NOVEL ASSAY TO CHARACTERIZE NEUTROPHIL RESPONSES TO ORAL BIOFILMS.

Neutrophils, the most numerous leukocytes, play an important role in maintaining oral health through interactions with oral microbial biofilms. Both neutrophil hyperactivity and the bacterial subversion of neutrophil responses can cause inflammation-mediated tissue damage as seen in periodontal disease. We describe here an assay that assesses neutrophil activation responses to mono-species biofilms bacteria in vitro based on surface expression of cluster of differentiation (CD) markers associated with various neutrophil functions. Most of what we know about neutrophil responses to bacteria is based on in vitro assays that use planktonic bacteria and isolated/pre-activated neutrophils which make interpreting the neutrophil responses to bacteria a challenge to interpret. An understanding of how neutrophils differentially interact with and respond to commensal and pathogenic oral bacteria is necessary in order to further understand the neutrophil's role in maintaining oral health and the pathogenesis of periodontal disease. In this study, a flow cytometry-based in vitro assay was developed to characterize neutrophil activation states based on CD marker expressions in response to oral mono-species bacterial biofilms. Using this approach, changes in the CD marker expressions were assayed in response to specific prominent oral commensal and pathogenic bacteria. Several functional assays, including phagocytosis, production of ROS, activation of the transcription factor Nrf2, NET formation and myeloperoxidase release, were also performed to correlate neutrophil function with CD marker expression. Our results demonstrate that neutrophils display bacterial species-specific responses. This assay can be used to characterize how specific biofilms alter specific neutrophil pathways associated with their activation.