The Gastric Mucosa from Patients Infected with CagA+ or VacA+ Helicobacter pylori Has a Lower Level of Dual Oxidase-2 Expression than Uninfected or Infected with CagA−/VacA− H. pylori

Gastric biopsies were obtained from patients with alimentary tract symptoms who underwent endoscopic diagnosis at the First Affiliated Hospital of Henan University of Science and Technology. To ensure accurate histopathological diagnosis, minimally four biopsies of gastric mucosa were collected during endoscopy at 2.5-cm-circle area from the pylorus, as well as one from gastric incisura angularis for the study. More biopsies were taken from the abnormal areas for pathological diagnosis. One biopsy was used for rapid urease test (RUT). One biopsy was fixed in 10 % formalin, embedded in paraffin, and then sectioned. The sections were stained with hematoxylin and eosin (H&E) for pathological scoring and for IHC staining. The remaining samples were stored at −80 °C for RNA extraction and Western blotting. RNAlater (Qiagen, Hilden, Germany, Cat. No. 76106) was used to prevent RNA degradation. Patient serum was collected during endoscopy and was tested for CagA IgG and VacA using enzyme-linked immunosorbent assays (ELISA). The clinical data collected from each patient included age, gender, medical history, medication usage, and endoscopy diagnosis (Table 1). Patient exclusion criteria were as follows: (1) undergoing proton pump inhibitor and antibiotic treatment, such as amoxicillin and clarithromycin, within 1 month of biopsy; (2) having esophageal or gastric cancers; (3) upper gastrointestinal bleeding; (4) severe disease in the liver, kidney, cardiovascular or cerebrovascular system; and (5) pregnant or breast-feeding. Informed consent was obtained from all patients, and the study was approved by the Clinical Research Ethics Committee of the Hospital.

Both RUT and C¹³ or C¹⁴ urea breath test (UBT) were used to determine H. pylori infection without performing colony-forming assay. When either one was positive, the patient was considered colonized with H. pylori. The diagnosis of chronic superficial gastritis and atrophic gastritis was based on endoscopy and pathology [41, 42]. Intestinal metaplasia (IM) was diagnosed by the presence of goblet cells. The gastric and duodenal ulcers were diagnosed by endoscopy.

The criteria for inflammation scores in gastric mucosa were based on the Updated Sydney System [41]. The pathologic features included (1) neutrophil infiltration, (2) monocyte infiltration, (3) atrophy, and (4) IM. The score for each feature is the following: normal to very mild as 0, mild as 1, moderate as 2, and marked to severe as 3. The total scores for normal tissue are below 4. The visual analogue scale was applied to microscopic examination results [41, 43].

The serum CagA IgG and VacA protein levels were measured using a CagA IgG, TSZ ELISA kit (Biotang Inc., Lexington, MA, USA, Catalog no. HU9659) and a human VacA ELISA kit (Catalog no. HU8333) following manufacturer’s instructions. Briefly, CagA IgG and VacA were detected with biotinylated monoclonal antibodies. After incubation with streptavidin–horseradish peroxidase (HRP) for 30 min, the peroxidase activity was analyzed with the addition of chromogenic substrate 3,3-,5,5-tetramethyl benzidine and hydrogen peroxide. After 15 min at room temperature, the reaction was stopped with H₂SO₄ and the absorbance was measured at 450 nm. According to the manufacturer’s specifications, above 1.2 ng/mL CagA IgG or 142 pg/mL VacA is considered positive (+).

Four-micron sections of paraffin-embedded samples were mounted on poly-l-lysine-coated slides. IHC was performed using a modified biotin–peroxidase complex method as described [44]. Briefly, tissue sections were dewaxed in xylene, rehydrated through graded alcohol, and the antigen retrieval was done by microwave-boiling the slides for 10 min in 0.1 N sodium citrate, pH 6.0. The endogenous peroxidase was blocked by incubating in 3 % H₂O₂ for 10 min, and non-specific binding was blocked by 5 % bovine serum albumin (Sigma, USA) for 20 min. Sections were incubated overnight at 4 °C with a rabbit anti-DUOX2 Ab (raised against a KLH-conjugated 501–600 amino acids of human DUOX2; Bioss, Beijing, China, Cat. No. bs-11432R). A rabbit anti-MPO Ab (1:200 dilution, Abcam, UK, Cat. No ab9535) was used to detect MPO. The DUOX2-Ab and MPO-Ab complexes were detected with biotinylated goat anti-rabbit Ab (Boster Biological Technology Co. Ltd, Wuhan, China, Cat. No SA1020) and streptavidin–HRP, and visualized with 3,3-diaminobenzidine substrate. A goat anti-NOX2 Ab (1:100 dilution, Santa Cruz, CA, USA, Cat. No sc-5826) and a biotinylated rabbit anti-goat Ab (1:300 dilution) (Boster Biological Technology Co. Ltd, Wuhan, China, Cat. No SA1023) were used as to detect NOX2. The sections were counterstained with hematoxylin. The positive control for anti-DUOX2 Ab was thyroid gland tissues, and the negative controls were either omitting the primary Ab or using an unrelated rabbit Ab.

