Mechanism and intervention of murine transfusion-related acute lung injury caused by anti-CD36 antibodies.

Anti-CD36 Abs have been suggested to induce transfusion-related acute lung injury (TRALI) upon blood transfusion, particularly in Asian populations. However, little is known about the pathological mechanism of anti-CD36 Ab-mediated TRALI, and potential therapies have not yet been identified. Here, we developed a murine model of anti-CD36 Ab-mediated TRALI to address these questions. Administration of mouse mAb against CD36 (mAb GZ1) or human anti-CD36 IgG, but not GZ1 F(ab')2 fragments, induced severe TRALI in Cd36+/+ male mice. Predepletion of recipient monocytes or complement, but not neutrophils or platelets, prevented the development of murine TRALI. Moreover, plasma C5a levels after TRALI induction by anti-CD36 Abs increased more than 3-fold, implying a critical role of complement C5 activation in the mechanism of Fc-dependent anti-CD36-mediated TRALI. Administration of GZ1 F(ab')2, antioxidant (N-acetyl cysteine, NAC), or C5 blocker (mAb BB5.1) before TRALI induction completely protected mice from anti-CD36-mediated TRALI. Although no significant amelioration in TRALI was observed when mice were injected with GZ1 F(ab')2 after TRALI induction, significant improvement was achieved when mice were treated postinduction with NAC or anti-C5. Importantly, anti-C5 treatment completely rescued mice from TRALI, suggesting the potential role of existing anti-C5 drugs in the treatment of patients with TRALI caused by anti-CD36.

PLATELET SUPPRESSION BY TIROFIBAN AMELIORATES PULMONARY COAGULATION AND FIBRINOLYSIS ABNORMALITIES IN THE LUNGS OF MOUSE ANTIBODY-MEDIATED TRANSFUSION-RELATED ACUTE LUNG INJURY.

ABSTRACT: This study aimed to explore the ameliorating effects of the platelet surface glycoprotein IIb/IIIa receptor antagonist tirofiban on coagulation and fibrinolytic abnormalities in a mouse model of antibody-mediated transfusion-associated acute lung injury (ALI). This is important because ALI is a major cause of death attributable to the occurrence of adverse transfusion reactions. No information on a definite diagnosis or pathological mechanism exists, and targeted treatment options are not available. In this study, wild-type male Balb/c mice aged 8 to 10 weeks were randomly divided into the TRALI model, blank control, tirofiban intervention, and isotype control groups. After different treatment exposures, the mice were observed for 2 h before being killed, and lung tissue samples were collected. To explore the intervention effect of tirofiban, the degree of lung injury was quantified by estimating the lung wet/dry ratio, rectal temperature, survival rate, total protein, and myeloperoxidase and via hematoxylin-eosin staining. Furthermore, the coagulation, anticoagulation, and fibrinolysis assays were measured by automatic coagulation instrument and enzyme-linked immunosorbent assay kits, and the fluorescence densities of platelets and fibrin were quantified using immunofluorescence to analyze the effects of tirofiban on the platelet and fibrin interactions of TRALI. Compared with the TRALI model group, the lung injury indices in the tirofiban intervention group decreased significantly, and survival rates also improved. Furthermore, the level of coagulation and fibrinolytic abnormalities were obviously lower than those in the TRALI model group. In conclusion, our findings suggest that tirofiban might interfere with TRALI by inhibiting platelet activation and improving coagulation and fibrinolytic abnormalities.

Platelets play a dual role in the pathophysiology of transfusion-related acute lung injury.

