Date on Master's Thesis/Doctoral Dissertation

5-2020

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Microbiology and Immunology

Degree Program

Microbiology and Immunology, PhD

Committee Chair

Uriarte, Silvia

Committee Co-Chair (if applicable)

Lawrenz, Matthew

Committee Member

Graham, James E.

Committee Member

Alard, Pascale

Committee Member

Potempa, Jan

Author's Keywords

Neutrophil; periodontitis; filifactor alocis; emerging microbes; innate immunity

Abstract

Periodontitis is an irreversible, chronic inflammatory disease where pathogenic microbial communities accumulate in the gingival crevice. Advances in culture-independent techniques have facilitated the identification of new bacterial species in periodontal lesions, such as the Gram-positive anaerobe, Filifactor alocis. Neutrophils are a major component of the innate host response, and the outcome of their interaction with F. alocis may be a determinant of oral health status. While neutrophil functions typically protect the host against periodontal disease, oral pathogens have adapted to evade or disarm neutrophil microbicidal functions while promoting mechanisms that drive inflammation, which also provides a source of nutrients for growth. This study has two main goals: to determine how F. alocis interferes with microbicidal mechanisms to survive in neutrophils and to examine how F. alocis contributes to the chronicity of periodontitis by promoting inflammation. This dissertation characterizes human neutrophils global gene expression during infection with F. alocis (Chapter 2). Challenge of human neutrophils with F. alocis resulted in the differential expression of genes involved in multiple neutrophil effector functions such as chemotaxis, cytokine and chemokine signaling pathways, and apoptosis. F. alocis challenge also affected the expression of components from the TNFα and MAPK kinase signaling pathways. This resulted in transient but dampened p38 MAPK activation by secondary stimuli TNFα. Functionally, the F. alocis-mediated inhibition of p38 activation by TNFα resulted in decreased cytokine production but had no effect on priming of the respiratory burst response, or the delay of apoptosis by TNFα. Since this modulatory effect was characteristic of viable F. alocis only, this is one of F. alocis’ mechanisms to control neutrophils and their functional responses. I also examined neutrophil functional mechanisms in bone marrow neutrophils from TLR2-/- mice (Chapter 3). I found that compared to WT, TLR2-/- neutrophils were more efficient at killing F. alocis, but the increase in killing capacity was not due to a difference in phagocytosis or production of ROS. Instead, it was due to an increase in fusion of myeloperoxidase granules with the F. alocis phagosome. Meanwhile, F. alocis promotes inflammation by inducing the release of pro-inflammatory cytokines through TLR2 signaling. The TLR2 adaptor protein, MyD88, was also critical for cytokine production, but the events downstream of MyD88 follow non-canonical signaling. Apoptotic pathways were also showed significant changes in mRNA levels when neutrophils are challenged with F. alocis. Neutrophils had prolonged lifespans and higher functional capacity than unchallenged cells (Chapter 4). Despite their viability, F. alocis-challenged cells were significantly efferocytosed by macrophages, which developed a pro-inflammatory profile (Chapter 5). Collectively, this data confirms that F. alocis interferes with TLR2 signaling to promote its survival within neutrophils and promotes chronic inflammation by extending neutrophil lifespan and delaying the resolution of inflammation.

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