scholarly journals Neutrophil Extracellular Traps Promote Aberrant Macrophages Activation in Behçet’s Disease

2021 ◽  
Vol 11 ◽  
Author(s):  
Lu Li ◽  
Xin Yu ◽  
Jinjing Liu ◽  
Zhimian Wang ◽  
Chaoran Li ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Behcet’S Disease ◽  
Behçet's Disease ◽  
Neutrophil Extracellular Traps ◽  
Histone H4 ◽  
Behcet's Disease ◽  
Extracellular Traps ◽  
Tnf Α ◽  
Ifn Γ

Neutrophil extracellular traps (NETs) are upregulated and promote thrombosis in Behçet’s disease (BD). However, whether NETs promote autoinflammation in BD remains unclear. This study aimed to investigate the potential role of NETs in promoting macrophage activation in BD. Firstly, we quantified NETs by measuring double-stranded DNA (dsDNA) using PicoGreen and calculating the proportion of NETosis. Then macrophages were stimulated with BD- or healthy controls (HC)-derived NETs, and IL-8 and TNF-α production and IFN-γ+ CD4+ T cells differentiation were measured using ELISA and flow cytometry, respectively. The protein components in NETs were analyzed by western blot. Macrophages were stimulated with Histone H4 neutralized NETs, and IL-8 and TNF-α production were measured using ELISA. The level of 8-hydroxydeoxyguanosine (8-OHdG) DNA in NETs was measured using ELISA. The levels of reactive oxygen species (ROS) in serum and neutrophils were measured using ROS probes by a microplate reader and flow cytometry. We found that circulating NETs and neutrophil-derived NETs were significantly higher in BD than HC. BD NETs stimulated macrophages produced higher levels of IL-8 and TNF-α, and promoted IFN-γ+ CD4+ T cells differentiation. BD NETs were enriched in Histone H4, and neutralizing Histone H4 abrogated the BD NETs-mediated IL-8 production by macrophages, but not TNF-α. Also, BD neutrophils produced more 8-OHdG DNA than HC neutrophils, and the percentage of 8-OHdG DNA in dsDNA from BD neutrophils was also higher than that of HC neutrophils. The ROS levels in serum and neutrophils were both higher in BD than HC. Our findings suggested that excessive BD NETs promoted macrophages activation and facilitated IFN-γ+ CD4+ T cells differentiation. Higher levels of Histone H4 and oxidized DNA in BD NETs might mediate macrophages hyperactivation.

scholarly journals FRI0003 ELEVATED HISTONE H4 IN NEUTROPHIL EXTRACELLULAR TRAPS PROMOTES MACROPHAGE ACTIVATION IN BEHÇET’S DISEASE

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 572.1-573
Author(s):  
L. LI ◽  
X. Yu ◽  
J. Liu ◽  
H. Chen ◽  
W. Zheng
Keyword(s):  
Behçet’S Disease ◽  
Macrophage Activation ◽  
Behcet’S Disease ◽  
Behçet's Disease ◽  
Neutrophil Extracellular Traps ◽  
Histone H4 ◽  
Behcet's Disease ◽  
Extracellular Traps ◽  
Tnf Α

Background:Neutrophil-released neutrophil extracellular traps (NETs) are upregulated and promote autoinflammation and thrombosis in Behçet’s disease (BD), a multi-system inflammatory disease with unknown etiology1,2. However, whether NETs promote macrophage activation in BD remains unclear.Objectives:To investigate the potential role of NETs in promoting aberrant macrophage activation in BD.Methods:We quantified NETs by measuring dsDNA using ELISA and immunofluorescence. Macrophages were stimulated with BD- and healthy controls (HC)-derived NETs, and IL-8 and TNF-α production were measured by ELISA. NETs-stimulated macrophages were incubated with naive CD4+T cells, and Th1 cell differentiation was examined on day 7 by flow cytometry. Histones H1, H2A, H2B, H3, H4, S100A8 and neutrophil elastase in NETs were analyzed by western blot. Macrophages were stimulated with anti-Histone 4 antibody-treated NETs, and IL-8 production was measured by ELISA.Results:Circulating NETs (2336±534 ng/ml vs. 1472±549 ng/ml,P=0.0008) and neutrophil-derived NETs (909.2±485.2 ng/ml vs. 582.4±199.2 ng/ml,P=0.0108) were significantly higher in BD patients compared with those in HC. BD NETs stimulated macrophages to produce a higher level of IL-8 (17±4 ng/ml vs. 13±4 ng/ml,P=0.0474) and TNF-α (166±61 pg/ml vs. 102±48 pg/ml,P=0.0132) than HC NETs. Moreover, BD NETs promoted macrophages to facilitate Th1 differentiation than HC NETs (33±10% vs. 24±7%,P=0.0398). Western blot analysis revealed more Histone H4 (289076 (144365, 544038) IOD values vs. 42121 (6958, 129625) IOD values,P=0.0286), but not Histones H1, H2A, H2B, H3, S100A8 or neutrophil elastase in BD NETs compared to HC NETs. Importantly, neutralizing Histone H4 abrogated the BD NETs-stimulated IL-8 overproduction by macrophages (9.99±2.07 ng/ml vs. 13.95±2.91 ng/ml,P=0.021).Conclusion:BD NETs promoted macrophages activation, which might be mediated by a higher level of Histone H4.References:[1]Safi R., Kallas R., Bardawil T., et al. Neutrophils contribute to vasculitis by increased release of neutrophil extracellular traps in Behçet’s disease. J. Dermatol. Sci. 2018;92:143–150.[2]Le Joncour a, Martos r, Loyau s, et al. Critical role of neutrophil extracellular traps (NETs) in patients with Behcet’s disease. Ann Rheum Dis 2019;78:1274–1282.Disclosure of Interests:None declared

