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Dipeptidyl peptidase 4 (DPP4)/CD26 truncates certain proteins, and this posttranslational modification can influence their activity. Truncated (T) colony-stimulating factors (CSFs) are decreased in potency for stimulating proliferation of hematopoietic progenitor cells (HPCs). T-CXCL12, a modified chemokine, is inactive as an HPC chemotactic, survival, and enhancing factor for replating or ex-vivo expansion of HPCs. Moreover, T-CSFs and T-CXCL12 specifically downmodulates the positively acting effects of their own full-length molecule. Other chemokines have DPP4 truncation sites. In the present study, we evaluated effects of DPP4 inhibition (by Diprotin A) or gene deletion of HPC on chemokine inhibition of multicytokine-stimulated HPC, and on chemokine-enhancing effects on single CSF-stimulated HPC proliferation, as well as effects of DPP4 treatment of a number of chemokines. Myelosuppressive effects of chemokines with, but not without, a DPP4 truncation site were greatly enhanced in inhibitory potency by pretreating target bone marrow (BM) cells with Diprotin A, or by assaying their activity on dpp4/cd26(-/-) BM cells. DPP4 treatment of myelosuppressive chemokines containing a DPP4 truncation site produced a nonmyelosuppressive molecule, but one which had the capacity to block suppression by that unmodified chemokine both in vitro and in vivo. Additionally, DPP4 treatment ablated the single cytokine-stimulated HPC-enhancing activity of CCL3/MIP-1α and CCL4/MIP-1β, and blocked the enhancing activity of each unmodified molecule, in vitro and in vivo. These results highlight the functional posttranslational modulating effects of DPP4 on chemokine activities, and information offering additional biological insight into chemokine regulation of hematopoiesis.

Hematopoietic stem cells (HSCs) reside in hypoxic niches within bone marrow and cord blood. Yet, essentially all HSC studies have been performed with cells isolated and processed in non-physiologic ambient air. By collecting and manipulating bone marrow and cord blood in native conditions of hypoxia, we demonstrate that brief exposure to ambient oxygen decreases recovery of long-term repopulating HSCs and increases progenitor cells, a phenomenon we term extraphysiologic oxygen shock/stress (EPHOSS). Thus, true numbers of HSCs in the bone marrow and cord blood are routinely underestimated. We linked ROS production and induction of the mitochondrial permeability transition pore (MPTP) via cyclophilin D and p53 as mechanisms of EPHOSS. The MPTP inhibitor cyclosporin A protects mouse bone marrow and human cord blood HSCs from EPHOSS during collection in air, resulting in increased recovery of transplantable HSCs. Mitigating EPHOSS during cell collection and processing by pharmacological means may be clinically advantageous for transplantation., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Simple efforts are needed to enhance cord blood (CB) transplantation. We hypothesized that short-term exposure of CD34(+) CB cells to 39.5°C would enhance their response to stromal-derived factor-1 (SDF-1), by increasing lipid raft aggregation and CXCR4 expression, thus leading to enhanced engraftment. Mild hyperthermia (39.5°C) significantly increased the percent of CD34(+) CB that migrated toward SDF-1. This was associated with increased expression of CXCR4 on the cells. Mechanistically, mild heating increased the percent of CD34(+) cells with aggregated lipid rafts and enhanced colocalization of CXCR4 within lipid raft domains. Using methyl-β-cyclodextrin (MβCD), an agent that blocks lipid raft aggregation, it was determined that this enhancement in chemotaxis was dependent upon lipid raft aggregation. Colocalization of Rac1, a GTPase crucial for cell migration and adhesion, with CXCR4 to the lipid raft was essential for the effects of heat on chemotaxis, as determined with an inhibitor of Rac1 activation, NSC23766. Application-wise, mild heat treatment significantly increased the percent chimerism as well as homing and engraftment of CD34(+) CB cells in sublethally irradiated non-obese diabetic severe combined immunodeficiency IL-2 receptor gamma chain d (NSG) mice. Mild heating may be a simple and inexpensive means to enhance engraftment following CB transplantation in patients., (© 2015 AlphaMed Press.)

