Defining the proteomic landscape of cultured macrophages and their polarization continuum.

Macrophages have previously been characterized based on phenotypical and functional differences into suggested simplified subtypes of MØ, M1, M2a and M2c. These macrophage subtypes can be generated in a well-established primary monocyte culture model that produces cells expressing accepted subtype surface markers. To determine how these subtypes retain functional similarities and better understand their formation, we generated all four subtypes from the same donors. Comparative whole-cell proteomics confirmed that four distinct macrophage subtypes could be induced from the same donor material, with > 50% of 5435 identified proteins being significantly altered in abundance between subtypes. Functional assessment highlighted that these distinct protein expression profiles are primed to enable specific cell functions, indicating that this shifting proteome is predictive of meaningful changes in cell characteristics. Importantly, the 2552 proteins remained consistent in abundance across all macrophage subtypes examined, demonstrating maintenance of a stable core proteome that likely enables swift polarity changes. We next explored the cross-polarization capabilities of preactivated M1 macrophages treated with dexamethasone. Importantly, these treated cells undergo a partial repolarization toward the M2c surface markers but still retain the M1 functional phenotype. Our investigation of polarized macrophage subtypes therefore provides evidence of a sliding scale of macrophage functionality, with these data sets providing a valuable benchmark resource for further studies of macrophage polarity, with relevance for cell therapy development and drug discovery.

Development and evaluation of low-volume tests to detect and characterize antibodies to SARS-CoV-2.

Low-volume antibody assays can be used to track SARS-CoV-2 infection rates in settings where active testing for virus is limited and remote sampling is optimal. We developed 12 ELISAs detecting total or antibody isotypes to SARS-CoV-2 nucleocapsid, spike protein or its receptor binding domain (RBD), 3 anti-RBD isotype specific luciferase immunoprecipitation system (LIPS) assays and a novel Spike-RBD bridging LIPS total-antibody assay. We utilized pre-pandemic (n=984) and confirmed/suspected recent COVID-19 sera taken pre-vaccination rollout in 2020 (n=269). Assays measuring total antibody discriminated best between pre-pandemic and COVID-19 sera and were selected for diagnostic evaluation. In the blind evaluation, two of these assays (Spike Pan ELISA and Spike-RBD Bridging LIPS assay) demonstrated >97% specificity and >92% sensitivity for samples from COVID-19 patients taken >21 days post symptom onset or PCR test. These assays offered better sensitivity for the detection of COVID-19 cases than a commercial assay which requires 100-fold larger serum volumes. This study demonstrates that low-volume in-house antibody assays can provide good diagnostic performance, and highlights the importance of using well-characterized samples and controls for all stages of assay development and evaluation. These cost-effective assays may be particularly useful for seroprevalence studies in low and middle-income countries.

Macrophage Reprogramming with Anti-miR223-Loaded Artificial Protocells Enhances In Vivo Cancer Therapeutic Potential.

Several immune cell-expressed miRNAs (miRs) are associated with altered prognostic outcome in cancer patients, suggesting that they may be potential targets for development of cancer therapies. Here, translucent zebrafish (Danio rerio) is utilized to demonstrate that genetic knockout or knockdown of one such miR, microRNA-223 (miR223), globally or specifically in leukocytes, does indeed lead to reduced cancer progression. As a first step toward potential translation to a clinical therapy, a novel strategy is described for reprogramming neutrophils and macrophages utilizing miniature artificial protocells (PCs) to deliver anti-miRs against the anti-inflammatory miR223. Using genetic and live imaging approaches, it is shown that phagocytic uptake of anti-miR223-loaded PCs by leukocytes in zebrafish (and by human macrophages in vitro) effectively prolongs their pro-inflammatory state by blocking the suppression of pro-inflammatory cytokines, which, in turn, drives altered immune cell-cancer cell interactions and ultimately leads to a reduced cancer burden by driving reduced proliferation and increased cell death of tumor cells. This PC cargo delivery strategy for reprogramming leukocytes toward beneficial phenotypes has implications also for treating other systemic or local immune-mediated pathologies.

The EHA Research Roadmap: Transfusion Medicine.