Critical role of neutrophil extracellular traps (NETs) in patients with Behcet’s disease

2019 ◽  
Vol 78 (9) ◽  
pp. 1274-1282 ◽  
Author(s):  
Alexandre Le Joncour ◽  
Raphael Martos ◽  
Stephane Loyau ◽  
Nicolas Lelay ◽  
Antoine Dossier ◽  
...  
Keyword(s):  
Behçet’S Disease ◽  
Thrombin Generation ◽  
Behcet’S Disease ◽  
Behçet's Disease ◽  
Neutrophil Extracellular Traps ◽  
Dna Complexes ◽  
Thrombotic Risk ◽  
Behcet's Disease ◽  
Extracellular Traps

ObjectivesBehçet’s disease (BD) is a chronic systemic vasculitis. Thrombosis is a frequent and life-threatening complication. The pathogenesis of BD is poorly understood and evidence supporting a role for primed neutrophils in BD-associated thrombotic risk is scant. To respond to inflammatory insults, neutrophils release web-like structures, known as neutrophil extracellular traps (NETs), which are prothrombotic. We evaluated the role of NETs and markers of NETs in BD.MethodsBlood samples were collected from patients with BD, according to the International Study Group Criteria for Behçet's disease, and healthy donors (HD). NET components, including cell-free DNA (CfDNA) and neutrophil enzymes myeloperoxidase (MPO), were assessed in serum or in purified neutrophils from patients with BD and HD.ResultsPatients with active BD had elevated serum cfDNA levels and MPO-DNA complexes compared with patients with inactive BD and to HD. In addition, levels of cfDNA and MPO-DNA complexes were significantly higher in patients with BD with vascular involvement compared with those without vascular symptoms. Purified neutrophils from patients with BD exhibited spontaneous NETosis compared with HD. Thrombin generation in BD plasma was significantly increased and positively correlated with the levels of MPO-DNA complexes and cfDNA. Importantly, DNAse treatment significantly decreased thrombin generation in BD plasma but not in HD plasma. In addition, biopsy materials obtained from patients with BD showed NETs production in areas of vasculitic inflammation and thrombosis.ConclusionsOur data show that NETs and markers of NETS levels are elevated in patients with BD and contribute to the procoagulant state. Targeting NETs may represent a potential therapeutic target for the reduction or prevention of BD-associated thrombotic risk.

Decreased expression of A20 is associated with ocular Behcet’s disease (BD) but not with Vogt-Koyanagi-Harada (VKH) disease

2018 ◽  
Vol 102 (8) ◽  
pp. 1167-1172 ◽  
Author(s):  
Yue He ◽  
Chaokui Wang ◽  
Guannan Su ◽  
Bolin Deng ◽  
Zi Ye ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Behçet’S Disease ◽  
Behcet’S Disease ◽  
Mononuclear Cells ◽  
Behçet's Disease ◽  
Normal Subjects ◽  
Peripheral Blood Mononuclear ◽  
Behcet's Disease ◽  
Vkh Disease

