Neutrophil extracellular traps regulate ischemic stroke brain injury

Ischemic stroke prompts a strong inflammatory response, which is associated with exacerbated outcomes. In this study, we investigated mechanistic regulators of neutrophil extracellular trap (NET) formation in stroke and whether they contribute to stroke outcomes. NET-forming neutrophils were found throughout brain tissue of ischemic stroke patients, and elevated plasma NET biomarkers correlated with worse stroke outcomes. Additionally, we observed increased plasma and platelet surface–expressed high-mobility group box 1 (HMGB1) in stroke patients. Mechanistically, platelets were identified as the critical source of HMGB1 that caused NETs in the acute phase of stroke. Depletion of platelets or platelet-specific knockout of HMGB1 significantly reduced plasma HMGB1 and NET levels after stroke, and greatly improved stroke outcomes. We subsequently investigated the therapeutic potential of neonatal NET-inhibitory factor (nNIF) in stroke. Mice treated with nNIF had smaller brain infarcts, improved long-term neurological and motor function, and enhanced survival after stroke. nNIF specifically blocked NET formation without affecting neutrophil recruitment after stroke. Importantly, nNIF also improved stroke outcomes in diabetic and aged mice and was still effective when given 1 hour after stroke onset. These results support a pathological role for NETs in ischemic stroke and warrant further investigation of nNIF for stroke therapy.     

Role of neutrophils,platelets, and extracellular vesicles and their interactions in COVID-19-associated thrombopathy

The COVID-19 pandemic extended all around the world causing millions of deaths. In addition to acute respiratory distress syndrome, many patients with severe COVID-19 develop thromboembolic complications associated to multiorgan failure and death. Here, we review evidence for the contribution of neutrophils, platelets, and extracellular vesicles (EVs) to the thromboinflammatory process in COVID-19. We discuss how the immune system, influenced by pro-inflammatory molecules, EVs, and neutrophil extracellular traps (NETs), can be caught out in patients with severe outcomes. We highlight how the deficient regulation of the innate immune system favors platelet activation and induces a vicious cycle amplifying an immunothrombogenic environment associated with platelet/NET interactions. In light of these considerations, we discuss potential therapeutic strategies underlining the modulation of purinergic signaling as an interesting target.     

Transfusion-Related Acute Lung Injury: Incidence, Pathogenesis and the Role of Multicomponent Apheresis in Its Prevention

SUMMARY: Although transfusion-related acute lung injury (TRALI) is now appreciated as the most common cause of death from transfusion, its incidence remains unknown. The most frequently cited figure is 1:5,000 plasma-containing components. Certain patient groups may be at significantly higher risk. TRALI is both underdiagnosed and un-derreported. It is misdiagnosed as transfusion-associated circulatory overload. Several mechanisms have been proposed for its pathogenesis-leukocyte antibodies and the 2-hit model. These may overlap, and both involve transfusion of leukocyte antibodies. Passive transfusion of leukocyte antibodies is strongly associated with TRALI; these are identified in 60-85% of cases. Multiparous blood donors are the most frequent source of these antibody-containing components. The antibodies are HLA class I and II and/or granulocyte-specific. In 50% of cases the antibody corresponds to an epitope in the patient. HLA class I antibodies have been shown to prime and activate neutrophils. Clinical reports and animal models link HNA-3a antibodies with severe lung injury. A number of TRALI prevention and risk mitigation strategies have been proposed. In the UK and the USA, these strategies have centered upon excluding 'high risk' (HLA/HNA antibody containing) plasma from fresh frozen plasma and platelet products. Multicomponent apheresis collection of platelets, plasma and red blood cells is a means of accomplishing this objective.     

The pathogenesis of transfusion-related acute lung injury and how to avoid this serious adverse reaction of transfusion.

