Project description:CD71+ erythroid cells (CECs) have a wide range of immunomodulatory properties. Here, we show that CECs are expanded in the peripheral blood of HIV patients, with a positive correlation between their frequency and the plasma viral load. CECs from HIV patients and human cord blood/placenta exacerbate HIV-1 infection/replication when cocultured with CD4+ T cells, and that preexposure of CD4+ T cells to CECs enhances their permissibility to HIV infection. However, mature red blood cells (RBCs) do not enhance HIV replication when cocultured with CD4+ T cells. We also found CECs express substantial levels of the NOX2 gene and via a mitochondrial reactive oxygen species (ROS)-dependent mechanism possibly upregulate NF-?B in CD4+ T cells once cocultured, which affects the cell cycle machinery to facilitate HIV-1 replication. The complement receptor-1 (CD35) and the Duffy antigen receptor for chemokines (DARC) as potential HIV target molecules are expressed significantly higher on CECs compared to mature red blood cells. Blocking CD35 or DARC substantially abolishes HIV-1 transmission by RBCs to uninfected CD4+ T cells but not by CECs. In contrast, we observed CECs bind to HIV-1 via CD235a and subsequently transfer the virus to uninfected CD4+ T cells, which can be partially blocked by the anti-CD235a antibody. More importantly, we found that CECs from HIV-infected individuals in the presence of antiretroviral therapy harbor infective viral particles, which mediate HIV-1 trans-infection of CD4+ T cells. Therefore, our findings provide a novel insight into the role of CECs in HIV pathogenesis as potential contributing cells in viral persistence and transmission.IMPORTANCE Immature red blood cells (erythroid precursors or CD71+ erythroid cells) have a wide range of immunomodulatory properties. In this study, we found that these erythroid precursors are abundant in the human cord blood/placental tissues, in the blood of HIV-infected and anemic individuals. We observed that these cells exacerbate HIV-1 replication/infection in target cells and even make HIV target cells more permissible to HIV infection. In addition, we found that HIV gets a free ride by binding on the surface of these cells and thus can travel to different parts of the body. In agreement, we noticed a positive correlation between the plasma viral load and the frequency of these cells in HIV patients. More importantly, we observed that infective HIV particles reside inside these erythroid precursors but not mature red blood cells. Therefore, these cells by harboring HIV can play an important role in HIV pathogenesis.
Project description:Cell-surface transferrin receptor (CD71+) erythroid cells are abundant in newborns with immunomodulatory properties. Here, we show that neonatal CD71+ erythroid cells express significant levels of V-domain Immunoglobulin (Ig) Suppressor of T Cell Activation (VISTA) and, via constitutive production of transforming growth factor (TGF)- ?, play a pivotal role in promotion of naïve CD4+ T cells into regulatory T cells (Tregs). Interestingly, we discovered that CD71+VISTA+ erythroid cells produce significantly higher levels of TGF-? compared to CD71+VISTA- erythroid cells and CD71+ erythroid cells from the VISTA knock-out (KO) mice. As a result, CD71+VISTA+ erythroid cells-compared to CD71+VISTA- and CD71+ erythroid cells from the VISTA KO mice-significantly exceed promotion of naïve CD4+ T cells into induced Tregs (iTreg) via TGF-? in vitro. However, depletion of CD71+ erythroid cells had no significant effects on the frequency of Tregs in vivo. Surprisingly, we observed that the remaining and/or newly generated CD71+ erythroid cells following anti-CD71 antibody administration exhibit a different gene expression profile, evidenced by the up-regulation of VISTA, TGF-?1, TGF-?2, and program death ligand-1 (PDL-1), which may account as a compensatory mechanism for the maintenance of Treg population. We also observed that iTreg development by CD71+ erythroid cells is mediated through the inhibition of key signaling molecules phosphorylated protein kinase B (phospho-Akt) and phosphorylated mechanistic target of rapamycin (phospho-mTOR). Finally, we found that elimination of Tregs using forkhead box P3 (FOXP3)-diptheria toxin receptor (DTR) mice resulted in a significant expansion in the frequency of CD71+ erythroid cells in vivo. Collectively, these studies provide a novel, to our knowledge, insight into the cross-talk between CD71+ erythroid cells and Tregs in newborns. Our results highlight the biological role of CD71+ erythroid cells in the neonatal period and possibly beyond.
Project description:<h4>Background</h4>Myeloid cells are key players in the recognition and response of the host against invading viruses. Paradoxically, upon HIV-1 infection, myeloid cells might also promote viral pathogenesis through trans-infection, a mechanism that promotes HIV-1 transmission to target cells via viral capture and storage. The receptor Siglec-1 (CD169) potently enhances HIV-1 trans-infection and is regulated by immune activating signals present throughout the course of HIV-1 infection, such as interferon α (IFNα).<h4>Results</h4>Here we show that IFNα-activated dendritic cells, monocytes and macrophages have an enhanced ability to capture and trans-infect HIV-1 via Siglec-1 recognition of viral membrane gangliosides. Monocytes from untreated HIV-1-infected individuals trans-infect HIV-1 via Siglec-1, but this capacity diminishes after effective antiretroviral treatment. Furthermore, Siglec-1 is expressed on myeloid cells residing in lymphoid tissues, where it can mediate viral trans-infection.<h4>Conclusions</h4>Siglec-1 on myeloid cells could fuel novel CD4(+) T-cell infections and contribute to HIV-1 dissemination in vivo.
