Transcription factor Nrf2 activation regulates NETosis, endothelial injury, and kidney disease in myeloperoxidase-positive antineutrophil cytoplasmic antibody-associated vasculitis.

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a systemic autoimmune disease pathologically characterized by vascular necrosis with inflammation. During AAV development, activated neutrophils produce reactive oxygen species (ROS), leading to the aberrant formation of neutrophil extracellular traps (NETs) via NETosis and subsequent fibrinoid vascular necrosis. Nuclear factor-erythroid 2-related factor 2 (Nrf2) functions as an intracellular defense system to counteract oxidative stress by providing antioxidant properties. Herein, we explored the role of Nrf2 in the pathogenesis of AAV. The role and mechanism of Nrf2 in ANCA-stimulated neutrophils and subsequent endothelial injury were evaluated in vitro using Nrf2 genetic deletion and Nrf2 activator treatment. In corresponding in vivo studies, the role of Nrf2 in ANCA-transfer AAV and spontaneous AAV murine models was examined. Pharmacological activation of Nrf2 in vitro suppressed ANCA-induced NET formation via the inhibition of ROS. In contrast, NET formation was enhanced in Nrf2-deficient neutrophils. Furthermore, Nrf2 activation protected endothelial cells from ANC-induced NETs-mediated injury. In vivo, Nrf2 activation ameliorated glomerulonephritis in two AAV models by upregulating antioxidants and inhibiting ROS-mediated NETs. Furthermore, Nrf2 activation restrained the expansion of splenic immune cells, including T lymphocytes and limited the infiltration of Th17 cells into the kidney. In contrast, Nrf2 genetic deficiency exacerbated vasculitis in a spontaneous AAV model. Thus, the pathophysiological process in AAV may be downregulated by Nrf2 activation, potentially leading to a new therapeutic strategy by regulating NETosis.

Decreased proteasome function increases oxidative stress in the early stage of pressure ulcer development.

The aging process in the elderly results in heightened skin fragility associated with various disorders, including pressure ulcers (PUs). Despite the high incidence of PUs in the elderly population, there is a limited body of research specifically examining the impact of aging on the development of pressure ulcers. Therefore, investigating age-related physiological abnormalities is essential to elucidate the pathogenesis of PUs. Ischemia-reperfusion (I/R) injury and the subsequent oxidative stress caused by reactive oxygen species (ROS) play essential roles in the early stage of PUs. In this study, we used a mouse model of proteasomal dysfunction with an age-related phenotype to examine the role of proteasome activity in cutaneous I/R injury in vivo. Decreased proteasome function did not affect the expression of inflammatory cytokines and adhesion molecules in the I/R area in transgenic mice; however, proteasome inhibition increased oxidative stress that was not attenuated by activation of the oxidative stress response mediated by NF-E2-related factor 2 (Nrf2). In dermal fibroblasts (FCs) subjected to hypoxia-reoxygenation (H/R), proteasome inhibition induced oxidative stress and ROS production, and Nrf2 activation did not adequately upregulate antioxidant enzyme expression, possibly leading to antioxidant/oxidant imbalance. The free radical scavenger edaravone had protective effects against I/R injury in vivo and decreased oxidative stress in FCs treated with a proteasome inhibitor and subjected to H/R in vitro. The results suggest that the age-related decline in proteasome activity promotes cutaneous I/R injury-induced oxidative stress, and free radical scavengers may exert protective effects by preventing oxidative stress in the early stage of PUs.

Decreased Proteasomal Function Exacerbates Endoplasmic Reticulum Stress-Induced Chronic Inflammation in Obese Adipose Tissue.