Transfusion-related acute lung injury (TRALI) is a serious, life-threatening complication of blood transfusion. Available evidence strongly suggests that leukocyte antibodies present in donor plasma are the predominant mechanism in TRALI. These antibodies lead to recipient neutrophil activation, with activated neutrophils inducing endothelial and alveolar damage in the lungs. These mechanisms are discussed in detail as are the alternative mechanisms that have been proposed. Preventive strategies that may help to reduce TRALI are presented.     

输血相关急性肺损伤及其预防与控制策略的研究进展

输血相关急性肺损伤(TRALI)是指输血后6h内发生的急性肺损伤(ALI),是输血导致患者死亡的主要原因之一.目前对TRALI发病机制的假说包括“二次打击”学说与“阈值模型”学说等.超过80％ TRALI是由输注的血液制品中含特异性抗人类白细胞抗原(HLA)或人类中性粒细胞抗原(HNA)抗体介导的,并且导致TRALI发生的血液制品,主要来源于有多次妊娠史的女性献血者.目前,多个国家实行以男性献血者为主体,供应高血浆含量血液制品的临床预防、控制TRALI策略,以效降低TRALI发生率.笔者拟就TRALI的定义、发病机制、诊断、预防控制策略进行综述.     

Analysis of Transfusion-Related Acute Lung Injury and Possible Transfusion-Related Acute Lung Injury Reported to the French Hemovigilance Network From 2007 to 2013

Using the French Hemovigilance Network database from 2007 to 2013, we provide information on demographics, incidence, and risk factors of reported transfusion-related acute lung injury (TRALI) and possible TRALI, analyze TRALI mitigation efforts for fresh frozen plasma and platelet concentrates, and consider the impact of platelet additive solutions on TRALI incidence. We applied the Toronto consensus conference definitions for TRALI and possible TRALI. Two TRALI subgroups were considered: “antibody positive” when a donor has human leukocyte antigen (class I or II) and/or human neutrophil antigen antibodies and the recipient has cognate antigen, and “antibody negative” when immunological investigation is negative or not done. The analysis targeted 378 cases, divided into antibody-positive TRALI (n = 75), antibody-negative TRALI (n = 100), and possible TRALI (n = 203). TRALI patients were younger and received more blood components than the general population of transfused patients. Moreover, we identified the following clinical conditions where patients seemed to be at higher risk to develop TRALI: postpartum hemorrhage, acute myeloid leukemia, liver transplantation, allogeneic and autologous hematopoietic stem cells transplantation, polytrauma, and thrombotic microangiopathy. Policy measures intended to reduce antibody-positive TRALI were found effective for apheresis platelet concentrates and fresh frozen plasma but not for whole blood–derived platelet concentrates. The use of platelet additive solutions was associated with a significant reduction in the incidence of TRALI following transfusion of buffy coat–derived platelet concentrates but not following transfusion of apheresis platelets. Our data reinforce the concept that possible TRALI and TRALI, as defined in the Canadian consensus conference, share many characteristics. No specific policy measures are currently directed at mitigation of possible TRALI despite its impact on transfusion safety. Despite TRALI mitigation measures, the overall incidence of TRALI cases reported to the French Hemovigilance system was not significantly reduced. Therefore, additional research is needed to reduce, if not eradicate, all TRALI categories.     

Evaluation of Platelet Responses in Transfusion-Related Acute Lung Injury (TRALI)

Platelets are versatile cells which are capable of eliciting nonhemostatic immune functions, especially under inflammatory conditions. Depending on the specific setting, platelets may be either protective or pathogenic in acute lung injury and acute respiratory distress syndrome (ARDS). Their role in transfusion-related acute lung injury (TRALI) is less well defined; however, it has been hypothesized that recipient platelets and transfused platelets both play a pathogenic role in TRALI. Overall, despite conflicting findings, it appears that recipient platelets may play a pathogenic role in antibody-mediated TRALI; however, their contribution appears to be limited. It is imperative to first validate the involvement of recipient platelets by standardizing the animal models, methods, reagents, and readouts for lung injury and taking the animal housing environment into consideration. For the involvement of transfused platelets in TRALI, it appears that predominantly lipids such as ceramide in stored platelets are able to induce TRALI in animal models. These studies will also need to be validated, and moreover, the platelet-derived lipid-mediated mechanisms leading to TRALI will need to be investigated.     

