Biology subjects

Zimring, J. C.

Publications and source records attributed to Zimring, J. C..

16 recordsLinked to original sources

bioRxiv2026

Genetic architecture of the murine serum metabolome reveals carboxyl esterases as master regulators of circulating fatty acid metabolism

BackgroundThe systemic biochemical diversity of circulating metabolites and lipids reflects the integrated effects of genetic variation and environmental exposure. Metabolite quantitative trait locus (mQTL) studies in humans have established gene-metabolite associations, but genetic contributions can be obscured by sex, diet, age, medication use, and environmental exposures. Genetically diverse model systems offer a powerful complementary strategy to isolate genetic contributions to the biochemical diversity of the circulating metabolome. Methodology/Principal FindingsWe applied mass spectrometry profiling to serum samples in 541 mice from the Diversity Outbred (DO) population and identified 1,933 mQTL across 240 metabolites, 561 lipids, 43 oxylipins, and 4,465 MS/MS features. Co-mapping QTL, i.e., QTL hotspots, on chromosomes 8 and 17 implicated carboxyl esterase gene clusters (Ces1 and Ces2) as major regulators of circulating lipid remodeling and demonstrated genetic control of circulating protein/peptide-like features at the major histocompatibility complex and complement C3 loci. QTL hotspots on chromosomes 9 and 10 revealed previously unknown genetic drivers of lipid and amino acid metabolism. Comparisons with matched red blood cell mQTL revealed widespread compartment-specific genetic control. Conclusions/SignificanceCollectively, these findings provide a high-resolution map of the genetic regulation of the circulating metabolome, offering mechanistic insights that complement and extend human metabolic genetics.

genomics↗

bioRxiv2025

Deep Red Blood Cell Proteome Defines the Band 3 N-Terminus Interactome as a Regulator of Hypoxic Adaptation via BLVRB-Dependent S-Nitroso Transfer

Red blood cells (RBCs) have long been regarded as passive oxygen carriers, yet growing evidence reveals a complex, dynamic proteome independent of de novo gene expression. Here, we define the erythrocyte as an oxygen-responsive system organized around a Band 3 (SLC4A1)-centered metabolon. Using deep proteomics of ultra-pure RBCs and cross-linking interactomics, we identify biliverdin reductase B (BLVRB) as a previously unrecognized Band 3 interactor that binds the N-terminal cytosolic domain under normoxia and dissociates under hypoxia, when band 3-deoxyhemoglobin interactions increase threefold. This reversible interaction forms an oxygen-sensitive switch coupling structural, redox, and metabolic remodeling. In humanized mice, truncation of the Band 3 N-terminus disrupted glycolytic activation, reduced 2,3-bisphosphoglycerate synthesis, and impaired exercise tolerance despite preserved cardiopulmonary function, establishing the physiological relevance of this module. Population-scale proteome quantitative trait locus (pQTL) analyses revealed coordinated variation of SLC4A1 and BLVRB abundance but minimal association of biliverdin levels with BLVRB genotype, suggesting alternative functions beyond heme catabolism. Mechanistically, BLVRB Cys109 acts as a nitric oxide (NO) relay, trans-nitrosating glycolytic enzymes such as GAPDH at active site Cys152, transiently inhibiting glycolysis. This S-nitrosation-mediated feedback mirrors conserved mechanisms in plants, where GAPDH-SNO redirects carbon flow toward the Calvin-Benson cycle under nitrosative stress, revealing an evolutionary convergence in gas-responsive metabolic control. Collectively, our findings define a Band 3-BLVRB-hemoglobin axis that links oxygen sensing, NO signaling, and redox homeostasis, providing a unifying model for how an anucleate cell achieves environmental adaptability through reversible protein-protein interactions and post-translational chemistry. Graphic abstractIssaian et al. define the most comprehensive proteome of ultra-pure human red blood cells (3,775 proteins) and map the O2-dependent interactome, revealing a Band 3-BLVRB-hemoglobin module that links oxygen sensing to metabolic remodeling via reversible inhibitory S-nitrosation of GAPDH C152. In plants this redirects carbon toward photosynthesis, illustrating a conserved NO-dependent metabolic reprogramming mechanism across oxygen-regulated systems. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/691178v1_ufig1.gif" ALT="Figure 1"> View larger version (89K): org.highwire.dtl.DTLVardef@122468org.highwire.dtl.DTLVardef@114428dorg.highwire.dtl.DTLVardef@11d40c4org.highwire.dtl.DTLVardef@1870fb7_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIDeep proteomics defines a complete, contamination-free RBC proteome (3,775 proteins) C_LIO_LICross-linking proteomics maps an oxygen-sensitive Band 3-centered interactome C_LIO_LIO2-dependent BLVRB-Band 3 binding regulates metabolism via S-nitrosation of GAPDH C_LIO_LIBand 3 N-terminus is required for hypoxic remodeling and exercise tolerance in vivo C_LI

