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Despite the identification of the high-incidence red cell antigen Era nearly 40 years ago, the molecular background of this antigen, together with the other 2 members of the Er blood group collection, has yet to be elucidated. Whole exome and Sanger sequencing of individuals with serologically defined Er alloantibodies identified several missense mutations within the PIEZO1 gene, encoding amino acid substitutions within the extracellular domain of the Piezo1 mechanosensor ion channel. Confirmation of Piezo1 as the carrier molecule for the Er blood group antigens was demonstrated using immunoprecipitation, CRISPR/Cas9-mediated gene knockout, and expression studies in an erythroblast cell line. We report the molecular bases of 5 Er blood group antigens: the recognized Era, Erb, and Er3 antigens and 2 novel high-incidence Er antigens, described here as Er4 and Er5, establishing a new blood group system. Anti-Er4 and anti-Er5 are implicated in severe hemolytic disease of the fetus and newborn. Demonstration of Piezo1, present at just a few hundred copies on the surface of the red blood cell, as the site of a new blood group system highlights the potential antigenicity of even low-abundance membrane proteins and contributes to our understanding of the in vivo characteristics of this important and widely studied protein in transfusion biology and beyond.

19 p.-6 fig., Mitochondria and peroxisomes are closely related metabolic organelles, both in terms of origin and in terms of function. Mitochondria and peroxisomes can also be turned over by autophagy, in processes termed mitophagy and pexophagy, respectively. However, despite their close relationship, it is not known if both organelles are turned over under similar conditions, and if so, how this might be coordinated molecularly. Here, we find that multiple selective autophagy pathways are activated upon iron chelation and show that mitophagy and pexophagy occur in a BNIP3L/NIX-dependent manner. We reveal that the outer mitochondrial membrane-anchored NIX protein, previously described as a mitophagy receptor, also independently localises to peroxisomes and drives pexophagy. We show this process happens in vivo, with mouse tissue that lacks NIX having a higher peroxisomal content. We further show that pexophagy is stimulated under the same physiological conditions that activate mitophagy, including cardiomyocyte and erythrocyte differentiation. Taken together, our work uncovers a dual role for NIX, not only in mitophagy but also in pexophagy, thus illustrating the interconnection between selective autophagy pathways., This work was funded by an EMBO Long-Term Fellowship (LPW; ALTF 1077-2018), a grant from the Medical Research Council, UK (IGG; MC_UU_00018/2) and by Ministerio de Ciencia, Innovación y Universidades (MCIU), Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (FEDER) PGC2018-098557-B-I00 to PB.

The SARS‐CoV‐2 virus causes COVID‐19, an infection capable of causing severe disease and death but which can also be asymptomatic or oligosymptomatic. We investigated whether ABO blood group or secretor status was associated with COVID‐19 severity. We investigated secretor status because expression of ABO glycans on secreted proteins and non‐erythroid cells are controlled by a fucosyltransferase (FUT2), and inactivating FUT2 mutations result in a non‐secretor phenotype which protects against some viral infections. Data combined from healthcare records and our own laboratory tests (n = 275) of hospitalized SARS‐CoV‐2 polymerase chain reaction positive patients confirmed higher than expected numbers of blood group A individuals compared to O (RR = 1.24, CI 95% [1.05, 1.47], p = 0.0111). There was also a significant association between group A and COVID‐19‐related cardiovascular complications (RR = 2.56, CI 95% [1.43, 4.55], p = 0.0011) which is independent of gender. Molecular analysis revealed that group A non‐secretors are significantly less likely to be hospitalized than secretors. Testing of convalescent plasma donors, among whom the majority displayed COVID‐19 symptoms and only a small minority required hospitalization, group A non‐secretors were slightly over‐represented. Our findings showed that group A non‐secretors are not resistant to infection by SARS‐CoV‐2, but are more likely to experience a less severe form of associated disease.

