ABSTRACT
In recent years, general hypoxia-inducible factor (HIF)-prolyl hydroxylase (PHD) enzyme inhibitors have been developed for the treatment of anemia due to renal disease and osteoporosis. However, it remains a challenge to target the HIF signaling pathway without dysregulating the skeletal and hematopoietic system. Here, we examined the effects of Vhl deletion in bone by performing longitudinal analyses of Vhl cKO mice at 3, 6, 10, and 24 weeks of age, where at 10 and 24 weeks of age, high bone mass and splenomegaly are present. Using flow cytometry, we observed increased frequency (%) of CD71 lo TER119 hi FSC lo orthochromatophilic erythroblasts and reticulocytes in 10- and 24-week-old Vhl cKO bone marrow (BM), which correlated with elevated erythropoietin levels in the BM and increased number of red blood cells in circulation. The absolute numbers of myeloerythroid progenitors (MEPs) in the BM were significantly reduced at 24 weeks. Bulk RNA-Seq of the MEPs showed upregulation of Epas1 ( Hif1a) and Efnb2 ( Hif2a) in Vhl cKO MEPs, consistent with a response to hypoxia, and genes involved in erythrocyte development, actin filament organization, and response to glucose. Additionally, histological analysis of Vhl cKO spleens revealed red pulp hyperplasia and the presence of megakaryocytes, both of which are features of extramedullary hematopoiesis (EMH). EMH in the spleen was correlated with the presence of mature stress erythroid progenitors, suggesting that stress erythropoiesis is occurring to compensate for the BM microenvironmental irregularities. Our studies implicate that HIF-driven alterations in skeletal homeostasis can accelerate erythropoiesis. Key Points: ⢠Dysregulation of HIF signaling in Dmp1+ bone cells induces stress erythropoiesis.⢠Skeletal homeostasis modulates erythropoiesis.
ABSTRACT
The contributions of skeletal cells to the processes of B cell development in the bone marrow (BM) have not been completely described. The von-Hippel Lindau protein (VHL) plays a key role in cellular responses to hypoxia. Previous work showed that Dmp1-Cre;Vhl conditional knockout mice (VhlcKO), which deletes Vhl in subsets of mesenchymal stem cells, late osteoblasts and osteocytes, display dysregulated bone growth and reduction in B cells. Here, we investigated the mechanisms underlying the B cell defects using flow cytometry and high-resolution imaging. In the VhlcKO BM, B cell progenitors were increased in frequency and number, whereas Hardy Fractions B-F were decreased. VhlcKO Fractions B-C cells showed increased apoptosis and quiescence. Reciprocal BM chimeras confirmed a B cell-extrinsic source of the VhlcKO B cell defects. In support of this, VhlcKO BM supernatant contained reduced CXCL12 and elevated EPO levels. Intravital and ex vivo imaging revealed VhlcKO BM blood vessels with increased diameter, volume, and a diminished blood-BM barrier. Staining of VhlcKO B cells with an intracellular hypoxic marker indicated the natural existence of distinct B cell microenvironments that differ in local oxygen tensions and that the B cell developmental defects in VhlcKO BM are not initiated by hypoxia. Our studies identify novel mechanisms linking altered bone homeostasis with drastic BM microenvironmental changes that dysregulate B cell development.