biochemistry↗

bioRxiv2025

Genetic variation of human G6PD impacts Red Blood Cell transfusion efficacy

Glucose-6-phosphate dehydrogenase (G6PD) deficiency, the most common human enzymopathy, affects 6% of the global population, yet its impact on blood storage and transfusion efficacy remains undefined. We integrated genome-metabolome-proteome analyses of 13,091 blood donors (362 G6PD SNPs), validated in a recalled cohort (n=643), linked donor-recipient databases, humanized mouse models (canonical, African A- [V68M+N126D], Mediterranean [S188F]), and a prospective sickle cell disease study. Common G6PD variants reduced protein abundance, reprogrammed redox metabolism, and increased storage hemolysis. In mice, G6PD-deficient RBCs showed lower post-transfusion recovery, higher oxidative stress, and impaired renal oxygenation. Clinically, recipients of G6PD-deficient units exhibited smaller hemoglobin increments and reduced RBC L{superscript 1}Cr-survival (-8% at 24 h; -12% at 4 weeks). Structural studies revealed kinetic fragility for A- and thermodynamic fragility for Med-, linking genotype to protein instability and transfusion outcome. These findings identify donor G6PD genotype as a determinant of transfusion efficacy, supporting genotype-aware inventory-management strategies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/689741v1_ufig1.gif" ALT="Figure 1"> View larger version (76K): org.highwire.dtl.DTLVardef@1897489org.highwire.dtl.DTLVardef@1420587org.highwire.dtl.DTLVardef@178e8ddorg.highwire.dtl.DTLVardef@1003b44_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Red Blood Cell Transfusion is a Non-Canonical Immune Stimulus Characterized by the Suboptimal Induction of CD4+ T Cell Help

Red blood cell (RBC) alloimmunization to non-ABO antigens is a major clinical complication for chronically transfused patients. When exposed to transfused RBCs carrying foreign antigens, some patients generate IgG antibodies that target these antigens, creating potential barriers to future transfusions. Interestingly, other patients produce only IgM antibodies against the same non-ABO antigens, which generally have fewer clinical consequences. Despite the stark differences in their impact, the factors regulating IgM versus IgG production in response to transfused RBCs remain poorly understood. This study explores the balance between IgM and IgG production following transfusion, comparing it to the well-characterized antibody response induced by vaccination in mouse models. By directly assessing antibody levels following RBC transfusion versus Alum-adjuvanted vaccination, we demonstrate that transfusion of RBCs expressing a model antigen is a relatively weak inducer of IgG class switching. Additionally, loss-of-function experiments using CD40L blockade and CD4 depletion confirmed that T cell help is essential for class switching after transfusion but has no effect on IgM production. Most notably, providing supra-physiological levels of T cell help enhanced class switching in a dose-dependent manner after transfusion, whereas vaccination-induced class switching remained unaffected. These findings support a model in which the limited IgG class switching following transfusion stems from suboptimal T cell help compared to vaccination. Furthermore, they suggest that transfusion activates T cells through a non-canonical pathway, distinct from the mechanisms driving immune responses to standard Alum vaccination.

immunology↗

bioRxiv2025

Caffeine Impairs Red Blood Cell Storage Quality by Dual Inhibition of ADORA2b Signaling and G6PD Activity

