Invasion of human dental pulp fibroblasts by Porphyromonas gingivalis leads to autophagy via the phosphoinositide 3-kinase/Akt/mammalian target of rapamycin signaling pathway

Abstract

Objectives

Porphyromonas gingivalis is a pathogenic bacterium that causes periodontitis and dental pulp infection. Autophagy is a potential mechanism involved in inflammatory disease. This study established an in vitro model of P. gingivalis intracellular infection in human dental pulp fibroblasts (HDPFs) to investigate the effects of live P. gingivalis on HDPFs.

Methods

Morphological and quantification techniques such as fluorescence microscopy, transmission electron microscopy (TEM), indirect immunofluorescence analysis, enzyme-linked immunosorbent assay (ELISA), real-time polymerase chain reaction (PCR), and western blotting were used in this study.

Results

After cell invasion, P. gingivalis is mainly localized in the cytoplasm and lysosomes. Additionally, P. gingivalis activates autophagy in HDPFs by upregulating the expression of autophagy-related gene Beclin-1, activate autophagy-related gene12 (ATG12), and microtubule-associated protein light chain 3 (LC3). Furthermore, the invasion of P. gingivalis leads to increased phosphorylation of PI3K, Akt, and mTOR with the addition of rapamycin, whereas the addition of wortmannin decreased phosphorylation. This invasion of P. gingivalis, also causes an inflammatory response, leading to the upregulation of IL-1β, IL-6, and TNF-α. Rapamycin helps decrease levels of pro-inflammatory cytokines, but the addition of wortmannin increases them. These results show that the invasion of P. gingivalis can cause excessive inflammation and promote the autophagy of HDPFs, which is regulated by PI3K/Akt/mTOR.

Conclusions

P. gingivalis escapes the immune system by inducing autophagy in the host cells, causing excessive inflammation. P. gingivalis regulates autophagy in HDPFs through the phosphoinositide 3-kinase/Akt/mammalian target of rapamycin pathway.