Hulzebos, J. (2015) Rheumatoid Arthritis & Periodontitis: The role of neutrophils and Porphyromonas gingivalis. Master's Thesis / Essay, Biology.
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Abstract
Introduction: Rheumatoid Arthritis (RA) is a chronic systemic autoimmune disease characterized by synovial inflammation resulting in damage to articular cartilage and the underlying bone. The formation of anti-citrullinated proteins (ACPAs) is a hallmark of RA. ACPAs target proteins which have been citrullinated by Peptidyl-Arginine Deiminase (PAD), such as PAD4 in neutrophils. Periodontitis (PD) is caused by periodontal pathogens, such as Porphyromonas gingivalis (PG). PG is the only known bacterium to possess its own PAD enzyme; PPAD. Furthermore, neutrophils attracted to the inflamed periodontium can perform a process named Neutrophil Extracellular Trap formation; (NET)osis. During NETosis, neutrophils eject intracellular compartments, which are citrullinated by their own PAD. It is hypothesized that PPAD in the periodontium is able to hypercitrullinate these excreted extracellular components. Due to this hypercitrullination, susceptible individuals initiate a loss of tolerance to citrullinated proteins and begin to generate ACPAs. This ACPA formation can result in the induction of RA. The aim of this study was to assess the role of neutrophils when they come into contact with PG and PPAD in terms of inducing NETosis and citrullinating NET-constituents. Methods: NETosis of stimulated or unstimulated healthy donor neutrophils was quantified using a SYTOX assay which measures extracellular DNA. PAD4 protein production has been visualized using the Western Blot method. Immunofluorescence Microscopy has been performed on coverslips to visualize NETosis and citrullination of histone H3. After optimization of these methods, co-cultures of neutrophils with several PG strains (PG-ATCC, PG-W83) and their PPAD mutants (ΔPG-ATCC, ΔPG-W83) have been performed. An in-house made PAD-assay was used to measure PAD4 activity. During these experiments, the effects of Cl-Amidine (CLA), a pan-PAD inhibitor, have been assessed. Results: Neutrophil PAD4 protein production is upregulated after stimulation. CLA inhibits this effect and is also able to inhibit direct PAD-enzyme activity, but not NETosis itself. When PG-ATCC or ΔPG-ATCC is co-cultured with neutrophils, it gets phagocytosed. However, when neutrophils were pre-stimulated with PMA to form NETs and subsequently addition of PG-ATCC or ΔPG-ATCC occurred, PG-ATCC – but not ΔPG-ATCC – would hypercitrullinate histone H3 within NETs. PG-W83 and ΔPG-W83 induced NETosis – and possibly hypercitrullination – when co-cultured with neutrophils. Discussion & Conclusion: A clear difference in interaction between different PG strains and neutrophils is observed in vitro. PG-ATCC and its mutant were both phagocytosed by neutrophils, whereas both PG-W83 strains induced NETosis and supposedly hypercitrullination of histone H3. After pre-stimulation of neutrophils, even clearer hypercitrullination of histone H3 was observed. However, a larger diversity of PG strains should be used in these experiments to determine more differences in virulence. Moreover, if NETs – and preferably hypercitrullination – could be shown in periodontal tissue of PD/RA patients, the hypothesis would become more plausible. Even though no clear conclusion could be drawn due to inconsistency of the data, it can be stated that PG is probably able to citrullinate human proteins and is highly likely to be capable of hypercitrullinating NETs and could therefore very well be the causative pathogen of the pathogenesis of RA.
| Item Type: | Thesis (Master's Thesis / Essay) |
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| Degree programme: | Biology |
| Thesis type: | Master's Thesis / Essay |
| Language: | English |
| Date Deposited: | 15 Feb 2018 08:08 |
| Last Modified: | 15 Feb 2018 08:08 |
| URI: | https://fse.studenttheses.ub.rug.nl/id/eprint/13345 |
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