Patients who underwent esophagogastroduodenoscopy and were diagnosed with gastritis, gastric ulcer, or gastric cancer at the Chung-Ang University Hospital, Seoul, Korea were included in the study. Six patients were enrolled, two patients for each disease type. H. pylori strains were isolated from gastric antral mucosal biopsy specimens collected from the enrolled patients. The study was approved by the Institutional Review Board of Chung-Ang University Hospital (10-080-12-23). Written informed consent was obtained from all study subjects.

Two human gastric epithelial cell lines (AGS and MKN-45) and a human monocytic cell line (U-937) were cultured in Roswell Park Memorial Institute (RPMI) 1680 medium supplemented with 10% fetal bovine serum. Cell monolayers were cultured in petri dishes at 37.5°C in a humidified atmosphere containing 5% CO₂.

H. pylori strains were cultured under microaerophilic conditions (37°C, 5% O₂, 10% CO₂, 85% N₂). In the H. pylori group, the cells were infected with H. pylori at a multiplicity of infection of 200:1 (bacterium-to-cell ratio of 200:1). In the OMV group, 10 ng/mL of OMVs, purified as mentioned below, were added. Before assessing the induction of inflammation in response to H. pylori-derived OMVs in gastric epithelial cells, we conducted a preliminary test using U-937, a monocyte cell line, to confirm whether OMVs could induce interleukin 8 (IL-8) secretion. U-937 cells were treated with different concentrations of OMVs (10, 100, or 1,000 ng/mL). Recombinant human IL-1 (PeproTech, Rocky Hill, NJ, USA) was used as the positive control.

H. pylori were cultured on blood agar plates for 72 hours in an atmosphere containing 10% CO₂. The cultured bacteria were resuspended in phosphate buffered saline (PBS). The resuspended H. pylori was inoculated in 1-L Brain Heart Infusion liquid media containing 10% horse serum, and cultured under microaerobic and shaking conditions until the optical density reached 1.0. The cultured bacteria were centrifuged twice for 15 minutes at 5,000 × g and 4°C to remove the H. pylori cells and the supernatant fraction was filtered using a 0.45-μm vacuum filter. One liter of the filtrate was concentrated by ultrafiltration (QuixStand, GE Healthcare, Piscataway, NJ, USA; 100 kDa hollow-fiber membrane) to a final volume of 50 mL. The retentate was filtered again using a 0.22-μm vacuum filter to remove any remaining cells. The resulting filtrate was subjected to ultracentrifugation (Optima XL-100K, Beckman, Brea, CA, USA) at 150,000 ×g for 3 hours at 4°C. The pellets were resuspended in 5-mL PBS. Next, 15 mL of 30% iodixanol and 5 mL of 10% iodixanol solution were added consecutively in the 25-mL tube and centrifuged at 200,000 ×g for 2 hours at 4°C and the whole fraction appearing between the PBS and 30% iodixanol layers was harvested. The fraction was resuspended in 25-mL PBS and centrifuged at 150,000 ×g for 3 hours at 4°C. The resultant pellet was resuspended in 0.2 mL of PBS. Purified OMVs were quantified using the Bradford dye assay (Bio-Rad Laboratories, Hercules, CA, USA) and stored at −72°C until use. This protocol of OMV purification was generously provided by Professor YG Kim (Department of Life Science, Pohang University of Science and Technology) [12].

For transmission electron microscopy (TEM) studies, the OMVs were diluted to a concentration of 50 μg/mL using PBS and 10 mL of the mixture was placed on 300-mesh copper grids (EMS, Hatfield, PA, USA). Next, it was stained with 2% uranyl acetate. We imaged OMVs using the JEM-1011 electron microscope (JEOL, Eching, München, Germany). For dynamic light scattering experiments, the OMVs were diluted to a concentration of 5 μg/mL in PBS, and 1 mL of the mixture was added into the cuvette. The diameters of the vesicles were measured using the Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK). The data were analyzed using the Dynamic V6 software (Protein Solutions, Lakewood, NJ, USA).

RNA was extracted from each cell line that had been stimulated with H. pylori (bacterium-to-cell ratio of 200:1), OMVs (10, 100, or 1,000 ng/mL), and IL-1 (positive control). We used IL-1 as a positive control as it is known to be a positive regulator of IL-8 expression [13]. The RNA extracted from the untreated cells was used as a control. Total cellular RNA was extracted using an RNeasy mini kit (Qiagen, Valencia, CA, USA) and was treated with RNase-free DNase to remove any contaminating genomic DNA. The RNA pellet obtained was resuspended in 30 μL of diethyl pyrocarbonate-treated water and stored at −75°C.

