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Cellular and molecular responses of periodontal connective tissue cells to Actinobacillus actinomycetemcomitans cytolethal distending toxin
Umeå University, Faculty of Medicine, Odontology, Oral Microbiology. Umeå University, Faculty of Medicine, Odontology, Oral Cell Biology.
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Actinobacillus actinomycetemcomitans is present in elevated proportions and numbers in dental bacterial biofilms of patients with localized aggressive periodontitis. This variant of periodontal disease, occurring in adolescents and young adults, is characterized by rapid and severe destruction of the connective tissues and bone supporting the teeth, eventually culminating in tooth loss. The cytolethal distending toxin (Cdt) is a newly discovered bacterial protein toxin, uniquely present in A. actinomycetemcomitans among all known to-date oral bacterial species. The Cdt has the capacity to inhibit mammalian cell growth, but its putative role in the pathogenesis of the disease is unclear. The aim of this in vitro work has been to study the effects of A. actinomycetemcomitans on periodontal connective tissue cell cultures, and to evaluate the possible involvement of its Cdt.

A. actinomycetemcomitans inhibited the proliferation of gingival and periodontal ligament fibroblasts, as a result of a combined arrest at the G1 and G2/M phases of the cell cycle. This growth inhibition was non-lethal and the cells remained metabolically active, although their DNA synthesis was reduced. The intoxicated cells exhibited increased size and irregular structure, characterized by distension and elongation. This cellular enlargement occurred in both G1 and G2/M phase arrested cells. The Cdt of A. actinomycetemcomitans was responsible for the observed growth inhibition, as well as the concomitant morphological alterations.

The possible induction of inflammatory cytokines related to bone resorption was investigated in response to A. actinomycetemcomitans, and the involvement of Cdt was evaluated. Extensive focus was given to the study of receptor activator of NF-κB ligand (RANKL) expression, a membrane-bound ligand that signals osteoclast progenitors to differentiate and fuse into mature osteoclasts, activating bone resorption. It was demonstrated that A. actinomycetemcomitans induced RANKL mRNA and protein expression in the cells studied, but did not affect the expression of its decoy receptor, osteoprotegerin. This induction was solely attributed to its Cdt, as demonstrated by the use of a cdt-knockout A. actinomycetemcomitans strain, purified recombinant Cdt, and antibodies blocking the Cdt. In addition, this event was not mediated by pro-inflammatory cytokines known to stimulate RANKL. Interleukin-6 mRNA and protein expression were also enhanced by A. actinomycetemcomitans, but Cdt had limited involvement in this enhancement.

In conclusion, two distinct mechanisms by which A. actinomycetemcomitans Cdt may be involved in the pathogenesis of localized aggressive periodontitis are proposed. Firstly, the growth arrest of the resident fibroblasts may impair the physiological connective tissue remodelling equilibrium and lead to connective tissue attachment loss. Secondly, the induction of RANKL by these cells, residing in the proximity of the alveolar bone, may locally stimulate osteoclastogenesis and promote alveolar bone resorption. This work also provides further insights to the understanding of Cdt mechanisms of action, contributing to the global characterization of the toxin’s virulence.

Place, publisher, year, edition, pages
Umeå: Odontologi , 2004. , 49 p.
Series
Umeå University odontological dissertations, ISSN 0345-7532 ; 88
Keyword [en]
Medical sciences, Actinobacillus actinomycetemcomitans, cytolethal distending toxin, periodontal connective tissue cells, localized aggressive periodontitis, growth arrest, bone resorption, receptor activator of NF-κB ligand, osteoprotegerin, pro-inflammatory cytokines
Keyword [sv]
MEDICIN OCH VÅRD
National Category
Medical and Health Sciences
Research subject
Odontology
Identifiers
URN: urn:nbn:se:umu:diva-345ISBN: 91-7305-746-0 (print)OAI: oai:DiVA.org:umu-345DiVA: diva2:143178
Public defence
2004-12-10, Sal 933, 3A, Norrlands Universitessjukhus, Umeå, 13:00 (English)
Opponent
Supervisors
Available from: 2004-11-01 Created: 2004-11-01 Last updated: 2009-08-24Bibliographically approved
List of papers
1. Inhibited proliferation of human periodontal ligament cells and gingival fibroblasts by Actinobacillus actinomycetemcomitans: involvement of the cytolethal distending toxin
Open this publication in new window or tab >>Inhibited proliferation of human periodontal ligament cells and gingival fibroblasts by Actinobacillus actinomycetemcomitans: involvement of the cytolethal distending toxin
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2002 (English)In: European Journal of Oral Sciences, ISSN 0909-8836, Vol. 110, no 5, 366-373 p.Article in journal (Refereed) Published
Abstract [en]

