scholarly journals Repeated Social Defeat Exaggerates Fibrin-Rich Clot Formation by Enhancing Neutrophil Extracellular Trap Formation via Platelet–Neutrophil Interactions

Cells ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 3344
Author(s):  
Takeshi Sugimoto ◽  
Hiroyuki Yamada ◽  
Naotoshi Wada ◽  
Shinichiro Motoyama ◽  
Makoto Saburi ◽  
...  
Keyword(s):  
Flow Cytometric Analysis ◽  
Social Defeat ◽  
Arterial Injury ◽  
Physical Contact ◽  
Clot Formation ◽  
Neutrophil Extracellular Trap ◽  
Positive Staining ◽  
Antibody Treatment ◽  
Extracellular Trap ◽  
The Impact

Depression is an independent risk factor for cardiovascular disease (CVD). We have previously shown that repeated social defeat (RSD) exaggerates atherosclerosis development by enhancing neutrophil extracellular trap (NET) formation. In this study, we investigated the impact of RSD on arterial thrombosis. Eight-week-old male wild-type mice (C57BL/6J) were exposed to RSD by housing with larger CD-1 mice in a shared home cage. They were subjected to vigorous physical contact daily for 10 consecutive days. After confirming depression-like behaviors, mice underwent FeCl3-induced carotid arterial injury and were analyzed after 3 h. Although the volume of thrombi was comparable between the two groups, fibrin(ogen)-positive areas were significantly increased in defeated mice, in which Ly-6G-positive cells were appreciably co-localized with Cit-H3-positive staining. Treatment with DNase I completely diminished exaggerated fibrin-rich clot formation in defeated mice. Flow cytometric analysis showed that neutrophil CD11b expression before FeCl3 application was significantly higher in defeated mice than in control mice. In vitro NET formation induced by activated platelets was significantly augmented in defeated mice, which was substantially inhibited by anti-CD11b antibody treatment. Our findings demonstrate that RSD enhances fibrin-rich clot formation after arterial injury by enhancing NET formation, suggesting that NET can be a new therapeutic target in depression-related CVD.

Abstract 14458: Repeated Social Defeat Exaggerates Fibrin-rich Clot Formation in Fecl3 Induced Arterial Thrombosis Mice Model by Enhancing Nets Formation

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Takeshi Sugimoto ◽  
Hiroyuki Yamada ◽  
Hiroshi Kubota ◽  
Daisuke Miyawaki ◽  
Noriyuki Wakana ◽  
...  
Keyword(s):  
Arterial Thrombosis ◽  
Flow Cytometric Analysis ◽  
Social Defeat ◽  
Arterial Injury ◽  
Physical Contact ◽  
Clot Formation ◽  
Positive Staining ◽  
Double Positive ◽  
The Impact

Background and Objective: Depression is an independent risk factor of cardiovascular disease (CVD). We have recently shown that repeated social defeat (RSD) exaggerates atherosclerosis development by enhancing neutrophil extracellular traps (NETs) formation. Here, we investigated the impact of RSD on arterial thrombosis. Methods and Results: Eight-week-old male WT mice were exposed to RSD by housing with a larger CD-1 mouse in a shared home cage. They were subjected to vigorous physical contact daily for 10 consecutive days. Control mice were housed in the same gage without physical contact. After social interaction test to confirm depressive-like behaviors, defeated mice (19 of 31) and control mice (12 of 14) were underwent arterial injury at 10 wks of age. A filter paper saturated with 10% FeCl3 was applied on the adventitial surface of left carotid artery for 3 min and analyzed 3 hrs later. The volume of thrombi was comparable between the two groups. However, fibrinogen/fibrin-positive areas in immunofluorescent images significantly increased in defeated mice (48.8% vs. 27.8%, p < 0.01). The number of Ly-6G-positive cells in thrombi was markedly higher in defeated mice (878/mm2 vs. 144/mm2, p < 0.05). Further, Ly-6G-positive cells were co-localized with neutrophil elastase, Cit-H3, and CD42b-positive staining. Percentage of CD42b-positive area in thrombi and in vitro platelets aggregations induced by ADP or collagen were comparable between the two groups. Treatment with DNase I completely diminished the exaggerated fibrin-rich clot formation in defeated mice to an extent similar to that in control mice (22.3% vs. 25.7%, p = ns), although the number of Ly-6G-positive cells in thrombi was not affected. We therefore examined the vulnerability to NETs formation induced by thrombin-activated platelets. Flow cytometric analysis showed that in vitro NETs formation assessed by Cit-H3/MPO double-positive cells was significantly higher in defeated mice (20.7% vs 12.5%, p < 0.01). Conclusions: Our findings demonstrate that RSD enhances fibrin-rich clot formation after arterial injury by enhancing NETs formation via platelet-neutrophil interactions, suggesting that NETosis could be a new therapeutic target in depression-related CVD development.