Caffeine is the most widely consumed psychoactive substance globally, yet its peripheral physiological effects remain incompletely understood. Leveraging comprehensive data from 13,091 blood donors in the REDS RBC-Omics study, we identify caffeine as a significant modulator of red blood cell (RBC) storage quality and transfusion outcomes. Elevated caffeine levels were reproducible across multiple donations from 643 recalled donors, selected based on their extremes in hemolytic propensity. Both in the screening and recalled cohorts, higher caffeine levels were associated with disrupted RBC metabolism, characterized by reduced glycolysis, depletion of adenylate pools or 2,3-bisphosphoglycerate, and increased markers of oxidative stress and osmotic fragility, including kynurenine accumulation. These observations were recapitulated in plasma and RBCs of eight volunteers upon consumption of a cup of coffee independently of brewing method (Chemex vs espresso). Clinically, elevated caffeine correlated with increased hemolysis and lower post-transfusion hemoglobin increments, especially pronounced in recipients transfused with RBCs from donors carrying common polymorphisms in the ADORA2b gene, a key regulator of RBC metabolism in hypoxia. These human findings were mechanistically validated using a murine model deficient in ADORA2b, which demonstrated impaired glycolytic flux, compromised antioxidant defenses - including caffeine-dependent direct inhibition of recombinantly-expressed glucose 6-phosphate dehydrogenase, and decreased transfusion efficacy (lower hemoglobin increments, higher bilirubin post-transfusion), effects further exacerbated by caffeine exposure during storage. Our study positions caffeine consumption as a modifiable factor in blood transfusion practice, advocating for precision strategies that integrate genetic and exposome factors, and identifies metabolic interventions to enhance blood quality and clinical outcomes. One sentence summaryCaffeine consumption and genetic variants in the ADORA2b receptor synergistically impair red blood cell metabolism and transfusion efficacy, revealing a modifiable exposome-gene interaction for precision transfusion medicine.

biochemistry↗

bioRxiv2025

Genetic architecture of the red blood cell proteome in genetically diverse mice reveals central role of hemoglobin beta cysteine redox status in maintaining circulating glutathione pools

Red blood cells (RBCs) transport oxygen but accumulate oxidative damage over time, reducing function in vivo and during storage--critical for transfusions. To explore genetic influences on RBC resilience, we profiled proteins, metabolites, and lipids from fresh and stored RBCs obtained from 350 genetically diverse mice. Our analysis identified over 6,000 quantitative trait loci (QTL). Compared to other tissues, prevalence of trans genetic effects over cis reflects the absence of de novo protein synthesis in anucleated RBCs. QTL hotspots at Hbb, Hba, Mon1a, and storage-specific Steap3 linked ferroptosis to hemolysis. Proteasome components clustered at multiple loci, underscoring the importance of degrading oxidized proteins. Post-translational modifications (PTMs) mapped predominantly to hemoglobins, particularly cysteine residues. Loss of reactive C93 in humanized mice (HBB C93A) disrupted redox balance, affecting glutathione pools, protein glutathionylation, and redox PTMs. These findings highlight genetic regulation of RBC oxidation, with implications for transfusion biology and oxidative stress-dependent hemolytic disorders. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/640676v1_ufig1.gif" ALT="Figure 1"> View larger version (98K): org.highwire.dtl.DTLVardef@14a3cc2org.highwire.dtl.DTLVardef@daf8b4org.highwire.dtl.DTLVardef@1fe306forg.highwire.dtl.DTLVardef@11de734_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2025

Splenic MARCO+ marginal zone macrophages regulate rapid production of MCP-1 and KC but are dispensable for alloantibody generation in response to stored RBCs in a murine model.