Low-grade chronic inflammation contributes to both aging and the pathogenesis of age-related diseases. White adipose tissue (WAT) in obese individuals exhibits chronic inflammation, which is associated with obesity-related disorders. Aging exacerbates obesity-related inflammation in WAT; however, the molecular mechanisms underlying chronic inflammation and its exacerbation by aging remain unclear. Age-related decline in activity of the proteasome, a multisubunit proteolytic complex, has been implicated in age-related diseases. This study employed a mouse model with decreased proteasomal function that exhibits age-related phenotypes to investigate the impact of adipocyte senescence on WAT inflammation. Transgenic mice expressing proteasomal subunit ß5t with weak chymotrypsin-like activity experience reduced lifespan and develop age-related phenotypes. Mice fed with a high-fat diet and experiencing proteasomal dysfunction exhibited increased WAT inflammation, increased infiltration of proinflammatory M1-like macrophages, and increased proinflammatory adipocytokine-like monocyte chemoattractant protein-1, plasminogen activator inhibitor-1, and tumor necrosis factor-α, which are all associated with activation of endoplasmic reticulum (ER) stress-related pathways. Impaired proteasomal activity also activated ER stress-related molecules and induced expression of proinflammatory adipocytokines in adipocyte-like cells differentiated from 3T3-L1 cells. Collective evidence suggests that impaired proteasomal activity increases ER stress and that subsequent inflammatory pathways play pivotal roles in WAT inflammation. Because proteasomal function declines with age, age-related proteasome impairment may be involved in obesity-related inflammation among elderly individuals.

Anti-neutrophil cytoplasmic antibody-negative granulomatosis with polyangiitis manifesting through pulmonary nodules and a genital lesion.

A 72-year-old female presented with bilateral pulmonary nodules before undergoing surgery for hysteroptosis. Transbronchial biopsy did not lead to a definitive diagnosis. The right mass in the upper lobe was resected through video-assisted thoracic surgery. Pathological findings showed granulomatosis with polyangiitis. However, the patient was negative for serum proteinase 3-anti-neutrophil cytoplasmic antibody. Although the nodule in the left lower lobe progressed, the serum inflammatory reaction yielded negative results. Resection of the nodule in the left lower lobe revealed identical pathological findings with those of the right pulmonary mass. Following total hysterectomy for hysteroptosis, the pathological findings indicated granulomatosis with polyangiitis.

Methylprednisolone pulse-enhanced neutrophil extracellular trap formation in mice with imiquimod-induced lupus-like disease, resulting in ischaemia of the femoral head cartilage.

OBJECTIVES: Methylprednisolone (mPSL) pulse therapy is an essential option for patients with active systemic lupus erythematosus, but there is a risk of adverse events related to microcirculation disorders, including idiopathic osteonecrosis of the femoral head (ONFH). Recent studies have revealed that excessive neutrophil extracellular traps (NETs) are involved in microcirculation disorders. This study aimed to demonstrate that mPSL pulse could induce NETs in lupus mice and identify the factors contributing to this induction. METHODS: Six mice with imiquimod (IMQ)-induced lupus-like disease and six normal mice were intraperitoneally injected with mPSL on days 39 to 41, and five mice with IMQ-induced lupus-like disease and six normal mice were injected with phosphate-buffered saline. Pathological examinations were conducted to evaluate the ischaemic state of the femoral head and tissue infiltration of NET-forming neutrophils. Proteome analysis was performed to extract plasma proteins specifically elevated in mPSL-administered mice with IMQ-induced lupus-like disease, and their effects on NET formation were assessed in vitro. RESULTS: Mice with IMQ-induced lupus-like disease that received mPSL pulse demonstrated ischaemia of the femoral head cartilage with tissue infiltration of NET-forming neutrophils. Proteome analysis suggested that prenylcysteine oxidase 1 (PCYOX1) played a role in this phenomenon. The reaction of PCYOX1-containing very low-density lipoproteins (VLDL) with its substrate farnesylcysteine (FC) induced NETs in vitro. The combined addition of IMQ and mPSL synergistically enhanced VLDL-plus-FC-induced NET formation. CONCLUSION: PCYOX1 and related factors are worthy of attention to understand the underlying mechanisms and create novel therapeutic strategies for mPSL-mediated microcirculation disorders, including ONFH.

Bruton's tyrosine kinase is a possible therapeutic target in microscopic polyangiitis.