scholarly journals Repeated social defeat exaggerates fibrin-rich clot formation in FeCl3-induced arterial thrombosis mouse model by enhancing NETs formation via modulation of neutrophil functional properties

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
T Sugimoto ◽  
H Yamada ◽  
H Kubota ◽  
D Miyawaki ◽  
M Saburi ◽  
...  
Keyword(s):  
Functional Properties ◽  
Arterial Thrombosis ◽  
Social Defeat ◽  
Arterial Injury ◽  
Physical Contact ◽  
Clot Formation ◽  
Positive Staining ◽  
Double Positive ◽  
The Impact

Abstract Background and objective Depression is an independent risk factor of cardiovascular disease (CVD). We have recently shown that repeated social defeat (RSD) precipitates depressive-like behaviors in apoE−/− mice and exaggerates atherosclerosis development by enhancing neutrophil extracellular traps (NETs) formation. Here, we investigated the impact of RSD on arterial thrombosis. Methods and results Eight-week-old male WT mice were exposed to RSD by housing with a larger CD-1 mouse in a shared home cage. They were subjected to vigorous physical contact daily for 10 consecutive days. Control mice were housed in the same gage without physical contact. After social interaction test to confirm depressive-like behaviors, defeated mice (19 of 31) and control mice (12 of 14) were underwent arterial injury at 10 wks of age. A filter paper saturated with 10% FeCl3 was applied on the adventitial surface of left carotid artery for 3 min and analyzed 3 hrs later. The volume of thrombi was comparable between the two groups. However, fibrinogen/fibrin-positive areas in immunofluorescent images significantly increased in defeated mice (27.8% vs. 48.8%, p&lt;0.01). The number of Ly-6G-positive cells in thrombi was markedly higher in defeated mice (144/mm2 vs. 878/mm2, p&lt;0.05). Further, Ly-6G-positive cells were almost accumulated at the inner surface of injured artery, which were co-localized with neutrophil elastase, Cit-H3, and CD41-positive staining. Treatment with DNase I completely diminished the exaggerated fibrin-rich clot formation in defeated mice to an extent similar to that in control mice (25.7% vs. 22.3%, p = ns), without affecting the volume of thrombi and accumulation of Ly-6G-positive cells. Given that platelet aggregations induced by ADP or collagen were comparable between the two groups, neutrophil functional properties primarily contribute to the exaggerated fibrin-rich clot formation in defeated mice. We then examined neutrophil subset and vulnerability to NETs formation. At 3 hrs after FeCl3 application, the numbers of immature neutrophils (Ly6Glo/+CXCR2-) were comparable between the two groups in both bone marrow (BM) and peripheral blood (PB). In contrast, the number of PB mature neutrophils (Ly6G+CXCR2+) was markedly higher in defeated mice than control mice (580±68 /μl vs. 1265±114, p&lt;0.01). We next examined in vitro NETs formation upon PMA in BM mature neutrophils by FACS and nucleic acid staining. The percentage of double-positive cells (Cit-H3, MPO) was significantly higher in defeated mice (7.5% vs. 10.2%, p&lt;0.05), as well as SYTOX green-positive cells expelling DNA fibers (8.1% vs. 11.8%, p&lt;0.05). Conclusions Our findings demonstrate for the first time that repeated social defeat enhances fibrin-rich clot formation after arterial injury by enhancing NETs formation via modulation of neutrophil functional properties, suggesting that NETosis could be a new therapeutic target in depression-related CVD development. Funding Acknowledgement Type of funding source: None

P740Repeated social defeat exaggerates fibrin-rich clot formation in FeCl3-induced arterial thrombosis mice model by enhancing NETs formation

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
T Sugimoto ◽  
H Yamada ◽  
H Kubota ◽  
D Miyawaki ◽  
S Motoyama ◽  
...  
Keyword(s):  
Arterial Thrombosis ◽  
Social Defeat ◽  
Arterial Injury ◽  
Physical Contact ◽  
Clot Formation ◽  
Positive Staining ◽  
Mice Model ◽  
Cross Sectional ◽  
Atherosclerosis Development ◽  
The Impact

