bs_bs_banner
doi:10.1111/jgh.12663
GASTROENTEROLOGY
Helicobacter pylori vacuolating cytotoxin induces apoptosis via activation of endoplasmic reticulum stress in dendritic cells Jung Mogg Kim,* Joo Sung Kim,† Nayoung Kim,†,‡ Su Hyuk Ko,* Jong Ik Jeon* and Young-Jeon Kim§ *Department of Microbiology, Hanyang University College of Medicine, †Department of Internal Medicine, Seoul National University College of Medicine, Seoul, ‡Department of Internal Medicine, Seoul National University Bundang Hospital, Seongnam, and §Department of Biotechnology, Joongbu University, Choongnam, Korea
Key words apoptosis, dendritic cells, ER stress, H. pylori vacuolating cytotoxin. Accepted for publication 22 May 2014. Correspondence Professor Jung Mogg Kim, Department of Microbiology, Hanyang University College of Medicine, 17 Haengdang-dong, Sungdong-gu, Seoul 133-791, Korea. Email: [email protected]
Abstract Background and Aim: Dendritic cells (DCs) are observed on the Helicobacter pyloriinfected gastric mucosa. DCs generally play an important role in the regulation of inflammation. Although stimulation of gastric epithelial cells with H. pylori vacuolating cytotoxin (VacA) has been reported to induce apoptosis and endoplasmic reticulum (ER) stress, the effects of VacA on the DC apoptotic response have not been well elucidated. This study was conducted to investigate the role of H. pylori VacA on the apoptotic process and ER stress in DCs. Methods: Murine and human DCs were generated from specific pathogen-free C57BL/6 mice and human peripheral blood mononuclear cells, respectively. DCs were incubated with purified VacA, after which Bax activation, cytochrome c release, and DNA fragmentation for apoptosis were measured by fluorescent microscopy, immunoblot, and ELISA. ER stress-related molecules such as GRP78 and CHOP were analyzed by immunoblot. Results: Treatment of DCs with purified H. pylori VacA resulted in the induction of apoptosis. DC stimulation with VacA led to the translocation of cytoplasmic Bax to mitochondria and cytochrome c release from mitochondria. H. pylori VacA induced signals for ER stress early during the stimulation process in DCs. Furthermore, suppression of ER stress resulted in a significant inhibition of the VacA-induced apoptosis in DCs. Conclusion: These results suggest that ER stress is critical for regulation of DC apoptotic process in response to VacA stimulation.
Introduction Helicobacter pylori is a Gram-negative bacterium that colonizes the stomach mucus layer and is the causative agent of diseases such as chronic gastritis, peptic ulcers, gastric cancers, and gastric MALT lymphoma.1 In addition, H. pylori infection is associated with a variety of motility, endocrine and acid-secretory abnormalities that could drive the symptoms of functional dyspepsia.2 In a meta-analysis of randomized controlled trials, H. pylori eradication had a small but statistically significant effect in controlling the symptoms of functional dyspepsia, indicating that H. pylori is involved in the pathogenesis of functional dyspepsia.2 Although the bacteria do not invade the gastric lamina propria, the infection leads to the infiltration of several immune cells.3 Vacuolating cytotoxin (VacA) is one of the major virulence factors in the pathogenesis of H. pylori-related diseases.4 VacA can induce apoptosis in gastric epithelial cells and eosinophils, which can lead to gastric mucosal inflammation.5–7 Dendric cells (DCs) are professional antigen (Ag)-presenting cells that play a critical
role in the induction of immunity. Since H. pylori VacA downregulates DC maturation,8,9 it is possible that VacA may be one of the factors that influence the fate of DCs during the infection. Previous report showed that the exposure of monocytederived DCs to H. pylori itself did not induce apoptosis,10 but this study performed using the live bacteria. Therefore, apoptosis induced by stimulation of DCs with purified VacA may be different from that by exposure of live bacteria. Endoplasmic reticulum (ER) stress-mediated apoptosis is implicated in the pathophysiology of human diseases.11,12 Both mitochondria-dependent and independent cell death pathways have been described to mediate apoptosis in response to ER stress.13 The available evidence suggests that transcriptional induction of the C/EBP homologous protein (CHOP) is critical in ER stress-induced apoptosis through suppression of Bcl-2 activation.14 ER stress can also induce mitochondrial dysfunction and apoptosis through organelle crosstalk between the ER and mitochondria.13 Recently, Akazawa et al. demonstrated that VacA-induced apoptosis in gastric epithelial cells was associated with ER stress.15
Journal of Gastroenterology and Hepatology 30 (2015) 99–108 © 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd
99
Vacuolating cytotoxin-induced apoptosis
JM Kim et al.
However, little information is available on DC apoptosis and ER stress in response to stimulation with H. pylori VacA. The goal of this study was to investigate the role of VacA in modulating the apoptotic process of DCs. H. pylori VacA is shown herein to induce DC apoptosis through ER stress.
and IL-4 (20 ng/mL, PeproTech) for 6 days. In some experiments, DCs were pretreated with the indicated concentration of salubrinal (Sigma Chemical Co., St. Louis, MO, USA) or 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF, Sigma) for 1 h, and then stimulated with VacA (10 μg/mL) for another 24 h.
Methods
Generation of murine bone marrow-derived DCs and DC2.4 cell culture. Femurs and tibias of specific pathogen-free C57BL/6 mice (4–12 weeks old, Orient Experimental Animal, Seoungnam, Korea) were harvested and bone marrow cells were obtained as previously described.8,18,19 For DC generation, RPMI 1640 media supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco BRL, Gaithersburg, MD, USA), 1% antibiotics, L-glutamine (2 mM), 2-mercaptoethanol (55 μM), murine recombinant GM-CSF (10 ng/mL, PeproTech, Rocky Hill, NJ, USA), and murine recombinant interleukin (IL)-4 (10 ng/mL, PeproTech) was used. After 6 days of culture, DCs were harvested and stimulated with VacA. All animal procedures were approved by Hanyang University College of Medicine. CD11c+ cell purities of greater than 95% were routinely achieved as determined by flow cytometric analysis. DC2.4, an immature murine DC cell line, was cultured in RPMI 1640 supplemented with 10% FBS, 1% antibiotics, L-glutamine (2 mM), sodium pyruvate (1 mM), and nonessential amino acids (2 mM). These cells were grown at 37°C with 5% CO2 as previously described.8
Analysis of apoptosis. For a morphologic assessment of cells undergoing apoptosis, cells were stained with the DNA dye Hoechst 33258 (Calbiochem, San Diego, CA, USA).6 To detect morphologic changes in the mitochondrial inner-membrane electrochemical potential in living cells, a mitochondrial staining kit (Sigma catalog #CS0390) was used according to the manufacturer’s instructions.6 Briefly, cells were cultured in Lab-Tek II 4 well-chamber slide (culture area 1.7 cm2; Nunc, Rochester, NY, USA). For mitochondrial stain, the commercially prepared fluorescent dye JC-1 (5,5′,6,6′-tetrachloro-1,1′3,3′-tetraethyl benzimidazol carbocyanine iodide) was added to the cultured chamber (0.5 mL/chamber slide), and cells were then incubated for 20 min at 37°C in a humidified atmosphere containing 5% CO2. Cell morphology was observed using a fluorescence microscope DMI4000B (Leica Microsystems GmbH, Wetzlar, Germany). In normal cells, the JC-1 dye concentrates in the mitochondrial matrix where it forms red fluorescent aggregates. During apoptosis, the mitochondrial membrane dissipates and the dye disperses throughout the entire cell, leading to a shift from JC-aggregates (red) to JC-1 monomers (green fluorescence).6 To assess the number of cells undergoing apoptosis, DCs were incubated with FITC-conjugated annexin V and propidium iodide (PI) according to the manufacturer’s instructions (Apoptosis Detection Kit; R&D Systems, Minneapolis, MN, USA) and then analyzed by flow cytometry (Becton Dickinson, San Jose, CA, USA).20 The mitochondrial transmembrane potential was assessed using tetramethylrhodamine ethyl ester (TMRE; Invitrogen-Molecular Probes, Eugene, Oregon, USA) and flow cytometry, as previously described.6 To assess DNA fragmentation, oligonucleosome release into the cytoplasm was assayed using the Cell Death Detection ELISAPLUS kit (Roche Diagnostics, Mannheim, Germany).6 The amount of cytosolic cytochrome c (human or mouse) in the cell lysates was quantified using an ELISA-based cytochrome c assay kit according to the manufacturer’s instructions (Quantikine, R&D Systems). All assays were performed in triplicate.