For reverse transcription-polymerase chain reaction (RT-PCR), 1 μg of the total cellular RNA was reverse transcribed into complementary DNA (cDNA) using oligo dT primers, SuperScript^®II Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA; 50 units; 4 μL), and 2-mM dNTP, followed by incubation at 37°C for 70 minutes. Subsequently, the reaction mixture was heat inactivated for 10 minutes at 85°C. The primers for β-actin and IL-8 were as follows (Bioneer, Daedeok, Daejeon, Korea): β-actin sense primer 5′-TGACGGGGTCACCCACACTGTGCCCATCTA-3′ and β-actin antisense primer 5′-CTAGAAGCATTGCGGTGGACGATGGAGGG-3′; IL-8 sense primer 5′-ATGACTTCCAAGCTGGCCGTGGCT-3′ and IL-8 antisense primer 5′-TCTCAGCCCTCTTCAAAAACTTCTC-3′. After a hot start, the amplification program for β-actin was as follows: 45-second denaturation at 95°C and 45-second annealing and extension at 72°C for 25 cycles; the amplification program for IL-8 was as follows: 60-second denaturation at 95°C and 2.5-minute annealing and extension at 60°C for 35 cycles. Samples (10 μL) of each amplified product were electrophoresed on a 1% agarose gel.

For real-time RT-PCR, 1 μL of the cDNA was amplified on a 7300 real-time RT-PCR system (Applied Biosystems, Foster City, CA, USA) using 10 μL 2x SYBR Green master mix (Qiagen GmbH, Hilden, Germany) and a sample (25 μL) of each amplified product, including 10 pM of primer. DNA amplification was performed using 40 cycles of 95°C for 15 seconds, followed by 60°C for 60 seconds. The β-actin primers included the sense primer 5′-AAGATGACCCAGATCATGTT-3′ and the antisense primer 5′-GCGACATAGCACAGCTTCT-3′, and the IL-8 primers included the sense primer 5′-ACATGACTTCCAAGCTGGCC-3′ and the antisense primer 5′-CAGAAATCAGGAAGGCTGCC-3′. All experiments were performed in triplicate and repeated thrice.

The quantity of IL-8 secreted into the cell culture medium was measured using enzyme-linked immunosorbent assay (ELISA) with a monoclonal anti-human IL-8 antibody (Endogen, Woburn, MA, USA) for capture. This antibody was coated onto a flat-bottomed 96-well NUNC-immuno plate (Nalge Nunc International, Roskilde, Denmark) and incubated overnight at room temperature and was subsequently cultured for 2 hours with a blocking buffer and then washed thrice. After washing, a biotin-labeled detecting antibody and horseradish peroxidase (HRP)-conjugated streptavidin were cultured sequentially for an hour, and then the plate was washed thrice. Tetramethylbenzidine substrate solution was added, followed by culturing for 30 minutes at room temperature. The response was terminated using 0.18-M H₂SO₄. The IL-8 content was estimated by measuring absorbance at 450 nm using an ELISA reader and was expressed in pg/mL. All experiments were performed in triplicate and repeated thrice.

To determine the nuclear factor kappa B (NF-κB) activity, inhibitor of NF-κB alpha (IκB-α) phosphorylation and degradation in the AGS cell line were assessed using Western blotting. The proteins were extracted from the AGS cells that had been stimulated with 10 μg of H. pylori-derived OMVs from each patient in a time-dependent manner. After harvesting, the cells were lysed with RIPA buffer and were centrifuged at 4°C for 30 minutes and 15,000 × g, following which the supernatant was harvested. The extracted proteins were quantified using the Bradford dye assay (Bio-Rad Laboratories) and boiled at 100°C for 5 minutes with 5× sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) buffer. After separation at 80 V in stacking gel and at 100 V in separation gel, the samples were transferred to a Nitrocellulose membrane (Whatman GmbH, Dassel, Germany). Nonspecific binding was prevented by blocking with 5% non-fat milk for 2 hours at room temperature. Primary antibodies, anti-IκB-α antibody (Santa Cruz Biotech, Dallas, TX, USA), and antiphospho-IκB-α antibody (Santa Cruz Biotech) diluted with 5% non-fat milk, were used to probe the membranes. After incubation, the membranes were washed thrice with PBS containing 0.05% Tween-20. The secondary antibody, anti-mouse IgG HRP (Santa Cruz Biotech), was diluted with 5% non-fat milk and used to probe the membrane for 2 hours. Following three washes with PBS, the protein bands were visualized by enhanced chemiluminescence (Santa Cruz Biotech) using the Bio-Imaging LAS 1000 plus (Fugifilm, Tokyo, Japan). Next, the ImageJ software version 1.47 (National Institutes of Health, Bethesda, MD, USA) was used to quantify the results of Western blotting.

The expression of the IL-8 mRNA and protein is expressed as mean ± standard error of the mean. Continuous variables were compared using the Mann-Whitney U test. The Kruskal-Wallis test was used to evaluate the differences among three groups. Statistical significance was defined by p < 0.05. Statistical analysis was performed using the SPSS software version 15.0 (SPSS Inc., Chicago, IL, USA).