Abstract Background and objective Depression is an independent risk factor of cardiovascular disease (CVD). We have recently shown that repeated social defeat (RSD) precipitates depressive-like behaviorsin apoE−/− mice and exaggerates atherosclerosis development by enhancing neutrophil extracellular traps (NETs) formation (BBRC 2018; 500:490). Here, we investigated the impact of RSD on arterial thrombosis. Methods and results Eight-week-old male WT mice were exposed to RSDby housing with a larger CD-1 mouse in a shared home cage. They were subjected to vigorous physical contact daily for 10 consecutive days. Control mice were housed in the same gage without physical contact. After social interaction testto confirm depressive-like behaviors, defeated mice (19 of 31) and control mice (12 of 14) were underwent arterial injury at 10 wks of age. A filter paper saturated with 10% FeCl3was applied on the adventitial surface of left carotid artery for 3 min and analyzed 3 hrs later. The volume of thrombi calculated by summing8–15 frozen cross-sectional images, each separated by 200 μm, was comparable between the 2 groups. However, fibrinogen/fibrin-positive areas in immunofluorescent images were significantly increased in defeated mice (27.8% vs. 48.8%, Control vs. Defeat, P<0.01).The numberof Ly-6G-positive cells in thrombi was markedly higher in defeated mice (144/mm2 vs. 878/mm2, Control vs. Defeat, P<0.05). Further, Ly-6G-positive cells were almost accumulated at the inner surface of injured artery, which were co-localized with neutrophil elastase, Cit-H3, and CD41-positive staining. Treatment with DNase Icompletely diminished the exaggerated fibrin-rich clot formation in defeated miceto a similar extent of control mice (25.7% vs. 22.3%, Control vs. Defeat, P= NS), while the volume of thrombi and number of Ly-6G-positive cells in thrombi were comparable between the 2 groups even afterDNase I treatment. Platelet aggregations induced by ADP or collagen were comparable between the 2 groups, suggesting that NETs formation primarily contributes to the exaggerated fibrin-rich clot formation in defeated mice. Conclusions Our findings demonstrate for the first time that repeated social defeat enhances fibrin-rich clot formation after arterial injury by enhancing NETs formation, suggesting that NETosis could be a new therapeutic target in depression-related CVD development.

scholarly journals A reverse translational study on the effect of rituximab, rituximab plus belimumab, or bortezomib on the humoral autoimmune response in SLE

Rheumatology ◽  
2020 ◽  
Vol 59 (10) ◽  
pp. 2734-2745 ◽  
Author(s):  
Laura S van Dam ◽  
Zgjim Osmani ◽  
Sylvia W A Kamerling ◽  
Tineke Kraaij ◽  
Jaap A   Bakker ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Randomized Clinical Trials ◽  
Ex Vivo ◽  
Neutrophil Extracellular Trap ◽  
Autoimmune Response ◽  
High Avidity ◽  
Trap Formation ◽  
Extracellular Trap ◽  
The Impact

Abstract Objectives SLE is a severe autoimmune disease characterized by autoreactive B cells and IC formation, which causes systemic inflammation. B cell–targeted therapy could be a promising treatment strategy in SLE patients; nevertheless, randomized clinical trials have not always been successful. However, some groups have demonstrated beneficial effects in severe SLE patients with off-label rituximab (RTX) with belimumab (BLM), or bortezomib (BTZ), which targeted different B cells subsets. This study assembled sera from SLE cohorts treated with RTX+BLM (n = 15), BTZ (n = 11) and RTX (n = 16) to get an in-depth insight into the immunological effects of these therapies on autoantibodies and IC formation. Methods Autoantibodies relevant for IC formation and the avidity of anti-dsDNA were determined by ELISA. IC-mediated inflammation was studied by complement levels and ex vivo serum-induced neutrophil extracellular trap formation. Results Reductions in autoantibodies were observed after all approaches, but the spectrum differed depending upon the treatment. Specifically, only RTX+BLM significantly decreased anti-C1q. Achieving seronegativity of ≥1 autoantibody, specifically anti-C1q, was associated with lower disease activity. In all SLE patients, the majority of anti-dsDNA autoantibodies had low avidity. RTX+BLM significantly reduced low-, medium- and high-avidity anti-dsDNA, while RTX and BTZ only significantly reduced medium avidity. IC-mediated inflammation, measured by C3 levels and neutrophil extracellular trap formation, improved after RTX+BLM and RTX but less after BTZ. Conclusion This study demonstrated the impact of different B cell–targeted strategies on autoantibodies and IC formation and their potential clinical relevance in SLE.

