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Gingipains from Porphyromonas gingivalis play a significant role in induction and regulation of CXCL8 in THP-1 cells
Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.ORCID iD: 0000-0001-7105-0425
Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
Örebro University, School of Medicine, Örebro University, Sweden.
2014 (English)In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 14, 193Article in journal (Refereed) Published
Abstract [en]

Background: Porphyromonas gingivalis is an important bacterial etiological agent involved in periodontitis. The bacterium expresses two kinds of cysteine proteases called gingipains: arginine gingipains (RgpA/B) and lysine gingipain (Kgp). This study evaluated the interaction between P. gingivalis and THP-1 cells, a widely used monocytic cell line, in vitro with a focus on CXCL8 at the gene and protein levels and its fate thereafter in cell culture supernatants. THP-1 cells were stimulated with viable and heat-killed wild-type strains ATCC 33277 or W50 or viable isogenic gingipain mutants of W50, E8 (Rgp mutant) or K1A (Kgp mutant), for 24 hours.

Results: ELISA and qPCR results show an elevated CXCL8 expression and secretion in THP-1 cells in response to P. gingivalis, where the heat-killed ATCC33277 and W50 induced higher levels of CXCL8 in comparison to their viable counterparts. Furthermore, the Kgp-deficient mutant K1A caused a higher CXCL8 response compared to the Rgp-deficient E8. Chromogenic quantification of lipopolysaccharide (LPS) in supernatant showed no significant differences between viable and heat killed bacteria except that W50 shed highest levels of LPS. The wild-type strains secreted relatively more Rgp during the co-culture with THP-1 cells. The CXCL8 degradation assay of filter-sterilized supernatant from heat-killed W50 treated cells showed that Rgp was most efficient at CXCL8 hydrolysis. Of all tested P. gingivalis strains, adhesion and internalization in THP-1 cells was least conspicuous by Rgp-deficient P. gingivalis (E8), as demonstrated by confocal imaging.

Conclusions: W50 and its Kgp mutant K1A exhibit a higher immunogenic and proteolytic function in comparison to the Rgp mutant E8. Since K1A differs from E8 in the expression of Rgp, it is rational to conclude that Rgp contributes to immunomodulation in a more dynamic manner in comparison to Kgp. Also, W50 is a more virulent strain when compared to the laboratory strain ATCC33277.

Place, publisher, year, edition, pages
2014. Vol. 14, 193
Keyword [en]
Porphyromonas gingivalis, THP-1 cells, Gingipains, Mutants, CXCL8 degradation
National Category
Microbiology
Research subject
Microbiology
Identifiers
URN: urn:nbn:se:oru:diva-36171DOI: 10.1186/1471-2180-14-193ISI: 000339837900001OAI: oai:DiVA.org:oru-36171DiVA: diva2:742932
Funder
Swedish Heart Lung Foundation
Note

Funding Agencies:

Foundation of Olle Engkvist

Knowledge Foundation

Available from: 2014-09-03 Created: 2014-08-28 Last updated: 2017-10-17Bibliographically approved
In thesis
1. Monocyte and Neutrophil Inflammatory Responses to the Periodontopathogen Porphyromonas gingivalis
Open this publication in new window or tab >>Monocyte and Neutrophil Inflammatory Responses to the Periodontopathogen Porphyromonas gingivalis
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Periodontitis is one of the most common adult infections. Duing bacteremia in healthy individuals or patients with chronic periodontitis, a number of oral bacteria such as Porphyromonas gingivalis encounter inflammatory cells in the blood eg. platelets, neutrophils and monocytes. Although several studies have suggested an association between periodontitis and cardiovascular diseases, the infection and inflammatory mechanisms are poorly understood. Hence, the aim of this thesis was to elucidate the mechanisms that are involved in P. gingivalis interaction with blood leukocytes, in order to further understand the molecular pathogenesis that renders periodontitis as a risk factor for several systemic conditions. We have demonstrated that P. gingivalis induces ROS production in neutrophils, THP1 cells and in whole blood, through activation of pattern recognition receptors, such as toll-like receptors, nuclear oligomerizing domains and protease- activated receptors. Besides, we have also shown that monocytes secrete IL-1β and CXCL8 in response to P. gingivalis. Both these cytokines prime neutrophils, endothelial cells and other vascular cells in an autocrine and paracrine manner. P. gingivalis has a plethora of virulence factors of which gingipains are very unique. In addition to activating inflammatory signalling pathways in cells, gingipains also regulate CXCL8 and IL-1β, thereby curtailing the host defence strategies. We demonstrated that oxidized LDL, but not native LDL, induces IL-1β release and CD36 expression on THP1 cells. Furthermore, LDL mildly modifies P. gingivalis-induced inflammatory responses as well as CD36 expression in THP1 cells. We also observed that P. gingivalis is eliminated mainly by phagocytosis in neutrophils. In summary, these studies clarify the mechanisms of interaction between P. gingivalis and leukocytes, which can increase the understanding of the pathogenesis of periodontitis and associated systemic disorders.

Place, publisher, year, edition, pages
Örebro: Örebro university, 2016. 63 p.
Series
Örebro Studies in Medicine, ISSN 1652-4063 ; 138
Keyword
Monocytes, Neutrophils, Porphyromonas gingivalis, Gingipains
National Category
Other Basic Medicine
Research subject
Biomedicine
Identifiers
urn:nbn:se:oru:diva-48228 (URN)978-91-7529-122-2 (ISBN)
Public defence
2016-04-15, Universitetssjukhuset, hörsal C2, Södra Grev Rosengatan, Örebro, 09:00 (English)
Opponent
Supervisors
Available from: 2016-02-12 Created: 2016-02-12 Last updated: 2017-10-17Bibliographically approved

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Jayaprakash, KartheyaeneKhalaf, HazemBengtsson, Torbjörn

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