ABSTRACT
Macrophages respond to their environment by adopting a predominantly inflammatory or anti-inflammatory profile, depending on the context. The polarization of the subsequent response is regulated by a combination of intrinsic and extrinsic signals and is associated with alterations in macrophage metabolism. Although macrophages are important producers of Wnt ligands, the role of Wnt signaling in regulating metabolic changes associated with macrophage polarization remains unclear. Wnt4 upregulation has been shown to be associated with tissue repair and suppression of age-associated inflammation, which led us to generate Wnt4-deficient bone marrow-derived macrophages to investigate its role in metabolism. We show that loss of Wnt4 led to modified mitochondrial structure, enhanced oxidative phosphorylation, and depleted intracellular lipid reserves, as the cells depended on fatty acid oxidation to fuel their mitochondria. Further we found that enhanced lipolysis was dependent on protein kinase C-mediated activation of lysosomal acid lipase in Wnt4-deficient bone marrow-derived macrophages. Although not irreversible, these metabolic changes promoted parasite survival during infection with Leishmania donovani. In conclusion, our results indicate that enhanced macrophage fatty acid oxidation impairs the control of intracellular pathogens, such as Leishmania. We further suggest that Wnt4 may represent a potential target in atherosclerosis, which is characterized by lipid storage in macrophages leading to them becoming foam cells.
Subject(s)
Atherosclerosis , Oxidative Phosphorylation , Atherosclerosis/metabolism , Fatty Acids/metabolism , Humans , Ligands , Lipids , Macrophages/metabolism , Mitochondria/metabolism , Wnt4 Protein/metabolismABSTRACT
Although intracellular Wnt signaling pathways need to be tightly regulated to promote hematopoietic stem cell self-renewal, the source and identity of important Wnt ligands in the bone marrow is still largely unknown. The noncanonical ligand Wnt4 is expressed in the bone marrow as well as in the stroma, and its overexpression in fetal liver cells facilitates thymic recovery; however, its impact on adult hematopoietic stem cell function remains unclear. Here, we report that the deletion of Wnt4 from hematopoietic cells in mice (Wnt4Δ/Δ ) resulted in decreased lymphopoiesis at steady state. This was likely at least in part due to the increased proinflammatory environment present in the bone marrow of Wnt4Δ/Δ mice. Wnt4Δ/Δ hematopoietic stem cells displayed reduced reconstitution capacity in serial transplants, thus demonstrating defective self-renewal, and they expanded poorly in response to lipopolysaccharide stimulation. This appeared to be the result of the absence of Wnt4 in stem/progenitor cells, as myeloid-restricted Wnt4 deletion had no notable effect. Finally, we observed that Wnt4Δ/Δ stem/progenitor cells were more quiescent, presenting enhanced levels of stress-associated JNK phosphorylation and p16INK4a expression, likely contributing to the reduced expansion observed in transplants. In conclusion, our results identify a new, largely autocrine role for Wnt4 in hematopoietic stem cell self-renewal, suggesting that regulation of Wnt signaling in hematopoiesis may not need Wnt secretion and could be independent of morphogen gradients.
Subject(s)
Hematopoiesis , Hematopoietic Stem Cell Transplantation , Animals , Cell Differentiation , Cell Self Renewal , Hematopoietic Stem Cells/metabolism , Lymphopoiesis , Mice , Wnt4 Protein/genetics , Wnt4 Protein/metabolismABSTRACT
Cells of the immune system are derived from hematopoietic stem cells (HSCs) residing in the bone marrow. HSCs become activated in response to stress, such as acute infections, which adapt the bone marrow output to the needs of the immune response. However, the impact of infection-adapted HSC activation and differentiation on the persistence of chronic infections is poorly understood. We have examined here the bone marrow outcome of chronic visceral leishmaniasis and show that the parasite Leishmania donovani induces HSC expansion and skews their differentiation towards non-classical myeloid progenitors with a regulatory phenotype. Our results further suggest that emergency hematopoiesis contributes to the pathogenesis of visceral leishmaniasis, as decreased HSC expansion results in a lower parasite burden. Conversely, monocytes derived in the presence of soluble factors from the infected bone marrow environment are more permissive to infection by Leishmania. Our results demonstrate that L. donovani is able to subvert host bone marrow emergency responses to facilitate parasite persistence, and put forward hematopoiesis as a novel therapeutic target in chronic infections.
