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.
Highlights
Polymorphonuclear leukocytes (PMNs) are terminally differentiated innate immune cells that descend from hematopoietic stem cells in the bone marrow and transit through the peripheral blood circulation as circulatory polymorphonuclear leukocyte (PMN) [1, 2]
The strength of the movement, evidenced by the length of cell tracks in spider plots and the distribution of dots in rose plots (Figures 1A,C), is evenly distributed indicating non-directional, minimal random movement of cells in response to PBS. circulatory PMN (cPMN) show obvious chemotactic responses to fMLP compared to control treated cells, as previously described by Roberts et al [16] (Figure 1B). cPMNs showed a strong response evidenced by longer cell tracks, with the largest proportion of cells moving North, showing a “Northern Hemisphere” distribution of cells and a median strength of 90◦ toward fMLP
The predominant immune cell that is constitutively recruited into the oral cavity is the PMN, being a protective and antimicrobial innate immune responder
Summary
Polymorphonuclear leukocytes (PMNs) are terminally differentiated innate immune cells that descend from hematopoietic stem cells in the bone marrow and transit through the peripheral blood circulation as circulatory PMNs (cPMNs) [1, 2]. CPMNs exit the vasculature and migrate to sites of tissue damage, inflammation, and infection to perform various protective and antimicrobial functions contributing to the neutralization and elimination of pathogens and damaged cells [3]. OPMNs form a wall between the epithelium and the dental biofilm to protect the periodontal tissue and to maintain periodontal tissue homeostasis [6,7,8]. It has been suggested that in the oral cavity, PMNs carry out a unique immune surveillance function and symbiotically interact with the commensal oral microflora in order to maintain homeostasis and oral health [10]. In cases of chronic inflammation, such as in periodontitis, a multifactorial chronic inflammatory disease of the periodontium leading to alveolar bone loss [11], an increased influx of oPMNs with a hyperactive phenotype extravasate into the oral cavity [12]
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