High-mobility group box 1 potentiates antineutrophil cytoplasmic antibody-inducing neutrophil extracellular traps formation

Background Recent studies found that the circulating high-mobility group box 1 (HMGB1) levels could reflect the disease activity of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). HMGB1 could prime neutrophils by increasing ANCA antigens translocation for ANCA-mediated respiratory burst and degranulation. The current study aimed to investigate whether HMGB1 participates in ANCA-induced neutrophil extracellular traps (NETs) formation, which is one of the most important pathogenic aspects in the development of AAV. Methods NETs were induced by treating neutrophils with HMGB1 and ANCA-positive IgG in vitro. NETs formation was assessed using immunofluorescence microscopy and fluorescence probe. Antagonist for relevant receptors Toll-like receptor (TLR)2, TLR4 and the receptor for advanced glycation end products (RAGE), as well as NADPH oxidase molecules were employed. Results The percentage of NETs formation was significantly higher in neutrophils stimulated with HMGB1 plus ANCA-positive IgG than that in neutrophils incubated with HMGB1 or ANCA-positive IgG alone. Consistently, compared with the nonstimulated neutrophils, the cell-free DNA (cfDNA) concentration of NETs was significantly increased from 334.09 ± 46.89 ng/ml to 563.32 ± 122.07 ng/ml in the neutrophils incubated with HMGB1 plus MPO-ANCA-positive IgG (P < 0.001), and from 303.44 ± 37.14 ng/ml to 563.79 ± 145.94 ng/ml in the neutrophils incubated with HMGB1 plus PR3-ANCA-positive IgG (P < 0.001). The aforementioned effect was significantly attenuated by antagonist for relevant receptors TLR2, TLR4 and RAGE, as well as blocking NADPH oxidase. Conclusions HMGB1 can potentiate ANCA-inducing NETs formation and may be involved in the pathogenesis of AAV. HMGB1 exerts effects on NETs formation through interaction with TLR2, TLR4 and RAGE, and the process is NADPH oxidase dependent. Electronic supplementary material The online version of this article (doi:10.1186/s13075-015-0903-z) contains supplementary material, which is available to authorized users.


Background
Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) encompasses granulomatosis with polyangiitis (GPA) and eosinophilic granulomatosis with polyangiitis (EGPA) and microscopic polyangiitis (MPA) [1]. The serological markers for the aforementioned primary small vessel vasculitis were ANCAs, which direct against granule proteins of neutrophils, in particular, proteinase 3 (PR3) and myeloperoxidase (MPO). Accumulated evidence from in vitro studies, animal models, and clinical observations suggests that ANCAs play a critical role in the vascular damage of AAV [2,3].
Neutrophils are the primary effector cells in the pathogenesis of AAV [4]. Cytokines or other pro-inflammatory mediators, such as tumor necrosis factor alpha (TNF-α) and interleukin (IL)-18, could prime neutrophils by inducing upregulation of the ANCA antigens expression on the surface of neutrophils. Thus, ANCAs could further activate primed neutrophils to undergo a respiratory burst and degranulation, which plays a crucial role in the development of vasculitis [5,6]. Recently, neutrophil extracellular traps (NETs), generated by ANCA-activated neutrophils, are increasingly recognized as another important aspect in the pathogenesis of AAV [7,8]. NETs consist of decondensed chromatin modification with cytoplasmic proteins such as MPO and PR3, which are induced by a wide range of stimuli including pathogens (bacteria, fungi), activated platelets and cytokines [8,9]. NETs can stick to the endothelium and cause tissue damage during inflammation similar to neutrophil-induced injury of capillaries in AAV [10]. Recent studies showed that the abnormal regulation of NETs is involved in the pathogenesis of AAV [7,11]. On the other hand, NETs are associated with thrombosis in AAV patients because histones and DNA within NETs can bind platelets and blood coagulants [12,13].
High-mobility group box 1 protein (HMGB1), a ubiquitous nuclear protein involved in nucleosome stabilization and gene transcription, has potent pro-inflammatory actions and can be classified as a danger-associated molecular pattern mediator when placed extracellularly [14]. HMGB1 can interact with Toll-like receptor (TLR)-2, TLR-4 and the receptor for advanced glycation end products (RAGE) in established cell lines and animal models, leading to a downstream translocation of nuclear factor (NF)-κB, inducing pro-inflammatory and chemotactic responses [15,16].
Recent studies found that circulating HMGB1 levels are associated the disease activity and renal involvement of AAV [17][18][19], although it remains controversial in some other studies [20]. Our further study found that HMGB1 could prime neutrophils by increasing ANCA antigens translocation, and the primed neutrophils could be further induced by ANCA, resulting in the respiratory burst and degranulation [21]. Therefore, in the current study, we hypothesized that HMGB1 can contribute to ANCAinduced NETs formation. Furthermore, we also investigated receptors and signaling molecules involved in HMGB1-promoted NETs formation in the presence of ANCA.