scholarly journals The impact of neutrophil extracellular trap from patients with systemic lupus erythematosus on the viability, CD11b expression and oxidative burst of healthy neutrophils

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Alimohammad Fatemi ◽  
Razieh Alipour ◽  
Hossein Khanahmad ◽  
Fereshteh Alsahebfosul ◽  
Alireza Andalib ◽  
...  
Keyword(s):  
Systemic Lupus Erythematosus ◽  
Lupus Erythematosus ◽  
Oxidative Burst ◽  
Neutrophil Extracellular Trap ◽  
Systemic Lupus ◽  
Cd11b Expression ◽  
Cell Mortality ◽  
Extracellular Trap ◽  
The Impact

Abstract Background NET (neutrophil extracellular trap) has been shown to directly influence inflammation; in SLE (systemic lupus erythematosus), it is reportedly a plausible cause for the broken self-tolerance that contributes to this pathology. Meanwhile, the role of NET is not easily explicable, and there is a serious discrepancy in the role of NET in SLE pathology and generally inflammation; in particular, the interactions of neutrophils with NET have been rarely inspected. This study evaluates the effect of NET on neutrophils in the context of SLE. The neutrophils were incubated by the collected NET (from SLE patients and healthy controls) and their expression of an activation marker, viability and oxidative burst ability were measured. Results The level of cell mortality, CD11b expression and the oxidative burst capacity were elevated in NET-treated neutrophils. Also, the elevation caused by the SLE NET was higher than that produced by the healthy NET. Conclusion The decreased neutrophil viability was not due to the increase in apoptosis; rather, it was because of the augmentation of other inflammatory cell-death modes. The upregulation of CD11b implies that NET causes neutrophils to more actively contribute to inflammation. The increased oxidative burst capacity of neutrophils can play a double role in inflammation. Overall, the effects induced by NET on neutrophils help prolong inflammation; accordingly, the NET collected from SLE patients is stronger than the NET from healthy individuals.

scholarly journals Platelet count reduction during in vitro membrane oxygenation affects platelet activation, neutrophil extracellular trap formation and clot stability, but does not prevent clotting

Perfusion ◽  
2021 ◽  
pp. 026765912198923
Author(s):  
Patrick Winnersbach ◽  
Jan Rossaint ◽  
Eva M. Buhl ◽  
Smriti Singh ◽  
Jonas Lölsberg ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Thrombus Formation ◽  
Clot Formation ◽  
Neutrophil Extracellular Trap ◽  
Bleeding Complications ◽  
In Vitro Test ◽  
Membrane Oxygenation ◽  
The Impact ◽  
Clot Stability

Introduction: Due to improved technology and increased application the mortality during extracorporeal membrane oxygenation (ECMO) is constantly declining. Nevertheless, complications including haemorrhage or thrombus formation remain frequent. Local mitigation of coagulation within an ECMO system to prevent thrombus formation on ECMO components and optimizing systemic anticoagulation is an approach to reduce clotting and bleeding complications at once. Foreign surfaces of ECMO systems, activate platelets (PLTs), which besides their major role in coagulation, can trigger the formation of neutrophil extracellular traps (NETs) contributing to robust thrombus formation. The impact of a reduced PLT count on PLT activation and NET formation is of paramount importance and worth investigating. Methods: In this study platelet poor (PLT–) and native (PLT+) heparinized human blood was circulated in two identical in vitro test circuits for ECMO devices for 6 hours. PLT reduction was achieved by a centrifugation protocol prior to the experiments. To achieve native coagulation characteristics within the test circuits, the initial heparin dose was antagonized by continuous protamine administration. Results: The PLT– group showed significantly lower platelet activation, basal NET formation and limited clot stability measured via thromboelastometry. Fluorescent and scanning electron microscope imaging showed differences in clot composition. Both groups showed equal clot formation within the circuit. Conclusions: This study demonstrated that the reduction of PLTs within an ECMO system is associated with limited PLT activation and NET formation, which reduces clot stability but is not sufficient to inhibit clot formation per se.

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