Generation of human monocyte-derived DCs. Peripheral blood mononuclear cells (PBMCs) were separated from the peripheral blood of volunteers using Ficoll-Paque (Histopaque-1077) density-gradient centrifugation. The Ethics Committee of Seoul National University Hospital approved this study. Briefly, CD14+ cells were purified from PBMCs using a magnetic cell separation system (Miltenyi Biotec, Bergisch Gladbach, Germany). The purity of CD14+ cells counted by fluorescein isothiocyanate (FITC-conjugated mouse anti-CD14 mAb, and flow cytometry was > 95%. For the generation of human DCs, purified CD14+ cells were plated in ultralow-adherence 24-well plates (Costar) at a density of 5 × 105 cell/mL and cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 1% antibiotics, L-glutamine (2 mM), 2-mercaptoethanol (55 μM), human recombinant GM-CSF (50 ng/mL, PeproTech),
Immunoblot analyses. Preparation of cytosolic and mitochondrial protein was performed as previously described.21,22 Fifteen to fifty micrograms of protein/lane were size-fractionated on polyacrylamide minigels (Mini-PROTEIN II; Bio-Rad) and electrophoretically transferred to a nitrocellulose membrane (0.1-μm pore size). Specific proteins were detected using primary antibodies (Abs) with 1: 1000 dilution, and the primary Abs immunoreactive proteins were visualized using goat anti-rabbit or anti-mouse secondary Abs conjugated to horseradish peroxidase, followed by enhanced chemiluminescence (ECL system; Amersham Life Science, Buckinghamshire, England) and exposure to X-ray film.21,22 Abs against cytochrome c, cytochrome c oxidase subunit IV (COX IV), CHOP, and actin were acquired from Cell Signaling Technology, Inc. (Beverly, MA, USA). Abs against glucose-regulated protein 78 (GRP78) and Bax, and anti-mouse,
Purification of H. pylori VacA. VacA-producing H. pylori strain 60190 (ATCC 49503, CagA+, vacA s1a/m1) was grown in sulfite-free Brucella broth containing 0.5% charcoal (untreated, granular 8–20 mesh) at 37°C under microaerophilic conditions. VacA was purified from broth culture supernatants according to previously described.6,8,16 Professor Patrice Boquet, Laboratoire de bacteriologie, Hopital de l’Archet 2, France, kindly supported the VacA protein purification method.17 Immediately before use in cells, the purified VacA protein was activated through the addition of 250 mM HCl until the pH reached 2.0. NH4Cl (5 mM) was also added to the medium to enhance the VacA activity.7,15
100
Journal of Gastroenterology and Hepatology 30 (2015) 99–108 © 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd
JM Kim et al.
anti-rabbit, and anti-goat secondary Abs conjugated to horseradish peroxidase were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The active form of specific anti-Bax-NT was from Upstate Biotech (Temecula, CA, USA) and Vectashield mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) was from Vector Laboratories (Burlingame, CA, USA). Immunofluorescence microscopy for Bax and cytochrome c. Immunofluorescence microscopy was analyzed using mAbs against active Bax and cytochrome c (Santa Cruz), as described previously.6 Briefly, DCs were seeded (5 × 104 cells in 0.2 mL of RPMI 1640/well) on 8-well poly-D-lysinecoated culture microslides (Santa Cruz). After treatment with VacA, cells were incubated with 100 nM Mitotracker Red CMXRos (Invitrogen-Molecular Probes, dilution, 1: 1000) for 20 min at 37°C in a humidified atmosphere containing 5% CO2 and fixed with 100% methanol for 10 min. For permeabilization, cells were treated with 0.3% Triton X-100 in PBS for 30 min at room temperature, after which the cells were incubated with the active form of specific anti-Bax-NT or anti-cytochrome c Abs (Upstate Biotech; dilution, 1: 1000) for 2 h. Cells were then treated with Alexa fluor 488-conjugated secondary Ab against rabbit IgG for 1 h. Images were captured using a fluorescence microscope DMI4000B (Leica Microsystems GmbH). Transfection assay. A Bax-specific silencing small interfering RNA (siRNA) was constructed as previously described.5 Negative control non-silencing RNA was purchased from Invitrogen. Predesigned siRNA against CHOP and control-scrambled siRNA were purchased from Santa Cruz Biotechnology.23 Transfection was performed according to the procedure described previously.8,24 Statistical analyses. Data are presented as the mean ± standard deviation (SD) or mean ± standard error of the means (SEM). The Wilcoxon’s rank sum test was used for statistical analysis.
Results H. pylori VacA induces apoptosis of DCs. Mouse DCs were treated with purified VacA, and apoptosis was assessed by several methods. Figure 1a shows nuclear fragmentation characteristics of apoptosis in VacA-stimulated DCs stained with the
Vacuolating cytotoxin-induced apoptosis
DNA dye Hoechst 33258. The fluorescent dye JC-1 was used to stain mitochondria. In normal cells, the JC-1 dye concentrated in the mitochondrial matrix where it forms red fluorescent aggregates. During apoptosis induced by VacA stimulation, the mitochondrial membrane dissipated, and the dye dispersed throughout the entire cell, leading to a shift from red (JC aggregates) to green fluorescence (JC-1 monomers) (Fig. 1b). Cells stained with FITC annexin V to detect the externalization of phosphatidylserine to the outer leaflet of the cell membrane were evaluated by flow cytometry (Fig. 1c). In addition, staining with TMRE was performed to detect reduced mitochondrial transmembrane potential (Fig. 1d). Results showed that the numbers of apoptotic cells in the VacA-stimulated cultures were higher than the control cultures (Fig. 1e,f,g).
Cytochrome c release from mitochondria and mitochondrial trafficking of activated Bax in DCs stimulated with H. pylori VacA. We next investigated whether VacA could induce cytochrome c release from mitochondria in DCs. As shown in Figure 2a, treatment with H. pylori VacA increased levels of cytochrome c in the cytosolic fraction but decreased in the mitochondrial fraction, indicating that VacA induces the release of cytochrome c from the mitochondria. Concurrently, activated Bax signals were concentrated within the mitochondrial fraction after stimulation with purified VacA. We measured cytochrome c release from mouse DCs using ELISA. Results showed that a significant release of cytochrome c was first observed at 24 h in mouse DCs (Fig. 2b). To confirm this result, immunofluorescent microscopy was performed. The distribution of cytochrome c was diffuse in the cytoplasm of VacA-treated DCs. In contrast, the distribution of cytochrome c was only localized within the mitochondria of untreated cells (Fig. 2c). In addition, the mitochondrial trafficking of activated Bax was shown in VacA-treated cells (Fig. 2d). We next determined whether VacA might influence apoptosis in human monocyte-derived DCs. Results using human monocytederived DCs were shown similar to those using murine DCs. Thus, the cytochrome c in the cytosolic fraction was increased, and in the mitochondrial fraction, cytochrome c was decreased but activated Bax signal was increased (Fig. 3a). In this experimental system, a significant release of cytochrome c in cytoplasm began 18 h after VacA stimulation (Fig. 3b). Consistent with this, an increase in DNA fragmentation was observed during the first 18 h after VacA
▶ Figure 1 Apoptosis in VacA-stimulated DCs. DCs generated from C57BL/6 mice were incubated with VacA (10 μg/mL) for 24 h. (a) Cells were fixed with 2% paraformaldehyde and stained with Hoechst dye 33258 (x 400). Apoptotic bodies are shown in VacA-treated cells (arrowhead). (b) Cells were stained with JC-1 dye and visualized under a fluorescent microscope (x 400). Normal cells show red, granular mitochondrial staining, whereas apoptotic cells show green, diffuse cytoplasmic staining. (c) Cells were incubated with FITC-conjugated annexin V and propidium iodide (PI), and analyzed by flow cytometry. Viable cells have low FITC-annexin V and low PI staining (left lower quadrant), and apoptotic cells have high FITC-annexin V and low PI staining (right lower quadrant, R1). Data are representative of three separate experiments. (d) Cells were incubated with 75 nM TMRE for 30 min at 37°C and analyzed by flow cytometry. The area marked as M1 to the left of the major peak contains the apoptotic cell population. Data are representative of three separate experiments. (e, f, and g) Quantification of apoptotic DCs in response to stimulation with VacA. (e and f) Quantitative data for R1 and M1. Culture conditions are the same as in Figure 1c,d. Values are the means ± SEM (n = 3). (g) Apoptosis in VacA-stimulated DCs. DCs generated from C57BL/6 mice were incubated with VacA (10 μg/mL) for 24 h. Cell death detection ELISA was measured as a fold increase in stimulated cells compared with each control (mean ± SEM, n = 5). *P < 0.05 versus untreated control. DCs, dendritic cells; FITC, fluorescein isothiocyanate; SEM, standard error of the means; TMRE, tetramethylrhodamine ethyl ester; VacA, vacuolating cytotoxin.
Journal of Gastroenterology and Hepatology 30 (2015) 99–108 © 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd
101
Vacuolating cytotoxin-induced apoptosis
102
JM Kim et al.
Journal of Gastroenterology and Hepatology 30 (2015) 99–108 © 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd
JM Kim et al.
Journal of Gastroenterology and Hepatology 30 (2015) 99–108 © 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd
Vacuolating cytotoxin-induced apoptosis
103
Vacuolating cytotoxin-induced apoptosis
JM Kim et al.