Actinobacillus actinomycetemcomitans can inhibit fibroblast proliferation. The objective of this study was to characterize the early proliferative responses of human periodontal ligament cells (PDLC) and gingival fibroblasts (GF) to A. actinomycetemcomitans components and to investigate the possible involvement of the cytolethal distending toxin (cdt) produced by this bacterium. The PDLC and GF were challenged with surface components of A. actinomycetemcomitans. Both DNA and protein synthesis as well as cell lysis or apoptosis were assayed for a 6-h period after addition of the bacterial extract. Unlike the controls, inhibition of DNA synthesis had already occurred in the challenged cells at the end of the initial 3- to 6-h period. No lysis or apoptosis was detected, and the total protein synthesis remained unaffected. The persistence of the effect on cell growth was confirmed after a 72-h period of challenge, during which the cells remained viable but exhibited an elongated and distended cell body. No significant differences were observed between PDLC and GF. When a cdt-knockout strain of A. actinomycetemcomitans was used almost no inhibitory effect on cell proliferation was observed. It was concluded that A. actinomycetemcomitans causes a non-lethal inhibition of proliferation in PDLC and GF as a result of an early arrest of DNA synthesis. Cytolethal distending toxin is responsible for most of this effect. This bacterial property may compromise tissue homeostasis in the periodontium.

Identifiers
urn:nbn:se:umu:diva-4181 (URN)10.1034/j.1600-0722.2002.21350.x (DOI)12664467 (PubMedID)
Available from: 2004-11-01 Created: 2004-11-01Bibliographically approved
2. Cell cycle arrest of human gingival fibroblasts and periodontal ligament cells by Actinobacillus actinomycetemcomitans: involvement of the cytolethal distending toxin
Open this publication in new window or tab >>Cell cycle arrest of human gingival fibroblasts and periodontal ligament cells by Actinobacillus actinomycetemcomitans: involvement of the cytolethal distending toxin
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2004 (English)In: Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS), ISSN 0903-4641, Vol. 112, no 10, 674-685 p.Article in journal (Refereed) Published
Abstract [en]

The cytolethal distending toxin (Cdt) is produced by several Gram-negative bacterial species and causes growth arrest and morphological alterations in mammalian cells. Actinobacillus actinomycetemcomitans, which is involved in the pathogenesis of localized aggressive periodontitis, also produces a Cdt that affects periodontal connective tissue cells. The aim of this study was to investigate in which phase of the cell cycle these cells are arrested and enlarged when challenged with A. actinomycetemcomitans, and to evaluate the involvement of its Cdt. Human gingival fibroblasts and periodontal ligament cells were challenged with A. actinomycetemcomitans extract, or with purified Cdt, and cell cycle analysis was performed by propidium iodide staining and flow cytometry. Cells exposed to an A. actinomycetemcomitans wild-type strain, or to purified Cdt, were arrested in both G1 and G2/M phases, and appeared enlarged compared to the corresponding controls. The cellular enlargement occurred in both G1 and G2/M arrested cells. In contrast, cells exposed to an A. actinomycetemcomitans cdt-knockout mutant strain showed cell cycle phase distribution and size similar to the controls. In conclusion, A. actinomycetemcomitans causes a combined G1 and G2/M growth arrest and enlargement in periodontal connective tissue cells, which is attributed to its Cdt.

