Arginine metabolism regulates human erythroid differentiation through hypusination of eIF5A.

Metabolic programs contribute to hematopoietic stem and progenitor cell (HSPC) fate, but it is not known whether the metabolic regulation of protein synthesis controls HSPC differentiation. Here, we show that SLC7A1/cationic amino acid transporter 1-dependent arginine uptake and its catabolism to the polyamine spermidine control human erythroid specification of HSPCs via the activation of the eukaryotic translation initiation factor 5A (eIF5A). eIF5A activity is dependent on its hypusination, a posttranslational modification resulting from the conjugation of the aminobutyl moiety of spermidine to lysine. Notably, attenuation of hypusine synthesis in erythroid progenitors, by the inhibition of deoxyhypusine synthase, abrogates erythropoiesis but not myeloid cell differentiation. Proteomic profiling reveals mitochondrial translation to be a critical target of hypusinated eIF5A, and accordingly, progenitors with decreased hypusine activity exhibit diminished oxidative phosphorylation. This affected pathway is critical for eIF5A-regulated erythropoiesis, as interventions augmenting mitochondrial function partially rescue human erythropoiesis under conditions of attenuated hypusination. Levels of mitochondrial ribosomal proteins (RPs) were especially sensitive to the loss of hypusine, and we find that the ineffective erythropoiesis linked to haploinsufficiency of RPS14 in chromosome 5q deletions in myelodysplastic syndrome is associated with a diminished pool of hypusinated eIF5A. Moreover, patients with RPL11-haploinsufficient Diamond-Blackfan anemia as well as CD34+ progenitors with downregulated RPL11 exhibit a markedly decreased hypusination in erythroid progenitors, concomitant with a loss of mitochondrial metabolism. Thus, eIF5A-dependent protein synthesis regulates human erythropoiesis, and our data reveal a novel role for RPs in controlling eIF5A hypusination in HSPCs, synchronizing mitochondrial metabolism with erythroid differentiation.

Vitamin C deficiency reveals developmental differences between neonatal and adult hematopoiesis.

Hematopoiesis, a process that results in the differentiation of all blood lineages, is essential throughout life. The production of 1x1012 blood cells per day, including 200x109 erythrocytes, is highly dependent on nutrient consumption. Notably though, the relative requirements for micronutrients during the perinatal period, a critical developmental window for immune cell and erythrocyte differentiation, have not been extensively studied. More specifically, the impact of the vitamin C/ascorbate micronutrient on perinatal as compared to adult hematopoiesis has been difficult to assess in animal models. Even though humans cannot synthesize ascorbate, due to a pseudogenization of the L-gulono-γ-lactone oxidase (GULO) gene, its generation from glucose is an ancestral mammalian trait. Taking advantage of a Gulo-/- mouse model, we show that ascorbic acid deficiency profoundly impacts perinatal hematopoiesis, resulting in a hypocellular bone marrow (BM) with a significant reduction in hematopoietic stem cells, multipotent progenitors, and hematopoietic progenitors. Furthermore, myeloid progenitors exhibited differential sensitivity to vitamin C levels; common myeloid progenitors and megakaryocyte-erythrocyte progenitors were markedly reduced in Gulo-/- pups following vitamin C depletion in the dams, whereas granulocyte-myeloid progenitors were spared, and their frequency was even augmented. Notably, hematopoietic cell subsets were rescued by vitamin C repletion. Consistent with these data, peripheral myeloid cells were maintained in ascorbate-deficient Gulo-/- pups while other lineage-committed hematopoietic cells were decreased. A reduction in B cell numbers was associated with a significantly reduced humoral immune response in ascorbate-depleted Gulo-/- pups but not adult mice. Erythropoiesis was particularly sensitive to vitamin C deprivation during both the perinatal and adult periods, with ascorbate-deficient Gulo-/- pups as well as adult mice exhibiting compensatory splenic differentiation. Furthermore, in the pathological context of hemolytic anemia, vitamin C-deficient adult Gulo-/- mice were not able to sufficiently increase their erythropoietic activity, resulting in a sustained anemia. Thus, vitamin C plays a pivotal role in the maintenance and differentiation of hematopoietic progenitors during the neonatal period and is required throughout life to sustain erythroid differentiation under stress conditions.

