The ten-eleven translocation (TET) family of proteins plays important roles in a wide range of biological processes by oxidizing 5-methylcytosine (5mC) to 5-hydroxy-methylcytosine. However, their ...function in erythropoiesis has remained unclear. We show here that TET2 and TET3 but not TET1 are expressed in human erythroid cells, and we explore the role of these proteins in erythropoiesis. Knockdown experiments revealed that TET2 and TET3 have different functions. Suppression of TET3 expression in human CD34+ cells markedly impaired terminal erythroid differentiation, as reflected by increased apoptosis, the generation of bi/multinucleated polychromatic/orthochromatic erythroblasts, and impaired enucleation, although without effect on erythroid progenitors. In marked contrast, TET2 knockdown led to hyper-proliferation and impaired differentiation of erythroid progenitors. Surprisingly, knockdown of neither TET2 nor TET3 affected global levels of 5mC. Thus, our findings have identified distinct roles for TET2 and TET3 in human erythropoiesis, and provide new insights into their role in regulating human erythroid differentiation at distinct stages of development. Moreover, because knockdown of TET2 recapitulates certain features of erythroid development defects characteristic of myelodysplastic syndromes (MDSs), and the TET2 gene mutation is one of the most common mutations in MDS, our findings may be relevant for improved understanding of dyserythropoiesis of MDS.
•TET3 knockdown impairs terminal erythroid differentiation, whereas TET2 knockdown leads to accumulation of erythroid progenitors.•Global levels of 5mC are not altered by knockdown of either TET2 or TET3.
Mammalian erythropoiesis occurs within erythroblastic islands (EBIs), niches where maturing erythroblasts interact closely with a central macrophage. While it is generally accepted that EBI ...macrophages play an important role in erythropoiesis, thorough investigation of the mechanisms by which they support erythropoiesis is limited largely by inability to identify and isolate the specific macrophage sub-population that constitute the EBI. Early studies utilized immunohistochemistry or immunofluorescence to study EBI morphology and structure, while more recent efforts have used flow cytometry for high-throughput quantitative characterization of EBIs and their central macrophages. However, these approaches based on the expectation that EBI macrophages are a homogeneous population (F4/80
/CD169
/VCAM-1
for example) provide an incomplete picture and potentially overlook critical information about the nature and biology of the islands and their central macrophages. Here, we present a novel method for analysis of EBI macrophages from hematopoietic tissues of mice and rats using multispectral imaging flow cytometry (IFC), which combines the high-throughput advantage of flow cytometry with the morphological and fluorescence features derived from microscopy. This method provides both quantitative analysis of EBIs, as well as structural and morphological details of the central macrophages and associated cells. Importantly, the images, combined with quantitative software features, can be used to evaluate co-expression of phenotypic markers which is crucial since some antigens used to identify macrophages (e.g., F4/80 and CD11b) can be expressed on non-erythroid cells associated with the islands instead of, or in addition to the central macrophage itself. We have used this method to analyze native EBIs from different hematopoietic tissues and evaluated the expression of several markers that have been previously reported to be expressed on EBI macrophages. We found that VCAM-1, F4/80, and CD169 are expressed heterogeneously by the central macrophages within the EBIs, while CD11b, although abundantly expressed by cells within the islands, is not expressed on the EBI macrophages. Moreover, differences in the phenotype of EBIs in rats compared to mice point to potential functional differences between these species. These data demonstrate the usefulness of IFC in analysis and characterization of EBIs and more importantly in exploring the heterogeneity and plasticity of EBI macrophages.
Obligate intracellular Apicomplexa parasites share a unique invasion mechanism involving a tight interaction between the host cell and the parasite surfaces called the moving junction (MJ). The MJ, ...which is the anchoring structure for the invasion process, is formed by secretion of a macromolecular complex (RON2/4/5/8), derived from secretory organelles called rhoptries, into the host cell membrane. AMA1, a protein secreted from micronemes and associated with the parasite surface during invasion, has been shown in vitro to bind the MJ complex through a direct association with RON2. Here we show that RON2 is inserted as an integral membrane protein in the host cell and, using several interaction assays with native or recombinant proteins, we define the region that binds AMA1. Our studies were performed both in Toxoplasma gondii and Plasmodium falciparum and although AMA1 and RON2 proteins have diverged between Apicomplexa species, we show an intra-species conservation of their interaction. More importantly, invasion inhibition assays using recombinant proteins demonstrate that the RON2-AMA1 interaction is crucial for both T. gondii and P. falciparum entry into their host cells. This work provides the first evidence that AMA1 uses the rhoptry neck protein RON2 as a receptor to promote invasion by Apicomplexa parasites.