Subject(s)
Cell Differentiation/immunology , Hematopoietic Stem Cells/immunology , Leishmaniasis, Visceral/immunology , Animals , Disease Models, Animal , Flow Cytometry , Hematopoiesis/immunology , Hematopoietic Stem Cells/parasitology , Mice , Mice, Inbred C57BLABSTRACT
Leishmania donovani is known to induce myelopoiesis and to dramatically increase extramedullary myelopoiesis. This results in splenomegaly, which is then accompanied by disruption of the splenic microarchitecture, a chronic inflammatory environment, and immunosuppression. Chronically inflamed tissues are typically hypoxic. The role of hypoxia on myeloid cell functions during visceral leishmaniasis has not yet been studied. Here we show that L. donovani promotes the output from the bone marrow of monocytes with a regulatory phenotype that function as safe targets for the parasite. We also demonstrate that splenic myeloid cells acquire MDSC-like function in a HIF-1α-dependent manner. HIF-1α is also involved in driving the polarization towards M2-like macrophages and rendering intermediate stage monocytes more susceptible to L. donovani infection. Our results suggest that HIF-1α is a major player in the establishment of chronic Leishmania infection and is crucial for enhancing immunosuppressive functions and lowering leishmanicidal capacity of myeloid cells.
Subject(s)
Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Leishmaniasis, Visceral , Macrophages/metabolism , Myeloid Cells/metabolism , Animals , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Immune Tolerance/drug effects , Interferon-gamma/pharmacology , Macrophages/parasitology , Mice , Monocytes/metabolism , Myeloid Cells/parasitology , Spleen/parasitologyABSTRACT
Despite studies based on deletion or activation of intracellular components of the canonical Wingless related (Wnt) pathway, the role of Wnts in hematolymphopoiesis remains controversial. Using gain-of-function and loss-of-function models, we found that Wnt4 differentially affected diverse subsets of hematopoietic stem and progenitor cells. Bone-marrow and thymic Lin(-)Sca1(+)Kit(hi) cells (LSKs) were the key targets of Wnt4. In adult mice, Wnt4-induced expansion of Flt3(+) bone-marrow LSKs (lymphoid-primed multipotent progenitors) led to a sizeable accumulation of the most immature thymocyte subsets (upstream of beta-selection) and a major increase in thymopoiesis. Conversely, Wnt4(-/-) neonates showed low frequencies of bone-marrow LSKs and thymic hypocellularity. We provide compelling evidence that Wnt4 activates noncanonical (beta-catenin-independent) signaling and that its effects on hematopoietic cells are mainly non-cell-autonomous. Our work shows that Wnt4 overexpression has a unique ability to expand Flt3(+) LSKs in adults and demonstrates that noncanonical Wnt signaling regulates thymopoiesis.
