Patients and specimens
The gastric biopsy specimens and blood were collected from 92 H. pylori-infected and 32 uninfected patients who underwent upper esophagogastroduodenoscopy for dyspeptic symptoms at XinQiao Hospital (Supplementary Table 1). H. pylori infection was determined by [14C] urea breath test and rapid urease test of biopsy specimens taken from the antrum, and subsequently conformed by real-time PCR for 16s rDNA and serology test for specific anti-H. pylori antibodies (Abs) . For isolation of human primary GECs, fresh non-tumor gastric tissues (at least 5-cm distant from the tumor site) were obtained from gastric cancer patients who underwent surgical resection and were determined as H. pylori-negative individuals as above at the Southwest Hospital. None of these patients had received chemotherapy or radiotherapy before sampling. Individuals with atrophic gastritis, hypochlorhydria, antibiotics treatment, autoimmune disease, infectious diseases and multi-primary cancer were excluded. The study was approved by the Ethics Committee of XinQiao Hospital and Southwest Hospital of Third Military Medical University. A written informed consent was obtained from each subject.
Antibodies and other reagents
(Supplementary Table 2)
All breeding and experiments were undertaken with review and approval from the Animal Ethical and Experimental Committee of Third Military Medical University. C57BL/6 Rev-erbα+/- mice were obtained from the Jackson Laboratory (Bar Harbor, USA). C57BL/6 Rev-erbα-/- mice and their littermate control (wild-type, WT) mice were generated by breeding between C57BL/6 Rev-erbα+/- mice. C57BL/6 interferon-γ-/- (IFN-γ-/-) mice were kindly provided by Dr. Richard A. Flavell (Yale University). All mice used in experiments were viral Ab negative for pathogenic murine viruses, negative for pathogenic bacteria including Helicobacter spp. and parasites, and were maintained under specific pathogen free (SPF) conditions in a barrier sustained facility and provided with sterile food and water.
Bacteria culture and infection of mice with bacteria
H. pylori NCTC 11637 (cagA positive) (WT H. pylori) and cagA-knockout mutant H. pylori NCTC 11637 (ΔcagA) or H. pylori 26695 were grown in brain-heart infusion plates containing 10% rabbit blood at 37°C under microaerophilic conditions. For infecting mouse, bacteria were propagated in Brucella broth with 5 % fetal bovine serum (FBS) with gentle shaking at 37°C under microaerobic conditions. After culture for 1 d, live bacteria were collected and adjusted to 109 colony-forming units (CFU)/ml. The mice were fasted overnight and orogastrically inoculated twice at a 1 d interval with 3×108 CFU bacteria. H. pylori infection status and H. pylori-induced gastritis in murine experiments were confirmed using real-time PCR of H. pylori 16s rDNA, urease biopsy assays, Warthin-Starry staining and immunohistochemical staining for H. pylori, and evaluation of inflammation by haematoxylin and eosin (H&E) staining [33, 34].
Generation of bone marrow (BM) chimera mice
The following BM chimeric mice were created: male WT BM→female WT mice, male WT BM→female Rev-erbα-/- mice, male Rev-erbα-/- BM→female WT mice, and male Rev-erbα-/- BM→female Rev-erbα-/- mice. BM cells were collected from the femurs and tibia of donor mice by aspiration and flushing, and were suspended in PBS at the concentration of 5×107/ml. The BM in recipient mice was ablated with lethal irradiation (8 Gy). Then, the animals received intravenously 1.5×107 BM cells from donor mice in a volume of 300 μl sterile PBS under the anaesthesia. Thereafter, the transplanted BM was allowed to reconstitute for 8 weeks before subsequent experimental procedures. To verify successful engraftment and reconstitution of the BM in the host mice, genomic DNA was isolated from tail tissues of each chimera mouse 8 weeks after BM transplantation. Quantitative PCR was performed to detect the Sry gene present in the Y chromosome (primers seen in Supplementary Table 3) and mouse β2-microglobulin gene as an internal control. The chimeric rates were calculated on the assumption that the ratio of the Sry to β2-microglobulin gene was 100% in male mice. We confirmed that the chimeric rates were consistently higher than 90%. After BM reconstitution was confirmed, mice were infected with bacteria as described above.
