Akihiro Ishizu

Abnormal Conformation and Impaired Degradation of Propylthiouracil-Induced Neutrophil Extracellular Traps Implications of Disordered Neutrophil Extracellular Traps in a Rat Model of Myeloperoxidase Antineutrophil Cytoplasmic Antibody-Associated Vasculitis

OBJECTIVE Neutrophil extracellular traps (NETs) are composed of DNA and antimicrobial proteins, including myeloperoxidase (MPO). Recent studies have demonstrated that impaired regulation of NETs could trigger an autoimmune response. Propylthiouracil (PTU), an antithyroid drug, is associated with a risk of MPO antineutrophil cytoplasmic antibody (ANCA) production and MPO ANCA-associated vasculitis (MPO AAV). This study was undertaken to clarify the mechanism of MPO ANCA production, using the PTU-induced model of MPO AAV. METHODS NETs were induced by treating human neutrophils with phorbol myristate acetate (PMA) in vitro. We examined whether the addition of PTU influenced the NET formation induced by PMA and the degradation of NETs by DNase I, which is regarded as a regulator of NETs. Furthermore, we examined whether NETs generated by the combination of PMA and PTU induced MPO ANCA and MPO AAV in vivo in rats. RESULTS When NETs were induced by PMA with PTU using human neutrophils in vitro, abnormal conformation of NETs was observed. Interestingly, the abnormal NETs were hardly digested by DNase I. Moreover, rats immunized with the abnormal NETs, which had been induced by PMA with PTU using rat neutrophils, produced MPO ANCA and developed pulmonary capillaritis. When rats were given oral PTU with intraperitoneal injection of PMA, pauci-immune glomerulonephritis and pulmonary capillaritis occurred with MPO ANCA production in the serum. CONCLUSION Our findings indicate that abnormal conformation and impaired degradation of NETs induced by PTU are involved in the pathogenesis of PTU-induced MPO ANCA production and MPO AAV. These findings suggest that disordered NETs can be critically implicated in the pathogenesis of MPO AAV.

Enhanced Formation and Disordered Regulation of NETs in Myeloperoxidase-ANCA–Associated Microscopic Polyangiitis

Microscopic polyangiitis (MPA) is an ANCA-associated vasculitis that affects small vessels, especially renal glomeruli. We recently demonstrated that the abnormal formation and impaired degradation of neutrophil extracellular traps (NETs) may be crucially involved in the generation of myeloperoxidase (MPO)-ANCA and subsequent development of MPA. This study assessed the formation and regulation of NETs in patients with MPO-ANCA-associated MPA. Peripheral blood samples were obtained from 38 patients with MPO-ANCA-associated MPA, 23 patients with systemic lupus erythematosus (SLE), and 8 healthy controls. IgG eluted from MPO-ANCA-associated MPA sera demonstrated the highest ability to induce NETs, and this ability correlated with disease activity and paralleled ANCA affinity for MPO. Moreover, addition of recombinant human MPO to these IgG samples reduced NET induction. Additionally, MPO-ANCA-associated MPA sera exhibited lower rates of NET degradation that recovered partially upon depletion of IgG. The activity of DNase I, an important regulator of NETs, was also lower in MPO-ANCA-associated MPA and SLE sera. IgG depletion from MPO-ANCA-associated MPA sera partially restored the rate of NET degradation, and addition of DNase I synergistically enhanced this restoration. Addition of anti-MPO antibodies did not inhibit DNase I activity, and some MPO-ANCA-associated MPA sera contained anti-NET antibodies at levels not correlated with MPO-ANCA titers, suggesting the involvement of unidentified autoantibodies as well. The collective evidence suggests a vicious cycle involving MPO-ANCA and the regulation of NETs could be critically involved in the pathogenesis of MPO-ANCA-associated MPA.

Decreased proteasomal activity causes age-related phenotypes and promotes the development of metabolic abnormalities.