Hereditary hemorrhagic telangiectasia (HHT) is a potentially life-threatening genetic vascular disorder caused by loss-of-function mutations in the genes encoding activin receptor-like kinase 1 ...(ALK1), endoglin, Smad4, and bone morphogenetic protein 9 (BMP9). Injections of mouse neonates with BMP9/10 blocking antibodies lead to HHT-like vascular defects in the postnatal retinal angiogenesis model. Mothers and their newborns share the same immunity through the transfer of maternal antibodies during lactation. Here, we investigated whether the transmammary delivery route could improve the ease and consistency of administering anti-BMP9/10 antibodies in the postnatal retinal angiogenesis model. We found that anti-BMP9/10 antibodies, when intraperitoneally injected into lactating dams, are efficiently transferred into the blood circulation of lactationally-exposed neonatal pups. Strikingly, pups receiving anti-BMP9/10 antibodies via lactation displayed consistent and robust vascular pathology in the retina, which included hypervascularization and defects in arteriovenous specification, as well as the presence of multiple and massive arteriovenous malformations. Furthermore, RNA-Seq analyses of neonatal retinas identified an increase in the key pro-angiogenic factor, angiopoietin-2, as the most significant change in gene expression triggered by the transmammary delivery of anti-BMP9/10 antibodies. Transmammary-delivered BMP9/10 immunoblocking in the mouse neonatal retina is therefore a practical, noninvasive, reliable, and robust model of HHT vascular pathology.
Biogenesis of mammalian red blood cells requires nuclear expulsion by orthochromatic erythoblasts late in terminal differentiation (enucleation), but the mechanism is largely unexplained. Here, we ...employed high-resolution confocal microscopy to analyze nuclear morphology and F-actin rearrangements during the initiation, progression, and completion of mouse and human erythroblast enucleation in vivo. Mouse erythroblast nuclei acquire a dumbbell-shaped morphology during enucleation, whereas human bone marrow erythroblast nuclei unexpectedly retain their spherical morphology. These morphological differences are linked to differential expression of Lamin isoforms, with primary mouse erythroblasts expressing only Lamin B and primary human erythroblasts only Lamin A/C. We did not consistently identify a continuous F-actin ring at the cell surface constriction in mouse erythroblasts, nor at the membrane protein-sorting boundary in human erythroblasts, which do not have a constriction, arguing against a contractile ring-based nuclear expulsion mechanism. However, both mouse and human erythroblasts contain an F-actin structure at the rear of the translocating nucleus, enriched in tropomodulin 1 (Tmod1) and nonmuscle myosin IIB. We investigated Tmod1 function in mouse and human erythroblasts both in vivo and in vitro and found that absence of Tmod1 leads to enucleation defects in mouse fetal liver erythroblasts, and in CD34+ hematopoietic stem and progenitor cells, with increased F-actin in the structure at the rear of the nucleus. This novel structure, the “enucleosome,” may mediate common cytoskeletal mechanisms underlying erythroblast enucleation, notwithstanding the morphological heterogeneity of enucleation across species.
•Morphological dissection of the progression of nuclear expulsion reveals complex F-actin rearrangements in primary erythroblasts.•Enucleation depends upon a novel, conserved, F-actin/myosin IIB/Tmod1 structure (the “enucleosome”) at the rear of the translocating nucleus.