Figure 2 Cytochrome c release and Bax translocation in VacA-stimulated DCs. (a) DCs generated from C57BL/6 mice were incubated with VacA (10 μg/mL) for the indicated time period, and cytosolic and mitochondrial fractions were prepared. The signals of cytochrome c and activated Bax were detected by each mAb. Abs against actin or COX IV were used to demonstrate equal protein loading. Data are representative of three or more independent experiments. (b) Cytosolic fractions were prepared and released cytochrome c was measured using an ELISA kit. Data represent as the fold increase in stimulated cells compared with the unstimulated control (mean ± SEM, n = 5). (c and d) DCs were incubated with or without VacA (10 μg/mL) for 24 h, and immunofluorescent microscopy were performed. (c) The cells were stained with anti-cytochrome c Ab (green), Mitotracker Red (red, mitochondria), and DAPI (blue, nucleus). VacA-treated DCs show diffuse distribution of cytochrome c in the cytoplasm and untreated control cells show a localized pattern of cytochrome c within mitochondria. (d) The cells were stained with the active form-specific anti-Bax-NT Ab (green), Mitotracker RED (red, mitochondria), and DAPI (blue, nucleus). Signals of activated Bax are shown in the mitochondria of VacA-treated DCs. The data are representative of at least five experiments. DAPI, 4′,6-diamidino-2-phenylindole; DCs, dendritic cells; SEM, standard error of the means; VacA, vacuolating cytotoxin. ◀
Figure 3 Effects of VacA on apoptosis in human monocyte-derived DCs. (a) Human monocyte-derived DCs were incubated with VacA (10 μg/mL) for the indicated time period, and cytosolic and mitochondrial fractions were prepared. Cytochrome c and activated Bax signals were detected by immunoblots. Abs against actin or COX IV were used to demonstrate equal protein loading. Data are representative of three or more independent experiments. (b and c) Time course of cytochrome c and DNA fragmentation in human monocyte-derived DCs treated with VacA was same. Human DCs were stimulated with VacA (10 μg/mL) for the indicated time period. (b) Cytosolic fractions were prepared and released cytochrome c was measured using an ELISA kit. Data represent as the fold increase in stimulated cells compared with the unstimulated control (mean ± SEM, n = 5). (c) Apoptosis was assessed using a cell death detection ELISA. Data represent as a fold increase in stimulated cells compared with unstimulated control (mean ± SEM, n = 5). *P < 0.05 versus untreated control (d) Human DCs were stimulated with the indicated concentration of VacA for 24 h. Apoptosis was assessed using a cell death detection ELISA. Data represent as a fold increase in stimulated cells compared with unstimulated control (mean ± SEM, n = 5). *P < 0.05 versus untreated control. Abs, antibodies; DCs, dendritic cells; SEM, standard error of the means; VacA, vacuolating cytotoxin.
stimulation, and apoptosis continued to increase over the 48 h post-stimulation (Fig. 3c). The magnitude of DNA fragmentation was dependent on the concentration of VacA used for stimulation (Fig. 3d). The concentration of VacA that gives half-maximal response (EC50) was 5.08 μg/mL calculated by SigmaPlot 10.0 104
software (Systat Software Inc., San Jose, CA, USA). In the present study, we used 10 μg/mL of VacA because more than 10 μg/mL of VacA showed maximal induction of apoptosis. To evaluate whether Bax activation might be related to cytochrome c release and DNA fragmentation, the transfection of a
Journal of Gastroenterology and Hepatology 30 (2015) 99–108 © 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd
JM Kim et al.
Figure 4 Cytochrome c release and DNA fragmentation in Baxsuppressed DCs stimulated with VacA. DC2.4 cells were transfected with Bax siRNA or non-silencing control siRNA (NS-RNA) for 48 h. The data are representative of three independent experiments. (a) The transfected cells were either untreated or stimulated with VacA (10 μg/mL) for 18 h. Immunoblot analysis for the active form of Bax-NT in mitochondrial fractions was performed. (b) The transfected cells were stimulated with VacA (10 μg/mL) for 24 h. Cytosolic levels of cytochrome c and oligonucleosome were measured using each ELISA kit. Data are expressed as mean fold-induction ± SEM of each parameter relative to unstimulated controls (n = 5). *P < 0.05. DCs, dendritic cells; SEM, standard error of the means; siRNA, short interfering RNA; VacA, vacuolating cytotoxin.
DC2.4 cell line with siRNA was used. Transfection with Bax siRNA reduced the mitochondrial levels of phospho-Bax in VacA-treated cells (Fig. 4a). In this experimental system, cells transfected with siRNA against Bax showed significant decreases in cytochrome c release and DNA fragmentation compared to control cells (Fig. 4b).
Vacuolating cytotoxin-induced apoptosis
Figure 5 VacA-induced ER stress is associated with apoptosis in DCs. (a) Human monocyte-derived DCs were incubated with VacA (10 μg/mL) for the indicated time period. Signals of CHOP and GRP78 were measured by immunoblot analysis. Results are representative of three independent experiments. (b) DCs were pretreated with the indicated concentration of salubrinal or AEBSF for 1 h, and then stimulated with VacA (10 μg/mL) for another 24 h. Data are expressed as the percent inhibition of cytochrome c release relative to unstimulated controls ± SEM (n = 5). *P < 0.05 versus unstimulated control (c) ER stress inhibitors attenuate VacA-induced cytochrome c release and DNA fragmentation. Human monocyte-derived DCs were treated with salubrinal (25 μM) and AEBSF (300 μM) prior to VacA stimulation. Cytosolic levels of cytochrome c and oligonucleosome after 24 h stimulation with VacA (10 μg/mL) were measured using ELISA kits. Data are expressed as the mean fold-induction ± SEM of each parameter relative to unstimulated controls (n = 5). *P < 0.05. AEBSF, 4-(2-aminoethyl) benzenesulfonyl fluoride; CHOP, C/EBP homologous protein; DCs, dendritic cells; ER, endoplasmic reticulum; SEM, standard error of the means; VacA, vacuolating cytotoxin.
H. pylori VacA-induced apoptotic response associated with ER stress. Stimulation of human DCs with VacA increased CHOP and GPR78 in (Fig. 5a). Compared to the
Journal of Gastroenterology and Hepatology 30 (2015) 99–108 © 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd
105
Vacuolating cytotoxin-induced apoptosis
JM Kim et al.
fact that cytochrome c release and DNA fragmentation were first observed 18 h poststimulation of VacA in human DCs (Fig. 3b,c), the induction of ER stress seems to be an earlier response than the apparent apoptotic phenomena. To evaluate the role of ER stress and VacA-induced apoptosis, VacA was added to human DCs following pretreatment with salubrinal or AEBSF. Pretreatment of DCs with salubrinal (≥ 10 μM) or AEBSF (≥ 100 μM) for 1 h significantly inhibited the VacA-induced release of cytochrome c (Fig. 5b). In addition, pretreatment with salubrinal or AEBSF significantly inhibited cytochrome c release and DNA fragmentation induced by VacA stimulation (Fig. 5c). Increased expression of CHOP protein was also observed in DC2.4 cells (Fig. 6a). To confirm the relationship between ER stress and VacA-induced apoptosis, we evaluated whether suppression of CHOP signals by siRNA transfection could affect apoptotic markers. Transfection with CHOP siRNA reduced CHOP signal in VacA-stimulated cells (Fig. 6b). In this experimental system, significant decreased cytochrome c release and DNA fragmentation occurred in cells transfected with siRNA against CHOP compared to control cells under VacA-stimulated conditions (Fig. 6c). These results suggest that ER stress is associated with DC apoptotic phenomenon in response to VacA stimulation.
Discussion In the present study, we demonstrated that one of the early responses to stimulation of DCs with H. pylori VacA is the induction of ER stress, which leads to apoptosis as a relatively late response. At the molecular level, mitochondria plays an important role in transmitting apoptotic signals through the expression of Bax and cytochrome c, which are considered key proteins in apoptosis.6 The present study showed that VacA increased levels of activated Bax in the mitochondrial fraction, increased levels of cytochrome c in the cytosolic fraction, and decreased levels of cytochrome c in the mitochondrial fraction. In addition, transfection with siRNA against Bax significantly decreased cytochrome c release and DNA fragmentation. These results suggest that the activated response of Bax signal to H. pylori VacA can lead to cytochrome c release and apoptosis in DCs. The cytochrome c release and the apparent apoptotic phenomena were observed 18 h after VacA stimulation in DCs, which seems to be a relatively late response. Based on these observations, we postulated that the ability of VacA to induce apoptosis might result from the induction of certain molecular events during early responses. Galgani et al. demonstrated that the exposure of monocytederived DCs to H. pylori itself did not induce apoptosis.10 In addition, DC activation and maturation were independent of the cag PAI and VacA status of H. pylori.25 However, these two studies performed using the live bacteria. Therefore, apoptosis induced by treatment with purified VacA may be different from that by exposure of live bacteria to DCs. Although a recent paper showed that VacA-induced apoptosis in gastric epithelial cells was associated with ER stress,15 the specific mechanisms involved in ER stress-induced apoptosis have not been reported in DCs. In the present study, stimulation of human DCs with H. pylori VacA increased signals of GRP78 and CHOP 1 h after stimulation. In murine DCs, expression of CHOP protein was observed 6 h after stimulation. Compared to the fact that the 106
Figure 6 Suppression of CHOP attenuates cytochrome c release and DNA fragmentation in VacA-stimulated DCs. (a) DC2.4 cells were incubated with VacA (10 μg/mL) for the indicated time period. CHOP and actin proteins were measured by immunoblot analysis. Results are representative of three independent experiments. (b) DC2.4 cells were transfected with CHOP siRNA or non-silencing control siRNA (nsRNA). After 48 h, the transfected cells were either untreated or stimulated with VacA (10 μg/mL) for 24 h. CHOP and actin proteins were measured by immunoblot analysis. These results are representative of three independent experiments. (c) The transfected cells with each siRNA were stimulated with VacA (10 μg/mL) for 24 h. Cytosolic levels of cytochrome c and oligonucleosome release were measured using ELISA kits. Data are expressed as the mean fold-induction ± SEM of each parameter relative to unstimulated controls (n = 5). *P < 0.05. CHOP, C/EBP homologous protein; DCs, dendritic cells; NS-RNA, non-silencing control siRNA; SEM, standard error of the means; siRNA, short interfering RNA; VacA, vacuolating cytotoxin.