Subject(s)
Lymphopoiesis , Mesenchymal Stem Cells , Animals , B-Lymphocytes , Bone Marrow , Extracellular Matrix Proteins , Hypoxia , Lymphopoiesis/genetics , Mice , Von Hippel-Lindau Tumor Suppressor ProteinABSTRACT
Romosozumab, a humanized monoclonal antibody specific for sclerostin (SOST), has been approved for treatment of postmenopausal women with osteoporosis at a high risk for fracture. Previous work in sclerostin global knockout (Sost-/-) mice indicated alterations in immune cell development in the bone marrow (BM), which could be a possible side effect in romosozumab-treated patients. Here, we examined the effects of short-term sclerostin depletion in the BM on hematopoiesis in young mice receiving sclerostin antibody (Scl-Ab) treatment for 6 weeks, and the effects of long-term Sost deficiency on wild-type (WT) long-term hematopoietic stem cells transplanted into older cohorts of Sost-/- mice. Our analyses revealed an increased frequency of granulocytes in the BM of Scl-Ab-treated mice and WTâSost-/- chimeras, indicating myeloid-biased differentiation in Sost-deficient BM microenvironments. This myeloid bias extended to extramedullary hematopoiesis in the spleen and was correlated with an increase in inflammatory cytokines TNFα, IL-1α, and MCP-1 in Sost-/- BM serum. Additionally, we observed alterations in erythrocyte differentiation in the BM and spleen of Sost-/- mice. Taken together, our current study indicates novel roles for Sost in the regulation of myelopoiesis and control of inflammation in the BM.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Bone Marrow/pathology , Inflammation/etiology , Adaptor Proteins, Signal Transducing/genetics , Animals , Antibodies, Monoclonal , Bone Marrow/physiology , Cytokines , Female , Gene Knockout Techniques , Hematopoietic Stem Cells , Inflammation/chemically induced , Male , Mice , Mice, Knockout , MyelopoiesisABSTRACT
Previous studies of NK cell inhibitory Ly-49 genes showed their expression is stochastic. However, relatively few studies have examined the mechanisms governing acquisition of inhibitory receptors in conjunction with activating Ly-49 receptors and NK cell development. We hypothesized that the surface expression of activating Ly-49 receptors is nonrandom and is influenced by inhibitory Ly-49 receptors. We analyzed NK cell "clusters" defined by combinatorial expression of activating (Ly-49H and Ly-49D) and inhibitory (Ly-49I and Ly-49G2) receptors in C57BL/6 mice. Using the product rule to evaluate the interdependencies of the Ly-49 receptors, we found evidence for a tightly regulated expression at the immature NK cell stage, with the highest interdependencies between clusters that express at least one activating receptor. Further analysis demonstrated that certain NK clusters predominated at the immature (CD27+CD11b-), transitional (CD27+CD11b+), and mature (CD27-CD11b-) NK cell stages. Using parallel in vitro culture and in vivo transplantation of sorted NK clusters, we discovered nonrandom expression of Ly-49 receptors, suggesting that prescribed pathways of NK cluster differentiation exist. Our data infer that surface expression of Ly-49I is an important step in NK cell maturation. Ki-67 expression and cell counts confirmed that immature NK cells proliferate more than mature NK cells. We found that MHC class I is particularly important for regulation of Ly-49D and Ly-49G2, even though no known MHC class I ligand for these receptors is present in B6 mice. Our data indicate that surface expression of both activating and inhibitory Ly-49 receptors on NK cell clusters occurs in a nonrandom process correlated to their maturation stage.
Subject(s)
Cell Differentiation/genetics , Killer Cells, Natural/immunology , NK Cell Lectin-Like Receptor Subfamily A/genetics , Adoptive Transfer , Animals , Cell Differentiation/immunology , Cell Proliferation/genetics , Female , Gene Expression Regulation/immunology , Killer Cells, Natural/metabolism , Killer Cells, Natural/transplantation , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , NK Cell Lectin-Like Receptor Subfamily A/metabolismABSTRACT
PURPOSE OF REVIEW: We reviewed recent progress on the role of sclerostin (SOST) and its effects on the immune system in order to summarize the current state of knowledge in osteoimmunology, in regard to hematopoiesis, lymphopoiesis, and inflammation. RECENT FINDINGS: Changes in sclerostin levels affect distinct niches within the bone marrow that support hematopoietic stem cells and B cell development. Sclerostin's regulation of adipogenesis could also be important for immune cell maintenance with age. Surprisingly, B cell development in the bone marrow is influenced by Sost produced by mesenchymal stem cells and osteoblasts, but not by osteocytes. Additionally, extramedullary hematopoiesis in the spleen and increased pro-inflammatory cytokine levels in the bone marrow are observed in global Sost-/- mice. In addition to changes in bone marrow density, sclerostin depletion affects B lymphopoiesis and myelopoiesis, as well as other changes within the bone marrow cavity that could affect hematopoiesis. It is therefore important to monitor for hematopoietic changes in patients receiving sclerostin-depleting therapies.