BACKGROUND: Bruton's tyrosine kinase (Btk) is an enzyme expressed in leukocytes other than T lymphocytes and plasma cells and involved in B-cell receptor- and Fcγ receptor (FcγR)-mediated signal transduction. Btk inhibitors potentially suppress autoantibody production due to the expected inhibitory ability of B lymphocyte differentiation into antibody-producing plasma cells and reduce FcγR-mediated neutrophil activation, including the release of neutrophil extracellular traps (NETs). Microscopic polyangiitis (MPA) is a systemic small-vessel vasculitis characterized by the pathogenic autoantibody, antineutrophil cytoplasmic antibody (ANCA) that reacts with myeloperoxidase (MPO). MPO and MPO-ANCA immune complex (IC)-induced FcγR-mediated NETs are critically involved in MPA pathogenesis. This study aimed to demonstrate the therapeutic efficacy of the Btk inhibitor tirabrutinib on MPA. METHODS: Various doses of tirabrutinib or vehicle were orally administered to Sprague-Dawley rats daily. Four weeks later, the number of peripheral B lymphocytes was counted, and Btk phosphorylation in B lymphocytes was evaluated by flow cytometry. Human peripheral blood neutrophils were stimulated by MPO and anti-MPO antibody ICs (MPO and anti-MPO-ICs), and Btk and its downstream Vav phosphorylation were assessed by western blotting. The effects of tirabrutinib on MPO and anti-MPO-IC-induced NET formation were examined in vitro. Wistar Kyoto rats were immunized with human MPO to induce experimental MPA and given drug-free or tirabrutinib-containing feed (0.0037% or 0.012%) from day 0 or 28. All rats were euthanized on day 42 for serological and histological evaluation. RESULTS: Tirabrutinib inhibited Btk phosphorylation without decreasing B lymphocytes in vivo. Neutrophil Btk and Vav were phosphorylated when stimulated with MPO and anti-MPO-ICs. Tirabrutinib suppressed MPO and anti-MPO-IC-induced NET formation in vitro and ameliorated experimental MPA in a dose-dependent manner in vivo. Although MPO-ANCA production was not affected, NET-forming neutrophils in the blood were significantly reduced by tirabrutinib. CONCLUSIONS: The Btk inhibitor tirabrutinib suppressed MPO and anti-MPO-IC-induced NET formation in vitro and ameliorated experimental MPA by reducing NET-forming neutrophils but not decreasing MPO-ANCA titer in vivo. This study suggests that Btk is a possible therapeutic target in MPA.

Demonstration of equivocal anti-glomerular basement membrane antibody positivity as a non-specific reaction through multiple immunologic assays in a case of pediatric asymptomatic hematuria.

BACKGROUND: Anti-glomerular basement membrane (anti-GBM) antibody is essential for the diagnosis of anti-GBM disease. The major epitope consists of the α3 subunits of type IV collagen non-collagenous domain (α 3(IV)NC1). There have been only a few reports of patients false-positive for anti-GBM antibody. CASE REPORT: We experienced an 8-year-old boy who presented with asymptomatic hematuria followed by positivity for anti-GBM antibody as evaluated by a commercially available chemiluminescent enzyme immunoassay (CLEIA). While his condition remained stable other than continuing hematuria, his anti-GBM antibody titer increased. Further examination of another anti-GBM antibody assay (fluoroenzyme immunoassay) showed negative results. Thus, evaluation of the accuracy of his positivity for anti-GBM antibody was required. We conducted the following examinations: A) enzyme-linked immunosorbent assay, B) immunoblotting for recombinant α 1-5(IV)NC1, and C) immunohistochemical analysis of normal kidney tissue sections. Specimens used for the analysis were sera in A and IgG from the patient in B and C, respectively. As a result, no anti-GBM antibody was detected in A. In B, no band specific to α 1-5(IV)NC1 was observed. In C, the kidney tissue was not stained. Taken together, these results led us to judge the positive anti-GBM result in CLEIA of our patient to be a non-specific reaction. CONCLUSION: The commercial assays for anti-GBM antibody can lead to false-positive results. We recommend confirmation of anti-GBM antibody positivity through the use of multiple assays in patients demonstrating an atypical clinical course for anti-GBM disease.

