Intravenous Administration of Mesenchymal Stem Cell-Derived Exosome Alleviates Spinal Cord Injury by Regulating Neutrophil Extracellular Trap Formation through Exosomal miR-125a-3p.

Spinal cord injury (SCI) leads to devastating sequelae, demanding effective treatments. Recent advancements have unveiled the role of neutrophil extracellular traps (NETs) produced by infiltrated neutrophils in exacerbating secondary inflammation after SCI, making it a potential target for treatment intervention. Previous research has established that intravenous administration of stem cell-derived exosomes can mitigate injuries. While stem cell-derived exosomes have demonstrated the ability to modulate microglial reactions and enhance blood-brain barrier integrity, their impact on neutrophil deactivation, especially in the context of NETs, remains poorly understood. This study aims to investigate the effects of intravenous administration of MSC-derived exosomes, with a specific focus on NET formation, and to elucidate the associated molecular mechanisms. Exosomes were isolated from the cell supernatants of amnion-derived mesenchymal stem cells using the ultracentrifugation method. Spinal cord injuries were induced in Sprague-Dawley rats (9 weeks old) using a clip injury model, and 100 µg of exosomes in 1 mL of PBS or PBS alone were intravenously administered 24 h post-injury. Motor function was assessed serially for up to 28 days following the injury. On Day 3 and Day 28, spinal cord specimens were analyzed to evaluate the extent of injury and the formation of NETs. Flow cytometry was employed to examine the formation of circulating neutrophil NETs. Exogenous miRNA was electroporated into neutrophil to evaluate the effect of inflammatory NET formation. Finally, the biodistribution of exosomes was assessed using 64Cu-labeled exosomes in animal positron emission tomography (PET). Rats treated with exosomes exhibited a substantial improvement in motor function recovery and a reduction in injury size. Notably, there was a significant decrease in neutrophil infiltration and NET formation within the spinal cord, as well as a reduction in neutrophils forming NETs in the circulation. In vitro investigations indicated that exosomes accumulated in the vicinity of the nuclei of activated neutrophils, and neutrophils electroporated with the miR-125a-3p mimic exhibited a significantly diminished NET formation, while miR-125a-3p inhibitor reversed the effect. PET studies revealed that, although the majority of the transplanted exosomes were sequestered in the liver and spleen, a notably high quantity of exosomes was detected in the damaged spinal cord when compared to normal rats. MSC-derived exosomes play a pivotal role in alleviating spinal cord injury, in part through the deactivation of NET formation via miR-125a-3p.

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.

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.

Spontaneously regressed granulomatosis with polyangiitis: A case report.

A 71-year-old woman presented with chest pain, cough, and back pain. A chest roentgenogram showed multiple nodular shadows in both lungs. She was diagnosed with granulomatosis with polyangiitis (GPA). The multiple nodular shadows in both lungs regressed spontaneously in a few months. There are few reports of spontaneous regression of GPA, and the underlying mechanism is unclear. Neutrophil extracellular traps (NETs) have been recently shown to be involved in GPA. NETs may also be related to the natural regression of GPA.

Fluvastatin prevents the development of arthritis in env-pX rats via up-regulation of Rho GTPase-activating protein 12.

The pleiotropic effects of statins, including an antiarthritic potential, have been noted. This study aimed to determine the efficacy of statins on rheumatoid arthritis (RA) and clarify how statins affect its pathogenesis. Fluvastatin (500 µg/kg/day) or vehicle was given per os to env-pX rats, which carry the human T-cell leukemia virus type I env-pX gene and spontaneously develop destructive arthritis mimicking RA, for 30 days. Blood sampling and ultrasonography (US) of the ankle joints were conducted on days 0, 10, 20, and 30. On day 30, all rats were euthanized, and the ankle joints were subjected to histological analysis. To clarify how fluvastatin affects the pathogenesis of RA, comprehensive serum exosomal microRNA (miRNA) analysis was performed. Gene expression in the primary culture of synovial fibroblasts derived from arthritic rat and human and non-arthritic rat periarticular tissues was determined quantitatively by real-time reverse transcription-polymerase chain reaction (RT-PCR). As a result, the development of arthritis in env-pX rats was significantly suppressed by fluvastatin, which was evident from the viewpoints of serology, US imaging, and histology. Comprehensive serum exosomal miRNA analysis suggested that the expression of Rho GTPase-activating protein 12 (Arhgap12) was decreased in arthritic env-pX rats but increased with the administration of fluvastatin. Corresponding results were obtained by quantitative RT- PCR using primary culture of synovial fibroblasts. The collective findings suggest that fluvastatin prevents the development of arthritis in env-pX rats via the up-regulation of ARHGAP12. This study suggests that ARHGAP12 can be a possible therapeutic target of RA.