This paper reflects on collaboration between an artist, a scientist, and two social scientists involved in research on cultured red blood cells. Cell culture is part of a suite of methods used by bioscientists to study cellular processes outside of the living organism. The production of laboratory-grown blood is at the cutting-edge of cell culture and regenerative medicine, with hopes for significant therapeutic benefit in the future, particularly for patients with rare blood types or with conditions that require frequent blood transfusions. We reflect on our collaboration and on artistic experimentation with spatial dimensions of cells and scaffolds used in red blood cell culture, highlighting our efforts to generate knowledge that cuts across our respective disciplinary locations. We situate our work together in the context of the increasing molecularization of the body in science and medicine, and on the efforts to ‘open up’ scientific practice to multiple publics.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare autosomal metabolic disorder caused by thymidine phosphorylase (TP) deficiency. Successful therapeutic interventions for this disease rely on a means for efficient and long-lasting circulation of the TP enzyme. In this study we exploit lentiviral transduction of hematopoietic stem cells and an erythroid cell line (BEL-A) to generate reticulocytes that contain active TP. Significant loss of overexpressed TP during erythroid differentiation can be reduced by addition of the ubiquitination inhibitor MG132. However, the ubiquitination sites are located in the substrate binding site in human TP, and their removal abolished enzyme activity. Examination of the TP structure and mechanism suggested that these sites are only exposed in the absence of substrate. We show that supplementation of culture media with thymidine during differentiation reduces enzyme degradation, doubling the amount of TP retained in reticulocytes. This study provides proof of principle that therapeutic reticulocytes expressing TP can be generated in vitro and that ubiquitin-mediated degradation can be subverted through masking ubiquitination sites to ensure retention of human TP in reticulocytes following erythroid differentiation.

[No abstract]

Severe COVID-19 appears rare in children. This is unexpected, especially in young infants, who are vulnerable to severe disease caused by other respiratory viruses. We evaluate convalescent immune responses in 4 infants under 3 months old with confirmed COVID-19 who presented with mild febrile illness, alongside their parents, and adult controls recovered from confirmed COVID-19. Although not statistically significant, compared to seropositive adults, infants have high serum levels of IgG and IgA to SARS-CoV-2 spike protein, with a corresponding functional ability to block SARS-CoV-2 cellular entry. Infants also exhibit robust saliva anti-spike IgG and IgA responses. Spike-specific IFN-γ production by infant peripheral blood mononuclear cells appears restrained, but the frequency of spike-specific IFN-γ- and/or TNF-α-producing T cells is comparable between infants and adults. On principal-component analysis, infant immune responses appear distinct from their parents. Robust functional antibody responses alongside restrained IFN-γ production may help protect infants from severe COVID-19., Graphical abstract, Goenka et al. demonstrate that the SARS-CoV-2 immune response of young infants appears distinct compared with their parents. They show that infants exhibit relatively high serum (and saliva) levels of anti-spike IgG associated with robust SARS-CoV-2 neutralization but restrained cellular spike-specific IFN-γ production.

The SARS-CoV-2 virus causes COVID-19, an infection capable of causing severe disease and death but which may also be asymptomatic or oligosymptomatic in many individuals. While several risk factors, including age, have been described, the mechanisms of this variation are poorly understood. Several studies have described associations between blood group and COVID-19 severity, while others do not. Expression of ABO glycans on secreted proteins and non-erythroid cells is controlled by a fucosyltransferase (FUT2). Inactivating mutations result in a non-secretor phenotype which is known to protect against some viral infections. We investigated whether ABO or secretor status was associated with COVID-19 severity. Data combined from healthcare records and laboratory tests (n=275) of SARS-CoV-2 PCR positive patients hospitalised with COVID-19, confirmed higher than expected numbers of blood group A individuals compared to O (RR=1.24, CI 95% [1.05,1.47], P=0.0111). There was also a significant association between group A and COVID-19-related cardiovascular complications (RR=2.56, CI 95% [1.43,4.55], P=0.0011) which is independent of gender. Molecular analysis of phenotype revealed that group A patients who are non-secretors are significantly less likely to be hospitalised than secretors. In a larger cohort of 1000 convalescent plasma donors, among whom the majority displayed COVID-19 symptoms and only a small minority required hospitalisation, group A non-secretors were slightly over-represented. Our findings indicate that group A non-secretors are not resistant to infection by SARS-CoV-2, but they are likely to experience a less severe form of its associated disease.Key PointsBlood group type A is associated with an increased risk of cardiovascular complications in COVID-19 patients.FUT2 “non-secretor” status reduces the risk of severe COVID-19 outcomes in patients with blood group A.