Inhibition of Toll-like receptor 4 and Interleukin-1 receptor prevent SARS-CoV-2 mediated kidney injury.

Acute kidney injury (AKI) is a common and severe complication of the coronavirus disease 2019 (COVID-19). Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) directly affects the glomerular and tubular epithelial cells to induce AKI; however, its pathophysiology remains unclear. Here, we explored the underlying mechanisms and therapeutic targets of renal involvement in COVID-19. We developed an in vitro human kidney cellular model, including immortalized tubular epithelial and endothelial cell lines, demonstrating that SARS-CoV-2 directly triggers cell death. To identify the molecular targets in the process of SARS-CoV-2-mediated cell injury, we performed transcriptional analysis using RNA sequencing. Tubular epithelial cells were more prone to dying by SARS-CoV-2 than endothelial cells; however, SARS-CoV-2 did not replicate in renal cells, distinct from VeroE6/transmembrane protease serine 2 cells. Transcriptomic analysis revealed increased inflammatory and immune-related gene expression levels in renal cells incubated with SARS-CoV-2. Toll-like receptor (TLR) 3 in renal cells recognized viral RNA and underwent cell death. Furthermore, analysis of upstream regulators identified several key transcriptional regulators. Among them, inhibition of the interleukin-1 receptor (IL-1R) and TLR4 pathways protects tubular epithelial and endothelial cells from injury via regulation of the signal transducer and activator of transcription protein-3/nuclear factor-kB pathway. Our results reveal that SARS-CoV-2 directly injures renal cells via the proinflammatory response without viral replication, and that IL-1R and TLR4 may be used as therapeutic targets for SARS-CoV-2 mediated kidney injury.

Polyarteritis nodosa diagnosed in a young male after COVID-19 vaccine: A case report.

In response to the coronavirus disease 2019 pandemic, the coronavirus disease 2019 vaccine was rapidly developed and the effectiveness of the vaccine has been established. However, various adverse effects have been reported, including the development of autoimmune diseases. We report a case of new-onset polyarteritis nodosa in a 32-year-old male following the coronavirus disease 2019 vaccination. The patient developed limb pain, fever, pulmonary embolism, multiple subcutaneous nodules, and haematomas. Skin biopsy revealed necrotising inflammation accompanied by fibrinoid necrosis and high inflammatory cell infiltration in the walls of medium to small arteries. The symptoms resolved following corticosteroid treatment. Although it is difficult to prove a relationship between the vaccine and polyarteritis nodosa, similar cases have been reported and further reports and analyses are therefore necessary.

CD47 blockade ameliorates autoimmune vasculitis via efferocytosis of neutrophil extracellular traps.

Neutrophil extracellular trap (NET) formation contributes to immune defense and is a distinct form of cell death. Excessive NET formation is found in patients with anti-neutrophil cytoplasmic antibody-associated (ANCA-associated) vasculitis (AAV), contributing to disease progression. The clearance of dead cells by macrophages, a process known as efferocytosis, is regulated by the CD47-mediated "don't eat me" signal. Hence, we hypothesized that pathogenic NETs in AAV escape from efferocytosis via the CD47 signaling pathway, resulting in the development of necrotizing vasculitis. Immunostaining for CD47 in human renal tissues revealed high CD47 expression in crescentic glomerular lesions of patients with AAV. In ex vivo studies, ANCA-induced netting neutrophils increased the expression of CD47 with the reduction of efferocytosis. After efferocytosis, macrophages displayed proinflammatory phenotypes. The blockade of CD47 in spontaneous crescentic glomerulonephritis-forming/Kinjoh (SCG/Kj) mice ameliorated renal disease and reduced myeloperoxidase-ANCA (MPO-ANCA) titers with a reduction in NET formation. Thus, CD47 blockade would protect against developing glomerulonephritis in AAV via restored efferocytosis of ANCA-induced NETs.