Preparation of IgG
Normal immunoglobulin G (IgG) and ANCA-positive IgG were prepared from plasma of normal volunteers and patients with active PR3-ANCA-or MPO-ANCApositive primary small vessel vasculitis. Plasma was filtered through a 0.22 mm syringe filter (Gelman Sciences, Ann Arbor, MI, USA) and applied to a HiTrap Protein G column on an AKTA-FPLC system (GE Biosciences, South San Francisco, CA, USA). Preparation of IgG was performed according to the methods described previously [22,23].

Neutrophil isolation
Neutrophils were isolated as described previously [24]. Briefly speaking, venous human blood for neutrophil isolation was obtained from healthy donors by venipuncture and anticoagulated with EDTA. Neutrophils were isolated by density gradient centrifugation on Lymphoprep (Nycomed, Oslo, Norway). Erythrocytes were lysed with ice-cold ammonium chloride buffer, and neutrophils were washed with phosphate-buffered saline (PBS) (Beijing Chemical Reagents, Beijing, China). The purity of the neutrophils was above 95 %. Neutrophils were then suspended in RPMI 1640 containing 0.5 % heat-inactivated fetal bovine serum (FBS). We obtained written informed consent from all participants. The research was in compliance of the Declaration of Helsinki and approved by the ethics committee of the Peking University First Hospital.

NETs induction
NETs induction was according to the protocols described previously, with some minor modifications [25,26]. Isolated neutrophils were seeded on 13 mm glass coverslips in 24-well plates in 400 μl of RPMI 1640 supplemented with 0.5 % heat-inactivated FBS at a density of 1 × 10 5 cells per well. The plates were incubated for 30-60 min at 37°C to allow adhesion of the cells. Then neutrophils were incubated with the buffer control or HMGB1 at a concentration of 10 ng/ml, which was comparable to the circulating HMGB1 level in active AAV patients, as demonstrated in our previous study [18], for 30 min at 37°C. Then we stimulated the pretreated neutrophils with a concentration of patient-derived ANCApositive IgG at 300 μg/ml in an incubator containing 5 % CO 2 at 37°C for 3 h. In order to investigate the role of candidate receptors through which HMGB1 exerted its effect, neutrophils were first incubated with blocking antibodies (anti-TLR2 at 10 μg/ml; anti-TLR4 at 10 μg/ml; RAGE-Fc at 1 μg/ml) for 30 min on ice. The above time set and inhibiting concentrations were according to our previous study [21]. We used phorbol myristate acetate (PMA) at a concentration of 100 nM in an incubator containing 5 % CO 2 at 37°C for 3.5 h as the positive control. Then we fixed the cells with 4 % paraformaldehyde (PFA) for further immunocellular chemistry (ICC).

NETs immunofluorescence
We detected NETs by immunolabeling as described previously [26]. Coverslips with the fixed cells were removed from the plates and processed by floating on drops kept on a parafilm sheet covering a test tube stand. The samples were permeabilized for 1 min with 0.5 % Triton X-100 after washing with PBS, washed again with PBS. Unspecific binding sites were blocked in PBS containing 5 % donkey serum. The samples were then incubated for 60 min respectively with the primary antibody as follows: anti-human myeloperoxidase mouse monoclonal antibody (diluted 1:100) and anti-human Cit-H3 rabbit polyclonal antibody (diluted 1:100). After washing with PBS, each primary antibody was visualized using secondary antibodies coupled to AF488-labeled donkey anti-rabbit IgG and Cy3-labeled donkey antimouse IgG (both 1:400, Jackson ImmunoResearch, West Grove, PA, USA) were applied for 60 min. The primary and secondary antibodies were diluted with PBS. After incubation for 60 min with the secondary antibodies, the specimens were washed with PBS, and the DNA was stained with DAPI for 5 min. All procedures were performed at room temperature. In negative controls, primary antibodies were replaced by PBS. Confocal images were captured with a Zeiss LSM 780 confocal microscope (Zeiss, Jena, Germany). Five images taken randomly from different regions of each coverslip in the experiment were taken with the 10× lens on a fluorescence microscope [25]. Exposure times of each channel were kept constant over the whole series in the experiment after calibrating on a bright representative sample to avoid saturated pixels. The image files were analyzed with the Image-Pro Plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA). The NET percentage was calculated as follows: NET rate [%] = 100 × number of neutrophils displaying expanded nuclei and releasing DNA fibers/total number of neutrophils.

