Anti‐β2GPI/β2GPI induces human neutrophils to generate NETs by relying on ROS

Neutrophils participate in the regulation of pathogens by phagocytosis as well as by generating neutrophil extracellular traps (NETs). Antiphospholipid antibodies, particularly those targeting beta‐2‐glycoprotein I (β2GPI), stimulate monocytes, platelets, and endothelial cells with prothrombotic participation. This study aimed to explore NET generation in response to anti‐β2GPI/β2GPI. A series of experiments involving the separation of primary human leukocytes, NETosis quantification using propidium iodide, exploration of NETosis by fluorescence microscopy, western blotting, examination of free Zn2+ using FluoZin‐3, and reactive oxygen species (ROS) examination with dihydrorhodamine 123 were performed in this study. We found that anti‐β2GPI/β2GPI triggered NETosis, resembling phorbol 12‐myristate 13‐acetate (PMA)‐induced NETosis in magnitude and morphology. The anti‐β2GPI/β2GPI complex in isolation stimulated NETs without relying on p38, protein kinase B (AKT), extracellular signal‐related kinase (ERK) 1/2, and zinc signals. NET generation was unaffected by the NADPH oxidase suppressor DP1. The anti‐β2GPI/β2GPI complex stimulated ROS generation without relying on NADPH oxidase, which may participate in NET generation triggered via the anti‐β2GPI/β2GPI complex. In summary, our results indicate that the anti‐β2GPI/β2GPI complex reinforced NET generation by relying on ROS. The significance of the paper in the context of current knowledge Neutrophils as one of the first lines of defence and essential in the response to pathogen invasion. They eradicate bacteria via phagocytosis or by releasing antimicrobial proteins in degranulation. In this study, we explored the capability of anti‐β2GPI/β2GPI to stimulate NETosis, demonstrating that anti‐β2GPI/β2GPI is a promising method for triggering NET. Anti‐β2GPI/β2GPI induced ROS generation without relying on NADPH oxidase, which contributes to NETosis independently of ERK1/2, Zn2+, or AKT. Our results showed that anti‐β2GPI/β2GPI triggered NETosis, resembling PMA‐induced NETosis in magnitude as well as morphology. The anti‐β2GPI/β2GPI complex in isolation stimulated NETs without relying on p38, AKT, ERK1/2, or zinc signals. The anti‐β2GPI/β2GPI complex stimulated ROS generation without relying on NADPH oxidase, which may participate in NET generation triggered via the anti‐β2GPI/β2GPI complex.


| INTRODUCTION
Neutrophils represent the most abundant cells in the immune system and are responsible for 50-70% of leucocytes in human blood. 1 As one of the first lines of defence, they are essential in the response to pathogen invasion. 2 They eradicate bacteria via phagocytosis or by releasing antimicrobial proteins in degranulation. 3 Neutrophils receive an innovative pattern of apoptosis programing signals known as NETosis that induces the release of neutrophil extracellular traps (NETs), which are composed of double-stranded DNA resembling a net with a histone coating as well as antimicrobial agents including myeloperoxidase. 4,5 NETs attract and eradicate bacteria and counteract viruses. 6 NETosis occurs in reaction to multiple fungal as well as bacterial pathogens. Initially, phorbol 12-myristate 13-acetate (PMA) stimulates NETosis. 7 As a stimulator of protein kinase C (PKC), PMA stimulates several downstream pathways triggering NADPH oxidase, which generates the reactive oxygen species (ROS) necessary for NETosis. 8 NETs are related to multiple autoimmune disorders such as smallvessel vasculitis, rheumatoid arthritis, psoriasis, and systemic lupus erythematosus (SLE). 9 Consequently, the release of chromatin during the generation of NETs is a source of autoantigens. 10 If NETs are produced during viral infections, they are particularly effective in influencing tolerance and inducing autoimmunity, because viruses trigger the delivery of inflammatory cytokines including Type I interferon, which participates in SLE. 11 Antiphospholipid antibodies identify not only thrombin, but also β2GPI. 12,13 Anti-β2GPI antibodies are commonly used for clinical assays. 14 The understanding of reactions downstream of β2GPI is more detailed. As a cationic protein that binds to lipids, β2GPI shows elevated concentrations in blood and can be generated via the liver, monocytes, endothelial cells, and trophoblasts. 15 Although several studies have suggested that β2GPI affects NET delivery, 16 its aetiology is unclear.

| Separation of primary human leukocytes (PHL)
PHL including monocytes, lymphocytes, and granulocytes were separated from blood subjected to heparinization and obtained from healthy participants. Participants were maily women (only one man) 22-42 years of age. Subjects who smoked, drank more than a moderate quantity of alcohol, displayed contemporary infection, and had a correlated clinical history were excluded. Fully informed consent was acquired from participants. To separate white blood cells (WBCs), 6% hydroxyethyl starch solution was added in a 2-fold volume to the blood. Phosphate-buffered saline (PBS) was used to wash the cells twice before sedimentation, which was conducted at room temperature (RT) for 45-60 min. Hypotonic lysis was carried out utilizing the remaining red blood cells. Ethical approval was acquired from the institutional ethics review board of The First Affiliated Hospital of Harbin Medical University.