In 2016, the European Hematology Association (EHA) published the EHA Roadmap for European Hematology Research 1 aiming to highlight achievements in the diagnostics and treatment of blood disorders, and to better inform European policy makers and other stakeholders about the urgent clinical and scientific needs and priorities in the field of hematology. Each section was coordinated by 1-2 section editors who were leading international experts in the field. In the 5 years that have followed, advances in the field of hematology have been plentiful. As such, EHA is pleased to present an updated Research Roadmap, now including eleven sections, each of which will be published separately. The updated EHA Research Roadmap identifies the most urgent priorities in hematology research and clinical science, therefore supporting a more informed, focused, and ideally a more funded future for European hematology research. The 11 EHA Research Roadmap sections include Normal Hematopoiesis; Malignant Lymphoid Diseases; Malignant Myeloid Diseases; Anemias and Related Diseases; Platelet Disorders; Blood Coagulation and Hemostatic Disorders; Transfusion Medicine; Infections in Hematology; Hematopoietic Stem Cell Transplantation; CAR-T and Other Cell-based Immune Therapies; and Gene Therapy.

Expression and Retention of Thymidine Phosphorylase in Cultured Reticulocytes as a Novel Treatment for MNGIE.

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.

Ephrin/Eph receptor interaction facilitates macrophage recognition of differentiating human erythroblasts.

Erythropoiesis is one of the most efficient cellular processes in the human body producing approximately 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 cultured macrophages 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 do not affect integrin activation. This study demonstrates for the first time that EPHB4 expression is required on erythroblasts to facilitate the initial recognition and subsequent interaction with macrophages, alongside the presence of active integrins.

Live imaging of wound angiogenesis reveals macrophage orchestrated vessel sprouting and regression.

Wound angiogenesis is an integral part of tissue repair and is impaired in many pathologies of healing. Here, we investigate the cellular interactions between innate immune cells and endothelial cells at wounds that drive neoangiogenic sprouting in real time and in vivo Our studies in mouse and zebrafish wounds indicate that macrophages are drawn to wound blood vessels soon after injury and are intimately associated throughout the repair process and that macrophage ablation results in impaired neoangiogenesis. Macrophages also positively influence wound angiogenesis by driving resolution of anti-angiogenic wound neutrophils. Experimental manipulation of the wound environment to specifically alter macrophage activation state dramatically influences subsequent blood vessel sprouting, with premature dampening of tumour necrosis factor-α expression leading to impaired neoangiogenesis. Complementary human tissue culture studies indicate that inflammatory macrophages associate with endothelial cells and are sufficient to drive vessel sprouting via vascular endothelial growth factor signalling. Subsequently, macrophages also play a role in blood vessel regression during the resolution phase of wound repair, and their absence, or shifted activation state, impairs appropriate vessel clearance.

Glucocorticoids induce differentiation of monocytes towards macrophages that share functional and phenotypical aspects with erythroblastic island macrophages.

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.

Polyurethane scaffolds seeded with CD34(+) cells maintain early stem cells whilst also facilitating prolonged egress of haematopoietic progenitors.

We describe a 3D erythroid culture system that utilises a porous polyurethane (PU) scaffold to mimic the compartmentalisation found in the bone marrow. PU scaffolds seeded with peripheral blood CD34(+) cells exhibit a remarkable reproducibility of egress, with an increased output when directly compared to human bone scaffolds over 28 days. Immunofluorescence demonstrated the persistence of CD34(+) cells within the scaffolds for the entirety of the culture. To characterise scaffold outputs, we designed a flow cytometry panel that utilises surface marker expression observed in standard 2D erythroid and megakaryocyte cultures. This showed that the egress population is comprised of haematopoietic progenitor cells (CD36(+)GPA(-/low)). Control cultures conducted in parallel but in the absence of a scaffold were also generally maintained for the longevity of the culture albeit with a higher level of cell death. The harvested scaffold egress can also be expanded and differentiated to the reticulocyte stage. In summary, PU scaffolds can behave as a subtractive compartmentalised culture system retaining and allowing maintenance of the seeded "CD34(+) cell" population despite this population decreasing in amount as the culture progresses, whilst also facilitating egress of increasingly differentiated cells.

CFTR potentiators partially restore channel function to A561E-CFTR, a cystic fibrosis mutant with a similar mechanism of dysfunction as F508del-CFTR.

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.

Ex-vivo generation of human red cells for transfusion.

