Reactive oxygen species (ROS) collectively refer to a large group of highly reactive derivatives of oxygen generated as a consequence of metabolic processes or during host stress response. Although associated with oxidative damage, when produced at low regulated levels, oxidants are essential for redox modulation of cellular pathways, immune effector function, cell signaling and anti-microbial responses.The majority of ROS generated in a cell is by the activation of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidases or NOX enzyme complexes. NOX enzyme family members NOX1, NOX2, NOX3, NOX4, NOX5 and the dual oxidase (DUOX) enzymes DUOX1, and DUOX2 are membrane associated hetero-oligomeric complexes that are dedicated to generation of ROS by using oxygen as its substrate. NOX enzymes are expressed in a cell specific or tissue specific manner and are rapidly activated in response to external stressors to generate large amounts of ROS. NOX2 or gp91 phox is the main catalytic subunit of the leukocyte NADPH oxidase complex. It is highly expressed in phagocytes (neutrophils, monocytes, macrophages, dendritic cells) and at much lower levels in lymphocytes, endothelial cells and colonic epithelial cells. 1,2 NOX2 derived oxidants modulate multiple cellular processes independent of their anti-microbial function. For instance, NADPH oxidase activation is essential for antigen presentation in B cells 3 , T cell receptor stimulation 4

Filifactor alocis is a recently recognized periodontal pathogen; however, little is known regarding its interactions with the immune system. As the first-responder phagocytic cells, neutrophils are recruited in large numbers to the periodontal pocket, where they play a crucial role in the innate defense of the periodontium. Thus, in order to colonize, successful periodontal pathogens must devise means to interfere with neutrophil chemotaxis and activation. In this study, we assessed major neutrophil functions, including degranulation and cell migration, associated with the p38 mitogen-activated protein kinase (MAPK) signaling pathway upon challenge with F. alocis. Under conditions lacking a chemotactic gradient, F. alocischallenged neutrophils had increased migration compared to uninfected cells, indicating that F. alocis increases chemokinesis in human neutrophils. In addition, neutrophil chemotaxis induced by interleukin-8 was significantly enhanced when cells were challenged with F. alocis, compared to noninfected cells. Similar to live bacteria, heat-killed F. alocis induced both random and directed migration of human neutrophils. The interaction of F. alocis with Toll-like receptor 2 induced granule exocytosis along with a transient ERK1/2 and sustained p38 MAPK activation. Moreover, F. alocis-induced secretory vesicle and specific granule exocytosis were p38 MAPK dependent. Blocking neutrophil degranulation with TAT-SNAP23 fusion protein significantly reduced the chemotactic and random migration induced by F. alocis. Therefore, we propose that induction of random migration by F. alocis will prolong neutrophil traffic time in the gingival tissue, and subsequent degranulation will contribute to tissue damage.

Filifactor alocis is a newly appreciated pathogen in periodontal diseases. Neutrophils are the predominant innate immune cell in the gingival crevice. In this study, we examined modulation of human neutrophil antimicrobial functions by F. alocis. Both non-opsonised and serum-opsonised F. alocis were engulfed by neutrophils but were not efficiently eliminated. Challenge of neutrophils with either non-opsonised or serum-opsonised F. alocis induced a minimal intracellular as well as extracellular respiratory burst response compared to opsonised Staphylococcus aureus and fMLF, respectively. However, pretreatment or simultaneous challenge of neutrophils with F. alocis did not affect the subsequent oxidative response to a particulate stimulus, suggesting that the inability to trigger the respiratory response was only localised to F. alocis phagosomes. In addition, although neutrophils engulfed live or heat-killed F. alocis with the same efficiency, heat-killed F. alocis elicited a higher intracellular respiratory burst response compared to viable organisms, along with decreased surface expression of CD35, a marker of secretory vesicles. F. alocis phagosomes remained immature by delayed and reduced recruitment of specific and azurophil granules, respectively. These results suggest that F. alocis withstands neutrophil antimicrobial responses by preventing intracellular ROS production, along with specific and azurophil granule recruitment to the bacterial phagosome.

Neutrophils operate at the site of injury or inflammation in the periodontal pocket to ensure periodontal health and clearance of bacterial pathogens. Filifactor alocis is recently identified as a potential periodontal pathogen, and in this study, we assessed the formation of neutrophil extracellular traps (NETs), in response to the presence of the organism . NET formation by human neutrophils was not induced when challenged with F. alocis, independent of opsonization, viability, time, or bacterial dose. F. alocis also failed to induce NETs from TNF-α-primed neutrophils and did not induce the release of extracellular neutrophil elastase. However, significant NET induction was observed when neutrophils were challenged with Streptococcus gordonii or Peptoanaerobacter stomatis, In addition, co-infection studies revealed that the presence of F. alocis with S. gordonii or P. stomatis does not enhance or reduce NETs. Additionally, F. alocis failed to impact pre-formed NETs induced by either S. gordonii or P. stomatis. Pretreatment with F. alocis prior to stimulation with phorbol 12-myristate 13-acetate (PMA), S. gordonii, or P. stomatis revealed that the bacterium is capable of reducing only PMA but not S. gordonii or P. stomatis NET formation. These results indicate that F. alocis manipulates neutrophils, inhibiting the triggering of NET induction.

No abstract