Role of interleukin-32 in Helicobacter pylori-induced gastric inflammation

Abstract

Helicobacter pylori infection is associated with gastritis and gastric cancer. An H. pylori virulence factor, the cag pathogenicity island (PAI), is related to host cell cytokine induction and gastric inflammation. Since elucidation of the mechanisms of inflammation is important for therapy, the associations between cytokines and inflammatory diseases have been investigated vigorously. Levels of interleukin-32 (IL-32), a recently described inflammatory cytokine, are increased in various inflammatory diseases, such as rheumatoid arthritis and Crohn's disease, and in malignancies, including gastric cancer. In this report, we examined IL-32 expression in human gastric disease. We also investigated the function of IL-32 in activation of the inflammatory cytokines in gastritis. IL-32 expression paralleled human gastric tissue pathology, with low IL-32 expression in H. pylori-uninfected gastric mucosa and higher expression levels in gastritis and gastric cancer tissues. H. pylori infection increased IL-32 expression in human gastric epithelial cell lines. H. pylori-induced IL-32 expression was dependent on the bacterial cagPAI genes and on activation of nuclear factor κB (NF-κB). IL-32 expression induced by H. pylori was not detected in the supernatant of AGS cells but was found in the cytosol. Expression of the H. pylori-induced cytokines CXCL1, CXCL2, and IL-8 was decreased in IL-32-knockdown AGS cell lines compared to a control AGS cell line. We also found that NF-κB activation was decreased in H. pylori-infected IL-32-knockdown cells. These results suggest that IL-32 has important functions in the regulation of cytokine expression in H. pylori-infected gastric mucosa.

Fig 1

Expression of IL-32 in human gastric tissues. (A) Immunoblot analysis of IL-32 protein in healthy human gastric tissues, gastritis tissues, and gastric cancer tissues. (B) (Left) Mouse IgG1 isotype antibody as the negative control. (Right) Representative immunohistochemical analysis of IL-32 expression in human gastritis tissue. (C) Typical examples of immunohistochemical analysis of IL-32 expression in healthy human gastric tissue (left), gastritis tissue (center), and gastric cancer tissue (right). (D) Graphic representation of IL-32 expression in human gastric samples, including healthy gastric mucosa, gastritis tissues, and gastric cancer tissues. Expression was quantified by ELISA. Each box plot indicates the median (horizontal line), interquartile range (the box itself), and the sample minimum and maximum (bars). *, P < 0.05 by Tukey's HSD test. (E) (Left) The full-length coding region of IL-32 cDNA, synthesized from RNA extracted from human gastritis tissue and AGS cells infected with H. pylori, was amplified by PCR. (Right) Known structures of IL-32 isoforms (splice variants) and numbers of clones detected in human gastritis tissue. The exon numbers are shown beneath the boxes.

Fig 2

H. pylori infection induces IL-32 expression in gastric cancer cells. (A) AGS cells were infected with cagPAI-positive H. pylori strain TN2 for 24 h or stimulated with IL-1β or TNF-α for 24 h at the indicated concentrations. IL-32 expression was detected by immunoblot analysis. (B) AGS, TMK-1, and MKN45 gastric cancer cells were cocultured with H. pylori for the indicated times (hours), and IL-32 expression was evaluated by immunoblot analysis. (C) IL-32 and IL-8 mRNA levels in AGS cells after infection with H. pylori for 6 h were determined by real-time RT-PCR. Data shown are means and standard errors of the means (SEM) (n = 3). *, P < 0.05 by Student's t test.

Fig 3

H. pylori infection induces IL-32 production in AGS cells via the NF-κB signaling pathway. (A) Levels of IL-32 in AGS cells infected with wild-type H. pylori (WT), TN2-ΔcagPAI, or TN2-ΔcagE for the indicated times were determined by immunoblot analysis. Anti-urease staining was used to control for H. pylori strain variations. (B) H. pylori-induced IL-32 mRNA expression, with or without chemical inhibitors, was examined by real-time RT-PCR. The IKKβ inhibitor SC-514 (SC) and the p38 inhibitor SB203580 (SB) were added at a concentration of 20 μM 1 h before H. pylori infection. Cells were cocultured with H. pylori for 24 h. Data are means and SEM (n = 3). *, P < 0.05 by Dunnett's multiple-comparison test; NS, not significant. (C) H. pylori-induced IL-32 expression in AGS cells treated with control siRNA and IKKβ siRNA, as determined by immunoblot analysis. AGS cells were transfected with the siRNAs for 48 h and then infected with H. pylori for the indicated times.