PURPOSE OF REVIEW: The present article reviews the recent data concerning the generation of red blood cells from haematopoietic stem cells using laboratory culture and discusses the potential for generating cultured red cells in sufficient quantity for use in transfusion practice. RECENT FINDINGS: Functional human reticulocytes have been generated from adult peripheral blood haematopoietic stem cells in laboratory culture without the use of heterologous feeder cells and their viability was demonstrated in vivo. Human erythroid progenitor cells lines have been produced from cord and human induced pluripotent stem cell (hiPSC) haematopoietic progenitors. SUMMARY: Availability of cultured human red cells from haematopoietic stem cells in the quantities required for transfusion therapy would have a major impact on healthcare provision worldwide. Recent studies provide cause for optimism that this ambitious goal is achievable. Functional adult reticulocytes have been made in culture and shown to survive in vivo. Erythroid progenitor cell lines have been derived from cord blood and from human induced pluripotent stem cells, suggesting that large-scale culture of erythroid cell lines and their differentiation to reticulocytes will be possible. Significant problems remain. More efficient enucleation and induction of maturation to an adult phenotype will be required in order to exploit high proliferative capacity of human embryonic stem cells and hiPSCs. Novel bioengineering solutions will be required to generate cultured red cells in the large quantities required, and in this context, use of synthetic three-dimensional scaffolds to mimic the bone marrow niche holds great promise for the future.

Critical band 3 multiprotein complex interactions establish early during human erythropoiesis.

Band 3, the major anion transport protein of human erythrocytes, forms the core of a multiprotein complex in the erythrocyte membrane. Here we studied the spatiotemporal mechanisms of band 3 multiprotein complex assembly during erythropoiesis. Significant pools of intracellular band 3 and Rh-associated glycoprotein (RhAG) were found in the basophilic erythroblast. These intracellular pools decreased in the polychromatic erythroblast, whereas surface expression increased and were lowest in the orthochromatic erythroblast and reticulocytes. Protease treatment of intact cells to remove extracellular epitopes recognized by antibodies to band 3 and RhAG was used to study surface delivery kinetics and intracellular complex composition from the proerythroblast stage to the enucleated reticulocyte. Newly synthesized band 3 and protein 4.2 interact initially in the early stages of the secretory pathway and are found associated at the plasma membrane from the basophilic stage of erythropoiesis. Although we could successfully coimmunoprecipitate Rh with RhAG from plasma membrane pools at a similar stage, no intracellular interaction between these proteins was detectable. Knockdown of RhAG during early erythropoiesis was accompanied by a concomitant drop in membrane expression of Rh polypeptides. These data are consistent with assembly of major components of the band 3 macrocomplex at an early stage during erythropoiesis.

RhCG is the major putative ammonia transporter expressed in the human kidney, and RhBG is not expressed at detectable levels.

Rhesus glycoprotein homologs RhAG, RhBG, and RhCG comprise a recently identified branch of the Mep/Amt ammonia transporter family. Animal studies have shown that RhBG and RhCG are present in the kidney distal tubules. Studies in mouse and rat tissue suggest a basolateral localization for RhBG in cells of the distal tubules including the alpha-intercalated cells (alpha-IC), but no localization of RhBG has been reported in human tissue. To date RhCG localization has been described as exclusively apical plasma membrane in mouse and rat kidney, or apical and basolateral in humans, and some mouse and rat tissue studies. We raised novel antibodies to RhBG and RhCG to examine their localization in the human kidney. Madin-Darby canine kidney (MDCKI) cell lines stably expressing human green fluorescent protein-tagged RhBG or RhCG and human tissue lysates were used to demonstrate the specificity of these antibodies for detecting RhBG and RhCG. Using immunoperoxidase staining and antigen liberation techniques, both apical and basolateral RhCG localization was observed in the majority of the cells of the distal convoluted tubule and IC of the connecting tubule and collecting duct. Confocal microscopic imaging of normal human kidney cryosections showed that RhCG staining was predominantly localized to the apical membrane in these cells with some basolateral and intracellular staining evident. A proportion of RhCG staining labeled kAE1-positive cells, confirming that RhCG is localized to the alpha-IC cells. Surprisingly, no RhBG protein was detectable in the human kidney by Western blot analysis of tissue lysates, or by immunohistochemistry or confocal microscopy of tissue sections. The same antibodies, however, could detect RhBG in rat tissue. We conclude that under normal conditions, RhCG is the major putative ammonia transporter expressed in the human kidney and RhBG is not expressed at detectable levels.

The human NBCe1-A mutant R881C, associated with proximal renal tubular acidosis, retains function but is mistargeted in polarized renal epithelia.