A hyaluronic-acid-rich node and duct system (HAR-NDS) was found on the surface of internal organs of mice, and inside their blood and lymph vessels. The nodes (HAR-Ns) were filled with immune cells of the innate system and were especially enriched with mast cells and histiocytes. They also contained hematopoietic progenitor cells (HPCs), such as granulocyte-macrophage, erythroid, multipotential progenitors, and mast cell progenitors (MCPs). MCPs were the most abundant among the HPCs in HAR-Ns. Their frequency was fivefold higher than that of the MCPs in bone marrow. In addition, the system contained pluripotent stem cells (PSCs) capable of producing CD45(-)Flk1(+) hemangioblast-like cells, which subsequently generated various types of HPCs and differentiated blood cells. Although HAR-Ns did not appear to harbor enough number of cells capable of long-term reconstitution or short-term radioprotection of lethally irradiated recipients, bone marrow cells were able to engraft in the HAR-NDS and reconstitute hematopoietic potentials of the system. PSCs and HPCs were consistently found in intravenous, intralymphatic, and intestinal HAR-ND. We infer that PSCs and HPCs reside in the HAR-ND and that this novel system may serve as an alternative means to traffic immature and mature blood cells throughout the body.

Hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) play a vital role in replenishment of blood cells. In addition to growth factors, energy metabolism plays an important role in cellular proliferation. Oxidative phosphorylation that occurs in the mitochondria is the major source of ATP. In this study, we have investigated the role of peroxisome proliferator-activated-γ coactivator-1α (PGC-1α), a major regulator of mitochondrial biogenesis, in hematopoiesis. PGC-1α is expressed in HSC/HPCs. Loss of PGC-1α minimally affects basal hematopoiesis; however, it significantly impairs stress hematopoiesis. Recovery of hematopoiesis poststress involves rapid proliferation of HSC/HPCs. Growth factors stimulate HSC/HPC proliferation in a dose-dependent manner and this response is modulated by oxygen tension. Although severe hypoxic conditions inhibit HSC/HPC proliferation, mild hypoxia enhances the clonogenic potential; however, the mechanism underlying this phenomenon remains largely unknown. Our studies demonstrate that PGC-1α-mediated mitochondrial biogenesis is critical for the increased clonogenic potential of progenitors under mild hypoxia. Metabolic programming and increased glucose uptake can drive rapid progenitor cell proliferation under relatively low oxygen tension only if the HPC has the capacity to increase PGC-1α expression and mitochondrial biogenesis. Loss of PGC-1α also impairs the long-term repopulating potential of HSCs. Our findings may have therapeutic applications for rapid recovery of blood cells following myeloablation.

Enhancement of hematopoietic recovery after radiation, chemotherapy, or hematopoietic stem cell (HSC) transplantation is clinically relevant. Dipeptidylpeptidase (DPP4) cleaves a wide variety of substrates, including the chemokine stromal cell-derived factor-1 (SDF-1). In the course of experiments showing that inhibition of DPP4 enhances SDF-1-mediated progenitor cell survival, ex vivo cytokine expansion and replating frequency, we unexpectedly found that DPP4 has a more general role in regulating colony-stimulating factor (CSF) activity. DPP4 cleaved within the N-termini of the CSFs granulocyte-macrophage (GM)-CSF, G-CSF, interleukin-3 (IL-3) and erythropoietin and decreased their activity. Dpp4 knockout or DPP4 inhibition enhanced CSF activities both in vitro and in vivo. The reduced activity of DPP4-truncated versus full-length human GM-CSF was mechanistically linked to effects on receptor-binding affinity, induction of GM-CSF receptor oligomerization and signaling capacity. Hematopoiesis in mice after radiation or chemotherapy was enhanced in Dpp4(-/-) mice or mice receiving an orally active DPP4 inhibitor. DPP4 inhibition enhanced engraftment in mice without compromising HSC function, suggesting the potential clinical utility of this approach.