Platelet depletion and aspirin treatment protect mice in a two-event model of transfusion-related acute lung injury

Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion-associated mortality in the US. Previously, we established an immune-mediated TRALI mouse model, wherein mice with cognate antigen were challenged with MHC class I mAb. In this study, when mice housed in a rodent, specific pathogen–free barrier room were challenged with MHC I mAb, there was significant protection from TRALI compared with nonbarrier mice. Priming mice with LPS restored lung injury with mAb challenge. Using TLR4-deficient bone marrow chimeras, the priming phenotype was restricted to animals with WT hematopoietic cells, and depletion of either neutrophils or platelets was protective. Both neutrophils and platelets were sequestered in the lungs of mice with TRALI, and retention of platelets was neutrophil dependent. Interestingly, treatment with aspirin prevented lung injury and mortality, but blocking the P selectin or CD11b/CD18 pathways did not. These data suggest a 2-step mechanism of TRALI: priming of hematopoietic cells, followed by vascular deposition of activated neutrophils and platelets that then mediate the severe lung injury. Furthermore, our data offer an explanation for the increased incidence of TRALI in patients with immune priming conditions, and we suggest what we believe to be a novel therapeutic approach.     

How much residual plasma may cause TRALI?

Although passive infusion of plasma-rich components containing white blood cell (WBC) antibodies are responsible for majority of the reported transfusion-related acute lung injury (TRALI) cases, the minimum volume of residual plasma, which might trigger TRALI, is not known. We report three cases of TRALI where the implicated donor component contained between 10 and 20 mL of residual plasma. Two cases were related to transfusion of red blood cells prepared in optimal additive solution, and the other was related to transfusion of pooled buffy coat platelets. In the latter case, WBC antibodies that matched the patient's human leucocyte antigen (HLA) antigens were only found in one buffy coat donor (female) who contributed a buffy coat for pooled platelets preparation. Plasma prepared from pooling platelets was collected from a male donor. Laboratory investigation confirmed that in all three cases, the donors' serum contained three to four different HLA class 1-specific and class 11-specific antibodies that matched with the patient's HLA type. Our cases suggest that the residual plasma volume as small as 10-20 mL containing donor derived WBC antibodies may cause TRALI. The risk of TRALI remains, despite providing pooled platelets suspended in male donor plasma. The significance of multiple HLA antigen/antibody matching between donor and recipient in immune TRALI warrants further study.     

