Peptoanaerobacter stomatis Primes Human Neutrophils and Induces Granule Exocytosis

Abstract

Peptoanaerobacter stomatis is a newly appreciated taxon associated with periodontal diseases; however, little is known about the organism's pathogenic potential or its interaction with the host immune response. Neutrophils are the most abundant innate immune cell present in the gingival tissue and function to constrain the oral microbial challenge. However, some periodontal pathogens have developed strategies to evade phagocytosis and killing by neutrophils. Therefore, to begin to understand the role of P. stomatis in periodontitis, we studied its interactions with human neutrophils. Our data showed that after 30 min of incubation, neutrophils failed to engulf P. stomatis efficiently; however, when P. stomatis was internalized, it was promptly eradicated. P. stomatis challenge induced a robust intracellular respiratory burst; however, this response did not contribute to bacterial killing. Minimal superoxide release was observed by direct bacterial challenge; however, P. stomatis significantly increased N-formyl-methionyl-leucyl phenylalanine (fMLF)-stimulated superoxide release to an extent similar to that of cells primed with tumor necrosis factor alpha (TNF-␣). When neutrophils were challenged with P. stomatis, 52% of the bacterium-containing phagosomes were enriched for the specific granule marker lactoferrin and 82% with the azurophil granule marker elastase. P. stomatis challenge stimulated exocytosis of the four neutrophil granule subtypes. Moreover, P. stomatis susceptibility to extracellular killing could be attributed to the exocytosis of antimicrobial components present in neutrophil granules. Priming neutrophils for an enhanced respiratory burst together with promoting granule content release could contribute to the chronic inflammation and tissue destruction that characterize periodontal diseases.

Filifactor alocismodulates human neutrophil antimicrobial functional responses

“…3B). Recently our laboratory reported that another emerging periodontal pathogen, Peptoanaerobacter stomatis , is able to elicit a robust intracellular ROS production by human neutrophils (Jimenez Flores, Tian, Sizova, Epstein, Lamont, & Uriarte, 2017). Hence, we next examined if F. alocis could modulate the ROS production induced by P. stomatis (Fig.…”

“…Another study compared the activation of the respiratory burst response when neutrophils were challenged with different periodontal pathogens; Fusobacterium nucleatum showed the highest intracellular and extracellular generation of ROS compared to P. gingivalis and Aggregatibacter actinomycetemcomitans (Katsuragi, Ohtake, Kurasawa, & Saito, 2003). Recently our laboratory showed that P. stomatis , an emerging oral pathogen, induces robust intracellular ROS production in human neutrophils, albeit the oxygen dependent response was not responsible for the antimicrobial activity (Jimenez Flores, Tian, Sizova, Epstein, Lamont, & Uriarte, 2017). In contrast, our results show that phagocytosis of F. alocis causes minimal intracellular and extracellular generation of reactive oxygen species; a phenotype, which to the best of our knowledge, is unique to F. alocis compared to other periodontal pathogens.…”

“…Several periodontal pathogens induce release of MMP-9 and elastase (Ding, Haapasalo, Kerosuo, Lounatmaa, Kotiranta, & Sorsa, 1997). Recently, we reported that P. stomatis induced exocytosis of the four neutrophil granules in a time and dose dependent manner (Jimenez Flores, Tian, Sizova, Epstein, Lamont, & Uriarte, 2017). In the present study we show that both live and heat-killed F. alocis are able to induce release of active MMP-9.…”

“…Serum opsonization was performed by incubation of F. alocis at 37°C for 20 min in different percentages, 2-5-10%, of normal human serum (Complement Technology, Inc.) For fluorescence immunostaining assays, F. alocis was labeled with carboxyfluorescein succinimidyl ester (CFSE; 40 ng/μl) for 30 min at room temperature in the dark, and washed 3 times with PBS prior to use. Peptoanaerobacter stomatis strain CM2 was cultured anaerobically at 37°C in Tryptic Soy Broth supplemented with 20 g/L yeast extract, 1% hemin and 1% reducing agent (37.5 g/L NH 4 Cl, 25 g/L MgCl 2 × 6H 2 O, 5 g/L CaCl 2 × 2H 2 O, 50 g/L L-cysteine HCl, 5 g/L FeCl 2 × 4H 2 O) as previously described (Jimenez Flores, Tian, Sizova, Epstein, Lamont, & Uriarte, 2017). …”

“…Specific and azurophil granule fusion to bacteria-containing phagosomes was tested by immunostaining using confocal microscopy as previously described (Jimenez Flores, Tian, Sizova, Epstein, Lamont, & Uriarte, 2017; Johnson & Criss, 2013b). Neutrophils (2 × 10 6 cells/ml) were unstimulated, or challenged with opsonized CFSE- labeled F. alocis , opsonized CFSE-labeled HK- F. alocis , or AlexaFluor 488 labeled heat-killed S. aureus for 30 and 120 min.…”