[No Abstract]

The process of maturation of reticulocytes into fully mature erythrocytes that occurs in the circulation is known to be characterized by a complex interplay between loss of cell surface area and volume, removal of remnant cell organelles and redundant proteins, and highly selective membrane and cytoskeletal remodeling. However, the mechanisms that underlie and drive these maturational processes in vivo are currently poorly understood and, at present, reticulocytes derived through in vitro culture fail to undergo the final transition to erythrocytes. Here, we used high-throughput proteomic methods to highlight differences between erythrocytes, cultured reticulocytes and endogenous reticulocytes. We identify a cytoskeletal protein, non-muscle myosin IIA (NMIIA) whose abundance and phosphorylation status differs between reticulocytes and erythrocytes and localized it in the proximity of autophagosomal vesicles. An ex vivo circulation system was developed to simulate the mechanical shear component of circulation and demonstrated that mechanical stimulus is necessary, but insufficient for reticulocyte maturation. Using this system in concurrence with non-muscle myosin II inhibition, we demonstrate the involvement of non-muscle myosin IIA in reticulocyte remodeling and propose a previously undescribed mechanism of shear stress-responsive vesicle clearance that is crucial for reticulocyte maturation.

Purpose of review Current research on the human band 3 glycoprotein, the red cell chloride/bicarbonate anion exchanger (AE1), is highlighted and placed within a structural context. Recent Findings The determination of the crystal structure of the membrane domain of human band 3, the founding member of the solute carrier 4 (SLC4) family of bicarbonate transporters, is a major breakthrough toward understanding the mechanism of action of this membrane transport protein, its interaction with partner proteins, and how mutations linked to disease affect its ability to fold and function. Summary Band 3 contains 14 transmembrane segments arranged in a 7+7 transmembrane inverted repeat topology common to all members of the SLC4 family and the unrelated SLC26 anion transporter family. A functional feature of this fold is the presence of a core and a gate domain: The core domain contains two short transmembrane helices (TM3 and 10) that face each other in the middle of the membrane with the positive N-terminal helix dipoles creating the anion-binding site, whereas the gate domain forms the dimer interface. During transport, the movement of these two domains relative to each other provides the intracellular and extracellular compartments with alternating access to the central anion-binding site.

The classical central macrophage found in erythroblastic islands plays an important role in erythroblast differentiation, proliferation and enucleation in the bone marrow. Convenient human in vitro models to facilitate the study of erythroid-macrophage interactions are desired. Recently, we demonstrated that cultured monocytes/macrophages enhance in vitro erythropoiesis by supporting hematopoietic stem and progenitor cell survival. Herein, we describe that these specific macrophages also support erythropoiesis. Human monocytes cultured in serum-free media supplemented with stem cell factor, erythropoietin, lipids and dexamethasone differentiate towards macrophages expressing CD16, CD163, CD169, CD206, CXCR4 and the phagocytic TAM-receptor family. Phenotypically, they resemble both human bone marrow and fetal liver resident macrophages. This differentiation is dependent on glucocorticoid receptor activation. Proteomic studies confirm that glucocorticoid receptor activation differentiates monocytes to anti-inflammatory tissue macrophages with a M2 phenotype, termed GC-macrophages. Proteins involved in migration, tissue residence and signal transduction/receptor activity are upregulated whilst lysosome and hydrolase activity GO-categories are downregulated. Functionally, we demonstrate that GC-macrophages are highly mobile and can interact to form clusters with erythroid cells of all differentiation stages and phagocytose the expelled nuclei, recapitulating aspects of erythroblastic islands. In conclusion, glucocorticoid-directed monocyte differentiation to macrophages represents a convenient model system to study erythroid-macrophage interactions.