中性粒细胞胞外诱捕网标志物游离DNA水平与脓毒症血小板活化的相关性

目的研究中性粒细胞胞外诱捕网（NETs）标志物游离DNA（cfDNA）水平与血小板（PLT）活化之间的相关性。 方法根据纳入标准、排除标准将2018年9至12月中国医科大学附属第一医院ICU收治的51例患者分为脓毒症组（29例）和对照组（22例）。用酶联免疫吸附测定（ELISA）方法检测患者外周血浆的cfDNA、血小板因子4（PF4）、P-选择素浓度。采用SPSS 25.0进行统计学分析，比较2组患者入ICU 24 h内生化指标、急性生理学与慢性健康状况（APACHEⅡ）评分、序贯器官衰竭评估（SOFA）评分、国际血栓与止血委员会（ISTH）评分、日本危重病协会（JAAM）评分、cfDNA以及PLT标志物（PF4、P-选择素）的差异性。同时将脓毒症组cfDNA和PLT、PLT标志物（PF4、P-选择素）、炎症指标［中性粒细胞计数（NE）、C反应蛋白（CRP）、降钙素原（PCT）］、凝血指标［凝血酶原时间（PT）、活化部分凝血酶时间（APTT）、D-二聚体（D-D）、抗凝血酶Ⅲ活性（AT-Ⅲ）］、疾病严重程度（APACHEⅡ评分、SOFA评分、ISTH评分、JAAM评分）等分别进行相关性分析。 结果脓毒症组患者cfDNA水平明显高于对照组，差异有统计学意义［（174.499±76.596）ng/ml vs （114.354±19.319）ng/ml，P＜0.05］，但2组P-选择素、PF4水平比较，差异无统计学意义（P＞0.05）。脓毒症组中cfDNA水平与P-选择素/PLT、D-D、CRP、APACHEⅡ评分、JAAM评分之间具有相关性（r=0.3849、0.3872、0.6211、0.4542、0.4451，P＜0.05）；与NE、PLT计数、PLT标志物（PF4、P-选择素）、ISTH评分等无明显相关性（P＞0.05）。 结论脓毒症组患者cfDNA明显升高。脓毒症组中cfDNA与D-D之间具有明显正相关性，提示NETs可能与继发性纤溶过程关系更加密切。尽管脓毒症组中cfDNA与P-选择素/PLT之间有正相关性，但因为P-选择素并不是特异性的PLT活化标志物，所以NETs与脓毒症PLT活化的关系仍需进一步探究。     

Experimental models of transfusion-related acute lung injury

Transfusion-related acute lung injury (TRALI) is defined clinically as acute lung injury occurring within six hours of the transfusion of any blood product. It is the leading cause of transfusion-related death in the United States, but under-recognition and diagnostic uncertainty have limited clinical research to smaller case control studies. In this review we discuss the contribution of experimental models to the understanding of TRALI pathophysiology and potential therapeutic approaches. Experimental models suggest that TRALI occurs when a host, with a primed immune system, is exposed to an activating agent such as anti-leukocyte antibody or a biologic response modifier such as lysophosphatidylcholines. Recent work has suggested a critical role for platelets in antibody-based experimental models and identified potential therapeutic strategies for TRALI.     

Roles of the neutrophil and other mediators in adult respiratory distress syndrome

The adult respiratory distress syndrome (ARDS) and transfusion-related acute lung injury (TRALI) are characterized by diffuse, acute lung injury. Most likely, TRALI is a type of ARDS although it is associated with a much lower morbidity and mortality than found with classic ARDS. For years, the pathogenesis of ARDS has been explained by the complement hypothesis in which pulmonary neutrophilic sequestration and degranulation follow complement-mediated neutrophil chemotaxis. A definitive role for the neutrophil in diffuse, acute lung injury, however, has not been established. Although numerous chemoattractants for neutrophils are generated in the lungs and, through degranulation and formation of toxic oxygen free radicals, the neutrophil is fully capable of causing tissue injury, substantial evidence refutes the requirement for neutrophils in diffuse, acute lung injury. Other potential factors in the pathogenesis of ARDS include primary endothelial cell injury, alveolar macrophage activity, and hemostatic disorders.     

Neutrophil extracellular traps sequester circulating tumor cells and promote metastasis

The majority of patients with cancer undergo at least one surgical procedure as part of their treatment. Severe postsurgical infection is associated with adverse oncologic outcomes; however, the mechanisms underlying this phenomenon are unclear. Emerging evidence suggests that neutrophils, which function as the first line of defense during infections, facilitate cancer progression. Neutrophil extracellular traps (NETs) are extracellular neutrophil-derived DNA webs released in response to inflammatory cues that trap and kill invading pathogens. The role of NETs in cancer progression is entirely unknown. We report that circulating tumor cells become trapped within NETs in vitro under static and dynamic conditions. In a murine model of infection using cecal ligation and puncture, we demonstrated microvascular NET deposition and consequent trapping of circulating lung carcinoma cells within DNA webs. NET trapping was associated with increased formation of hepatic micrometastases at 48 hours and gross metastatic disease burden at 2 weeks following tumor cell injection. These effects were abrogated by NET inhibition with DNAse or a neutrophil elastase inhibitor. These findings implicate NETs in the process of cancer metastasis in the context of systemic infection and identify NETs as potential therapeutic targets.     