Anti-PLA2R Antibody Development During NELL1-Associated Membranous Glomerulonephritis Treatment: A Case Report.

A Japanese man in his early 70s was referred to our hospital because of massive proteinuria. Analysis of his kidney biopsy demonstrated glomerular subepithelial immune deposits containing immunoglobulin (Ig)G, which was dominant for the IgG1 subclass. Immunoperoxidase staining for neural epidermal growth factor-like 1 protein (NELL1) was positive on the glomerular capillary walls, whereas neither serum anti-phospholipase A2 receptor (PLA2R) antibodies nor immunofluorescence staining for PLA2R was positive. Detailed investigation revealed no associated conditions, including underlying malignancies, and thus he was diagnosed as having NELL1-associated idiopathic membranous nephropathy (MN). The patient was treated with steroids, which substantially improved his nephrotic syndrome. Interestingly, serum anti-NELL1 as well as anti-PLA2R antibodies became positive during his clinical course. Serology-based approaches are currently proposed for the treatment of patients suspected of having MN; however, an accurate diagnosis of the present patient would have been difficult if such an approach was performed only at a later phase of the disease. Several target antigens for the glomerular immune deposits observed in patients with MN have recently been identified, and dual positivity of antibodies to these antigens reportedly occurs in some patients. Further accumulation and analyses of such patients are needed to establish more appropriate diagnostic approaches for MN.

Transcriptional dynamics of granulocytes in direct response to incubation with SARS-CoV-2.

Severe coronavirus disease 2019 (COVID-19) is characterized by acute respiratory distress syndrome and multiple organ dysfunction, in which the host immune response plays a pivotal role. Excessive neutrophil activation and subsequent superfluity of neutrophil extracellular traps (NETs) can lead to tissue damage, and several studies have shown the involvement of neutrophils in severe COVID-19. However, the detailed responses of each neutrophil subset to SARS-CoV-2 infection has not been fully described. To explore this issue, we incubated normal-density granulocytes (NDGs) and low-density granulocytes (LDGs) with different viral titers of SARS-CoV-2. NDGs form NETs with chromatin fibers in response to SARS-CoV-2, whereas LDGs incubated with SARS-CoV-2 display a distinct morphology with condensed nuclei and moderate transcriptional changes. Based on these transcriptional changes, we suggest that AGO2 possibly plays a role in LDG regulation in response to SARS-CoV-2.

Regulation of NETosis and Inflammation by Cyclophilin D in Myeloperoxidase-Positive Antineutrophil Cytoplasmic Antibody-Associated Vasculitis.

OBJECTIVE: Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is pathologically characterized by focal fibrinoid necrosis, in which ANCA-mediated neutrophil extracellular trap (NET) formation and subsequent endothelial cell necrosis occur. Cyclophilin D (CypD) plays an important role in mediation of cell necrosis and inflammation via the opening of mitochondrial permeability transition pores. This study was undertaken to examine the role of CypD in AAV pathogenesis. METHODS: We assessed the role and mechanism of CypD in ANCA-stimulated neutrophils in vitro by immunostaining and electron microscopy observation. We performed a comprehensive RNA-sequencing analysis on ANCA-treated murine neutrophils. To investigate the role of CypD in vivo, we assessed disease features in CypD-knockout mice and wild-type mice using 2 different murine AAV models: anti-myeloperoxidase IgG transfer-induced AAV and spontaneous AAV. RESULTS: In vitro experiments showed that pharmacologic and genetic inhibition of CypD suppressed ANCA-induced NET formation via the suppression of reactive oxygen species and cytochrome c release from the mitochondria. RNA-sequencing analyses in ANCA-treated murine neutrophils revealed the involvement of inflammatory responses, with CypD deficiency reducing ANCA-induced alterations in gene expression. Furthermore, analyses of upstream regulators revealed the relevance of intracellular calcium (CypD activator) and cyclosporin (CypD inhibitor) in ANCA stimulation, indicating that the CypD-dependent opening of mitochondrial permeability transition pores is associated with ANCA-induced neutrophil activation and NETosis. In both AAV mouse models, the genetic deletion of CypD ameliorated crescentic glomerulonephritis via the inhibition of CypD-dependent neutrophil and endothelial necrosis. CONCLUSION: CypD targeting is a novel and specific therapeutic strategy for AAV via the resolution of necrotizing vasculitis.