Formation and Disordered Degradation of Neutrophil Extracellular Traps in Necrotizing Lesions of Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis.

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is characterized by the production of ANCAs and systemic necrotizing vasculitis in small vessels. Disordered regulation of neutrophil extracellular traps (NETs) is critically involved in the pathogenesis of AAV. NETs are web-like DNA decorated with antimicrobial proteins; they are extruded from activated neutrophils. The principal degradation factor of NETs in vivo is DNase I; however, NETs resistant to DNase I can persist in tissues and can lead to the production of ANCAs. Deposition of NETs has been demonstrated in glomerular crescents and necrotizing vasculitis in AAV. Here, the amount of NETs in formalin-fixed, paraffin-embedded tissue sections was examined, and the results for AAV were compared with the results for diseases that should be distinguished from AAV. NETs were more abundant in necrotizing vasculitis of AAV than in non-ANCA-associated vasculitis, or in granulomatous angiitis. Pulmonary granulomas in AAV and non-ANCA-associated diseases were further studied. The amount of NETs was significantly greater in necrotizing granulomas of AAV than in granulomas of sarcoidosis without necrosis. Although NETs were formed in necrotizing granulomas of tuberculosis equivalently to those formed in AAV, they were more susceptible to degradation by DNase I than were NETs in AAV. The formation and disordered degradation of NETs in necrotizing lesions are characteristics of AAV and are possibly related to its pathogenesis.

Pathogenesis and therapeutic interventions for ANCA-associated vasculitis.

Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) affects systemic small vessels and is accompanied by the presence of ANCAs in the serum. This disease entity includes microscopic polyangiitis, granulomatosis with polyangiitis, eosinophilic granulomatosis with polyangiitis and drug-induced AAV. Similar to other autoimmune diseases, AAV develops in patients with a predisposing genetic background who have been exposed to causative environmental factors. The mechanism by which ANCAs cause vasculitis involves ANCA-mediated excessive activation of neutrophils that subsequently release inflammatory cytokines, reactive oxygen species and lytic enzymes. In addition, this excessive activation of neutrophils by ANCAs induces formation of neutrophil extracellular traps (NETs). Although NETs are essential elements in innate immunity, excessive NET formation is harmful to small vessels. Moreover, NETs are involved not only in ANCA-mediated vascular injury but also in the production of ANCAs themselves. Therefore, a vicious cycle of NET formation and ANCA production is considered to be involved in the pathogenesis of AAV. In addition to this role of NETs in AAV, some other important discoveries have been made in the past few years. Incorporating these new insights into our understanding of the pathogenesis of AAV is needed to fully understand and ultimately overcome this disease.

Brain-derived neurotrophic factor induces angiogenin secretion and nuclear translocation in human umbilical vein endothelial cells.

Brain-derived neurotrophic factor (BDNF) is a well-known humoral protein that induces growth of neurons. Recent studies have suggested that BDNF could act as an angiogenesis inducer similar to vascular endothelial growth factor (VEGF). Angiogenin is a strong mediator of angiogenesis. It has particular characteristics both as a secreted protein and a transcription factor. After being incorporated into the cytoplasm, angiogenin is immediately transferred to the nucleus and then mediates the angiogenic effects of angiogenesis inducers, including VEGF. The aim of this study is to determine the association between BDNF and angiogenin. At first, we determined the secretion of angiogenin from human umbilical vein endothelial cells (HUVEC) induced by BDNF with enzyme-linked immunosorbent assay. Next, we determined BDNF-induced nuclear translocation of angiogenin by immunofluorescent staining. In addition, we examined the mRNA expression of angiogenin in HUVEC before and after BDNF stimulation by quantitative reverse transcriptase-polymerase chain reaction. As a result, we noted that BDNF induced angiogenin secretion and nuclear translocation without an increase in the mRNA expression in HUVEC. Furthermore, we demonstrated that BDNF-induced HUVEC proliferation was significantly suppressed when neomycin, a specific inhibitor of nuclear translocation of angiogenin, was administered. These findings indicate that nuclear translocation of angiogenin is critically involved in BDNF-induced proliferation of HUVEC. In conclusion, angiogenin contributes to angiogenesis induced by BDNF.

CD1d-Restricted Type II NKT Cells Reactive With Endogenous Hydrophobic Peptides.