LIX1-mediated changes in mitochondrial metabolism control the fate of digestive mesenchyme-derived cells.

YAP1 and TAZ are transcriptional co-activator proteins that play fundamental roles in many biological processes, from cell proliferation and cell lineage fate determination to tumorigenesis. We previously demonstrated that Limb Expression 1 (LIX1) regulates YAP1 and TAZ activity and controls digestive mesenchymal progenitor proliferation. However, LIX1 mode of action remains elusive. Here, we found that endogenous LIX1 is localized in mitochondria and is anchored to the outer mitochondrial membrane through S-palmitoylation of cysteine 84, a residue conserved in all LIX1 orthologs. LIX1 downregulation altered the mitochondrial ultrastructure, resulting in a significantly decreased respiration and attenuated production of mitochondrial reactive oxygen species (mtROS). Mechanistically, LIX1 knock-down impaired the stability of the mitochondrial proteins PHB2 and OPA1 that are found in complexes with mitochondrial-specific phospholipids and are required for cristae organization. Supplementation with unsaturated fatty acids counteracted the effects of LIX1 knock-down on mitochondrial morphology and ultrastructure and restored YAP1/TAZ signaling. Collectively, our data demonstrate that LIX1 is a key regulator of cristae organization, modulating mtROS level and subsequently regulating the signaling cascades that control fate commitment of digestive mesenchyme-derived cells.

Human erythroid differentiation requires VDAC1-mediated mitochondrial clearance.

Erythroblast maturation in mammals is dependent on organelle clearance throughout terminal erythropoiesis. We studied the role of the outer mitochondrial membrane protein voltage-dependent anion channel-1 (VDAC1) in human terminal erythropoiesis. We show that short hairpin (shRNA)-mediated downregulation of VDAC1 accelerates erythroblast maturation. Thereafter, erythroblasts are blocked at the orthochromatic stage, exhibiting a significant decreased level of enucleation, concomitant with an increased cell death. We demonstrate that mitochondria clearance starts at the transition from basophilic to polychromatic erythroblast, and that VDAC1 downregulation induces the mitochondrial retention. In damaged mitochondria from non-erythroid cells, VDAC1 was identified as a target for Parkin-mediated ubiquitination to recruit the phagophore. Here, we showed that VDAC1 is involved in phagophore's membrane recruitment regulating selective mitophagy of still functional mitochondria from human erythroblasts. These findings demonstrate for the first time a crucial role for VDAC1 in human erythroblast terminal differentiation, regulating mitochondria clearance.

Regulatory T cell differentiation is controlled by αKG-induced alterations in mitochondrial metabolism and lipid homeostasis.

Suppressive regulatory T cell (Treg) differentiation is controlled by diverse immunometabolic signaling pathways and intracellular metabolites. Here we show that cell-permeable α-ketoglutarate (αKG) alters the DNA methylation profile of naive CD4 T cells activated under Treg polarizing conditions, markedly attenuating FoxP3+ Treg differentiation and increasing inflammatory cytokines. Adoptive transfer of these T cells into tumor-bearing mice results in enhanced tumor infiltration, decreased FoxP3 expression, and delayed tumor growth. Mechanistically, αKG leads to an energetic state that is reprogrammed toward a mitochondrial metabolism, with increased oxidative phosphorylation and expression of mitochondrial complex enzymes. Furthermore, carbons from ectopic αKG are directly utilized in the generation of fatty acids, associated with lipidome remodeling and increased triacylglyceride stores. Notably, inhibition of either mitochondrial complex II or DGAT2-mediated triacylglyceride synthesis restores Treg differentiation and decreases the αKG-induced inflammatory phenotype. Thus, we identify a crosstalk between αKG, mitochondrial metabolism and triacylglyceride synthesis that controls Treg fate.

Comprehensive phenotyping of erythropoiesis in human bone marrow: Evaluation of normal and ineffective erythropoiesis.