The capacity to undergo substantial deformation is a defining characteristic of the red blood cell (RBC), facilitating transit through the splenic interendothelial slits and microvasculature. Establishment of this remarkable property occurs during a process of reticulocyte maturation that begins with egress through micron-wide pores in the bone marrow and is completed within the circulation. The requirement to undertake repeated cycles of deformation necessitates that both reticulocytes and erythrocytes regulate membrane-cytoskeletal protein interactions in order to maintain cellular stability. In the absence of transcriptional activity, modulation of these interactions in RBCs is likely to be achieved primarily through specific protein posttranslational modifications, which at present remain undefined. In this study, we use high-throughput methods to define the processes that underlie the response to deformation and shear stress in both reticulocytes and erythrocytes. Through combination of a bead-based microsphiltration assay with phosphoproteomics we describe posttranslational modification of RBC proteins associated with deformation. Using microsphiltration and microfluidic biochip-based assays, we explore the effect of inhibiting kinases identified using this dataset. We demonstrate roles for GSK3 and Lyn in capillary transit and maintenance of membrane stability following deformation and show that combined inhibition of these kinases significantly decreases reticulocyte capacity to undergo repeated deformation. Finally, we derive a comprehensive and integrative phosphoproteomic dataset that provides a valuable resource for further mechanistic dissection of the molecular pathways that underlie the RBC’s response to mechanical stimuli and for the study of reticulocyte maturation.

Erythropoiesis is one of the most efficient cellular processes in the human body producingapproximately 2.5 million red blood cells every second. This process occurs in a bone marrow niche comprised of a central resident macrophage surrounded by differentiating erythroblasts, termed an erythroblastic island. It is not known what initially attracts the macrophage to erythroblasts to form these islands. The ephrin/EPH receptor family are known to regulate heterophilic cell-cell adhesion. We find that human VCAM1+ and VCAM1- bone marrow macrophages and in vitro culturedmacrophages are ephrin-B2 positive, whereas differentiating human erythroblasts express EPHB4, EPHB6 and EPHA4. Furthermore, we detect a rise in integrin activation on erythroblasts at the stage at which the cells bind which is independent of EPH receptor presence. Using a live cell imaging assay, we show that specific inhibitory peptides or shRNA depletion of EPHB4 cause a significant reduction in the ability of macrophages to interact with erythroblasts but does not affect integrinactivation. This study demonstrates for the first time that EPHB4 expression is required onerythroblasts to facilitate the initial recognition and subsequent interaction with macrophages,alongside the presence of active integrins.

Investigating the role that host erythrocyte proteins play in malaria infection is hampered by the genetic intractability of this anucleate cell. Here we report that reticulocytes derived through in vitro differentiation of an enucleation-competent immortalized erythroblast cell line (BEL-A) support both successful invasion and intracellular development of the malaria parasite Plasmodium falciparum. Using CRISPR-mediated gene knockout and subsequent complementation, we validate an essential role for the erythrocyte receptor basigin in P. falciparum invasion and demonstrate rescue of invasive susceptibility by receptor re-expression. Successful invasion of reticulocytes complemented with a truncated mutant excludes a functional role for the basigin cytoplasmic domain during invasion. Contrastingly, knockout of cyclophilin B, reported to participate in invasion and interact with basigin, did not impact invasive susceptibility of reticulocytes. These data establish the use of reticulocytes derived from immortalized erythroblasts as a powerful model system to explore hypotheses regarding host receptor requirements for P. falciparum invasion., Here, the authors show that reticulocytes derived from immortalized erythroblasts support invasion and development of Plasmodium falciparum and use CRISPR-mediated gene knockout and complementation of an invasion receptor to demonstrate utility of this model system for research in malaria invasion.