The proteasome is a multicatalytic enzyme complex responsible for the degradation of both normal and damaged proteins. An age-related decline in proteasomal activity has been implicated in various age-related pathologies. The relevance of decreased proteasomal activity to aging and age-related diseases remains unclear, however, because suitable animal models are not available. In the present study, we established a transgenic (Tg) mouse model with decreased proteasomal chymotrypsin-like activity. Tg mice exhibited a shortened life span and developed age-related phenotypes. In Tg mice, polyubiquitinated and oxidized proteins accumulated, and the expression levels of cellular proteins such as Bcl-xL and RNase L were altered. When Tg mice were fed a high-fat diet, they developed more pronounced obesity and hepatic steatosis than did wild-type mice. Consistent with its role in lipid droplet formation, the expression of adipose differentiation-related protein (ADRP) was elevated in the livers of Tg mice. Of note, obesity and hepatic steatosis induced by a high-fat diet were more pronounced in aged than in young wild-type mice, and aged wild-type mice had elevated levels of ADRP, suggesting that the metabolic abnormalities present in Tg mice mimic those in aged mice. Our results provide the first in vivo evidence that decreased proteasomal chymotrypsin-like activity affects longevity and aggravates age-related metabolic disorders, such as obesity and hepatic steatosis.

Abundant neutrophil extracellular traps in thrombus of patient with microscopic polyangiitis

This is a case study of a patient diagnosed with microscopic polyangiitis (MPA) and complicated with deep vein thrombosis (DVT), who died of respiratory failure despite treatment. Autopsy revealed severe crescentic glomerulonephritis and massive alveolar hemorrhage. The thrombus contained abundant neutrophils. Although it is reported that patients with ANCA-associated vasculitis (AAV) have an increased risk of DVT, it remains elusive why they are prone to thrombosis. A recent study has demonstrated the presence of neutrophil extracellular traps (NETs), a newly recognized mode of neutrophil cell-death, in glomerular crescents of MPA patients. Interestingly, NETs were identified in the thrombus as well as in the glomerular crescents in the present case. When compared to other thrombi unrelated to MPA, the amount of NETs was significantly greater in the MPA patient. On the other hand, NETs are critically involved in thrombogenesis because histones within NETs can bind platelets and blood coagulants. Although this is important in regard to containment of microbes within NETs, excessive NETs could cause thrombosis. The collective findings suggest the possibility that thrombosis could be critically associated with MPA via NETs, and that NETs could be a therapeutic target in MPA patients.

Exclusive expression of proteasome subunit β5t in the human thymic cortex

The ubiquitin-proteasome pathway, which degrades intracellular proteins, is involved in numerous cellular processes, including the supply of immunocompetent peptides to the antigen presenting machinery. Proteolysis by proteasomes is conducted by three beta subunits, beta1, beta2, and beta5, of the 20S proteasome. Recently, a novel beta subunit expressed exclusively in cortical thymic epithelial cells was discovered in mice. This subunit, designated beta5t, is a component of the thymoproteasome, a specialized type of proteasomes implicated in thymic positive selection. In this study, we show that, like its mouse counterpart, human beta5t is expressed exclusively in the thymic cortex. Human beta5t was expressed in approximately 80% of cortical thymic epithelial cells and some cortical dendritic cells. Human beta5t was incorporated into proteasomes with two other catalytically active beta subunits beta1i and beta2i, forming 20S proteasomes with subunit compositions characteristic of thymoproteasomes. The present study demonstrates, for the first time, the existence of thymoproteasomes in the human thymic cortex, indicating that thymoproteasome function is likely conserved between humans and mice.

The incidence and mechanism of sunitinib-induced thyroid atrophy in patients with metastatic renal cell carcinoma.