BACKGROUNDAlloimmunization to transfused red blood cells (RBCs) remains a significant clinical problem. However, the cells that initiate immune responses to transfused RBCs remain incompletely characterized. Recently published work has identified splenic marginal zone B (MZB) cells as being critically required for the production of anti-RBC alloantibodies in response to RBCs. In infectious models, MZB cell activation has been shown to depend on a unique population of marginal zone macrophages (MZMs). We hypothesized that MZMs would capture stored RBCs and present them to MZBs, and ultimately MZMs would be required for generation of anti-RBC alloantibodies in response to stored RBC transfusion. STUDY DESIGN AND METHODSStored GFP+ murine RBCs were utilized to determine the splenic localization and erythrophagocytosis by splenic macrophage populations. To determine the functional impact of MZMs, we compared LXR-KO mice, which have been reported to lack MZMs, with wild type mice. Both innate and adaptive immune responses to stored HOD allogenic RBC transfusion were measured in LXR-KO and wild type mice. RESULTSRBC storage leads to a significant increase in the phagocytosis of transfused RBCs by splenic MZMs. LXR-KO mice demonstrated a lack of MZMs and had significantly decreased rapid phase production of cytokines MCP-1 and KC, but similar levels of IL-6. Surprisingly, anti-RBC alloantibody levels were unaffected by the absence of splenic MZMs. CONCLUSIONSSplenic MZMs are involved in the innate response to transfused stored HOD RBCs, contributing to both MCP-1 and KC cytokine production. However, MZMs are dispensable for anti-RBC alloantibody production.

immunology↗

bioRxiv2025

Transfusion of allogenic murine HOD red blood cells preferentially induces low-affinity, short-lived IgG antibodies that are germinal center independent

Transfusion-induced anti-red blood cell (RBC) alloantibodies pose a significant risk to patients who require chronic transfusions. Anti-RBC alloantibodies can be remarkably short-lived (i.e. evanescent), leading to clinically relevant alloantibodies that are not detected in later pre-transfusion antibody screens. Subsequent transfusion of alloantigen-positive RBCs stimulates a rapid memory antibody response that may induce a delayed hemolytic transfusion reaction (DHTR), causing morbidity and occasional mortality in chronically transfused patients. It is unclear why transfusions favor evanescent antibody responses over long-lived antibodies typically observed upon infections and vaccinations. We therefore turned to the HOD mouse model of RBC alloimmunization to elucidate regulators of antibody persistence in response to allogenic transfusions. By following antibody responses over time in transfused mice, we found that HOD-specific alloantibodies rapidly decay within three months while vaccination-induced antibodies remain constant. Thus, the HOD model recapitulates RBC antibody evanescence. The rapid antibody evanescence suggests that transfusion is a poor inducer of germinal centers (GCs), specialized immunological structures where B cells differentiate into germinal center B (GC B) cells and undergo iterative rounds of affinity maturation, ultimately differentiating into long-lived plasma cells that can produce antibodies for decades. Consistent with this hypothesis, we failed to observe an increase in GC B cell formation in response to transfusion, and the majority of anti-RBC alloantibodies were low affinity when compared to vaccination. To formally test the functional requirement for GCs in anti-RBC alloantibody production, we employed two orthogonal approaches to disrupt GC formation: i) day 4 CD40L blockade and ii) genetic disruption of the GC-transcription factor BCL6 selectively in B cells. Both approaches fully blocked GC formation, yet anti-RBC alloantibody production was unchanged. Collectively, our data demonstrate that anti-HOD RBC alloantibodies are GC-independent, low affinity and short-lived. The GC-independence of HOD RBC IgG responses has important implications for understanding the cellular and molecular pathways that regulate the humoral immune response to transfused RBCs, potentially explaining anti-RBC alloantibody evanescence patterns in patients.

