Metronidazole-tolerant Porphyromonas gingivalis persisters dampen cytokine expression in HGECs

Abstract

Objectives: Our group has recently demonstrated the existence of metronidazole-tolerant Porphyromonas gingivalis persisters. This study further investigated the effects of P. gingivalis persisters on the cytokine expression in primary human gingival epithelial cells (HGECs). Methods: P. gingivalis cells (ATCC 33277) were cultured in broth containing 1, 5, or 10 μg/ml of hemin to stationary phase, and subsequently treated with lethal doses of metronidazole (MTZ) for 6 hours. HGECs were pre-treated with IL-1β for 24 hours, and then stimulated with P. gingivalis, P. gingivalis persisters, heat-killed P. gingivalis and heat-killed P. gingivalis persisters respectively for 1, 6 and 24 hours. HGECs without treatments and those with IL-1β stimulation alone served as the blank and negative controls. The samples were collected for TEM, cytokine arrays, ELISA and qPCR. Results: TEM showed that P. gingivalis and P. gingivalis persisters had similar invasion abilities to HGECs. Over 10 cytokines, chemokines or growth factors, detected by cytokine arrays, were downregulated in both P. gingivalis and P. gingivalis persisters infected HGECs. Moreover, P. gingivalis and P. gingivalis persisters inhibited the expression of IL-6 and IL-8 mRNAs and proteins in a time-dependent manner. Whereas, no such effects were observed in the HGECs stimulated by heat-killed P. gingivalis and heat-killed P. gingivalis persisters. Furthermore, P. gingivalis maintenance in broth containing 1 or 10 μg/ml hemin showed compatible effects on the cytokine expression of HGECs. Conclusions: Our current work suggests that metronidazole-tolerant P. gingivalis persisters retain their ability to dampen cytokine expression in HGECs, and may critically account for their survival and virulence in the pathogenesis of P. gingivalis-related diseases.