EBV LMP1 in Gingival Epithelium Potentially Contributes to Human Chronic Periodontitis via Inducible IL8 Production

Abstract

Background/Aim: Human chronic periodontitis is a major health problem. Although some oral bacteria have been reported to be putative pathogens, Epstein-Barr virus (EBV) is reported to be associated with the progression of periodontitis. However, the role of EBV in the aetiology of periodontitis is unknown. Therefore, we investigated periodontal pathogenesis of EBV to confirm whether EBVencoded latent membrane protein 1 (LMP1) induces Interleukin-8 (IL8) production in human gingival cells. Materials and Methods: Real-time polymerase chain reaction, luciferase assay, enzyme-linked immunosorbent assay (ELISA), and western blotting were performed for determining IL8 mRNA expression, nuclear factor kappa B (NF-ĸB) transcription, IL8 production, and the phosphorylation of NF-ĸB p65 and Inhibitor of kappa B alpha (IĸBα), respectively, in Ca9-22 human gingival epithelial cells. Two LMP1 mutants lacking C-terminal activating region (CATR) domains responsible for activating NF-ĸB were used. Results: Extremely high IL8 production was induced by LMP1 in time-and dose-dependent manner, where simultaneous phosphorylation of NF-κB p65 and IĸBα and transcription of NF-ĸB were observed. On the contrary, IL8 production and NF-ĸB transcription were drastically inhibited by dominant negative mutant of IĸBα. Moreover, the LMP1 mutants failed to induce IL8 production. Conclusion: Our findings suggest that due to CATR domains, LMP1 contributes to the progression of periodontitis via IL8 production attributable to NF-ĸB activation. Chronic periodontitis, a chronic inflammatory and infectious disease causing the destruction of the periodontium including the alveolar bone, is prevalent worldwide (1, 2). Mounting evidence has indicated that chronic periodontitis is a risk factor for pre-term birth, heart disease, diabetes, and atherosclerosis (1, 2). Over the past decade, neutrophil infiltration in the periodontium has been revealed to be the major aetiology for periodontitis (2). Some oral endogenous bacteria are believed to trigger periodontitis via host-parasite interactions (2, 3). However, periodontopathic bacteria, such as Porphyromonas gingivalis, are not always detected in periodontal lesions (4-6); therefore, the conventional theory based on bacterial aetiology alone cannot fully explain the aetiology of periodontitis. A positive association has been reported between chronic periodontitis and Epstein-Barr virus (EBV) infection (7-11). EBV, a member of the herpesvirus family, infects many adults. During primary EBV infection, the virus undergoes lytic replication in B-cells and epithelial cells of the upper aerodigestive tract, where it later establishes latency (12, 13). EBV can be reactivated and is commonly found in the saliva of infected people (9, 10, 14). Many reports have demonstrated that the amount of EBV DNA detected in 1793 This article is freely accessible online.

Association between Epstein-Barr virus and periodontitis: A meta-analysis

“…Another study indicated the mechanism of EBV infection, which correlates with the severity of chronic periodontitis. EBV-encoded latent membrane protein 1 (LMP1) induces interleukin-2 (IL-8) production in human gingival cells [ 57 , 58 ], and the presence of IL-8 as well as proinflammatory cytokine levels in the gingival crevicular fluid are closely associated with the severity of inflammation and periodontal destruction [ 59 ].…”

How Does Epstein–Barr Virus Contribute to Chronic Periodontitis?

“…Few studies are thus far available that describe de novo etiological significance for the onset or progression of chronic periodontitis with latent EBV infection in the periodontium. We therefore introduce herein some progress in understanding EBV-related periodontal etiology from Doglio's group and ours [17,48,49].…”

“…To further investigate mechanisms pertaining to pathogenesis of EBV latently infected periodontium, we recently examined potential periodontal pathogenesis associated with LMP1 [49]. C-terminal activating region (CATR) domains of LMP1 bind to TNF receptor-associated factor and TNF receptor-associated death domain protein.…”

Butyric Acid in Saliva of Chronic Periodontitis Patients Induces Transcription of the EBV Lytic Switch Activator BZLF1: A Pilot Study

“…Immunoblot assay. The experimental procedures for immunoprecipitation and immunoblotting were performed according to previously published protocols (22,23). Briefly, cells were harvested with lysis buffer [25 mM HEPES-NaOH (pH 7.9), 150 mM NaCl, 1.5 mM MgCl 2 , 0.2 mM EDTA, 0.3% NP-40, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride], the proteins were separated by SDS-PAGE and transferred to a polyvinylidene fluoride membrane (EMD Millipore Corporation, Billerica, MA, USA).…”