The transcriptional repressor Bcl6 is a critical arbiter of Th cell fate, promoting the follicular Th lineage while repressing other Th cell lineages. Bcl6-deficient (Bcl6(-/-)) mice develop a spontaneous and severe Th2-type inflammatory disease, thus warranting assessment of Bcl6 in regulatory T cell (Treg) function. Bcl6(-/-) Tregs were competent at suppressing T cell proliferation in vitro and Th1-type colitogenic T cell responses in vivo. In contrast, Bcl6(-/-) Tregs strongly exacerbated lung inflammation in a model of allergic airway disease and promoted higher Th2 responses, including systemic upregulation of microRNA-21. Further, Bcl6(-/-) Tregs were selectively impaired at controlling Th2 responses, but not Th1 and Th17 responses, in mixed chimeras of Bcl6(-/-) bone marrow with Foxp3(-/-) bone marrow. Bcl6(-/-) Tregs displayed increased levels of the Th2 transcription factor Gata3 and other Th2 and Treg genes. Bcl6 potently repressed Gata3 transcriptional transactivation, providing a mechanism for the increased expression of Th2 genes by Bcl6(-/-) Tregs. Gata3 has a critical role in regulating Foxp3 expression and functional fitness of Tregs; however, the signal that regulates Gata3 and restricts its transactivation of Th2 cytokines in Tregs has remained unexplored. Our results identify Bcl6 as an essential transcription factor regulating Gata3 activity in Tregs. Thus, Bcl6 represents a crucial regulatory layer in the Treg functional program that is required for specific suppression of Gata3 and Th2 effector responses by Tregs.

Nuclear transcription factor Stat3 is important for proper regulation of hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) proliferation, survival, and cytokine signaling responses. A new, noncanonical role for Stat3 in mitochondrial function has been discovered recently. However, there is little information on the role(s) of mitochondrial Stat3 in HSC/HPC function, especially potential effects of Stat3/mitochondrial dysregulation in human diseases. We investigated hematopoietic cell-targeted deletion of the STAT3 gene in HSCs/HPCs with a focus on mitochondrial function. We found that STAT3(-/-) mice, which have a very shortened lifespan, dysfunctional/dysregulated mitochondrial function and excessive reactive oxygen species production in HSCs/HPCs that coincides with pronounced defects in function. These animals have a blood phenotype with similarities to premature aging and to human diseases of myelodysplastic syndrome and myeloproliferative neoplasms such as erythroid dysplasia, anemia, excessive myeloproliferation, and lymphomyeloid ratio shifts. We show herein that the lifespan of STAT3(-/-) animals is lengthened by treatment with a reactive oxygen species scavenger, which lessened the severity of the blood phenotype. These data suggest a need for more detailed studies of role(s) of Stat3 in HSC/HPC mitochondrial function in human diseases and raise the idea that mitochondrial Stat3 could be used as a potential therapeutic target.

Cyclin dependent kinase inhibitors (CDKIs) influence proliferation of hematopoietic progenitor cells (HPCs), but little is known of how they influence proliferative responsiveness of HPCs to colony stimulating factors (CSFs), alone and in combination with other hematopoietically active factors, such as the potent co-stimulating cytokine stem cell factor (SCF), or inhibition by myelosuppressive chemokines. Using mice with deletions in p18(INK4c), p21(CIP1/WAF1), or p27(KIP1) genes, and in mice with double gene deletions for either p18/p21 or p18/p27, we determined effects of absence of these CDKIs and their interactions on functional HPC numbers in vivo, and HPC proliferative responsiveness in vitro. There is a decrease in bone marrow HPC proliferation in p18(-/-) mice commensurate with decreased numbers of HPC, suggesting a positive role for p18 on HPC in vivo, similar to that for p21. These positive effects of p18 dominate negative effects of p27 gene deletion. Moreover, the CDKIs differentially regulate responsiveness of granulocyte macrophage (GM) progenitors to synergistic cell proliferation in response to GM-CSF plus SCF, which is considered important for normal hematopoiesis. Responsiveness of HPCs to inhibition by myelosuppressive chemokines is directly related to the capacity of HPCs to respond to synergistic stimulation, and their cell cycle status. P18(INK4c) gene deletion rescued the loss of chemokine suppression of synergistic proliferation due to deletion of p21(CIP1/WAF1). These findings underscore the complex interplay of cell cycle regulators in HPC, and demonstrate that loss of one can sometimes be compensated by loss of another CDKI in both, a pro- or anti-proliferative context.

