Murine Transfusion-related Acute Lung Injury Research Articles

Preservation of recipient plasma sphingosine-1-phosphate levels reduces transfusion-related acute lung injury.

Cold-stored (CS) platelets are once again being reintroduced for clinical use. Transfused CS platelets offer benefits over room temperature-stored (RTS) platelets such as increased hemostatic effects and prolongation of shelf-life. Despite these advantages little is known about their association with transfusion-related acute lung injury (TRALI). TRALI is associated with prolonged storage of RTS platelets and has a mortality of >15%. Determining the safety of CS platelets is important considering their proposed use in TRALI-vulnerable populations with inflammation such as surgical patients or patients with trauma. Donor platelet-derived ceramide causes TRALI, whereas donor platelet sphingosine-1-phosphate (S1P) is barrier protective. Females have higher plasma levels of S1P than males. Cold temperatures increase S1P levels in cells. Therefore, we hypothesized that female (donors or recipients) and/or CS platelets would decrease TRALI. To test this, we compared how male and female donor and recipient allogeneic platelet transfusions of CS (4°C) versus RTS (23°C) platelets stored for 5 days influence murine TRALI. Transfusion of CS platelets significantly reduced recipient lung tissue wet-to-dry ratios, bronchoalveolar lavage total protein, lung tissue myeloperoxidase enzyme activity, histological lung injury scores, and increased plasma sphingosine-1-phosphate (S1P) levels compared with RTS platelet transfusions. Female as opposed to male recipients had less TRALI and higher plasma S1P levels. Female donor mouse platelets had higher S1P levels than males. Mouse and human CS platelets had increased S1P levels compared with RTS platelets. Higher recipient plasma S1P levels appear protective considering females, and males receiving platelets from females or male CS platelets had less TRALI.NEW & NOTEWORTHY Transfusion-related acute lung injury (TRALI) though relatively rare represents a severe lung injury. The sphingolipid sphingosine-1-phosphate (S1P) regulates the severity of platelet-mediated TRALI. Female platelet transfusion recipient plasmas or stored platelets from female donors have higher S1P levels than males, which reduces TRALI. Cold storage of murine platelets preserves platelet-S1P, which reduces TRALI in platelet-transfused recipients.

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.

Fc-Mediated Complement Activation Is Associated with Macrophage Trafficking and Induction of Neutrophil Extracellular Traps in Transfusion-Related Acute Lung Injury