H. pylori-derived OMVs were separated using density gradients and centrifugation. The OMVs were detected in two particle bands, one band appeared between the PBS and 10% iodixanol layers, and the other appeared between the 10% and 30% iodixanol layers (Fig. 1A). TEM studies identified that round to ovoid-shaped vesicles were derived from H. pylori in all specimens regardless of the underlying gastric disease (Fig. 1B). The vesicle size was approximately 10 to 100 nm regardless of the underlying gastric disease (Fig. 1C).

First, induction of IL-8 mRNA expression in the U-937 cell line in response to different quantities of H. pylori-derived OMVs (10, 100, or 1,000 ng) was analyzed using RT-PCR and real-time RT-PCR. The expression of IL-8 mRNA was observed in response to 10 ng or higher amounts of OMVs (Fig. 2A). As the quantity of OMVs increased, the IL-8 expression also increased in a dose-dependent manner (Fig. 2B). In a follow-up study, we determined that a dose of 10 ng OMVs was adequate to induce a significant increase in IL-8 mRNA expression.

As shown in Fig. 3A, IL-8 mRNA expression increased in the U-937 cell line upon exposure to H. pylori or OMVs (10 ng). IL-8 mRNA expression was higher in cells exposed to H. pylori than that in cells exposed to OMVs. Conventional RT-PCR revealed that vesicles derived from H. pylori in patients with gastric cancer or gastric ulcer may induce higher level of IL-8 mRNA expression than those from patients with gastritis. However, real-time RT-PCR did not reveal any significant difference in the IL-8 mRNA expression in U-937 cells when the cells were exposed to H. pylori derived from patients with different underlying gastric diseases (p = 0.874). In contrast, the IL-8 mRNA expression was significantly higher in cells exposed to OMVs from gastric cancer patient-derived strains than in those exposed to OMVs from gastritis patient-derived strains (p = 0.021).

IL-8 mRNA expression also increased in AGS cells exposed to either H. pylori or to OMVs (10 ng). The expression was higher in H. pylori-exposed cells than that in the OMV-exposed cells. The expression of IL-8 mRNA did not differ based on the underlying gastric disease in AGS cells exposed to bacteria (p = 0.626) or OMVs (p = 0.122) (Fig. 3B).

Similar results were observed in the MKN-45 cell line exposed to either H. pylori or OMVs (10 ng). The expression of IL-8 mRNA was also higher in cells exposed to the bacteria than in those exposed to OMVs. In the cells exposed to either bacteria or OMVs, IL-8 mRNA expression did not differ based on the underlying gastric disease (bacteria, p = 0.995; OMVs, p = 0.241) (Fig. 3C).

Enhanced IL-8 production in the U-937 cell line exposed to H. pylori and OMVs (10 ng) was detected using ELISA. IL-8 secretion was higher in cells exposed to bacteria than in those exposed to OMVs. IL-8 production in cells exposed to bacteria was highest in case of gastric ulcer-patient-derived strains, followed by gastric cancer patient- and gastritis patient-derived strains (p = 0.001). ELISA also revealed that the IL-8 levels were higher in cells exposed to OMVs from gastric cancer or gastric ulcer patient-derived strains than in those exposed to OMVs from gastritis patient-derived strains (p = 0.018) (Fig. 4A).

IL-8 levels also increased in AGS cells exposed to either H. pylori or OMVs (10 ng). The expression was stronger in the H. pylori-exposed cells than that in the OMV-exposed cells. The IL-8 levels in H. pylori-exposed cells did not vary significantly based on the associated gastric disease (p = 0.271). However, in case of cells exposed to OMVs, OMVs derived from gastric cancer patient-derived strains induced higher levels of IL-8 production than did those derived from gastric ulcer or gastritis patient-derived strains (p = 0.018) (Fig. 4B).

As shown in Fig. 4C, the IL-8 levels also increased in the MKN-45 cell line exposed to either H. pylori or OMVs (10 ng). The IL-8 mRNA expression levels were higher in cells exposed to bacteria than in those exposed to OMVs derived from patients with gastric cancer or gastric ulcer. Moreover, the bacteria derived from gastric cancer or gastric ulcer patients tended to induce higher IL-8 expression than those from gastritis patients (p = 0.011). Among the OMV-exposed cells, the vesicles from gastric cancer patient-derived strains induced higher levels of IL-8 expression than those from gastric ulcer or gastritis patient-derived strains (p = 0.002). In all three cell lines, the OMVs from gastric cancer patient-derived strains tended to induce higher levels of IL-8 expression than those from gastritis patient-derived strains.

Western blotting revealed that IκB-α degradation reduced significantly, while IκB-α phosphorylation increased significantly 30 minutes after the AGS cells were exposed to H. pylori-derived OMVs. In particular, IκB-α degradation was significantly reduced (by 0.2-fold) in the cells treated with OMVs from gastric cancer patient-derived strains compared with that in the control (0-minute) (Fig. 5A). IκB-α phosphorylation was significantly enhanced (by 6.2-fold) in cells treated with OMVs from gastric cancer patient-derived strains compared with that in the control (0-minute) (Fig. 5B).