Journal of Gastroenterology and Hepatology 30 (2015) 99–108 © 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd
JM Kim et al.
cytochrome c release and the apparent apoptotic phenomena were observed 18 h post-stimulation of VacA in DCs, the induction of ER stress may be earlier than that of apoptosis. In the present study, we used salubrinal and AEBSF to inhibit ER stress signaling. Salubrinal acts as a selective inhibitor of eIF2α dephosphorylation to protect cells from ER stress,26 and the serine protease inhibitor AEBSF is reported to inhibit ER stressinduced proteolysis of ATF6, resulting in inhibition of ATF6target gene transcription by ER stress.13,27 Pretreatment of DCs with the chemical inhibitors salubrinal or AEBSF significantly inhibited the VacA-induced release of cytochrome c and DNA fragmentation. In addition, cells transfected with CHOP siRNA significantly decreased cytochrome c release and DNA fragmentation compared to control cells. These results suggest that ER stress is critical for regulation of DC apoptotic process in response to VacA stimulation. Tsugawa et al. demonstrated that autophagy and cytotoxinassociated gene A degradation are induced by the H. pylori VacA, which acted via decreasing levels of intracellular glutathione, causing reactive oxygen species (ROS) accumulation and Akt activation.28 The unfolded protein response (UPR) is a collection of adaptive signaling pathways that evolved to resolve protein misfolding, in which the ROS production is known to be associated with the UPR and ER stress.11,13 Therefore, it is possible that VacA may induce the decreasing glutathione levels and ROS accumulation, leading to ER stress in DCs. Our study used pharmacologic doses of VacA to induce ER stress and apoptosis in DCs. Since the in vitro pharmacologic dose used in the present study is presumed to be higher than the physiologic concentration in gastric mucosa infected with toxigenic H. pylori, further study is needed to investigate the relationship between in vitro pharmacologic doses and in vivo physiologic concentration of VacA. In summary, we demonstrated that exposure of DCs to VacA results in the induction of ER stress signaling; ER stress signaling then mediates the release of cytochrome c and DNA fragmentation. Based on these findings, we propose that the induction of ER stress by VacA exposure may lead to increased apoptotic cell death and distorted clearance of H. pylori, despite that many DCs exist in gastric mucosal layers infected with VacA-producing toxigenic H. pylori.
Acknowledgments We thank Dr Patrice Boquet for VacA purification. This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (MEST) (No. 2013R1A1A2A-10004404) and a grant from the NRF of Korea Grant funded by the Korean Government (MEST) (MRC Program no. NRF-2008-0062287).
References 1 Polk DB, Peek RM Jr. Helicobacter pylori: gastric cancer and beyond. Nat. Rev. Cancer. 2010; 10: 403–14. 2 Suzuki H, Moayyedi P. Helicobacter pylori infection in functional dyspepsia. Nat. Rev. Gastroenterol. Hepatol. 2013; 10: 168–74.
Vacuolating cytotoxin-induced apoptosis
3 Necchi V, Manca R, Ricci V et al. Evidence for transepithelial dendritic cells in human H. pylori active gastritis. Helicobacter. 2009; 14: 208–22. 4 Kusters JG, van Vliet AH, Kuipers EJ. Pathogenesis of Helicobacter pylori infection. Clin. Microbiol. Rev. 2006; 19: 449–90. 5 Calore F, Genisset C, Casellato A et al. Endosome-mitochondria juxtaposition during apoptosis induced by H. pylori VacA. Cell. Death. Differ. 2010; 17: 1707–16. 6 Kim JM, Kim JS, Lee JY et al. Dual effects of Helicobacter pylori vacuolating cytotoxin on human eosinophil apoptosis in early and late periods of stimulation. Eur. J. Immunol. 2010; 40: 1651–62. 7 Yamasaki E, Wada A, Kumatori A et al. Helicobacter pylori vacuolating cytotoxin induces activation of the proapoptotic proteins Bax and Bak, leading to cytochrome c release and cell death, independent of vacuolation. J. Biol. Chem. 2006; 281: 11250–9. 8 Kim JM, Kim JS, Yoo DY et al. Stimulation of dendritic cells with Helicobacter pylori vacuolating cytotoxin negatively regulates their maturation via the restoration of E2F1. Clin. Exp. Immunol. 2011; 166: 34–45. 9 Oertli M, Noben M, Engler DB et al. Helicobacter pylori γ-glutamyl transpeptidase and vacuolating cytotoxin promote gastric persistence and immune tolerance. Proc. Natl. Acad. Sci. USA. 2013; 110: 3047–52. 10 Galgani M, Busiello I, Censini S et al. Helicobacter pylori induces apoptosis of human monocytes but not monocyte-derived dendritic cells: role of the cag pathogenicity island. Infect. Immun. 2004; 72: 4480–5. 11 Song CH. Endoplasmic reticulum stress responses and apoptosis. J. Bacteriol. Virol. 2012; 42: 196–202. 12 Oyadomari S, Mori M. Roles of CHOP/GADD153 in endoplasmic reticulum stress. Cell. Death. Differ. 2004; 11: 381–9. 13 Vannuvel K, Renard P, Raes M et al. Functional and morphological impact of ER stress on mitochondria. J. Cell. Physiol. 2013; 228: 1802–18. 14 Szegezdi E, Logue SE, Gorman AM et al. Mediators of endoplasmic reticulum stress-induced apoptosis. EMBO. Rep. 2006; 7: 880–5. 15 Akazawa Y, Isomoto H, Matsushima K et al. Endoplasmic reticulum stress contributes to Helicobacter pylori VacA-induced apoptosis. PLoS. ONE. 2013; 8: e82322. 16 Kim JM, Kim JS, Lee JY et al. Vacuolating cytotoxin in Helicobacter pylori water-soluble proteins upregulates chemokine expression in human eosinophils via Ca2+ influx, mitochondrial reactive oxygen intermediates, and NF-kappaB activation. Infect. Immun. 2007; 75: 3373–81. 17 Gauthier NC, Monzo P, Kaddai V et al. Helicobacter pylori VacA cytotoxin: a probe for a clathrin-independent and Cdc42-dependent pinocytic pathway routed to late endosomes. Mol. Biol. Cell. 2005; 16: 4852–66. 18 Cho A, Seok SH. Ethical guidelines for use of experimental animals in biomedical research. J. Bacteriol. Virol. 2013; 43: 18–26. 19 Lee JY, Kim H, Cha MY et al. Clostridium difficile toxin A promotes dendritic cell maturation and chemokine CXCL2 expression through p38, IKK, and the NF-kappaB signaling pathway. J. Mol. Med. 2009; 87: 169–80. 20 Kim JM, Lee JY, Kim YJ. Inhibition of apoptosis in Bacteroides fragilis enterotoxin-stimulated intestinal epithelial cells through the induction of c-IAP-2. Eur. J. Immunol. 2008; 38: 2190–9. 21 Roh HC, Yoo do Y, Ko SH et al. Bacteroides fragilis enterotoxin upregulates intercellular adhesion molecule-1 in endothelial cells via an aldose reductase-, MAPK-, and NF-kappaB-dependent pathway, leading to monocyte adhesion to endothelial cells. J. Immunol. 2011; 187: 1931–41.
Journal of Gastroenterology and Hepatology 30 (2015) 99–108 © 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd
107
Vacuolating cytotoxin-induced apoptosis
JM Kim et al.
22 Ko SH, Jeon JI, Kim H et al. Mitogen-activated protein kinase/IκB kinase/NF-κB-dependent and AP-1-independent CX3CL1 expression in intestinal epithelial cells stimulated with Clostridium difficile toxin A. J. Mol. Med. 2014; 92: 411–27. 23 Choi JH, Choi AY, Yoon H et al. Baicalein protects HT22 murine hippocampal neuronal cells against endoplasmic reticulum stress-induced apoptosis through inhibition of reactive oxygen species production and CHOP induction. Exp. Mol. Med. 2010; 42: 811–22. 24 Yoo do Y, Ko SH, Jung J et al. Bacteroides fragilis enterotoxin upregulates lipocalin-2 expression in intestinal epithelial cells. Lab. Invest. 2013; 93: 384–96.
108
25 Kranzer K, Söllner L, Aigner ML et al. Impact of Helicobacter pylori virulence factors and compounds on activation and maturation of human dendritic cells. Infect. Immun. 2005; 73: 4180–9. 26 Boyce M, Bryant KF, Jousse C et al. A selective inhibitor of eIF2alpha dephosphorylation protects cells from ER stress. Science. 2005; 307: 935–9. 27 Tabas I, Ron D. Integrating the mechanisms of apoptosis induced by endoplasmic reticulum stress. Nat. Cell. Biol. 2011; 13: 184–90. 28 Tsugawa H, Suzuki H, Saya H et al. Reactive oxygen species-induced autophagic degradation of Helicobacter pylori CagA is specifically suppressed in cancer stem-like cells. Cell. Host. Microbe. 2012; 12: 764–77.