Platelets are increasingly recognized as key regulators of inflammatory and immune responses, through their interaction with endothelium and immune cells. Therefore they might have a role in transfusion-related acute lung injury (TRALI), in which endothelial cells and neutrophils are the key players. In this study, by a classic TRALI animal model, combining a custom-designed system for intravital confocal microscopy of pulmonary microvasculature and a platelet tracking technique, we found that thrombin-activated platelets transfusion aggravated TRALI while resting platelets transfusion alleviated TRALI. Promoting endogenous platelets activation also aggravated TRALI while inhibiting endogenous platelets activation alleviated TRALI. Activated platelets interfered with the stability of endothelial barrier function while resting platelets modulated the activation of neutrophils. Anti-thrombin could alleviate TRALI, which was not reproduced upon anti-GPIIbIIIa or anti-P-selectin In conclusion, platelets might play a dual role (protective and pathogenic) in TRALI, the balance between the two roles is highly dependent on whether platelets are activated by thrombin or not. This might explain the conflicting results of previous researches studying the contribution of platelets in TRALI by platelet depletion technology, in which the induction of TRALI and the condition of animals were different, hence the state of platelets during TRALI was different. Moreover, anti-platelet-activation (such as anti-thrombin) might be a better approach than anti-activated-platelets (such as anti-P-selectin) to search for potential therapies in TRALI. Considering the involvement of thrombin-activated platelets in TRALI, anti-thrombin might be needed when blood component transfusion is performed.

Crosstalk between neutrophil extracellular traps and immune regulation: insights into pathobiology and therapeutic implications of transfusion-related acute lung injury.

Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion-associated death, occurring during or within 6 hours after transfusion. Reports indicate that TRALI can be categorized as having or lacking acute respiratory distress syndrome (ARDS) risk factors. There are two types of TRALI in terms of its pathogenesis: antibody-mediated and non-antibody-mediated. The key initiation steps involve the priming and activation of neutrophils, with neutrophil extracellular traps (NETs) being established as effector molecules formed by activated neutrophils in response to various stimuli. These NETs contribute to the production and release of reactive oxygen species (ROS) and participate in the destruction of pulmonary vascular endothelial cells. The significant role of NETs in TRALI is well recognized, offering a potential pathway for TRALI treatment. Moreover, platelets, macrophages, endothelial cells, and complements have been identified as promoters of NET formation. Concurrently, studies have demonstrated that the storage of platelets and concentrated red blood cells (RBC) can induce TRALI through bioactive lipids. In this article, recent clinical and pre-clinical studies on the pathophysiology and pathogenesis of TRALI are reviewed to further illuminate the mechanism through which NETs induce TRALI. This review aims to propose new therapeutic strategies for TRALI, with the hope of effectively improving its poor prognosis.

Pulmonary coagulation and fibrinolysis abnormalities that favor fibrin deposition in the lungs of mouse antibody-mediated transfusion-related acute lung injury.

Transfusion­related acute lung injury (TRALI) is a life­threatening disease caused by blood transfusion. However, its pathogenesis is poorly understood and specific therapies are not available. Experimental and clinical studies have indicated that alveolar fibrin deposition serves a pathological role in acute lung injuries. The present study investigated whether pulmonary fibrin deposition occurs in a TRALI mouse model and the possible mechanisms underlying this deposition. The TRALI model was established by priming male Balb/c mice with lipopolysaccharide (LPS) 18 h prior to injection of an anti­major histocompatibility complex class I (MHC­I) antibody. Untreated mice and mice administered LPS plus isotype antibody served as controls. At 2 h after TRALI induction, blood and lung tissue were collected. Disease characteristics were assessed based on lung tissue histology, inflammatory responses and alterations in the alveolar­capillary barrier. Immunofluorescence staining was used to detect pulmonary fibrin deposition, platelets and fibrin­platelet interactions. Levels of plasminogen activator inhibitor­1 (PAI­1), thrombin­antithrombin complex (TATc), tissue factor pathway inhibitor (TFPI), coagulation factor activity and fibrin degradation product (FDP) in lung tissue homogenates were measured. Severe lung injury, increased inflammatory responses and a damaged alveolar­capillary barrier in the LPS­primed, anti­MHC­I antibody­administered mice indicated that the TRALI model was successfully established. Fibrin deposition, fibrin­platelet interactions and platelets accumulation in the lungs of mouse models were clearly promoted. Additionally, levels of TATc, coagulation factor V (FV), TFPI and PAI­1 were elevated, whereas FDP level was decreased in TRALI mice. In conclusion, both impaired fibrinolysis and enhanced coagulation, which might be induced by boosted FV activity, increased pulmonary platelets accumulation and enhanced fibrin­platelet interactions and contributed to pulmonary fibrin deposition in TRALI mice. The results provided a therapeutic rationale to target abnormalities in either coagulation or fibrinolysis pathways for antibody­mediated TRALI.