In the present study, surface CD1d, which is involved in immune cell interactions, was assessed for effects on hematopoiesis. Mouse BM hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) express CD1d. The numbers and cycling status of HPCs in the BM and spleen of different strains of cd1d(-/-) mice were enhanced significantly, suggesting that CD1d is a negative regulator of HPCs. In support of this, CD1d was required for the SCF and Flt3 ligand synergistic enhancement of CSF induction of HPC colony formation and for HPC response to myelosuppressive chemokines. Colony formation by immature subsets of HPCs was greatly enhanced when normal, but not cd1d(-/-), BM cells were pretreated with CD1d Abs in vitro. These effects required the full CD1d cytoplasmic tail. In contrast, long-term, but not short-term, repopulating HSC engraftment was impaired significantly, an effect that was minimally influenced by the presence of a truncated CD1d cytoplasmic tail. Pretreatment of normal BM cells with CD1d Abs greatly enhanced their engraftment of HSCs. The results of the present study implicate CD1d in a previously unrecognized regulatory role of normal and stressed hematopoiesis.

DEK is a biochemically distinct protein that is generally found in the nucleus, where it is vital to global heterochromatin integrity. However, DEK is also secreted by cells (eg, macrophages) and influences other adjacent cells (eg, acts as a chemoattractant for certain mature blood cells). We hypothesized that DEK may modulate functions of hematopoietic stem (HSCs) and progenitor (HPCs) cells. C57Bl/6 mice were used to demonstrate that absolute numbers and cycling status of HPCs (colony forming unit-granulocyte macrophage [CFU-GM], burst forming unit-erythroid [BFU-E], and colony forming unit-granulocyte erythroid macrophage megakaryocyte [CFU-GEMM]) in bone marrow (BM) and spleen were significantly enhanced in DEK -/- as compared with wild-type (WT) control mice. Moreover, purified recombinant DEK protein inhibited colony formation in vitro by CFU-GM, BFU-E, and CFU-GEMM from WT BM cells and human cord blood (CB) cells in a dose-dependent fashion, demonstrating that DEK plays a negative role in HPC proliferation in vitro and in vivo. Suppression was direct acting as determined by inhibition of proliferation of single isolated CD34(+) CB cells in vitro. In contrast, DEK -/- BM cells significantly demonstrated reduced long term competitive and secondary mouse repopulating HSC capacity compared with WT BM cells, demonstrating that DEK positively regulates engrafting capability of self-renewing HSCs. This demonstrates that DEK has potent effects on HSCs, HPCs, and hematopoiesis, information of biological and potential clinical interest.

