Properties of saliva-induced NETs. (A) NETs were induced with saliva, PMA, or bacteria. Microscopy images were taken to determine the area of NETs. The NETs were subsequently incubated with saliva or supernatant from S. pygogenes (AP1) with DNase activity and the area of NETs after treatment was determined by microscopy. The area of NETs before degradation was set to 100. Results are shown from 3 independent experiments. Representative images are found in supplementary Figure 5. (B) NETs were induced by saliva, sialyl Lewis^X in RPMI 1640 or saliva buffer (SB), and PMA in RPMI 1640 or SB. NET degradation was subsequently quantitated after treatment with saliva or AP1 supernatant. Results are shown from four independent experiments quantified as in A. (C) NETs were induced by saliva, PMA or bacteria and subsequently incubated with S. aureus. The total amount and amount of dead bacteria were examined by bacterial viability staining. Quantitative data from three independent experiments are presented. Representative images are found in supplementary Figure 5. (D) NETs were induced by saliva, sialyl Lewis^X and PMA and subsequently incubated with S. aureus. The total amount and amount of dead bacteria were examined by bacterial viability staining. Quantitative data from four independent experiments are presented. (E) NETs were induced by saliva and PMA and subsequently incubated with oral bacteria cultured from saliva. The total amount and amount of dead bacteria were determined by bacterial viability staining. Quantitative data from four independent experiments are presented where the oral bacteria were from 4 different donors. (F) Neutrophils were stimulated to phagocytose S. pyogenes either opsonized with saliva (saliva-opsonized bacteria SB) or plasma (plasma-opsonized bacteria). Extracellular bacteria were killed by gentamycin. NETosis was induced by saliva or sialyl Lewis^X. The NETs were disrupted by high concentration of DNase and colony counts performed on cell lysates. The survival of bacteria in polymorphonuclear leukocytes not stimulated to NETosis was set to 100. Results are shown from 4 independent experiments. (G) Similar experiments (as in F) were performed with oral bacteria cultured from saliva and opsonized with saliva prior to phagocytosis. Results are shown from 4 independent experiments in which oral bacteria were from 4 different donors. Columns denote average values and error bars standard deviations. N.S., not significant. *P < .05; **P < .01; ***P < .001; ****P < .0001 refers to nominal significance.