Identification of stage-specific erythroid cells is critical for studies of normal and disordered human erythropoiesis. While immunophenotypic strategies have previously been developed to identify cells at each stage of terminal erythroid differentiation, erythroid progenitors are currently defined very broadly. Refined strategies to identify and characterize BFU-E and CFU-E subsets are critically needed. To address this unmet need, a flow cytometry-based technique was developed that combines the established surface markers CD34 and CD36 with CD117, CD71, and CD105. This combination allowed for the separation of erythroid progenitor cells into four discrete populations along a continuum of progressive maturation, with increasing cell size and decreasing nuclear/cytoplasmic ratio, proliferative capacity and stem cell factor responsiveness. This strategy was validated in uncultured, primary erythroid cells isolated from bone marrow of healthy individuals. Functional colony assays of these progenitor populations revealed enrichment of BFU-E only in the earliest population, transitioning to cells yielding BFU-E and CFU-E, then CFU-E only. Utilizing CD34/CD105 and GPA/CD105 profiles, all four progenitor stages and all five stages of terminal erythroid differentiation could be identified. Applying this immunophenotyping strategy to primary bone marrow cells from patients with myelodysplastic syndrome, identified defects in erythroid progenitors and in terminal erythroid differentiation. This novel immunophenotyping technique will be a valuable tool for studies of normal and perturbed human erythropoiesis. It will allow for the discovery of stage-specific molecular and functional insights into normal erythropoiesis as well as for identification and characterization of stage-specific defects in inherited and acquired disorders of erythropoiesis.

The equilibrative nucleoside transporter ENT1 is critical for nucleotide homeostasis and optimal erythropoiesis.

The tight regulation of intracellular nucleotides is critical for the self-renewal and lineage specification of hematopoietic stem cells (HSCs). Nucleosides are major metabolite precursors for nucleotide biosynthesis and their availability in HSCs is dependent on their transport through specific membrane transporters. However, the role of nucleoside transporters in the differentiation of HSCs to the erythroid lineage and in red cell biology remains to be fully defined. Here, we show that the absence of the equilibrative nucleoside transporter (ENT1) in human red blood cells with a rare Augustine-null blood type is associated with macrocytosis, anisopoikilocytosis, an abnormal nucleotide metabolome, and deregulated protein phosphorylation. A specific role for ENT1 in human erythropoiesis was demonstrated by a defective erythropoiesis of human CD34+ progenitors following short hairpin RNA-mediated knockdown of ENT1. Furthermore, genetic deletion of ENT1 in mice was associated with reduced erythroid progenitors in the bone marrow, anemia, and macrocytosis. Mechanistically, we found that ENT1-mediated adenosine transport is critical for cyclic adenosine monophosphate homeostasis and the regulation of erythroid transcription factors. Notably, genetic investigation of 2 ENT1null individuals demonstrated a compensation by a loss-of-function variant in the ABCC4 cyclic nucleotide exporter. Indeed, pharmacological inhibition of ABCC4 in Ent1-/- mice rescued erythropoiesis. Overall, our results highlight the importance of ENT1-mediated nucleotide metabolism in erythropoiesis.

An IDH1-vitamin C crosstalk drives human erythroid development by inhibiting pro-oxidant mitochondrial metabolism.

The metabolic changes controlling the stepwise differentiation of hematopoietic stem and progenitor cells (HSPCs) to mature erythrocytes are poorly understood. Here, we show that HSPC development to an erythroid-committed proerythroblast results in augmented glutaminolysis, generating alpha-ketoglutarate (αKG) and driving mitochondrial oxidative phosphorylation (OXPHOS). However, sequential late-stage erythropoiesis is dependent on decreasing αKG-driven OXPHOS, and we find that isocitrate dehydrogenase 1 (IDH1) plays a central role in this process. IDH1 downregulation augments mitochondrial oxidation of αKG and inhibits reticulocyte generation. Furthermore, IDH1 knockdown results in the generation of multinucleated erythroblasts, a morphological abnormality characteristic of myelodysplastic syndrome and congenital dyserythropoietic anemia. We identify vitamin C homeostasis as a critical regulator of ineffective erythropoiesis; oxidized ascorbate increases mitochondrial superoxide and significantly exacerbates the abnormal erythroblast phenotype of IDH1-downregulated progenitors, whereas vitamin C, scavenging reactive oxygen species (ROS) and reprogramming mitochondrial metabolism, rescues erythropoiesis. Thus, an IDH1-vitamin C crosstalk controls terminal steps of human erythroid differentiation.

p53 activation during ribosome biogenesis regulates normal erythroid differentiation.