Background:To elucidate the incidence and mechanisms of sunitinib-induced thyroid atrophy, we investigated serial volumetric and functional changes, and evaluated histological changes of the thyroid gland in metastatic renal cell carcinoma patients who received sunitinib.Methods:Thyroid volume (by computed tomography volumetry) and thyroid function were measured at baseline, during the treatment, and at post-treatment periods. Histological evaluation of the thyroid gland was performed in four autopsied patients.Results:The median reduction rate in thyroid volume at last evaluation during sunitinib treatment was 30% in all 17 patients. The incidence of hypothyroidism during sunitinib treatment was significantly higher in the high reduction rate group (n=8; more than 50% reduction in volume) than in the low reduction rate group (n=9; less than 50% reduction in volume). Half of the patients in the high reduction rate group exhibited a transient thyroid-stimulating hormone suppression, suggesting thyrotoxicosis during sunitinib treatment. Histological evaluation demonstrated atrophy of thyroid follicles and degeneration of follicular epithelial cells without critical diminution of vascular volume in the thyroid gland.Conclusion:Thyroid atrophy is frequently observed following sunitinib treatment and may be brought about by sunitinib-induced thyrotoxicosis or the direct effects of sunitinib that lead to degeneration of thyroid follicular cells.

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.

Dexamethasone Increased Plasminogen Activator Inhibitor-1 Expression on Human Umbilical Vein Endothelial Cells: An Additive Effect to Tumor Necrosis Factor-α

Objective: In patients with autoimmune diseases, blood vessels may be critically involved at the site of inflammation. For these patients, glucocorticoids (GC) are often used as therapeutics. The aim of this study is to determine the effects of GC on vascular endothelial cells which are under inflammatory conditions. Methods: We examined the molecular expressions in human umbilical vein endothelial cells (HUVEC) induced by dexamethasone (Dex) and tumor necrosis factor (TNF)-α. Live cell number of HUVEC under exposure to Dex and TNF-α was also assayed. Results: The cDNA array analysis showed that a number of genes were upregulated, but only a few were downregulated by Dex and TNF-α, respectively. Among them, thrombomodulin (TM) gene showed the least fold change when HUVEC were stimulated by TNF-α. Since TM inhibits blood coagulation, we took notice of molecules associated with coagulation and fibrinolysis. The quantitative real-time RT-PCR revealed that the expression of plasminogen activator inhibitor-1 (PAI-1) gene increased when HUVEC were exposed to Dex and TNF-α, respectively, and the corresponding augmentation of its protein expression was confirmed by immunohistochemistry. The expression of PAI-1 gene additively increased when Dex and TNF-α were added to stimulate HUVEC. Under our experimental conditions, TNF-α induced proliferation of HUVEC. On the other hand, Dex did not change the number of live cells regardless of stimulation by TNF-α. Conclusion: TNF-α can induce proliferation of vascular endothelial cells with downregulation of the anticoagulation molecule, TM, and upregulation of the anti-fibrinolysis molecule, PAI-1. Dex further increased the expression of PAI-1 gene in the cells stimulated by TNF-α, and did not reduce the effect on cell proliferation induced by TNF-α. These findings suggest that the balance of blood coagulation versus fibrinolysis may incline to coagulation when Dex and TNF-α cooperate on vascular endothelial cells.

Vaccination of fusion cells of rat dendritic and carcinoma cells prevents tumor growth in vivo

Several reports on immunotherapy using dendritic cells‐based vaccine have been published. We investigated findings using fusion cells (FCs) generated from rat dendritic cells and a syngeneic hepatic cancer cell line with regard to inducing anti‐tumor immunity. Vaccination of rats using FCs protected against growth of the subcutaneously implanted tumor in vivo and induced infiltration of CD8+ T cells into the tumor. At the site of CD8+ T cell infiltration, there were apoptotic tumor cells. T cells from spleen of FCs‐vaccinated rats with protective ability against tumor growth included tumor specific cytotoxic CD8+ T cells restricted to major histocompatibility complex Class I. In addition, adaptive transfer of in vitro re‐stimulated splenic T cells with FCs was effective in preventing tumor growth and in vivo vaccinations of rats with FCs after resection of the subcutaneous implanted tumor inhibited local tumor recurrences. Immunotherapy using FCs appears to be an effective method if used in combination with surgical or other anti‐cancer therapies.

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.