Neutrophil Extracellular Traps Promote the Formation of Canine Dental Calculus

Dental calculus is a highly prevalent oral condition in dogs and is widely recognized as an important risk factor for gingival inflammation and periodontal disease. Effective strategies for its prevention and treatment remain limited, highlighting the significance of exploring novel mechanisms underlying its formation. Neutrophil extracellular traps (NETs), a key component of innate immunity, have been found in various diseases. To investigate the relationship between NETs and canine dental calculus formation, NET-associated markers were assessed in the oral cavities of dogs with dental calculus and healthy controls. Based on previously published full-length 16S rRNA amplicon sequencing data of canine dental calculus, Porphyromonas gulae was selected as a candidate NET-inducing bacterium for subsequent validation experiments. Subsequent neutrophil stimulation experiments were conducted to explore the effects of NETs and related factors on dental calculus formation. Collectively, our findings demonstrate the presence of NETs within canine dental calculus and reveal that P. gulae present in canine dental calculus is capable of inducing NET formation. The level of myeloperoxidase-DNA complex in gingival crevicular fluid was significantly elevated in dogs with dental calculus. NETs promoted aggregation and microcrystal formation from calcium and phosphate ions under both physiological and supersaturated concentrations. By adhering to the surface of dental calculus, NETs facilitated calculus accumulation. This effect showed positive correlation with neutrophil counts and administration frequency, but was independent of the concentration of administered calcium and phosphate solutions. IL-1β promoted the formation of aggregated NETs but did not enhance calculus accumulation. DNase I inhibited this process by degrading NET-DNA. In conclusion, dental calculus and the calculus-inhabiting P. gulae could stimulate oral neutrophils to release NETs, which participate in and facilitate the initial formation, aggregation, and subsequent accumulation of canine dental calculus.