Biology subjects

Toye, A. M.

Publications and source records attributed to Toye, A. M..

6 recordsLinked to original sources

bioRxiv2025

Choice of lipid supplementation for in vitro erythroid cell culture impacts reticulocyte yield and characteristics

Lipids, particularly cholesterol, are critical components of red blood cell (RBC) membranes, influencing protein function, cell stability, and deformability. Reticulocytes (young RBC) derived from in vitro erythroid cultures have been reported to possess less cholesterol than their native counterparts, compromising their functional integrity and lifespan. However, variability in starting materials and culture protocols between studies has hindered deQnitive conclusions regarding the nature and consequences of this lipid deQciency. Here, we evaluated the influence of lipid sources on reticulocyte quality using a well-established CD34 erythroid culture system. We compared the use of human AB-serum and Octaplas (solvent/detergent-treated pooled plasma) as lipid sources. Our results reveal that detergent-treated plasma leads to cholesterol-deQcient reticulocytes with impaired characteristics, including reduced Qltration yield, heightened osmotic fragility, and altered PIEZO1 activity. In contrast, AB-serum supported the generation of functionally stable reticulocytes, with cholesterol supplementation required to rescue the defects observed with plasma. Importantly, this study provides the Qrst integrated lipidomic, metabolomic, and proteomic characterisation of in vitro-derived reticulocytes cultured under distinct lipid conditions. These multi-omic datasets offer new insights into the consequences of reduced lipid availability during erythroid culture and offer new insights into how culture media affects the development and functionality of lab grown blood.

cell biology↗

bioRxiv2024

The Immobilization of Hyaluronic Acid in 3D Hydrogel Scaffolds Modulates Macrophage Polarization

Macrophages are key modulators of immunity, tissue homeostasis and disease development. As our understanding of macrophage biology and their tissue specific behaviors grow the necessity to model macrophages within a 3D biomimetic environment becomes increasingly apparent. Numerous hydrogels have been developed and explored for this purpose, extracellular matrix (ECM) mimicking hydrogels gaining a special interest. In this study, we present the use of such a hydrogel composed of collagen and hyaluronic acid (HA), two of the major ECM components, for the 3D culture of macrophages to model their role in different tissues and diseases. We demonstrate the ability to tailor the mechanical properties of the hydrogel through formulation modulation. Human macrophages retain morphology, viability, and expression of key cell surface markers when 3D cultured within the hydrogel. Interestingly, we demonstrate in this work, that independent of mechanical properties, by adjusting the composition of the hydrogel, specifically HA molecular weight, we can steer macrophage polarization towards either a pro-inflammatory or anti-inflammatory phenotype. This HA-dependent modulation of macrophage behavior is nullified if the HA is chemically crosslinked, shedding light on the impact of one of the most commonly used preparation methods for collagen-HA hydrogels on macrophage behavior. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=84 SRC="FIGDIR/small/588451v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1cbe667org.highwire.dtl.DTLVardef@1837e58org.highwire.dtl.DTLVardef@1dbdbc6org.highwire.dtl.DTLVardef@139d65b_HPS_FORMAT_FIGEXP M_FIG C_FIG Hyaluronic acid (HA) is an extracellular matrix component, which can modulate the polarization of macrophages. The chemical crosslinking of HA to hydrogel scaffolds counteracts the cell signaling role of this molecule, preventing HA from modulating the immune polarization of macrophages within the model. Key pointsO_LICollagen-HA hydrogels form suitable biomimetic niches for the 3D culture of human macrophages in which hydrogel composition modulates macrophage phenotype, specifically the presence and molecular weight of HA. C_LIO_LIPolarization of macrophage phenotype by HA is nullified if HA is chemically cross-linked within the hydrogel. C_LI

bioengineering↗

bioRxiv2024

Complete absence of GLUT1 does not impair human terminal erythroid differentiation

