Oxidative Stress Inhibits Endotoxin Tolerance and May Affect Periodontitis.

Periodontal disease is caused by dysbiosis of the dental biofilm and the host inflammatory response. Various pathogenic factors, such as proteases and lipopolysaccharides (LPSs) produced by bacteria, are involved in disease progression. Endotoxin tolerance is a function of myeloid cells, which sustain inflammation and promote tissue regeneration upon prolonged stimulation by endotoxins such as LPS. The role of endotoxin tolerance is gaining attention in various chronic inflammatory diseases, but its role in periodontal disease remains elusive. Oxidative stress, one of the major risk factors for periodontal disease, promotes disease progression through various mechanisms, of which only some are known. The effect of oxidative stress on endotoxin tolerance has not yet been studied, and we postulated that endotoxin tolerance regulation may be an additional mechanism through which oxidative stress influences periodontal disease. This study aimed to reveal the effect of oxidative stress on endotoxin tolerance and that of endotoxin tolerance on periodontitis progression. The effect of oxidative stress on endotoxin tolerance was analyzed in vitro using peritoneal macrophages of mice and hydrogen peroxide (H2O2). The results showed that oxidative stress inhibits endotoxin tolerance induced by Porphyromonas gingivalis LPS in macrophages, at least partially, by downregulating LPS-elicited negative regulators of Toll-like receptor (TLR) signaling. A novel oxidative stress mouse model was established using SMP30KO mice incapable of ascorbate biosynthesis. Using this model, we revealed that oxidative stress impairs endotoxin tolerance potential in macrophages in vivo. Furthermore, gingival expression of endotoxin tolerance-related genes and TLR signaling negative regulators was decreased, and symptoms of ligature-induced periodontitis were aggravated in the oxidative stress mouse model. Our findings suggest that oxidative stress may contribute to periodontitis progression through endotoxin tolerance inhibition.

Decreased expression of E-cadherin by Porphyromonas gingivalis-lipopolysaccharide attenuates epithelial barrier function.

BACKGROUND AND OBJECTIVE: The gingival epithelium is a first line of defense against bacterial challenge. E-cadherin (E-cad) plays an important role in cell-cell adhesion as a barrier in the epithelium. Recently, a decrease in the expression of E-cad has been observed in inflamed gingival tissue. The aims of this study were to clarify the changes in E-cad expression and barrier function in human gingival epithelial cells stimulated with Porphyromonas gingivalis-lipopolysaccharide (P. gingivalis-LPS) and to evaluate the influence of these changes on the inflammatory reaction. Furthermore, to clarify the mechanism of the E-cad changes induced by P. gingivalis-LPS, we focused on reactive oxygen species (ROS) that are reported to induce a decrease in E-cad expression. MATERIAL AND METHODS: Human gingival epithelial cells were incubated in Humedia-KG2 in the presence or absence of P. gingivalis-LPS and antioxidants to analyze ROS involvement in P. gingivalis-LPS-induced E-cad changes. E-cad protein expression was analyzed by immunofluorescence staining. To investigate barrier function and inflammatory changes, we performed transport and cytokine assays using gingival epithelial cells and macrophages co-culture model in transwell plates. Medium containing 10 µg/mL P. gingivalis-LPS (transport substance) was added to the upper compartment, which harvested gingival epithelial cells, and medium without P. gingivalis-LPS was added to the lower compartment, which harvested macrophages. In the transport assay, P. gingivalis-LPS penetration was analyzed using the Limulus amebocyte lysate test. In the cytokine assay, we examined the change in tumor necrosis factor-α (TNF-α) production from the macrophages in the lower compartment using enzyme-linked immunosorbent assay. RESULTS: Expression of E-cad in human gingival epithelial cells was decreased by P. gingivalis-LPS, and the decrease in E-cad accelerated the penetration of P. gingivalis-LPS through the monolayer. In addition, the concentration of TNF-α was higher under the E-cad reduced monolayer. Antioxidants, particularly vitamin E and l-ascorbic acid 2-phosphate magnesium salt, inhibited the decrease in E-cad expression, penetration of P. gingivalis-LPS and increase in TNF-α. CONCLUSION: These results suggest that the decrease in E-cad caused by P. gingivalis-LPS leads to destruction of the epithelial barrier function in human gingival epithelial cells, and finally accelerates the inflammatory reaction under the barrier. Antioxidants, particularly vitamin E and l-ascorbic acid 2-phosphate magnesium salt, may restore the impaired function by scavenging ROS, which are related to the decrease in E-cad expression by P. gingivalis-LPS.

Elicitor action via cell membrane of a cultured rice cell demonstrated by the single-cell transient assay.

In order to analyze intracellular signal transduction, we investigated the mechanism of chemical elicitor action by single-cell transient assay using green fluorescent protein (GFP) as a reporter gene. When the elicitor was applied from outside the cell into which the chitinase promoter and GFP reporter were introduced beforehand, fluorescence emission of GFP was observed. In contrast, when the elicitor was introduced in the cell to let the elicitor act from inside, no emission was observed. Addition of further elicitor from outside, however, did cause GFP emission. Therefore, it is clear that the elicitor does not act after entering the cell but that its signal is transduced into the cell via the cell membrane.