The protein levels were evaluated blindly in 10 fields under 400× magnification from each slide. One hundred cells per field were categorized as follows: “−,” 0 %, no staining; “+,” >25 % cells were stained; “++,” 26–50 % cells were stained; and “+++,” >50 % cells stained.

Total RNA was extracted using TRIzol Reagent (Invitrogen, USA). Two μg of total RNA was used for cDNA synthesis using PrimeScript™ RT Master Mix (Takara, Japan) in a 40-μl reaction mixture, following these steps: 37 °C for 15 min,85 °C for 5 s, and 4 °C for 10 min. The primer sequences for DUOX2, NOX1, NOX2, LPO, MPO, and β-actin (Table 2) were designed by using Primer3.0 software [45] and synthesized by Sangon Biotechnology (Zhengzhou, China). Real-time qPCR was done with a CFX96™ Real-Time PCR system (Bio-Rad Labs, USA). In 25-µl reaction mixture contained 2 μl of cDNA, 12.5 μl of 2 × SYBR Premix Ex Taq II (Takara, Japan), 8.5 μl of H₂O, and 2 ul of 0.4 µM primers. A two-step method was used for DUOX2, NOX1, LPO, and β-actin cDNAs detection because of the 60 °C annealing temperature; the reaction had an initial step of 95 °C for 30 s and 40 cycles of 95 °C for 5 s plus 60 °C for 30 s. A three-step method (40 cycles of 95 °C for 5 s, 57 °C for 30 s, and 72 °C for 30 s) was used for NOX2 cDNA, which had 57 °C annealing temperature. Each sample was assayed in triplicates. The efficiency of PCR amplification was 97–105 %. A melting curve was performed to evaluate product specificity. RNA levels were quantified using the Ct (2^−ΔΔCt) method and normalized to β-actin. The gene is considered as not expressed when the Ct value is higher than 35.

Protein lysates were prepared from tissues by homogenization in RIPA lysis buffer (Soliarbio Co., Beijing, China) on ice with a grinder. The supernatant was collected after centrifugation for 15 min at 12,000 rpm and determined for protein concentrations with bicinchoninic acid (BCA, Solarbio Company, Beijing). Thirty µg of protein was resolved by 10 % SDS-PAGE and then transferred onto polyvinylidene difluoride membranes (Millipore; USA). A rabbit anti-DUOX2 Ab (200×, Abcam, UK; Cat. No. ab65813; raised against 400–500 a.a. of human DUOX2) and rabbit anti-GAPDH r Ab (10,000×, Abcam; UK, Cat. No. ab37168) were used as primary Ab. After hybridization with the HRP-conjugated anti-rabbit IgG (300X, Boster Biological Technology Co. Ltd, Wuhan, China, Cat. No. BA1054), the Ag/Ab complex was detected by an enhanced chemiluminescence reagent (Pierce; Minneapolis, MN, USA). The image was captured by ChemiDoc XRS (Bio-Rad, USA), and the intensity was quantified with ImageJ 1.48v program (NIH, USA).

Student’s t test and Mann–Whitney U test were used to assess the difference in each pair. One-way ANOVA and Kruskal–Wallis tests were used to compare the data among at least three groups. Significant difference was defined as P < 0.05. Data were reported as mean ± standard error of the mean (SEM). All statistical analysis was performed by using the SPSS 19.0 statistics package (SPSS Inc., Chicago, IL, USA).

Among the 200 patients recruited in the study, 113 patients were male and 87 were female (Table 1). Besides the higher disease incidence in males than females, there was no gender difference in disease characteristics. The average ages of male and female were 53.5 and 52.7 years. Most patients (66 %, 132/200) had chronic superficial gastritis; other patients had atrophic gastritis (2.5 %, 5/200), gastric ulcer (21.5 %, 43/200), and duodenal ulcer (10 %, 20/200). While only one of the five atrophic gastritis patients was positive for H. pylori infection, 52 % (68/132), 63 % (27/43), and 75 % (15/20) of patients with superficial gastritis, gastric ulcer, and duodenal ulcer, respectively, were positive for H. pylori infection. The total H. pylori infection rate was 55.5 % (111/200) (Table 1). This distribution pattern of H. pylori infection is similar to other studies in China [46].