| Exploration of NETosis using fluorescence microscopy
Anti-β 2 GPI/β 2 GPI or PMA was applied to stimulate the cells at 37°C in cultivation media. SYTOX green was added at 1 μM followed by incubation for 4 h. Cells were placed on glass slides after centrifugation in a cytospin at 300 × g for 5 min. A Zeiss Axioskop was utilized to observe fluorescence (Oberkochen, Germany). A Nikon Coolpix 4500 digital camera was used to capture the images at 10X magnification (Tokyo, Japan).

| Examination of free Zn 2+ using FluoZin-3
Free Zn 2+ inside the cells was examined as previously described. 17 Briefly, WBCs were treated for 1 h with anti-β2GPI/β2GPI in measur-

| ROS examination with DHR123
For ROS examination, cells were subjected to 30-min loading using

| Statistical analysis
Data are presented as the mean ± SD unless otherwise indicated. The data were analysed by Student t test or a one-way analysis of variance using Prism version 6 (GraphPad, Inc., La Jolla, CA, USA). Values of P < 0.05 were considered significant.
NET generation was quantified by examining the DNA outside the cells by PI staining (Figure 1). Fluorescence was noticeably increased with anti-β2GPI/β2GPI, suggesting the generation of NETs. Independent supplementation with β2GPI or anti-β2GPI did not affect the fluorescence. Additional procedures were conducted using anti-β2GPI/β2GPI. The effect of anti-β2GPI/β2GPI was dependent on the time and concentration and showed a similar effect as PMA, a known stimulator of NETosis ( Figure 2). Anti-β2GPI/β2GPI-induced NETs were confirmed by SYTOXgreen staining (Figure 3). Briefly, anti-β2GPI/β2GPI triggered NETosis resembling PMA-induced NETosis in magnitude and morphology.

| Kinase phosphorylation
In order to examine the aetiology of how anti-β2GPI/β2GPI triggered NETosis, WB was applied to explore AKT function with the help of antibodies counteracting AKT serine phosphorylation ( Figure 4A).

FIGURE 1
Induction of NETosis by anti-β2GPI/β2GPI. Primary human leukocytes were treated with anti-β2GPI/β2GPI complex, isotype control for 4 h at 37°C. extracellular NET-DNA was quantified. Data are presented as the mean ± SD of three independent experiments. ***, P < 0.001

FIGURE 2
Induction of NETosis by anti-β2GPI/β2GPI. A, Primary human leukocytes (PHL) were treated with anti-β2GPI/β2GPI for 4 h at 37°C and extracellular NET-DNA was quantified. B, PHL were treated with anti-β2GPI/β2GPI at the indicated concentration for 4 h at 37°C and extracellular NET-DNA was quantified. Data are presented as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001

| Zn 2+ delivery
Zn 2+ delivery was reinforced in lymphocytes and monocytes in response to anti-β2GPI/β2GPI rather than in WBC granulocytes ( Figure 5A).
Chelation of free Zn 2+ inside the cells using TPEN, a Zn 2+ -selective chelator that can penetrate membranes, failed to influence NETosis triggered by anti-β2GPI/β2GPI ( Figure 5B). This indicates that Zn 2+ delivery did not participate in NETosis triggered by anti-β2GPI/β2GPI. Role of Zn 2+ in anti-β2GPI/β2GPI-induced NETosis. A, Leukocytes were treated with anti-β2GPI/β2GPI for 1 h, followed by loading with Fluo-Zin-3. Zinc-dependent fluorescence was measured. B, Leukocytes were pre-treated with TPEN, followed by incubation with anti-β2GPI/ β2GPI for 4 h. extracellular NET-DNA was quantified. Data are presented as the mean ± SD from three independent experiments. *, P < 0.05

| ROS
A previous study showed that ROS generation is crucial in NETosis. 10 The pro-fluorophore DHR123, which is sensitive to redox, displayed similar fluorescence subsequent to supplementation with anti-β2GPI/β2GPI and PMA, despite its weakness compared with the strongest activation by H 2 O 2 ( Figure 6A). The NADPH oxidase inhibitor diphenylene iodonium (DPI) notably suppressed NETosis triggered via PMA, but not in the presence of anti-β2GPI/β2GPI ( Figure 6B). N-Acetylcysteine counteracted oxidation and suppressed NETosis triggered via anti-β2GPI/β2GPI, indicating the influence of ROS on these reactions ( Figure 6C). Our findings indicate that anti-β2GPI/β2GPI stimulated NETosis by activating ROS generation independently of NADPH oxidase.

| DISCUSSION
NETosis is a crucial reaction used by WBCs to eliminate microorganisms. 19  Evaluation of free Zn 2+ in the reaction to anti-β 2 GPI/β 2 GPI revealed noticeably increased concentrations of lymphocytes and monocytes, which was similar to the results of previous studies. 26 Zinc functions downstream of ROS and is crucial but insufficient for NETosis. 17,27 Consequently, anti-β 2 GPI/β 2 GPI replaced Zn 2+ with ROS, eliminating the requirement for Zn 2+ .
NETosis is associated with the stimulation of NADPH oxidase, which generates ROS crucial for NET generation. 28 Several studies showed that NADPH oxidase does not always participate in NETosis and relies on other sources of ROS. 29,30 However, the pro-fluorophore DHR123, which is sensitive to oxidation, displayed similar fluorescence subsequent to activation by PMA and anti-β 2 GPI/β 2 GPI, indicating similar ROS generation, although they arose from different sources. Notably, mercurial compounds alter the process of ROS arising from NADPH oxidase. Not only organic but also inorganic patterns of mercury cause mitochondrial injury following ROS delivery.