immunology↗

bioRxiv2024

Ferroptosis regulates hemolysis in stored murine and human red blood cells

Red blood cell (RBC) metabolism regulates hemolysis during aging in vivo and in the blood bank. Here, we leveraged a diversity outbred mouse population to map the genetic drivers of fresh/stored RBC metabolism and extravascular hemolysis upon storage and transfusion in 350 mice. We identify the ferrireductase Steap3 as a critical regulator of a ferroptosis-like process of lipid peroxidation. Steap3 polymorphisms were associated with RBC iron content, in vitro hemolysis, and in vivo extravascular hemolysis both in mice and 13,091 blood donors from the Recipient Epidemiology and Donor evaluation Study. Using metabolite Quantitative Trait Loci analyses, we identified a network of gene products (FADS1/2, EPHX2 and LPCAT3) - enriched in donors of African descent - associated with oxylipin metabolism in stored human RBCs and related to Steap3 or its transcriptional regulator, the tumor protein TP53. Genetic variants were associated with lower in vivo hemolysis in thousands of single-unit transfusion recipients. HighlightsO_LISteap3 regulates lipid peroxidation and extravascular hemolysis in 350 diversity outbred mice C_LIO_LISteap3 SNPs are linked to RBC iron, hemolysis, vesiculation in 13,091 blood donors C_LIO_LImQTL analyses of oxylipins identified ferroptosis-related gene products FADS1/2, EPHX2, LPCAT3 C_LIO_LIFerroptosis markers are linked to hemoglobin increments in transfusion recipients C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/598512v1_ufig1.gif" ALT="Figure 1"> View larger version (117K): org.highwire.dtl.DTLVardef@150fec2org.highwire.dtl.DTLVardef@859c43org.highwire.dtl.DTLVardef@1d60156org.highwire.dtl.DTLVardef@f1b91e_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

IgG hexamers initiate acute lung injury

Antibodies can initiate lung injury in a variety of disease states such as autoimmunity, transfusion reactions, or after organ transplantation, but the key factors determining in vivo pathogenicity of injury-inducing antibodies are unclear. A previously overlooked step in complement activation by IgG antibodies has been elucidated involving interactions between IgG Fc domains that enable assembly of IgG hexamers, which can optimally activate the complement cascade. Here, we tested the in vivo relevance of IgG hexamers in a complement-dependent alloantibody model of acute lung injury. We used three approaches to block alloantibody hexamerization (antibody carbamylation, the K439E Fc mutation, or treatment with domain B from Staphylococcal protein A), all of which reduced acute lung injury. Conversely, Fc mutations promoting spontaneous hexamerization made a harmful alloantibody into a more potent inducer of acute lung injury and rendered an innocuous alloantibody pathogenic. Treatment with a recombinant Fc hexamer decoy therapeutic protected mice from lung injury, including in a model with transgenic human FCGR2A expression that exacerbated pathology. These results indicate a direct in vivo role of IgG hexamerization in initiating acute lung injury and the potential for therapeutics that inhibit or mimic hexamerization to treat antibody-mediated diseases. Brief summaryIgG antibodies can form hexamers. This study shows that hexamer assembly is an important event determining the ability of IgG to trigger acute lung injury. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/577129v1_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@15b0346org.highwire.dtl.DTLVardef@33f39eorg.highwire.dtl.DTLVardef@bf0f4dorg.highwire.dtl.DTLVardef@185b5bf_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2023

Biological and Genetic Determinants of Red Blood Cell Glycolysis

Mature red blood cells (RBCs) lack mitochondria, and thus exclusively rely on glycolysis to generate adenosine triphosphate (ATP) during aging in vivo or storage in the blood bank. Here we leveraged 13,029 volunteers from the Recipient Epidemiology and Donor Evaluation Study to identify an association between end-of-storage levels of glycolytic metabolites and donor age, sex, and ancestry-specific genetic polymorphisms in regions encoding phosphofructokinase 1, platelet (detected in mature RBCs), hexokinase 1, ADP-ribosyl cyclase 1 and 2 (CD38/BST1). Gene-metabolite associations were validated in fresh and stored RBCs from 525 Diversity Outbred mice, and via multi-omics characterization of 1,929 samples from 643 human RBC units during storage. ATP and hypoxanthine levels - and the genetic traits linked to them - were associated with hemolysis in vitro and in vivo, both in healthy autologous transfusion recipients and in 5,816 critically ill patients receiving heterologous transfusions, suggesting their potential as markers to improve transfusion outcomes. eTOC and Highlights O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/557250v4_ufig1.gif" ALT="Figure 1"> View larger version (87K): org.highwire.dtl.DTLVardef@1a556b7org.highwire.dtl.DTLVardef@1e02b13org.highwire.dtl.DTLVardef@2bfab1org.highwire.dtl.DTLVardef@15787e3_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIBlood donor age and sex affect glycolysis in stored RBCs from 13,029 volunteers; C_LIO_LIAncestry, genetic polymorphisms in PFKP, HK1, CD38/BST1 influence RBC glycolysis; C_LIO_LIModeled PFKP effects relate to preventing loss of the total AXP pool in stored RBCs; C_LIO_LIATP and hypoxanthine are biomarkers of hemolysis in vitro and in vivo. C_LI