The role of ribosome biogenesis in erythroid development is supported by the recognition of erythroid defects in ribosomopathies in both Diamond-Blackfan anemia and 5q- syndrome. Whether ribosome biogenesis exerts a regulatory function on normal erythroid development is still unknown. In the present study, a detailed characterization of ribosome biogenesis dynamics during human and murine erythropoiesis showed that ribosome biogenesis is abruptly interrupted by the decline in ribosomal DNA transcription and the collapse of ribosomal protein neosynthesis. Its premature arrest by the RNA Pol I inhibitor CX-5461 targeted the proliferation of immature erythroblasts. p53 was activated spontaneously or in response to CX-5461, concomitant to ribosome biogenesis arrest, and drove a transcriptional program in which genes involved in cell cycle-arrested, negative regulation of apoptosis, and DNA damage response were upregulated. RNA Pol I transcriptional stress resulted in nucleolar disruption and activation of the ATR-CHK1-p53 pathway. Our results imply that the timing of ribosome biogenesis extinction and p53 activation is crucial for erythroid development. In ribosomopathies in which ribosome availability is altered by unbalanced production of ribosomal proteins, the threshold downregulation of ribosome biogenesis could be prematurely reached and, together with pathological p53 activation, prevents a normal expansion of erythroid progenitors.

Downregulation of Mitochondrial TSPO Inhibits Mitophagy and Reduces Enucleation during Human Terminal Erythropoiesis.

Translocator protein (TSPO) and voltage dependent anion channels (VDAC) are two proteins forming a macromolecular complex in the outer mitochondrial membrane that is involved in pleiotropic functions. Specifically, these proteins were described to regulate the clearance of damaged mitochondria by selective mitophagy in non-erythroid immortalized cell lines. Although it is well established that erythroblast maturation in mammals depends on organelle clearance, less is known about mechanisms regulating this clearance throughout terminal erythropoiesis. Here, we studied the effect of TSPO1 downregulation and the action of Ro5-4864, a drug ligand known to bind to the TSPO/VDAC complex interface, in ex vivo human terminal erythropoiesis. We found that both treatments delay mitochondrial clearance, a process associated with reduced levels of the PINK1 protein, which is a key protein triggering canonical mitophagy. We also observed that TSPO1 downregulation blocks erythroblast maturation at the orthochromatic stage, decreases the enucleation rate, and increases cell death. Interestingly, TSPO1 downregulation does not modify reactive oxygen species (ROS) production nor intracellular adenosine triphosphate (ATP) levels. Ro5-4864 treatment recapitulates these phenotypes, strongly suggesting an active role of the TSPO/VDAC complex in selective mitophagy throughout human erythropoiesis. The present study links the function of the TSPO/VDAC complex to the PINK1/Parkin-dependent mitophagy induction during terminal erythropoiesis, leading to the proper completion of erythroid maturation.

A predictable conserved DNA base composition signature defines human core DNA replication origins.

DNA replication initiates from multiple genomic locations called replication origins. In metazoa, DNA sequence elements involved in origin specification remain elusive. Here, we examine pluripotent, primary, differentiating, and immortalized human cells, and demonstrate that a class of origins, termed core origins, is shared by different cell types and host ~80% of all DNA replication initiation events in any cell population. We detect a shared G-rich DNA sequence signature that coincides with most core origins in both human and mouse genomes. Transcription and G-rich elements can independently associate with replication origin activity. Computational algorithms show that core origins can be predicted, based solely on DNA sequence patterns but not on consensus motifs. Our results demonstrate that, despite an attributed stochasticity, core origins are chosen from a limited pool of genomic regions. Immortalization through oncogenic gene expression, but not normal cellular differentiation, results in increased stochastic firing from heterochromatin and decreased origin density at TAD borders.

Steroid resistance in Diamond Blackfan anemia associates with p57Kip2 dysregulation in erythroid progenitors.