Subject(s)
Hematopoietic Stem Cells/cytology , Multipotent Stem Cells/cytology , Signal Transduction/immunology , Thymus Gland/growth & development , Wnt Proteins/metabolism , beta Catenin/metabolism , Animals , Cell Differentiation/immunology , Flow Cytometry , Hematopoietic Stem Cells/immunology , Hematopoietic Stem Cells/metabolism , Immunoblotting , Mice , Multipotent Stem Cells/immunology , Multipotent Stem Cells/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Thymus Gland/cytology , Thymus Gland/immunology , Wnt Proteins/immunology , Wnt4 Protein , beta Catenin/immunologyABSTRACT
Adult hematopoietic stem/progenitor cell (HSPC) numbers remain stable in the absence of external stressors. After bone marrow (BM) transplant, HSPCs need to expand substantially to repopulate the BM and replenish the peripheral blood cell pool. In this study, we show that a noncanonical Wnt receptor, Frizzled-6 (Fzd6), regulates HSPC expansion and survival in a hematopoietic cell-intrinsic manner. Fzd6 deficiency increased the ratio of Flt3(hi) multipotent progenitors to CD150(+) stem cells in the mouse BM, suggesting defective stem cell maintenance. Competitive transplantation experiments demonstrated that Fzd6(-) (/) (-) HSPCs were able to home to the BM but were severely impaired in their capacity to reconstitute a lethally irradiated host. Lack of Fzd6 resulted in a strong activation of caspase-3 and a gradual loss of donor HSPCs and peripheral blood granulocytes. Fzd6 was also necessary for the efficient HSPC expansion during emergency hematopoiesis. Mechanistically, Fzd6 is a negative regulator of Cdc42 clustering in polarized cells. Furthermore, ß-catenin-dependent signaling may be disinhibited in Fzd6(-) (/) (-) HSPCs. Collectively, our data reveal that Fzd6 has an essential role in HSPC maintenance and survival. Noncanonical Wnt-Fzd6 signaling pathway could thus present an interesting target for promoting HSPC expansion and multilineage hematopoietic recovery after transplant.
Subject(s)
Cell Proliferation , Frizzled Receptors/immunology , Hematopoiesis/immunology , Hematopoietic Stem Cells/immunology , Animals , Bone Marrow Cells/immunology , Bone Marrow Cells/metabolism , Bone Marrow Transplantation/methods , Cell Lineage/genetics , Cell Lineage/immunology , Cell Survival/genetics , Cell Survival/immunology , Cells, Cultured , Flow Cytometry , Frizzled Receptors/genetics , Frizzled Receptors/metabolism , Hematopoiesis/genetics , Hematopoietic Stem Cells/metabolism , JNK Mitogen-Activated Protein Kinases/immunology , JNK Mitogen-Activated Protein Kinases/metabolism , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Signal Transduction/genetics , Signal Transduction/immunology , Time Factors , beta Catenin/immunology , beta Catenin/metabolism , cdc42 GTP-Binding Protein/immunology , cdc42 GTP-Binding Protein/metabolismABSTRACT
Wnt4 is a key signal that channels the developmental fate of the indifferent mammalian gonad toward the ovary, but whether Wnt4 has later roles during ovary development remains unknown. To investigate this, we inactivated the Wnt4 gene by crossing Amhr2Cre and doxycycline-inducible Rosa(rtTA)-knock-in Cre mice with mice carrying a floxed Wnt4 allele and used a novel Wnt4(mCherry)-knock-in mouse. In these models, ovarian folliculogenesis was compromised, and female fertility was severely reduced, and Wnt4 deficiency eventually led to premature ovarian failure. These anomalies were associated with cell polarity defects in the follicle. Within the follicle, laminin and type IV collagen assembled ectopic basement membrane-like structures, the cell adherens junction components N-cadherin and ß-catenin lost their polarized expression pattern, and expression of the gap junction protein connexin 43 was reduced by ~30% when compared with that of the controls. Besides these changes, expression of antimüllerian hormone (Amh) was inhibited in the absence of Wnt4 signaling in vivo. Consistent with this, Wnt4 signaling up-regulated Amh gene expression in KK1 cells in vitro. Thus, Wnt4 signaling is necessary during maturation of the ovarian follicles, where it coordinates expression of Amh, cell survival, and polarized organization of the follicular cells.