Identifiers
urn:nbn:se:umu:diva-6487 (URN)10.1111/j.1600-0463.2004.apm1121006.x (DOI)15601319 (PubMedID)
Available from: 2007-12-13 Created: 2007-12-13 Last updated: 2009-12-08Bibliographically approved
3. The cytolethal distending toxin induces receptor activator of NF-κB ligand expression in human gingival fibroblasts and periodontal ligament cells
Open this publication in new window or tab >>The cytolethal distending toxin induces receptor activator of NF-κB ligand expression in human gingival fibroblasts and periodontal ligament cells
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2005 (English)In: Infection and Immunity, ISSN 0019-9567, Vol. 73, no 1, 342-351 p.Article in journal (Refereed) Published
Abstract [en]

Actinobacillus actinomycetemcomitans is associated with localized aggressive periodontitis, a disease characterized by rapid loss of the alveolar bone surrounding the teeth. Receptor activator of NF-kappaB Ligand (RANKL) and osteoprotegerin (OPG) are two molecules that regulate osteoclast formation and bone resorption. RANKL induces osteoclast differentiation and activation, whereas OPG blocks this process by acting as a decoy receptor for RANKL. The purpose of this study was to investigate the effect of A. actinomycetemcomitans on the expression of RANKL and OPG in human gingival fibroblasts and periodontal ligament cells. RANKL mRNA expression was induced in both cell types challenged by A. actinomycetemcomitans extract, whereas OPG mRNA expression remained unaffected. Cell surface RANKL protein was also induced by A. actinomycetemcomitans, whereas there was no change in OPG protein secretion. A cytolethal distending toxin (Cdt) gene-knockout strain of A. actinomycetemcomitans did not induce RANKL expression, in contrast to its wild-type strain. Purified Cdt from Haemophilus ducreyi alone, or in combination with extract from the A. actinomycetemcomitans cdt mutant strain, induced RANKL expression. Pretreatment of A. actinomycetemcomitans wild-type extract with Cdt antiserum abolished RANKL expression. In conclusion, A. actinomycetemcomitans induces RANKL expression in periodontal connective tissue cells. Cdt is crucial for this induction and may therefore be involved in the pathological bone resorption during the process of localized aggressive periodontitis.

Identifiers
urn:nbn:se:umu:diva-18058 (URN)10.1128/IAI.73.1.342-351.2005 (DOI)15618171 (PubMedID)
Available from: 2007-12-13 Created: 2007-12-13 Last updated: 2009-12-08Bibliographically approved
4. Cytokine responses of human gingival fibroblasts to Actinobacillus actinomycetemcomitans cytolethal distending toxin
Open this publication in new window or tab >>Cytokine responses of human gingival fibroblasts to Actinobacillus actinomycetemcomitans cytolethal distending toxin
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2005 (English)In: Cytokine, ISSN 1043-4666, Vol. 30, no 2, 56-63 p.Article in journal (Refereed) Published
Abstract [en]

Actinobacillus actinomycetemcomitans is implicated in the pathogenesis of localized aggressive periodontitis, and has the capacity to express a cytolethal distending toxin (Cdt). Gingival fibroblasts (GF) are resident cells of the periodontium, which can express several osteolytic cytokines. The aims of this study were a) to investigate the role of Cdt in A. actinomycetemcomitans-induced expression of osteolytic cytokines and their cognate receptors in GF and b) to determine if the previously demonstrated induction of receptor activator of NFkappaB ligand (RANKL) by A. actinomycetemcomitans is mediated by these pro-inflammatory cytokines or by prostaglandin E(2) (PGE(2)). A. actinomycetemcomitans clearly induced interleukin (IL)-6, IL-1beta, and to a minimal extent, tumor necrosis factor (TNF)-alpha mRNA expression. At the protein level, IL-6 but not IL-1beta or TNF-alpha expression was stimulated. The mRNA expression of the different receptor subtypes recognizing IL-6, IL-1beta and TNF-alpha was not affected. A cdt-knockout strain of A. actinomycetemcomitans had similar effects on cytokine and cytokine receptor mRNA expression, compared to its parental wild-type strain. Purified Cdt stimulated IL-6, but not IL-1beta or TNF-alpha protein biosynthesis. Antibodies neutralizing IL-6, IL-1 or TNF-alpha, and the PGE(2) synthesis inhibitor indomethacin, did not affect A. actinomycetemcomitans-induced RANKL expression. In conclusion, a) A. actinomycetemcomitans induces IL-6 production in GF by a mechanism largely independent of its Cdt and b) A. actinomycetemcomitans-induced RANKL expression in GF occurs independently of IL-1, IL-6, TNF-alpha, or PGE(2).

Identifiers
urn:nbn:se:umu:diva-18083 (URN)10.1016/j.cyto.2004.11.008 (DOI)15804596 (PubMedID)
Available from: 2007-12-13 Created: 2007-12-13 Last updated: 2009-08-21Bibliographically approved

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