Despite the effective clinical use of steroids for the treatment of Diamond Blackfan anemia (DBA), the mechanisms through which glucocorticoids regulate human erythropoiesis remain poorly understood. We report that the sensitivity of erythroid differentiation to dexamethasone is dependent on the developmental origin of human CD34+ progenitor cells, specifically increasing the expansion of CD34+ progenitors from peripheral blood (PB) but not cord blood (CB). Dexamethasone treatment of erythroid-differentiated PB, but not CB, CD34+ progenitors resulted in the expansion of a newly defined CD34+CD36+CD71hiCD105med immature colony-forming unit-erythroid (CFU-E) population. Furthermore, proteomics analyses revealed the induction of distinct proteins in dexamethasone-treated PB and CB erythroid progenitors. Dexamethasone treatment of PB progenitors resulted in the specific upregulation of p57Kip2, a Cip/Kip cyclin-dependent kinase inhibitor, and we identified this induction as critical; shRNA-mediated downregulation of p57Kip2, but not the related p27Kip1, significantly attenuated the impact of dexamethasone on erythroid differentiation and inhibited the expansion of the immature CFU-E subset. Notably, in the context of DBA, we found that steroid resistance was associated with dysregulated p57Kip2 expression. Altogether, these data identify a unique glucocorticoid-responsive human erythroid progenitor and provide new insights into glucocorticoid-based therapeutic strategies for the treatment of patients with DBA.

GLUT1 protects prostate cancer cells from glucose deprivation-induced oxidative stress.

Glucose, chief metabolic support for cancer cell survival and growth, is mainly imported into cells by facilitated glucose transporters (GLUTs). The increase in glucose uptake along with tumor progression is due to an increment of facilitative glucose transporters as GLUT1. GLUT1 prevents cell death of cancer cells caused by growth factors deprivation, but there is scarce information about its role on the damage caused by glucose deprivation, which usually occurs within the core of a growing tumor. In prostate cancer (PCa), GLUT1 is found in the most aggressive tumors, and it is regulated by androgens. To study the response of androgen-sensitive and insensitive PCa cells to glucose deprivation and the role of GLUT1 on survival mechanisms, androgen-sensitive LNCaP and castration-resistant LNCaP-R cells were employed. Results demonstrated that glucose deprivation induced a necrotic type of cell death which is prevented by antioxidants. Androgen-sensitive cells show a higher resistance to cell death triggered by glucose deprivation than castration-resistant cells. Glucose removal causes an increment of H2O2, an activation of androgen receptor (AR) and a stimulation of AMP-activated protein kinase activity. In addition, glucose removal increases GLUT1 production in androgen sensitive PCa cells. GLUT1 ectopic overexpression makes PCa cells more resistant to glucose deprivation and oxidative stress-induced cell death. Under glucose deprivation, GLUT1 overexpressing PCa cells sustains mitochondrial SOD2 activity, compromised after glucose removal, and significantly increases reduced glutathione (GSH). In conclusion, androgen-sensitive PCa cells are more resistant to glucose deprivation-induced cell death by a GLUT1 upregulation through an enhancement of reduced glutathione levels.

Developmental differences between neonatal and adult human erythropoiesis.

Studies of human erythropoiesis have relied, for the most part, on the in vitro differentiation of hematopoietic stem and progenitor cells (HSPC) from different sources. Here, we report that despite the common core erythroid program that exists between cord blood (CB)- and peripheral blood (PB)-HSPC induced toward erythroid differentiation in vitro, significant functional differences exist. We undertook a comparative analysis of human erythropoiesis using these two different sources of HSPC. Upon in vitro erythroid differentiation, CB-derived cells proliferated 4-fold more than PB-derived cells. However, CB-derived cells exhibited a delayed kinetics of differentiation, resulting in an increased number of progenitors, notably colony-forming unit (CFU-E). The phenotypes of early erythroid differentiation stages also differed between the two sources with a significantly higher percentage of IL3R- GPA- CD34+ CD36+ cells generated from PB- than CB-HSPCs. This subset was found to generate both burst-forming unit (BFU-E) and CFU-E colonies in colony-forming assays. To further understand the differences between CB- and PB-HSPC, cells at eight stages of erythroid differentiation were sorted from each of the two sources and their transcriptional profiles were compared. We document differences at the CD34, BFU-E, poly- and orthochromatic stages. Genes exhibiting the most significant differences in expression between HSPC sources clustered into cell cycle- and autophagy-related pathways. Altogether, our studies provide a qualitative and quantitative comparative analysis of human erythropoiesis, highlighting the impact of the developmental origin of HSPCs on erythroid differentiation.