Subject(s)
Anti-Mullerian Hormone/genetics , Basement Membrane/metabolism , Cell Polarity/genetics , Oocytes/metabolism , Ovarian Follicle/metabolism , Wnt4 Protein/genetics , Animals , Animals, Newborn , Anti-Mullerian Hormone/metabolism , Cells, Cultured , Female , Gene Expression Regulation, Developmental , Granulosa Cells/metabolism , Granulosa Cells/ultrastructure , Immunohistochemistry , In Situ Hybridization , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Microscopy, Electron, Transmission , Oocytes/growth & development , Oocytes/ultrastructure , Ovarian Follicle/cytology , Ovarian Follicle/growth & development , Reverse Transcriptase Polymerase Chain Reaction , Wnt Signaling Pathway/genetics , Wnt4 Protein/metabolismABSTRACT
Thymus atrophy is the most common immunopathology in humans, and its occurrence is hastened by several factors that coalesce in patients receiving chemotherapy and most of all in recipients of hematopoietic cell transplantation. We have shown previously that posthematopoietic cell transplantation thymic function was improved by retroviral overexpression of Wnt4 in donor hematopoietic cells. Here, by using both conventional and conditional null mutant mice, we show that Wnt4 regulates steady-state thymic cellularity by a thymic epithelial cell (TEC)-dependent mechanism. The absence of Wnt4 suppressed fetal and postnatal thymic expansion and resulted in decreased TEC numbers, an alteration of the medullary-to-cortical TEC ratio, and a disproportionate loss of the most immature cKit(hi) thymocyte precursors. Wnt4 also is implicated in the maintenance of adult thymopoiesis, although the impact of its deletion once thymic involution has been initiated is more subtle. Together, our results show that Wnt4 controls thymic size by modulating TEC expansion and the earliest, TEC-dependent steps of thymocyte development both in the fetal and postnatal thymus. Wnt4 and its downstream signaling pathways could thus represent interesting candidates to improve thymic output in subjects with thymic atrophy.
Subject(s)
Lymphopoiesis/physiology , Thymus Gland/cytology , Thymus Gland/physiology , Wnt4 Protein/physiology , Animals , Embryonic Stem Cells/cytology , Epithelial Cells/cytology , Female , Hematopoietic Stem Cells/cytology , Humans , Lymphopoiesis/genetics , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Models, Biological , Pregnancy , Thymus Gland/embryology , Wnt4 Protein/deficiency , Wnt4 Protein/geneticsABSTRACT
Gene expression profiling of human donor T cells before allogeneic hematopoietic cell transplantation revealed that expression of selected genes correlated with the occurrence of graft-versus-host disease (GVHD) in recipients. The gene with the best GVHD predictive accuracy was SMAD3, a core component of the transforming growth factor-ß signaling pathway, whose expression levels vary more than a 6-fold range in humans. The putative role of SMAD3 in the establishment of graft-host tolerance remained elusive. We report that SMAD3-KO mice present ostensibly normal lymphoid and myeloid cell subsets. However, the lack of SMAD3 dramatically increased the frequency and severity of GVHD after allogeneic hematopoietic cell transplantation into major histocompatibility complex-identical recipients. Lethal GVHD induced by SMAD3-KO donors affected mainly the intestine and resulted from massive tissue infiltration by T-bet(+) CD4 T cells and granulocytes that caused tissue damage by in situ release of Th1 cytokines and oxidative-nitrosative mediators, respectively. Our report reveals the nonredundant roles of SMAD3 in the development of tolerance to the host. Furthermore, our data support the concept that SMAD3 levels in donor cells dictate the risk of GVHD and that SMAD3 agonists would be attractive for prevention of GVHD.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , Cell Differentiation , Colon/pathology , Graft vs Host Disease/prevention & control , Granulocytes/metabolism , Smad3 Protein/physiology , Th1 Cells/cytology , Animals , Blotting, Western , Bone Marrow Transplantation , Cell Proliferation , Cells, Cultured , Colon/immunology , Colon/metabolism , Graft vs Host Disease/immunology , Granulocytes/cytology , Hematopoiesis , Humans , Lymphocyte Activation , Mice , Mice, Inbred BALB C , Mice, Knockout , Neutrophils/metabolism , Th1 Cells/immunology , Th1 Cells/metabolism , Transplantation, HomologousABSTRACT