We evaluated H. pylori virulence with CagA IgG and VacA ELISA on 82 of 111 patient sera. We found that 58.5 % of patients (n = 48) were negative for both CagA and VacA (CagA−/VacA−), 9.7 % (n = 8) were CagA−/VacA+, 13.4 % (n = 11) were CagA+/VacA−, and 18.3 % (n = 15) were double positive (CagA+/VacA+). The CagA IgG+ rate in our study was also similar to other studies conducted in China [47].

We analyzed the inflammation scores and compared the scores between the H. pylori-negative (Hp−) and H. pylori-positive (Hp+) groups (Table 3). The pathology scores were based on inflammation, atrophy, and intestinal metaplasia (IM). The inflammation, but not atrophy or IM, was significantly higher in the Hp+ group.

We also compared the inflammation scores between the Hp+ and Hp− groups as well as among the subgroups of Hp+ patients based on CagA IgG and VacA in their serum (Fig. 1a, b). The inflammation scores in gastric mucosa of Hp+ patients were significantly higher than Hp- group. Also, the inflammation scores of patients of CagA+/VacA+ group are significantly higher than CagA−/VacA− group (Fig. 1b). However, the inflammation scores of patients positive for CagA IgG or VacA+ H. pylori infection were not different from any other infected groups.

NOX2 protein is highly expressed in the neutrophils and macrophages, which are recruited into the inflammatory sites by H. pylori infection [48]. The NOX2 mRNA level is twofold higher in the gastric mucosa of Hp+ patients than in Hp− patients, and no difference is found among the infected groups stratified with CagA and VacA status (Fig. 2a and Supplementary Figure 1A).

Because DUOX2 protected against Helicobacter felis infection in mouse stomach [40], we analyzed DUOX2 mRNA levels in our patients. Unexpectedly, the DUOX2 RNA level is decreased in the gastric mucosa of patients who are infected with the H. pylori expressing either of the virulence factors compared to Hp + patients without either virulence factors and in Hp- patients (Fig. 2b and Supplementary Figure 1A). The DUOX2 mRNA level of the CagA−/VacA− group is similar to that in the Hp- group.

Because DUOX2 and LPO have been postulated to form a defense mechanism against bacterial infection and LPO is expressed in the intestine [22, 34], we also analyzed LPO gene expression in the gastric mucosa. No LPO mRNA can be detected in both Hp+ and Hp− patients (n = 20 for each group; data not shown). Furthermore, because NOX1 plays a role in wound-healing [49] and promotes inflammation in a mouse model of ileocolitis [24], we also analyzed NOX1 mRNA levels in the gastric mucosa. Similar to LPO, no NOX1 mRNA was detected in the gastric mucosa (n = 20 each for Hp+ and Hp− groups, as well as two IM samples (data not shown).

We quantified DUOX protein levels with Western blotting (Fig. 3a, b). Consistent with DUOX2 mRNA levels, twofold higher DUOX protein level was present in Hp− gastric mucosa than Hp+ samples. After confirming the Ab specificity by IHC of thyroid tissues (data not shown), this antibody was used to compare DUOX2 protein levels in the gastric mucosa among the Hp+ subgroups and Hp− group by IHC. A stronger DUOX staining was detected in Hp− patients especially at the apical surface, although it is also present in the cytoplasm of the epithelial cells (Fig. 3c). Consistent with DUOX2 mRNA levels, mucosa of CagA+ or VacA+ group had weaker DUOX2 staining than the CagA−/VacA− and Hp- groups (Table 4). Also, the CagA−/VacA− group had similar DUOX2 staining intensity as the Hp− group.

NOX2 IHC was performed on the gastric mucosa of Hp+ and Hp− groups of patients (n = 20 for each group, Fig. 3d). In the Hp+ groups of patients, a stronger NOX2 staining was detected in the infiltrating inflammatory cells compared to that in Hp− gastric mucosa (IHC scores not shown). Similarly to NOX2 IHC pattern, a higher level of MPO+ inflammatory cells is detected in the Hp+ groups than in the Hp− patients (n = 15 for each group, data not shown).