Journal of Gastroenterology and Hepatology 30 (2015) 99–108 © 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd
doi:10.1111/jgh.12663
GASTROENTEROLOGY
Helicobacter pylori vacuolating cytotoxin induces apoptosis via activation of endoplasmic reticulum stress in dendritic cells Jung Mogg Kim,* Joo Sung Kim,† Nayoung Kim,†,‡ Su Hyuk Ko,* Jong Ik Jeon* and Young-Jeon Kim§ *Department of Microbiology, Hanyang University College of Medicine, †Department of Internal Medicine, Seoul National University College of Medicine, Seoul, ‡Department of Internal Medicine, Seoul National University Bundang Hospital, Seongnam, and §Department of Biotechnology, Joongbu University, Choongnam, Korea
Key words apoptosis, dendritic cells, ER stress, H. pylori vacuolating cytotoxin. Accepted for publication 22 May 2014. Correspondence Professor Jung Mogg Kim, Department of Microbiology, Hanyang University College of Medicine, 17 Haengdang-dong, Sungdong-gu, Seoul 133-791, Korea. Email: [email protected]
Abstract Background and Aim: Dendritic cells (DCs) are observed on the Helicobacter pyloriinfected gastric mucosa. DCs generally play an important role in the regulation of inflammation. Although stimulation of gastric epithelial cells with H. pylori vacuolating cytotoxin (VacA) has been reported to induce apoptosis and endoplasmic reticulum (ER) stress, the effects of VacA on the DC apoptotic response have not been well elucidated. This study was conducted to investigate the role of H. pylori VacA on the apoptotic process and ER stress in DCs. Methods: Murine and human DCs were generated from specific pathogen-free C57BL/6 mice and human peripheral blood mononuclear cells, respectively. DCs were incubated with purified VacA, after which Bax activation, cytochrome c release, and DNA fragmentation for apoptosis were measured by fluorescent microscopy, immunoblot, and ELISA. ER stress-related molecules such as GRP78 and CHOP were analyzed by immunoblot. Results: Treatment of DCs with purified H. pylori VacA resulted in the induction of apoptosis. DC stimulation with VacA led to the translocation of cytoplasmic Bax to mitochondria and cytochrome c release from mitochondria. H. pylori VacA induced signals for ER stress early during the stimulation process in DCs. Furthermore, suppression of ER stress resulted in a significant inhibition of the VacA-induced apoptosis in DCs. Conclusion: These results suggest that ER stress is critical for regulation of DC apoptotic process in response to VacA stimulation.
Introduction Helicobacter pylori is a Gram-negative bacterium that colonizes the stomach mucus layer and is the causative agent of diseases such as chronic gastritis, peptic ulcers, gastric cancers, and gastric MALT lymphoma.1 In addition, H. pylori infection is associated with a variety of motility, endocrine and acid-secretory abnormalities that could drive the symptoms of functional dyspepsia.2 In a meta-analysis of randomized controlled trials, H. pylori eradication had a small but statistically significant effect in controlling the symptoms of functional dyspepsia, indicating that H. pylori is involved in the pathogenesis of functional dyspepsia.2 Although the bacteria do not invade the gastric lamina propria, the infection leads to the infiltration of several immune cells.3 Vacuolating cytotoxin (VacA) is one of the major virulence factors in the pathogenesis of H. pylori-related diseases.4 VacA can induce apoptosis in gastric epithelial cells and eosinophils, which can lead to gastric mucosal inflammation.5–7 Dendric cells (DCs) are professional antigen (Ag)-presenting cells that play a critical
role in the induction of immunity. Since H. pylori VacA downregulates DC maturation,8,9 it is possible that VacA may be one of the factors that influence the fate of DCs during the infection. Previous report showed that the exposure of monocytederived DCs to H. pylori itself did not induce apoptosis,10 but this study performed using the live bacteria. Therefore, apoptosis induced by stimulation of DCs with purified VacA may be different from that by exposure of live bacteria. Endoplasmic reticulum (ER) stress-mediated apoptosis is implicated in the pathophysiology of human diseases.11,12 Both mitochondria-dependent and independent cell death pathways have been described to mediate apoptosis in response to ER stress.13 The available evidence suggests that transcriptional induction of the C/EBP homologous protein (CHOP) is critical in ER stress-induced apoptosis through suppression of Bcl-2 activation.14 ER stress can also induce mitochondrial dysfunction and apoptosis through organelle crosstalk between the ER and mitochondria.13 Recently, Akazawa et al. demonstrated that VacA-induced apoptosis in gastric epithelial cells was associated with ER stress.15
Journal of Gastroenterology and Hepatology 30 (2015) 99–108 © 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd
99
Vacuolating cytotoxin-induced apoptosis
JM Kim et al.
However, little information is available on DC apoptosis and ER stress in response to stimulation with H. pylori VacA. The goal of this study was to investigate the role of VacA in modulating the apoptotic process of DCs. H. pylori VacA is shown herein to induce DC apoptosis through ER stress.
and IL-4 (20 ng/mL, PeproTech) for 6 days. In some experiments, DCs were pretreated with the indicated concentration of salubrinal (Sigma Chemical Co., St. Louis, MO, USA) or 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF, Sigma) for 1 h, and then stimulated with VacA (10 μg/mL) for another 24 h.
Methods
Generation of murine bone marrow-derived DCs and DC2.4 cell culture. Femurs and tibias of specific pathogen-free C57BL/6 mice (4–12 weeks old, Orient Experimental Animal, Seoungnam, Korea) were harvested and bone marrow cells were obtained as previously described.8,18,19 For DC generation, RPMI 1640 media supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco BRL, Gaithersburg, MD, USA), 1% antibiotics, L-glutamine (2 mM), 2-mercaptoethanol (55 μM), murine recombinant GM-CSF (10 ng/mL, PeproTech, Rocky Hill, NJ, USA), and murine recombinant interleukin (IL)-4 (10 ng/mL, PeproTech) was used. After 6 days of culture, DCs were harvested and stimulated with VacA. All animal procedures were approved by Hanyang University College of Medicine. CD11c+ cell purities of greater than 95% were routinely achieved as determined by flow cytometric analysis. DC2.4, an immature murine DC cell line, was cultured in RPMI 1640 supplemented with 10% FBS, 1% antibiotics, L-glutamine (2 mM), sodium pyruvate (1 mM), and nonessential amino acids (2 mM). These cells were grown at 37°C with 5% CO2 as previously described.8
Analysis of apoptosis. For a morphologic assessment of cells undergoing apoptosis, cells were stained with the DNA dye Hoechst 33258 (Calbiochem, San Diego, CA, USA).6 To detect morphologic changes in the mitochondrial inner-membrane electrochemical potential in living cells, a mitochondrial staining kit (Sigma catalog #CS0390) was used according to the manufacturer’s instructions.6 Briefly, cells were cultured in Lab-Tek II 4 well-chamber slide (culture area 1.7 cm2; Nunc, Rochester, NY, USA). For mitochondrial stain, the commercially prepared fluorescent dye JC-1 (5,5′,6,6′-tetrachloro-1,1′3,3′-tetraethyl benzimidazol carbocyanine iodide) was added to the cultured chamber (0.5 mL/chamber slide), and cells were then incubated for 20 min at 37°C in a humidified atmosphere containing 5% CO2. Cell morphology was observed using a fluorescence microscope DMI4000B (Leica Microsystems GmbH, Wetzlar, Germany). In normal cells, the JC-1 dye concentrates in the mitochondrial matrix where it forms red fluorescent aggregates. During apoptosis, the mitochondrial membrane dissipates and the dye disperses throughout the entire cell, leading to a shift from JC-aggregates (red) to JC-1 monomers (green fluorescence).6 To assess the number of cells undergoing apoptosis, DCs were incubated with FITC-conjugated annexin V and propidium iodide (PI) according to the manufacturer’s instructions (Apoptosis Detection Kit; R&D Systems, Minneapolis, MN, USA) and then analyzed by flow cytometry (Becton Dickinson, San Jose, CA, USA).20 The mitochondrial transmembrane potential was assessed using tetramethylrhodamine ethyl ester (TMRE; Invitrogen-Molecular Probes, Eugene, Oregon, USA) and flow cytometry, as previously described.6 To assess DNA fragmentation, oligonucleosome release into the cytoplasm was assayed using the Cell Death Detection ELISAPLUS kit (Roche Diagnostics, Mannheim, Germany).6 The amount of cytosolic cytochrome c (human or mouse) in the cell lysates was quantified using an ELISA-based cytochrome c assay kit according to the manufacturer’s instructions (Quantikine, R&D Systems). All assays were performed in triplicate.