The Glucose transporter 1 (GLUT1) is one of the most abundant proteins within the erythrocyte membrane and is required for glucose and dehydroascorbic acid (Vitamin C precursor) transport. It is widely recognized as a key protein for red cell structure, function, and metabolism. Previous reports highlighted the importance of GLUT1 activity within these uniquely glycolysis-dependent cells, in particular for increasing antioxidant capacity needed to avoid irreversible damage from oxidative stress in humans. However, studies of glucose transporter roles in erythroid cells are complicated by species-specific differences between humans and mice. Here, using CRISPR-mediated gene editing of immortalized erythroblasts and adult CD34+ hematopoietic progenitor cells, we generate committed human erythroid cells completely deficient in expression of GLUT1. We show that absence of GLUT1 does not impede human erythroblast proliferation, differentiation, or enucleation. This work demonstrates for the first-time generation of enucleated human reticulocytes lacking GLUT1. The GLUT1-deficient reticulocytes possess no tangible alterations to membrane composition or deformability in reticulocytes. Metabolomic analyses of GLUT1-deficient reticulocytes reveal hallmarks of reduced glucose import, downregulated metabolic processes and upregulated AMPK-signalling, alongside alterations in antioxidant metabolism, resulting in increased osmotic fragility and metabolic shifts indicative of higher oxidant stress. Despite detectable metabolic changes in GLUT1 deficient reticulocytes, the absence of developmental phenotype, detectable proteomic compensation or impaired deformability comprehensively alters our understanding of the role of GLUT1 in red blood cell structure, function and metabolism. It also provides cell biological evidence supporting clinical consensus that reduced GLUT1 expression does not cause anaemia in GLUT1 deficiency syndrome. Key PointsO_LIGLUT1 knockout does not affect erythroid differentiation and minimally impacts reticulocyte membrane composition C_LIO_LIMetabolic adaptation facilitates reticulocyte tolerance of GLUT1 absence C_LI

cell biology↗

bioRxiv2023

Basigin mediation of Plasmodium falciparum red blood cell invasion does not require its interaction with monocarboxylate transporter 1

Plasmodium falciparum invasion of the red blood cell is reliant upon the essential interaction of PfRh5 with the host receptor protein basigin. Basigin exists as part of one or more multiprotein complexes, most notably through interaction with the monocarboxylate transporter MCT1. However, the potential requirement for basigin association with MCT1 and the wider role of basigin host membrane context and lateral protein associations during merozoite invasion has not been established. Using genetically manipulated in vitro derived reticulocytes, we demonstrate the ability to uncouple basigin ectodomain presentation from the transmembrane domain-mediated interaction with MCT1. Merozoite invasion of reticulocytes is unaffected by disruption of basigin MCT1 interaction and by removal or replacement of the basigin transmembrane helix. Therefore, presentation of the basigin ectodomain at the red blood cell surface, independent of its native association with MCT1 or other interactions mediated by the transmembrane domain, is sufficient to facilitate merozoite invasion.

cell biology↗

bioRxiv2022

Characterizing the polarization continuum of macrophage subtypes M1, M2a and M2c

Macrophages are vital components of the inflammatory response and exhibit phenotypical plasticity through active conversion between pro- and anti-inflammatory cell subtypes, a feature which can be reproduced in ex vivo culture. We employed a multifaceted approach utilizing proteomics, flow cytometry, activity assays and livecell microscopy imaging to characterize four cultured macrophage subtypes: unstimulated MO, classically activated M1, alternatively activated M2a, and deactivated M2c macrophages. Whole cell proteomics identified a total of 5435 proteins, with >50% of these proteins exhibiting significant alterations in abundance between the different subtypes. This confirms that four distinct macrophage subtypes are induced from the same originating donor material through stimulation with specific cytokines. Additional surfaceome analysis revealed that M2c macrophages significantly upregulate pro-inflammatory markers compared to the MO baseline and thus appear to be activated or primed to activate, similar to M1. Surface protein expression provided further subtype characterization, in particular distinguishing between the M2a and M2c macrophages. We next explored the re-polarization capabilities of macrophages using dexamethasone, an anti-inflammatory glucocorticoid known to induce macrophage polarization towards the M2c de-activated phenotype. We show that activated M1 macrophages treated with dexamethasone for 48-hours upregulate the levels of CD163 and CD206, markers synonymous with a phenotypical shift from M1 to M2c yet retain key surface markers and display the functional phenotype of M1 macrophages. The observed repolarization of M1 pro-inflammatory macrophages provides a potential mechanism through which dexamethasone treatment improves COVID-19 prognosis and constitutes evidence of partial repolarization along the macrophage continuum. These proteomic and functional ex vivo macrophage datasets provide a valuable resource for studying macrophage polarity and the impact of dexamethasone on macrophage phenotype and function.

immunology↗

bioRxiv2018

Stable knockout and complementation of receptor expression using in vitro cell line derived reticulocytes for dissection of host malaria invasion requirements

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.

cell biology↗