PurposeA20 is a ubiquitously expressed and inducible cytosolic protein, which plays an important role in the negative regulation of inflammation and immunity. In this study, we investigated the role of A20 in Behcet’s disease (BD) and Vogt-Koyanagi-Harada (VKH) disease.MethodsThe levels of A20 in peripheral blood mononuclear cells (PBMCs) and dendritic cells (DCs) were detected in BD patients with active and inactive uveitis, VKH patients with active and inactive uveitis, and normal subjects, respectively, by real-time PCR. The effect of A20 silencing was performed by transduction of DCs with adenovirus containing an A20 shRNA vector. The effect of A20 silencing on the maturation of DCs was measured by flow cytometry. The effect of A20 silencing of DCs on cytokine production by DCs and CD4+ T cells was analysed by ELISA. The phosphorylation levels of JNK, p38 and ERK1/2 were detected by flow cytometry.ResultsThe expression of A20 was markedly decreased in PBMCs and DCs obtained from BD patients with active uveitis, but not in patients with VKH disease as compared with normal controls. Silencing of A20 significantly increased the levels of interleukin (IL)-1β and IL-6 and suppressed the expression of the anti-inflammatory cytokines IL-10 and IL-27. Downregulation of A20 also led to an increase in IL-17 production by CD4+ T cells. However, downregulation of A20 in DCs did not have an effect on cell surface markers such as CD40, CD80, CD83, CD86 and HLA-DR. Silencing of A20 caused an increased expression of phospho-JNK and phospho-MAPK p38 but not phospho-ERK1/2.ConclusionsThis study showed that the expression of A20 was decreased in BD patients with active uveitis but not in VKH disease. Decreased expression of A20 may lead to an enhanced activation of proinflammatory Th17 cells, causing a reactivation of BD.

scholarly journals Saliva and Serum Cytokine Profiles During Oral Ulceration in Behçet’s Disease

2021 ◽  
Vol 12 ◽  
Author(s):  
Tanya Novak ◽  
Mojgan Hamedi ◽  
Lesley Ann Bergmeier ◽  
Farida Fortune ◽  
Eleni Hagi-Pavli
Keyword(s):  
Behcet’S Disease ◽  
Behçet's Disease ◽  
Serum Cytokine ◽  
Oral Ulceration ◽  
Behcet's Disease ◽  
Cytokine Profiles ◽  
Oral Ulcers ◽  
Tnf Α ◽  
Cytokine Levels ◽  
Ifn Γ

Behçet’s disease (BD) is a chronic, multi-systemic disorder of unknown aetiology typified by recurrent oral and genital mucocutaneous lesions, uveitis and vasculitis. Innate and adaptive immune system dysregulation has been implicated in pathogenesis with alterations in serum cytokine profiles. Few studies have investigated salivary cytokines in BD, despite more than 90% of BD patients first presenting with oral ulceration. The aim of this pilot study was twofold; firstly to investigate whether cytokine levels in matched serum and saliva samples show a differential profile in BD (with and without oral ulcers), recurrent aphthous stomatitis (RAS) and healthy controls (HCs), and secondly, to explore if any differential profiles in serum and/or saliva could provide a panel of cytokines with diagnostic and therapeutic potential for BD. Concentrations of 12 cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-17A, IFN-γ, TNF-α, TNF-β) were measured using the Human Th1/Th2 11-Plex FlowCytomix™ kit with IL-17A, in BD (N=20), RAS (N=6) and HCs (N=10). A differential range of cytokines was detected in serum and saliva with the majority of cytokine levels higher in saliva. The most prevalent salivary cytokines were IL-1β, IL-2, IL-8, IL-10 and TNF-α present in all samples in contrast to serum where the most prevalent cytokine detected was IL-8 (91.9%). The least abundant cytokine was IFN-γ in both saliva (43.2%) and serum (2.7%). After normalizing saliva for protein content, BD patients with oral ulcers (BD-MA) had significantly higher levels of salivary IL-1β (p=0.01), IL-8 (p=0.02), TNF-α (p=0.004) and IL-6 (p=0.01) than HCs. Notably, BD patients without oral ulcers (BD-MQ) also had significantly higher salivary IL-1β, IL-8 and TNF-α (p ≤ 0.05) than HCs. During relapsed (BD-RE) and quiet (BD-Q) systemic episodes, salivary IL-β and TNF-α were also significantly increased with IL-8 significantly higher only in BD-Q (p=0.02). BD oral ulcers signify a potential reactivation of systemic inflammation. Identifying cytokines released during asymptomatic episodes and oral ulceration might lead to targeted drug therapy to prevent recurrent oral ulcers and possible disease relapse. This is the first study to report salivary cytokine levels in BD. The detectable levels suggests cytokine profiling of BD saliva may provide an alternative, less invasive, sensitive procedure for frequent monitoring of disease activity and progression.

