Yoshihiro Kusunoki

The responses of macrophages in interaction with neutrophils that undergo NETosis.

Neutrophil extracellular traps (NETs) are net-like chromatin fibers decorated with antimicrobial proteins, which are released from dying neutrophils. The death of neutrophils with NET formation is called NETosis. Although NETs play important roles in the innate immunity, especially in the elimination of microbes, the extracellular release of DNA and intra-cytoplasmic/nuclear proteins can, on the other hand, result in diverse adversities to the hosts. Therefore, NETosis is adequately regulated in vivo. Currently, two mechanisms, namely DNase I-dependent digestion and phagocytosis by macrophages, have been shown as such regulatory mechanisms. In this study, we focused on the interaction of macrophages and neutrophils that underwent NETosis. Results demonstrated that macrophages displayed a phenotype-dependent response after degradation of NETs. Several hours after the interaction, M2 macrophages induced a pro-inflammatory response, while M1 macrophages underwent cell death with nuclear decondensation. This nuclear decondensation of M1 macrophages occurred in a peptidylarginine deiminase 4-dependent manner and resulted in a local release of extracellular DNA. Thereafter, M1 macrophages degraded DNA derived from themselves in a caspase-activated DNase-dependent manner resulting in the clearance of extracellular DNA within 24 h. This transient increase and subsequent clearance mechanism of extracellular DNA seems very reasonable in terms of the double-edged sword-like property of NETs. The collective findings demonstrate a novel phenotype- and time-dependent regulation of NETosis by macrophages.

Peptidylarginine Deiminase Inhibitor Suppresses Neutrophil Extracellular Trap Formation and MPO-ANCA Production

Myeloperoxidase-antineutrophil cytoplasmic antibody (MPO-ANCA)-associated vasculitis is a systemic small-vessel vasculitis, wherein, MPO-ANCA plays a critical role in the pathogenesis. Neutrophil extracellular traps (NETs) released from activated neutrophils are composed of extracellular web-like DNA and antimicrobial proteins, including MPO. Diverse stimuli, such as phorbol myristate acetate (PMA) and ligands of toll-like receptors (TLR), induce NETs. Although TLR-mediated NET formation can occur with preservation of living neutrophilic functions (called vital NETosis), PMA-stimulated neutrophils undergo cell death with NET formation (called suicidal NETosis). In the process of suicidal NETosis, histones are citrullinated by peptidylarginine deiminase 4 (PAD4). Since this step is necessary for decondensation of DNA, PAD4 plays a pivotal role in suicidal NETosis. Although NETs are essential for elimination of microorganisms, excessive formation of NETs has been suggested to be implicated in MPO-ANCA production. This study aimed to determine if pan-PAD inhibitors could suppress MPO-ANCA production in vivo. At first, NETs were induced in peripheral blood neutrophils derived from healthy donors (1 × 106/ml) by stimulation with 20 nM PMA with or without 20 μM propylthiouracil (PTU), an anti-thyroid drug. We then determined that the in vitro NET formation was inhibited completely by 200 μM Cl-amidine, a pan-PAD inhibitor. Next, we established mouse models with MPO-ANCA production. BALB/c mice were given intraperitoneal (i.p.) injection of PMA (50 ng at days 0 and 7) and oral PTU (2.5 mg/day) for 2 weeks. These mice were divided into two groups; the first group was given daily i.p. injection of PBS (200 μl/day) (n = 13) and the other group with daily i.p. injection of Cl-amidine (0.3 mg/200 μl PBS/day) (n = 7). Two weeks later, citrullination as an indicator of NET formation in the peritoneum and serum MPO-ANCA titer was compared between the two groups. Results demonstrated that citrullination in the peritoneum was significantly reduced in the Cl-amidine-treated mice compared with the vehicle-injected control mice (38% reduction). Additionally, the serum MPO-ANCA titer of the Cl-amidine-treated mice (32.3 ± 31.0 ng/ml) was significantly lower than that in the vehicle-injected mice (132.1 ± 41.6 ng/ml). The collective findings indicate that excessive formation of NETs may be implicated in MPO-ANCA production in vivo.

