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.

Plasmacytoid Dendritic Cell Activation and IFN-α Production Are Prominent Features of Murine Autoimmune Pancreatitis and Human IgG4-Related Autoimmune Pancreatitis

“…6D, 6E). Presumably the latter can occur because, as shown previously, the neutrophils are capable of producing LF that can then react with anti-LF Ab to form NET-stimulating immune complexes (46,47). It should be noted, however, that IgG4 subclass isolated from patient serum did not have the capacity to induce NETs in vitro, probably because the concentration of anti-LF Ab in this preparation was too low (data not shown).…”

Neutrophil Extracellular Traps Downregulate Lipopolysaccharide-Induced Activation of Monocyte-Derived Dendritic Cells

“…PMN kill pathogens through various strategies, including phagocytosis, degranulation, the oxidative burst, and release of neutrophil extracellular traps (NETs), a process called netosis (1,2). NETs are released by activated PMN in response to pathogenic bacteria, fungi, viruses, and protozoa (1,(3)(4)(5) [reviewed in (6,7)] and also in response to endogenous mediators of inflammation (3,4) and pharmacological agents such as PMA and calcium ionophore (1,3,(5)(6)(7). Netosis was initially described as a suicide-like mechanism, but a concept of "vital netosis" is also emerging (8).…”

Characterization of Filifactor alocis and its immune evasion strategies employed against human neutrophils.

“…It has been determined by many studies that NETs can be induced and/or kill a large range of pathogens including bacteria (both Gram-positive and Gram-negative), fungi, protozoan parasites and viruses (165)(166)(167). Also, NETs can be formed in the presence of physiological inducers, like lipopolysaccharide (LPS) non-physiological agents like phorbol 12-myristate 13-acetate (PMA) (168).…”

“…3-4 A) or from 4 independent experiments ( Fig. 3-4 (165,168,361,362). It is essential to strictly regulate NET formation in a time and dose-dependent fashion to ensure production and clearance processes occur when it is most beneficial to the host (168).…”

“…Neutrophils will be stimulated with PMA, S. gordonii or P. stomatis to induce NETs and these isolated NETs will be introduced to an F. alocis challenge setting and BacLight viability staining will be used to determine if it has antimicrobial properties towards F. alocis (165).…”