The control of tyrosine phosphorylation depends on the fine balance between kinase and phosphatase activities. Protein tyrosine phosphatase 1B (PTP-1B) and T cell protein tyrosine phosphatase (TC-PTP) are 2 closely related phosphatases known to control cytokine signaling. We studied the functional redundancy of PTP-1B and TC-PTP by deleting 1 or both copies of these genes by interbreeding TC-PTP and PTP-1B parental lines. Our results indicate that the double mutant (tcptp(-/-)ptp1b(-/-)) is lethal at day E9.5-10.5 of embryonic development with constitutive phosphorylation of Stat1. Mice heterozygous for TC-PTP on a PTP-1B-deficient background (tcptp(+/-)ptp1b(-/-)) developed signs of inflammation. Macrophages from these animals were highly sensitive to IFN-gamma, as demonstrated by increased Stat1 phosphorylation and nitric oxide production. In addition, splenic T cells demonstrated increased IFN-gamma secretion capacity. Mice with deletions of single copies of TC-PTP and PTP-1B (tcptp(+/-)ptp1b(+/-)) exhibited normal development, confirming that these genes are not interchangeable. Together, these data indicate a nonredundant role for PTP-1B and TC-PTP in the regulation of IFN signaling.
Subject(s)
Interferon-gamma/metabolism , Macrophages/cytology , Protein Tyrosine Phosphatase, Non-Receptor Type 1/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 2/metabolism , Signal Transduction , Animals , Embryo, Mammalian/immunology , Macrophages/metabolism , Mice , Protein Tyrosine Phosphatase, Non-Receptor Type 1/genetics , Protein Tyrosine Phosphatase, Non-Receptor Type 2/genetics , STAT1 Transcription Factor/metabolismABSTRACT
Emergency hematopoiesis involves the activation of bone marrow hematopoietic stem/progenitor cells (HSPCs) in response to systemic inflammation by a combination of cell-autonomous and stroma-dependent signals and leads to their release from bone marrow and migration to periphery. We have previously shown that FZD6 plays a pivotal role in regulating HSPC expansion and long-term maintenance. Now we sought to better understand the underlying mechanisms. Using lipopolysaccharide (LPS)-induced emergency granulopoiesis as a model, we show that failed expansion was intrinsic to FZD6-deficient HSPCs but also required a FZD6-deficient environment. FZD6-deficient HSPCs became more strongly activated, but their mobilization to peripheral blood was impaired and they were more susceptible to inflammatory cell death, leading to enhanced release of pro-inflammatory cytokines in the marrow. These studies indicate that FZD6 has a protective effect in the bone marrow to prevent an overactive inflammatory response and further suggest that mobilization improves HSPC survival during bone marrow inflammation.
Subject(s)
Lipopolysaccharides , Myelopoiesis , Cytokines/metabolism , Hematopoietic Stem Cells/metabolism , Humans , Inflammation/metabolismABSTRACT
Hematopoietic stem/progenitor cells (HSPC) are responsible for the generation of most immune cells throughout the lifespan of the organism. Inflammation can activate bone marrow HSPCs, leading to enhanced myelopoiesis to replace cells, such as neutrophils, which are attracted to inflamed tissues. We have previously shown that HSPC activation promotes parasite persistence and expansion in experimental visceral leishmaniasis through the increased production of permissive monocytes. However, it is not clear if the presence of the parasite in the bone marrow was required for infection-adapted myelopoiesis. We therefore hypothesized that persistent forms of Leishmania major (cutaneous leishmaniasis) could also activate HSPCs and myeloid precursors in the C57Bl/6 mouse model of intradermal infection in the ear. The accrued influx of myeloid cells to the lesion site corresponded to an increase in myeloid-biased HSPCs in the bone marrow and spleen in mice infected with a persistent strain of L. major, together with an increase in monocytes and monocyte-derived myeloid cells in the spleen. Analysis of the bone marrow cytokine and chemokine environment revealed an attenuated type I and type II interferon response in the mice infected with the persistent strain compared to the self-healing strain, while both strains induced a rapid upregulation of myelopoietic cytokines, such as IL-1ß and GM-CSF. These results demonstrate that an active infection in the bone marrow is not necessary for the induction of infection-adapted myelopoiesis, and underline the importance of considering alterations to the bone marrow output when analyzing in vivo host-pathogen interactions.