Quantification of DNA release from neutrophils
NETs formation was quantified using PicoGreen as described previously [27]. Lambda DNA of known concentration was serially diluted with Tris-EDTA (TE) buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5) to create standard DNA samples. To follow NETs formation, 100 μl fresh neutrophils (1 × 10 5 cells) were seeded in Costar 96-well black plates (Corning, Tewksbury, MA, USA) in the presence of 0.5 % FBS. Neutrophils were pretreated with buffer or 10 ng/ml HMGB1 for 30 min followed by stimulation with MPO-ANCA-positive IgG, or PR3-ANCA-positive IgG, or normal IgGs in an incubator containing 5 % CO 2 at 37°C for 3 h, respectively. For assay of the role of candidate receptors, certain groups of neutrophils were preincubated with relevant reagents for 30 min on ice before being pretreated with HMGB1. Some 100 μl of the PicoGreen dye diluted 1:200 in TE buffer was added to the microplate in order to make a final volume of 200 μl per well. Following incubation in the dark for 5 min at room temperature, the fluorescent signal of the sample was measured using the microplate fluorescence reader (TriStar Multimode Microplate Reader LB941, Berthold Technologies, Bad Wildbad, Germany), at an excitation wavelength of 480 nm and an emission wavelength of 530 nm.

Statistical analysis
Differences between the two sets of data were analyzed using t tests. When the differences between more than two sets of data were analyzed, we used the one-way analysis of variance. A P value < 0.05 was considered to be statistically significant. Reported values were expressed as mean ± standard deviation (SD). Analyses were performed on SPSS version 13.0 for Windows (SPSS Inc, Chicago, IL, USA).

Results
Neutrophils pretreated with HMGB1 showed greater ability to produce NETs in the presence of ANCA We investigated the effects of HMGB1 on ANCAinduced NETs formation. ANCA-IgG were prepared from two patients with active PR3-ANCA-positive vasculitis, five patients with active MPO-ANCA-positive vasculitis and three healthy volunteers, respectively. Neutrophils of the abovementioned nine healthy donors were analyzed. The NETs were quantified by measuring cell-free DNA (cfDNA) concentration with the Quant-iT PicoGreen fluorescence probe. Compared with the buffer control, the cell-free DNA concentration increased significantly in neutrophils incubated with HMGB1 plus MPO-ANCA-positive IgG or PR3-ANCA-positive IgG (334.09 ± 46.89 vs. 563.32 ± 122.07, P < 0.001; 303.44 ± 37.14 vs. 563.79 ± 145.94, P < 0.001, respectively). Compared with neutrophils incubated with HMGB1 or ANCApositive IgG alone, the cell-free DNA concentration of NETs increased significantly from 337.29 ± 99.06 ng/ml and 430.05 ± 43.79 ng/ml to 563.32 ± 122.07 ng/ml in the neutrophils incubated with HMGB1 plus MPO-ANCApositive IgG (P < 0.001, P = 0.017, respectively), from 359.82 ± 76.86 ng/ml and 407.25 ± 89.90 ng/ml to 563.79 ± 145.94 ng/ml in the neutrophils incubated with HMGB1 plus PR3-ANCA-positive IgG (P = 0.002, P = 0.018, respectively) (Fig. 1). No obvious NET formation was observed with HMGB1, ANCA-IgG alone, normal IgG alone or HMGB1 plus normal IgG, either. PMA was used as a positive control.

The effects of HMGB1 and ANCA on NETs formation were dose dependent
Neutrophils were pretreated with various concentrations of HMGB1 (0, 1, 2, 5, 10, 100 and 1000 ng/ml, respectively), then were stimulated with ANCA-positive IgG at a concentration of 300 μg/ml. The results showed that the effect of HMGB1 potentiating ANCA-inducing NETs formation was dose dependent (Fig. 3a).
On the other hand, neutrophils were pretreated with the same concentrations of HMGB1 (10 ng/ml), then were stimulated by various concentration of ANCApositive IgG (0, 50, 100, 300, 500, and 700 μg/ml, respectively). The results showed that the effects of PR3-and MPO-ANCA-positive IgG-inducing NETs formation were both dose dependent (Fig. 3b and c).
To further confirm the receptors through which HMGB1 exerts its effects, we used TLR2−/− and TLR4 −/− mice [28]. However, RAGE−/− mice are not commercially available. These results were in line with our data of human neutrophils with inhibitors and blocking antibodies to block the activity of corresponding receptors. For detailed information, see Additional file 1.
Collectively, these results indicated that TLR2, TLR4 and RAGE were required in the process of HMGB1 promoting NETs formation in the presence of ANCApositive IgG.

