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Lipopolysaccharides From Non-Helicobacter pylori Gastric Bacteria Potently Stimulate Interleukin-8 Production in Gastric Epithelial Cells.

ABSTRACT: BACKGROUND:Gastric acid secretion is compromised in chronic Helicobacter pylori (H. pylori) infection allowing overgrowth of non-H. pylori gastric bacteria (NHGB) in the stomach. METHODS:NHGB were isolated from gastric mucosa in selective media and further characterized with biochemical methods and 16S rRNA gene sequencing. Human gastric tissues were studied with indirect immunofluorescence with antibodies against H. pylori and Neisseria subflava (N. subflava). Gastric epithelial cell lines were cocultured with bacteria or incubated with lipopolysaccharides isolated from NHGB, and interleukin-8 released in the media was measured by enzyme-linked immunosorbent assay. Expression of Toll-like receptor (TLR)2, TLR4, it's coreceptor myeloid differentiation factor 2 (MD2), and CD14 in gastric cells was investigated by immunofluorescence microscopy and reverse transcriptase-polymerase chain reaction. RESULTS:Haemophilus species, Neisseria species, Fusobacterium species, and Veillonella species were predominant Gram-negative bacteria coinfected with H. pylori. Lipopolysaccharides from N. subflava potently stimulated interleukin-8 secretion in MKN45 cells which was cancelled by preincubation with polymyxin B. TLR2, TLR4, CD14, and myeloid differentiation factor 2 were expressed in MKN45 cells, though their levels of expression were low. N. subflava adhered to MKN45 cells in vitro and colocalized with H. pylori in the human gastric mucosa. CONCLUSIONS:Our data suggest that N. subflava colonized in the gastric mucosa contribute to gastric inflammation during chronic H. pylori gastritis. TRANSLATIONAL IMPACT:NHGB may perpetuate gastric inflammation and accelerate neoplastic progression in the hypochlorhydric stomach.

SUBMITTER: Miyata N

PROVIDER: S-EPMC6445647 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Lipopolysaccharides From Non-Helicobacter pylori Gastric Bacteria Potently Stimulate Interleukin-8 Production in Gastric Epithelial Cells.

Miyata Natsumi N Hayashi Yoshikazu Y Hayashi Shunji S Sato Kiichi K Hirai Yoshikazu Y Yamamoto Hironori H Sugano Kentaro K

Clinical and translational gastroenterology 20190301 3

<h4>Background</h4>Gastric acid secretion is compromised in chronic Helicobacter pylori (H. pylori) infection allowing overgrowth of non-H. pylori gastric bacteria (NHGB) in the stomach.<h4>Methods</h4>NHGB were isolated from gastric mucosa in selective media and further characterized with biochemical methods and 16S rRNA gene sequencing. Human gastric tissues were studied with indirect immunofluorescence with antibodies against H. pylori and Neisseria subflava (N. subflava). Gastric epithelial ...[more]

PMID: 30913125

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Project description:BackgroundHelicobacter pylori colonises the stomach of approximately 50% of the global population. Cytotoxin-associated gene A protein (CagA) is one of the important virulent factors responsible for the increased inflammation and increases the risk of developing peptic ulcers and gastric carcinoma. The cytokine interleukin-6 (IL-6) has particularly important roles in the malignant transformation of gastric and intestinal epithelial cells as it is upregulated in H. pylori-infected gastric mucosa. In this study, we investigated the underlying mechanisms of CagA-induced IL-6 up-regulation during H. pylori infection. AGS cells, a human gastric adenocarcinoma cell line, lacking eEF1A1 were infected with CagA+ H. pylori (NCTC11637), CagA- H. pylori (NCTC11637ΔcagA), or transduced by Ad-cagA/Ad-GFP. The expression and production of IL-6 were measured by quantitative real-time reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The interactions among CagA, eukaryotic translation elongation factor 1-alpha 1 (eEF1A1), protein kinase Cδ (PKCδ), and signal transducer and activator of transcription 3 (STAT3) were determined by western blot or co-immunoprecipitation.ResultsDuring H. pylori infection, CagA-M (residues 256‒871aa) was found to interact with eEF1A1-I (residues 1‒240aa). NCTC11637 increased the expression of IL-6 in AGS cells compared with NCTC11637ΔcagA whereas knockdown of eEF1A1 in AGS cells completely abrogated these effects. Moreover, the CagA-eEF1A1 complex promoted the expression of IL-6 in AGS cells. CagA and eEF1A1 cooperated to mediate the expression of IL-6 by affecting the activity of p-STATS727 in the nucleus. Further, CagA-eEF1A1 affected the activity of STAT3 by recruiting PKCδ. However, blocking PKCδ inhibited the phosphorylation of STAT3S727 and induction of IL-6 by CagA.ConclusionsCagA promotes the expression of IL-6 in AGS cells by recruiting PKCδ through eEF1A1 in the cytoplasm to increase the phosphorylation of STAT3S727 in the nucleus. These findings provide new insights into the function of CagA-eEF1A1 interaction in gastric adenocarcinoma.

Dataset Information

Lipopolysaccharides From Non-Helicobacter pylori Gastric Bacteria Potently Stimulate Interleukin-8 Production in Gastric Epithelial Cells.

Publications

Lipopolysaccharides From Non-Helicobacter pylori Gastric Bacteria Potently Stimulate Interleukin-8 Production in Gastric Epithelial Cells.

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