In vitro induction of NETosis: Comprehensive live imaging comparison and systematic review

Abstract

Background: Multiple inducers of in vitro Neutrophil Extracellular Trap (NET) formation (NETosis) have been described. Since there is much variation in study design and results, our aim was to create a systematic review of NETosis inducers and perform a standardized in vitro study of NETosis inducers important in (cardiac) wound healing.

Methods: In vitro NETosis was studied by incubating neutrophils with PMA, living and dead bacteria (S. aureus and E. coli), LPS, (activated) platelets (supernatant), glucose and calcium ionophore Ionomycin using 3-hour periods of time-lapse confocal imaging.

Results: PMA is a consistent and potent inducer of NETosis. Ionomycin also consistently resulted in extrusion of DNA, albeit with a process that differs from the NETosis process induced by PMA. In our standardized experiments, living bacteria were also potent inducers of NETosis, but dead bacteria, LPS, (activated) platelets (supernatant) and glucose did not induce NETosis.

Conclusion: Our systematic review confirms that there is much variation in study design and results of NETosis induction. Our experimental results confirm that under standardized conditions, PMA, living bacteria and Ionomycin all strongly induce NETosis, but real-time confocal imaging reveal different courses of events.

Fig 1. PRISM chart of the systematic literature review.

Fig 2. NETosis induction for the different inducers.

NETosis was defined as the ratio between the number of Hoechst and PI positive cells. PMA induced NETosis when compared to unstimulated neutrophils, p<0.001 repeated measures ANOVA post-hoc Bonferroni (*) (none n = 5, PMA n = 7, LPS n = 7, glucose n = 5). Error Bars +/- SEM.

Fig 3. NETs formed by S. aureus and E. coli 20 minutes after stimulation for one hour.

DNA (Hoechst, blue, 405) and Extracelullar DNA (PI, red, 561) were stained.

Fig 4. NETosis per inducer comparing the effect of platelets and the effect of activated platelets supernatant (n = 5, n = 7 respectively).

NETosis was defined as the ratio between Hoechst and PI positive cells. Neutrophils stimulated by platelets were compared against neutrophils stimulated by LPS, platelets + LPS, activated platelets, activated platelets + LPS, activated platelets supernatant, activated platelets supernatant + LPS using repeated measures ANOVA post-hoc Bonferroni (*). No significant differences were found (p>0.05 all). Error Bars +/- SEM.

Fig 5. The effect of Ionomycin compared to PMA.

(A) Time lapse images of PMA and Ionomycin at different time frames. The arrows indicate the decondensation of the nuclei before (Hoechst, 405) and after (PI, 561) DNA extrusion. (B) The amount of Hoechst and PI positive cells in PMA and Ionomycin stimulated cells and unstimulated cells, show the difference in the process of NETosis. In the PMA stimulated cells, the number of Hoechst positive cells go down as the PI positive cells (NETosis) go up. In the Ionomycin stimulated cells, the number of PI positive cells go up very rapidly, but the Hoechst positive cells remain similar. In unstimulated cells, the intensity of Hoechst staining remains high and no PI staining was detected.