Response of periodontitis and healthy patients in a Porphyromonas gingivalis-stimulated whole-blood model.

AIM: To investigate the inflammatory responses of periodontitis patients and healthy patients in a whole-blood model stimulated with Porphyromonas gingivalis. METHODS: Whole blood collected from 17 periodontitis patients and six healthy patients was stimulated with Porphyromonas gingivalis cells. The secretion of cytokines and matrix metalloproteinases was quantified by enzyme-linked immunosorbent assay. An analysis of covariance with the ancova model was used to evaluate the significance of differences in secreted host molecules by whole blood from the periodontitis and healthy groups. RESULTS: Porphyromonas gingivalis induced the secretion of interleukin-1ß, interleukin-6, interleukin-8, tumor necrosis factor-α, monocyte chemoattractant protein-1, interferon inducible protein-10 by whole blood from patients in the periodontitis and healthy groups. Matrix metalloproteinase-8 and -9 levels secreted by whole blood also increased following stimulation. No significant differences (P < 0.05) in the amounts of secreted host molecules were observed between periodontitis and healthy patients. CONCLUSION: This study suggests that Porphyromonas gingivalis can provoke an inflammatory response and promote the progression of periodontitis by inducing the secretion of high levels of cytokines and matrix metalloproteinases by a mixed leukocyte population. However, the whole-blood model did not reveal any significant differences in the inflammatory response between periodontitis patients (n = 17) and periodontally-healthy patients (n = 6).

Tetracyclines and chemically modified tetracycline-3 (CMT-3) modulate cytokine secretion by lipopolysaccharide-stimulated whole blood.

In addition to their bacteriostatic effect, tetracyclines, which are often used in the treatment of periodontitis, also present anti-inflammatory properties. In the present study, we investigated the effects of tetracycline (TC), doxycycline (doxy), and chemically modified tetracycline-3 (CMT-3) on the production of pro-inflammatory mediators and matrix metalloproteinases (MMPs) in an ex vivo human whole blood (WB) model stimulated with Porphyromonas gingivalis lipopolysaccharide (LPS). WB samples obtained from three periodontitis patients and six healthy subjects were stimulated with P. gingivalis LPS in the absence and presence of TC, doxy, or CMT-3. The secretion of interleukin-1beta (IL-1beta), interleukin-6 (IL-6), interleukin-8 (IL-8), MMP-8, and MMP-9 by the WB samples was determined using enzyme-linked immunosorbent assays. P. gingivalis LPS significantly increased the secretion of all cytokines and MMPs tested. While we observed inter-patient variations, TC, doxy, and CMT-3 caused reductions of LPS-induced cytokine secretion to various degrees. TC, doxy, and CMT-3 had no significant effect on MMP-8 and MMP-9 secretion by LPS-stimulated WB samples. In conclusion, we used a human WB model that takes into consideration relevant in vivo immune cell interactions in the presence of plasma proteins to show that TC, doxy, and CMT-3 can reduce the production of pro-inflammatory mediators. This property may contribute to the clinically proven benefits of these molecules in the treatment of periodontitis and other chronic inflammatory diseases.

Doxycycline reduces lipopolysaccharide-induced inflammatory mediator secretion in macrophage and ex vivo human whole blood models.

BACKGROUND: Tetracyclines have been extensively used as adjuncts in the treatment of some forms of periodontitis. The aim of this study was to evaluate the capacity of doxycycline to influence the secretion of inflammatory mediators in macrophage and ex vivo human whole blood models stimulated with periodontopathogen lipopolysaccharides (LPS). METHODS: Monocyte-derived macrophages were treated with various concentrations of doxycycline prior to being stimulated with Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans) LPS. The capacity of doxycycline to mediate the inflammatory response was also tested in an ex vivo whole blood model (whole blood isolated from periodontitis patients and healthy subjects) stimulated with Porphyromonas gingivalis LPS. The secretion of interleukin (IL)-1beta, -6, and -8 and tumor necrosis factor-alpha (TNF-alpha) in both models was assessed by enzyme-linked immunosorbent assays (ELISA). Changes in phosphorylation state of kinases induced by A. actinomycetemcomitans LPS and doxycycline in the macrophage model were characterized by a multiplex ELISA analysis. RESULTS: The secretion of IL-1beta and -8 and TNF-alpha by macrophages decreased significantly (P <0.05) when they were pretreated with 2 microM doxycycline, whereas a concentration of 10 microM was required to significantly reduce IL-6 secretion. Pretreatment of macrophages with 10 microM doxycycline prior to A. actinomycetemcomitans LPS stimulation resulted in a marked decrease in the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 (-76%). In the whole blood model, doxycycline, more particularly at 10 microM, was also a potent inhibitor of the proinflammatory cytokine response. CONCLUSION: These two models provided clear evidence that some of the clinically proven benefits of doxycycline may be related to its ability to regulate inflammatory mediator release by host cells.