During the infection process, Apicomplexa discharge their secretory organelles called micronemes, rhoptries and dense granules to sustain host cell invasion, intracellular replication and to modulate ...host cell pathways and immune responses. Herein, we describe the Toxoplasma gondii Deg-like serine protein (TgDegP), a rhoptry protein homologous to High temperature requirement A (HtrA) or Deg-like family of serine proteases. TgDegP undergoes processing in both types I and II strains as most of the rhoptries proteins. We show that genetic disruption of the degP gene does not impact the parasite lytic cycle in vitro but affects virulence in mice. While in a type I strain DegPI appears dispensable for the establishment of an infection, removal of DegPII in a type II strain dramatically impairs the virulence. Finally, we show that KO-DegPII parasites kill immunodeficient mice as efficiently as the wild-type strain indicating that the protease might be involved in the complex crosstalk that the parasite engaged with the host immune response. Thus, this study unravels a novel rhoptry protein in T. gondii important for the establishment of lethal infection.
Anemia commonly occurs in systemic lupus erythematosus (SLE), a disease characterized by innate immune activation by nucleic acids. Overactivation of cytoplasmic sensors by self-DNA or RNA can cause ...erythroid cell death while sparing other hematopoietic cell lineages. However, little is currently known about the impact of nucleic acid sensing innate receptors on the bone marrow (BM) erythropoietic niche.The ssRNA endosomal receptors TLR7 and human TLR8 both recognize ssRNA. TLR7 overexpression causes a lupus syndrome that includes the development of mild to moderate anemia (Hb >10) and thrombocytopenia and is associated with spleen histiocytosis, autoimmune hemolysis, erythrophagocytosis and compensatory stress erythropoiesis in the spleen. A recent study demonstrated that patients with TLR8 gain of function present with immunodeficiency, inflammation and bone marrow failure. However, the role of TLR8 in SLE has been difficult to study in mice because it has a 5 amino acid deletion that attenuates its RNA binding capacity.To address the role of TLR8 in SLE, we overexpressed human TLR8 in a lupus mouse model (huTLR8tg.Sle1.Yaa) using a BAC transgene. 50% of homozygous huTLR8tg.Sle1.Yaa mice developed severe anemia (Hb < 9) resulting in early mortality starting at 3–4.5 months of age. This phenotype required both the Sle1 and Yaa loci that promote the formation of high titer anti-chromatin and anti-RNA antibodies and onset of nephritis at > 6 months of age. There was no difference in autoantibody titers between Sle1.Yaa wt and huTLR8tg mice and early death in the transgenic mice was not due to premature onset of renal disease. All mice had normal RBC indices prior to 10 weeks of age. Anemia was associated with an increase in bone marrow (BM) trabecular bone and a decrease in erythromyeloblastic islands (EMBI) in the BM with compensatory stress erythropoiesis leading to reticulocytosis and vast splenomegaly. RBC half- life decreased in severely anemic mice as a pre-terminal event and was due to hemophagocytosis by 3 subsets of phagocytic red pulp macrophages.Flow cytometry of BMs showed a block in CFU-E to proerythroblast differentiation that was confirmed by single cell RNASeq of bone marrow EMBIs. The erythroblast cluster proximal to the block had a signature of mitochondrial stress and decreased proliferation. We found 6 closely related clusters of EMBI central macrophages in the BM, most of which displayed an inflammatory and Type 1 IFN signature. One cluster (M2) expressed all the classical central macrophage phenotypic markers was characterized in transgenic mice by downregulation of multiple phagocytic receptors and a 5-fold decrease of VCAM1 expression. Loss of VCAM1 and downregulation of Mertk and CD169 in BM central macrophages was confirmed by flow cytometry. By contrast spleen central macrophages from transgenic mice retained VCAM1 and CD169 expression (figures 1–3).Together, these results suggest that erythropoiesis in the BM of huTLR8tg SLE-prone mice fails due to a block in differentiation from CFU-E to proerythroblasts in the BM and is associated with an inflammatory phenotype specifically in BM erythroblastic island central macrophages and down regulation of adhesion and phagocytic receptors. These data implicate the endosomal RNA sensor TLR8 as an additional innate receptor whose overactivation causes acquired failure of erythropoiesis via myeloid cell dysregulation. Compensatory stress erythropoiesis in the spleens is associated with expansion of several subsets of macrophages with phagocytic properties and fatal anemia is associated with a decrease in red blood cell half-life, suggesting that excessive RBC phagocytosis, coupled with insufficient erythroblast progenitors, eventually exceeds the capability of stress erythropoiesis to replace the RBC mass.Abstract 801 Figure 1Premature death in huTLR8tg Sle1.Yaa mice is due to severe anemia that is due to loss of RBC precursors at the proerythroblast stage as shown by flow cytometry of bone marrowAbstract 801 Figure 2Single cell analysis of isolated BM EMBIs shows a decrease in central macrophage adhesion molecules, confirmed by flow cytometryAbstract 801 Figure 3Decrease in RBC half-life in anemic mice (A) is acquired when RBC from young mice are transferred to anemic mice (B). Hemophagocytic macrophages (C) from anemic mice comprise 3 subsets including iHPCs and red pulp macrophages (D).
