Gingipains from Porphyromonas gingivalis play a significant role in induction and regulation of CXCL8 in THP-1 cells.

BACKGROUND: Porphyromonas gingivalis is an important bacterial etiological agent involved in periodontitis. The bacterium expresses two kinds of cysteine proteases called gingipains: arginine gingipains (RgpA/B) and lysine gingipain (Kgp). This study evaluated the interaction between P. gingivalis and THP-1 cells, a widely used monocytic cell line, in vitro with a focus on CXCL8 at the gene and protein levels and its fate thereafter in cell culture supernatants. THP-1 cells were stimulated with viable and heat-killed wild-type strains ATCC 33277 or W50 or viable isogenic gingipain mutants of W50, E8 (Rgp mutant) or K1A (Kgp mutant), for 24 hours. RESULTS: ELISA and qPCR results show an elevated CXCL8 expression and secretion in THP-1 cells in response to P. gingivalis, where the heat-killed ATCC33277 and W50 induced higher levels of CXCL8 in comparison to their viable counterparts. Furthermore, the Kgp-deficient mutant K1A caused a higher CXCL8 response compared to the Rgp-deficient E8. Chromogenic quantification of lipopolysaccharide (LPS) in supernatant showed no significant differences between viable and heat killed bacteria except that W50 shed highest levels of LPS. The wild-type strains secreted relatively more Rgp during the co-culture with THP-1 cells. The CXCL8 degradation assay of filter-sterilized supernatant from heat-killed W50 treated cells showed that Rgp was most efficient at CXCL8 hydrolysis. Of all tested P. gingivalis strains, adhesion and internalization in THP-1 cells was least conspicuous by Rgp-deficient P. gingivalis (E8), as demonstrated by confocal imaging. CONCLUSIONS: W50 and its Kgp mutant K1A exhibit a higher immunogenic and proteolytic function in comparison to the Rgp mutant E8. Since K1A differs from E8 in the expression of Rgp, it is rational to conclude that Rgp contributes to immunomodulation in a more dynamic manner in comparison to Kgp. Also, W50 is a more virulent strain when compared to the laboratory strain ATCC33277.

The periodontal pathogen Porphyromonas gingivalis changes the gene expression in vascular smooth muscle cells involving the TGFbeta/Notch signalling pathway and increased cell proliferation.

BACKGROUND: Porphyromonas gingivalis is a gram-negative bacterium that causes destructive chronic periodontitis. In addition, this bacterium is also involved in the development of cardiovascular disease. The aim of this study was to investigate the effects of P. gingivalis infection on gene and protein expression in human aortic smooth muscle cells (AoSMCs) and its relation to cellular function. RESULTS: AoSMCs were exposed to viable P. gingivalis for 24 h, whereafter confocal fluorescence microscopy was used to study P. gingivalis invasion of AoSMCs. AoSMCs proliferation was evaluated by neutral red assay. Human genome microarray, western blot and ELISA were used to investigate how P. gingivalis changes the gene and protein expression of AoSMCs. We found that viable P. gingivalis invades AoSMCs, disrupts stress fiber structures and significantly increases cell proliferation. Microarray results showed that, a total of 982 genes were identified as differentially expressed with the threshold log2 fold change > |1| (adjust p-value <0.05). Using bioinformatic data mining, we demonstrated that up-regulated genes are enriched in gene ontology function of positive control of cell proliferation and down-regulated genes are enriched in the function of negative control of cell proliferation. The results from pathway analysis revealed that all the genes belonging to these two categories induced by P. gingivalis were enriched in 25 pathways, including genes of Notch and TGF-beta pathways. CONCLUSIONS: This study demonstrates that P. gingivalis is able to invade AoSMCs and stimulate their proliferation. The activation of TGF-beta and Notch signaling pathways may be involved in the bacteria-mediated proliferation of AoSMCs. These findings further support the association between periodontitis and cardiovascular diseases.

Porphyromonas gingivalis-induced inflammatory responses in THP1 cells are altered by native and modified low-density lipoproteins in a strain-dependent manner.

Strong epidemiological evidence supports an association between cardiovascular and periodontal disease and furthermore, the periodontopathogen Porphyromonas gingivalis has been identified in blood and from atheromatous plaques. Blood exposed to P. gingivalis shows an increased protein modification of low-density lipoprotein (LDL). In this study, we investigate the inflammatory responses of THP1 cells incubated with P. gingivalis and the effects of native or modified LDL on these responses. Reactive oxygen species (ROS) and IL-1ß were observed in THP1 cells following infection with P. gingivalis ATCC33277 and W50. Caspase 1 activity was quantified in THP1 cells and correlated with IL-1ß accumulation. Oxidized LDL (oxLDL) induced IL-1ß release and CD36 expression on THP1 cells. Modified LDL co-stimulated with ATCC33277 exhibited regulatory effects on caspase 1 activity, IL-1ß release and CD36 expression in THP1 cells, whereas W50 induced more modest responses in THP1 cells. In summary, we show that P. gingivalis is capable of inducing pro-inflammatory responses in THP1 cells, and native and modified LDL could alter these responses in a dose- and strain-dependent manner. Strain-dependent differences in THP1 cell responses could be due to the effect of P. gingivalis proteases, presence or absence of capsule and proteolytic transformation of native and modified LDL.

PKC, ERK/p38 MAP kinases and NF-κB targeted signalling play a role in the expression and release of IL-1ß and CXCL8 in Porphyromonas gingivalis-infected THP1 cells.

Porphyromonas gingivalis is a keystone pathogen in periodontitis and is gaining importance in cardiovascular pathogenesis. Protease-activated receptors (PARs), toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD) on monocytes recognize the structural components on P. gingivalis, inducing inflammatory intermediates. Here, we elucidate the modulation of PARs, TLRs, NODs, and the role of MAPK and NF-κB in IL-1ß and CXCL8 release. THP1 cells were stimulated with P. gingivalis wild-type W50 and its isogenic gingipain mutants: Rgp mutant E8 and Kgp mutant K1A. We observed modulation of PARs, TLRs, NOD, IL-1ß and CXCL8 expression by P. gingivalis. Gingipains hydrolyse IL-1ß and CXCL8, which is more evident for IL-1ß accumulation at 24 h. Inhibition of PKC (protein kinase C), p38 and ERK (extracellular signal-regulated kinases) partially reduced P. gingivalis-induced IL-1ß at 6 h, whereas PKC and ERK reduced CXCL8 at both 6 and 24 h. Following NF-κB inhibition, P. gingivalis-induced IL-1ß and CXCL8 were completely suppressed to basal levels. Overall, TLRs, PARs and NOD possibly act in synergy with PKC, MAPK ERK/p38 and NF-κB in P. gingivalis-induced IL-1ß and CXCL8 release from THP1 cells. These pro-inflammatory cytokines could affect leucocytes in circulation and exacerbate other vascular inflammatory conditions such as atherosclerosis.