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Monocyte and Neutrophil Inflammatory Responses to the Periodontopathogen Porphyromonas gingivalis
Örebro University, School of Medical Sciences.ORCID iD: 0000-0001-7105-0425
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 [en]
Monocytes, Neutrophils, Porphyromonas gingivalis, Gingipains
National Category
Other Basic Medicine
Research subject
Biomedicine
Identifiers
URN: urn:nbn:se:oru:diva-48228ISBN: 978-91-7529-122-2 (print)OAI: oai:DiVA.org:oru-48228DiVA: diva2:902884
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: 2016-03-23Bibliographically approved
List of papers
1. Gingipains from Porphyromonas gingivalis play a significant role in induction and regulation of CXCL8 in THP-1 cells
Open this publication in new window or tab >>Gingipains from Porphyromonas gingivalis play a significant role in induction and regulation of CXCL8 in THP-1 cells
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.

Keyword
Porphyromonas gingivalis, THP-1 cells, Gingipains, Mutants, CXCL8 degradation
National Category
Microbiology
Research subject
Microbiology
Identifiers
urn:nbn:se:oru:diva-36171 (URN)10.1186/1471-2180-14-193 (DOI)000339837900001 ()
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: 2016-08-10Bibliographically approved
2. PKC, ERK/p38 MAP kinases and NF-κB targeted signalling plays a crucial role in expression and release of IL-1β and CXCL8 in Porphyromonas gingivalis infected monocytes
Open this publication in new window or tab >>PKC, ERK/p38 MAP kinases and NF-κB targeted signalling plays a crucial role in expression and release of IL-1β and CXCL8 in Porphyromonas gingivalis infected monocytes
Show others...
(English)Manuscript (preprint) (Other academic)
National Category
Other Basic Medicine
Research subject
Biomedicine
Identifiers
urn:nbn:se:oru:diva-49455 (URN)
Available from: 2016-03-22 Created: 2016-03-22 Last updated: 2016-03-22Bibliographically approved
3. Porphyromonas gingivalis induced release of reactive oxygen species and interleukin-1 beta and the effects of low density lipoproteins in monocytes and whole blood
Open this publication in new window or tab >>Porphyromonas gingivalis induced release of reactive oxygen species and interleukin-1 beta and the effects of low density lipoproteins in monocytes and whole blood
(English)Manuscript (preprint) (Other academic)
National Category
Other Basic Medicine
Research subject
Biomedicine
Identifiers
urn:nbn:se:oru:diva-49456 (URN)
Available from: 2016-03-22 Created: 2016-03-22 Last updated: 2016-03-22Bibliographically approved
4. The role of phagocytosis, oxidative burst and neutrophil extracellular traps in the interaction between neutrophils and the periodontal pathogen Porphyromonas gingivalis
Open this publication in new window or tab >>The role of phagocytosis, oxidative burst and neutrophil extracellular traps in the interaction between neutrophils and the periodontal pathogen Porphyromonas gingivalis
2015 (English)In: Molecular Oral Microbiology, ISSN 2041-1006, Vol. 30, no 5, 361-375 p.Article in journal (Refereed) Published
Abstract [en]

Neutrophils are regarded as the sentinel cells of innate immunity and are found in abundance within the gingival crevice. Discovery of neutrophil extracellular traps (NETs) within the gingival pockets prompted us to probe the nature of the interactions of neutrophils with the prominent periopathogen Porphyromonas gingivalis. Some of the noted virulence factors of this Gram-negative anaerobe are gingipains: arginine gingipains (RgpA/B) and lysine gingipain (Kgp). The aim of this study was to evaluate the role of gingipains in phagocytosis, formation of reactive oxygen species, NETs and CXCL8 modulation by using wild-type strains and isogenic gingipain mutants. Confocal imaging showed that gingipain mutants K1A (Kgp) and E8 (RgpA/B) induced extracellular traps in neutrophils, whereas ATCC33277 and W50 were phagocytosed. The viability of both ATCC33277 and W50 dwindled as the result of phagocytosis and could be salvaged by cytochalasin D, and the bacteria released high levels of lipopolysaccharide in the culture supernatant. Porphyromonas gingivalis induced reactive oxygen species and CXCL8 with the most prominent effect being that of the wild-type strain ATCC33277, whereas the other wild-type strain W50 was less effective. Quantitative real-time polymerase chain reaction revealed a significant CXCL8 expression by E8. All the tested P.gingivalis strains increased cytosolic free calcium. In conclusion, phagocytosis is the primary neutrophil response to P.gingivalis, although NETs could play an accessory role in infection control. Although gingipains do not seem to directly regulate phagocytosis, NETs or oxidative burst in neutrophils, their proteolytic properties could modulate the subsequent outcomes such as nutrition acquisition and survival by the bacteria.

Keyword
neutrophils, neutrophil extracellular traps, periodontitis, phagocytosis, reactive oxygen species
National Category
Dentistry
Identifiers
urn:nbn:se:oru:diva-46034 (URN)10.1111/omi.12099 (DOI)000361007200003 ()25869817 (PubMedID)2-s2.0-84941023684 (Scopus ID)
Funder
Swedish Heart Lung FoundationSwedish Research Council
Note

Funding Agency:

Foundation of Olle Engkvist

Available from: 2015-10-07 Created: 2015-10-07 Last updated: 2016-03-22Bibliographically approved

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