Invasion of and intracellular development within the red blood cell by malaria parasites requires interaction with a multitude of host proteins expressed at the surface of or inside the cell. Perhaps the biggest obstacle to dissection of specific functions of host proteins during invasion is the inability to manipulate protein expression in the genetically intractable anucleate red blood cell. Whilst genetic manipulation and subsequent in vitro differentiation of nucleated erythroid precursors have facilitated progress in this area, such approaches are limited by the finite proliferative capacity of primary hematopoietic stem cells, and a failure of erythroleukemic cell lines to enucleate, respectively. Here, we report that reticulocytes derived through in vitro differentiation of an enucleation competent immortalized erythroblast cell line (BEL-A) support both successful invasion and growth by Plasmodium falciparum. Using CRISPR-mediated gene knockout and lentiviral expression of open reading frames, we validate an essential role for the erythrocyte receptor basigin in P. falciparum invasion and, for the first time, demonstrate that this can be rescued by re-expression of the receptor or of a mutant thereof. Specifically, using sustainable edited clones derived from this line, we exclude a functional role for the cytoplasmic domain of basigin during invasion, and challenge the reported requirement of its associated receptor cyclophilin B. These data establish the use of reticulocytes derived from immortalized erythroblasts as a crucial model system to explore specific hypotheses regarding host receptor requirements and involvement in P. falciparum invasion.

[No abstract]

Regular blood transfusion is the cornerstone of care for patients with red blood cell (RBC) disorders such as thalassaemia or sickle‐cell disease. With repeated transfusion, alloimmunisation often occurs due to incompatibility at the level of minor blood group antigens. We use CRISPR‐mediated genome editing of an immortalised human erythroblast cell line (BEL‐A) to generate multiple enucleation competent cell lines deficient in individual blood groups. Edits are combined to generate a single cell line deficient in multiple antigens responsible for the most common transfusion incompatibilities: ABO (Bombay phenotype), Rh (Rhnull), Kell (K0), Duffy (Fynull), GPB (S−s−U−). These cells can be differentiated to generate deformable reticulocytes, illustrating the capacity for coexistence of multiple rare blood group antigen null phenotypes. This study provides the first proof‐of‐principle demonstration of combinatorial CRISPR‐mediated blood group gene editing to generate customisable or multi‐compatible RBCs for diagnostic reagents or recipients with complicated matching requirements.

Hemoglobin (Hb) E/β-thalassemia has a wide spectrum of clinical manifestations that cannot be explained purely by its genetic background. Circulating extracellular vesicles (EVs) are one factor that likely contributes to disease severity. This study has explored the differences in protein composition and quantity between EVs from HbE/β-thalassemic patients and normal healthy individuals. We used Tandem Mass Tag (TMT) labeling mass spectrometry to analyze the EV proteins isolated from the plasma of 15 patients compared to the controls. To reduce biological variation between individuals, the EV proteins isolated from randomly assigned groups of 5 HbE/β-thalassemic patients were pooled and compared to 5 pooled age, and gender-matched controls, in 3 separate experiments. Alpha hemoglobin stabilizing protein had the highest fold increase. Catalase, superoxide dismutase, T-complex proteins, heat shock proteins, transferrin receptor, ferritin, and cathepsin S were also up-regulated in thalassemic circulating EVs. Importantly, haptoglobin and hemopexin were consistently reduced in patients’ EVs across all data sets, in keeping with the existing hemolysis that occurs in thalassemia. The proteomic data analysis of EVs samples isolated from 6 individual HbE/β-thalassemic patients and western blotting results corroborated these findings. In conclusion, we have successfully identified consistent alterations of protein quantity between EVs from HbE/β-thalassemic and normal individuals. This work highlights haptoglobin, hemopexin, and cathepsin S as potential clinically relevant biomarkers for levels of hemolysis and inflammation. Monitoring of these plasma proteins could help in the clinical management of thalassemia.