MHC class I-specific antibody binding to nonhematopoietic cells drives complement activation to induce transfusion-related acute lung injury in mice

Transfusion-related acute lung injury (TRALI), a form of noncardiogenic pulmonary edema that develops during or within 6 h after a blood transfusion, is the most frequent cause of transfusion-associated death in the United States. Because development of TRALI is associated with donor antibodies (Abs) reactive with recipient major histocompatibility complex (MHC), a mouse model has been studied in which TRALI-like disease is caused by injecting mice with anti-MHC class I monoclonal Ab (mAb). Previous publications with this model have concluded that disease is caused by FcR-dependent activation of neutrophils and platelets, with production of reactive oxygen species that damage pulmonary vascular endothelium. In this study, we confirm the role of reactive oxygen species in the pathogenesis of this mouse model of TRALI and show ultrastructural evidence of pulmonary vascular injury within 5 min of anti-MHC class I mAb injection. However, we demonstrate that disease induction in this model involves macrophages rather than neutrophils or platelets, activation of complement and production of C5a rather than activation of FcγRI, FcγRIII, or FcγRIV, and binding of anti-MHC class I mAb to non-BM-derived cells such as pulmonary vascular endothelium. These observations have important implications for the prevention and treatment of TRALI.     

Transfusion-related acute lung injury

Transfusion-related acute lung injury (TRALI) is characterized by the sudden development of noncardlogenic pulmonary edema (acute lung Injury) after transfusion of blood products. Poor awareness of TRALI outside of the blood transfusion medicine community has led to a serious underestimation of this condition, currently the most Important severe complication of blood transfusion. Concern for the transfer of donor antileukocyte antibodies has prompted major changes in the management of the blood supply in some countries; however, recent studies have suggested alternative pathophyslological mechanisms for TRALI related to the shelf life of cellular blood products. Although all blood products have been implicated, most reported cases were associated with fresh frozen plasma, red blood cell, and platelet transfusions. Because many patients have additional predisposing factors for acute lung injury, carefully designed prospective studies are needed to fully assess attributable risk related to transfusion. The treatment of TRALI is supportive, and the prognosis is generally better than for other causes of acute lung Injury. As many as one third of all patients who develop acute lung injury have been exposed to blood products. TRALI may be an important and potentially preventable cause of acute lung injury.     

Acute and transient decrease in neutrophil count in transfusion-related acute lung injury: cases at one hospital

BACKGROUND: Transfusion-related acute lung injury (TRALI) is a rare but serious complication of blood transfusion. The syndrome is characterized by new acute lung injury developing during or within 6 hours of blood transfusion. STUDY DESIGN AND METHODS: The study design was observational in nature. RESULTS: All three cases of TRALI were associated with acute but transient decrease in the white blood cell (WBC) count. Implicated donors had HLA antibodies that matched the recipients' HLA antigens. The implicated units were a plateletpheresis in one case and fresh frozen plasma units in the other two. All implicated donors were multiparous women. The implicated antibody specificities were anti-HLA Class I and Class II in one case and anti-HLA Class II in the other two cases. Interestingly, patient neutrophil counts decreased by 80 to 90 percent in all three cases, including the two cases associated with HLA Class II antibodies. CONCLUSION: An acute and transient decrease in WBC count may be a previously underrecognized feature of TRALI. A drop in the neutrophil count can occur even when the implicated antibodies have specificities to HLA Class II antigens, although they are expressed only on stimulated neutrophils. Based on the observations in these cases, it is recommended that a complete blood count and differential be obtained when TRALI is suspected. Further investigations into the mechanisms of the decrease in circulating neutrophils that is associated with infusion of HLA Class II antibody may yield new insights into the mechanism of TRALI.     

