A Critical Reappraisal of Neutrophil Extracellular Traps and NETosis Mimics Based on Differential Requirements for Protein Citrullination

Abstract

NETosis, an antimicrobial form of neutrophil cell death, is considered a primary source of citrullinated autoantigens in rheumatoid arthritis (RA) and immunogenic DNA in systemic lupus erythematosus (SLE). Activation of the citrullinating enzyme peptidylarginine deiminase type 4 (PAD4) is believed to be essential for neutrophil extracellular trap (NET) formation and NETosis. PAD4 is therefore viewed as a promising therapeutic target to inhibit the formation of NETs in both diseases. In this review, we examine the evidence for PAD4 activation during NETosis and provide experimental data to suggest that protein citrullination is not a universal feature of NETs. We delineate two distinct biological processes, leukotoxic hypercitrullination (LTH) and defective mitophagy, which have been erroneously classified as "NETosis." While these NETosis mimics share morphological similarities with NETosis (i.e., extracellular DNA release), they are biologically distinct. As such, these processes can be readily classified by their stimuli, activation of distinct biochemical pathways, the presence of hypercitrullination, and antimicrobial effector function. NETosis is an antimicrobial form of cell death that is NADPH oxidase-dependent and not associated with hypercitrullination. In contrast, LTH is NADPH oxidase-independent and not bactericidal. Rather, LTH represents a bacterial strategy to achieve immune evasion. It is triggered by pore-forming pathways and equivalent signals that cumulate in calcium-dependent hyperactivation of PADs, protein hypercitrullination, and neutrophil death. The generation of citrullinated autoantigens in RA is likely driven by LTH, but not NETosis. Mitochondrial DNA (mtDNA) expulsion, the result of a constitutive defect in mitophagy, represents a second NETosis mimic. In the presence of interferon-α and immune complexes, this process can generate highly interferogenic oxidized mtDNA, which has previously been mistaken for NETosis in SLE. Distinguishing NETosis from LTH and defective mitophagy is paramount to understanding the role of neutrophil damage in immunity and the pathogenesis of human diseases. This provides a framework to design specific inhibitors of these distinct biological processes in human disease.

Keywords: NADPH oxidase; NETosis; citrullination; leukotoxic hypercitrullination; mitophagy; peptidylarginine deiminase; rheumatoid arthritis; systemic lupus erythematosus.

Figure 1

Calcium ionophores induce cellular hypercitrullination in neutrophils. After informed consent, peripheral blood neutrophils were isolated from healthy donors as previously described (58), resuspended in HBSS/10 mM Hepes/1.5 mM CaCl₂, and incubated with 1 μM ionomycin or 5 μM A23187 at 37°C for 30–180 min. Neutrophils incubated for 3 h in the absence of stimulus were used as negative control. General protein citrullination was visualized by anti-modified citrulline (AMC) immunoblotting (59) (upper panel) and histone H3 citrullination (Cit-H3) using antibodies against citrullinated histone H3 (citrulline 2 + 8 + 17) (Abcam, Cat# ab5103) (middle panel). Histone H3 (H3) was detected as loading control (lower panel) (anti-histone H3, Abcam, Cat# ab1791). The experiments were performed on at least three separate occasions with similar results.

Figure 2

Calcium ionophores and the pore-forming toxin PVL from S. aureus, but not NETosis-inducing stimuli, activate cellular hypercitrullination in neutrophils. Purified human neutrophils in HBSS/10 mM Hepes/1.5 mM CaCl₂ were incubated (A) in the absence or presence of 100 nM PMA, 500 ng/mL LPS, 1 μM ionomycin, or 5 μM A23187 at 37°C for 3 h, or (B) in the absence or presence of PLV from S. aureus (recombinant LukS and LukF from IBT BioServices) at 30 or 100 nM for 90 min at 37°C. Total citrullinated proteins (AMC), citrullinated histone H3 (citH3), and histone H3 (H3) (loading control) were detected by immunoblotting. The experiments were performed on at least two separate occasions with similar results.

Figure 3

Purified perforin induces PAD activation and the release of extracellular DNA in neutrophils. Human neutrophils (5 × 10⁵ cells/50 μL) in HBSS/10 mM Hepes/1.5 mM CaCl₂ were plated onto standard microscope slides coated with poly-d-lysine (Sigma) and incubated for 30 min at 37°C to allow for cell attachment. Neutrophils were then incubated in the absence or presence of sublytic amounts (500 ng/mL) of purified perforin (Enzo Life Science) for 1 h at 37°C, as previously described (58). Then, the cells were fixed, permeabilized, and stained with anti-histone H3 (citrulline 2 + 8 + 17) antibodies (H3cit) and Alexa Fluor 594 goat anti-rabbit F(ab′)₂ fragment (Invitrogen, Cat# A-11072). Cells were mounted with ProLong Gold (Molecular Probes) plus DAPI. Samples were analyzed using a Zeiss axioscope microscope. Excitation filter: 510–560 nm for Alexa Fluor 594 and excitation filter: 300–390 nm for DAPI. Merged images of DAPI (blue) and H3cit (red) staining are shown. Perforin induces PAD activation (detected by citrullination of histone H3) and the extracellular release of neutrophil DNA (arrowheads). The experiments were performed on at least four separated occasions, with similar results.

Figure 4

Theoretical models of biological mechanisms of DNA release in neutrophils. The three models are depicted and illustrate their potential triggers, biochemical pathways, and biological consequences. (A) NETosis is initiated by stimuli that potently activate NADPH oxidase and ROS production, but decreases PAD4 activation and likely mtROS production. NETosis is antimicrobial, potentially anti-inflammatory, and has limited protein citrullination. (B) Leukotoxic hypercitrullination is triggered by stimuli that generate prominent and sustained increases in cytosolic calcium, such as ionophores and proteins that damage the cellular membrane (e.g., pore-forming proteins). PAD hyperactivation and protein hypercitrullination are characteristic of this process, but not ROS production by NADPH oxidase. While mtROS may have some role in the release of DNA by calcium ionophores in neutrophils (32), the relevance of this pathway in neutrophil damage induced by pore-forming proteins is unknown. Antimicrobial functions and the immunogenicity of self-antigens are affected as a result of cellular hypercitrullination. (C,D) To compensate a constitutive defect in mitophagy, neutrophils normally extrude mitochondrial components, including mtDNA, as a mechanism for mitochondria clearance. (C) The process of mtDNA expulsion (MDEX) may be enhanced by inflammatory signals such LPS or C5a following neutrophil priming with GM-CSF and requires NADPH oxidase activity. (D) IFN-I and RNP-ICs alter the normal disposal of mitochondria in neutrophils and promote the expulsion of ox-mtDNA (OMEX). This process is dependent on mtROS, but not NADPH oxidase, and may be predisposed by factors that decrease NADPH oxidase activity. Different to mtDNA, ox-mtDNA is pro-inflammatory and highly interferogenic. LDGs from patients with SLE have spontaneous OMEX. The role of citrullination in (C,D) is unknown. The question marks (?) reflect pathways/functions that require further experimental confirmation.