NFkB-dependent Gene Expression of CCL20 in Gingival Epithelial Cells

Objectives: Antimicrobial peptides, such as C-C chemokine ligand 20 (CCL20), play an important role in innate immune processes. In addition to direct microbicidal effects against Gram-negative and Gram-positive bacteria, CCL20 also exhibits cytokine-like mediator functions that are critical during activation of immature dendritic cells. The aim of the present study was to investigate whether the gene expression of CCL20 in response to Porphyromonas gingivalis is dependent on nuclear factor kappa B (NFkB) mediated pathways.

Methods: Primary human gingival keratinocytes (GECs) from a total of 5 donors and the immortalized oral keratinocyte cell line OKF6/TERT-2 were stimulated with P. gingivalis for 16 hours using a multiplicity of infection of 50:1. Prior to stimulation, GECs and OKF6/TERT-2 cells were pretreated with specific inhibitors for NFkB, mitogen-activated protein kinase (MAPK), phospholipase C (PLC, U73122 and U73343 as control), and phosphatidylinositol-3-kinase (PI3K, LY294002). The gene expression analysis for CCL20 and IL-8 was performed using real-time PCR technology. In GECs and OKF6/TERT-2 cells, specific activation of NFkB was examined after exposure to P. gingivalis for 14, 30, 45, and 60 minutes. Statistical analysis was performed using the t-test for unpaired samples, and a P-value of ≤0.05 was considered as significant.

Results: In GECs and OKF6/TERT-2 cells, the gene expression of CCL20 and IL-8 was significantly induced in response to P. gingivalis (P≤0.05) compared to unstimulated control cells. This induction was specifically blocked when cells were preincubated with inhibitors for NFkB, MAPK, and PLC (P≤0.05), but not for PI3K. After 30, 45, and 60 minutes, specific activation of NFkB was shown in P. gingivalis-stimulated GECs and OKF6/TERT-2 cells (P≤0.05).

Conclusions: The results of the present study indicate for the first time that the gene expression of CCL20 is NFkB- and MAPK-dependent in oral keratinocytes when stimulated with P. gingivalis.

NIDCR R01DE16961