Putative Periodontal Pathogens, Filifactor alocis and Peptoanaerobacter stomatis, Induce Differential Cytokine and Chemokine Production by Human Neutrophils

Abstract

Periodontitis is a highly prevalent infectious disease that affects ~ 50% of the adults in the USA alone. Two Gram-positive anaerobic oral bacteria, Filifactor alocis and Peptoanaerobacter stomatis, have emerged as important periodontal pathogens. Neutrophils are a major component of the innate host response in the gingival tissue, and the contribution of neutrophil-derived cytokines and chemokines plays a central role in disease progression. The pattern of cytokines and chemokines released by human neutrophils upon stimulation with newly appreciated periodontal bacteria compared to the keystone oral pathogen Porphyromonas gingivalis was investigated. Our results showed that both F. alocis and P. stomatis triggered TLR2/6 activation. F. alocis induced significant changes in gene expression of cytokines and chemokines in human neutrophils compared to unstimulated cells. However, except for IL-1ra, neutrophils released lower levels of cytokines and chemokines in response to F. alocis compared to P. stomatis. Furthermore, bacteria-free conditioned supernatant collected from neutrophils challenged with P. stomatis, but not from P. gingivalis or F. alocis, was chemotactic towards both neutrophils and monocytes. Elucidating stimuli-specific modulation of human neutrophil effector functions in the context of dysbiotic microbial community constituents provides valuable information for understanding the pathogenesis of periodontal diseases.

Whole Transcriptome Analysis Reveals That Filifactor alocis Modulates TNFα-Stimulated MAPK Activation in Human Neutrophils

“…Together, this data shows that F. alocis challenge induces temporal changes in neutrophil functional mechanisms like cytokine production, chemotaxis, vesicular trafficking, and degranulation, as well as neutrophil biochemical mechanisms like the regulation of signaling pathways and metabolism. This coincides with previous data from our laboratory that shows that F. alocis affects neutrophil cytokine production, chemotaxis, vesicle trafficking, and degranulation functions (21,22).…”

“…Using Metacore, we divided the significant pathways into the top 20 upregulated ( Figure 3A) and downregulated (Figure 3B) plots. The list of upregulated pathways supports published data that shows initial contact with F. alocis results in the early transcription and production of cytokines (22). At 1 h, cytokine-related pathways such as cytokine-cytokine receptor interaction, TNF signaling pathway, and chemokine signaling pathway were also the most prominent pathways by KEGG analysis ( Table 3).…”

“…Two major bacterial recognition receptor signaling pathways, NOD-like receptor and Toll-like receptor signaling, were identified in our data set. These receptor pathways align with published data on F. alocis-induced cytokine production, where NOD1 is activated during challenge with heat-killed F. alocis to produce IL-6 in monocytes (50), and TLR2/6 activation of neutrophils leads to the production and release of cytokines and chemokines (22). Using Metacore, we divided the significant pathways into the top 20 upregulated ( Figure 3A) and downregulated (Figure 3B) plots.…”

“…We recently demonstrated that F. alocis survives within human neutrophils by inducing minimal ROS production and blocking granule recruitment to the bacteria-containing phagosome (21). Despite F. alocis causing significant changes in the mRNA expression of different neutrophil-derived cytokines and chemokines, lower levels of these inflammatory mediators are released when compared to the response elicited by other oral pathogens (22). Therefore, a systems biology-level approach was used to define global changes in human neutrophil transcriptome modulated by F. alocis.…”

“…Previous work from our laboratory has shown that despite efficient phagocytosis by neutrophils, F. alocis survives within neutrophils by inducing minimal production of intracellular ROS and curtailing the fusion of antimicrobial granules with its phagosome (20,21). However, in comparison to the keystone oral pathogen, P. gingivalis, and another emerging oral pathogen, Peptoanaerobacter stomatis, challenge with F. alocis resulted in a mild release of neutrophil-derived proinflammatory cytokines, which resulted in limited recruitment of monocytes and other neutrophils (22). Thus, we hypothesize that F. alocis may modulate neutrophil signaling events to interrupt pro-inflammatory cytokine production and alter immune cell recruitment and communication.…”