Antibodies/CCL21/Reg3b/β-defensin-1/Rev-erbα agonist/Rev-erbα antagonist administration
One day after infection with WT H. pylori as described above, WT mice were injected intraperitoneally with recombinant mouse CCL21 (25 μg) or Reg3b and/or β-defensin-1 (50 μg), or anti-mouse CCL21 and/or anti-mouse CCR7 or isotype control Abs (100 μg), or Rev-erbα agonist SR9009 (100 mg/kg, dissolved in cremophor) or cremophor control, or Rev-erbα antagonist SR8278 (25 mg/kg, dissolved in DMSO) or DMSO control and repeated every week until the mice were sacrificed.
T-cell adoptive transfer
One day before infection with WT H. pylori, WT or Rev-erbα-/- mice were injected intravenously (1×106 cells/mouse) with purified spleen CD4+ T cells (StemCell Technologies) from uninfected WT mice, or WT H. pylori-infected WT mice or WT H. pylori-infected IFN-γ-/- mice (8 week post infection (p.i.)). Then the recipient mice were infected with bacteria as described above and sacrificed for bacteria colonization evaluation at week 8 p.i..
Evaluation of bacteria colonization
The mice were sacrificed at the indicated time points. The stomach was cut open from the greater curvature and half of the tissue was cut into four parts for RNA extraction, DNA extraction, protein extraction, and tissue fixation for immunohistochemistry or immunofluorescence staining, respectively. DNA of the biopsy specimens were extracted with QIAamp DNA Mini Kit. As previously described , H. pylori colonization was quantified by measuring H. pylori-specific 16s rDNA using specific primer and probe (Supplementary Table 3) by the TaqMan method. The amount of mouse β2-microglobulin DNA in the same specimen was used to normalize the data. According to a previous study , the density of H. pylori was shown as the number of bacterial genomes per nanogram of host genomic DNA. Another half of stomach was used for isolation of single cells as described below. The isolated single cells were collected and analyzed by flow cytometry staining.
Isolation of single cells from tissues
Fresh tissues were washed three times with Hank’s solution containing 1% FBS, cut into small pieces, collected in RPMI 1640 containing 1 mg/ml collagenase IV and 10 mg/ml DNase I, and then mechanically dissociated by using the gentle MACS Dissociator (Miltenyi Biotec). Dissociated cells were further incubated for 0.5-1 h at 37°C under continuous rotation. The cell suspensions were then filtered through a 70-μm cell strainer (BD Labware).
Cell/tissue culture, transfection and stimulation
Primary GECs were purified from gastric tissue single-cell suspensions from uninfected donors or mice with a MACS column purification system using anti-human or mouse CD326 magnetic beads. The sorted primary GECs were used only when their viability was determined >90% and their purity was determined >95%. For human GEC lines (AGS cells, GES-1 cells, HGC-27 cells, SGC-7901 cells, BGC-823 cells), 3×105 cells per well in 12-well cell culture plate (for real-time PCR) or 1×106 cells per well in 6-well cell culture plate (for western blot and ELISA) were starved in DMEM (Dulbecco’s Modified Eagle Medium)/F-12 medium supplemented with penicillin (100 U/ml) and streptomycin (100 μg/ml) for 6 h in a humidified environment containing 5% CO2 at 37 °C. Then the cells were incubated in antibiotic-free DMEM/F-12 medium supplemented with 10% FBS instead. The cell lines were used when their viability was determined >90%. Human GEC lines, primary GECs, or primary gastric mucosa tissues from uninfected donors or mice were infected with WT H. pylori, ΔcagA or H. pylori 26695 at a multiplicity of infection (MOI) of 100 for 24 h. AGS cells were also infected with WT H. pylori at different MOI (24 h) or at the indicated time points (MOI=100). For signal pathway inhibition experiments, AGS cells were pre-treated with 5 μl (20 μM) U0126 (an MEK-1 and MEK-2 inhibitor), SB203580 (a p38 MAPK inhibitor), SP600125 (a JNK inhibitor), FLLL32 (an STAT3 inhibitor), AG490 (a JAK inhibitor), Wortmannin (a PI3K-AKT inhibitor), PP2 (a cagA EPIYA motif phosphorylation inhibitor) or DMSO control for 2 h. For Rev-erbα activation or inhibition experiments, AGS cells were pre-treated with 5 μl (10, 20 μM) Rev-erbα agonist SR9009 or cremophor control, or Rev-erbα antagonist SR8278 or DMSO control for 2 h, or were pre-treated with Rev-erbα siRNA, non-specific control siRNA (NC) (40 nM), or lipofectamine 2000 only (Mock) for 24 h. In some cases, AGS cells were transfected with plasmids pcDNA3.1 or cagA-pcDNA3.1 by using lipofectamine 2000 according to the manufacturer’s protocols. At 24 h after transfection, cells were treated with or without U0126 (5 μl, 20 μM) or DMSO control for 2 h and cultured for an additional 24 h. After co-culture, cells were collected for microarray, immunofluorescence, real-time PCR, and western blot, and the culture supernatants were harvested for ELISA.