Transfusion-related acute lung injury (TRALI) is a leading cause of blood transfusion-related fatalities without available therapies. The pulmonary endothelium is damaged in TRALI, through incompletely understood pathogenic mechanisms, resulting in pulmonary edema. Generally, anti-leukocyte antibodies or biological response modifiers in the transfusion product, in combination with predisposing risk factors in the transfused recipient (e.g. inflammation), are responsible for initiation of TRALI. Remarkably, not all anti-leukocyte antibodies cause TRALI. In a previous in vitro study, we identified increased Fc-mediated complement activation to be a key feature of murine TRALI-inducing antibody 34-1-2S (anti-H-2K d) compared to TRALI-resistant antibody SF1.1.10 (anti-H-2K d) (Zeeuw van der Laan et al, Blood Adv 2020).In the current study, we further explored antibody-mediated TRALI mechanisms in vivo using our previously established TRALI mouse model in which mice are pre-depleted of protective CD4+ T cells and primed with LPS, followed by infusion of antibody 34-1-2S (Kapur et al, Blood 2017, Blood Adv 2018, Blood 2019). A key read-out for TRALI was the lung wet/dry weight ratio (Lung W/D, measure for pulmonary edema). We found that in vitro antibody-mediated complement activation was associated with in vivo antibody-mediated TRALI. 34-1-2S caused severe TRALI (complement activation: +++, Lung W/D: 7.4 ± 0.21), while SF1.1.10 did not cause TRALI (complement activation: +, Lung W/D: 4.68 ± 0.16). Furthermore, Fc-deglycosylated 34-1-2S still caused significant TRALI (complement activation: ++, in vivo Lung W/D: 5.16 ± 0.52). TRALI development was fully Fc-dependent as 34-1-2S-Fab did not cause any TRALI (no complement activation, Lung W/D: 4.35 ± 0.18). Importantly, we found significantly increased levels of complement C1q-C4 complexes in plasma samples of TRALI patients (n=46) compared to healthy controls (n=25): 6.49 ± 5.27 vs 3.84 ± 2.27 AU/ml, respectively, P: 0.0005,***. Similarly, C5a levels were significantly elevated in plasma samples of TRALI patients (n=53) compared to healthy controls (n=30): 2.52 ± 2.17 vs 1.37 ± 1.08 ng/ml, respectively, P: 0.0006,***. To further dissect the effect of the Fc-part of 34-1-2S in TRALI-induction, we aim to investigate the contribution of Fc-mediated complement activation vs Fc-receptor interaction. We therefore successfully generated chimeric variants of 34-1-2S with a humanized IgG1 Fc-domain containing mutations making them functionally complement dead (K322A), Fc-receptor dead (dG236) or both complement and Fc-receptor dead (PG LALA). We are currently investigating the effects of these variants on in vivo TRALI induction.We next investigated how the in vivo murine TRALI reaction was related to numbers of macrophages, monocytes and neutrophils in blood and lungs. We found that 34-1-2S-TRALI was associated with significantly decreased levels of macrophages in the lungs and increased levels in blood, compared to mice infused with PBS, SF1.1.10 or 34-1-2S-Fab, suggesting that Fc-mediated complement activation and TRALI induction is related to macrophage trafficking from lungs to blood.We did not observe any significant differences between blood and lung neutrophil levels of 34-1-2S-TRALI mice compared to SF1.1.10 TRALI-resistant mice. We hypothesized that in TRALI neutrophils undergo formation of neutrophil extracellular traps (NETs) induced by complement. We observed that C5a enabled potent neutrophil-chemotaxis in vitro (P: 0.0048,**). In addition, using direct immunofluorescence staining of extracellular DNA and Citrullinated histone H3, we observed that both LPS and C5a on their own could induce NET formation in vitro, which was synergistically increased with a combination of both LPS and C5a (P: 0.0417,*), as occurring during TRALI. Furthermore, we found increased levels of NETs to be present in plasma samples of TRALI patients (n=53) compared to healthy controls (n=30): 1.64 ± 0.97 vs 0.80 ± 0.34 MPO-DNA OD, respectively, P: 0.0002,***.Finally, we targeted the C5a-receptor (C5aR) using a C5aR-antagonist in our TRALI mouse model. Surprisingly, this did not prevent but even worsened TRALI (P: 0.0398,*), with elevated levels of blood monocytes and macrophages. This suggests that an approach directly targeting complement components may be a more promising therapeutic strategy to explore in combatting TRALI. DisclosuresNo relevant conflicts of interest to declare.

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

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. 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. 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.

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

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. 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. 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. 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.

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

AbstractTransfusion-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.

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

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. 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. 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. 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.

Osteopontin Mediates Murine Transfusion-Related Acute Lung Injury through Stimulation of Pulmonary Neutrophil Accumulation

