ALBERT

Description: Post-operative cognitive dysfunction (POCD) is associated with increased cost of care, morbidity, and mortality. However, its pathogenesis remains largely to be determined. Specifically, it is unknown why elderly patients are more likely to develop POCD and whether POCD is dependent on general anesthesia. We therefore set out to investigate the effects of peripheral surgery on the cognition and Alzheimer-related neuropathology in mice with different ages. Abdominal surgery under local anesthesia was established in the mice. The surgery induced post-operative elevation in brain β-amyloid (Aβ) levels and cognitive impairment in the 18 month-old wild-type and 9 month-old Alzheimer's disease transgenic mice, but not the 9 month-old wild-type mice. The Aβ accumulation likely resulted from elevation of beta-site amyloid precursor protein cleaving enzyme and phosphorylated eukaryotic translation initiation factor 2α. γ-Secretase inhibitor compound E ameliorated the surgery-induced brain Aβ accumulation and cognitive impairment in the 18 month-old mice. These data suggested that the peripheral surgery was able to induce cognitive impairment independent of general anesthesia, and that the combination of peripheral surgery with aging- or Alzheimer gene mutation-associated Aβ accumulation was needed for the POCD to occur. These findings would likely promote more research to investigate the pathogenesis of POCD. Scientific Reports 4 doi: 10.1038/srep03766

Description: Erythroblastic island (EBI), composed of a central macrophage and surrounding erythroid cells, is the first hematopoietic niche discovered for erythropoiesis. Yet, the identity of the central macrophage has so far remained elusive. Based on the previous findings that F4/80, VCAM1 and CD169 are potential mouse central macrophage markers, we first calculated the number of F4/80+VCAM1+CD169+ mouse macrophages in the mouse bone marrow and compared it to the number of Ter119+ erythroblasts. We found that the ratio of F4/80+VCAM1+CD169+ macrophage and erythroblasts is about 1:2. Given the fact that one central macrophage is surrounded by multiple erythroblasts, the above finding suggests that it is unlikely that all the F4/80+VCAM1+CD169+ macrophages are central macrophages. Erythropoietin (Epo) is essential for erythropoiesis. It has been reported that the Epo receptor (Epor) is expressed in peritoneal macrophages. These findings promoted us to speculate that EBI central macrophages may express Epor so that Epo acts on both erythroid cells and the central macrophages simultaneously in the niche to ensure efficient and optimal red cell production. To test this notion, we first examined whether mouse bone marrow and fetal liver macrophages express Epor using the Epor-GFPcre knockin mouse model. We found that ~5% of bone marrow F4/80+ macrophages and ~35% of fetal liver F4/80+ macrophages express Epor-GFP. As negative control, no Epor-GFP macrophages are noted in wild type F4/80+ macrophages. Importantly, ImageStream analyses revealed the native EBIs in bone marrow and fetal liver are formed by Epor+ but not Epor- macrophages. Bioinformatics analyses of RNA-seq data on the sorted Epor+ and Epor- macrophage populations revealed that molecules involved in central macrophage-erythroblast association such as VCAM1, CD169, and molecules known to be important for central macrophage function such as Dnase2a, ferroportin, are highly expressed in Epor+ macrophages. In marked contrast, highly expressed pathways in Epor- macrophages are associated with immune responses including antigen process and presentation. Intriguingly, the immune related pathways are dramatically downregulated in the Epor+ macrophages, suggesting that the Epor+ macrophages in bone marrow and fetal liver have evolved a specialized function in supporting erythropoiesis. To examine whether expression of Epor in EBI central macrophages is a conserved feature across species, we generated Epor-GFPcre knockin rat using the CRISP/Cas9 technology. Using CD163 as rat macrophage marker, we found that a subpopulation of rat bone marrow CD163+ macrophages expresses Epor-GFP. As a negative control, no Epor-GFP macrophages are noted in wild type CD163+ macrophages. To examine whether EPOR is expressed in human EBI central macrophages, antibody specificity for human EPOR is critical. To this end, we employed CRISP/Cas9 approach to knock out EPOR in K562 and Hela cell lines and validated the specificity of a commercially available anti-human EPOR antibody. Using CD163, CD169 as human macrophage markers, we found that EPOR is also expressed in a subpopulation of human macrophages. Moreover, in vitro EBI formation assay revealed that human EPOR+ but not EPOR- macrophages form EBIs with erythroid cells and that the EBI formation is enhanced by EPO. In summary, we for the first time, after discovery of the EBIs 60 years ago, have identified Epor+ macrophages in mouse bone marrow and fetal liver as EBI central macrophages. Our findings provide solid foundation for studying the mechanisms by which erythropoieis is supported EBI central macrophages. A better understanding of such mechanisms will provide extensive new knowledge on basic biology of erythropoiesis. It is also important to understand the pathology of erythropoietic disorders as well as to improve ex vivo erythrocyte production. Disclosures No relevant conflicts of interest to declare.