RASA3 is a Ras GTPase activating protein that plays a critical role in blood formation. The autosomal recessive mouse model
scat
(severe combined anemia and thrombocytopenia) carries a missense ...mutation in
Rasa3
. Homozygotes present with a phenotype characteristic of bone marrow failure that is accompanied by alternating episodes of crisis and remission. The mechanism leading to impaired erythropoiesis and peripheral cell destruction as evidenced by membrane fragmentation in
scat
is unclear, although we previously reported that the mislocalization of RASA3 to the cytosol of reticulocytes and mature red cells plays a role in the disease. In this study, we further characterized the bone marrow failure in
scat
and found that RASA3 plays a central role in cell cycle progression and maintenance of reactive oxygen species (ROS) levels during terminal erythroid differentiation, without inducing apoptosis of the precursors. In
scat
mice undergoing crises, there is a consistent pattern of an increased proportion of cells in the G
0
/G
1
phase at the basophilic and polychromatophilic stages of erythroid differentiation, suggesting that RASA3 is involved in the G
1
checkpoint. However, this increase in G
1
is transient, and either resolves or becomes indiscernible by the orthochromatic stage. In addition, while ROS levels are normal early in erythropoiesis, there is accumulation of superoxide levels at the reticulocyte stage (DHE increased 40% in
scat; p
= 0.02) even though mitochondria, a potential source for ROS, are eliminated normally. Surprisingly, apoptosis is significantly decreased in the
scat
bone marrow at the proerythroblastic (15.3%;
p
= 0.004), polychromatophilic (8.5%;
p
= 0.01), and orthochromatic (4.2%;
p
= 0.02) stages. Together, these data indicate that ROS accumulation at the reticulocyte stage, without apoptosis, contributes to the membrane fragmentation observed in
scat
. Finally, the cell cycle defect and increased levels of ROS suggest that
scat
is a model of bone marrow failure with characteristics of aplastic anemia.
Apicomplexan parasites secrete and inject into the host cell the content of specialized secretory organelles called rhoptries, which take part into critical processes such as host cell invasion and ...modulation of the host cell immune response. The rhoptries are structurally and functionally divided into two compartments. The apical duct contains rhoptry neck (RON) proteins that are conserved in Apicomplexa and are involved in formation of the moving junction (MJ) driving parasite invasion. The posterior bulb contains rhoptry proteins (ROPs) unique to an individual genus and, once injected in the host cell act as effector proteins to co-opt host processes and modulate parasite growth and virulence. We describe here two new RON proteins of Toxoplasma gondii, RON9 and RON10, which form a high molecular mass complex. In contrast to the other RONs described to date, this complex was not detected at the MJ during invasion and therefore was not associated to the MJ complex RON2/4/5/8. Disruptions of either RON9 or RON10 gene leads to the retention of the partner in the ER followed by subsequent degradation, suggesting that the RON9/RON10 complex formation is required for proper sorting to the rhoptries. Finally, we show that the absence of RON9/RON10 has no significant impact on the morphology of rhoptry, on the invasion and growth in fibroblasts in vitro or on virulence in vivo. The conservation of RON9 and RON10 in Coccidia and Cryptosporidia suggests a specific relation with development in intestinal epithelial cells.
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