MiR-144-induced KLF2 inhibition and NF-kappaB/CXCR1 activation promote neutrophil extracellular trap-induced transfusion-related acute lung injury.

Transfusion-related acute lung injury (TRALI) is a clinical syndrome which is associated with the formation of neutrophil extracellular trap (NET). Recent studies have demonstrated the roles of microRNAs (miRNAs) in the pathophysiological process of TRALI. Here, the study focused on the role of miR-144 and the molecular mechanisms in NET-induced TRALI. Up-regulated miR-144 and under-expressed KLF2 were determined in patients with TRALI. In the mouse model of a two-event TRALI induced by intraperitoneal injections with lipopolysaccharide and anti-H-2Kd mAb, we determined expression patterns of miR-144, Krüppel-like factor 2 (KLF2), chemokine (C-X-C motif) receptor 1 (CXCR1) and nuclear factor kappa-B (NF-kappaB) p65. The results confirmed that miR-144 was highly expressed, while KLF2 was poorly expressed in mice with TRALI. Dual-luciferase reporter gene assay identified that miR-144 could target KLF2. Using gain- and loss-of-function approaches, we analysed the effects of miR-144 and its interaction with KLF2 on TRALI. Enforced expression of miR-144 was found to aggravate NET-induced TRALI by down-regulating KLF2 and activating the NF-kappaB/CXCR1 signalling pathway in TRALI mice. Collectively, miR-144-targeted inhibition of KLF2 and activation of NF-kappaB/CXCR1 are possible mechanisms responsible for NET-caused TRALI. These findings aid in the development of therapeutic modalities for the treatment of TRALI.

Transfusion-Associated Circulatory Overload and Transfusion-Related Acute Lung Injury.

OBJECTIVES: To review the new current diagnostic criteria of transfusion-associated circulatory overload (TACO) and transfusion-related acute lung injury (TRALI) from the literature while highlighting distinguishing features. We provide comprehensive understanding of the importance of hemovigilance and its role in appropriately identifying and reporting these potentially fatal transfusion reactions. METHODS: A review of the English language literature was performed to analyze TACO and TRALI while providing further understanding of the rationale behind the historical underrecognition and underreporting. RESULTS: Our review demonstrates the new 2018 and 2019 case definitions for TACO and TRALI, respectively. With more comprehensive diagnostic strategies, adverse transfusion events can be better recognized from mimicking events and underlying disease. In addition, there are mitigation strategies in place to help prevent complications of blood product transfusion, with emphasis on the prevention of TACO and TRALI. CONCLUSIONS: TACO and TRALI are potentially fatal adverse complications of blood transfusion. Both have been historically underrecognized and underreported due to poor defining criteria and overlapping symptomatology. Developing a thorough clinical understanding between these two entities can improve hemovigilance reporting and can contribute to risk factor identification and preventative measures.

Update on the pathophysiology of transfusion-related acute lung injury.

PURPOSE OF REVIEW: The aim of this study was to discuss recent advances regarding the pathogenesis of transfusion-related acute lung injury (TRALI), which highlight the pathogenic role of macrophages. RECENT FINDINGS: TRALI remains a leading cause of transfusion-related fatalities, despite the success of the mitigation strategy, and therapeutic approaches are unavailable. Neutrophils (PMNs) are recognized pathogenic cells in TRALI. Macrophages have previously also been suggested to be pathogenic in mice via binding of C5a to their C5a-receptor, producing reactive oxygen species (ROS), which damages the pulmonary endothelium. Recent work has further highlighted the role of macrophages in the TRALI-pathogenesis. It has been shown that the protein osteopontin (OPN) released by macrophages is critical for pulmonary PMN recruitment in mice suffering from TRALI and that targeting OPN prevents the occurrence of TRALI. Another recent study demonstrated the importance of M1-polarized alveolar macrophages in murine TRALI induction by showing that α1-antitrypsin (AAT) overexpression prevented TRALI in mice through decreasing the polarization of alveolar macrophages towards the M1 phenotype. SUMMARY: Apart from PMNs, macrophages also appear to be important in the pathogenesis of TRALI. Targeting the pathogenic functions of macrophages may be a promising therapeutic strategy to explore in TRALI.

Complement activation on endothelium initiates antibody-mediated acute lung injury.