Luciferase reporter assay
Promoter constructs containing the region from -2000 to 0, -1000 to 0, -500 to 0, -250 to 0, -100 to 0, and other 250 bp fragments of Rev-erbα gene were amplified from human genomic DNA using specifically designed primers (Supplementary Table 4) by PCR. The amplified full-length or fragmented sequences were cloned into the NheI and HindIII sites of the pGL3-basic vector respectively. Promoter constructs containing the region from -2000 to 0 of CCL21 and β-defensin-1 gene, and mutants of predicted Rev-erbα binding site of CCL21 and β-defensin-1 gene sequences were also synthesized with primers (Supplementary Table 5) and cloned into the NheI and HindIII sites of the pGL3-basic vector respectively. The above constructs were sequence verified. Plasmids over-expressing Rev-erbα (Rev-erbα-pcDNA3.1) were constructed by inserting target gene into plasmid pcDNA3.1, these plasmids were constructed and produced by Sangon Biotech (Shanghai) Co., Ltd. Cells were transfected with various combinations of the reporter plasmid, which contains the internal control pRL-TK (Promega) or the expression plasmid. Cells were harvested at 24 h (H. pylori infection assay) or 48 h (cagA-pcDNA3.1 or Rev-erbα-pcDNA3.1 plasmid transfection assay) after transfection. Luciferase activities of the lysates were measured using the Dual-Luciferase Reporter assay Kit following the manufacturer’s protocol. Luciferase activity was normalized to Renilla luciferase activity.
Chromatin Immunoprecipitation (ChIP)
AGS cells infected with H. pylori (MOI=100, 24 h) were treated at room temperature for 10 min with 1% formaldehyde in cell culture media. Glycine (11 % in media) solution was then gently mixed in at room temperature for 5 min to terminate cross-linking. Cells were washed twice with ice-cold PBS and palleted at 3000g for 5 min. Membrane Extraction Buffer containing protease/phosphatase inhibitors was then added to each palleted sample. Cell lysates were pulse-sonicated on ice; supernatants containing the digested chromatin were collected into two tubes for input and immunoprecipitation. Anti-NF-κB p65 or anti-Rev-erbα Ab was added and IP reactions conducted overnight at 4°C with agitation. ChIP grade protein A/G magnetic beads were then added to each IP reaction. Two hours later beads were collected, washed, bounded IP materials eluted with 5 M NaCl containing 20 μg/ml Proteinase K. The cross-linking was reversed by heating up to 65°C for 1.5 h and DNA was purified. Purified DNA samples were analyzed by PCR with designed primers (Supplementary Table 6).