NETosis markers: Quest for specific, objective, and quantitative markers

More than 10years have passed since the discovery of neutrophil extracellular traps (NETs) in 2004. NETs are extracellular web-like DNA decorated with antimicrobial proteins, which are released from activated neutrophils. The state of neutrophils with NET formation is called NETosis. It has been realized that NETosis includes suicidal NETosis and vital NETosis. The former state means cell death of neutrophils, whereas the latter state preserves living neutrophilic functions. Although both suicidal and vital NETosis play essential roles in elimination of microorganisms, excessive formation of NETs, especially the ones derived from suicidal NETosis, can harm the hosts. Therefore, the discovery of NETosis markers and development of evaluation methods are important. In this review, we compare the methods for evaluating NETosis, including immunocytological and immunohistological detection of co-localized neutrophil-derived proteins and extracellular DNA, and citrullinated histones, detection of NET remnants in fluid samples, and flow cytometric detection of cell-appendant NET components, with focus on the specificity, objectivity, and quantitativity. Since the gold standard marker of NETosis or method of NET detection has not been established yet, researchers should choose the most appropriate marker or method in each situation based on the knowledge of the respective virtues and faults.

Circulating Neutrophil Extracellular Trap Levels in Well-Controlled Type 2 Diabetes and Pathway Involved in Their Formation Induced by High-Dose Glucose

Objectives: Although intensive therapy for type 2 diabetes (T2D) prevents microvascular complications, 10% of well-controlled T2D patients develop microangiopathy. Therefore, the identification of risk markers for microvascular complications in well-controlled T2D patients is important. Recent studies have demonstrated that high-dose glucose induces neutrophil extracellular trap (NET) formation, which can be a risk for microvascular disorders. Thus, we attempted to determine the correlation of circulating NET levels with clinical/laboratory parameters in well-controlled T2D patients and to reveal the mechanism of NET formation induced by high-dose glucose. Methods: Circulating NET levels represented by myeloperoxidase (MPO)-DNA complexes in the serum of 11 well-controlled T2D patients and 13 healthy volunteers were determined by enzyme-linked immunosorbent assay. The pathway involved in the NET formation induced by high-dose glucose was determined using specific inhibitors. Results: Serum MPO-DNA complex levels were significantly higher in some well-controlled T2D patients in correlation with the clinical/laboratory parameters which have been regarded as risk markers for microvascular complications. The aldose reductase inhibitor, ranirestat, could inhibit the NET formation induced by high-dose glucose. Conclusions: Elevated levels of circulating NETs can be a risk marker for microvascular complications in well-controlled T2D patients. The polyol pathway is involved in the NET formation induced by high-dose glucose.

Measurement of NET formation in vitro and in vivo by flow cytometry.

Neutrophil extracellular traps (NETs) are extracellular chromatin fibers adorned with antimicrobial proteins, such as myeloperoxidase (MPO), which are extruded from activated neutrophils. NETosis is the metamorphosis of neutrophils with NET formation that follows decondensation of DNA and rupture of the plasma membrane. Although NETs play important roles in innate immunity, excessive formation of NETs can be harmful to the hosts. Until now, various methods for evaluation of NETs have been reported. Although each has a virtue, the gold standard has not been established. Here we demonstrate a simple, objective, and quantitative method to detect NETs using flow cytometry. This method uses a plasma membrane‐impermeable DNA‐binding dye, SYTOX Green. SYTOX Green‐positive cells were detected in human peripheral polymorphonuclear cells exposed to a NET inducer, phorbol 12‐myristate 13‐acetate (PMA). The number of SYTOX Green‐positive cells was increased depending on the exposure duration and concentrations of PMA. Furthermore, co‐localization of MPO and plasma membrane‐appendant DNA of SYTOX Green‐positive cells was demonstrated. Moreover, a NET inhibitor, diphenylene iodonium, could significantly reduce the number of SYTOX Green‐positive cells induced by PMA. The collective evidence suggests that SYTOX Green‐positive cells include neutrophils that formed NETs. The established method could detect neutrophils that underwent NETosis but not early apoptosis with equivalence in quantification to another well‐used image analysis, which is based on fluorescent staining. Additionally, NETs that were formed in vivo were also detectable by this method. It is conceivable that the established method will bring us better understanding of the relation between NETosis and human diseases.