Antibodies targeting human leukocyte antigen (HLA)/major histocompatibility complex (MHC) proteins limit successful transplantation and transfusion, and their presence in blood products can cause lethal transfusion-related acute lung injury (TRALI). It is unclear which cell types are bound by these anti-leukocyte antibodies to initiate an immunologic cascade resulting in lung injury. We therefore conditionally removed MHC class I (MHC I) from likely cellular targets in antibody-mediated lung injury. Only the removal of endothelial MHC I reduced lung injury and mortality, related mechanistically to absent endothelial complement fixation and lung platelet retention. Restoration of endothelial MHC I rendered MHC I-deficient mice susceptible to lung injury. Neutrophil responses, including neutrophil extracellular trap (NET) release, were intact in endothelial MHC I-deficient mice, whereas complement depletion reduced both lung injury and NETs. Human pulmonary endothelial cells showed high HLA class I expression, and posttransfusion complement activation was increased in clinical TRALI. These results indicate that the critical source of antigen for anti-leukocyte antibodies is in fact the endothelium, which reframes our understanding of TRALI as a rapid-onset vasculitis. Inhibition of complement activation may have multiple beneficial effects of reducing endothelial injury, platelet retention, and NET release in conditions where antibodies trigger these pathogenic responses.

Convalescent Plasma Therapy in Coronavirus Disease 2019: a Case Report and Suggestions to Overcome Obstacles.

Coronavirus disease 2019 (COVID-19) is rapidly spreading around the world, causing much morbidity and mortality everywhere. However, effective treatments or vaccines are still not available. Although convalescent plasma (CP) therapy can be useful in the treatment of COVID-19, it has not been widely used in Korea because of the concerns about adverse effects and the difficulty in matching patients to donors. The use of ABO-incompatible plasma is not contraindicated in treatment, but can be hesitated due to the lack of experience of physicians. Here, we describe a 68-year old man with COVID-19 who was treated ABO-incompatible plasma therapy; additionally, we comment on the acute side effects associated with ABO mismatch transfusion. To overcome the obstacles of donor-recipient connections (schedule and distance), we propose the storage of frozen plasma, modification of the current Blood Management Law, and the establishment of a CP bank. We suggest that experience gained in CP therapy will be useful for not only the treatment of COVID-19, but also for coping with new emerging infectious diseases.

rIL-35 prevents murine transfusion-related acute lung injury by inhibiting the activation of endothelial cells.

BACKGROUND: Transfusion-related acute lung injury (TRALI) is an important cause of death associated with transfusion, and no specific clinical treatments are available. Endothelial cells are believed to play an important role in the development of TRALI. This study investigated whether IL-35, an endothelial stabilizing cytokine could regulate the severity of antibody-mediated TRALI in vivo. STUDY DESIGN AND METHODS: Human microvascular endothelial cells (HMVECs) were cultured in vitro, rIL-35(2 µg/mL) was added before HMVECs activation, and HMVECs were fully activated by LPS (0.5 µg/mL). Then cells were collected for flow cytometry analysis. We used a previously established "two-event" mouse model of TRALI with naive and lipopolysaccharide (LPS)-injected mice as controls. rIL-35(100 µg/kg) was injected into the tail vein for 3 consecutive days before the induction of the TRALI model. Samples were collected 2 hours after TRALI induction and tested for lung tissue myeloperoxidase activity, total protein levels, lung tissue histology, endothelial cell activation assay, and cytokine assay. RESULTS: In vitro culture of HMVECs with rIL-35 verified that rIL-35 inhibited endothelial cells. In a mouse model, prophylactic administration of rIL-35 prevented pulmonary edema, increased lung protein levels, and reduced polymorphonuclear neutrophil accumulation in the lung. CONCLUSIONS: This work suggests that antibody-mediated murine TRALI can be prevented by rIL-35, and that rIL-35 appears to work by inhibiting the activation of lung endothelial cells.

An animal model of transfusion-related acute lung injury and the role of soluble CD40 ligand.

