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 bacteria in vitro based 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 on in vitro assays 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-based in vitro assay 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.

Keywords: CD markers; biofilm; commensal bacteria; immunology; microbiology; neutrophil; pathogens.

FIG 1

Upregulation of neutrophil CD markers is induced by isolation procedures. Whole blood or neutrophils isolated by conventional gradient centrifugation procedures were fixed and analyzed by multicolor flow cytometry. (a) The mean MFI ± SEM is shown for each marker. Significant differences were determined by paired Student's t test (n = 5). *, P ≤ 0.05. (b) The isolation procedures induced a greater variation within the population for each CD marker and also in the forward scatter and side scatter than it did with whole blood, confirming procedure-induced perturbations in cell activation.

FIG 2

Neutrophil CD markers are upregulated differentially by monospecies biofilms of commensal and pathogenic bacteria. Human blood was incubated with commensal and pathogenic bacterial biofilms for 1 h at 37°C. The fold increase of surface CD marker expressions of gated neutrophil populations relative to that for unstimulated controls was determined. The mean fold increase ± SEM is shown for each CD marker (n = 9). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. Abbreviations: unstim, unstimulated; So, S. oralis; Ssang, S. sanguinis; Ssal, S. salivarius; Sm, S. mutans; Aa, A. actinomycetemcomitans; Pg, P. gingivalis.

FIG 3

Mixed biofilms of S. oralis and S. salivarius result in a lower level of neutrophil activation than the monospecies biofilm of S. oralis, whereas the biofilm mixture of S. oralis and P. gingivalis results in a higher level of neutrophil activation than the monospecies biofilm of S. oralis. Each sample was incubated with 100 μl human whole blood for 1 h. Flow cytometry was performed, and the mean fold increase ± SEM is shown for each CD marker (n = 3). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (a) The activation of neutrophils through CD16 and CD64 is higher by S. oralis than by S. salivarius. However, in mixed biofilms, this expression is reduced. (b) There is greater neutrophil activation through CD63, CD66, and CD18 by a mixed biofilm of S. oralis and P. gingivalis than by the anaerobically grown monospecies S. oralis. Abbreviations: So, S. oralis; Ssal, S. salivarius; Pg, P. gingivalis.

FIG 4

Only the S. oralis biofilm induces neutrophil ROS production. Isolated human blood neutrophils were incubated with the biofilms for 1 h at 37°C. The fold increase in the level of intracellular ROS production relative to that for unstimulated controls was determined (n = 4). *, P ≤ 0.05. Abbreviations: unstim, unstimulated; So, S. oralis; Ssang, S. sanguinis; Ssal, S. salivarius; Sm, S. mutans; Aa, A. actinomycetemcomitans; Pg, P. gingivalis.

FIG 5

S. oralis biofilm induces nuclear translocation/activation of Nrf2. Isolated human blood neutrophils were incubated with biofilms for 1 h at 37°C. The densitometry of nuclear Nrf2 was determined by Western blotting. Cytoplasmic Nrf2 was also detected, and there were no significant differences between the samples. Actin was used as a protein control (n = 4). **, P ≤ 0.01. Abbreviations: unstim, unstimulated; So, S. oralis; Ssal or Sal, S. salivarius; Aa, A. actinomycetemcomitans.

FIG 6

S. oralis and S. sanguinis induce expression of the NETosis marker H3Cit. Only S. sanguinis results in a significant release of MPO. Human blood neutrophils were incubated with biofilms for 1 h at 37°C. Using flow cytometry, the fold increase in the levels of surface H3Cit (a) and MPO (b) on gated neutrophil populations relative to that for unstimulated controls was determined (n = 6). *, P ≤ 0.05; ***, P ≤ 0.001. Abbreviations: unstim, unstimulated; So, S. oralis; Ssang, S. sanguinis; Ssal, S. salivarius; Sm, S. mutans; Aa, A. actinomycetemcomitans; Pg, P. gingivalis.

FIG 7

S. oralis and S. sanguinis induce neutrophil phagocytosis. Biofilms of monospecies were labeled with pHrodo Red for 1 h. Neutrophils were then incubated with labeled bacteria for 1 h at 37°C. The mean fluorescent intensity of pHrodo Red was measured using flow cytometry. S. oralis and S. sanguinis resulted in more neutrophil phagocytosis (n = 3). *, P ≤ 0.05; **, P ≤ 0.01. Abbreviations: unstim, unstimulated; So, S. oralis; Ssang, S. sanguinis; Ssal, S. salivarius; Sm, S. mutans; Aa, A. actinomycetemcomitans; Pg, P. gingivalis.

FIG 8

P. gingivalis lacking gingipains activates neutrophils through CD14, whereas P. gingivalis does not. This suggests that gingipains play an important role in cleaving CD14. Human blood was incubated with P. gingivalis and P. gingivalis gingipain mutant (KDP131 and KDP132) biofilms for 1 h at 37°C. The fold increase in the level of surface CD14 expressions of gated neutrophil populations relative to that for unstimulated controls was determined. The mean fold increase ± SEM is shown for CD14 (n = 4). ***, P ≤ 0.001. Abbreviations: unstim, unstimulated; Pg, P. gingivalis; KDP131, P. gingivalis Arg-gingipain A mutant; KDP132, P. gingivalis Arg-gingipain B mutant.