Transfusion-related acute lung injury (TRALI) is a syndrome of respiratory distress which develops within 6 hours of blood transfusion. It is the leading cause of transfusion-related deaths and the pathogenesis is complex and incompletely understood. In the majority of the cases, anti-leukocyte antibodies present in the transfused blood product, in combination with recipient predisposing risk-factors such as inflammation, are implicated to be responsible for the onset of TRALI. Unfortunately, no therapies are available for TRALI.Osteopontin (OPN) is an extracellular matrix protein with multiple biological functions. OPN is involved in normal physiological processes, such as cell migration and adhesion, but has also been implicated in a wide range of disease states, including cancer, atherosclerosis, glomerulonephritis, and several chronic inflammatory diseases. Interestingly, OPN is upregulated at sites of inflammation and tissue remodeling. As inflammation is an important risk factor for TRALI development, and as neutrophils (PMNs) are known effector cells in the pathogenesis of TRALI which migrate and accumulate in the lungs during TRALI development, we investigated the potential contribution of OPN in the onset of antibody-mediated TRALI.We utilized a previously established murine TRALI model (Kapur et al, Blood 2017, Blood Advances 2018) in which C57BL/6 mice were first primed with a low dose of lipopolysaccharide (LPS) and depleted of their CD4+ T cells in vivo followed by injection of anti-major histocompatibility complex (MHC) class I antibodies (clones 34-1-2s and AF6-88.5.5.3). The TRALI response was analyzed after 90 minutes by analysis of pulmonary edema (lung wet-to-dry weight ratios, W/Ds) and the levels of pulmonary neutrophils. Wildtype (WT) mice suffered from antibody-mediated TRALI compared to untreated naïve mice, as was shown by their significantly increased lung W/Ds (4.72 vs 4.50, respectively, P<0.0001). This also corresponded to significantly increased levels of pulmonary PMNs compared to untreated naïve mice (34% vs 5%, P<0.0001). In contrast, C57BL/6 OPN knock-out mice were resistant to antibody-mediated TRALI induction as they did not display any significant increase in lung W/Ds levels or pulmonary PMNs compared to untreated naïve OPN knock-out mice (lung W/Ds 4.83 vs 4.75, and pulmonary PMNs 18% vs 16%, respectively). Strikingly, administration of purified recombinant OPN during TRALI induction in C57BL/6 OPN knock-out mice, significantly induced a TRALI reaction (lung W/Ds 5.12 vs 4.75 as compared to untreated naïve OPN knock-out mice, P<0.05). Mechanistically, this TRALI inducing effect of OPN administration to OPN knock-out mice was associated with increased levels of pulmonary PMNs (38% vs 16%, as compared to untreated naïve OPN knock-out mice, P<0.0001). In vivo blocking of OPN in WT mice with an anti-OPN antibody demonstrated decreased lung W/Ds as compared to treatment with an isotype antibody (4.48 vs 4.69, respectively, P<0.05). The OPN blocking response during TRALI was associated with a decreased level of pulmonary PMN accumulation as compared to treatment with an isotype antibody (14% vs 34%, respectively, P<0.0001). As the PMN-chemoattractant macrophage inflammatory protein (MIP)-2 has previously been described to be upregulated in murine antibody-mediated TRALI, we investigated if the OPN-associated pulmonary PMN accumulation and TRALI induction could be related to the levels of MIP-2. We found that plasma MIP-2 levels were increased in mice that were infused with anti-MHC class I antibodies as compared to naïve controls, but that addition or blocking of OPN did not affect these MIP-2 levels. This indicates that the OPN-related PMN responses in TRALI are independent of plasma MIP-2 levels.Collectively, these data indicate OPN as a novel pathogenic factor which enhances antibody-mediated murine TRALI through stimulation of PMN migration towards the lungs, independent of MIP-2. This may suggest that blocking OPN (using an anti-OPN antibody) may prevent TRALI by impairing pulmonary PMN accumulation and could be a therapeutic avenue to explore in combatting this serious adverse complication of blood transfusion. DisclosuresNo relevant conflicts of interest to declare.

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

AbstractTransfusion-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.

Gastrointestinal Flora Dictates the Biological Response in Murine Transfusion Related Acute Lung Injury (TRALI)