NET formation was dependent on NADPH oxidase in the presence of HMGB1 and ANCA-positive IgG
Recent studies demonstrated that NETosis can occur in a reactive oxygen species (ROS)-independent manner, such as Candida albicans and uric acid-inducing NETs formation [29,30]. In addition, a study by Tadie et al. showed that HMGB1 induced NETs formation also independent of NADPH oxidase ROS production [28]. Our study showed that neutrophils incubated with HMGB1 Bars represent mean ± SD of repeated measurements on neutrophils of 8-9 independent experiments and 9 donors. ANCA antineutrophil cytoplasmic antibody, HMGB1 high-mobility group box 1, IgG immunoglobulin G, MPO myeloperoxidase, NETs neutrophil extracellular traps, PR3 proteinase 3, RAGE receptor for advanced glycation end products, TLR Toll-like receptor plus MPO-ANCA-positive IgG or PR3-ANCA-positive IgG, the cell-free DNA concentration decreased from 553.66 ± 118.10 ng/ml and 577.93 ± 121.69 ng/ml to 458.33 ± 136.59 ng/ml and 450.93 ± 107.54 ng/ml, respectively, by preincubating with DPI (P = 0.010 and P = 0.001, respectively) (Fig. 5). Consistently, in neutrophils incubated with HMGB1 plus MPO-ANCA-positive IgG or PR3-ANCA-positive IgG, the percentage of NET formation was decreased from 14.41 ± 2.48 % and 14.99 ± 6.55 % to 7.42 ± 0.51 % and 7.09 ± 2.57 %, respectively, by preincubating with DPI (P = 0.003 and P = 0.005, respectively) (Fig. 5). These results suggested that NETs formation in neutrophils incubated with HMGB1 plus ANCA was also dependent on NADPH oxidase ROS production.

Discussion
In the study by Tadie et al., it was shown that HMGB1 alone contributes to NETs formation in vitro or under relevant in vivo conditions, but the concentrations of HMGB1 was 300 ng/ml in their study, which is much higher than the pathophysiological concentration of circulating HMGB1 in active AAV patients [28]. In the current study, we demonstrated that 10 ng/ml HMGB1 can contribute to ANCA-induced NETs formation. Our previous study found that HMGB1 could prime neutrophils, and further result in the respiratory burst and degranulation in the presence of ANCA [21]. The findings of the current study extended the role of HMGB1 in activating neutrophils, and thus, may contribute to the development of AAV. Although the concentration of HMGB1 used in our study was comparable to the circulating HMGB1 level in active AAV patients [18], we cannot exclude that local concentrations of HMGB1 may be higher.
Extracellular HMGB1 induces several responses, including the release of pro-inflammatory cytokines, cell proliferation and cell migration [31,32]. Several receptors have been implicated in HMGB1-mediated functions, including RAGE and TLR2 and TLR4 [33][34][35][36]. It is not fully clear which of these receptors are required for the different bio-function of HMGB1. The current study showed that HMGB1-dependent engagement of TLR2, TLR4 and RAGE contributed to ANCA-induced NETs formation. Our previous study found that HMGB1 exerts priming effects on neutrophils by increasing ANCA antigens translocation, which was TLR4 and RAGE dependent, not TLR2 dependent [21], while Tadie's study showed that the NETs formation induced by HMGB1 alone was TLR4 dependent, not TLR2 or RAGE dependent [28]. Thus, the process of HMGB1 potentiating ANCA-induced NETs formation may be distinct from facilitating neutrophil respiratory burst and directly inducing NETs formation.
The mechanisms responsible for NETs formation is not completely delineated yet. Despite that previous studies showed that NETs formation requires activation of NADPH oxidase and production of ROS [37], there is now growing evidence suggesting that some stimuli induce NETs formation independent of NADPH oxidase [30]. Indeed, the study by Tadie et al. showed that HMGB1-inducing NETs formation was independent of ROS generated by NADPH oxidase [28], which is consistent with rapid NETs formation in response to Candida albicans and uric acid-inducing NETs formation [29,30]. However, our results indicated that the HMGB1 plus ANCA-IgG-inducing NETs formation was dependent on ROS generation. These findings suggested that HMGB1 contributing to NETs formation may involve heterogeneous mechanisms in the context of the dependency on NADPH oxidase and production of ROS.

Conclusions
Our study demonstrated that HMGB1 can potentiate ANCA-inducing NETs formation. HMGB1 exerts effects on NETs formation through the interaction with TLR2, TLR4 and RAGE, and the process is NADPH oxidase dependent. Blockade of HMGB1 might limit inflammatory damage caused by ANCA-induced NETs formation.