Project description:Neonatal CD71+ erythroid cells are thought to have immunosuppressive functions. Recently, we demonstrated that CD71+ erythroid cells from neonates born to women who underwent spontaneous preterm labor (PTL) are reduced to levels similar to those of term neonates; yet, their functional properties are unknown. Herein, we investigated the functionality of CD71+ erythroid cells from neonates born to women who underwent spontaneous preterm or term labor. CD71+ erythroid cells from neonates born to women who underwent PTL displayed a similar mRNA profile to that of those from term neonates. The direct contact between preterm or term neonatal CD71+ erythroid cells and maternal mononuclear immune cells, but not soluble products from these cells, induced the release of proinflammatory cytokines and a reduction in the release of TGF-?. Moreover, PTL-derived neonatal CD71+ erythroid cells (1) modestly altered CD8+ T cell activation; (2) inhibited conventional CD4+ and CD8+ T-cell expansion; (3) suppressed the expansion of CD8+ regulatory T cells; (4) regulated cytokine responses mounted by myeloid cells in the presence of a microbial product; and (5) indirectly modulated T-cell cytokine responses. In conclusion, neonatal CD71+ erythroid cells regulate neonatal T-cell and myeloid responses and their direct contact with maternal mononuclear cells induces a proinflammatory response. These findings provide insight into the biology of neonatal CD71+ erythroid cells during the physiologic and pathologic processes of labor.
Project description:Reticulocytes shed CD71 from the cell membrane and eliminate mitochondria during terminal maturation, but it is unknown whether these two events are coordinated. We demonstrate that timely removal of CD71 is coupled with mitochondrial clearance, which can be disrupted by null mutation of immediate early response gene X-1 (IEX-1), leading to generation of aberrant CD71-positive and mitochondria-negative (CD71+Mito-) reticulocytes. CD71+Mito- reticulocytes were also present in a subset of patients with myelodysplastic syndromes (MDS) in direct proportion to reduced mitochondrial membrane potential (??m). Mitochondrial abnormality caused by either IEX-1 deficiency or agents that dissipate ??m could trigger premature clearance of mitochondria in reticulocytes. Premature clearance of mitochondria or addition of anti-oxidants lowered intracellular reactive oxygen species (ROS) that in turn hindered CD71 shedding and reticulocyte maturation. In contrast, introduction of ROS accelerated CD71 shedding via release of exosomes that contained a high proportion of Fe3+ over Fe2+, suggesting dual functions of CD71 shedding both in removal of toxic Fe3+ from reticulocytes and in limiting importation of Fe3+ into the cells. These observations emphasize the coordination of mitochondrial and CD71 clearance in erythroid terminal maturation and offer new insights into a role for mitochondrial degeneration in the pathogenesis of some MDS-associated anemia.
Project description:Erythropoiesis requires rapid and extensive hemoglobin production. Heme activates globin transcription and translation; therefore, heme synthesis must precede globin synthesis. As free heme is a potent inducer of oxidative damage, its levels within cellular compartments require stringent regulation. Mice lacking the heme exporter FLVCR1 have a severe macrocytic anemia; however, the mechanisms that underlie erythropoiesis dysfunction in these animals are unclear. Here, we determined that erythropoiesis failure occurs in these animals at the CFU-E/proerythroblast stage, a point at which the transferrin receptor (CD71) is upregulated, iron is imported, and heme is synthesized--before ample globin is produced. From the CFU-E/proerythroblast (CD71(+) Ter119(-) cells) stage onward, erythroid progenitors exhibited excess heme content, increased cytoplasmic ROS, and increased apoptosis. Reducing heme synthesis in FLVCR1-defient animals via genetic and biochemical approaches improved the anemia, implying that heme excess causes, and is not just associated with, the erythroid marrow failure. Expression of the cell surface FLVCR1 isoform, but not the mitochondrial FLVCR1 isoform, restored normal rbc production, demonstrating that cellular heme export is essential. Together, these studies provide insight into how heme is regulated to allow effective erythropoiesis, show that erythropoiesis fails when heme is excessive, and emphasize the importance of evaluating Ter119(-) erythroid cells when studying erythroid marrow failure in murine models.
