Helicobacter pylori induces CCL20 expression

Abstract

CCL20 attracts immature dendritic cells and memory T cells and plays a role on mucosal surfaces in inflammation. However, whether Helicobacter pylori infection induces CCL20 in human gastric epithelial cells remains to be determined. The aim of this study was to analyze the molecular mechanism of H. pylori-induced CCL20 expression. Expression of CCL20 mRNA was assessed by reverse transcription-PCR. Five normal and five H. pylori-infected gastric tissue samples were stained immunohistochemically for CCL20. A luciferase assay was used to monitor activation of the CCL20 gene promoter, and an electrophoretic mobility shift assay was used to explore the binding of transcription factors to this promoter. The CCL20 expression in epithelial cells of H. pylori-positive tissues was higher than that in H. pylori-negative tissues. H. pylori induced CCL20 expression in gastric epithelial cell lines, and the induction was dependent on an intact cag pathogenicity island. Activation of the CCL20 promoter by H. pylori occurred through the action of NF-kappaB. Transfection of IkappaB kinase and NF-kappaB-inducing kinase dominant negative mutants inhibited H. pylori-mediated activation of CCL20. Treatment with an inhibitor of Hsp90 suppressed H. pylori-induced CCL20 mRNA due to deactivation of NF-kappaB. Collectively, these results suggest that H. pylori activates NF-kappaB through an intracellular signaling pathway that involves IkappaB kinase and NF-kappaB-inducing kinase, leading to CCL20 gene transcription, and that Hsp90 is a crucial regulator of H. pylori-induced CCL20 expression, presumably contributing to the immune response in H. pylori.

FIG. 1.

Expression of CCL20 in H. pylori-infected gastric mucosa. (A) RT-PCR analysis of CCL20 in human gastric tissues. Lanes 1 to 5, normal mucosa; lanes 6 to 10, H. pylori-positive gastritis; lane M, markers. β-Actin expression served as a control. Representative results of three similar experiments are shown. (B to E) Immunohistochemical detection of CCL20 in tissues of patients with H. pylori-positive gastritis. The serial sections of gastric biopsy specimens were stained with goat polyclonal antibody to CCL20. Sections were counterstained with methyl green. (B and C) Representative examples of normal mucosa. (D and E) Representative examples of mucosa from patients with H. pylori-positive gastritis. Note the positive staining for CCL20 in the epithelial cells of the mucosa from patients with H. pylori-positive gastritis. Original magnification, ×400.

FIG. 2.

H. pylori-induced CCL20 mRNA expression in gastric epithelial cells. (A) Dynamics of H. pylori-induced CCL20 mRNA expression. Total RNA was extracted from MKN45 and MKN28 cells infected with H. pylori ATCC 49503 for the indicated times and used for RT-PCR. The bacterium-to-cell ratio was 20:1. (B) cag PAI-positive and cag PAI-negative H. pylori strains differ in the ability to induce CCL20 expression. (C) cag PAI of H. pylori is required for induction of CCL20 expression in MNK45 cells. Total RNA was extracted from the cells infected with H. pylori for 6 h and used for RT-PCR. β-Actin expression served as a control. Representative results of three similar experiments in each panel are shown. Lane M contained markers. WT, wild type.

FIG. 3.

Increased secretion of CCL20 into the supernatants of MKN45 and MKN28 cultures in response to H. pylori infection at 24 h. Cells were infected with various densities of H. pylori ATCC 49503. CCL20 concentrations in the supernatants were determined by ELISA. The data are means ± standard deviations of three experiments.

FIG. 4.

H. pylori infection activates the CCL20 promoter in gastric epithelial cells. (A) H. pylori infection increased CCL20 promoter activity in a dose-dependent fashion. Either pGL2-CCL20 or pGL2 (promoterless luciferase vector) was transfected into MKN45 cells, and the cells were subsequently infected with H. pylori ATCC 49503 for 6 h. The activities are expressed relative to that of cells transfected with pGL2-CCL20 without further treatment, which was defined as 1. (B) cag PAI is required for induction of CCL20 promoter activity. pGL2-CCL20, pGL2, or κB-LUC was transfected into MKN45 cells, and the cells were subsequently infected with wild-type strain 26695 (WT) or the isogenic mutant Δcag PAI (Δcag) for 6 h. The bacterium-to-cell ratio was 20:1. The activities are expressed relative to that of cells transfected with pGL2-CCL20 without further treatment, which was defined as 1. The data are means ± standard deviations of three independent experiments.

FIG. 5.

H. pylori activates the CCL20 promoter through the NF-κB binding site. (A) Schematic diagram of the CCL20 reporter constructs containing the wild-type (pGL2-CCL20) and mutant (pGL2-CCL20/κBM) NF-κB sites. Underlined letters indicate substituted base pairs. (B) NF-κB element in the CCL20 promoter is critical for H. pylori-induced activity. Either pGL2-CCL20 or pGL2-CCL20/κBM was transfected into MKN45 cells, and the cells were subsequently infected with H. pylori ATCC 49503 for 6 h. The bacterium-to-cell ratio was 20:1. The activities are expressed relative to that of cells transfected with pGL2-CCL20 without further treatment, which was defined as 1. The data are means ± standard deviations of three independent experiments. WT, wild type.

