Date on Senior Honors Thesis
Senior Honors Thesis
College of Arts and Sciences
neutrophil; periodontal disease; Filifactor alocis; respiratory burst; reactive oxygen species; phagosome maturation
Almost 50% of adult Americans suffer from periodontitis which is a bacterially induced inflammation of the tissue that surround and support the tooth. The accumulation of neutrophils, a critical cell component of the innate immune system, in the gingival crevice contributes to tissue damage. Filifactor alocis is a newly appreciated pathogen present in oral biofilms at periodontal disease sites. Studying the interactions between neutrophils and F. alocis will provide valuable information for delineating the role of this bacterium in periodontal disease and enhance our understanding of bacterial strategies to evade leukocytes’ antimicrobial mechanisms. The hypothesis that F. alocis modulates human neutrophil antimicrobial functions was tested. One of several antimicrobial mechanisms employed by the neutrophil is the respiratory burst response with production of reactive oxygen species within bacteria-containing phagosomes. Previous studies in our lab showed that human neutrophils challenged for 30 min with either non-opsonized or serum opsonized F. alocis at a multiplicity of infection (MOI) of 10 failed to induce a robust respiratory burst response. In addition, serum opsonized F. alocis failed to induce the respiratory burst response even after 60 and 120 min post neutrophil challenge. Interestingly, neutrophils challenged for 30 min with the serum opsonized heat-killed organism at an MOI of 10 elicited a 2.5 fold higher intracellular respiratory burst response compared to viable F. alocis. To determine if the failure to induce the respiratory burst was mediated by secreted bacterial products, neutrophils were exposed to the F. alocis culture supernatant and the oxidative burst response was determined. The culture supernatant by itself did not induce a respiratory burst response; however, it primed the S. aureus-stimulated response. The recruitment of specific granules to the phagosome assists in the accumulation of NADPH oxidase complexes because they contain approximately 60% of the membrane-bound subunits; 4 therefore, a possible strategy to avoid/delay killing would be to hinder specific granule recruitment post-phagocytosis. After 30 min of bacteria challenge, only 35% of viable F. alociscontaining phagosomes were enriched for lactoferrin positive granules, compared to 66-70% of heat-killed F. alocis or S. aureus-containing phagosomes. Diverting granule exocytosis to the plasma membrane instead of the bacterial phagosome could prevent bacterial killing and contribute to the tissue damage characteristic of periodontitis. Serum opsonized F. alocis induced significant secretory vesicle and specific granule exocytosis; whereas the heat-killed bacteria significantly reduced secretory vesicle exocytosis but stimulated specific granule exocytosis to the same extent as the viable bacteria. F. alocis also induced gelatinase granule exocytosis, but did not cause azurophil granule release. These data demonstrate previously unexplored aspects of the new oral pathogen F. alocis and how this species modulates neutrophil function. By examining the differences between viable and heat-killed F. alocis and the effector molecules released by the pathogen during growth, I will begin to characterize F. alocis’ role in the pathogenesis of periodontitis.
Edmisson, Jacob S., "Filifactor alocis, a newly appreciated oral pathogen, fails to induce the respiratory burst response of human neutrophils." (2016). College of Arts & Sciences Senior Honors Theses. Paper 109.
Periodontal disease affects numerous people worldwide, and it is correlated with many other inflammatory diseases. This disease is caused by an imbalance between the oral bacterial community and the host immune system. Neutrophils are white blood cells that regularly enter the gingival tissues to keep the bacteria populations under control. In periodontal disease, the neutrophils add to the tissue damage without resolving the infection. One of the species, Filifactor alocis has recently been recognized. To determine its role in the disease, F. alocis is studied with human neutrophils. In this study, I found the F. alocis inhibits neutrophil killing mechanisms while simultaneously increasing release of certain tissue damaging agents.