Metabolic orchestration of T lineage differentiation and function.

T cells are stimulated by the engagement of antigen, cytokine, pathogen, and hormone receptors. While research performed over many years has focused on deciphering the molecular components of these pathways, recent data underscore the importance of the metabolic environment in conditioning responses to receptor engagement. The ability of T cells to undergo a massive proliferation and cytokine secretion in response to receptor signals requires alterations to their bioenergetic homeostasis, allowing them to meet new energetic and biosynthetic demands. The metabolic reprogramming of activated T cells is regulated not only by changes in intracellular nutrient uptake and utilization but also by nutrient and oxygen concentrations in the extracellular environment. Notably, the extracellular environment can be profoundly altered by pathological conditions such as infections and tumors, thereby perturbing the metabolism and function of antigen-specific T lymphocytes. This review highlights the interplay between diverse metabolic networks and the transcriptional/epigenetic states that condition T-cell differentiation, comparing the metabolic features of T lymphocytes with other immune cells. We further address recent discoveries in the metabolic pathways that govern T-cell function in physiological and pathological conditions.

Cell surface Glut1 levels distinguish human CD4 and CD8 T lymphocyte subsets with distinct effector functions.

CD4 and CD8 T lymphocyte activation requires the generation of sufficient energy to support new biosynthetic demands. Following T cell receptor (TCR) engagement, these requirements are met by an increased glycolysis, due, at least in part, to induction of the Glut1 glucose transporter. As Glut1 is upregulated on tumor cells in response to hypoxia, we assessed whether surface Glut1 levels regulate the antigen responsiveness of human T lymphocytes in both hypoxic and atmospheric oxygen conditions. Notably, Glut1 upregulation in response to TCR stimulation was significantly higher in T lymphocytes activated under hypoxic as compared to atmospheric oxygen conditions. Furthermore, TCR-stimulated human T lymphocytes sorted on the basis of Glut1-Lo and Glut1-Hi profiles maintained distinct characteristics, irrespective of the oxygen tension. While T cells activated in hypoxia divided less than those activated in atmospheric oxygen, Glut1-Hi lymphocytes exhibited increased effector phenotype acquisition, augmented proliferation, and an inverted CD4/CD8 ratio in both oxygen conditions. Moreover, Glut1-Hi T lymphocytes exhibited a significantly enhanced ability to produce IFN-γ and this secretion potential was completely dependent on continued glycolysis. Thus, Glut1 surface levels identify human T lymphocytes with distinct effector functions in both hypoxic and atmospheric oxygen tensions.

Metabolic regulation of hematopoietic stem cell commitment and erythroid differentiation.

PURPOSE OF REVIEW: Hematopoietic stem cell (HSC) renewal and lineage differentiation are finely tuned processes, regulated by cytokines, transcription factors and cell-cell contacts. However, recent studies have shown that fuel utilization also conditions HSC fate. This review focuses on our current understanding of the metabolic pathways that govern HSC self-renewal, commitment and specification to the erythroid lineage. RECENT FINDINGS: HSCs reside in a hypoxic bone marrow niche that favors anaerobic glycolysis. Although this metabolic pathway is required for stem cell maintenance, other pathways also play critical roles. Fatty acid oxidation preserves HSC self-renewal by promoting asymmetric division, whereas oxidative phosphorylation induces lineage commitment. Committed erythroid progenitors support the production of 2.4 million erythrocytes per second in human adults via a synchronized regulation of iron, amino acid and glucose metabolism. Iron is indispensable for heme biosynthesis in erythroblasts; a process finely coordinated by at least two hormones, hepcidin and erythroferrone, together with multiple cell surface iron transporters. Furthermore, hemoglobin production is promoted by amino acid-induced mTOR signaling. Erythropoiesis is also strictly dependent on glutamine metabolism; under conditions where glutaminolysis is inhibited, erythropoietin-signaled progenitors are diverted to a myelomonocytic fate. Indeed, the utilization of both glutamine and glucose in de-novo nucleotide biosynthesis is a sine qua non for erythroid differentiation. SUMMARY: Diverse metabolic networks function in concert with transcriptional, translational and epigenetic programs to regulate HSC potential and orient physiological as well as pathological erythroid differentiation.