Subject(s)
Adaptor Proteins, Signal Transducing/immunology , Adipogenesis/immunology , Hematopoiesis, Extramedullary/immunology , Lymphopoiesis/immunology , Animals , B-Lymphocytes , Bone Marrow/immunology , Cytokines/immunology , Hematopoiesis/immunology , Hematopoietic Stem Cells , Humans , Mesenchymal Stem Cells/immunology , Mice , Mice, Knockout , Myelopoiesis/immunology , Osteoblasts/immunology , Osteocytes/immunologyABSTRACT
NK cells are innate-like lymphocytes that eliminate virally infected and cancerous cells, but the mechanisms that control NK cell development and cytotoxicity are incompletely understood. We identified roles for sclerostin domain-containing-1 (Sostdc1) in NK cell development and function. Sostdc1-knockout (Sostdc1 -/-) mice display a progressive accumulation of transitional NK cells (tNKs) (CD27+CD11b+) with age, indicating a partial developmental block. The NK cell Ly49 repertoire in Sostdc1 -/- mice is also changed. Lower frequencies of Sostdc1 -/- splenic tNKs express inhibitory Ly49G2 receptors, but higher frequencies express activating Ly49H and Ly49D receptors. However, the frequencies of Ly49I+, G2+, H+, and D+ populations were universally decreased at the most mature (CD27-CD11b+) stage. We hypothesized that the Ly49 repertoire in Sostdc1 -/- mice would correlate with NK killing ability and observed that Sostdc1-/- NK cells are hyporesponsive against MHC class I-deficient cell targets in vitro and in vivo, despite higher CD107a surface levels and similar IFN-γ expression to controls. Consistent with Sostdc1's known role in Wnt signaling regulation, Tcf7 and Lef1 levels were higher in Sostdc1 -/- NK cells. Expression of the NK development gene Id2 was decreased in Sostdc1-/- immature NK and tNK cells, but Eomes and Tbx21 expression was unaffected. Reciprocal bone marrow transplant experiments showed that Sostdc1 regulates NK cell maturation and expression of Ly49 receptors in a cell-extrinsic fashion from both nonhematopoietic and hematopoietic sources. Taken together, these data support a role for Sostdc1 in the regulation of NK cell maturation and cytotoxicity, and identify potential NK cell niches.
Subject(s)
Bone Morphogenetic Proteins/immunology , Immunity, Cellular , Killer Cells, Natural/immunology , Wnt Signaling Pathway/immunology , Adaptor Proteins, Signal Transducing , Animals , Bone Morphogenetic Proteins/genetics , Hepatocyte Nuclear Factor 1-alpha/genetics , Hepatocyte Nuclear Factor 1-alpha/immunology , Inhibitor of Differentiation Protein 2/genetics , Inhibitor of Differentiation Protein 2/immunology , Killer Cells, Natural/cytology , Lymphoid Enhancer-Binding Factor 1/genetics , Lymphoid Enhancer-Binding Factor 1/immunology , Mice , Mice, Knockout , T-Box Domain Proteins/genetics , T-Box Domain Proteins/immunology , Wnt Signaling Pathway/geneticsABSTRACT
PURPOSE OF REVIEW: We reviewed the current literature on the roles of the Wnt antagonists sclerostin (Sost) and sclerostin-containing domain protein 1 (Sostdc1) on bone homeostasis, the relationship of the hypoxia-inducible factor (Hif) and von Hippel-Lindau (Vhl) pathways on Sost expression, and how changes in bone induced by depletion of Sost, Sostdc1, and Vhl affect hematopoietic cells. RECENT FINDINGS: B cell development is adversely affected in Sost-knockout mice and is more severely affected in Vhl-knockout mice. Inflammation in the Sost-/- bone microenvironment could alter hematopoietic stem cell behavior. Sostdc1-/- mice display defects in natural killer cell development and cytotoxicity. Depletion of Sost and Sostdc1 have effects on immune cell function that warrant investigation in patients receiving Wnt antagonist-depleting therapies for treatment of bone diseases. Additional clinical applications for manipulation of Wnt antagonists include cancer immunotherapies, stem cell transplantation, and directed differentiation to immune lineages.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Bone and Bones/metabolism , Hematopoiesis/drug effects , Osteoporosis/drug therapy , Wnt Signaling Pathway/drug effects , Animals , Bone and Bones/drug effects , Hematopoiesis/physiology , Hematopoietic Stem Cells , Homeostasis/drug effects , Homeostasis/physiology , Immune System/cytology , Immune System/drug effects , Immune System/metabolism , Mice , Skeleton/cytology , Skeleton/drug effects , Skeleton/metabolismABSTRACT
Tissue oxygen (O2) levels vary during development and disease; adaptations to decreased O2 (hypoxia) are mediated by hypoxia-inducible factor (HIF) transcription factors. HIFs are active in the skeleton, and stabilizing HIF-α isoforms cause high bone mass (HBM) phenotypes. A fundamental limitation of previous studies examining the obligate role for HIF-α isoforms in the skeleton involves the persistence of gene deletion as osteolineage cells differentiate into osteocytes. Because osteocytes orchestrate skeletal development and homeostasis, we evaluated the influence of Vhl or Hif1a disruption in osteocytes. Osteocytic Vhl deletion caused HBM phenotype, but Hif1a was dispensable in osteocytes. Vhl cKO mice revealed enhanced canonical Wnt signaling. B cell development was reduced while myelopoiesis increased in osteocytic Vhl cKO, revealing a novel influence of Vhl/HIF-α function in osteocytes on maintenance of bone microarchitecture via canonical Wnt signaling and effects on hematopoiesis.