Description: The erythroblastic island (EBI), composed of a central macrophage and surrounding erythroid cells, was the first hematopoietic niche discovered. The identity of EBI macrophages has thus far remained elusive. Given that Epo is essential for erythropoiesis and that Epor is expressed in numerous nonerythroid cells, we hypothesized that EBI macrophages express Epor so that Epo can act on both erythroid cells and EBI macrophages simultaneously to ensure efficient erythropoiesis. To test this notion, we used Epor-eGFPcre knockin mouse model. We show that in bone marrow (BM) and fetal liver, a subset of macrophages express Epor-eGFP. Imaging flow cytometry analyses revealed that 〉90% of native EBIs comprised F4/80+Epor-eGFP+ macrophages. Human fetal liver EBIs also comprised EPOR+ macrophages. Gene expression profiles of BM F4/80+Epor-eGFP+ macrophages suggest a specialized function in supporting erythropoiesis. Molecules known to be important for EBI macrophage function such as Vcam1, CD169, Mertk, and Dnase2α were highly expressed in F4/80+Epor-eGFP+ macrophages compared with F4/80+Epor-eGFP− macrophages. Key molecules involved in iron recycling were also highly expressed in BM F4/80+Epor-eGFP+ macrophages, suggesting that EBI macrophages may provide an iron source for erythropoiesis within this niche. Thus, we have characterized EBI macrophages in mouse and man. Our findings provide important resources for future studies of EBI macrophage function during normal as well as disordered erythropoiesis in hematologic diseases such as thalassemia, polycythemia vera, and myelodysplastic syndromes.

Description: Anemia, characterized by a decreased number of circulating red blood cells (RBCs) and/or hemoglobin, is a major public health problem worldwide. It can be caused by different pathophysiologic processes, prognoses and preventive or therapeutic approaches. As the commonest hematological manifestation of cancer, anemia afflicts 40-64% of patients treated for malignancies and leads to decline in the quality of life, poor tolerance to chemotherapeutic drugs, and shortened survival. However, few agents have been developed to treat this disease in the clinic, especially applying in malignancies. Erythropoietin (EPO) as a common agent is used to treat for a various of anemia. Nevertheless, several reports have showed that it can aggravate the proliferation and metastasis of some cancer cells, including breast cancer and ovarian cancer. Therefore, it is urgent to develop new efficient agents to manage and treat caner-related anemia. Carbon dots (CDs) as a new class of zero-dimensional carbon nano materials with size less than 10 nm, have shown enormous potentials for biomedical and optoelectronic applications owing to their outstanding characteristics such as good biocompatibility, low cytotoxicity, photostability as well as unique tunable photoluminescence and exceptional physicochemical properties. Herein, we have designed one kind of distinctive CDs with sp2/sp3 carbon and oxygen/nitrogen-based groups via a hydrothermal process, which are efficient to treat anemia without triggering tumor proliferation and migration. We first showed that the CDs significantly promote the proliferation of erythroid cells, using an in vitro erythroid differentiation system. To explore the mechanisms responsible for increased cell growth, we examined the effects of CDs on cell-cycle progression, and found that CDs led to an increased S phase in conjunction with decreased G1/G0 phase. Interestingly, CDs remarkably increased the rate of erythroblast enucleation, the essential step of human RBCs production, without affecting erythroid progenitor development and terminal erythroid differentiation. We then checked the effect of CDs on erythropoiesis in vivo. Compared with the control group, intraperitoneal injection of CDs in normal mice caused higher percentage of reticulocytes, RBCs and hemoglobin in peripheral blood, which are similar with those of intraperitoneal injection of EPO. To further investigate the functions of CDs on stress erythropoiesis, we performed the phenylhydrazine-induced anemia mice model, and found that CDs, similar with EPO, could quickly rescue the typical anemia phenotypes, such as compensatory splenomegaly, decreased RBCs, reduced hemoglobin and lower hematocrit. Moreover, flow cytology analysis indicated that CDs and EPO led to elevation of orthochromatic erythroblasts and reticulocytes in bone marrow and lower rates of these populations in spleen, implying CDs' potential function to treat anemia. Macrophages have been proved to be important for erythropoiesis and associated with tumorigenesis. Intriguingly, we found that while EPO induced more CD169+F4/80+ macrophages in bone marrow, treatment of CDs had no influence on macrophages compared with control group. To examine whether the CDs have effects on tumor directly, we tested breast cancer cell line MD-231 and ovarian cancer cell line A2780. When EPO obviously promoted the proliferation and migration of these tumor cells, which is consistent with previous reports, CDs had no discernible effect on the tumor cell growth and migration. Taken together, our study demonstrates that CDs could induce efficient erythropoiesis without affecting tumour growth, representing a promising agent for caner-related anemia. Disclosures No relevant conflicts of interest to declare.