ABSTRACT
Regulation of hematopoietic stem cell (HSC) self-renewal and differentiation is essential for their maintenance, and HSC polarity has been shown to play an important role in this regulation. Vangl2, a key component of the Wnt/polarity pathway, is expressed by fetal and adult HSCs, but its role in hematopoiesis and HSC function is unknown. Here we show the deletion of Vangl2 in mouse hematopoietic cells impairs HSC expansion and hematopoietic recovery post-transplant. Old Vangl2-deficient mice showed increased expansion of myeloid-biased multipotent progenitor cells concomitant with splenomegaly. Moreover, Vangl2-deficient cells were not able to effectively reconstitute the recipient bone marrow in serial transplants, or when coming from slightly older donors, demonstrating impaired self-renewal or expansion. Aged Vangl2-deficient HSCs displayed increased levels of cell cycle inhibitor p16INK4a and active ß-catenin, which could contribute to their impaired function. Overall, our findings identify Vangl2 as a new regulator of hematopoiesis.
ABSTRACT
Hematopoietic stem cells have the ability to produce all blood cells. When hematological malignancies occur, transplant of compatible blood or bone marrow cells from a healthy donor to the patient is an efficient solution to restore normal hematopoiesis. Bone marrow transplant in a mouse model is often used to study HSC function and capacity to repopulate an irradiated recipient. This protocol details the different steps of a competitive bone marrow transplant experiment, beginning with total body irradiation of the recipient mice; preparation and administration of the donor and competitor bone marrow samples; peripheral blood analysis to follow reconstitution posttransplant; and finally, the analysis of recipient bone marrow and secondary transplants to evaluate long-term HSC function. Different formulas used to establish transplant efficiency are explained. All the steps are discussed in detail, including tips, variations, and alternative procedures with their advantages and disadvantages.
Subject(s)
Bone Marrow Transplantation , Flow Cytometry , Transplantation Chimera/blood , Transplantation Conditioning , Whole-Body Irradiation , Animals , MiceABSTRACT
Although vertebrate hematopoiesis is known to occur in a variety of locations, the differentiation of T lymphocytes takes place only in the thymus. Conversely, the only known function of the thymus is to produce T lymphocytes. As thymic function progressively diminishes with age in all individuals, and decreased thymic capacity has been linked to an impaired resistance to infections and cancer, increasing thymic function and/or extrathymic T-cell development could thus play an important role in health management in elderly subjects. However, it is currently unclear why the thymus is essential for T-cell development and precisely what is necessary to make a functional T lymphopoietic organ. In this review, we shall discuss the current literature on the similarities and differences between thymic and extrathymic T cells. A major conclusion is that innate T cells can be generated extrathymically, but only the thymus can support the development of classic adaptive T cells. Functional differences between thymic and extrathymic T cells provide a rationale for conservation of the thymus as the sole primary T lymphoid organ. These differences also have implications for the management of thymus involution.