The Presence of Anti-Lactoferrin Antibodies in a Subgroup of Eosinophilic Granulomatosis with Polyangiitis Patients and Their Possible Contribution to Enhancement of Neutrophil Extracellular Trap Formation

Lactoferrin (Lf) is one of the antigens of antineutrophil cytoplasmic antibodies (ANCA) and functions as an endogenous suppressor of neutrophil extracellular trap (NET) formation. However, the prevalence and pathogenicity of anti-lactoferrin antibodies (aLf) in ANCA-associated vasculitis (AAV) remain unrevealed. This study aimed to examine the significance of aLf in AAV, initially. Sixty-five sera from AAV patients, including 41 microscopic polyangiitis, 5 granulomatosis with polyangiitis, and 19 eosinophilic granulomatosis with polyangiitis (EGPA) patients, were subjected to aLf detection using enzyme-linked immunosorbent assay. Clinical characteristics were compared between aLf-positive and aLf-negative patients. Neutrophils from healthy donors were exposed to suboptimal dose (10 nM) of phorbol myristate acetate (PMA) with aLf followed by evaluation of NET formation. Results demonstrated that 4 out of 65 AAV sera (6.2%) were positive for aLf. All of them were EGPA sera (4/19, 21.1%). In EGPA, the frequency of renal involvement, serum CRP levels, and Birmingham Vasculitis Activity Score (BVAS) in the aLf-positive patients was significantly higher than those in the aLf-negative patients, and the aLf titer correlated positively with the serum CRP level and BVAS. The NET formation was particularly enhanced by combined stimulation of 10 nM PMA and 1 µg/mL aLf. IgG isolated from sera of the aLf-positive EGPA patients (250 µg/mL) enhanced NET formation induced by 10 nM of PMA, and the effect was abolished completely by absorption of the aLf. This pilot study suggests that aLf enhance NET formation induced by PMA and are associated with disease activity of EGPA.

Detection of Autoreactive Type II NKT Cells: A Pilot Study of Comparison Between Healthy Individuals and Patients with Vasculitis: Detection of Autoreactive Type II NKT Cells: A Pilot Study of Comparison Between Healthy Individuals and Patients with Vasculitis

NKT cells are defined as T cells that recognize hydrophobic antigens presented by class I MHC‐like molecules, including CD1d. Among CD1d‐restricted NKT cells, type I and type II subsets have been noted. CD1d‐restricted type I NKT cells are regarded as pro‐inflammatory cells in general. On the contrary, accumulated evidence has demonstrated an anti‐inflammatory property of CD1d‐restricted type II NKT cells. In our earlier study using a rat model with vasculitis, we demonstrated the pro‐inflammatory function of CD1d‐restricted type II NKT cells and identified that one such cell recognized P518–532 of rat sterol carrier protein 2 (rSCP2518–532), which appeared on vascular endothelial cells presented by CD1d. Based on this evidence, we attempted to detect human CD1d‐restricted type II NKT cells in peripheral blood using hSCP2518–532, the human counterpart of rSCP2518–532, together with a CD1d tetramer in flow cytometry. First, we determined the binding of hSCP2518–532 to CD1d. Next, we detected CD3‐positive hSCP2518–532‐loaded CD1d (hSCP2518–532/CD1d) tetramer‐binding cells in peripheral blood of healthy donors. The abundance of TGF‐β‐producing cells rather than TNF‐α‐producing cells in CD3‐positive hSCP2518–532/CD1d tetramer‐binding cells suggests the anti‐inflammatory property of SCP2‐loaded CD1d (SCP2/CD1d) tetramer‐binding type II NKT cells in healthy individuals. Furthermore, we compared cytokine profile between healthy individuals and patients with vasculitis in a pilot study. Interestingly, the percentage of TGF‐β‐producing cells in SCP2/CD1d tetramer‐binding type II NKT cells in vasculitic patients was significantly lower than that in healthy controls despite the greater number of these cells. Although further studies to clarify the mechanism and significance of this phenomenon are needed, SCP2/CD1d tetramer‐binding type II NKT cells in peripheral blood should be examined in more detail to understand the pathophysiology of vasculitides in humans.