FIG. 6.

H. pylori infection induced NF-κB binding activity. (A) Time course of NF-κB activation in MKN45 and MKN28 cells infected with H. pylori, evaluated by EMSA. Nuclear extracts from cells infected with H. pylori ATCC 49503 for the indicated times were mixed with ³²P-labeled NF-κB probe. The bacterium-to-cell ratio was 20:1. (B) Sequence specificity of NF-κB binding activity and characterization of NF-κB proteins that bound to the NF-κB binding site of the CCL20 gene. Competition assays were performed with nuclear extracts from MKN45 and MKN28 cells infected with H. pylori ATCC 49503 for 1 h. Where indicated, 100-fold excess amounts of each specific competitor oligonucleotide were added to the reaction mixture with labeled probe NF-κB (lanes 2 to 4). A supershift assay of NF-κB DNA binding complexes in the same nuclear extracts was also performed. Where indicated, appropriate antibodies (Ab) were added to the reaction mixture before addition of the ³²P-labeled probe (lanes 5 to 9). The arrows indicate the specific complexes, while the arrowheads indicate the DNA binding complexes supershifted by antibodies. (C) cag PAI products of H. pylori are required for induction of NF-κB binding activity in MKN45 cells. Nuclear extracts from MKN45 cells cocultured with different densities of wild-type strain 26695 or the isogenic mutant Δcag PAI were analyzed for NF-κB. Representative results of three similar experiments in each panel are shown. WT, wild type.

FIG. 7.

NF-κB signal is essential for activation of CCL20 expression by H. pylori. (A) H. pylori infection leads to IκBα phosphorylation and degradation. MKN45 cells were infected with H. pylori ATCC 49503 for the indicated times. The bacterium-to-cell ratio was 20:1. The cells were then lysed and analyzed by immunoblotting with phospho-specific IκBα, IκBα, and actin antibodies. Representative results of three similar experiments are shown. (B) Functional effects of IκBα, IκBβ, and IKKγ dominant interfering mutants and kinase-deficient IKKα, IKKβ, and NIK mutants on H. pylori-induced activation of the CCL20 promoter. MKN45 cells were transfected with pGL2-CCL20 and the indicated mutant plasmids or empty vector (pCMV4) and then infected with H. pylori ATCC 49503 for 6 h. The open bar indicates the luciferase activity of pGL2-CCL20 and pCMV4 without H. pylori infection. All values were first calculated as fold induction values relative to the basal level measured in uninfected cells. The data are means ± standard deviations of three independent experiments. (C) Bay 11-7082 and LLnL inhibit CCL20 mRNA expression induced by H. pylori. MKN45 cells were pretreated with Bay 11-7082 (20 μM) and LLnL (20 μM) for 1 h prior to H. pylori infection. They were subsequently infected with H. pylori for 6 h. CCL20 mRNA expression on harvested cells was analyzed by RT-PCR. Representative results of three similar experiments are shown. (D) Bay 11-7082 and LLnL inhibit H. pylori-induced NF-κB DNA binding. MKN45 cells were pretreated with Bay 11-7082 (20 μM) and LLnL (20 μM) for 1 h prior to H. pylori infection. They were subsequently infected with H. pylori for 1 h. Nuclear extracts from harvested cells were analyzed for NF-κB. Representative results of three similar experiments are shown.

FIG. 8.

Inhibitory effects of 17-AAG on H. pylori-induced CCL20 expression. (A) H. pylori infection does not affect Hsp90 expression. MKN45 cells were infected with H. pylori ATCC 49503 for the indicated times. The bacterium-to-cell ratio was 20:1. The cells were then lysed and analyzed by immunoblotting with Hsp90 and actin antibodies. (B) MKN45 cells were incubated with 1 μM 17-AAG for 16 h prior to infection with different densities of H. pylori for 6 h. RT-PCR was performed to check the changes in CCL20 mRNA expression after 17-AAG treatment in H. pylori-infected MKN45 cells. (C) Attenuation of H. pylori-induced NF-κB DNA binding by 17-AAG treatment. MKN45 cells were treated (+) or not treated (−) with 17-AAG for 16 h prior to infection with H. pylori for 1 h. The nuclear extracts were isolated from MKN45 cells infected with H. pylori and analyzed for NF-κB. (D) Hsp90 protects IKKα and IKKβ from proteasomal degradation. MKN45 cells either were pretreated with the proteasomal inhibitor LLnL (20 μM) for 1 h, followed or not followed by addition of 17-AAG (1 μM) and incubation for 16 h, or were treated with 17-AAG for 16 h or left untreated in the absence of H. pylori as indicated. Samples were analyzed for each protein by Western blotting. Representative results of three similar experiments are shown in each panel.