Neutrophils contribute to vasculitis by increased release of neutrophil extracellular traps in Behçet's disease

2018 ◽  
Vol 92 (2) ◽  
pp. 143-150 ◽  
Author(s):  
Rémi Safi ◽  
Romy Kallas ◽  
Tara Bardawil ◽  
Carl Joe Mehanna ◽  
Ossama Abbas ◽  
...  
Keyword(s):  
Behçet’S Disease ◽  
Behcet’S Disease ◽  
Behçet's Disease ◽  
Neutrophil Extracellular Traps ◽  
Behcet's Disease ◽  
Extracellular Traps

scholarly journals AB0531 GOLIMUMAB IN THE TREATMENT OF SEVERE AND/OR REFRACTORY VASCULO-BEHÇET’S DISEASE: A SINGLE-CENTRE EXPERIENCE IN CHINA

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 1562.1-1563
Author(s):  
L. Sun ◽  
J. Liu ◽  
W. Zheng
Keyword(s):  
Aortic Regurgitation ◽  
Behçet’S Disease ◽  
Behcet’S Disease ◽  
Behçet's Disease ◽  
High Dose ◽  
Efficacy And Safety ◽  
Behcet's Disease ◽  
Severe Aortic Regurgitation ◽  
Tnf Α

Background:Vascular involvement is one of the leading causes of mortality and morbidity in Behcet’s Disease (BD)1. Surgical treatments are difficult for Vaculo-BD (VBD) patients due to the high risk of serious postoperative complications without effective and promptly perioperative immunotherapy2, 3. Anti-tumor necrosis factor alpha (TNF-α) therapy has been reported as a potential treatment in severe VBD, e.g. infliximab (IFX) and adalimumab (ADA). However, only few case reports are available regarding the fully humanized monoclonal antibody to TNF-α, golimumab (GOL), in the management of VBD4.Objectives:The objective of this study was to report the efficacy and safety of GOL for the treatment of severe and/or refractory VBD.Methods:We retrospectively analyzed the efficacy and safety profile of patients with severe and/or refractory VBD treated with GOL in our medical center between 2018 to 2020.Results:Nine VBD patients (8 male and 1 female) were enrolled, with a mean age and median course of 37±8.6 years and 72 months (range 12 to 300), respectively. Cardiac involvements (severe aortic regurgitation secondary to BD) were presented in 7 patients, including 2 patients with post-operative paravalvular leakage (PVL) after aortic valve replacement surgery. Multiple vascular lesions were documented in the other 2 patients, including one patient with life-threatening multiple pulmonary aneurysms, pulmonary thromboembolism and recurrent deep vein thrombosis, and another patient with abdominal aortic pseudoaneurysm and multiple artery stenosis and occlusion. Prior to GOL therapy, all patients experienced disease progression despite high-dose glucocorticoids combined with multiple immunosuppressants. Moreover, seven patients required effective and fast control of inflammation and a decrease of glucocorticoid dose during the perioperative period. They were treated with GOL, 50mg every 4 weeks, in combination with background low-or medium-dose glucocorticoids and immunosuppressants, for a median of 6 (range 3-15) months. After a mean duration of follow-up of 10 (range 2-6) months, all patients achieved improvement both in clinical symptoms and serum inflammation markers. The ESR level [4.88±4.94 mm/h vs 31.13±31.78mm/h, P<0.01] and CRP level [1.9 (0.11-3.73)mg/L vs 24.3 (0.4-85.57)mg/L, P<0.01] significantly decreased. The dosage of glucocorticoid[10 (0-15) vs 40 (0-100)mg/d, P<0.01] effectively tapered, indicating a potential steroid-sparing effect. No newly-onset aneurysm and recurrent venous thrombosis were observed. Also, one patient had a marked reduction in size and number of pulmonary aneurysms. No post-operative PVL was observed in the five patients after Bentall operation with a median follow-up of 10 months. One patient with severe aortic regurgitation remained stable and without surgical intervention with the treatment of GOL for 16 months. No severe complication occurred in one patient after underwent endovascular repair of abdominal aorta for 8 months. GOL was well-tolerated, and no serious adverse event was observed.Conclusion:Our results suggested that GOL is safe and effective for the treatment of patients with severe and / or refractory VBD. Further controlled studies are warranted to confirm the therapeutic potential of GOL in VBD patients.Disclosure of Interests:None declared

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