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

Abstract

To explore the presence of the cGAS-STING inflammatory pathway in human pulp tissue and human dental pulp cells (HDPCs). 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) were performed to assess IFN-β, TNF and IL-6. Human dental pulp cells 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 knock down 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 were 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. IFN-β, TNF and IL-6 were up-regulated 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. Knock-down 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. Knock-down 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. Human dental pulp cells expressed an intact cGAS-STING signalling axis. The cGAS-STING signalling axis may play an important role in pulp inflammation and immune defence.