Differential activation of NF-kappaB and gene expression in oral epithelial cells by periodontal pathogens

Abstract

To investigate the molecular effects of the periodontopathogens Fusobacterium nucleatum (FN) and Porphyromonas gingivalis (PG) on the oral epithelium, the H400 oral epithelial cell line was cultured in the presence of non-viable bacteria. Following confirmation of the presence of transcripts for the bacterial pattern recognition receptors in H400 cells, Toll-like receptors -2, -4 and -9, and components of the NF-kappaB signalling pathway, immunocytochemical analyses were performed showing that NF-kappaB was activated within 1 h of exposure to both periodontopathogens. A significantly greater number of NF-kappaB nuclear translocations were apparent following H400 cell exposure to FN as compared with PG. Gene expression analyses indicated that transcripts known to be regulated by the NF-kappaB pathway, including cytokines/chemokines TNF-alpha, IL-1beta, IL-8, MCP-1/CCL2 and GM-CSF, were up-regulated following 4 and 24 h of exposure to both periodontopathogens. In addition, H400 periodontopathogen exposure resulted in differential regulation of transcripts for several cytokeratin gene family members. Consistent with the immunocytochemical data, microarray results indicated that FN induced a greater number of gene expression changes than PG following 24 h of exposure, 609 and 409 genes, respectively. Ninety-one genes were commonly differentially expressed by both periodontopathogens and represented biological processes commonly associated with periodontitis. Gene expression analyses by reserve transcriptase-polymerase chain reaction (RT-PCR) of molecules identified from the microarray data sets, including Heme oxygenase-1, lysyl oxidase, SOD2, CCL20 and calprotectin components, confirmed their differential expression profiles induced by the two periodontopathogens. FN and PG have clearly different molecular effects on oral epithelial cells, potentially highlighting the importance of the composition of the plaque biofilm in periodontitis pathogenesis.

Fig. 1

Semi-quantitative RT-PCR analysis of selected genes involved in the detection of bacterial products and downstream intracellular signalling cascade, including TLR and NF-κB molecules. H400 cells were exposed to media alone (M) or 10⁹ cfu/ml of PG or FN for 4 and 24 h. (a) Representative gel images of duplicate analyses are shown. (b) Densitometric analysis of gel products. Expression levels are shown as percentage of the highest level detected. Amplified product values were normalized to GAPDH housekeeping gene levels.

Fig. 2

Immunocytochemical analysis of NF-κB activity in H400 cells following 60 min of exposure to stimuli. (a) Histological images of H400 cells in media alone (controls (i) (iii) (v) & (vii)) or exposed to the peridontopathogens PG (ii), FN (iv) and E.coli LPS controls 10 µg/ml (vi) and 20 µg/ml (viii). Arrows indicate either p65 cytoplasmic ((i) (iii) (v) & (vii)) or nuclear ((ii) (iv) (vi) & viii)) localization in H400 cells. (b) Quantitative immunocytochemical analysis of p65 nuclear staining following exposure to PG, FN and 10 or 20 µg/ml of E.coli LPS. U = unexposed H400 cells; E = exposed H400 cells. Asterisks indicate statistically significant differences (P< 0·001).

Fig. 3

Semi-quantitative RT-PCR analysis of selected cytokines/chemokines (a) and cytokeratin genes (b). H400 cells were exposed to media alone (M) or 10⁹ cfu/ml of PG or FN for 4 and 24 h. (i) Representative gel images of duplicate analyses are shown. (ii) Densitometric analysis of gel products. Expression levels are shown as percentage of the highest level detected. Amplified product values were normalized to GAPDH housekeeping gene levels. All semi-quantitative RT-PCR analyses were performed in duplicate.

Fig. 4

Microarray data analyses. (a) Venn diagram representing the number of gene probes differentially expressed (≥ two-fold) in H400 cells as a result of 24-h exposure to the periodontopathogens FN and PG as determined by dChip analysis of microarray hybridization experiments. (b) Pie chart representations of the biological process ontological groups identified using Onto-Express for the genes up-regulated in H400 OECs for (i) PG (170 genes), (ii) FN (247 genes) and the genes common to both data sets, (iii) PG/FN (46 genes). Groups for the 10 most represented gene ontologies are shown for each data set.

Fig. 5

Semi-quantitative RT-PCR analysis of genes identified from microarray data as being differentially expressed in H400 cells due to PG or FN exposure. H400 cells were exposed to media alone (M) or 10⁹ cfu/ml of PG or FN for 4 and 24 h. (a) Representative gel images of amplified products. (b) Densitometric analysis of gel products. Expression levels are shown as percentage of the highest level detected. Amplified product values were normalized to GAPDH housekeeping gene levels. All semi-quantitative RT-PCR analyses were performed in duplicate.