Subject(s)
Lymphopoiesis , T-Lymphocytes/physiology , Thymus Gland/cytology , Animals , Bone Marrow Cells/physiology , Cell Differentiation , Cell Lineage , Hematopoietic Stem Cells/physiology , Humans , Lymphocyte Activation , Stromal Cells/physiologyABSTRACT
The switch from fetal to adult hematopoietic stem/progenitor cells (HSPCs) is associated with profound changes in several genetic programs. Although HSPC ageing corresponds to alterations in Wnt signaling, relatively little is known about the relative roles of different Wnt signaling pathways in HSPC ontogeny. We hypothesized that proliferating fetal HSPCs would be more dependent on canonical ß-catenin-dependent Wnt signaling when compared to quiescent adult bone marrow HSPCs. We have compared here Wnt signaling activities in murine fetal and adult HSPCs and demonstrate a shift from Wnt/ß-catenin-dependent signaling in fetal liver HSPCs to more predominantly noncanonical Wnt/polarity signaling in adult HSPCs. ß-Catenin was selectively required for fetal HSPC competitiveness shortly after transplant, and protected cells from oxidative stress. Our results emphasize the complexity of Wnt signaling dynamics in HSPC maintenance and function.
Subject(s)
Fetal Tissue Transplantation , Fetus/cytology , Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells/cytology , Wnt Signaling Pathway , beta Catenin/metabolism , Animals , Cell Differentiation , Cell Proliferation , Cells, Cultured , Female , Hematopoiesis , Male , Mice , Mice, Inbred C57BLABSTRACT
Inflammation, although responsible for controlling infection, is often associated with the pathogenesis of chronic diseases. Leishmania donovani, the causative agent of visceral leishmaniasis, induces a strong inflammatory response that leads to splenomegaly and ultimately immune suppression. Inflamed tissues are typically characterized by low levels of oxygen, a microenvironment that triggers the hypoxia-inducible transcription factor 1α (HIF-1α). Although HIF-1α plays an integral role in dendritic cell function, its involvement in the generation of protective Th1 responses against Leishmania has not yet been studied. Here we demonstrate that HIF-1α inhibits IL-12 production in dendritic cells, limiting therefore Th1 cell development. Indeed, depletion of HIF-1α in CD11c+ cells resulted in higher and sustained expression of IL-12 and complete abrogation of IL-10. Moreover, CD11c-specific HIF-1α-deficient mice showed higher frequencies of IFN-γ-producing CD4 T cells in the spleen and bone marrow and, consequently, a significantly reduced parasite burden in both organs. Taken together, our results suggest that HIF-1α expression in dendritic cells largely contributes to the establishment of persistent Leishmania infection and may therefore represent a possible therapeutic target.
Subject(s)
Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Inflammation/genetics , Leishmania donovani/genetics , Leishmaniasis, Visceral/genetics , Animals , CD11 Antigens/genetics , CD11 Antigens/immunology , CD4-Positive T-Lymphocytes/immunology , Cellular Microenvironment/genetics , Cellular Microenvironment/immunology , Dendritic Cells/metabolism , Dendritic Cells/parasitology , Humans , Inflammation/pathology , Interferon-gamma/genetics , Interferon-gamma/immunology , Interleukin-10/genetics , Interleukin-10/immunology , Interleukin-12/genetics , Interleukin-12/immunology , Leishmania donovani/immunology , Leishmania donovani/pathogenicity , Leishmaniasis, Visceral/immunology , Leishmaniasis, Visceral/parasitology , Leishmaniasis, Visceral/pathology , Mice , Mice, Inbred C57BL , Spleen/immunology , Spleen/parasitology , Splenomegaly/genetics , Splenomegaly/immunology , Splenomegaly/parasitology , Splenomegaly/pathology , Th1 Cells/immunologyABSTRACT
Protein tyrosine phosphatase 1B (PTP1B) is involved in multiple signaling pathways by down-regulating several tyrosine kinases. For example, gene-targeting studies in mice have established PTP1B as a critical physiologic regulator of metabolism by attenuating insulin signaling. PTP1B is an important target for the treatment of diabetes, because the PTP1B null mice are resistant to diet-induced diabetes and obesity. On the other hand, despite the potential for enhanced oncogenic signaling in the absence of PTP1B, PTP1B null mice do not develop spontaneous tumors. Because the majority of human cancers harbor mutations in p53, we generated p53/PTP1B double null mice to elucidate the role of PTP1B in tumorigenesis. We show that genetic ablation of PTP1B in p53 null mice decreases survival rate and increases susceptibility towards the development of B lymphomas. This suggested a role for PTP1B in lymphopoiesis, and we report that PTP1B null mice have an accumulation of B cells in bone marrow and lymph nodes, which contributed to the increased incidence of B lymphomas. The mean time of tumor development and tumor spectrum are unchanged in p53-/-PTP1B+/- mice. We conclude that PTP1B is an important determinant of the latency and type of tumors in a p53-deficient background through its role in the regulation of B-cell development.