Expert perspectives on pathological findings in vasculitis.

Pathological findings are important in the diagnosis of vasculitis. However, due to the rarity of the disease, standard textbooks usually devote only a few pages to this topic, and this makes it difficult for clinicians not specializing in vasculitis to fully understand the pathological findings in vasculitis. To address the paucity of information, we present representative pathological findings in vasculitis classified in the 2012 Revised International Chapel Hill Consensus Conference Nomenclature of Vasculitides (CHCC2012). The CHCC2012 classifies 26 vasculitides into seven categories: (1) large-vessel vasculitis, (2) medium-vessel vasculitis, (3) small-vessel vasculitis, including antineutrophil cytoplasmic antibody-associated vasculitis and immune complex small-vessel vasculitis, (4) variable-vessel vasculitis, (5) single-organ vasculitis, (6) vasculitis associated with systemic disease, and (7) vasculitis associated with probable aetiology. Moreover, representative pathological findings of vasculitis-related diseases and non-inflammatory vasculopathy not mentioned in the CHCC2012 are also presented. This will be useful for clinicians to refer to typical pathological findings of vasculitis in daily practice.

Low disease activity of microscopic polyangiitis in patients with anti-myosin light chain 6 antibody that disrupts actin rearrangement necessary for neutrophil extracellular trap formation.

BACKGROUND: Neutrophil extracellular traps (NETs) are critically involved in microscopic polyangiitis (MPA) pathogenesis, and some patients with MPA possess anti-NET antibody (ANETA). Anti-myosin light chain 6 (MYL6) antibody is an ANETA that affects NETs. This study aimed to determine the significance of anti-MYL6 antibody in MPA. METHODS: The influence of anti-MYL6 antibody on NET formation and actin rearrangement necessary for NET formation was assessed by fluorescent staining. An enzyme-linked immunosorbent assay was established to detect serum anti-MYL6 antibody, and the prevalence of this antibody in MPA was determined. Furthermore, the disease activity and response to remission-induction therapy of MPA were compared between anti-MYL6 antibody-positive and anti-MYL6 antibody-negative MPA patients. RESULTS: Anti-MYL6 antibody disrupted G-actin polymerization into F-actin, suppressing phorbol 12-myristate 13-acetate-induced NET formation. Serum anti-MYL6 antibody was detected in 7 of 59 patients with MPA. The Birmingham vasculitis activity score (BVAS) of anti-MYL6 antibody-positive MPA patients was significantly lower than anti-MYL6 antibody-negative MPA patients. Among the nine BVAS evaluation items, the cutaneous, cardiovascular, and nervous system scores of anti-MYL6 antibody-positive MPA patients were significantly lower than anti-MYL6 antibody-negative MPA patients, although other items, including the renal and chest scores, were equivalent between the two groups. The proportion of patients with remission 6 months after initiation of remission-induction therapy in anti-MYL6 antibody-positive MPA patients was significantly higher than in anti-MYL6 antibody-negative MPA patients. CONCLUSIONS: Collective findings suggested that anti-MYL6 antibody disrupted actin rearrangement necessary for NET formation and could reduce the disease activity of MPA.

Typical cutaneous small-vessel vasculitis induced by combined injection of antiphosphatidylserine/prothrombin complex antibody and anti-LAMP-2 antibody in normal rats.