Peptidylarginine deiminase inhibition is immunomodulatory and vasculoprotective in murine lupus

Recent evidence suggests that enhanced neutrophil extracellular trap (NET) formation activates plasmacytoid dendritic cells and serves as a source of autoantigens in SLE. We propose that aberrant NET formation is also linked to organ damage and to the premature vascular disease characteristic of human SLE. Here, we demonstrate enhanced NET formation in the New Zealand mixed 2328 (NZM) model of murine lupus. NZM mice also developed autoantibodies to NETs as well as the ortholog of human cathelicidin/LL37 (CRAMP), a molecule externalized in the NETs. NZM mice were treated with Cl-amidine, an inhibitor of peptidylarginine deiminases (PAD), to block NET formation and were evaluated for lupus-like disease activity, endothelial function, and prothrombotic phenotype. Cl-amidine treatment inhibited NZM NET formation in vivo and significantly altered circulating autoantibody profiles and complement levels while reducing glomerular IgG deposition. Further, Cl-amidine increased the differentiation capacity of bone marrow endothelial progenitor cells, improved endothelium-dependent vasorelaxation, and markedly delayed time to arterial thrombosis induced by photochemical injury. Overall, these findings suggest that PAD inhibition can modulate phenotypes crucial for lupus pathogenesis and disease activity and may represent an important strategy for mitigating cardiovascular risk in lupus patients.     

Conversion to low transfusion-related acute lung injury (TRALI)-risk plasma significantly reduces TRALI

BACKGROUND: Transfusion‐related acute lung injury (TRALI) is an uncommon but serious transfusion reaction. Studies have shown that the transfusion of HLA and HNA antibodies in donor plasma can lead to TRALI. Female donors are more likely to have such antibodies due to alloantigen exposure during pregnancy. Many blood suppliers have now implemented various TRALI risk reduction strategies to unknown effect. A retrospective analysis of TRALI reactions in plasma recipients before and after the conversion to low‐TRALI‐risk plasma (all‐male donor plasma, male‐predominant plasma, nulliparous female plasma, and HLA antibody–tested plasma) is reported. STUDY DESIGN AND METHODS: Transfusion reaction reports at three large hospitals 16 months before and 16 months after the conversion to low‐TRALI‐risk plasma were analyzed. Respiratory reactions were categorized as TRALI, possible TRALI, or other (e.g., transfusion‐associated circulatory overload or allergic reactions). Reactions were reported as a percentage of total units transfused and rates for the two time periods were compared. Trends in reaction rates for other components were also compared. RESULTS: A total of 2156 transfusion reactions in association with 461,598 transfused blood components were reviewed. The incidence of combined TRALI or possible TRALI reactions, due to the transfusion of plasma, decreased from 0.0084% to zero (p = 0.052). The rate of TRALI or possible TRALI reactions in red blood cell and platelet recipients did not change significantly. CONCLUSION: The conversion to low‐TRALI‐risk plasma has reduced the incidence of TRALI reactions in plasma recipients.     

Epidemiology of Transfusion Related Acute Lung Injury in France: Preliminary Results

SUMMARY: The French Hemovigilance Network has been established in 1994 and records all adverse events associated with the transfusion of a labile blood products (LBP) regardless of their severity. From 1994 to 2006 35,423,172 LBP were issued, 85,812 adverse transfusion reactions notified, and 139 cases of transfusion related acute lung injury (TRALI) observed. The LBP most at risk is fresh frozen plasma (FFP), followed by platelets concentrates (PC) and packed red cells (PRC). However, because the use of FFP is not frequent in France, it only accounts for about 10% of TRALI, whereas PRC and PC are involved in the remaining cases. In no case, pooled FFP treated with solvent-detergent were involved. Patients' profiles are peculiar with a high disease burden. Therefore, targeting a prevention policy only on FFP would result in a marginal reduction of TRALI in France.