Loss of lipopolysaccharide receptor CD14 from the surface of human macrophage-like cells mediated by Porphyromonas gingivalis outer membrane vesicles

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Abstract

Porphyromonas gingivalis, the major etiologic agent of chronic periodontitis, produces a broad spectrum of virulence factors, including outer membrane vesicles. In this study, we investigated the capacity of P. gingivalis vesicles to promote the shedding or cleavage of the lipopolysaccharide (LPS) receptor CD14 from the surface of human U937 macrophage-like cells. SDS-PAGE/Western immunoblotting analysis of gingival crevicular fluid samples from patients affected by moderate or advanced periodontitis revealed the presence of soluble CD14 and CD14 fragments, thus supporting the hypothesis of an in vivo shedding and cleavage of CD14 receptors. Flow cytometry analysis of macrophage-like cells treated with a vesicle-containing culture supernatant of P. gingivalis showed a significant decrease in the binding of anti-human CD14 to the cell surface. However, no accumulation of soluble CD14 or immunoreactive CD14 fragments in the assay supernatant could be demonstrated by ELISA. Treatment of macrophage-like cells with various concentrations of P. gingivalis vesicles substantially suppressed TNF-α production triggered by Escherichia coli LPS. This suppressive effect was much less important using heat-treated vesicles or in the presence of leupeptin, a gingipain inhibitor, during the treatment. Recombinant human CD14 receptors were found to be susceptible to proteolytic degradation by P. gingivalis vesicles. A purified Arg-gingipain preparation produced much more degradation than a Lys-gingipain preparation. This study provides evidence that P. gingivalis outer membrane vesicles contribute to the loss of membrane-bound CD14 receptors and that gingipains degrade this LPS receptor. Such a phenomenon, which results in an hyporesponsiveness of macrophages to LPS stimulation, may contribute to an increased capacity of P. gingivalis, and other periodontopathogens, to evade the host immune system mechanisms.

Introduction

Periodontitis is an inflammatory disorder that results in the destruction of the supporting structures of the teeth, including the alveolar bone. It is the most important cause of tooth loss among adults and is initiated by an accumulation of predominantly anaerobic Gram-negative bacteria in subgingival sites. Among the various bacterial species associated with the development of periodontitis, the Gram negative anaerobic bacterium Porphyromonas gingivalis is suspected to be one of the most important causative agents of the chronic form of this disease [1].

P. gingivalis produces a broad spectrum of virulence factors, including outer membrane vesicles, adhesins, lipopolysaccharides (LPS), hemolysins and proteinases [2], [3], [4], [5]. Arg- and Lys-gingipain cysteine proteinases are the main endopeptidases produced by P. gingivalis and have received considerable attention due to their strong ability to degrade a broad range of host proteins [5]. Two genes code for Arg-gingipain (rgpA and rgpB), and one codes for Lys-gingipain (kgp) [5]. Gingipains and LPS are present in large quantities on the surface of outer membrane vesicles, which are extracellular structures believed to contribute to host colonization, evasion of immune defense mechanisms and destruction of periodontal tissues [6].

P. gingivalis vesicles may be involved in shedding and cleavage processes of surface receptors present on host cells of various types, a phenomenon that may modify the capacity of these cells to respond to stimuli. Shedding implies the release of the ectodomain of a cell surface molecule that will keep its biological activity, whereas cleavage or hydrolysis is associated with production of several fragments usually resulting in inactivation of the molecule. Several types of human cell surface molecules may constitute a substrate for gingipains including CD14, the specific receptor for LPS. This 55 kDa glycoprotein is anchored in the plasma membrane by a phospholipid called glycosylphosphatidyl inositol and is strongly expressed on monocytes/macrophages [7]. Once present in the extracellular environment in a soluble and biologically active form, CD14 can interact with cells that do not normally express CD14, such as endothelial and epithelial cells [8], [9]. This enables LPS activation of these cells, which in turn increases proinflammatory cytokine production, amplifying the inflammatory process [8], [9]. On the other hand, shedding and cleavage of CD14 receptors result in a decreased capacity of these cells to respond to stimuli. In this study, we investigated the capacity of P. gingivalis outer membrane vesicles to promote the shedding or cleavage of the LPS receptor CD14 from the surface of human macrophage-like cells.

Section snippets

Results

In the first part of the study, gingival crevicular fluid (GCF) samples from patients with various periodontal conditions were analyzed by SDS-PAGE/Western immunoblotting in order to support the hypothesis of an in vivo shedding or cleavage of CD14 during periodontitis (Fig. 1). Three out of eight GCF samples from healthy periodontal sites showed traces of soluble CD14 whereas 9 out of 16 samples from patients affected by moderate or advanced periodontitis contained higher amount of soluble

Discussion

Outer membrane vesicles are produced by P. gingivalis and can act as a virulence factor [6]. More specifically, LPS as well as Arg- and Lys-gingipain cysteine proteinases, found in large quantities on the vesicle surface, may contribute to the pathogenic process of periodontitis by disrupting host defense mechanisms and promoting tissue destruction. In this study, we hypothesized that P. gingivalis vesicles may mediate the shedding or cleavage of the LPS receptor CD14 from the surface of human

Gingival crevicular fluid (GCF) collection and analysis for the presence of soluble CD14 receptor and CD14-derived fragments

GCF samples were obtained from 24 patients attending the dental clinic at Université Laval. The pocket depth of each site was measured using a Michigan periodontal probe. Patients were distributed into three categories: healthy (pocket depth <3 mm), moderate periodontitis (pocket depth between 4 and 6 mm) and advanced periodontitis (pocket depth >7 mm). Paper strips (2×8 mm; 3MM; Whatman, Clifton, NJ) were inserted into the subgingival site for 30 s. The strips were then placed in 250 μl of 100

Acknowledgements

We wish to thank C. Duchaı̂ne (Université Laval) for her assistance in the flow cytometric analysis. We also thank K. Nakayama (Nagasaki University) for providing the gingipain-deficient mutants of P. gingivalis and G. Grenier (Université Laval) for collecting GCF samples. This study was supported by the Canadian Institutes of Health Research.

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