NKT cells belong to a distinct subset of T cells that recognize hydrophobic antigens presented by major histocompatibility complex class I-like molecules, such as CD1d. Because NKT cells stimulated by antigens can activate or suppress other immunocompetent cells through an immediate production of a large amount of cytokines, they are regarded as immunological modulators. CD1d-restricted NKT cells are classified into two subsets, namely, type I and type II. CD1d-restricted type I NKT cells express invariant T cell receptors (TCRs) and react with lipid antigens, including the marine sponge-derived glycolipid α-galactosylceramide. On the contrary, CD1d-restricted type II NKT cells recognize a wide variety of antigens, including glycolipids, phospholipids, and hydrophobic peptides, by their diverse TCRs. In this review, we focus particularly on CD1d-restricted type II NKT cells that recognize endogenous hydrophobic peptides presented by CD1d. Previous studies have demonstrated that CD1d-restricted type I NKT cells usually act as pro-inflammatory cells but sometimes behave as anti-inflammatory cells. It has been also demonstrated that CD1d-restricted type II NKT cells play opposite roles to CD1d-restricted type I NKT cells; thus, they function as anti-inflammatory or pro-inflammatory cells depending on the situation. In line with this, CD1d-restricted type II NKT cells that recognize type II collagen peptide have been demonstrated to act as anti-inflammatory cells in diverse inflammation-induction models in mice, whereas pro-inflammatory CD1d-restricted type II NKT cells reactive with sterol carrier protein 2 peptide have been demonstrated to be involved in the development of small vessel vasculitis in rats.

Type II Natural Killer T Cells that Recognize Sterol Carrier Protein 2 Are Implicated in Vascular Inflammation in the Rat Model of Systemic Connective Tissue Diseases.

We previously generated a rat model that developed systemic connective tissue diseases, including synovitis, myositis, and small-vessel vasculitis (SVV), and established a vascular endothelial cell-reactive T-cell clone, VASC-1, from the model. VASC-1 was determined to be a type II natural killer T-cell clone. In this study, we attempted to identify the antigen recognized by VASC-1. The monkey-derived cell line COS-7 was used because VASC-1 does not bind naturally to COS-7, although the amino acid sequences are well conserved between monkey CD1d and rat CD1d. We generated 98 COS-7 clones transfected with miscellaneous rat cDNA and screened them for VASC-1 binding. Consequently, we found one clone, 4D2, which could bind to VASC-1. Sequencing identified the rat cDNA introduced into 4D2 as sterol carrier protein 2 (SCP2). When VASC-1 was co-cultured with SCP2 knockdown rat vascular endothelial cells, VASC-1 binding was reduced significantly. Moreover, we designed a series of rat SCP2 peptides and introduced them into COS-7 cells. On the basis of VASC-1 binding and proliferation, we revealed that the peptide rSCP2518-532 included the epitope recognized by VASC-1. Furthermore, immunization with rSCP2518-532 accelerated the development of SVV in the rat model. The collective findings suggest that type II natural killer T cells reactive with autologous SCP2 are implicated in vascular inflammation in the rat model.

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.

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.

Mechanism of Fcγ receptor-mediated trogocytosis-based false-positive results in flow cytometry.

The whole blood erythrocyte lysis method is the most common protocol of sample preparation for flow cytometry (FCM). Although this method has many virtues, our recent study has demonstrated false-positive results when surface markers of monocytes were examined by this method due to the phenomenon called Fcγ receptor (FcγR)-mediated trogocytosis. In the present study, similar FcγR-mediated trogocytosis-based false-positive results have been demonstrated when granulocytes were focused on instead of monocytes. These findings indicated that not only monocytes but also granulocytes, the largest population with FcγR expression in peripheral blood, could perform FcγR-mediated trogocytosis. Since the capacity of FcγR-mediated trogocytosis was different among blood samples, identification of factors that could regulate the occurrence of FcγR-mediated trogocytosis should be important for the quality control of FCM. Our studies have suggested that such factors are present in the serum. In order to identify the serum factors, we employed the in vitro model of FcγR-mediated trogocytosis using granulocytes. Investigation with this model determined the serum factors as heat-labile molecules with molecular weight of more than 100 kDa. Complements in the classical pathway were initially assumed as candidates; however, the C1 inhibitor did not yield an obvious influence on FcγR-mediated trogocytosis. On the other hand, although immunoglobulin ought to be resistant to heat inactivation, the inhibitor of human anti-mouse antibodies (HAMA) effectively blocked FcγR-mediated trogocytosis. Moreover, the inhibition rates were significantly higher in HAMA(high) serum than HAMA(low) serum. The collective findings suggested the involvement of heterophilic antibodies such as HAMA in the mechanism of false-positive results in FCM due to FcγR-mediated trogocytosis.