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Inflammatory cell death of human macrophages in response to Aggregatibacter actinomycetemcomitans leukotoxin
Umeå University, Faculty of Medicine, Department of Odontology, Periodontology. Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
Centre for Microbiological Preparedness, Swedish Institute for Infectious Disease Control, Solna, Sweden.
Umeå University, Faculty of Medicine, Department of Odontology, Oral Microbiology.
Centre for Microbiological Preparedness, Swedish Institute for Infectious Disease Control, Solna, Sweden ; Department of Laboratory Medicine, Division of Clinical Microbiology, Karolinska Institute, Stockholm, Sweden.
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(English)Manuscript (Other academic)
Abstract [en]

Aggregatibacter (Actinobacillus) actinomycetemcomitans is a facultative anaerobic gram-negative bacterium associated with severe forms of periodontitis. A leukotoxin, which belongs to the Repeats in Toxin (RTX) family, is believed to be one of its virulence factors and to play an important role in the bacterium's pathogenicity. This toxin selectively kills human leukocytes by inducing apoptosis and lysis. Here we report that leukotoxin-induced cell death of macrophages proceeded through a process that differs from the classical characteristics of apoptosis and necrosis. Interestingly, this process resembled pyroptosis, and resulted in an extensive leukotoxin-induced interleukin-1β (IL-1β) secretion. This activation was mainly mediated by caspase-1 activation, while the levels of mRNA for IL-1β were not affected by the leukotoxin. A similar pattern was seen for IL-18, but the level of that cytokine was about 30 times lower. Both of these cytokines are synthesized as biologically inactive precursors and need active caspase-1 for their activation and secretion. In conclusion, A. actinomycetemcomitans leukotoxin induces a pyroptosis-like cell death in human macrophages and that leads to a specific and excessive pro-inflammatory response. This novel virulence mechanism of the leukotoxin may play an important role in the pathogenic potential of this bacterium.

Keyword [en]
A. actinomycetemcomitans, leukotoxin, IL-1β, macrophages, pyroptosis
National Category
Dentistry
Identifiers
URN: urn:nbn:se:umu:diva-19143OAI: oai:DiVA.org:umu-19143DiVA: diva2:201467
Available from: 2009-03-09 Created: 2009-03-04 Last updated: 2014-10-09Bibliographically approved
In thesis
1. Inflammatory cell death of human macrophages induced by Aggregatibacter actinomycetemcomitans leukotoxin
Open this publication in new window or tab >>Inflammatory cell death of human macrophages induced by Aggregatibacter actinomycetemcomitans leukotoxin
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Aggregatibacter (Actinobacillus) actinomycetemcomitans is a bacterium mainly associated with aggressive forms of periodontitis. Among its virulence factors, a leukotoxin is suggested to play an important role in the pathogenicity. Periodontal infections with strains producing high levels of the leukotoxin are strongly associated with severe disease. Leukotoxin selectively kills human leukocytes and can disrupt the local defense mechanisms. Previous studies examining the role of the leukotoxin in host-parasite interactions have mainly focused on polymorphonuclear leukocytes (PMNs). In the inflamed periodontium, macrophages play a significant role in the regulation of the inflammatory reactions and the tissue breakdown and remodeling.

Thus, the aim of this dissertation was to investigate death mechanisms of human macrophages exposed to leukotoxin.

Human lymphocytes, PMNs, and monocytes/macrophages isolated from venous blood were exposed to purified leukotoxin or live A. actinomycetemcomitans strains producing variable levels or no leukotoxin. Different target cells were characterized by their expression of cell surface molecules. Cell death and viability were studied by examining cell membrane integrity and morphological alterations. Further, processes and cellular markers involved in apoptosis and necrosis were investigated. The expression and activation of pro-inflammatory cytokines of the leukotoxin-challenged leukocytes were examined at the mRNA and protein level. The biological activity of the secreted cytokines was investigated by testing the culture supernatants in a bone resorption assay. Additionally, different intracellular signaling pathways involved in the pro-inflammatory response from the macrophages were examined.

Monocytes/macrophages were the most sensitive leukocytes for A. actinomycetemcomitans leukotoxin-induced lysis. This process in monocytes/ macrophages involved caspase-1 activation, and in addition, leukotoxin triggered abundant activation and secretion of IL-1β from these cells. The secreted IL-1β was mainly the 17 kDa bioactive protein and stimulated bone resorption. This activity could be blocked by an IL-1 receptor antagonist. When live bacteria were used, the A. actinomycetemcomitans-induced IL-1β secretion from human macrophages was mainly caused by the leukotoxin. Closer examination of the macrophages exposed to leukotoxin revealed that the induced cell death proceeded through a process that differed from classical apoptosis and necrosis. Interestingly, this process resembled a newly discovered death mechanism termed pyroptosis. The extensive leukotoxin induced IL-1β secretion did not correlate to increased levels of mRNA for IL-1β. It was mainly mediated by caspase-1 activation, since blocking it by a specific inhibitor also abolished the secretion of IL-1β. A similar pattern, but at much lower level, was seen for IL-18.

In conclusion, these results show that A. actinomycetemcomitans leukotoxin induces a death process in human macrophages leading to a specific and excessive pro-inflammatory response. Our results indicate that this novel virulence mechanism of leukotoxin may play an important role in the pathogenic potential of A. actinomycetemcomitans.

Place, publisher, year, edition, pages
Umeå: Institutionen för odontologi , 2009. 80 p.
Series
Umeå University odontological dissertations, ISSN 0345-7532 ; 107
Keyword
Aggregatibacter actinomycetemcomitans, leukotoxin, IL-1β, caspase-1, inflammatory cell death, pyroptosis
National Category
Dentistry
Research subject
Odontology
Identifiers
urn:nbn:se:umu:diva-19204 (URN)978-91-7264-649-0 (ISBN)
Public defence
2009-03-27, Sal B, byggnad 1D, 9 tr, Norrlands universitetssjukhus, Norrlands universitetssjukhus, Umeå, 08:15 (English)
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Available from: 2009-03-09 Created: 2009-03-05 Last updated: 2009-03-11Bibliographically approved

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Kelk, PeymanClaesson, RolfSjöstedt, AndersJohansson, Anders

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