Novel Assay To Characterize Neutrophil Responses to Oral Biofilms

Abstract

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 like that seen in periodontal disease. We describe here an assay that assesses neutrophil activation responses to monospecies biofilm bacteriain vitrobased on the 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 onin vitroassays that use planktonic bacteria and isolated/preactivated neutrophils, which makes interpretation of the neutrophil responses to bacteria a challenge. 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-basedin vitroassay was developed to characterize neutrophil activation states based on CD marker expressions in response to oral monospecies bacterial biofilms. Using this approach, changes in CD marker expressions in response to specific prominent oral commensal and pathogenic bacteria were assayed. Several functional assays, including assays for phagocytosis, production of reactive oxygen species, activation of the transcription factor Nrf2, neutrophil extracellular trap 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.

Bioengineered Platforms for Chronic Wound Infection Studies: How Can We Make Them More Human-Relevant?

“…Therefore, experimental setups require freshly isolated neutrophils from human subjects (Kuhns et al, 2015). In spite of these technical limitations, interactions between neutrophils and biofilms at different sites, such as on dental and ocular surfaces, have been well-explored (Hirschfeld, 2014;Oveisi et al, 2019;Papayannopoulos, 2019;Thanabalasuriar et al, 2019), which could serve as a template for developing immune-infection interface studies for chronic wound biofilms.…”

“…A relatively simple way to do this would be to incorporate immune signaling factors, such as cytokines and antimicrobial peptides, at or near the biofilm, in the nutrient media or across a semi-permeable barrier (Gopalakrishnan et al, 2015). A more complex approach is to introduce freshly-isolated neutrophils across a semi-permeable membrane or within the biofilm matrix (Oveisi et al, 2019).…”

Oral Neutrophils: Underestimated Players in Oral Cancer

“…Several bacterial species are reported to modulate neutrophil functions in vitro. The effect of different oral microorganisms, both commensal ( S. oralis , S. sanguinis , S. salivarius ) and pathogenic ( S. mutans , A. actinomycetemcomitans , P. gingivalis ), on the activation status of cPMN was described by Oveisi et al ( 76 ). While CD63 and CD11b/CD18 markers were upregulated after exposure to both commensal and pathogenic bacteria, commensal microorganisms in biofilms induced the selective increase of CD66, CD64, CD55, while pathogenic bacteria induced the expression of lipopolysaccharide receptor CD14.…”

“…While CD63 and CD11b/CD18 markers were upregulated after exposure to both commensal and pathogenic bacteria, commensal microorganisms in biofilms induced the selective increase of CD66, CD64, CD55, while pathogenic bacteria induced the expression of lipopolysaccharide receptor CD14. Moreover, only commensal bacteria in biofilms stimulated degranulation, phagocytosis, ROS production and NET formation, while pathogenic bacteria showed no effect ( 76 ). Coexistence of F. alocis with other pathogens induced the secretion of proinflammatory cytokines from epithelial cells and promoted apoptosis of neutrophils ( 77 ).…”

The Neutrophil: Constant Defender and First Responder

“…These PMNs restrict biofilm growth through the release of toxic granule contents and ROS production, but cannot phagocytose the large biofilm structure. Although it would be biologically consistent for oral PMNs to preferentially kill pathogenic microbes during early colonization of the otherwise healthy oral cavity, there is evidence that some pathogens can, in fact, evade PMN mediated destruction within the oral cavity ( 2 , 92 ), which likely accounts for their pathogenic properties.…”