biochemistry↗

bioRxiv2023

Reticulocytes in donor RBC units enhance RBC alloimmunization

Although red blood cell (RBC) transfusions save lives, some patients develop clinically-significant alloantibodies against donor blood group antigens, which then have adverse effects in multiple clinical settings. Few effective measures exist to prevent RBC alloimmunization and/or eliminate alloantibodies in sensitized patients. Donor-related factors may influence alloimmunization; thus, there is an unmet clinical need to identify which RBC units are immunogenic. Repeat volunteer blood donors and donors on iron supplements have elevated reticulocyte counts compared to healthy non-donors. Early reticulocytes retain mitochondria and other components, which may act as danger signals in immune responses. Herein, we tested whether reticulocytes in donor RBC units could enhance RBC alloimmunization. Using a murine model, we demonstrate that transfusing donor RBC units with increased reticulocyte frequencies dose-dependently increase RBC alloimmunization rates and alloantibody levels. Transfusing reticulocyte-rich RBC units was associated with increased RBC clearance from the circulation and a robust proinflammatory cytokine response. As compared to previously reported post-transfusion RBC consumption patterns, erythrophagocytosis from reticulocyte-rich units was increasingly performed by splenic B cells. These data suggest that reticulocytes in a donated RBC unit impact the quality of blood transfused, are targeted to a distinct compartment, and may be an underappreciated risk factor for RBC alloimmunization.

immunology↗

bioRxiv2022

Genome-wide metabolite quantitative trait loci analysis (mQTL) in red blood cells from volunteer blood donors

The Red Blood Cell (RBC)-Omics study, part of the larger NHLBI-funded Recipient Epidemiology and Donor Evaluation Study (REDS-III), aims to understand the genetic contribution to blood donor RBC characteristics. Previous work identified donor demographic, behavioral, genetic and metabolic underpinnings to blood donation, storage, and - to a lesser extent - transfusion outcomes, but none have yet linked the genetic and metabolic bodies of work. We performed a Genome-Wide Association (GWA) analysis using RBC-Omics study participants with generated untargeted metabolomics data to identify metabolite quantitative trait loci (mQTL) in RBCs. We performed GWA analyses of 382 metabolites in 243 individuals imputed using the 1000 Genomes Project phase 3 all-ancestry reference panel. Analyses were conducted using ProbABEL and adjusted for sex, age, donation center, number of whole blood donations in the past two years, and first ten principal components of ancestry. Our results identified 423 independent genetic loci associated with 132 metabolites (p < 5x10-8). Potentially novel locus-metabolite associations were identified for FLVCR1 and choline, and for LPCAT3 and the lysophosphatidylserine 16.0, 18.0, 18.1, and 18.2; these associations are supported by published rare disease and mouse studies. We also confirmed previous metabolite GWA results for associations including N(6)-Methyl-L-lysine and PYROXD2, and various carnitines and SLC22A16. Association between pyruvate levels and G6PD polymorphisms was validated in an independent cohort and novel murine models of G6PD deficiency (African and Mediterranean variants). We demonstrate that it is possible to perform metabolomics-scale GWA analyses with a modest, trans-ancestry sample size. Key pointsO_LIMetabolite heterogeneity in fresh (<14 day old) RBCs donated by volunteer donors is linked to genetic polymorphisms; C_LIO_LIWe report 2,831 high-confidence SNP-metabolite linkages (p < 5.0 x 10-8). Pyruvate levels in fresh RBCs are associated with glucose-6-phosphate dehydrogenase (G6PD) status C_LI

biochemistry↗

bioRxiv2021

Innate and adaptive immunity to transfused allogenic red blood cells in mice requires MyD88

