An improved strategy to recover large fragments of functional human neutrophil extracellular traps

Abstract

Netosis is a recently described neutrophil function that leads to the release of neutrophil extracellular traps (NETs) in response to various stimuli. NETs are filaments of decondensed chromatin associated with granular proteins. In addition to their role against microorganisms, NETs have been implicated in autoimmunity, thrombosis, and tissue injury. Access to a standardized source of isolated NETs is needed to better analyze the roles of NETs. The aim of this study was to develop a procedure yielding soluble, well-characterized NET preparations from fresh human neutrophils. The calcium ionophore A23187 was chosen to induce netosis, and the restriction enzyme AluI was used to prepare large NET fragments. DNA and proteins were detected by electrophoresis and specific labeling. Some NET proteins [histone 3, lactoferrin (LF)] were quantified by western blotting, and double-stranded DNA (dsDNA) was quantified by immunofluorescence. Co-existence of dsDNA and neutrophil proteins confirmed the quality of the NET preparations. Their biological activity was checked by measuring elastase (ELA) activity and bacterial killing against various strains. Interindividual differences in histone 3, LF, ELA, and dsDNA relative contents were observed in isolated NETs. However, the reproducibility of NET preparation and characterization was validated, suggesting that this interindividual variability was rather related to donor variation than to technical bias. This standardized protocol is suitable for producing, isolating, and quantifying functional NETs that could serve as a tool for studying NET effects on immune cells and tissues.

Keywords: characterization; isolation; microbicidal activity; netosis; neutrophil; neutrophil extracellular traps; quantification.

Figure 1

Calcium ionophore A23187-activated PMN release dsDNA more rapidly than PMA-activated PMN. PMN (2 × 10⁵) were incubated in the presence or absence of increasing concentrations of PMA (A) or A23197 (B) for 240 min. The presence of extracellular dsDNA was detected at the indicated times with Sytox Green. Both graphs are representative of three identical experiments and show means ± SEM of duplicate samples (C) Comparison of extracellular dsDNA levels 3 h after PMA or A23187 activation; data are means ± SEM (n = 3). AU, arbitrary units.

Figure 2

A23187-stimulated PMN release NETs similar to those induced by PMA. PMN were treated with PMA (50 nM) or A23187 (5 μM) for 3 h. NETs were observed by immunofluorescence microscopy after DNA staining with DAPI (blue), and after elastase, LF, H3, or cit-H3 staining with specific Abs followed by an Alexa Fluor 488-labeled secondary antibody (green). These experiments were repeated at least six times with PMN from different healthy controls.

Figure 3

DNA nucleases induce NET digestion. (A) Migration profile of pure λDNA after digestion with 4 U/mL DNase, MNase, or Alu-I. (B) Alu-I, DNase, and MNase dose-effects on NET dsDNA obtained after A23187 stimulation of PMN. Incubation with the restriction enzymes lasted 20 min at 37°C. DNA migration took place in 0.8% agarose gel containing ethidium bromide.

Figure 4

NETs are recovered after AluI treatment of activated PMN. Unstimulated PMN and PMA- or A23187-stimulated PMN were treated with the restriction enzyme Alu-I. (A) NET samples migrated on agarose gel were stained with ethidium bromide: dsDNA fragments were revealed as a smearing pattern along the gel. (B) Visualization of NET protein content by silver staining. Few proteins were observed in the untreated sample, whereas numerous proteins were observed in PMA- and A23187-NET samples, with similar profiles. (C) Identification of three specific NET proteins (LF, H3, and cit-H3) by immunoblotting. Cit-H3 is a signature of netosis. These experiments were repeated at least six times with PMN from different healthy controls.

Figure 5

Quantification of dsDNA and three NET-associated proteins shows interindividual heterogeneity (10 healthy controls). (A) Representative western blot of different concentrations of purified H3 and LF immunoblotted in parallel with isolated NETs and non-stimulated (NS) PMN supernatants from one healthy control. The bands were quantified by densitometry and plotted against the standard curve. (B) dsDNA, H3, LF, and ELA concentrations of A23187-NET samples from 10 healthy donors. Results are expressed in μg/10⁶ PMN.

Figure 6

NET preparation and characterization are reproducible. dsDNA, H3, LF, and ELA concentrations of A23187-NET samples from four healthy donors. Each fresh blood sample was divided in three parts, and three independent NET isolations, and characterizations were done. Data are means ± SEM (n = 3). Results are expressed in μg/10⁶ PMN.

Figure 7

Alu-I-derived NETs retain microbicidal activity partially dependent on DNA integrity. S. aureus, E. coli C1845, S. flexneri, and S. enterica serovar Typhimurium SL1344 were incubated for 45 min in the presence of isolated NETs obtained after AluI treatment of A23187- or PMA-activated PMN. In some experiments, NET samples were pretreated with DNase to dismantle NETs. Bacterial viability was measured by a colony count assay (CFU/mL). Results are expressed as percentage bacterial viability, calculated from CFU/mL values of bacteria exposed to NETs relative to bacteria not exposed to NETs (control tube = C). *p < 0.05 versus bacterial viability in the absence of NETs. *p < 0.05 versus DNase-treated NETs.