Subject(s)
Bone and Bones/metabolism , Hematopoiesis , Osteocytes/metabolism , Von Hippel-Lindau Tumor Suppressor Protein/metabolism , Animals , Cancellous Bone/pathology , Cortical Bone/pathology , Gene Deletion , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Lymphopoiesis , Mice, Inbred C57BL , Organ Size , Wnt Signaling PathwayABSTRACT
Sclerostin (Sost) is a negative regulator of bone formation and blocking its function via antibodies has shown great therapeutic promise by increasing both bone mass in humans and animal models. Sclerostin deletion in Sost KO mice (Sost-/- ) causes high bone mass (HBM) similar to sclerosteosis patients. Sost-/- mice have been shown to display an up to 300% increase in bone volume/total volume (BV/TV), relative to age-matched controls. It has been postulated that the main source of skeletal sclerostin is the osteocyte. To understand the cell-type specific contributions to the HBM phenotype described in Sost-/- mice, as well as to address the endocrine and paracrine mode of action of sclerostin, we examined the skeletal phenotypes of conditional Sost loss-of-function (SostiCOIN/iCOIN ) mice with specific deletions in (1) the limb mesenchyme (Prx1-Cre; targets osteoprogenitors and their progeny); (2) midstage osteoblasts and their progenitors (Col1-Cre); (3) mature osteocytes (Dmp1-Cre); and (4) hypertrophic chondrocytes and their progenitors (ColX-Cre). All conditional alleles resulted in significant increases in bone mass in trabecular bone in both the femur and lumbar vertebrae, but only Prx1-Cre deletion fully recapitulated the amplitude of the HBM phenotype in the appendicular skeleton and the B-cell defect described in the global KO. Despite WT expression of Sost in the axial skeleton of Prx1-Cre deleted mice, these mice also had a significant increase in bone mass in the vertebrae, but the sclerostin released in circulation by the axial skeleton did not affect bone parameters in the appendicular skeleton. Also, both Col1 and Dmp1 deletion resulted in a similar 80% significant increase in trabecular bone mass, but only Col1 and Prx1 deletion resulted in a significant increase in cortical thickness. We conclude that several cell types within the Prx1-osteoprogenitor-derived lineages contribute significant amounts of sclerostin protein to the paracrine pool of Sost in bone. © 2018 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals, Inc.
Subject(s)
B-Lymphocytes/metabolism , Cell Lineage , Femur/pathology , Gene Deletion , Glycoproteins/genetics , Lumbar Vertebrae/pathology , Mesenchymal Stem Cells/metabolism , Adaptor Proteins, Signal Transducing , Animals , Bone Marrow/pathology , Cancellous Bone/diagnostic imaging , Cancellous Bone/pathology , Collagen Type X/metabolism , Extracellular Matrix Proteins/metabolism , Female , Femur/diagnostic imaging , Glycoproteins/metabolism , Homeodomain Proteins/metabolism , Integrases/metabolism , Intercellular Signaling Peptides and Proteins , Lumbar Vertebrae/diagnostic imaging , Lymphocytes/metabolism , Mice, Inbred C57BL , Organ Size , Osteoblasts/metabolism , Osteogenesis , Phenotype , X-Ray MicrotomographyABSTRACT
Understanding how embryonic stem cells and their derivatives interact with the adult host immune system is critical to developing their therapeutic potential. Murine embryonic stem cell-derived hematopoietic progenitors (ESHPs) were generated via coculture with the bone marrow stromal cell line, OP9, and then transplanted into NOD.SCID.Common Gamma Chain (NSG) knockout mice, which lack B, T, and natural killer cells. Compared to control mice transplanted with adult lineage-negative bone marrow (Lin- BM) progenitors, ESHP-transplanted mice attained a low but significant level of donor hematopoietic chimerism. Based on our previous studies, we hypothesized that macrophages might contribute to the low engraftment of ESHPs in vivo. Enlarged spleens were observed in ESHP-transplanted mice and found to contain higher numbers of host F4/80+ macrophages compared to BM-transplanted controls. In vivo depletion of host macrophages using clodronate-loaded liposomes improved the ESHP-derived hematopoietic chimerism in the spleen but not in the BM. F4/80+ macrophages demonstrated a striking propensity to phagocytose ESHP targets in vitro. Taken together, these results suggest that macrophages are a barrier to both syngeneic and allogeneic ESHP engraftment in vivo.