BACKGROUND AND OBJECTIVES: Transfusion-related acute lung injury (TRALI) is a life-threatening complication of transfusion and is one of leading causes of transfusion-associated fatalities. However, the pathogenesis of TRALI is still unclear. Soluble CD40 ligand (sCD40L) is a proinflammatory cytokine that accumulates during blood component storage and is involved in transfusion reactions. The objective of this study was to establish a clinically relevant TRALI animal model and to evaluate the role of sCD40L in TRALI. MATERIALS AND METHODS: Rats' red-blood-cell (RBC) suspensions were prepared, and the quality of RBC was evaluated. A trauma-haemorrhage-transfusion strategy was applied to build the animal model. Lung oedema was evaluated by histopathology examination, total bronchoalveolar lavage fluid (BALF) protein concentration, Evans blue dye (EBD) leakage and inflammatory cytokines. The sCD40L concentrations were measured. RESULTS: Storage lesions of RBCs gradually increased over time. Obvious histological evidence of lung injury of rats transfused with a 35-day RBC was observed. The total BALF protein concentration, EBD leakage, inflammatory cytokines concentration were increased significantly in the Day 35 group. The sCD40L concentration increased significantly in the storage RBC suspension over time but was slightly elevated in rat plasma. CONCLUSIONS: These findings indicated successful establishment of a TRALI animal model with trauma-haemorrhage-transfusion, in which sCD40L may play a minor role in the development of TRALI.

Osteopontin mediates murine transfusion-related acute lung injury via stimulation of pulmonary neutrophil accumulation.

Transfusion-related acute lung injury (TRALI) is one of the leading causes of transfusion-related fatalities and is characterized by the onset of acute respiratory distress within 6 hours upon blood transfusion. Specific therapies are unavailable. Preexisting inflammation is a risk factor for TRALI and neutrophils (polymorphonuclear neutrophils [PMNs]) are considered to be the major pathogenic cells. Osteopontin (OPN) is a multifunctional protein expressed at sites of inflammation and, for example, is involved in pulmonary disorders, can regulate cellular migration, and can function as a PMN chemoattractant. We investigated whether OPN is involved in TRALI induction by promoting PMN recruitment to the lungs. Using a previously established murine TRALI model, we found that in contrast to wild-type (WT) mice, OPN knockout (KO) mice were resistant to antibody-mediated PMN-dependent TRALI induction. Administration of purified OPN to the OPN KO mice, however, restored the TRALI response and pulmonary PMN accumulation. Alternatively, blockade of OPN in WT mice using an anti-OPN antibody prevented the onset of TRALI induction. Using pulmonary immunohistochemistry, OPN could be specifically detected in the lungs of mice that suffered from TRALI. The OPN-mediated TRALI response seemed dependent on macrophages, likely the cellular source of OPN and OPN polymerization, and independent from the OPN receptor CD44, interleukin 6 (IL-6), and other PMN chemoattractants including macrophage inflammatory protein-2 (MIP-2). These data indicate that OPN is critically required for induction of antibody-mediated murine TRALI through localization to the lungs and stimulation of pulmonary PMN recruitment. This suggests that anti-OPN antibody therapy may be a potential therapeutic strategy to explore in TRALI patients.

Slit2/Robo4 signaling pathway modulates endothelial hyper-permeability in a two-event in vitro model of transfusion-related acute lung injury.

Transfusion-related acute lung injury (TRALI) remains the leading cause of transfusion-related mortality. Endothelium semipermeable barrier function plays a critical role in the pathophysiology of transfusion-related acute lung injury (TRALI). Recently, Roundabout protein 4 (Robo4), interaction with its ligand Slit 2, was appreciated as a modulator of endothelial permeability and integrity. However, not much is known about the role of Slit2/Robo4 signaling pathway in the pathophysiology of TRALI. In this study, the TRALI model was performed by the "two-event" model of polymorphonuclear neutrophils (PMN)-mediated pulmonary microvascular endothelial cells (PMVECs) damage. We investigated the expression of Slit2/Robo4 and VE-cadherin and examined the pulmonary endothelial hyper-permeability in TRALI model. We found that the expression of Slit2/Robo4 and VE-cadherin were significantly decreased in a time-dependent manner, whereas the PMVECs permeability was gradually increased over time in TRALI model. Moreover, the treatment with Slit2-N, an active fragment of Slit2, increased the expression of Slit2/Robo4 and VE-cadherin to protect PMVECs from PMN-mediated pulmonary endothelial hyper-permeability. These results indicate that targeting Slit2/Robo4 signaling pathway may modulate the permeability as well as protect the integrity of endothelial barrier. In addition, Slit2-N appears to be a promising candidate for developing novel therapies against TRALI.

