Effect of Aqueous Ozone on the NF-κB System

Ozone has been proposed as an alternative oral antiseptic in dentistry, due to its antimicrobial power reported for gaseous and aqueous forms, the latter showing a high biocompatibility with mammalian cells. New therapeutic strategies for the treatment of periodontal disease and apical periodontitis should consider not only antibacterial effects, but also their influence on the host immune response. Therefore, our aim was to investigate the effect of aqueous ozone on the NF-κB system, a paradigm for inflammationassociated signaling/transcription. We showed that NF-κB activity in oral cells stimulated with TNF, and in periodontal ligament tissue from root surfaces of periodontally damaged teeth, was inhibited following incubation with ozonized medium. Under this treatment, IκBalpah proteolysis, cytokine expression, and κB-dependent transcription were prevented. Specific ozonized amino acids were shown to represent major inhibitory components of ozonized medium. In summary, our study establishes a condition under which aqueous ozone exerts inhibitory effects on the NF-κB system, suggesting that it has an antiinflammatory capacity

C/EBPß blocks p65 phosphorylation and thereby NF-kB-mediated transcription in TNF-tolerant cells.

TNF is a major mediator of inflammation, immunity, and apoptosis. Pre-exposure to TNF reduces sensitivity to restimulation, a phenomenon known as tolerance, considered as protective in sepsis, but also as a paradigm for immunoparalysis. Earlier experiments in TNF-tolerant cells display inhibition of NF-kappaB-dependent IL-8 gene expression at the transcriptional level with potential involvement of C/EBPbeta. In this study, we have shown that a kappaB motive was sufficient to mediate transcriptional inhibition under TNF tolerance conditions in monocytic cells. Furthermore, in tolerant cells, TNF-induced NF-kappaB p65 phosphorylation was markedly decreased, which was accompanied by the formation of C/EBPbeta-p65 complexes. Remarkably, in C/EBPbeta(-/-) cells incubated under the conditions of TNF tolerance, neither impairment of transcription nor inhibition of p65 phosphorylation was observed. Finally, we showed that C/EBPbeta overexpression reduced p65-mediated transactivation and that association of C/EBPbeta with p65 specifically prevented p65 phosphorylation. Our data demonstrate that C/EBPbeta is an essential signaling component for inhibition of NF-kappaB-mediated transcription in TNF-tolerant cells and suggest that this is caused by blockade of p65 phosphorylation. These results define a new molecular mechanism responsible for TNF tolerance in monocytic cells that may contribute to the unresponsiveness seen in patients with sepsis

Transcriptional Inhibition of Interleukin-8 Expression in Tumor Necrosis Factor-tolerant Cells.

There is some evidence that the potent cytokine tumor necrosis factor (TNF) is able to induce tolerance after repeated stimulation of cells. To investigate the molecular mechanisms mediating this phenomenon, the expression of interleukin-8 (IL-8), which is regulated by transcription factors NF-kappaB and C/EBPbeta, was monitored under TNF tolerance conditions. Pretreatment of monocytic cells for 72 h with low TNF doses inhibited TNF-induced (restimulation with a high dose) IL-8 promoter-dependent transcription as well as IL-8 production. Under these conditions neither activation of NF-kappaB nor IkappaB proteolysis was affected after TNF re-stimulation, albeit a slightly reduced IkappaB-alpha level was found in the TNF pretreated but not re-stimulated sample. Remarkably, in tolerant cells an increased binding of C/EBPbeta to its IL-8 promoter-specific DNA motif as well as an elevated association of C/EBPbeta protein with p65-containing NF-kappaB complexes was observed. Finally, overexpression of C/EBPbeta, but not p65 or Oct-1, markedly prevented TNF-induced IL-8 promoter-dependent transcription. Taken together, these data indicate that the expression of IL-8 is inhibited at the transcriptional level in TNF-tolerant cells and C/EBPbeta is involved under these conditions in mediating the negative-regulatory effects, a mechanism that may play a role in inflammatory processes such as sepsis