Subject(s)
B-Lymphocytes/immunology , Lymphoma, B-Cell/genetics , Protein Tyrosine Phosphatases/deficiency , Tumor Suppressor Protein p53/deficiency , Alleles , Animals , B-Lymphocytes/enzymology , Bone Marrow Cells/immunology , Bone Marrow Cells/pathology , Genetic Predisposition to Disease , Hematopoietic Stem Cells/enzymology , Hematopoietic Stem Cells/immunology , Lymph Nodes/immunology , Lymph Nodes/pathology , Lymphoma, B-Cell/enzymology , Lymphoma, B-Cell/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Protein Tyrosine Phosphatase, Non-Receptor Type 1 , Protein Tyrosine Phosphatases/genetics , Protein Tyrosine Phosphatases/immunology , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/immunologyABSTRACT
The gold standard definition of a hematopoietic stem cell (HSC) is a cell that when transferred into an irradiated recipient will have the ability to reestablish blood cell production for the lifespan of the recipient. This protocol explains how to set up a functional assay to compare the HSC capacities of two different populations of cells, such as bone marrow from mice of two different genotypes, and how to analyze the recipient mice by flow cytometry. The protocol uses HSC equivalents rather than cell sorting for standardization and discusses the advantages and disadvantages of both approaches. We further discuss different variations to the basic protocol, including serial transplants, limiting dilution assays, homing assays and non-competitive transplants, including the advantages and preferred uses of these varied approaches. These assays are central for the study of HSC function and could be used not only for the investigation of fundamental HSC intrinsic aspects of biology but also for the development of preclinical assays for bone marrow transplant and HSC expansion in culture.
ABSTRACT
BACKGROUND: While the role of canonical (ß-catenin-mediated) Wnt signaling in hematolymphopoiesis has been studied extensively, little is known of the potential importance of non-canonical Wnt signals in hematopoietic cells. Wnt4 is one of the Wnt proteins that can elicit non-canonical pathways. We have previously shown that retroviral overexpression of Wnt4 by hematopoietic cells increased thymic cellularity as well as the frequency of early thymic progenitors and bone marrow hematopoietic progenitor cells (HPCs). However, the molecular pathways responsible for its effect in HPCs are not known. METHODOLOGY/PRINCIPAL FINDINGS: Here we report that Wnt4 stimulation resulted in the activation of the small GTPase Rac1 as well as Jnk kinases in an HPC cell line. Jnk activity was necessary, while ß-catenin was dispensable, for the Wnt4-mediated expansion of primary fetal liver HPCs in culture. Furthermore, Jnk2-deficient and Wnt4 hemizygous mice presented lower numbers of HPCs in their bone marrow, and Jnk2-deficient HPCs showed increased rates of apoptosis. Wnt4 also improved HPC activity in a competitive reconstitution model in a cell-autonomous, Jnk2-dependent manner. Lastly, we identified Fz6 as a receptor for Wnt4 in immature HPCs and showed that the absence of Wnt4 led to a decreased expression of four polarity complex genes. CONCLUSIONS/SIGNIFICANCE: Our results establish a functional role for non-canonical Wnt signaling in hematopoiesis through a pathway involving Wnt4, Fz6, Rac1 and Jnk kinases.