Cryoglobulinemia as a Possible Primer for TRALI: Report of a Case.

Waldenström macroglobulinemia (WM) is a form of lymphoplasmacytic lymphoma that can cause hyperviscosity syndrome due to unchecked monoclonal antibody production. Some patients are also found to have associated cryoglobulinemia, which can cause systemic complications including vasculitis, renal disease, and pulmonary complications. Cryoglobulins can also serve as a source of interference with various laboratory assays. Therapeutic plasma exchange (TPE) is one of the recommended treatment modalities to manage hyperviscosity. Herein, we present the case of an 84-year-old female patient with Waldenström macroglobulinemia who presented with hyperviscosity syndrome and discrepant laboratory findings, and who then developed transfusion-related acute lung injury (TRALI) during TPE. This case is one of many in the emerging possible linkages observed between cryoglobulinemia and TRALI.

Preventing murine transfusion-related acute lung injury by expansion of CD4+ CD25+ FoxP3+ Tregs using IL-2/anti-IL-2 complexes.

BACKGROUND: Transfusion-related acute lung injury (TRALI) is one of the most serious adverse events following transfusion, and there is no specific treatment in clinical practice. However, regulatory T cells (Tregs) have been suggested to play a potential role in the treatment of TRALI. This study investigated whether interleukin (IL)-2 or IL-2/anti-IL-2 complexes (IL-2c), which are mediators of Treg expansion, can modulate the severity of antibody-mediated TRALI in vivo. STUDY DESIGN AND METHODS: This study utilized a mouse model of the "two-hit" mechanism: BALB/c mice were primed with lipopolysaccharide (LPS) as the first hit, and then TRALI was induced by injecting major histocompatibility complex Class I antibodies. Mice injected with LPS only or LPS combined with isotype control antibodies served as controls. For the Treg-depleted groups, mice were infused with anti-mouse IL-2Rα first and then subjected to the same treatments as the TRALI group. Regarding IL-2- and IL-2c-treated mice, recombinant murine IL-2 or IL-2c was intraperitoneally administered to mice for 5 consecutive days before induction of the TRALI model. Samples were collected 2 hours after TRALI induction. RESULTS: Prophylactic administration of IL-2 or IL-2c to mice prevented the onset of edema, pulmonary protein levels, and proinflammatory factors that inhibited polymorphonuclear neutrophil aggregation in the lungs. Furthermore, the percentage of CD4+ CD25+ FoxP3+ Tregs was expanded in vivo using IL-2 and IL-2c compared to TRALI mice, as was confirmed through analysis of the spleen, blood, and lung. CONCLUSION: This study validates that the protective mechanisms against TRALI involve CD4+ CD25+ FoxP3+ Tregs, which can be expanded in vivo by IL-2 and IL-2c. This results in increased IL-10 levels and decreased IL-17A, thereby prophylactically preventing antibody-mediated murine TRALI.

Gastrointestinal microbiota contributes to the development of murine transfusion-related acute lung injury.

Transfusion-related acute lung injury (TRALI) is a syndrome of respiratory distress upon blood transfusion and is the leading cause of transfusion-related fatalities. Whether the gut microbiota plays any role in the development of TRALI is currently unknown. We observed that untreated barrier-free (BF) mice suffered from severe antibody-mediated acute lung injury, whereas the more sterile housed specific pathogen-free (SPF) mice and gut flora-depleted BF mice were both protected from lung injury. The prevention of TRALI in the SPF mice and gut flora-depleted BF mice was associated with decreased plasma macrophage inflammatory protein-2 levels as well as decreased pulmonary neutrophil accumulation. DNA sequencing of amplicons of the 16S ribosomal RNA gene revealed a varying gastrointestinal bacterial composition between BF and SPF mice. BF fecal matter transferred into SPF mice significantly restored TRALI susceptibility in SPF mice. These data reveal a link between the gut flora composition and the development of antibody-mediated TRALI in mice. Assessment of gut microbial composition may help in TRALI risk assessment before transfusion.