We previously reported that IgA vasculitis and cutaneous arteritis could be dependently associated with the presence of the antiphosphatidylserine/prothrombin complex (anti-PS/PT) antibody and lysosomal-associated membrane protein-2 (LAMP-2). The copy number of LAMP-2 mRNA in skin tissue samples from rats with cutaneous vasculitis induced by intravenous administration of anti-PS/PT antibody after subcutaneous histone injection was significantly higher than in those without cutaneous vasculitis. We found LAMP-2 protein overexpression in neutrophils and vascular endothelial cells of the affected blood vessels in rats with cutaneous vasculitis induced by intravenous administration of anti-PS/PT antibody after cutaneous priming by histones. We found typical cutaneous small-vessel vasculitis in the skin of rats given intravenous injection of both anti-PS/PT antibody and anti-LAMP-2 antibody after cutaneous priming by histones. We suggested that the introduction of skin local histones and anti-PS/PT antibody in serum could move LAMP-2 to the cell surface of neutrophils and vascular endothelial cells, and that anti-LAMP-2 antibody could bridge these cells through antigen-specific binding in typical cutaneous small-vessel vasculitis.

Presence of neutrophil extracellular traps in superficial venous thrombosis of Behçet's disease.

Behçet's disease (BD) has a heterogeneous spectrum of disease manifestations featuring the involvement of different organs and can be characterized with different symptoms depending on the clinical department in charge. We retrospectively reviewed BD patients seen at our hospital and investigated the presence of neutrophils producing neutrophil extracellular traps (NET) in those patients. Immunolabeling of myeloperoxidase and histone citrullination proteins was performed on skin biopsies from three BD patients who had skin biopsy-proven superficial vein thrombophlebitis in their erythema nodosum-like lesions. We observed a higher proportion of female patients and a higher incidence of acne-like eruptions among BD patients seen at our dermatology department, while there was a higher incidence of ocular and gastrointestinal involvement among BD patients treated in other departments. We suggest that sex statistical trends could lead to the co-development of different manifestations and may help clinicians choose the best therapeutic approaches, tailoring them to the specific phenotype of the patient rather than one based on single disease manifestations. NET were found in neutrophils of panniculitis concurrent with superficial vein thrombophlebitis. We suggest that the pathogenesis of BD-related thrombosis could be associated with neutrophil activation and NET are released in the panniculitis of affected skin lesions, erythema nodosum-like lesions.

Phorbol 12-myristate 13-acetate stimulation under hypoxia induces nuclear swelling with DNA outflow but not extracellular trap formation of neutrophils.

Neutrophils stand sentinel over infection and possess diverse antimicrobial weapons, including neutrophil extracellular traps (NETs). NETs are composed of web-like extracellular DNA decorated with antimicrobial substances and can trap and eliminate invading microorganisms. Although phorbol 12-myristate 13-acetate (PMA) is a potent NET inducer, previous studies have demonstrated that not all neutrophils exhibit NET formation even if stimulated by PMA at high concentrations. This study first showed that some neutrophils stimulated by PMA displayed a swollen nucleus but not NET formation and that hypoxic environments suppressed the NET release. Next, characterization of PMA-stimulated neutrophils with a swollen nucleus was accomplished by differentiating between suicidal-type NETosis and apoptosis. Furthermore, the significance of the phenomenon was examined using formalin-fixed, paraffin-embedded human lung disease tissues with and without pneumonia. As a result, histone H3 citrullination, DNA outflow, propidium iodide labeling, resistance to DNase I, and suspended actin rearrangement were characteristics of PMA-stimulated neutrophils with a swollen nucleus distinct from neutrophils that underwent either suicidal-type NETosis or apoptosis. Neutrophils stimulated by PMA under hypoxic conditions secreted matrix metalloproteinase-9 cytotoxic to human lung-derived fibroblasts. Further, deposition of neutrophil-derived citrullinated histone H3+ chromatin substances in pulmonary lesions was greater in patients with pneumonia than in patients without pneumonia and positively correlated with hypoxia-inducible factor-1α expression. The collective findings suggested that neutrophils activated under hypoxic conditions could be putative modulators of hypoxia-related disease manifestations.