Date on Senior Honors Thesis

5-2023

Document Type

Senior Honors Thesis

Degree Name

B.S.

Department

Biology

Author's Keywords

Periodontitis; Phagocytosis; Priming; Respiratory Burst; Reactive Oxygen Species; Filifactor alocis

Abstract

Periodontitis is an irreversible, chronic inflammatory, infectious disease of the oral cavity that affects approximately half of all adults 30 years or older in the USA. The oral cavity is under high immune surveillance because of its constant exposure to microbes in the environment. The primary immune cell responsible for this surveillance is the neutrophil. Pathogens associated with periodontitis possess virulence factors and have evolved strategies to evade neutrophil antimicrobial responses to survive. One such pathogen is Filifactor alocis, whose presence is associated with the progression of periodontitis. F. alocis manipulates several neutrophil antimicrobial functional responses to avoid killing, an evasion strategy that might perpetuate the unresolved destructive inflammation that is the hallmark of periodontitis.

An essential aspect of the host immune response against a threat is the release of cytokines and chemokines, a wide array of small endogenous inflammatory mediators that communicate between cells and evoke different responses from the cells, contributing to the initiation and resolution of the inflammatory response. Specific inflammatory cytokines and chemokines are associated with inflammation of the periodontium, a specialized tissue that surrounds and supports the tooth structure. These inflammatory mediators can prime or preactivate quiescent neutrophils. A primed neutrophil will display an enhanced response to a secondary stimulus compared to a quiescent or unprimed cell. Besides endogenous priming agents, exogenous agents such as the lipopolysaccharide (LPS) from bacteria can prime neutrophils via pattern recognition toll-like receptors (TLRs).

This project aims to define if F. alocis modulates the antimicrobial responses of primed neutrophils to promote its survival and the progression of periodontitis. In this project, two endogenous priming agents, tumor necrosis (TNF)α and interleukin (IL)-8, and LPS as an exogenous priming agent, were tested. LPS stimulates TLR4, but if the preparation contains additional bacterial components like lipoproteins, it also stimulates TLR2. To assess the response of LPS-primed neutrophils toward F. alcois, three sources of LPS were used as priming agents for this project: Escherichia coli LPS-EK (containing lipoproteins), E. coli LPS-EK Ultrapure (no lipoproteins), and LPS from the keystone periodontal pathogen, Porphyromonas gingivalis. The data showed that F. alocis internalization was significantly increased in TNFa and both forms of tested E. coli LPS primed neutrophils compared to unprimed cells, both by flow cytometry and confocal microscopy.

In contrast, phagocytosis of F. alocis was similar between unprimed or IL-8 primed neutrophils. Interestingly, the respiratory burst response elicited by F. alocis was similar between unprimed and neutrophils primed by TNFα, IL-8, or E. coli LPS. However, P. gingivalis LPS was unable to prime neutrophils. These findings suggest that although TNFα and LPS primed neutrophils have enhanced phagocytosis towards F. alocis, it does not result in enhanced respiratory burst response. These results suggest that F. alocis can disable one of the phagocyte's critical antimicrobial functions, the respiratory burst response, to evade killing. Another exciting finding suggests that F. alocis might modulate the IL-8 signaling pathway to prevent internalization. Preliminary data also suggest that priming neutrophils with TNFα did not enhance the killing of F. alocis. In summary, the observed modulations of primed neutrophil antimicrobial responses by F. alocis may represent one of the organisms' evasion strategies to survive neutrophils in the hostile inflamed periodontal tissue.

Lay Summary

Periodontitis is an inflammatory disease of the gum tissue caused by certain oral pathogens' colonization of the gums. This disease is widespread, affecting hundreds of millions of people worldwide, and is associated with other systemic diseases such as diabetes and cardiovascular disease. Neutrophils are white blood cells of the immune system that patrol the mouth tissues in high numbers. Usually, neutrophils can effectively kill pathogens by employing several antimicrobial responses. However, pathogens associated with periodontitis, including the pathogen Filifactor alocis, can hijack these immune responses and harm the host by leading to tissue destruction and tooth loss. In the body, neutrophils can communicate with cells of the gingival tissue and are notified of infection by small proteins called cytokines. Exposure to cytokines causes an enhancement of their antimicrobial responses, a process known as "priming." Priming can also be induced by molecules derived from pathogens themselves. Previous characterizations of neutrophil interactions with F. alocis used unprimed cells. This project aimed to characterize the interactions of neutrophils primed with different priming agents with F. alocis to reflect more on what occurs in the body during infection. I found that F. alocis can modulate certain antimicrobial responses of primed neutrophils, which may contribute to its survival and the progression of periodontitis.

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