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Inflammatory cell death of human macrophages induced by Aggregatibacter actinomycetemcomitans leukotoxin
Umeå universitet, Medicinsk fakultet, Odontologi, Parodontologi. Umeå universitet, Medicinsk fakultet, Klinisk mikrobiologi, Klinisk bakteriologi.
2009 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
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.

sted, utgiver, år, opplag, sider
Umeå: Institutionen för odontologi , 2009. , 80 s.
Serie
Umeå University odontological dissertations, ISSN 0345-7532 ; 107
Emneord [en]
Aggregatibacter actinomycetemcomitans, leukotoxin, IL-1β, caspase-1, inflammatory cell death, pyroptosis
HSV kategori
Forskningsprogram
odontologi
Identifikatorer
URN: urn:nbn:se:umu:diva-19204ISBN: 978-91-7264-649-0 (tryckt)OAI: oai:DiVA.org:umu-19204DiVA: diva2:201566
Disputas
2009-03-27, Sal B, byggnad 1D, 9 tr, Norrlands universitetssjukhus, Norrlands universitetssjukhus, Umeå, 08:15 (engelsk)
Opponent
Veileder
Tilgjengelig fra: 2009-03-09 Laget: 2009-03-05 Sist oppdatert: 2009-03-11bibliografisk kontrollert
Delarbeid
1. Caspase 1 involvement in human monocyte lysis induced by Actinobacillus actinomycetemcomitans leukotoxin
Åpne denne publikasjonen i ny fane eller vindu >>Caspase 1 involvement in human monocyte lysis induced by Actinobacillus actinomycetemcomitans leukotoxin
Vise andre…
2003 (engelsk)Inngår i: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 71, nr 8, 4448-4455 s.Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Actinobacillus actinomycetemcomitans, an oral bacterium implicated in the etiology of periodontal diseases, produces a leukotoxin that selectively lyses primate neutrophils and monocytes, the major populations of defense cells in the periodontium. Though lysis requires expression of the receptor lymphocyte function-associated molecule 1 (LFA-1) on the cell surface, not all LFA-1-expressing leukocyte populations are equally susceptible to the toxin. In this study, the susceptibility of human leukocytes to leukotoxin-induced lysis is compared to their expression of LFA-1 and the activity of caspase 1. Cytolysis was determined by the activity of lactate dehydrogenase released from peripheral human leukocytes after 1-h exposure to leukotoxin. Monocytes were lysed at leukotoxin concentrations of > or = 5 ng/ml, while the corresponding values for neutrophils and lymphocytes were approximately 10 times greater. Similar LFA-1 expression was found in all susceptible cell populations irrespective of their degree of sensitivity to the toxin. Exposure of monocytes to leukotoxin increased their caspase 1 activity about fivefold within 10 to 20 min. Presence of the caspase 1 inhibitor Ac-YVAD-CMK significantly blocked the leukotoxin-induced lysis of monocytes only. At sublytic concentrations, leukotoxin induced no apoptotic activity in monocytes, as revealed by the lack of caspase 3 activation and DNA fragmentation. Monocytes are the most lysis-sensitive leukocytes for A. actinomycetemcomitans leukotoxin. Their lysis by this toxin depends on caspase 1 activation and proceeds through a process that differs from classical apoptosis.

