NET formation can occur independently of RIPK3 and MLKL signaling

Abstract

The importance of neutrophil extracellular traps (NETs) in innate immunity is well established but the molecular mechanisms responsible for their formation are still a matter of scientific dispute. Here, we aim to characterize a possible role of the receptor-interacting protein kinase 3 (RIPK3) and the mixed lineage kinase domain-like (MLKL) signaling pathway, which are known to cause necroptosis, in NET formation. Using genetic and pharmacological approaches, we investigated whether this programmed form of necrosis is a prerequisite for NET formation. NETs have been defined as extracellular DNA scaffolds associated with the neutrophil granule protein elastase that are capable of killing bacteria. Neither Ripk3-deficient mouse neutrophils nor human neutrophils in which MLKL had been pharmacologically inactivated, exhibited abnormalities in NET formation upon physiological activation or exposure to low concentrations of PMA. These data indicate that NET formation occurs independently of both RIPK3 and MLKL signaling.

Keywords: MLKL; NET formation; NETosis; Necroptosis; Neutrophils; RIPK.

Figure 1

The formation of mouse NETs is independent of RIPK3. Mature mouse neutrophils were isolated from bone marrow of wild‐type and Ripk3‐deficient mice. (A) Immunoblotting. Bone marrow cells from wild‐type and Ripk3‐deficient mice were analyzed for RIPK3 protein expression. Three mice per genotype are shown. (B) Quantification of NET‐forming neutrophils by confocal microscopy. NET formation following short‐term stimulation (total 45 min) of mouse neutrophils with the indicated triggers. The number of NET‐forming neutrophils was determined by counting the DNA‐releasing cells in ten high power fields (Supporting Information Fig. 1A and Supporting Information Movie 1). No statistical differences were observed between wild‐type and Ripk3‐deficient cells (n = 3). (C) Representative microscopy. Co‐localization (arrows) of elastase (green) with released DNA (PI, red) assessed by confocal microscopy. Bars, 10 μM. (D) Quantification of dsDNA in supernatants of activated neutrophils using PicoGreen fluorescent dye. A significant difference in dsDNA release was detected between control and activated cells, but not between wild‐type and Ripk3‐deficient neutrophils (n = 3). (E) Total ROS activity assessed by DHR123 fluorescence and flow cytometry (n = 3). (F) Quantification of H₂O₂ production upon activation of neutrophils was performed using luminescent ROS‐Glo that measures H₂O₂ levels directly in cell culture. Again, ROS activity is increased in activated mouse neutrophils, but no statistical differences were observed between wild‐type and Ripk3‐deficient cells (n = 3). (G) Bacterial killing by colony formation unit (cfu) assay (n = 3). Mouse neutrophils exert antibacterial activity against E. coli that can be partially blocked by 100 U/mL DNase I. All data are shown as mean ± SEM of the indicated number of independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001; one‐way ANOVA test.

Figure 2

The formation of human NETs occurs independently of MLKL signaling. Human blood neutrophils were isolated from healthy donors by Ficoll‐Hypaque centrifugation. (A) Confocal microscopy. NET formation following short‐term stimulation (total 45 min) of human neutrophils with the indicated triggers in the presence and absence of 5 μM NSA. The number of NET‐forming neutrophils was determined by counting the DNA‐releasing cells in ten high power fields (n = 5). Representative original data (right). Co‐localization of elastase (green) with released DNA (PI, red) is indicated by the yellow color. Bars, 10 μM. (B) Quantification of dsDNA in supernatants of activated neutrophils using PicoGreen fluorescent dye (n = 3). (C) Total ROS production by activated human neutrophils in the presence and absence of 5 μM NSA was measured by flow cytometry. During cell activation, cells were incubated with 1 μM DHR123 (n = 3). (D) Bacterial killing by colony forming unit (cfu) assay. Human neutrophils exert antibacterial activity against E. coli that can be partially blocked by 100 U/mL DNase I both in the presence and absence of 5 μM NSA (n = 3). (E) Viability of Jurkat cells was analyzed by ethidium bromide exclusion assay. In parallel experiments, 5 μM NSA blocked cell death induced by 20 ng/mL TNF‐α plus 100 nM Smac mimetic AT‐406 (cIAP1/2 selective IAP‐antagonist) in caspase‐8 deficient and 20 μM zVAD‐FMK–treated Jurkat cells (n = 3), demonstrating that the inhibitor was pharmacologically active. All data are shown as mean ± SEM of the indicated number of independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001; one‐way ANOVA test.