Oral Neutrophils Characterized: Chemotactic, Phagocytic, and Neutrophil Extracellular Trap (NET) Formation Properties

Abstract

Maintenance of oral health is in part managed by the immune-surveillance and antimicrobial functions of polymorphonuclear leukocytes (PMNs), which migrate from the circulatory system through the oral mucosal tissues as oral PMNs (oPMNs). In any microorganism-rich ecosystem, such as the oral cavity, PMNs migrate toward various exogenous chemoattractants, phagocytose bacteria, and produce neutrophil extracellular traps (NETs) to immobilize and eliminate pathogens. PMNs obtained from the circulation through venipuncture (hereafter called cPMNs) have been widely studied using various functional assays. We aimed to study the potential of oPMNs in maintaining oral health and therefore compared their chemotactic and antimicrobial functions with cPMNs. To establish chemotactic, phagocytic, and NET forming capacities, oPMNs and cPMNs were isolated from healthy subjects without obvious oral inflammation. Directional chemotaxis toward the chemoattractant fMLP was analyzed using an Insall chamber and video microscopy. fMLP expression was assessed by flow cytometry. Phagocytosis was analyzed by flow cytometry, following PMN incubation with heat-inactivated FITC-labeled micro-organisms. Furthermore, agar plate-based killing assays were performed with Escherichia coli ( Ec ). NET formation by oPMNs and cPMNs was quantified fluorimetrically using SYTOX™ Green, following stimulation with either PMA or RPMI medium (unstimulated control). In contrast to cPMNs, the chemotactic responses of oPMNs to fMLP did not differ from controls (mean velocity ± SEM of cPMNs: 0.79 ± 0.24; of oPMNs; 0.10 ± 0.07 micrometer/min). The impaired directional movement toward fMLP by oPMNs was explained by significantly lower fMLP receptor expression. Increased adhesion and internalization of various micro-organisms by oPMNs was observed. oPMNs formed 13 times more NETs than stimulated cPMNs, in both unstimulated and stimulated conditions. Compared to cPMNs, oPMNs showed a limited ability for intracellular killing of Ec . In conclusion, oPMNs showed exhausted capacity for efficient chemotaxis toward fMLP which may be the result of migration through the oral tissues into the oral cavity, being a highly “hostile” ecosystem. Overall, oPMNs' behavior is consistent with hyperactivity and frustrated killing. Nevertheless, oPMNs most likely contribute to maintaining a balanced oral ecosystem, as their ability to internalize microbes in conjunction with their abundant NET production remains after entering the oral cavity.

Neutrophil Subsets in Periodontal Health and Disease: A Mini Review

“…Interestingly, this group also investigated oral neutrophils in edentulous patients and found that they expressed low levels of all three activation markers and low constitutive ROS release (20). In a recent publication, these researchers characterized oral neutrophils as terminally migrated cells and reported that oral neutrophils had lost their ability to migrate in a coordinated directional manner (21). They showed that the fMLP receptor, crucial for fMLP-mediated chemotaxis, was detectable in only half of the neutrophils, compared to blood neutrophils.…”

Oral inflammatory load: Neutrophils as oral health biomarkers

“…The potential use of oPMN counts as a screening tool for periodontitis is coming at a time when research concerning the immunophysiologic characteristics of oPMNs is demonstrating the potential existence of peripheral and tissue‐resident neutrophil subsets in periodontal health and disease 12,19,72,73 . This suggests the potential of using both the quantification of oPMNs and the various peripheral and oPMN population subsets to provide even greater insight into establishing the diagnosis and grading the severity of periodontitis.…”

Oral Neutrophils: Underestimated Players in Oral Cancer

“…To immobilize and kill distantly localized microorganisms, neutrophils release NETs ( 75 ). Importantly, increased NET formation by oPMN, in comparison to cPMN, was reported ( 63 ), with para-inflammatory 2 subpopulation showing significantly higher release ( 73 ). This was in line with their elevated phagocytosis.…”

“…After their release from the bone marrow, neutrophils circulate for 5–9 h and then migrate into tissues where they survive 8–16 h before they die ( 63 ). Recently, a prolonged (up to 5 days) survival of activated neutrophils in tissues has been described ( 64 , 65 ), which ensures the presence of functional neutrophils at the site of inflammation ( 66 ).…”

“…Antibacterial functions of neutrophils include adhesion and internalization of bacteria (phagocytosis), production of reactive oxygen species (ROS) that damage membranes and genetical material, the release of neutrophil extracellular traps (NETs) capturing pathogens and the secretion of antibacterial proteins. oPMN isolated from healthy controls demonstrate slightly elevated adhesion to pathogens and significantly higher internalization of bacteria ( A. actinomycetemcomitans , P. gingivalis , E. coli ) in comparison to cPMN ( 63 ). Higher phagocytotic activity in para-inflammatory 2 oral neutrophils was also demonstrated, as compared to para-inflammatory 1 ( 73 ).…”