Generation of human monocyte-derived DCs. Peripheral blood mononuclear cells (PBMCs) were separated from the peripheral blood of volunteers using Ficoll-Paque (Histopaque-1077) density-gradient centrifugation. The Ethics Committee of Seoul National University Hospital approved this study. Briefly, CD14+ cells were purified from PBMCs using a magnetic cell separation system (Miltenyi Biotec, Bergisch Gladbach, Germany). The purity of CD14+ cells counted by fluorescein isothiocyanate (FITC-conjugated mouse anti-CD14 mAb, and flow cytometry was > 95%. For the generation of human DCs, purified CD14+ cells were plated in ultralow-adherence 24-well plates (Costar) at a density of 5 × 105 cell/mL and cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 1% antibiotics, L-glutamine (2 mM), 2-mercaptoethanol (55 μM), human recombinant GM-CSF (50 ng/mL, PeproTech),
Immunoblot analyses. Preparation of cytosolic and mitochondrial protein was performed as previously described.21,22 Fifteen to fifty micrograms of protein/lane were size-fractionated on polyacrylamide minigels (Mini-PROTEIN II; Bio-Rad) and electrophoretically transferred to a nitrocellulose membrane (0.1-μm pore size). Specific proteins were detected using primary antibodies (Abs) with 1: 1000 dilution, and the primary Abs immunoreactive proteins were visualized using goat anti-rabbit or anti-mouse secondary Abs conjugated to horseradish peroxidase, followed by enhanced chemiluminescence (ECL system; Amersham Life Science, Buckinghamshire, England) and exposure to X-ray film.21,22 Abs against cytochrome c, cytochrome c oxidase subunit IV (COX IV), CHOP, and actin were acquired from Cell Signaling Technology, Inc. (Beverly, MA, USA). Abs against glucose-regulated protein 78 (GRP78) and Bax, and anti-mouse,
Purification of H. pylori VacA. VacA-producing H. pylori strain 60190 (ATCC 49503, CagA+, vacA s1a/m1) was grown in sulfite-free Brucella broth containing 0.5% charcoal (untreated, granular 8–20 mesh) at 37°C under microaerophilic conditions. VacA was purified from broth culture supernatants according to previously described.6,8,16 Professor Patrice Boquet, Laboratoire de bacteriologie, Hopital de l’Archet 2, France, kindly supported the VacA protein purification method.17 Immediately before use in cells, the purified VacA protein was activated through the addition of 250 mM HCl until the pH reached 2.0. NH4Cl (5 mM) was also added to the medium to enhance the VacA activity.7,15
100
Journal of Gastroenterology and Hepatology 30 (2015) 99–108 © 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd
JM Kim et al.
anti-rabbit, and anti-goat secondary Abs conjugated to horseradish peroxidase were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The active form of specific anti-Bax-NT was from Upstate Biotech (Temecula, CA, USA) and Vectashield mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) was from Vector Laboratories (Burlingame, CA, USA). Immunofluorescence microscopy for Bax and cytochrome c. Immunofluorescence microscopy was analyzed using mAbs against active Bax and cytochrome c (Santa Cruz), as described previously.6 Briefly, DCs were seeded (5 × 104 cells in 0.2 mL of RPMI 1640/well) on 8-well poly-D-lysinecoated culture microslides (Santa Cruz). After treatment with VacA, cells were incubated with 100 nM Mitotracker Red CMXRos (Invitrogen-Molecular Probes, dilution, 1: 1000) for 20 min at 37°C in a humidified atmosphere containing 5% CO2 and fixed with 100% methanol for 10 min. For permeabilization, cells were treated with 0.3% Triton X-100 in PBS for 30 min at room temperature, after which the cells were incubated with the active form of specific anti-Bax-NT or anti-cytochrome c Abs (Upstate Biotech; dilution, 1: 1000) for 2 h. Cells were then treated with Alexa fluor 488-conjugated secondary Ab against rabbit IgG for 1 h. Images were captured using a fluorescence microscope DMI4000B (Leica Microsystems GmbH). Transfection assay. A Bax-specific silencing small interfering RNA (siRNA) was constructed as previously described.5 Negative control non-silencing RNA was purchased from Invitrogen. Predesigned siRNA against CHOP and control-scrambled siRNA were purchased from Santa Cruz Biotechnology.23 Transfection was performed according to the procedure described previously.8,24 Statistical analyses. Data are presented as the mean ± standard deviation (SD) or mean ± standard error of the means (SEM). The Wilcoxon’s rank sum test was used for statistical analysis.
Results H. pylori VacA induces apoptosis of DCs. Mouse DCs were treated with purified VacA, and apoptosis was assessed by several methods. Figure 1a shows nuclear fragmentation characteristics of apoptosis in VacA-stimulated DCs stained with the
Vacuolating cytotoxin-induced apoptosis
DNA dye Hoechst 33258. The fluorescent dye JC-1 was used to stain mitochondria. In normal cells, the JC-1 dye concentrated in the mitochondrial matrix where it forms red fluorescent aggregates. During apoptosis induced by VacA stimulation, the mitochondrial membrane dissipated, and the dye dispersed throughout the entire cell, leading to a shift from red (JC aggregates) to green fluorescence (JC-1 monomers) (Fig. 1b). Cells stained with FITC annexin V to detect the externalization of phosphatidylserine to the outer leaflet of the cell membrane were evaluated by flow cytometry (Fig. 1c). In addition, staining with TMRE was performed to detect reduced mitochondrial transmembrane potential (Fig. 1d). Results showed that the numbers of apoptotic cells in the VacA-stimulated cultures were higher than the control cultures (Fig. 1e,f,g).
Cytochrome c release from mitochondria and mitochondrial trafficking of activated Bax in DCs stimulated with H. pylori VacA. We next investigated whether VacA could induce cytochrome c release from mitochondria in DCs. As shown in Figure 2a, treatment with H. pylori VacA increased levels of cytochrome c in the cytosolic fraction but decreased in the mitochondrial fraction, indicating that VacA induces the release of cytochrome c from the mitochondria. Concurrently, activated Bax signals were concentrated within the mitochondrial fraction after stimulation with purified VacA. We measured cytochrome c release from mouse DCs using ELISA. Results showed that a significant release of cytochrome c was first observed at 24 h in mouse DCs (Fig. 2b). To confirm this result, immunofluorescent microscopy was performed. The distribution of cytochrome c was diffuse in the cytoplasm of VacA-treated DCs. In contrast, the distribution of cytochrome c was only localized within the mitochondria of untreated cells (Fig. 2c). In addition, the mitochondrial trafficking of activated Bax was shown in VacA-treated cells (Fig. 2d). We next determined whether VacA might influence apoptosis in human monocyte-derived DCs. Results using human monocytederived DCs were shown similar to those using murine DCs. Thus, the cytochrome c in the cytosolic fraction was increased, and in the mitochondrial fraction, cytochrome c was decreased but activated Bax signal was increased (Fig. 3a). In this experimental system, a significant release of cytochrome c in cytoplasm began 18 h after VacA stimulation (Fig. 3b). Consistent with this, an increase in DNA fragmentation was observed during the first 18 h after VacA
▶ Figure 1 Apoptosis in VacA-stimulated DCs. DCs generated from C57BL/6 mice were incubated with VacA (10 μg/mL) for 24 h. (a) Cells were fixed with 2% paraformaldehyde and stained with Hoechst dye 33258 (x 400). Apoptotic bodies are shown in VacA-treated cells (arrowhead). (b) Cells were stained with JC-1 dye and visualized under a fluorescent microscope (x 400). Normal cells show red, granular mitochondrial staining, whereas apoptotic cells show green, diffuse cytoplasmic staining. (c) Cells were incubated with FITC-conjugated annexin V and propidium iodide (PI), and analyzed by flow cytometry. Viable cells have low FITC-annexin V and low PI staining (left lower quadrant), and apoptotic cells have high FITC-annexin V and low PI staining (right lower quadrant, R1). Data are representative of three separate experiments. (d) Cells were incubated with 75 nM TMRE for 30 min at 37°C and analyzed by flow cytometry. The area marked as M1 to the left of the major peak contains the apoptotic cell population. Data are representative of three separate experiments. (e, f, and g) Quantification of apoptotic DCs in response to stimulation with VacA. (e and f) Quantitative data for R1 and M1. Culture conditions are the same as in Figure 1c,d. Values are the means ± SEM (n = 3). (g) Apoptosis in VacA-stimulated DCs. DCs generated from C57BL/6 mice were incubated with VacA (10 μg/mL) for 24 h. Cell death detection ELISA was measured as a fold increase in stimulated cells compared with each control (mean ± SEM, n = 5). *P < 0.05 versus untreated control. DCs, dendritic cells; FITC, fluorescein isothiocyanate; SEM, standard error of the means; TMRE, tetramethylrhodamine ethyl ester; VacA, vacuolating cytotoxin.
Journal of Gastroenterology and Hepatology 30 (2015) 99–108 © 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd
101
Vacuolating cytotoxin-induced apoptosis
102
JM Kim et al.
Journal of Gastroenterology and Hepatology 30 (2015) 99–108 © 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd
JM Kim et al.
Journal of Gastroenterology and Hepatology 30 (2015) 99–108 © 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd
Vacuolating cytotoxin-induced apoptosis
103
Vacuolating cytotoxin-induced apoptosis
JM Kim et al.
