first, 7^th, 14^th, and 21^st administrations of GCE (Figure 3A). The development of AHR and airway inflammation following GCE administration was attenuated by the systemic neutralization of TNF-α in these mice (Figure 3B and 3C). TNF-α blockade reduced intracellular TNF-α production in the macrophages as well as TNF-α secretion from the lung tissues of the long-term GCE exposure mice (Figure 3D–3F). TNF-α blockade also decreased Th2 cytokine production, serum IgE levels, goblet cell hyperplasia, and peri-bronchial fibrosis in the long-term GCE exposure model (Figure 4).

To confirm the effect of TNF-α blockade, as a therapeutic agent, we blocked TNF-α just before the last challenge in a short-term GCE exposure model. In this model, both AHR and inflammatory cell infiltration in the BAL fluid also improved (Figure S6), and kinetically increased of PAR-2 and TNF-α levels of alveolar macrophages were diminished in the TNF-α blockade group (Figure 2E–2G).

Serine protease activity of GCE develops allergic pulmonary inflammation

We identified whether the serine protease activity of GCE has a critical role in the pulmonary allergic inflammation in the short-term GCE exposure model. We incubated GCE with aprotinin (100 µg) and administered the GCE+aprotinin intranasally to the mice 3 times per week for 2 weeks (Figure 5A). When the serine protease activity of GCE was inhibited by aprotinin, AHR (Figure 5B) and airway inflammation (Figure 5C) was attenuated. The inhibition of GCE protease activity reduced the infiltrations of alveolar macrophages (CD11c⁺ and F4/80⁺ cells; Figure 5D and 5E) and the intracellular TNF-α production of the alveolar macrophages (TNF-α⁺, CD11c⁺ and F4/80⁺ cells; Figure 5F and 5G), whereas it did not affect the infiltration of interstitial macrophages (CD11c⁻ and F4/80⁺ cells) nor dendritic cells (CD11c⁺ and F4/80⁻ cells) in the short-term GCE exposure model (Figure 5D and Figure S7).

Activated alveolar macrophages and their TNF-α production promote GCE-induced pulmonary inflammation

According to our in vitro and in vivo studies, we demonstrated that serine protease activity in the GCE stimulated macrophages, which are the major source of TNF-α production. To examine the function of alveolar macrophages in the short-term GCE exposure models, we intranasally treated mice with Cl₂MDP-containing liposomes (50 µL/mouse) or control liposomes (50 µL/mouse) intranasally 12 hours before the first administration of GCE and then selectively eliminated alveolar macrophages (Figure 6A). The number of macrophages in the BAL fluid and the AHR induced by the GCE treatment were significantly reduced in the alveolar macrophage-depleted mice compared to the non-depleted animals (Figure 6B and 6C). Intracellular TNF-α levels in the lung cells of short-term GCE exposure mice were significantly decreased by depletion of the alveolar macrophages (Figure 6D and 6E).