Date on Senior Honors Thesis

5-2020

Document Type

Senior Honors Thesis

Degree Name

B.S.

Department

Biology

Degree Program

College of Arts and Sciences

Author's Keywords

periodontitis; MAPK; degranulation; phagocytosis

Abstract

Periodontitis is a multifactorial chronic inflammatory infectious disease that affects around 50% of the adult population in the USA. Neutrophils, which are the primary immune cells recruited to restore health at sites of infection, are profuse in the gingival space during oral infections. In a community-wide effort, pathogenic oral bacteria hijack neutrophil effector functions to evade killing, resulting in an uncontrollable inflammatory loop that leads into inflammation-mediated tissue injury. One of these harmful emerging oral pathogens, Filifactor alocis, has been shown to evade neutrophil killing and contribute to disease progression. The purpose of this study is to gain a better characterization of the strategies deployed by F. alocis to manipulate neutrophil functional responses to support an unresolving inflammation. It has been previously shown that F. alocis is efficiently internalized by human neutrophils and 60% of the internalized organism remains viable in the phagosome up to 4 h post-infection. As a measure of productive bacterial infection, the expression of F. alocis proteins in infected neutrophils was determined at different time points of infection. A high abundance of F. alocis proteins was detected at 1 and 3 h post infection with a decrease by 18 h. The presence of bacterial proteins in infected neutrophils after 18 h post infection led us to explore the functional capacity of those infected neutrophils upon additional stimulation. Interestingly, F. alocis-infected neutrophils were able to phagocytize another bacterial challenge more effectively than uninfected neutrophils.

Microbial pathogens can impact neutrophil functional responses both by direct interaction and by the release of bacterial-derived molecule(s). Preliminary results from the Uriarte laboratory showed that pre-treatment of human neutrophils with F. alocis-derived molecule(s), in filtered culture supernatants, inhibited both N-formyl peptide (fMLF) and Toll-like receptor (TLR)-induced granule exocytosis. To reach a better understanding of the ability of F. alocis-derived molecule(s) to manipulate granule exocytosis, their effect on p38 and ERK 1/2 MAPK signaling pathways associated with neutrophil functional responses was examined. Phosphorylation of p38 MAPK and ERK 1/2 was determined by immunoblotting. Pre-treatment of neutrophils with F. alocis-derived molecule(s) significantly reduced fMLF-induced ERK 1/2 phosphorylation but not p38 MAPK phosphorylation. Similarly, pre-exposure of neutrophils to the F. alocis-derived molecule(s) inhibited ERK 1/2, but not p38 MAPK, activation induced by opsonized F. alocis challenge. Furthermore, the inhibitory effect on ERK phosphorylation, provoked by F. alocis culture supernatant, was reversible. Moreover, treatment of the bacterial supernatant with DNAse, RNAse, Proteinase K or 2 h at 95 °C, had no impact on the inhibitory activity. Together, this data shows that the bacterial compound(s) released by F. alocis, which is neither a protein nor a nucleic acid, proactively inhibited ERK 1/2 activation. This inhibition may compromise several key neutrophil functions beyond granule exocytosis, putting the immune cell at a disadvantage in the fight against the dysbiotic microbial community present in periodontitis.

Lay Summary

Periodontitis is a severe gum disease that afflicts hundreds of millions of people worldwide. The disease is characterized by an alteration of the microbial community due to colonization of keystone pathogens that promotes exacerbated inflammation through a disruption in the host-microbe homeostasis. The resulting chronic inflammation leads to gingival tissue deterioration and tooth loss. Neutrophils, which are white blood cells that are usually efficient at fighting off infections, are instead manipulated by these pathogenic bacteria into an uncontrollable inflammatory state. Essentially, the microorganisms present in periodontitis force the host to harm itself by manipulating its own defense system. One of the pathogenic bacteria involved in this process is Filifactor alocis. This bacterium has been shown to manipulate certain neutrophil functional responses to promote bacterial survival, but the specific mechanisms induced by F. alocis remains to be determined. In this study, I further characterized F. alocis’ ability to survive the neutrophil assault and found that the bacteria, as well as their secreted molecules, are able to redirect certain neutrophil killing tactics.

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