Red blood cell (RBC) transfusion therapy is essential for the survival of patients with hematological disorders such as sickle cell anemia. A potentially fatal complication of transfusion is development of non-ABO alloantibodies to polymorphic RBC antigens, yet mechanisms of alloantibody formation remain unclear. Human and mouse RBCs acquire a "storage lesion" prior to transfusion, which in mice contributes to immunogenicity. We previously reported that mouse splenic dendritic cells (DCs) are required for RBC alloimmunization and are activated by sterile and leukoreduced mouse RBCs after storage. Yet how syngeneic RBCs activate innate immune pathways to induce DC activation is unknown. We now show that DC activation to transfused RBCs occurs regardless of alloantigen presence, suggesting that RBC damage induced during storage triggers innate immune receptors. We discovered an unexpected dependence of RBC alloimmunization on the Toll-like receptor (TLR) signaling adaptor molecule MyD88. TLRs are a class of pattern recognition receptors (PRRs) that regulate DC activation and signal through two adaptor molecules, MyD88 and TRIF. We show that the inflammatory cytokine response, DC activation, and the subsequent alloantibody response to transfused syngeneic RBCs require MyD88 but not TRIF, suggesting a restricted set of PRRs are responsible for sensing RBCs and triggering alloimmunization.

immunology↗

bioRxiv2021

Hematologic and systemic metabolic alterations due to Mediterranean type II G6PD deficiency in a novel murine model

Deficiency of Glucose 6 phosphate dehydrogenase (G6PD) is the single most common enzymopathy, present in approximately 400 million humans (e.g. 5% of humans). Its prevalence is hypothesized to be due to conferring resistance to malaria. However, G6PD deficiency also results in hemolytic sequelae from oxidant stress. Moreover, G6PD deficiency is associated with kidney disease, diabetes, pulmonary hypertension, immunological defects, and neurodegenerative diseases. To date, the only available mouse models have decreased levels of G6PD due to promoter mutations, but with stable G6PD. However, human G6PD mutations are missense mutations that result in decreased enzymatic stability. As such, this results in very low activity in red blood cells and platelets that cannot synthesize new protein. To generate a more accurate model, the human sequence for a severe form of G6PD deficiency (Med -) was knocked into the murine G6PD locus. As predicted, G6PD levels were extremely low in RBCs and deficient mice have increased hemolytic sequalae to oxidant stress. G6PD levels were mildly decreased in non-erythroid organs, consistent with what has been observed in humans. Juxtaposition of G6PD deficient and wild-type mice revealed altered lipid metabolism in multiple organ systems. Together, these findings both establish a new mouse model of G6PD deficiency that more accurately reflects human G6PD deficiency and also advance our basic understanding of altered metabolism in this setting.

biochemistry↗

bioRxiv2020

The interactome of the N-terminus of band 3 regulates red blood cell metabolism and storage quality

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, based on indirect evidence, 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 the first direct mechanistic evidence of its role in metabolic regulation and blood storage quality. Observations in mice phenocopied those in a human subject lacking expression of AE11-11 (band 3 Neapolis), while common polymorphisms in the region coding for AE11-56 increased susceptibility to osmotic hemolysis in healthy blood donors. Through thermal proteome profiling and cross-linking proteomics, we provide the first comprehensive analysis of the RBC interactome, with a focus on AE11-56 and validate recombinant AE1 interactions with glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Finally, 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, paving the way for the in vivo metabolic manipulation of RBCs facing oxidant stress - a landmark of many diseases. Key pointsO_LIGenetic ablation of N-term of band 3 results in significant metabolic aberrations and poor post-transfusion recoveries in mice and humans; C_LIO_LIStructural studies on the N-term of band 3 reveal a complex interactome with several enzymes, including GAPDH; C_LI

biochemistry↗