Subject(s)
Embryonic Stem Cells/cytology , Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells/cytology , Macrophages/immunology , Macrophages/metabolism , Animals , Antigens, Surface/metabolism , Graft Survival/immunology , Immunophenotyping , Mice , Phagocytosis/immunology , Transplantation Chimera , Transplantation ImmunologyABSTRACT
Bone marrow (BM) failure syndrome encompasses a group of disorders characterized by BM stem cell dysfunction, resulting in varying degrees of hypoplasia and blood pancytopenia, and in many patients is autoimmune and inflammatory in nature. The important role of T helper 1 (Th1) polarized CD4+ T cells in driving BM failure has been clearly established in several models. However, animal model data demonstrating a functional role for CD8+ T cells in BM dysfunction is largely lacking and our objective was to test the hypothesis that CD8+ T cells play a non-redundant role in driving BM failure. Clinical evidence implicates a detrimental role for CD8+ T cells in BM failure and a beneficial role for Foxp3+ regulatory T cells (Tregs) in maintaining immune tolerance in the BM. We demonstrate that IL-2-deficient mice, which have a deficit in functional Tregs, develop spontaneous BM failure. Furthermore, we demonstrate a critical role for CD8+ T cells in the development of BM failure, which is dependent on the cytokine, IFNγ. CD8+ T cells promote hematopoietic stem cell dysfunction and depletion of myeloid lineage progenitor cells, resulting in anemia. Adoptive transfer experiments demonstrate that CD8+ T cells dramatically expedite disease progression and promote CD4+ T cell accumulation in the BM. Thus, BM dysregulation in IL-2-deficient mice is mediated by a Th1 and IFNγ-producing CD8+ T cell (Tc1) response.
Subject(s)
Autoimmunity/immunology , Bone Marrow Cells/immunology , Bone Marrow/immunology , CD8-Positive T-Lymphocytes/immunology , Hematopoietic Stem Cells/immunology , Adoptive Transfer , Anemia/genetics , Anemia/immunology , Anemia/metabolism , Animals , Bone Marrow/metabolism , Bone Marrow/pathology , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/metabolism , Cell Proliferation , Flow Cytometry , Forkhead Transcription Factors , Interferon-gamma/immunology , Interferon-gamma/metabolism , Interleukin-2/deficiency , Interleukin-2/genetics , Interleukin-2/immunology , Mice, Inbred BALB C , Mice, Knockout , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolismABSTRACT
Loss of Sostdc1, a growth factor paralogous to Sost, causes the formation of ectopic incisors, fused molars, abnormal hair follicles, and resistance to kidney disease. Sostdc1 is expressed in the periosteum, a source of osteoblasts, fibroblasts and mesenchymal progenitor cells, which are critically important for fracture repair. Here, we investigated the role of Sostdc1 in bone metabolism and fracture repair. Mice lacking Sostdc1 (Sostdc1(-/-)) had a low bone mass phenotype associated with loss of trabecular bone in both lumbar vertebrae and in the appendicular skeleton. In contrast, Sostdc1(-/-) cortical bone measurements revealed larger bones with higher BMD, suggesting that Sostdc1 exerts differential effects on cortical and trabecular bone. Mid-diaphyseal femoral fractures induced in Sostdc1(-/-) mice showed that the periosteal population normally positive for Sostdc1 rapidly expands during periosteal thickening and these cells migrate into the fracture callus at 3days post fracture. Quantitative analysis of mesenchymal stem cell (MSC) and osteoblast populations determined that MSCs express Sostdc1, and that Sostdc1(-/-) 5day calluses harbor >2-fold more MSCs than fractured wildtype controls. Histologically a fraction of Sostdc1-positive cells also expressed nestin and α-smooth muscle actin, suggesting that Sostdc1 marks a population of osteochondral progenitor cells that actively participate in callus formation and bone repair. Elevated numbers of MSCs in D5 calluses resulted in a larger, more vascularized cartilage callus at day 7, and a more rapid turnover of cartilage with significantly more remodeled bone and a thicker cortical shell at 21days post fracture. These data support accelerated or enhanced bone formation/remodeling of the callus in Sostdc1(-/-) mice, suggesting that Sostdc1 may promote and maintain mesenchymal stem cell quiescence in the periosteum.
