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.
Synopsis
Live imaging studies in zebrafish, alongside mouse and in vitro human cell studies reveal how macrophages play essential roles in wound angiogenesis, coordinating both the initial blood vessel sprouting and subsequent vessel regression.
Pro‐inflammatory wound macrophages interact with blood vessel tips, driving sprouting angiogenesis at the wound site during early stages of repair by acting as a local source of vegf.
Neutrophils are transient at the wound site where they suppress sprouting angiogenesis; they are subsequently dismissed from vessel tips by macrophages to establish an angiogenic “permissive” area.
Anti‐inflammatory macrophages play a role in blood vessel regression and phagocytic clearance during wound resolution, and this function is impaired if macrophages remain pro‐inflammatory.
Imaging studies in zebrafish, mouse and human cells reveal how macrophages play essential roles in wound angiogenesis, coordinating both the initial blood vessel sprouting and subsequent vessel regression.
With increasing worldwide demand for safe blood, there is much interest in generating red blood cells in vitro as an alternative clinical product. However, available methods for in vitro generation ...of red cells from adult and cord blood progenitors do not yet provide a sustainable supply, and current systems using pluripotent stem cells as progenitors do not generate viable red cells. We have taken an alternative approach, immortalizing early adult erythroblasts generating a stable line, which provides a continuous supply of red cells. The immortalized cells differentiate efficiently into mature, functional reticulocytes that can be isolated by filtration. Extensive characterization has not revealed any differences between these reticulocytes and in vitro-cultured adult reticulocytes functionally or at the molecular level, and importantly no aberrant protein expression. We demonstrate a feasible approach to the manufacture of red cells for clinical use from in vitro culture.
Patients with inherited anemia and hemoglobinopathies (such as sickle cell disease and β-thalassemia) are treated with red blood cell (RBC) transfusions to alleviate their symptoms. Some of these ...patients may have rare blood group types or go on to develop alloimmune reactions, which can make it difficult to source compatible blood in the donor population. Laboratory-grown RBC represent a particularly attractive alternative which could satisfy an unmet clinical need. The challenge, however, is to produce - from a limited number of stem cells - the 2x1012 RBC required for a standard adult therapeutic dose. Encouraging progress has been made in RBC production from adult stem cells under good manufacturing practice. In 2011, the Douay group conducted a successful proof-of-principle mini-transfusion of autologous manufactured RBC in a single volunteer. In the UK, a trial is planned to assess whether manufactured RBC are equivalent to RBC produced naturally in donors, by testing an allogeneic mini-dose of laboratory-grown manufactured RBC in multiple volunteers. This review discusses recent progress in the erythroid culture field as well as opportunities for further scaling up of manufactured RBC production for transfusion practice.
The blood stage malaria parasite, the merozoite, has a small window of opportunity during which it must successfully target and invade a human erythrocyte. The process of invasion is nonetheless ...remarkably rapid. To date, mechanistic models of invasion have focused predominantly on the parasite actomyosin motor contribution to the energetics of entry. Here, we have conducted a numerical analysis using dimensions for an archetypal merozoite to predict the respective contributions of the host-parasite interactions to invasion, in particular the role of membrane wrapping. Our theoretical modeling demonstrates that erythrocyte membrane wrapping alone, as a function of merozoite adhesive and shape properties, is sufficient to entirely account for the first key step of the invasion process, that of merozoite reorientation to its apex and tight adhesive linkage between the two cells. Next, parasite-induced reorganization of the erythrocyte cytoskeleton and release of parasite-derived membrane can also account for a considerable energetic portion of actual invasion itself, through membrane wrapping. Thus, contrary to the prevailing dogma, wrapping by the erythrocyte combined with parasite-derived membrane release can markedly reduce the expected contributions of the merozoite actomyosin motor to invasion. We therefore propose that invasion is a balance between parasite and host cell contributions, evolved toward maximal efficient use of biophysical forces between the two cells.
Abstract
CRISPR-based DNA editing technologies enable rapid and accessible genome engineering of eukaryotic cells. However, the delivery of genetically encoded CRISPR components remains challenging ...and sustained Cas9 expression correlates with higher off-target activities, which can be reduced via Cas9-protein delivery. Here we demonstrate that baculovirus, alongside its DNA cargo, can be used to package and deliver proteins to human cells. Using protein-loaded baculovirus (pBV), we demonstrate delivery of Cas9 or base editors proteins, leading to efficient genome and base editing in human cells. By implementing a reversible, chemically inducible heterodimerization system, we show that protein cargoes can selectively and more efficiently be loaded into pBVs (spBVs). Using spBVs we achieved high levels of multiplexed genome editing in a panel of human cell lines. Importantly, spBVs maintain high editing efficiencies in absence of detectable off-targets events. Finally, by exploiting Cas9 protein and template DNA co-delivery, we demonstrate up to 5% site-specific targeted integration of a 1.8 kb heterologous DNA payload using a single spBV in a panel of human cell lines. In summary, we demonstrate that spBVs represent a versatile, efficient and potentially safer alternative for CRISPR applications requiring co-delivery of DNA and protein cargoes.
Graphical Abstract
Graphical Abstract
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 its transmembrane domain-mediated interactions, including 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.
The classical central macrophage found in erythroblastic islands plays an important role in erythroblast differentiation, proliferation and enucleation in the bone marrow. Convenient human
models to ...facilitate the study of erythroid-macrophage interactions are desired. Recently, we demonstrated that cultured monocytes/macrophages enhance
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.
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