Red blood cells (RBCs) are the most numerous cell type in the body and serve a vital purpose of delivering oxygen to essentially all tissues. In addition to the central role of RBCs in health and disease, RBC storage is a requirement for the >90 million units of RBC transfusions given to millions of recipients each year, worldwide. It is well known that there is genetic donor-to-donor variability in how human RBCs store, rendering blood a nonstandardized therapeutic with a wide range of biological properties from unit to unit, by the time it is transfused. As with humans, genetic variation exists in how murine RBCs, from different strains of mice, store and perform after transfusion. The genetic mechanisms for variation, in humans and mice, both remain obscure. Combining advanced metabolomics, genetics, and molecular and cellular biology approaches, we identify genetic variation in six-transmembrane epithelial antigen of prostate 3 (Steap3) expression as a critical and previously unrecognized mechanism of oxidative damage of RBCs during storage. Increased levels of Steap3 result in degradation of cellular membrane through lipid peroxidation, leading to failure of RBC homeostasis and hemolysis/clearance of RBCs. This article is the first report of a role of Steap3 in mature RBCs; it defines a new mechanism of redox biology in RBCs with a substantial effect upon RBC function and provides a novel mechanistic determinant of genetic variation of RBC storage.
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 antibodymediated 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 Ideficient 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.
Band 3 (anion exchanger 1 - AE1) is the most abundant membrane protein in red blood cells (RBCs), the most abundant cell in the human body. A compelling model posits that - at high oxygen saturation - the N-term cytosolic domain of AE1 binds to and inhibits glycolytic enzymes, thus diverting metabolic fluxes to the pentose phosphate pathway to generate reducing equivalents. Dysfunction of this mechanism occurs during RBC aging or storage under blood bank conditions, suggesting a role for AE1 in the regulation of blood storage quality and efficacy of transfusion – a life-saving intervention for millions of recipients worldwide. Here we leverage two murine models carrying genetic ablations of AE1 to provide mechanistic evidence of its role in the regulation of erythrocyte metabolism and storage quality. Metabolic observations in mice recapitulated those in a human subject lacking expression of AE11-11 (band 3 Neapolis), while common polymorphisms in the region coding for AE11-56 correlate with increased susceptibility to osmotic hemolysis in healthy blood donors. Through thermal proteome profiling and cross-linking proteomics, we provide a map of the RBC interactome, with a focus on AE11-56 and validate recombinant AE1 interactions with glyceraldehyde 3-phosphate dehydrogenase (GAPDH). As a proof-of-principle and further mechanistic evidence of the role of AE1 in the regulation of redox homeostasis of stored RBCs, we show that incubation with a cell-penetrating AE11-56 peptide can rescue the metabolic defect in glutathione recycling and boost post-transfusion recoveries of stored RBCs from healthy human donors and genetically ablated mice.
Antibodies are typically thought of as the endpoint of humoral immunity that occur as the result of an adaptive immune response. However, affinity-matured antibodies can be present at the initiation of a new immune response, most commonly because of passive administration as a medical therapy. The current paradigm is that immunoglobulin M (IgM), IgA, and IgE enhance subsequent humoral immunity. In contrast, IgG has a “dual effect” in which it enhances responses to soluble antigens but suppresses responses to antigens on red blood cells (RBCs) (eg, immunoprophylaxis with anti-RhD). Here, we report a system in which passive antibody to an RBC antigen promotes a robust cellular immune response leading to endogenous CD4+ T-cell activation, germinal center formation, antibody secretion, and immunological memory. The mechanism requires ligation of Fcγ receptors on a specific subset of dendritic cells that results in CD4+ T-cell activation and expansion. Moreover, antibodies cross-enhance responses to a third-party antigen, but only if it is expressed on the same RBC as the antigen recognized by the antibody. Importantly, these observations were IgG subtype specific. Thus, these findings demonstrate that antibodies to RBC alloantigens can enhance humoral immunity in an IgG subtype-specific fashion and provide mechanistic elucidation of the enhancing effects.
Computational models based on recent maps of the RBC proteome suggest that mature erythrocytes may harbor targets for common drugs. This prediction is relevant to RBC storage in the blood bank, in which the impact of small molecule drugs or other xenometabolites deriving from dietary, iatrogenic, or environmental exposures (“exposome”) may alter erythrocyte energy and redox metabolism and, in so doing, affect red cell storage quality and posttransfusion efficacy. To test this prediction, here we provide a comprehensive characterization of the blood donor exposome, including the detection of common prescription and over-the-counter drugs in blood units donated by 250 healthy volunteers in the Recipient Epidemiology and Donor Evaluation Study III Red Blood Cell–Omics (REDS-III RBC-Omics) Study. Based on high-throughput drug screenings of 1366 FDA-approved drugs, we report that approximately 65% of the tested drugs had an impact on erythrocyte metabolism. Machine learning models built using metabolites as predictors were able to accurately predict drugs for several drug classes/targets (bisphosphonates, anticholinergics, calcium channel blockers, adrenergics, proton pump inhibitors, antimetabolites, selective serotonin reuptake inhibitors, and mTOR), suggesting that these drugs have a direct, conserved, and substantial impact on erythrocyte metabolism. As a proof of principle, here we show that the antacid ranitidine — though rarely detected in the blood donor population — has a strong effect on RBC markers of storage quality in vitro. We thus show that supplementation of blood units stored in bags with ranitidine could — through mechanisms involving sphingosine 1–phosphate–dependent modulation of erythrocyte glycolysis and/or direct binding to hemoglobin — improve erythrocyte metabolism and storage quality.
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