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- Title
Overexpression of Tim-3 reduces Helicobacter pylori-associated inflammation through TLR4/NFκB signaling in vitro.
- Authors
FUCAI WANG; ZHIRONG MAO; DONGSHENG LIU; JING YU; YOUHUA WANG; WEN YE; DONGJIA LIN; NANJIN ZHOU; YONG XIE
- Abstract
The present study aimed to investigate the interaction between T-cell immunoglobulin and mucin-domain-containing molecule-3 (Tim-3) and Toll-like receptor 4 (TLR4)/nuclear factor κB (NF-κB) signaling in Helicobacter pylori-infected RAW264.7 macrophage cells. RAW264.7 cells were co-cultured with H. pylori SS1 at different bacteria/cell ratios, and subsequently the mRNA expression of Tim-3, TLR4, and myeloid differentiation factor 88 (MyD88) was measured by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Furthermore, the effect of Tim-3 overexpression was examined by transfection of RAW264.7 with pLVX-IRES-ZsGreen-Tim-3 and co-culturing with H. pylori. mRNA and protein expression levels were then analyzed for Tim-3, TLR4, MyD88, and phosphorylated (p-) NF-κB by RT-qPCR and western blot analysis respectively. The concentrations of pro-inflammatory cytokines [tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), interferon-γ (IFN-γ) and interleukin 10 (IL-10)] released in the culture supernatants were measured by ELISA. H. pylori stimulation resulted in a significant increase of Tim-3, TLR4, and MyD88 mRNA expression in RAW264.7 cells. H. pylori stimulation upregulated Tim-3 expression even in the Tim-3-overexpressing RAW264.7 cells compared with unstimulated cells. TLR4, MyD88, and pNF-κB protein expression and pro-inflammatory cytokines (TNF-α, IL-6, and IFN-γ) release levels were increased in the control RAW264.7 cells following H. pylori infection, but not in the Tim-3-overexpressing RAW264.7 cells. By contrast, IL-10 levels were decreased following H. pylori infection in both control and Tim-3-overexpressing RAW264.7 cells. Overexpression of Tim-3 reduced H. pylori-associated inflammation in RAW264.7 macrophages, by downregulating expression of proteins in the TLR4 pathway and release of pro-inflammatory cytokines. These findings suggest that Tim-3 serves a crucial role in the negative regulation of H. pylori-associated inflammation and may be a novel therapeutic target for H. pylori infection.
- Subjects
T cells; HELICOBACTER pylori infections; MUCINS; ANTIBODY-dependent cell cytotoxicity; GENETIC overexpression; ENZYME-linked immunosorbent assay
- Publication
Molecular Medicine Reports, 2017, Vol 15, Issue 5, p3252
- ISSN
1791-2997
- Publication type
Article
- DOI
10.3892/mmr.2017.6346