Fig 4

IL-32 is intracellular in H. pylori-infected cells. (A) (Left) IL-32 concentrations in supernatants were quantitated by ELISA. Supernatants were collected from AGS cells infected with H. pylori for 24 h and from the same cells that had been freeze-thawed to facilitate cell membrane destruction. (Right) IL-32 concentrations in AGS cell lysates, with or without H. pylori infection for 24 h, were determined by ELISA. Data shown are means and SEM (n = 3). *, P < 0.05 by Student's t test. (B) AGS cells were cocultured with H. pylori for 24 h, and IL-32 expression was analyzed by immunofluorescence staining. Nuclear staining was conducted using Hoechst 33342. (C) Immunoblots of the indicated proteins in AGS cells stimulated with TNF-α (1 ng/ml) or recombinant IL-32β (1 ng/ml) for 0, 15, 30, or 60 min.

Fig 5

IL-32 expression affects induction of other cytokines involved in gastritis. (A) Control (Ctrl) and stable IL-32-knockdown AGS cell lines (IL32KD1 and IL32KD2) were cultured with or without H. pylori for 6 h. IL-32β and IL-8 mRNA levels were determined by real-time RT-PCR. Data shown are means and SEM (n = 3). *, P < 0.05 by Dunnett's multiple-comparison test. (B) CXCL1, CXCL2, TNF-α, and IL-11 mRNA levels were evaluated by real-time RT-PCR analysis of AGS control cells (Ctrl) and IL32KD1 cells cultured with or without H. pylori for 6 h. Data shown are means and SEM (n = 3). *, P < 0.05 by Student's t test; NS, not significant. (C) ELISAs of IL-8, CXCL1, and CXCL2 expression in AGS cell supernatants of control (Ctrl) and IL32KD1 cells cultured with or without H. pylori for 24 h. Data shown are means and SEM (n = 3). *, P < 0.05 by Student's t test. (D) Two stable IL-32-overexpressing AGS cell lines (AGS IL-32 clone 15 and AGS IL-32 clone 19) were established. (Left) The increased IL-32 mRNA level was confirmed by real-time RT-PCR. (Right) IL-8 mRNA levels in the control (Ctrl), IL-32 clone 15, and IL-32 clone 19 cells were determined by real-time RT-PCR. Data shown are means and SEM (n = 3). *, P < 0.05 by Dunnett's multiple-comparison test. (E) IL-8 reporter activity was examined in AGS control (Ctrl), IL32KD1, and IL32KD1 cells transfected with IL-32β cDNA (pcDNA-mIL32β). Cells were left uninfected or infected with H. pylori for 24 h. Data shown are means and SEM (n = 3). *, P < 0.05 by Student's t test. The IL-32 level in each cell line is shown in the upper right panel.

Fig 6

Role of IL-32 in H. pylori-induced cell signaling. (A) (Top) Micrographs of uninfected AGS cells transfected with the nonsilencing control (AGS Ctrl), AGS Ctrl cells infected with H. pylori, and stable IL-32-knockdown AGS (AGS-IL32KD1) cells infected with H. pylori for 24 h. (Bottom) Cell lysates of uninfected AGS Ctrl cells, AGS Ctrl cells infected with H. pylori, and AGS-IL32KD1 cells infected with H. pylori for 24 h were immunoprecipitated (IP) using anti-CagA antibody, and the immunoprecipitates were immunoblotted (IB) using anti-phosphotyrosine and anti-CagA antibodies. (B) NF-κB activation induced by 24-h H. pylori infection of AGS Ctrl cells and AGS-IL32KD1 cells was determined using luciferase activity assays. Data shown are means and SEM (n = 3). *, P < 0.05 by Student's t test. (C) Immunoblot analysis of IL-32 and pIκBα levels in AGS Ctrl cells and AGS-IL32KD1 cells infected with H. pylori for the indicated times. Anti-urease staining was used to control the H. pylori load. (D) H. pylori-induced IL-32β mRNA (right) and IL-8 mRNA (left) levels in AGS cells treated with control siRNA (AGS Ctrl) and IL-8 siRNA (AGS-siIL8), as determined by real-time RT-PCR. AGS cells were transfected with the siRNAs for 48 h and then infected with H. pylori for an additional 24 h. Data shown are means and SEM (n = 3). *, P < 0.05 by Student's t test; NS, not significant. (E) Summary of the role of IL-32 in gastritis. H. pylori induces NF-κB activation in gastric epithelial cells in a cagPAI-dependent manner. NF-κB activation is required for IL-32 expression. Intracellular IL-32 expression amplifies both NF-κB activation and IL-8 expression.