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.