In vitro bactericidal assay
Two hundred microliters of 5×106 CFU/ml WT H. pylori suspension was incubated with 20 μg/ml mouse Reg3b and/or β-defensin-1 for 3, 6, or 12 h, or incubated with 5, 10, or 20 μg/ml mouse Reg3b and/or β-defensin-1 for 24 h. PBS was used as control. In another set of experiments, primary GECs from WT or Rev-erbα-/- mice were infected with WT H. pylori (MOI=100) for 24 h. The culture supernatants were filtered through 0.4-μm filters and collected. Then two hundred microliters of 5×106 CFU/ml WT H. pylori suspension were incubated with the above collected culture supernatants with or without anti-Reg3b and/or anti-β-defensin-1 Abs (20 μg/ml) for 24 h. Bacteria were serially diluted and plated on brain-heart infusion plates containing 10% rabbit blood and incubated for 3-5 days at 37°C under microaerophilic conditions and CFU was enumerated. The results was determined by counting CFU of alive bacteria with agar plating and expressed as the survival rate of WT H. pylori after incubation with Reg3b/β-defensin-1 or PBS, or was determined by counting CFU of alive bacteria with agar plating and expressed as the survival rate of WT H. pylori after incubation with supernatants from H. pylori-infected GECs of Rev-erbα-/- mice or WT mice with or without anti-Reg3b and/or anti-β-defensin-1 Abs.
Human CD45+CD11c-CD66b-CD11b+CD68- myeloid cells or mouse CD45+CD11c-Ly6G-CD11b+CD68- myeloid cells from blood of H. pylori-infected donors or WT H. pylori-infected mice (8 week p.i.) were sorted by fluorescence activating cell sorter (FACS) (FACSAria II; BD Biosciences). AGS cells were pre-treated with Rev-erbα siRNA or non-specific control siRNA (NC) (both at 40 nM) for 24 h, and then infected with WT H. pylori or ΔcagA (MOI=100) for 24 h. The culture supernatants were filtered through 0.4-μm filters, collected and used as source of chemoattractants in a human myeloid cell chemotaxis assay. In another set of experiments, mouse primary GECs were purified from gastric tissue single-cell suspensions of uninfected WT or Rev-erbα-/- mice with anti-mouse CD326-conjugated MACS magnetic beads, and then infected with WT H. pylori or ΔcagA (MOI=100) for 24 h. The culture supernatants were then collected mentioned above. These culture supernatants were then used as source of chemoattractants in a mouse myeloid cell chemotaxis assay.
In a chemotaxis assay, FACS-sorted myeloid cells (1×105) were transferred into the upper chambers of the transwells (5-μm pore). CCL21 (100 ng/ml) and culture supernatants from various cultures were placed in the lower chambers. After 6 h culture, migration was quantified by counting cells in the lower chamber and cells adhering to the bottom of the membrane. In some cases, blocking Ab for human/mouse CCL21 (20 μg/ml) or corresponding control IgG/IgG2a (20 μg/ml) were added into the culture supernatants, and blocking Ab for human/mouse CCR7 (20 μg/ml) or corresponding control IgG2a (20 μg/ml) were added into myeloid cell suspensions and incubated for 2 h before chemotaxis assays.
In vitro T cell culture system
Purified mouse peripheral or spleen CD4+ T cells from uninfected or WT H. pylori-infected WT mice (8 week p.i.) were labeled with carboxyfluorescein succinimidyl ester (CFSE) and co-cultured (1×105 cells/well) with FACS-sorted gastric CD45+CD11c-Ly6G-CD11b+CD68- myeloid cells from WT H. pylori-infected WT mice (8 week p.i.) at 2:1 ratio in 200 μl RPMI 1640 medium containing mouse recombinant IL-2 (20 IU/ml), anti-CD3 (2 μg/ml), and anti-CD28 (1 μg/ml) Abs. After a 5-d incubation, cells were collected and analyzed by intracellular cytokine staining, and the culture supernatants were harvested for ELISA.
Paraformaldehyde-fixed and paraffin-embedded samples were cut into 5 µm sections. For immunohistochemical staining, the sections were incubated with rabbit anti-human/mouse Rev-erbα, rabbit anti-human/mouse p-ERK, sheep anti-mouse Reg3b or rabbit anti-mouse β-defensin-1 followed by HRP-conjugated anti-rabbit IgG or HRP-conjugated anti-sheep IgG and later its substrate diaminobenzidine. All the sections were finally counterstained with haematoxylin and examined using a microscope (Nikon Eclipse 80i; Nikon).