Glutamine-dependent α-ketoglutarate production regulates the balance between T helper 1 cell and regulatory T cell generation.

T cell activation requires that the cell meet increased energetic and biosynthetic demands. We showed that exogenous nutrient availability regulated the differentiation of naïve CD4(+) T cells into distinct subsets. Activation of naïve CD4(+) T cells under conditions of glutamine deprivation resulted in their differentiation into Foxp3(+) (forkhead box P3-positive) regulatory T (Treg) cells, which had suppressor function in vivo. Moreover, glutamine-deprived CD4(+) T cells that were activated in the presence of cytokines that normally induce the generation of T helper 1 (TH1) cells instead differentiated into Foxp3(+) Treg cells. We found that α-ketoglutarate (αKG), the glutamine-derived metabolite that enters into the mitochondrial citric acid cycle, acted as a metabolic regulator of CD4(+) T cell differentiation. Activation of glutamine-deprived naïve CD4(+) T cells in the presence of a cell-permeable αKG analog increased the expression of the gene encoding the TH1 cell-associated transcription factor Tbet and resulted in their differentiation into TH1 cells, concomitant with stimulation of mammalian target of rapamycin complex 1 (mTORC1) signaling. Together, these data suggest that a decrease in the intracellular amount of αKG, caused by the limited availability of extracellular glutamine, shifts the balance between the generation of TH1 and Treg cells toward that of a Treg phenotype.

Glucose and glutamine metabolism regulate human hematopoietic stem cell lineage specification.

The metabolic state of quiescent hematopoietic stem cells (HSCs) is an important regulator of self-renewal, but it is unclear whether or how metabolic parameters contribute to HSC lineage specification and commitment. Here, we show that the commitment of human and murine HSCs to the erythroid lineage is dependent upon glutamine metabolism. HSCs require the ASCT2 glutamine transporter and active glutamine metabolism for erythroid specification. Blocking this pathway diverts EPO-stimulated HSCs to differentiate into myelomonocytic fates, altering in vivo HSC responses and erythroid commitment under stress conditions such as hemolytic anemia. Mechanistically, erythroid specification of HSCs requires glutamine-dependent de novo nucleotide biosynthesis. Exogenous nucleosides rescue erythroid commitment of human HSCs under conditions of limited glutamine catabolism, and glucose-stimulated nucleotide biosynthesis further enhances erythroid specification. Thus, the availability of glutamine and glucose to provide fuel for nucleotide biosynthesis regulates HSC lineage commitment under conditions of metabolic stress.

Glut1-mediated glucose transport regulates HIV infection.

Cell cycle entry is commonly considered to positively regulate HIV-1 infection of CD4 T cells, raising the question as to how quiescent lymphocytes, representing a large portion of the viral reservoir, are infected in vivo. Factors such as the homeostatic cytokine IL-7 have been shown to render quiescent T cells permissive to HIV-1 infection, presumably by transiently stimulating their entry into the cell cycle. However, we show here that at physiological oxygen (O(2)) levels (2-5% O(2) tension in lymphoid organs), IL-7 stimulation generates an environment permissive to HIV-1 infection, despite a significantly attenuated level of cell cycle entry. We identify the IL-7-induced increase in Glut1 expression, resulting in augmented glucose uptake, as a key factor in rendering these T lymphocytes susceptible to HIV-1 infection. HIV-1 infection of human T cells is abrogated either by impairment of Glut1 signal transduction or by siRNA-mediated Glut1 down-regulation. Consistent with this, we show that the susceptibility of human thymocyte subsets to HIV-1 infection correlates with Glut1 expression; single-round infection is markedly higher in the Glut1-expressing double-positive thymocyte population than in any of the Glut1-negative subsets. Thus, our studies reveal the Glut1-mediated metabolic pathway as a critical regulator of HIV-1 infection in human CD4 T cells and thymocytes.