Transfusion related acute lung injury (TRALI) is a syndrome of respiratory distress which occurs within 6 hours of blood transfusion. It is the leading cause of transfusion-related fatalities and the pathogenesis is incompletely understood. In the majority of the cases, anti-leukocyte antibodies present in the transfused blood product, in combination with recipient predisposing risk-factors such as inflammation, are implicated to be responsible for the onset of TRALI. Unfortunately, no therapies are available for TRALI. Recently, using novel murine models of TRALI, CD4+T cells were found to be important protector cells against antibody-mediated TRALI and administration of interleukin (IL)-10 was demonstrated to be a successful treatment strategy for TRALI, rescuing mice therapeutically from pulmonary edema, the hallmark of acute lung injury (Kapur et al, Blood 2017, 129(18):2557-2569). Whether the gut microbiome plays any role in the development of TRALI is currently unknown. For that purpose, we compared the biological TRALI response in mice housed in a barrier-free (BF) setting versus mice housed in a specific pathogen-free (SPF) environment. We utilized our TRALI model in which C57BL/6 mice were first depleted of CD4+ T cells in vivo followed by injection of anti-major histocompatibility complex class I antibodies (clones 34-1-2s and AF6-88.5.5.3). The TRALI response was analyzed after 90 minutes for several parameters including pulmonary edema (lung wet-to-dry weight ratios, W/Ds), rectal temperatures (indicative of systemic shock), plasma levels of macrophage inflammatory protein (MIP)-2 (murine homologue of IL-8, a neutrophil chemoattractant), levels of pulmonary neutrophils (major effector cells in TRALI) and lung histology. We observed that after one week of housing, baseline rectal temperatures of BF mice significantly increased from 38.7o C to 39.5° C (p<0.001) while SPF mice remained stable around 38.3 °C. When comparing BF versus SPF mice, the baseline temperature of BF mice was significantly higher than SPF mice after one week of housing (P<0.0001). The gut flora was subsequently depleted in BF mice via administration of broad-spectrum antibiotics through the drinking water. When both aerobic and anaerobic gut microbes were efficiently depleted after a week of antibiotic treatment, rectal temperatures of the treated BF mice significantly dropped again to 38.7 °C (P<0.05). When inducing TRALI by depleting CD4+ T cells and injecting TRALI-antibodies, the rectal temperatures of the untreated BF mice remained low while the rectal temperatures of SPF mice and gut flora depleted-BF mice normalized again from 60 minutes onwards (34.9 versus 36.8 versus 37.8 °C, respectively). Moreover, the untreated BF mice suffered from TRALI while the SPF mice and the gut flora depleted-BF mice were protected from TRALI development (lung W/Ds 5.77 versus 4.63 versus 4.49, respectively) which was also evident from lung histology-analyses. The prevention of TRALI in SPF mice and gut flora depleted BF mice was paralleled by decreased plasma MIP-2 levels compared to the untreated BF mice which suffered from TRALI (MIP-2: 0.09 versus 0.08 versus 82.94 pg/ml, respectively) which also corresponded to pulmonary neutrophil numbers. As previously shown, low IL-10 levels were associated with TRALI development and IL-10 KO mice were found to be susceptible to antibody-mediated murine TRALI (without prior in vivo cell-depletion). However, when IL-10 KO mice were housed under SPF conditions and injected with TRALI-inducing antibodies, they did not display increased lung W/Ds levels compared to naïve SPF mice indicating that the gut flora precedes and dictates the biological TRALI response. These data collectively link the gut flora to the development of antibody-mediated TRALI in mice and antibiotic treatment prevents TRALI and may perhaps prevent the onset of human TRALI which may help in TRALI risk-assessment prior to transfusion. DisclosuresSemple:Rigel: Consultancy; Novartis: Consultancy; Amgen: Consultancy; UCB: Consultancy.

Peripheral blood monocyte-derived chemokine blockade prevents murine transfusion-related acute lung injury (TRALI)

AbstractTransfusion-related acute lung injury (TRALI) is the leading cause of transfusion-related mortality and can occur with any type of transfusion. TRALI is thought to be primarily mediated by donor antibodies activating recipient neutrophils resulting in pulmonary endothelial damage. Nonetheless, details regarding the interactions between donor antibodies and recipient factors are unknown. A murine antibody-mediated TRALI model was used to elucidate the roles of the F(ab′)2 and Fc regions of a TRALI-inducing immunoglobulin G anti–major histocompatibility complex (MHC) class I antibody (34.1.2s). Compared with intact antibody, F(ab′)2 fragments significantly increased serum levels of the neutrophil chemoattractant macrophage inflammatory protein 2 (MIP-2); however, pulmonary neutrophil levels were only moderately increased, and no pulmonary edema or mortality occurred. Fc fragments did not modulate any of these parameters. TRALI induction by intact antibody was completely abrogated by in vivo peripheral blood monocyte depletion by gadolinium chloride (GdCl3) or chemokine blockade with a MIP-2 receptor antagonist but was restored upon repletion with purified monocytes. The results suggest a two-step process for antibody-mediated TRALI induction: the first step involves antibody binding its cognate antigen on blood monocytes, which generates MIP-2 chemokine production that is correlated with pulmonary neutrophil recruitment; the second step occurs when antibody-coated monocytes increase Fc-dependent lung damage.