Zebrafish display a distinct ability to regenerate their heart following injury. However, this ability is not shared by another teleost, the medaka. In order to identify cellular and molecular bases for this difference, we performed comparative transcriptomic analyses following cardiac cryoinjury. This comparison points to major differences in immune cell dynamics between these models. Upon closer examination, we observed delayed and reduced macrophage recruitment in medaka, along with delayed neutrophil clearance. To investigate the role of immune responses in cardiac regeneration, we delayed macrophage recruitment in zebrafish and observed compromised neovascularization, neutrophil clearance, cardiomyocyte proliferation and scar resolution. In contrast, stimulating Toll-like receptor signaling in medaka enhanced immune cell dynamics and promoted neovascularization, neutrophil clearance, cardiomyocyte proliferation and scar resolution. Altogether, these data provide further insight into the complex role of the immune response during regeneration, and serve as a platform to identify and test additional regulators of cardiac repair.DOI: http://dx.doi.org/10.7554/eLife.25605.001

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.This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

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.

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.

Dehydrated hereditary stomatocytosis 1 or hereditary xerocytosis (HX, OMIM 194380) is a rare hereditary autosomal dominant disorder characterized by hemolytic anemia and red blood cell (RBC) dehydration. The occurrence of HX is linked with gain-of-function mutations in PIEZO1, the gene encoding for the mechanosensitive non-specific cation channel PIEZO1(1, 2) which is activated by shear-stress and in concert with other ion channels (particularly the Gardos potassium calcium-activated channel, KCNN4) regulates cell volume homeostasis and metabolic activity in the RBC(3). Intriguingly, PIEZO1 gain-of-function mutations have recently been reported to occur at a much higher frequency within the population than had been previously described and also implicated in malaria resistance(4), suggesting that the mechanisms underpinning HX may merit further investigation. Since reticulocytosis is one of the hallmarks of HX(5), we sought to determine whether altered reticulocyte maturation could be a causative agent of this phenotype. We characterize reticulocytes and erythrocytes from 10 HX patients in comparison to healthy controls, revealing alterations in deformability and vesicle content that implicate a maturational defect in HX. We further demonstrate that HX patients suffer from impaired reticulocyte maturation as assayed through differences in the extent and rate of loss of CD71 and RNA content over time and that this effect can be recapitulated in healthy reticulocytes upon chemically

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.

Zebrafish display a distinct ability to regenerate their heart following injury. However, this ability is not shared by another teleost, the medaka. In order to identify cellular and molecular bases for this difference, we performed comparative transcriptomic analyses following cardiac cryoinjury. This comparison points to major differences in immune cell dynamics between these models. Upon closer examination, we observed delayed and reduced macrophage recruitment in medaka, along with delayed neutrophil clearance. To investigate the role of immune responses in cardiac regeneration, we delayed macrophage recruitment in zebrafish and observed compromised neovascularization, neutrophil clearance, cardiomyocyte proliferation and scar resolution. In contrast, stimulating Toll-like receptor signaling in medaka enhanced immune cell dynamics and promoted neovascularization, neutrophil clearance, cardiomyocyte proliferation and scar resolution. Altogether, these data provide further insight into the complex role of the immune response during regeneration, and serve as a platform to identify and test additional regulators of cardiac repair.

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 live-cell microscopy imaging to characterize four cultured macrophage subtypes: unstimulated M, 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 M 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.