Subject(s)
Bone Morphogenetic Proteins/deficiency , Fracture Healing , Mesenchymal Stem Cells/cytology , Periosteum/cytology , Actins/metabolism , Adaptor Proteins, Signal Transducing , Animals , Biomechanical Phenomena , Bone Morphogenetic Proteins/metabolism , Bony Callus/pathology , Calcification, Physiologic , Cancellous Bone/diagnostic imaging , Cancellous Bone/pathology , Cell Differentiation , Cell Proliferation , Cortical Bone/diagnostic imaging , Cortical Bone/pathology , Femur/pathology , Gene Deletion , Mice, Inbred C57BL , Nestin/metabolism , Organ Size , Osteoblasts/metabolism , Osteogenesis , Phenotype , Sp7 Transcription Factor/metabolism , Stem Cells/metabolism , Wnt Signaling Pathway , X-Ray MicrotomographyABSTRACT
Mounting evidence indicates that the immune system and the skeletal system share several regulatory nodes. B lymphocytes, which play key roles in immune homeostasis, are uniquely endowed with osteointeractive properties. From their early development to the plasma cell stage, they are in close proximity with the skeletal system and produce factors important for bone maintenance. Not surprisingly, perturbation of B lymphopoiesis affects bone mass. Reciprocally, inactivation of bone cell functions results in B cell development blocks. This new understanding is refining our insights into the pathogenesis of several diseases such as periodontitis and rheumatoid arthritis.
Subject(s)
Arthritis, Rheumatoid/pathology , B-Lymphocytes/pathology , Bone and Bones/pathology , Immune System/pathology , Osteoclasts/pathology , Periodontitis/pathology , Animals , Arthritis, Rheumatoid/immunology , B-Lymphocytes/immunology , Bone and Bones/immunology , Cell Communication , Humans , Immune System/immunology , Osteoclasts/immunology , Periodontitis/immunologyABSTRACT
The clinical use of embryonic stem cell (ESC)-derived hematopoietic progenitors (ESHPs) requires the generation of ESHPs that produce mature hematopoietic cells and do not induce immune rejection after transplantation. We compared the developmental maturity and immunogenicity of ESHPs generated using two methods: embryoid body (EB) formation and culture of ESCs with the OP9 bone marrow stromal cell line (ESC-OP9). ESHPs derived from EBs displayed an immature hematopoietic phenotype and were devoid of immunogenicity marker expression. In contrast, ESHPs derived via ESC-OP9 displayed a mature phenotype and expressed high levels of some immunostimulatory molecules. ESHPs alone could not stimulate CD4(+) T lymphocyte proliferation directly. However, preferential phagocytosis of ESHPs and T cell proliferation were observed in the presence of antigen-presenting cells, consistent with a model of indirect immune recognition of ESHPs. These results suggest that depletion of host CD4(+) T lymphocytes or antigen-presenting cells may be necessary for successful ESHP transplantation.
Subject(s)
Antigen-Presenting Cells/immunology , CD4-Positive T-Lymphocytes/immunology , Cell Proliferation , Embryonic Stem Cells/immunology , Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells/immunology , Animals , Antigen-Presenting Cells/cytology , Cell Line , Embryonic Stem Cells/cytology , Hematopoietic Stem Cells/cytology , Mice , Mice, Inbred BALB CABSTRACT
Semaphorins are important regulators of peripheral T and B-cell mediated immune responses in mice and humans. Modulatory roles of semaphorins in T cell development are also being characterized. We carefully analyzed the gene expression and protein levels of semaphorins 4A, 4D, and 7A at various developmental stages of T cell maturation in the thymus of C57BL/6 mice. Sema7a was expressed at very low levels, while Sema4d was abundant at all developmental stages of mouse thymocytes. We found the most interesting pattern of gene regulation and protein localization for semaphorin 4A. Both semaphorin 4A mRNA and protein were clearly detected on the earliest progenitors and were downregulated through thymic development. SEMA4A protein also showed a distinct cortico-medullary pattern of localization. Our findings contribute to an understanding of the complex roles played by semaphorins in the network of spatially and temporally regulated cues underpinning T cell development in the thymus.