Identifikatorer
urn:nbn:se:umu:diva-6485 (URN)10.1128/IAI.71.8.4448-4455.2003 (DOI)12874324 (PubMedID)
Tilgjengelig fra: 2007-12-13 Laget: 2007-12-13 Sist oppdatert: 2017-12-14bibliografisk kontrollert
2. Abundant secretion of bioactive interleukin-1beta by human macrophages induced by Actinobacillus actinomycetemcomitans leukotoxin
Åpne denne publikasjonen i ny fane eller vindu >>Abundant secretion of bioactive interleukin-1beta by human macrophages induced by Actinobacillus actinomycetemcomitans leukotoxin
Vise andre…
2005 (engelsk)Inngår i: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 73, nr 1, 453-458 s.Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Actinobacillus actinomycetemcomitans produces a leukotoxin that selectively kills human leukocytes. Recently, we reported that macrophages are highly sensitive to leukotoxin and that their lysis involves activation of caspase 1. In this study, we show that leukotoxin also induces the production and release of proinflammatory cytokines from human macrophages. The macrophages were challenged with leukotoxin or lipopolysaccharide (LPS) from A. actinomycetemcomitans or LPS from Escherichia coli, and the production and secretion of interleukin-1beta (IL-1beta), IL-6, and tumor necrosis factor alpha (TNF-alpha) were determined at the mRNA and protein levels by reverse transcription-PCR and enzyme-linked immunosorbent assay, respectively. Leukotoxin (1 to 30 ng/ml) induced abundant production and secretion of IL-1beta, while the effects on IL-6 and TNF-alpha production were limited. Leukotoxin (1 ng/ml) caused a 10-times-higher release of IL-1beta than did LPS (100 ng/ml). The secreted IL-1beta was mainly the bioactive 17-kDa protein. At higher concentrations (>30 ng/ml), leukotoxin caused secretion of mainly inactive cytokine, the 31-kDa pro-IL-1beta. The presence of specific antibodies to IL-1beta or of a caspase 1 inhibitor blocked the secretion and production of the cytokine. Supernatants of leukotoxin-challenged macrophages stimulated bone resorption when tested in a mouse calvarial model. The activity could be blocked by an IL-1 receptor antagonist or specific antibodies to IL-1beta. We concluded that A. actinomycetemcomitans leukotoxin can trigger abundant production and secretion of bioactive IL-1beta by human macrophages, which is mediated by activation of caspase 1.

Identifikatorer
urn:nbn:se:umu:diva-18061 (URN)10.1128/IAI.73.1.453-458.2005 (DOI)15618184 (PubMedID)
Tilgjengelig fra: 2007-12-13 Laget: 2007-12-13 Sist oppdatert: 2017-12-14bibliografisk kontrollert
3. IL-1beta secretion induced by Aggregatibacter (Actinobacillus) actinomycetemcomitans is mainly caused by the leukotoxin
Åpne denne publikasjonen i ny fane eller vindu >>IL-1beta secretion induced by Aggregatibacter (Actinobacillus) actinomycetemcomitans is mainly caused by the leukotoxin
Vise andre…
2008 (engelsk)Inngår i: International Journal of Medical Microbiology, ISSN 1438-4221, E-ISSN 1618-0607, Vol. 298, nr 5/6, 529-541 s.Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Aggregatibacter (Actinobacillus) actinomycetemcomitans forms a leukotoxin that selectively lyses primate neutrophils, monocytes and triggers apoptosis in promyeloic cells and degranulation of human neutrophils. Recently, we showed that the leukotoxin causes activation of caspase-1 and abundant secretion of bio-active IL-1beta from human macrophages. In this study, we show that high levels of IL-beta correlated with a high proportion of A. actinomycetemcomitans in clinical samples from a patient with aggressive periodontitis. To determine the relative contribution of leukotoxin to the overall bacteria-induced IL-1beta secretion, macrophages were isolated from peripheral blood and exposed to different concentrations of live A. actinomycetemcomitans strains with either no, low or high production of leukotoxin. Cell lysis and levels of IL-1beta, IL-6, TNF-alpha and caspase-1 were measured by ELISA and flow cytometry. Leukotoxin was the predominant cause of IL-1beta secretion from macrophages, even in the A. actinomycetemcomitans strain with low leukotoxin production. Macrophages exposed to non-leukotoxic bacteria accumulated cytosolic pro-IL-1beta, which was secreted by a secondary exposure to leukotoxic bacteria. In conclusion, the present study shows for the first time that A. actinomycetemcomitans-induced IL-1beta secretion from human macrophages in vitro is mainly caused by leukotoxin.

Identifikatorer
urn:nbn:se:umu:diva-6475 (URN)10.1016/j.ijmm.2007.06.005 (DOI)17888725 (PubMedID)
Tilgjengelig fra: 2007-12-13 Laget: 2007-12-13 Sist oppdatert: 2017-12-14bibliografisk kontrollert
4. Inflammatory cell death of human macrophages in response to Aggregatibacter actinomycetemcomitans leukotoxin
Åpne denne publikasjonen i ny fane eller vindu >>Inflammatory cell death of human macrophages in response to Aggregatibacter actinomycetemcomitans leukotoxin
Vise andre…
(engelsk)Manuskript (Annet vitenskapelig)
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.

Emneord
A. actinomycetemcomitans, leukotoxin, IL-1β, macrophages, pyroptosis
HSV kategori
Identifikatorer
urn:nbn:se:umu:diva-19143 (URN)
Tilgjengelig fra: 2009-03-09 Laget: 2009-03-04 Sist oppdatert: 2014-10-09bibliografisk kontrollert

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