The human electrogenic renal Na-HCO(3) cotransporter (NBCe1-A; SLC4A4) is localized to the basolateral membrane of proximal tubule cells. Mutations in the SLC4A4 gene cause an autosomal recessive proximal renal tubular acidosis (pRTA), a disease characterized by impaired ability of the proximal tubule to reabsorb HCO(3)(-) from the glomerular filtrate. Other symptoms can include mental retardation and ocular abnormalities. Recently, a novel homozygous missense mutant (R881C) of NBCe1-A was reported from a patient with a severe pRTA phenotype. The mutant protein was described as having a lower than normal activity when expressed in Xenopus oocytes, despite having normal Na(+) affinity. However, without trafficking data, it is impossible to determine the molecular basis for the phenotype. In the present study, we expressed wild-type NBCe1-A (WT) and mutant NBCe1-A (R881C), tagged at the COOH terminus with enhanced green fluorescent protein (EGFP). This approach permitted semiquantification of surface expression in individual Xenopus oocytes before assay by two-electrode voltage clamp or measurements of intracellular pH. These data show that the mutation reduces the surface expression rather than the activity of the individual protein molecules. Confocal microscopy on polarized mammalian epithelial kidney cells [Madin-Darby canine kidney (MDCK)I] expressing nontagged WT or R881C demonstrates that WT is expressed at the basolateral membrane of these cells, whereas R881C is retained in the endoplasmic reticulum. In summary, the pathophysiology of pRTA caused by the R881C mutation is likely due to a deficit of NBCe1-A at the proximal tubule basolateral membrane, rather than a defect in the transport activity of individual molecules.

Renal tubular acidosis: developments in our understanding of the molecular basis.

Renal tubular acidosis is a metabolic acidosis due to impaired acid excretion by the kidney. Hyperchloraemic acidosis with a normal anion gap and normal (or near normal) glomerular filtration rate, and in the absence of diarrhoea, defines this disorder. However, systemic acidosis is not always evident and renal tubular acidosis can present with hypokalaemia, medullary nephrocalcinosis and recurrent calcium phosphate stone disease, as well as growth retardation and rickets in children, or short stature and osteomalacia in adults. Renal dysfunction in renal tubular acidosis is not always confined to acid excretion and can be part of a more generalised renal tubule defect, as in the renal Fanconi syndrome. Isolated renal tubular acidosis is more usually acquired, due to drugs, autoimmune disease, post-obstructive uropathy or any cause of medullary nephrocalcinosis. Less commonly, it is inherited and may be associated with deafness, osteopetrosis or ocular abnormalities. The clinical classification of renal tubular acidosis has been correlated with our current physiological model of how the nephron excretes acid, and this has facilitated genetic studies that have identified mutations in several genes encoding acid and base ion transporters. In vitro functional studies of these mutant proteins in cell expression systems have helped to elucidate the molecular mechanisms underlying renal tubular acidosis, which ultimately may lead to new therapeutic options in what is still treatment only by giving an oral alkali.

An N-terminal GFP tag does not alter the functional expression to the plasma membrane of red cell and kidney anion exchanger (AE1) in mammalian cells.

Two isoforms of the band 3 anion exchanger are expressed in mammalian cells, a 911 residue protein (B3) in red cells, and a truncated protein (KB3) in the alpha-intercalated cells of the kidney. Mutants of both isoforms are known to be associated with human disease, and mistargeting of the mutated proteins has been suggested as the mechanism of pathogenesis in several cases but has been difficult to prove. The present study demonstrates the feasibility of using confocal microscopy for investigating the targeting of homozygous and heterozygous B3 and KB3 mutants. K562 erythroleukemia cells offer several advantages for the stable expression of B3, but have not previously been used for its visualization. A wide range of cell attachment factors, growth conditions, fixation reagents and primary antibodies were investigated to enable imaging of B3 and endogenous GPA by immunofluorescence confocal microscopy in stable K562/B3 clones. B3 co-localized with GPA at the cell surface and also in an intracellular compartment. Functional cell surface expression of KB3 in stable K562 clones was also obtained. Importantly, both B3 and KB3 could be expressed as stable fusion proteins tagged with green fluorescent protein (GFP) in K562 cells, and it was demonstrated that N-terminal GFP-tagging does not affect the targeting or chloride transport properties of B3 or KB3. The use of GFP as well as double-labelling methods developed for immunostaining will be invaluable for investigating the interactions of band 3 with itself and other proteins during its trafficking in erythroid and kidney cells. This will help elucidate how band 3 mutations can cause human diseases such as hereditary spherocytosis and distal renal tubular acidosis.