Student Theses, Dissertations and Capstones

Document Type

Thesis

Degree Name

Master of Science (M.S.) in Dentistry

Copyright Statement

All rights reserved. This publication is intended for use solely by faculty, students, and staff of Nova Southeastern University. No part of this publication may be reproduced, distributed, or transmitted in any form or by any means, now known or later developed, including but not limited to photocopying, recording, or other electronic or mechanical methods, without the prior written permission of the author or the publisher.

Department

College of Dental Medicine

First Advisor

Toshihisa Kawai

Publication Date / Copyright Date

2019

Publisher

Nova Southeastern University

Abstract

BACKGROUND: Periodontitis is an inflammatory disease caused by poly-microbial infection that leads to destruction of connective tissue and alveolar bone. It is well documented that bacteria-derived virulent factors that can act on Toll-like receptors (TLRs), represented by Lipopolysaccharides (LPS), are engaged in the initiation of inflammatory responses. However, while LPS is also produced by the bacteria colonized in the healthy periodontal tissue, inflammation is not induced by LPS in those periodontal healthy subjects, suggesting the requirement of additional factor to upregulate the LPS-mediated inflammatory response in periodontal tissue. Recent studies revealed that novel class of endogenous proinflammatory mediator, high mobility group box 1 (HMGB1) protein, can be released extracellularly from host cells in response to a variety of stimuli, such as pathogen invasion. Although, significantly elevated levels of HMGB1 are reported in gingival tissues and gingival crevicular fluid (GCF) of chronic periodontitis patients, its pathophysiological role in periodontitis is not clear. The herein study investigated the effects of HMGB1 on P. gingivalis-LPS (Pg-LPS)-elicited inflammation induced in macrophages using an in vitro assay system. OBJECTIVE: The present study examined the effects of the novel host danger alarming molecule, high mobility group box 1 (HMGB1), on pro-inflammatory cytokine production by P. gingivalis-LPS (Pg-LPS)-stimulated macrophages. HYPOTHESIS: We hypothesized that HMGB1 forms complex with Pg LPS which can induce hyper inflammatory response by macrophages. HMGB1-Pg-LPS complex is expected to induce production of more pro-inflammatory cytokines (i.e. Tumor Necrosis Factor alpha (TNF-alpha) and Interleukin 6 (IL-6)) than those induced by LPS or HMGB1 alone. MATERIALS AND METHODS: RAW264.7 macrophage cells (ATCC) were stimulated in vitro for 24 hours with HMGB1, P. gingivalis-LPS, E. coli-LPS or HMGB1 in a preformed complex with P. gingivalis-LPS or E. coli-LPS. Supernatants were collected and kept at -20°C until analysis. Chemical antagonists for TLR4, TLR2 and RAGE were applied to some cultures in the presence or absence of LPS and/or HMGB1. TNF-alpha and IL-6 levels in the culture supernatants were measured by enzyme-linked immunosorbent assay (ELISA). PAMP receptor genes analysis was performed to assess TLR2, TLR4 and RAGE mRNAs expressed in RAW264.7 cells by quantitative PCR technique. The levels of macrophage (RAW264.7) proliferations in response to stimulation to LPS with or without HMGB1 was determined by WST assay. RESULTS: Compared to E.coli-LPS that induced prominently elevated TNF-alpha production by RAW264.7 macrophages, Pg-LPS as well as HMGB1 showed significantly lower levels of TNF-alpha production. However, combination of HMGB1 with Pg-LPS, but not E.coli-LPS, showed a remarkable additive effect on TNF-alpha production by RAW264.7-macrophages which was abrogated by addition of TLR4-antagonist. Interestingly, additive effect was only found on production of TNF-alfa, but not IL-6. HMGB1-Pg-LPS complex also increased the proliferation of macrophages, whereas HMGB1-E.coli-LPS complex did not affect the proliferation of macrophages. According to the qPCR-based analysis of gene expressions for PAMP receptors, the macrophages used in this study expressed mRNAs for TLR2, TLR4 and RAGE as putative ligands for HMGB1. Pg-LPS alone or in combination with HMGB1 did not change the expression levels of all three PAMPs expressed by macrophages, indicating that elevated production of TNF-alpha by HMGB1-Pg-LPS complex was not mediated by modulation of PAMP receptors expressed on macrophages. CONCLUSION: These results demonstrated that HMGB1 can form a hyper-inflammatory complex with Pg-LPS, but not E. coli-LPS, that activates TLR4 and promotes TNF-alpha production from the macrophages, suggesting that locally released HMGB1 may up-regulate the pathogenic engagement of keystone pathogen, P. gingivalis, in periodontitis.

Disciplines

Dentistry

Keywords

High mobility group box 1, Inflammation, LPS, Macrophage, Periodontitis, Porphyromonas gingivalis

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