Subject(s)
Gene Expression Regulation, Developmental , Semaphorins/genetics , Thymocytes/metabolism , Thymus Gland/metabolism , Animals , CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/metabolism , Flow Cytometry , Fluorescent Antibody Technique , Gene Expression Profiling , Mice , Mice, Inbred C57BL , Precursor Cells, T-Lymphoid/cytology , Precursor Cells, T-Lymphoid/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Semaphorins/metabolism , Thymocytes/cytology , Thymus Gland/cytology , Thymus Gland/growth & development , Time FactorsABSTRACT
The ontogeny of hematopoietic stem cells (HSCs) is complex, with multiple sites of embryonic origin as well as several locations of expansion and maturation in the embryo and the adult. Hematopoietic progenitors (HPs) with diverse developmental potential are first found in the yolk sac, aorta-gonad-mesonephros region and placenta. These progenitors then colonize the fetal liver (FL), where they undergo expansion and maturation. HSCs from the FL colonize the fetal bone marrow (FBM), governed by a complex orchestration of transcription programs including migratory molecules with chemotactic activity, adhesion molecules, and molecules that modulate the extracellular matrix. Understanding the mechanisms that regulate the patterns of HSC migration between FL and FBM could improve the engraftment potential of embryonic stem cell-derived HPs, because these cells might display a migratory behavior more similar to early HPs than to adult HSCs. Understanding the changes in migratory behavior in the context of FL to FBM HSC migration could lead to new approaches in the treatment of blood malignancies. We will review the current knowledge in the field of FL to the FBM HSCs migration during development, focusing on changes in expression of molecules important for this process and exploring its clinical applications.
Subject(s)
Cell Movement , Embryonic Stem Cells/cytology , Hematopoietic Stem Cells/cytology , Liver/cytology , Bone Marrow/embryology , Cell Adhesion Molecules/metabolism , Cytokines/metabolism , Embryonic Stem Cells/metabolism , Hematopoietic Stem Cell Transplantation/methods , Hematopoietic Stem Cells/metabolism , Humans , Liver/embryology , Liver/metabolism , Models, BiologicalABSTRACT
Wingless and int (Wnt) proteins are secreted proteins that are important for regulating hematopoietic stem cell self-renewal and differentiation in the bone marrow microenvironment in mice. The mechanisms by which Wnt signaling regulates these hematopoietic cell fate decisions are not fully understood. Secreted Wnt antagonists, which are expressed in bone and bone marrow stromal cells, either bind to Wnt ligands directly or block Wnt receptors and co-receptors to halt Wnt-mediated signal transduction in both osteolineage and hematopoietic cell types. Secreted frizzled related proteins-1 and -2, Wnt inhibitory factor-1, Dickkopf-1, and Sclerostin are Wnt antagonists that influence hematopoietic cell fate decisions in the bone marrow niche. In this review, we compare and contrast the roles of these Wnt antagonists and their effects on hematopoietic development in mice, and also discuss the clinical significance of targeting Wnt antagonists within the context of hematopoietic disease.
Subject(s)
Cell Differentiation/drug effects , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/drug effects , Wnt Proteins/antagonists & inhibitors , Wnt Signaling Pathway/drug effects , Adaptor Proteins, Signal Transducing , Animals , B-Lymphocytes/physiology , Bone Morphogenetic Proteins/physiology , Calcium/metabolism , Genetic Markers/physiology , Glycoproteins/physiology , Hematopoiesis , Humans , Intercellular Signaling Peptides and Proteins/physiology , Intracellular Signaling Peptides and Proteins , JNK Mitogen-Activated Protein Kinases/physiology , Lymphoid Enhancer-Binding Factor 1/physiology , Membrane Proteins/physiology , Receptor Tyrosine Kinase-like Orphan Receptors/physiology , Wnt Proteins/physiologyABSTRACT
Increased osteoblast activity in sclerostin-knockout (Sost(-/-)) mice results in generalized hyperostosis and bones with small bone marrow cavities resulting from hyperactive mineralizing osteoblast populations. Hematopoietic cell fate decisions are dependent on their local microenvironment, which contains osteoblast and stromal cell populations that support both hematopoietic stem cell quiescence and facilitate B-cell development. In this study, we investigated whether high bone mass environments affect B-cell development via the utilization of Sost(-/-) mice, a model of sclerosteosis. We found the bone marrow of Sost(-/-) mice to be specifically depleted of B cells because of elevated apoptosis at all B-cell developmental stages. In contrast, B-cell function in the spleen was normal. Sost expression analysis confirmed that Sost is primarily expressed in osteocytes and is not expressed in any hematopoietic lineage, which indicated that the B-cell defects in Sost(-/-) mice are non-cell autonomous, and this was confirmed by transplantation of wild-type (WT) bone marrow into lethally irradiated Sost(-/-) recipients. WTâSost(-/-) chimeras displayed a reduction in B cells, whereas reciprocal Sost(-/-) âWT chimeras did not, supporting the idea that the Sost(-/-) bone environment cannot fully support normal B-cell development. Expression of the pre-B-cell growth stimulating factor, Cxcl12, was significantly lower in bone marrow stromal cells of Sost(-/-) mice, whereas the Wnt target genes Lef-1 and Ccnd1 remained unchanged in B cells. Taken together, these results demonstrate a novel role for Sost in the regulation of bone marrow environments that support B cells.