Figure 2 Cytochrome c release and Bax translocation in VacA-stimulated DCs. (a) DCs generated from C57BL/6 mice were incubated with VacA (10 μg/mL) for the indicated time period, and cytosolic and mitochondrial fractions were prepared. The signals of cytochrome c and activated Bax were detected by each mAb. Abs against actin or COX IV were used to demonstrate equal protein loading. Data are representative of three or more independent experiments. (b) Cytosolic fractions were prepared and released cytochrome c was measured using an ELISA kit. Data represent as the fold increase in stimulated cells compared with the unstimulated control (mean ± SEM, n = 5). (c and d) DCs were incubated with or without VacA (10 μg/mL) for 24 h, and immunofluorescent microscopy were performed. (c) The cells were stained with anti-cytochrome c Ab (green), Mitotracker Red (red, mitochondria), and DAPI (blue, nucleus). VacA-treated DCs show diffuse distribution of cytochrome c in the cytoplasm and untreated control cells show a localized pattern of cytochrome c within mitochondria. (d) The cells were stained with the active form-specific anti-Bax-NT Ab (green), Mitotracker RED (red, mitochondria), and DAPI (blue, nucleus). Signals of activated Bax are shown in the mitochondria of VacA-treated DCs. The data are representative of at least five experiments. DAPI, 4′,6-diamidino-2-phenylindole; DCs, dendritic cells; SEM, standard error of the means; VacA, vacuolating cytotoxin. ◀
Figure 3 Effects of VacA on apoptosis in human monocyte-derived DCs. (a) Human monocyte-derived DCs were incubated with VacA (10 μg/mL) for the indicated time period, and cytosolic and mitochondrial fractions were prepared. Cytochrome c and activated Bax signals were detected by immunoblots. Abs against actin or COX IV were used to demonstrate equal protein loading. Data are representative of three or more independent experiments. (b and c) Time course of cytochrome c and DNA fragmentation in human monocyte-derived DCs treated with VacA was same. Human DCs were stimulated with VacA (10 μg/mL) for the indicated time period. (b) Cytosolic fractions were prepared and released cytochrome c was measured using an ELISA kit. Data represent as the fold increase in stimulated cells compared with the unstimulated control (mean ± SEM, n = 5). (c) Apoptosis was assessed using a cell death detection ELISA. Data represent as a fold increase in stimulated cells compared with unstimulated control (mean ± SEM, n = 5). *P < 0.05 versus untreated control (d) Human DCs were stimulated with the indicated concentration of VacA for 24 h. Apoptosis was assessed using a cell death detection ELISA. Data represent as a fold increase in stimulated cells compared with unstimulated control (mean ± SEM, n = 5). *P < 0.05 versus untreated control. Abs, antibodies; DCs, dendritic cells; SEM, standard error of the means; VacA, vacuolating cytotoxin.
stimulation, and apoptosis continued to increase over the 48 h post-stimulation (Fig. 3c). The magnitude of DNA fragmentation was dependent on the concentration of VacA used for stimulation (Fig. 3d). The concentration of VacA that gives half-maximal response (EC50) was 5.08 μg/mL calculated by SigmaPlot 10.0 104
software (Systat Software Inc., San Jose, CA, USA). In the present study, we used 10 μg/mL of VacA because more than 10 μg/mL of VacA showed maximal induction of apoptosis. To evaluate whether Bax activation might be related to cytochrome c release and DNA fragmentation, the transfection of a
Journal of Gastroenterology and Hepatology 30 (2015) 99–108 © 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd
JM Kim et al.
Figure 4 Cytochrome c release and DNA fragmentation in Baxsuppressed DCs stimulated with VacA. DC2.4 cells were transfected with Bax siRNA or non-silencing control siRNA (NS-RNA) for 48 h. The data are representative of three independent experiments. (a) The transfected cells were either untreated or stimulated with VacA (10 μg/mL) for 18 h. Immunoblot analysis for the active form of Bax-NT in mitochondrial fractions was performed. (b) The transfected cells were stimulated with VacA (10 μg/mL) for 24 h. Cytosolic levels of cytochrome c and oligonucleosome were measured using each ELISA kit. Data are expressed as mean fold-induction ± SEM of each parameter relative to unstimulated controls (n = 5). *P < 0.05. DCs, dendritic cells; SEM, standard error of the means; siRNA, short interfering RNA; VacA, vacuolating cytotoxin.
DC2.4 cell line with siRNA was used. Transfection with Bax siRNA reduced the mitochondrial levels of phospho-Bax in VacA-treated cells (Fig. 4a). In this experimental system, cells transfected with siRNA against Bax showed significant decreases in cytochrome c release and DNA fragmentation compared to control cells (Fig. 4b).
Vacuolating cytotoxin-induced apoptosis
Figure 5 VacA-induced ER stress is associated with apoptosis in DCs. (a) Human monocyte-derived DCs were incubated with VacA (10 μg/mL) for the indicated time period. Signals of CHOP and GRP78 were measured by immunoblot analysis. Results are representative of three independent experiments. (b) DCs were pretreated with the indicated concentration of salubrinal or AEBSF for 1 h, and then stimulated with VacA (10 μg/mL) for another 24 h. Data are expressed as the percent inhibition of cytochrome c release relative to unstimulated controls ± SEM (n = 5). *P < 0.05 versus unstimulated control (c) ER stress inhibitors attenuate VacA-induced cytochrome c release and DNA fragmentation. Human monocyte-derived DCs were treated with salubrinal (25 μM) and AEBSF (300 μM) prior to VacA stimulation. Cytosolic levels of cytochrome c and oligonucleosome after 24 h stimulation with VacA (10 μg/mL) were measured using ELISA kits. Data are expressed as the mean fold-induction ± SEM of each parameter relative to unstimulated controls (n = 5). *P < 0.05. AEBSF, 4-(2-aminoethyl) benzenesulfonyl fluoride; CHOP, C/EBP homologous protein; DCs, dendritic cells; ER, endoplasmic reticulum; SEM, standard error of the means; VacA, vacuolating cytotoxin.
H. pylori VacA-induced apoptotic response associated with ER stress. Stimulation of human DCs with VacA increased CHOP and GPR78 in (Fig. 5a). Compared to the
Journal of Gastroenterology and Hepatology 30 (2015) 99–108 © 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd
105
Vacuolating cytotoxin-induced apoptosis
JM Kim et al.
fact that cytochrome c release and DNA fragmentation were first observed 18 h poststimulation of VacA in human DCs (Fig. 3b,c), the induction of ER stress seems to be an earlier response than the apparent apoptotic phenomena. To evaluate the role of ER stress and VacA-induced apoptosis, VacA was added to human DCs following pretreatment with salubrinal or AEBSF. Pretreatment of DCs with salubrinal (≥ 10 μM) or AEBSF (≥ 100 μM) for 1 h significantly inhibited the VacA-induced release of cytochrome c (Fig. 5b). In addition, pretreatment with salubrinal or AEBSF significantly inhibited cytochrome c release and DNA fragmentation induced by VacA stimulation (Fig. 5c). Increased expression of CHOP protein was also observed in DC2.4 cells (Fig. 6a). To confirm the relationship between ER stress and VacA-induced apoptosis, we evaluated whether suppression of CHOP signals by siRNA transfection could affect apoptotic markers. Transfection with CHOP siRNA reduced CHOP signal in VacA-stimulated cells (Fig. 6b). In this experimental system, significant decreased cytochrome c release and DNA fragmentation occurred in cells transfected with siRNA against CHOP compared to control cells under VacA-stimulated conditions (Fig. 6c). These results suggest that ER stress is associated with DC apoptotic phenomenon in response to VacA stimulation.
Discussion In the present study, we demonstrated that one of the early responses to stimulation of DCs with H. pylori VacA is the induction of ER stress, which leads to apoptosis as a relatively late response. At the molecular level, mitochondria plays an important role in transmitting apoptotic signals through the expression of Bax and cytochrome c, which are considered key proteins in apoptosis.6 The present study showed that VacA increased levels of activated Bax in the mitochondrial fraction, increased levels of cytochrome c in the cytosolic fraction, and decreased levels of cytochrome c in the mitochondrial fraction. In addition, transfection with siRNA against Bax significantly decreased cytochrome c release and DNA fragmentation. These results suggest that the activated response of Bax signal to H. pylori VacA can lead to cytochrome c release and apoptosis in DCs. The cytochrome c release and the apparent apoptotic phenomena were observed 18 h after VacA stimulation in DCs, which seems to be a relatively late response. Based on these observations, we postulated that the ability of VacA to induce apoptosis might result from the induction of certain molecular events during early responses. Galgani et al. demonstrated that the exposure of monocytederived DCs to H. pylori itself did not induce apoptosis.10 In addition, DC activation and maturation were independent of the cag PAI and VacA status of H. pylori.25 However, these two studies performed using the live bacteria. Therefore, apoptosis induced by treatment with purified VacA may be different from that by exposure of live bacteria to DCs. Although a recent paper showed that VacA-induced apoptosis in gastric epithelial cells was associated with ER stress,15 the specific mechanisms involved in ER stress-induced apoptosis have not been reported in DCs. In the present study, stimulation of human DCs with H. pylori VacA increased signals of GRP78 and CHOP 1 h after stimulation. In murine DCs, expression of CHOP protein was observed 6 h after stimulation. Compared to the fact that the 106
Figure 6 Suppression of CHOP attenuates cytochrome c release and DNA fragmentation in VacA-stimulated DCs. (a) DC2.4 cells were incubated with VacA (10 μg/mL) for the indicated time period. CHOP and actin proteins were measured by immunoblot analysis. Results are representative of three independent experiments. (b) DC2.4 cells were transfected with CHOP siRNA or non-silencing control siRNA (nsRNA). After 48 h, the transfected cells were either untreated or stimulated with VacA (10 μg/mL) for 24 h. CHOP and actin proteins were measured by immunoblot analysis. These results are representative of three independent experiments. (c) The transfected cells with each siRNA were stimulated with VacA (10 μg/mL) for 24 h. Cytosolic levels of cytochrome c and oligonucleosome release were measured using ELISA kits. Data are expressed as the mean fold-induction ± SEM of each parameter relative to unstimulated controls (n = 5). *P < 0.05. CHOP, C/EBP homologous protein; DCs, dendritic cells; NS-RNA, non-silencing control siRNA; SEM, standard error of the means; siRNA, short interfering RNA; VacA, vacuolating cytotoxin.