Several angiopoietin-like (ANGPTL) molecules have been implicated in enhancement of ex-vivo expansion of murine and human (hu) hematopoietic stem cells, but there are no reports on hematopoietic progenitor cells (HPCs). We assessed purified recombinant endotoxin-free hu ANGPTL-2 Coiled-Coil (CC), -3, -3CC, -3 fibrinogen-like domain (FLD), -4, -4CC, -5CC, -6 and -7 for effects on proliferation and survival of HPCs from hu cord blood (CB). None of the ANGPTL molecules stimulated CB HPC proliferation, or enhanced or inhibited colony formation of CB HPC stimulated by various growth factors. However, ANGPTL-2CC, -3, and -3CC significantly enhanced survival of HPC (CFU-GM, BFU-E, CFU-GEMM) subjected to delayed addition of growth factors. Survival enhancing effects of ANGPTL-3 were neutralized by purified anti-ANGPTL-3, but not by anti-ANGPTL-4, -6, or -7. ANGPTL-2CC, -3, and -3CC, but not -4, -6, or -7 also enhanced replating capacity of single CB CFU-GEMM colonies, an estimate of the self-renewal capabilities of HPCs, by greater than 2 fold. Effects of at least ANGPTL-3CC may in part be mediated through phosphorylation of ERK. The ANGPTL molecules did not influence ex-vivo expansion of hu CB CD34(+) cells, alone, or in combination with SCF, TPO, Flt3-ligand, with or without IL-3. Thus, among ANGPTL family members, ANGPTL-2 and -3 had enhancing activities on human HPC survival and replating activity, effects requiring the CC domain of the ANGPTL molecules. This information is of relevance to hu HPC regulation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Neurexin I α (NRXN1α) and Dystroglycan (DAG1) are membrane receptors which serve as mutual ligands in the neuronal system. Neurexophilins (NXPHs) bind NRXN1α. NRXN1α was expressed in primitive populations in human CB (huCB) and murine BM (muBM). DAG1 is ubiquitously expressed in hematopoietic tissue; however, osteoblasts appear to be sites of very high expression within muBM. High concentrations of NXPH were found in huCB plasma and murine lineage-positive splenocytes. We evaluated effects of these molecules on huCB and muBM hematopoietic progenitor cells (HPCs) and HSCs. At both a single and population cell level in vitro, we found that NXPH1 was a potent inhibitor of HPC proliferation acting through NRXN1α an effect down-modulated by DAG1. Injection of recombinant NXPH1 in vivo resulted in myelo- and lymphosuppression in the BM, with absolute numbers and cycling status of functional and phenotypically defined HPCs dose- and time-dependently decreased. Competitive HSC transplantations showed no change in the long-term repopulating activity of HSCs from mice exposed to recombinant NXPH1. These results demonstrate the presence and function of a regulated signaling axis in hematopoiesis centered on NRXN1α and its modulation by DAG1 and NXPH1.

The contribution of specific cell types to the production of cytokines that regulate hematopoiesis is still not well defined. We have previously identified T cell-dependent regulation of hematopoietic progenitor cell (HPC) numbers and cycling. In this report, we demonstrated that HPC activity is decreased in mice with STAT3-deficient T cells, a phenotype that is not because of decreased expression of IL-17 or RORγt. STAT3 expression in T cells was required for IL-21 production by multiple T helper subsets, and neutralization of IL-21 resulted in decreased HPC activity identical to that in mice with STAT3-deficient T cells. Importantly, injection of IL-21 rescued HPC activity in mice with STAT3-deficient T cells. Thus, STAT3-dependent IL-21 production in T cells is required for HPC homeostasis.

Intracellular factors are involved in and essential for hematopoiesis. HIV-1 Tat-interacting protein of 110 kDa (TIP110; p110(nrb)/SART3/p110) is an RNA-binding nuclear protein implicated in the regulation of HIV-1 gene and host gene transcription, pre-mRNA splicing, and cancer immunology. In the present study, we demonstrate a role for TIP110 in the regulation of hematopoiesis. TIP110 was expressed in human CD34(+) cells and decreased with differentiation of CD34(+) cells. TIP110 mRNA was also expressed in phenotyped mouse marrow hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs). Using TIP110 transgenic (TIP110(TG)) and haploinsufficient (TIP110(+/-)) mice, we found that increased TIP110 expression enhanced HPC numbers, survival, and cell cycling, whereas decreased TIP110 expression had the opposite effects. Moreover, TIP110(+/-) bone marrow HPCs responded more effectively, and TIP110(TG) HPCs less effectively, than those of wild-type control mice to recovery from the cell-cycle-active drug 5-fluorouracil (5-FU). Unexplained sex differences were noted in HSC competitive repopulating ability, but not HPC numbers, in TIP110(TG) mice. Intracellularly, TIP110 regulated CMYC and GATA2 expression at the transcriptional level, and TIP110 and CMYC reciprocally regulated the expression of each other. These results demonstrate a role for TIP110 in the regulation of hematopoiesis, effects that are likely linked to TIP110 regulation of CMYC.