Project description:Sepsis is a major cause of neonatal mortality and morbidity worldwide. A recent report suggested that murine neonatal host defense against infection could be compromised by immunosuppressive CD71(+) erythroid splenocytes. We examined the impact of CD71(+) erythroid splenocytes on murine neonatal mortality to endotoxin challenge or polymicrobial sepsis and characterized circulating CD71(+) erythroid (CD235a(+)) cells in human neonates. Adoptive transfer or an Ab-mediated reduction in neonatal CD71(+) erythroid splenocytes did not alter murine neonatal survival to endotoxin challenge or polymicrobial sepsis challenge. Ex vivo immunosuppression of stimulated adult CD11b(+) cells was not limited to neonatal splenocytes; it also occurred with adult and neonatal bone marrow. Animals treated with anti-CD71 Ab showed reduced splenic bacterial load following bacterial challenge compared with isotype-treated mice. However, adoptive transfer of enriched CD71(+) erythroid splenocytes to CD71(+)-reduced animals did not reduce bacterial clearance. Human CD71(+)CD235a(+) cells were common among cord blood mononuclear cells and were shown to be reticulocytes. In summary, a lack of effect on murine survival to polymicrobial sepsis following adoptive transfer or diminution of CD71(+) erythroid splenocytes under these experimental conditions suggests that the impact of these cells on neonatal infection risk and progression may be limited. An unanticipated immune priming effect of anti-CD71 Ab treatment, rather than a reduction in immunosuppressive CD71(+) erythroid splenocytes, was likely responsible for the reported enhanced bacterial clearance. In humans, the well-described rapid decrease in circulating reticulocytes after birth suggests that they may have a limited role in reducing inflammation secondary to microbial colonization.
Project description:Extramedullary hematopoietic cells are present in the liver of normal neonates in the first few days of life and persist in infants with biliary atresia. Based on a previous report that liver genes are enriched by erythroid pathways, we examined the liver gene expression pattern at diagnosis and found the top 5 enriched pathways are related to erythrocyte pathobiology in children who survived with the native liver beyond 2 years of age. Using immunostaining, anti-CD71 antibodies identified CD71+ erythroid cells among extramedullary hematopoietic cells in the livers at the time of diagnosis. In mechanistic experiments, the preemptive antibody depletion of hepatic CD71+ erythroid cells in neonatal mice rendered them resistant to rhesus rotavirus-induced (RRV-induced) biliary atresia. The depletion of CD71+ erythroid cells increased the number of effector lymphocytes and delayed the RRV infection of livers and extrahepatic bile ducts. In coculture experiments, CD71+ erythroid cells suppressed the activation of hepatic mononuclear cells. These data uncover an immunoregulatory role for CD71+ erythroid cells in the neonatal liver.
Project description:The simultaneous increases in blood lactic acid and erythrocytes after intense exercise could suggest a link between lactate and the erythropoiesis. However, the effects of lactic acid on erythropoiesis remain to be elucidated. Here, we utilized a mouse model to determine the role of lactic acid in this process in parallel with studies using leukaemic K562 cells. Treatment of K562 cells in vitro with lactic acid increased the mRNA and protein expression of haemoglobin genes and the frequency of GPA<sup>+</sup> cells. Also, increases in haematocrit and CD71<sup>-</sup>/Ter119<sup>+</sup> erythroid cells were observed in lactic acid-treated mice, which showed a physiological increase in blood lactate. Mouse bone marrow CD34<sup>+</sup>/CD117<sup>-</sup> cells showed an increase in erythroid burst-forming units after stimulation with lactic acid in vitro. Furthermore, lactic acid increased the intracellular reactive oxygen species (ROS) content in bone marrow and in K562 cells. Erythroid differentiation induced in Haematopoietic Stem Cells (HSCs) and K562 cells by lactic acid was abolished by reducing ROS levels with SOD or 2-mercaptoethanol, which suggests that ROS is a critical regulator of this process. These findings provide a better understanding of the role of lactic acid in cellular metabolism and physiological functions.
Project description:Determining the developmental pathway leading to erythrocytes and being able to isolate their progenitors are crucial to understanding and treating disorders of red cell imbalance such as anemia, myelodysplastic syndrome, and polycythemia vera. Here we show that the human erythrocyte progenitor (hEP) can be prospectively isolated from adult bone marrow. We found three subfractions that possessed different expression patterns of CD105 and CD71 within the previously defined human megakaryocyte/erythrocyte progenitor (hMEP; Lineage(-) CD34(+) CD38(+) IL-3Rα(-) CD45RA(-)) population. Both CD71(-) CD105(-) and CD71(+) CD105(-) MEPs, at least in vitro, still retained bipotency for the megakaryocyte (MegK) and erythrocyte (E) lineages, although the latter subpopulation is skewed in differentiation toward the erythroid lineage. Notably, the proliferative and differentiation output of the CD71(intermediate(int)/+) CD105(+) subset of cells within the MEP population was completely restricted to the erythroid lineage with the loss of MegK potential. CD71(+) CD105(-) MEPs are erythrocyte-biased MEPs (E-MEPs) and CD71(int/+) CD105(+) cells are EPs. These previously unclassified populations may facilitate further understanding of the molecular mechanisms governing human erythroid development and serve as potential therapeutic targets in disorders of the erythroid lineage.