Background and Purpose Dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel causes the genetic disease cystic fibrosis (CF). Towards the development of transformational drug therapies for CF, we investigated the channel function and action of CFTR potentiators on A561E, a CF mutation found frequently in Portugal. Like the most common CF mutation F508del, A561E causes a temperature-sensitive folding defect that prevents CFTR delivery to the cell membrane and is associated with severe disease. Experimental Approach Using baby hamster kidney cells expressing recombinant CFTR, we investigated CFTR expression by cell surface biotinylation, and function and pharmacology with the iodide efflux and patch-clamp techniques. Key Results Low temperature incubation delivered a small proportion of A561E-CFTR protein to the cell surface. Like F508del-CFTR, low temperature-rescued A561E-CFTR exhibited a severe gating defect characterized by brief channel openings separated by prolonged channel closures. A561E-CFTR also exhibited thermoinstability, losing function more quickly than F508del-CFTR in cell-free membrane patches and intact cells. Using the iodide efflux assay, CFTR potentiators, including genistein and the clinically approved small-molecule ivacaftor, partially restored function to A561E-CFTR. Interestingly, ivacaftor restored wild-type levels of channel activity (as measured by open probability) to single A561E- and F508del-CFTR Cl− channels. However, it accentuated the thermoinstability of both mutants in cell-free membrane patches. Conclusions and Implications Like F508del-CFTR, A561E-CFTR perturbs protein processing, thermostability and channel gating. CFTR potentiators partially restore channel function to low temperature-rescued A561E-CFTR. Transformational drug therapy for A561E-CFTR is likely to require CFTR correctors, CFTR potentiators and special attention to thermostability.

Haematopoiesis, the process of blood production, occurs from a tiny contingent of haematopoietic stem cells (HSC) in highly specialised three-dimensional niches located within the bone marrow. When haematopoiesis is replicated using in vitro two-dimensional culture, HSCs rapidly differentiate, limiting self-renewal. Emulsion-templated highly porous polyHIPE foam scaffolds were chosen to mimic the honeycomb architecture of human bone. The unmodified polyHIPE material supports haematopoietic stem and progenitor cell (HSPC) culture, with successful culture of erythroid progenitors and neutrophils within the scaffolds. Using erythroid culture methodology, the CD34+ population was maintained for 28 days with continual release of erythroid progenitors. These cells are shown to spontaneously repopulate the scaffolds, and the accumulated egress can be expanded and grown at large scale to reticulocytes. We next show that the polyHIPE scaffolds can be successfully functionalised using activated BM(PEG)2 (1,8-bismaleimido-diethyleneglycol) and then a Jagged-1 peptide attached in an attempt to facilitate notch signalling. Although Jagged-1 peptide had no detectable effect, the BM(PEG)2 alone significantly increased cell egress when compared to controls, without depleting the scaffold population. This work highlights polyHIPE as a novel functionalisable material for mimicking the bone marrow, and also that PEG can influence HSPC behaviour within scaffolds.

Congenital dyserythropoietic anemia type II is an autosomally recessive form of hereditary anemia caused by SEC23B gene mutations. Patients exhibit characteristic phenotypes including multinucleate erythroblasts, erythrocytes with hypoglycosylated membrane proteins and an apparent double plasma membrane. Despite ubiquitous expression of SEC23B, the effects of mutations in this gene are confined to the erythroid lineage and the basis of this erythroid specificity remains to be defined. In addition, little is known regarding the stage at which the disparate phenotypes of this disease manifest during erythropoiesis. We employ an in vitro culture system to monitor the appearance of the defining phenotypes associated with congenital dyserythropoietic anemia type II during terminal differentiation of erythroblasts derived from small volumes of patient peripheral blood. Membrane protein hypoglycosylation was detected by the basophilic stage, preceding the onset of multinuclearity in orthochromatic erythroblasts that occurs coincident with the loss of secretory pathway proteins including SEC23A during erythropoiesis. Endoplasmic reticulum remnants were observed in nascent reticulocytes of both diseased and healthy donor cultures but were lost upon further maturation of normal reticulocytes, implicating a defect of ER clearance during reticulocyte maturation in congenital dyserythropoietic anemia type II. We also demonstrate distinct isoform and species-specific expression profiles of SEC23 during terminal erythroid differentiation and identify a prolonged expression of SEC23A in murine erythropoiesis compared to humans. We propose that SEC23A is able to compensate for the absence of SEC23B in mouse erythroblasts, providing a basis for the absence of phenotype within the erythroid lineage of a recently described SEC23B knockout mouse.

In this issue of Blood, Lu et al report on the generation and characterizationof erythrocytes from a dematin knockout mouse and demonstrate thepreviously unrecognized centrality of this protein to the junctional proteincomplex–mediated maintenance of red blood cell (RBC) membrane integrity.