Paraformaldehyde-fixed cryostat tissue sections or AGS cells were washed in PBS, blocked for 30 min with 20% goat serum in PBS, stained for Rev-erbα, Rev-erbα and CD326, or CCL21 and CD326. Slides were examined with a confocal fluorescence microscope (LSM 510 META, Zeiss).
DNA of the biopsy specimens were extracted with QIAamp DNA Mini Kit and RNA of biopsy specimens and cultured cells were extracted with TRIzol reagent. The RNA samples were reversed transcribed into cDNA with PrimeScriptTM RT reagent Kit. Real-time PCR was performed on an IQ5 (Bio-Rad) with Real-time PCR Master Mix according to the manufacturer's specifications. The mRNA expression of 16s rDNA, cagA, Rev-erbα, chemokine, IFN-γ, β-defensin and Reg3 genes was measured using the TaqMan and/or SYBR green method with the relevant primers (Supplementary Table 3). For mouse samples, mouse β2-microglobulin mRNA level served as a normalizer, and its level in the stomach of uninfected or WT mice served as a calibrator. For human samples, human β-actin mRNA level served as a normalizer, and its level in the uninfected cells/tissues or stomach of uninfected donors served as a calibrator. The relative gene expression was expressed as fold change of relevant mRNA calculated by the ΔΔCt method.
Cell surface markers were stained with specific or isotype control Abs. For intracellular molecules measurements, the cells were stimulated for 5 h with PMA (50 ng/ml) plus ionomycin (1 μg/ml) in the presence of Golgistop. Intracellular cytokine staining was performed after fixation and permeabilization, using Perm/Wash solution. Then, the cells were analyzed by multicolor flow cytometry on a FACSCanto II (BD Biosciences). Data were analyzed with Flowjo software (TreeStar) or FACSDiva software (BD Biosciences).
Isolated human and mouse gastric tissues were homogenized in 1 ml sterile Protein Extraction Reagent, and centrifuged. Tissue supernatants were collected for ELISA. Concentrations of CCL21, Reg3b, β-defensin-1 or IFN-γ in the tissue supernatants; concentrations of CCL21, Reg3b or β-defensin-1 in the gastric epithelial cell culture supernatants; concentrations of IFN-γ in the T cell culture supernatants were determined using ELISA kits according to the manufacturer’s instructions.
Western blot analysis
Western blots were performed on 10%-15% SDS-PAGE gel transferred PVDF membranes with equivalent amounts of cell or tissue lysate protein for each sample. Five percent skim milk was used for blocking the PVDF membranes. Mouse Rev-erbα, ERK1/2, and p-ERK1/2 were detected with rabbit anti-Rev-erbα Ab, rabbit anti-ERK1/2 Ab, and rabbit anti-p-ERK1/2 Ab; human Rev-erbα, ERK1/2, and p-ERK1/2 were detected with rabbit anti-Rev-erbα Ab, rabbit anti-ERK1/2 Ab, and rabbit anti-p-ERK1/2 Ab; cagA was detected with rabbit anti-cagA Ab respectively. This was followed by incubation with HRP-conjugated secondary Abs. Bound proteins were visualized by using SuperSignal® West Dura Extended Duration Substrate kit.
Gene expression profiles of WT H. pylori-infected and uninfected AGS cells were analyzed with the human Exon 1.0 ST GeneChip (Affymetrix), strictly following the manufacturer’s protocol. Microarray experiments were performed at the Genminix Informatics (China) with the microarray service certified by Affymetrix.
Results are expressed as mean ± SEM. Student t test was generally used to analyze the differences between two groups, but when the variances differed, the Mann-Whitney U test was used. For multiple comparisons, the 1-way ANOVA was used. Correlations between parameters were assessed using Pearson correlation analysis and linear regression analysis, as appropriate. SPSS statistical software (version 13.0) was used for all statistical analysis. All data were analyzed using two-tailed tests, and P<0.05 was considered statistically significant. Microarray data analysis was performed with the assistance of Genminix Informatics. Raw data from each array were analyzed using TwoClassDif.