Dataset Information

Thrombospondin-1 production is enhanced by Porphyromonas gingivalis lipopolysaccharide in THP-1 cells.

ABSTRACT: Periodontitis is a chronic inflammatory disease caused by gram-negative anaerobic bacteria. Monocytes and macrophages stimulated by periodontopathic bacteria induce inflammatory mediators that cause tooth-supporting structure destruction and alveolar bone resorption. In this study, using a DNA microarray, we identified the enhanced gene expression of thrombospondin-1 (TSP-1) in human monocytic cells stimulated by Porphyromonas gingivalis lipopolysaccharide (LPS). TSP-1 is a multifunctional extracellular matrix protein that is upregulated during the inflammatory process. Recent studies have suggested that TSP-1 is associated with rheumatoid arthritis, diabetes mellitus, and osteoclastogenesis. TSP-1 is secreted from neutrophils, monocytes, and macrophages, which mediate immune responses at inflammatory regions. However, TSP-1 expression in periodontitis and the mechanisms underlying TSP-1 expression in human monocytic cells remain unknown. Here using real-time RT-PCR, we demonstrated that TSP-1 mRNA expression level was significantly upregulated in inflamed periodontitis gingival tissues and in P. gingivalis LPS-stimulated human monocytic cell line THP-1 cells. TSP-1 was expressed via Toll-like receptor (TLR) 2 and TLR4 pathways. In P. gingivalis LPS stimulation, TSP-1 expression was dependent upon TLR2 through the activation of NF-?B signaling. Furthermore, IL-17F synergistically enhanced P. gingivalis LPS-induced TSP-1 production. These results suggest that modulation of TSP-1 expression by P. gingivalis plays an important role in the progression and chronicity of periodontitis. It may also contribute a new target molecule for periodontal therapy.

SUBMITTER: Gokyu M

PROVIDER: S-EPMC4264871 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Thrombospondin-1 production is enhanced by Porphyromonas gingivalis lipopolysaccharide in THP-1 cells.

Gokyu Misa M Kobayashi Hiroaki H Nanbara Hiromi H Sudo Takeaki T Ikeda Yuichi Y Suda Tomonari T Izumi Yuichi Y

PloS one 20141212 12

Periodontitis is a chronic inflammatory disease caused by gram-negative anaerobic bacteria. Monocytes and macrophages stimulated by periodontopathic bacteria induce inflammatory mediators that cause tooth-supporting structure destruction and alveolar bone resorption. In this study, using a DNA microarray, we identified the enhanced gene expression of thrombospondin-1 (TSP-1) in human monocytic cells stimulated by Porphyromonas gingivalis lipopolysaccharide (LPS). TSP-1 is a multifunctional extra ...[more]

PMID: 25501558

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Project description:Porphyromonas gingivalis, a bacterial pathogen contributing to human periodontitis, exports and anchors cargo proteins to its surface, enabling the production of black pigmentation using a type IX secretion system (T9SS) and conjugation to anionic lipopolysaccharide (A-LPS). To determine whether T9SS components need to be assembled in situ for correct secretion and A-LPS modification of cargo proteins, combinations of nonpigmented mutants lacking A-LPS or a T9SS component were mixed to investigate in trans complementation. Reacquisition of pigmentation occurred only between an A-LPS mutant and a T9SS mutant, which coincided with A-LPS modification of cargo proteins detected by Western blotting and coimmunoprecipitation/quantitative mass spectrometry. Complementation also occurred using an A-LPS mutant mixed with outer membrane vesicles (OMVs) or purified A-LPS. Fluorescence experiments demonstrated that OMVs can fuse with and transfer lipid to P. gingivalis, leading to the conclusion that complementation of T9SS function occurred through A-LPS transfer between cells. None of the two-strain crosses involving only the five T9SS OM component mutants produced black pigmentation, implying that the OM proteins cannot be transferred in a manner that restores function and surface pigmentation, and hence, a more ordered temporal in situ assembly of T9SS components may be required. Our results show that LPS can be transferred between cells or between cells and OMVs to complement deficiencies in LPS biosynthesis and hemin-related pigmentation to reveal a potentially new mechanism by which the oral microbial community is modulated to produce clinical consequences in the human host.IMPORTANCEPorphyromonas gingivalis is a keystone pathogen contributing to periodontitis in humans, leading to tooth loss. The oral microbiota is essential in this pathogenic process and changes from predominantly Gram-positive (health) to predominantly Gram-negative (disease) species. P. gingivalis uses its type IX secretion system (T9SS) to secrete and conjugate virulence proteins to anionic lipopolysaccharide (A-LPS). This study investigated whether components of this secretion system could be complemented and found that it was possible for A-LPS biosynthetic mutants to be complemented in trans both by strains that had the A-LPS on the cell surface and by exogenous sources of A-LPS. This is the first known example of LPS exchange in a human bacterial pathogen which causes disease through complex microbiota-host interactions.

