The induction of inflammation by the cGAS-STING pathway in human dental pulp cells: a laboratory investigation.

AIM To explore the presence of the cGAS-STING inflammatory pathway in human pulp tissue and human dental pulp cells (HDPCs). METHODOLOGY Pulp tissue was collected from freshly extracted human healthy third molars or third molars with irreversible pulpitis. Quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunoassay (ELISA) was performed to assess IFN-β, TNF, and IL-6. HDPCs prepared from healthy human pulp tissues were transfected with interferon stimulatory DNA (ISD), bacterial genomic DNA, bacterial cyclic dinucleotides c-di-AMP, c-di-GMP, or host cyclic dinucleotide cGAMP. SiRNA was used to knockdown the endogenous cGAS or STING. G140 and H-151 were used to inhibit cGAS and STING, respectively. Amlexanox and BAY 11-7082 were used to inhibit TBK1 and NF-κB, respectively. qRT-PCR and ELISA was performed to detect the level of IFN-β, TNF, and IL-6. Western blot was performed to evaluate the TBK1, IRF3, and p65 phosphorylation. The Student's t-test and one-way ANOVA were used for statistical analysis RESULTS: IFN-β, TNF, and IL-6 were upregulated in the inflamed human dental pulp tissue. CGAS and STING mRNA were increased in the inflamed human dental pulp tissue and detected in HDPCs prepared from healthy human pulp tissues. ISD transfection induced TBK1, IRF3, and p65 phosphorylation as well as IFN-β, TNF, and IL-6 production. IFN-β, TNF, and IL-6 production were also induced by transfection of bacterial and host cyclic dinucleotides or bacteria DNA. ISD or bacteria DNA transfection elevated the intracellular levels of cGAMP. Knockdown of cGAS or STING, as well as using cGAS inhibitor G140 or STING inhibitor H-151 abolished the IFN-β, TNF, and IL-6 production induced by ISD transfection. Knockdown of STING or using STING inhibitor H-151 abolished the IFN-β, TNF, and IL-6 induction by transfection of bacterial and host cyclic dinucleotides. Both Amlexanox and BAY 11-7082 inhibited IFN-β, TNF, and IL-6 production triggered by ISD and cyclic dinucleotides transfection. CONCLUSIONS HDPCs expressed an intact cGAS-STING signaling axis. The cGAS-STING signaling axis may play an important role in pulp inflammation and immune defense.