Subject(s)
Bone and Bones/metabolism , Glycoproteins/physiology , Immune System/physiology , Adaptor Proteins, Signal Transducing , Animals , B-Lymphocytes/cytology , Bone and Bones/cytology , Cell Differentiation , Cell Lineage , Female , Glycoproteins/metabolism , Hematopoiesis , Intercellular Signaling Peptides and Proteins , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Osteocytes/cytology , PhenotypeABSTRACT
The epithelial cells (TECs) are microenvironmental niche cells which support T lymphocyte development in the thymus. Most studies of TEC biology have focused on TEC at the fetal stage of development, whereas the biology of adult-stage TECs is not as well-understood. Delineating the molecular mechanisms that control adult TEC differentiation has implications for the success of T-lymphocyte based therapies for autoimmune diseases and induction of immunological tolerance to stem cell-derived tissues. Detailed analysis of adult TECs is technically challenging due to their rarity, their diminishing numbers with age, and the limited number of markers to distinguish between unique TEC subpopulations. Here, we have devised an improved isolation protocol for adult mouse TECs and combined it with six-color multiparameter flow cytometry. Using these techniques, we have identified four distinct subsets of CD45- EpCAM+ TECs in adult mice: a) UEA1(low) CDR1(low) (UC(low)); b) UEA1(high) CDR1(high)(UC(high)); c) UEA1(low) CDR1(high) MHC(high) (cTEC); and d) UEA1(high)CDR1(low) MHC(int/high) (mTEC). PCR analysis verified that these TEC subsets differentially expressed known TEC genes. TEC subsets were further analyzed using high-throughput quantitative PCR arrays to reveal novel genes that could be important for TEC subset maintenance. Intracellular staining for keratin-5 and keratin-8 can also be added, but our results suggest that keratin expression alone cannot be used to distinguish adult TEC subsets. Our enhanced isolation allows for detailed analysis of rare TEC subpopulations in the adult mouse at the cellular and molecular levels.
Subject(s)
Cell Separation/methods , Flow Cytometry/methods , Gene Expression , Thymus Gland/cytology , Animals , Epithelial Cells/cytology , Epithelial Cells/metabolism , Mice , Mice, Inbred C57BL , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
BACKGROUND: Cellular interactions between thymocytes and thymic stromal cells are critical for normal T cell development. Thymic epithelial cells (TECs) are important stromal niche cells that provide essential growth factors, cytokines, and present self-antigens to developing thymocytes. The identification of genes that mediate cellular crosstalk in the thymus is ongoing. One candidate gene, Adam17, encodes a metalloprotease that functions by cleaving the ectodomain of several transmembrane proteins and regulates various developmental processes. In conventional Adam17 knockout mice, a non-cell autonomous role for ADAM17 in adult T cell development was reported, which strongly suggested that expression of ADAM17 in TECs was required for normal T cell development. However, knockdown of Adam17 results in multisystem developmental defects and perinatal lethality, which has made study of the role of Adam17 in specific cell types difficult. Here, we examined T cell and thymic epithelial cell development using a conditional knockout approach. METHODOLOGY/PRINCIPAL FINDINGS: We generated an Adam17 conditional knockout mouse in which floxed Adam17 is deleted specifically in TECs by Cre recombinase under the control of the Foxn1 promoter. Normal T cell lineage choice and development through the canonical αß T cell stages was observed. Interestingly, Adam17 deficiency in TECs resulted in reduced expression of the transcription factor Aire. However, no alterations in the patterns of TEC phenotypic marker expression and thymus morphology were noted. CONCLUSIONS/SIGNIFICANCE: In contrast to expectation, our data clearly shows that absence of Adam17 in TECs is dispensable for normal T cell development. Differentiation of TECs is also unaffected by loss of Adam17 based on phenotypic markers. Surprisingly, we have uncovered a novel genetic link between Adam17and Aire expression in vivo. The cell type in which ADAM17 mediates its non-cell autonomous impact and the mechanisms by which it regulates intrathymic T cell development remain to be identified.