Project description:ObjectiveEphrinA2-EphA2 and ephrinB2-EphB4 critically engage in bidirectional signalling to modulate alveolar bone remodelling. The present study aimed to investigate the effects of lipopolysaccharides (LPS) derived from Porphyromonas gingivalis on ephrin/Eph signalling in periodontal ligament fibroblasts (PDLFs).Material and methodsThe primary cultured PDLFs were incubated in the absence (as a control) or presence of P. gingivalisLPS at 0.001-10 μg/mL for 24 hours. The PDLFs were then stimulated with P. gingivalisLPS at the optimal concentration (0.1 μg/mL) for different periods (6-48 hours). The expression of ephrinA2, ephrinB2, EphA2 and EphB4 was assessed by quantitative reverse-transcription real-time polymerase chain reaction and western blotting. The osteoblastic markers alkaline phosphatase, osteocalcin and Runt-related transcription factor 2 (Runx2), and the osteoclastogenesis-related factors receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin were also evaluated.ResultsThe ephrinA2 and EphA2 expression was upregulated and EphB4 expression was downregulated by stimulation of P. gingivalisLPS. EphrinA2 mRNA expression in the PDLFs was significantly upregulated from 12 to 48 hours (P<.05), whereas EphA2 exhibited no change for the first 24 hours, after which there was a significant increase at 48 hours (P<.05). EphB4 exhibited lower mRNA expression at 12 and 24 hours than did the control (P<.05), but the change was insignificant at 48 hours. In contrast, the expression of ephrinB2 remained unchanged. The expressions of ephrinA2, EphA2, ephrinB2 and EphB4 at the protein level showed a similar pattern to that at the mRNA level. The expression of Runx2 and osteocalcin significantly decreased, whereas that of RANKL/osteoprotegerin increased.ConclusionThe present study suggest that P. gingivalisLPS would contribute to a dysregulation of bone remodelling, whereby ephrinA2/EphA2 expression is stimulated and EphB4 expression is inhibited.

Project description:Periodontitis is a very common oral inflammatory disease that results in the destruction of supporting connective and osseous tissues of the teeth. Although the exact etiology is still unclear, Gram-negative bacteria, especially Porphyromonas gingivalis in subgingival pockets are thought to be one of the major etiologic agents of periodontitis. Endothelin (ET) is a family of three 21-amino acid peptides, ET-1, -2, and -3, that activate G protein-coupled receptors, ETA and ETB. Endothelin is involved in the occurrence and progression of various inflammatory diseases. Previous reports have shown that ET-1 and its receptors, ETA and ETB are expressed in the periodontal tissues and, that ET-1 levels in gingival crevicular fluid are increased in periodontitis patients. Moreover, P. gingivalis infection has been shown to induce the production of ET-1 along with other inflammatory cytokines. Despite these studies, however, the functional significance of endothelin in periodontitis is still largely unknown. In this study, we explored the cellular and molecular mechanisms of ET-1 action in periodontitis using human gingival epithelial cells (HGECs). ET-1 and ETA, but not ETB, were abundantly expressed in HGECs. Stimulation of HGECs with P. gingivalis or P. gingivalis lipopolysaccharide increased the expression of ET-1 and ETA suggesting the activation of the endothelin signaling pathway. Production of inflammatory cytokines, IL-1?, TNF?, and IL-6, was significantly enhanced by exogenous ET-1 treatment, and this effect depended on the mitogen-activated protein kinases via intracellular Ca2+ increase, which resulted from the activation of the phospholipase C/inositol 1,4,5-trisphosphate pathway. The inhibition of the endothelin receptor-mediated signaling pathway with the dual receptor inhibitor, bosentan, partially ameliorated alveolar bone loss and immune cell infiltration. These results suggest that endothelin plays an important role in P. gingivalis-mediated periodontitis. Thus, endothelin antagonism may be a potential therapeutic approach for periodontitis treatment.

Dataset Information

Thrombospondin-1 production is enhanced by Porphyromonas gingivalis lipopolysaccharide in THP-1 cells.

Publications

Thrombospondin-1 production is enhanced by Porphyromonas gingivalis lipopolysaccharide in THP-1 cells.

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