Journal of Gastroenterology and Hepatology 30 (2015) 99–108 © 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd
JM Kim et al.
cytochrome c release and the apparent apoptotic phenomena were observed 18 h post-stimulation of VacA in DCs, the induction of ER stress may be earlier than that of apoptosis. In the present study, we used salubrinal and AEBSF to inhibit ER stress signaling. Salubrinal acts as a selective inhibitor of eIF2α dephosphorylation to protect cells from ER stress,26 and the serine protease inhibitor AEBSF is reported to inhibit ER stressinduced proteolysis of ATF6, resulting in inhibition of ATF6target gene transcription by ER stress.13,27 Pretreatment of DCs with the chemical inhibitors salubrinal or AEBSF significantly inhibited the VacA-induced release of cytochrome c and DNA fragmentation. In addition, cells transfected with CHOP siRNA significantly decreased cytochrome c release and DNA fragmentation compared to control cells. These results suggest that ER stress is critical for regulation of DC apoptotic process in response to VacA stimulation. Tsugawa et al. demonstrated that autophagy and cytotoxinassociated gene A degradation are induced by the H. pylori VacA, which acted via decreasing levels of intracellular glutathione, causing reactive oxygen species (ROS) accumulation and Akt activation.28 The unfolded protein response (UPR) is a collection of adaptive signaling pathways that evolved to resolve protein misfolding, in which the ROS production is known to be associated with the UPR and ER stress.11,13 Therefore, it is possible that VacA may induce the decreasing glutathione levels and ROS accumulation, leading to ER stress in DCs. Our study used pharmacologic doses of VacA to induce ER stress and apoptosis in DCs. Since the in vitro pharmacologic dose used in the present study is presumed to be higher than the physiologic concentration in gastric mucosa infected with toxigenic H. pylori, further study is needed to investigate the relationship between in vitro pharmacologic doses and in vivo physiologic concentration of VacA. In summary, we demonstrated that exposure of DCs to VacA results in the induction of ER stress signaling; ER stress signaling then mediates the release of cytochrome c and DNA fragmentation. Based on these findings, we propose that the induction of ER stress by VacA exposure may lead to increased apoptotic cell death and distorted clearance of H. pylori, despite that many DCs exist in gastric mucosal layers infected with VacA-producing toxigenic H. pylori.
Acknowledgments We thank Dr Patrice Boquet for VacA purification. This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (MEST) (No. 2013R1A1A2A-10004404) and a grant from the NRF of Korea Grant funded by the Korean Government (MEST) (MRC Program no. NRF-2008-0062287).
References 1 Polk DB, Peek RM Jr. Helicobacter pylori: gastric cancer and beyond. Nat. Rev. Cancer. 2010; 10: 403–14. 2 Suzuki H, Moayyedi P. Helicobacter pylori infection in functional dyspepsia. Nat. Rev. Gastroenterol. Hepatol. 2013; 10: 168–74.
Vacuolating cytotoxin-induced apoptosis
3 Necchi V, Manca R, Ricci V et al. Evidence for transepithelial dendritic cells in human H. pylori active gastritis. Helicobacter. 2009; 14: 208–22. 4 Kusters JG, van Vliet AH, Kuipers EJ. Pathogenesis of Helicobacter pylori infection. Clin. Microbiol. Rev. 2006; 19: 449–90. 5 Calore F, Genisset C, Casellato A et al. Endosome-mitochondria juxtaposition during apoptosis induced by H. pylori VacA. Cell. Death. Differ. 2010; 17: 1707–16. 6 Kim JM, Kim JS, Lee JY et al. Dual effects of Helicobacter pylori vacuolating cytotoxin on human eosinophil apoptosis in early and late periods of stimulation. Eur. J. Immunol. 2010; 40: 1651–62. 7 Yamasaki E, Wada A, Kumatori A et al. Helicobacter pylori vacuolating cytotoxin induces activation of the proapoptotic proteins Bax and Bak, leading to cytochrome c release and cell death, independent of vacuolation. J. Biol. Chem. 2006; 281: 11250–9. 8 Kim JM, Kim JS, Yoo DY et al. Stimulation of dendritic cells with Helicobacter pylori vacuolating cytotoxin negatively regulates their maturation via the restoration of E2F1. Clin. Exp. Immunol. 2011; 166: 34–45. 9 Oertli M, Noben M, Engler DB et al. Helicobacter pylori γ-glutamyl transpeptidase and vacuolating cytotoxin promote gastric persistence and immune tolerance. Proc. Natl. Acad. Sci. USA. 2013; 110: 3047–52. 10 Galgani M, Busiello I, Censini S et al. Helicobacter pylori induces apoptosis of human monocytes but not monocyte-derived dendritic cells: role of the cag pathogenicity island. Infect. Immun. 2004; 72: 4480–5. 11 Song CH. Endoplasmic reticulum stress responses and apoptosis. J. Bacteriol. Virol. 2012; 42: 196–202. 12 Oyadomari S, Mori M. Roles of CHOP/GADD153 in endoplasmic reticulum stress. Cell. Death. Differ. 2004; 11: 381–9. 13 Vannuvel K, Renard P, Raes M et al. Functional and morphological impact of ER stress on mitochondria. J. Cell. Physiol. 2013; 228: 1802–18. 14 Szegezdi E, Logue SE, Gorman AM et al. Mediators of endoplasmic reticulum stress-induced apoptosis. EMBO. Rep. 2006; 7: 880–5. 15 Akazawa Y, Isomoto H, Matsushima K et al. Endoplasmic reticulum stress contributes to Helicobacter pylori VacA-induced apoptosis. PLoS. ONE. 2013; 8: e82322. 16 Kim JM, Kim JS, Lee JY et al. Vacuolating cytotoxin in Helicobacter pylori water-soluble proteins upregulates chemokine expression in human eosinophils via Ca2+ influx, mitochondrial reactive oxygen intermediates, and NF-kappaB activation. Infect. Immun. 2007; 75: 3373–81. 17 Gauthier NC, Monzo P, Kaddai V et al. Helicobacter pylori VacA cytotoxin: a probe for a clathrin-independent and Cdc42-dependent pinocytic pathway routed to late endosomes. Mol. Biol. Cell. 2005; 16: 4852–66. 18 Cho A, Seok SH. Ethical guidelines for use of experimental animals in biomedical research. J. Bacteriol. Virol. 2013; 43: 18–26. 19 Lee JY, Kim H, Cha MY et al. Clostridium difficile toxin A promotes dendritic cell maturation and chemokine CXCL2 expression through p38, IKK, and the NF-kappaB signaling pathway. J. Mol. Med. 2009; 87: 169–80. 20 Kim JM, Lee JY, Kim YJ. Inhibition of apoptosis in Bacteroides fragilis enterotoxin-stimulated intestinal epithelial cells through the induction of c-IAP-2. Eur. J. Immunol. 2008; 38: 2190–9. 21 Roh HC, Yoo do Y, Ko SH et al. Bacteroides fragilis enterotoxin upregulates intercellular adhesion molecule-1 in endothelial cells via an aldose reductase-, MAPK-, and NF-kappaB-dependent pathway, leading to monocyte adhesion to endothelial cells. J. Immunol. 2011; 187: 1931–41.
Journal of Gastroenterology and Hepatology 30 (2015) 99–108 © 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd
107
Vacuolating cytotoxin-induced apoptosis
JM Kim et al.
22 Ko SH, Jeon JI, Kim H et al. Mitogen-activated protein kinase/IκB kinase/NF-κB-dependent and AP-1-independent CX3CL1 expression in intestinal epithelial cells stimulated with Clostridium difficile toxin A. J. Mol. Med. 2014; 92: 411–27. 23 Choi JH, Choi AY, Yoon H et al. Baicalein protects HT22 murine hippocampal neuronal cells against endoplasmic reticulum stress-induced apoptosis through inhibition of reactive oxygen species production and CHOP induction. Exp. Mol. Med. 2010; 42: 811–22. 24 Yoo do Y, Ko SH, Jung J et al. Bacteroides fragilis enterotoxin upregulates lipocalin-2 expression in intestinal epithelial cells. Lab. Invest. 2013; 93: 384–96.
108
25 Kranzer K, Söllner L, Aigner ML et al. Impact of Helicobacter pylori virulence factors and compounds on activation and maturation of human dendritic cells. Infect. Immun. 2005; 73: 4180–9. 26 Boyce M, Bryant KF, Jousse C et al. A selective inhibitor of eIF2alpha dephosphorylation protects cells from ER stress. Science. 2005; 307: 935–9. 27 Tabas I, Ron D. Integrating the mechanisms of apoptosis induced by endoplasmic reticulum stress. Nat. Cell. Biol. 2011; 13: 184–90. 28 Tsugawa H, Suzuki H, Saya H et al. Reactive oxygen species-induced autophagic degradation of Helicobacter pylori CagA is specifically suppressed in cancer stem-like cells. Cell. Host. Microbe. 2012; 12: 764–77.
Journal of Gastroenterology and Hepatology 30 (2015) 99–108 © 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd
