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1.
Nature ; 626(8000): 864-873, 2024 Feb.
Article in English | MEDLINE | ID: mdl-38326607

ABSTRACT

Macrophage activation is controlled by a balance between activating and inhibitory receptors1-7, which protect normal tissues from excessive damage during infection8,9 but promote tumour growth and metastasis in cancer7,10. Here we report that the Kupffer cell lineage-determining factor ID3 controls this balance and selectively endows Kupffer cells with the ability to phagocytose live tumour cells and orchestrate the recruitment, proliferation and activation of natural killer and CD8 T lymphoid effector cells in the liver to restrict the growth of a variety of tumours. ID3 shifts the macrophage inhibitory/activating receptor balance to promote the phagocytic and lymphoid response, at least in part by buffering the binding of the transcription factors ELK1 and E2A at the SIRPA locus. Furthermore, loss- and gain-of-function experiments demonstrate that ID3 is sufficient to confer this potent anti-tumour activity to mouse bone-marrow-derived macrophages and human induced pluripotent stem-cell-derived macrophages. Expression of ID3 is therefore necessary and sufficient to endow macrophages with the ability to form an efficient anti-tumour niche, which could be harnessed for cell therapy in cancer.


Subject(s)
Inhibitor of Differentiation Proteins , Kupffer Cells , Neoplasms , Animals , Humans , Mice , Bone Marrow Cells/cytology , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/immunology , Cell Lineage , Induced Pluripotent Stem Cells/cytology , Inhibitor of Differentiation Proteins/deficiency , Inhibitor of Differentiation Proteins/genetics , Inhibitor of Differentiation Proteins/metabolism , Killer Cells, Natural/cytology , Killer Cells, Natural/immunology , Kupffer Cells/cytology , Kupffer Cells/immunology , Kupffer Cells/metabolism , Liver/immunology , Liver/pathology , Macrophage Activation , Neoplasm Proteins , Neoplasms/immunology , Neoplasms/pathology , Neoplasms/therapy , Phagocytosis
2.
Arterioscler Thromb Vasc Biol ; 42(1): 6-18, 2022 01.
Article in English | MEDLINE | ID: mdl-34809449

ABSTRACT

OBJECTIVE: Neovascularization can maintain and even improve tissue perfusion in the setting of limb ischemia during peripheral artery disease. The molecular and cellular mechanisms mediating this process are incompletely understood. We investigate the potential role(s) for Id3 (inhibitor of differentiation 3) in regulating blood flow in a murine model of hindlimb ischemia (HLI). Approach and Results: HLI was modeled through femoral artery ligation and resection and blood flow recovery was quantified by laser Doppler perfusion imaging. Mice with global Id3 deletion had significantly impaired perfusion recovery at 14 and 21 days of HLI. Endothelial- or myeloid cell-specific deletion of Id3 revealed no effect on perfusion recovery while B-cell-specific knockout of Id3 (Id3BKO) revealed a significant attenuation of perfusion recovery. Flow cytometry revealed no differences in ischemia-induced T cells or myeloid cell numbers at 7 days of HLI, yet there was a significant increase in B-1b cells in Id3BKO. Consistent with these findings, ELISA (enzyme-linked immunoassay) demonstrated increases in skeletal muscle and plasma IgM. In vitro experiments demonstrated reduced proliferation and increased cell death when endothelial cells were treated with conditioned media from IgM-producing B-1b cells and tibialis anterior muscles in Id3BKO mice showed reduced density of total CD31+ and αSMA+CD31+ vessels. CONCLUSIONS: This study is the first to demonstrate a role for B-cell-specific Id3 in maintaining blood flow recovery during HLI. Results suggest a role for Id3 in promoting blood flow during HLI and limiting IgM-expressing B-1b cell expansion. These findings present new mechanisms to investigate in peripheral artery disease pathogenesis.


Subject(s)
B-Lymphocytes/metabolism , Immunoglobulin M/metabolism , Inhibitor of Differentiation Proteins/deficiency , Ischemia/metabolism , Muscle, Skeletal/blood supply , Neovascularization, Physiologic , Animals , Cell Proliferation , Cell Survival , Cells, Cultured , Disease Models, Animal , Female , Hindlimb , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Immunoglobulin M/genetics , Inhibitor of Differentiation Proteins/genetics , Ischemia/genetics , Ischemia/pathology , Ischemia/physiopathology , Male , Mice, Inbred C57BL , Mice, Knockout , Muscle, Skeletal/metabolism , Muscle, Skeletal/pathology , Recovery of Function , Regional Blood Flow , Time Factors
3.
Sci Rep ; 7(1): 3079, 2017 06 08.
Article in English | MEDLINE | ID: mdl-28596553

ABSTRACT

Inhibitor of DNA binding (Id) proteins play important roles in regulating cardiac development via paracrine signaling. Id1/Id3 knockout mice die at mid-gestation with multiple cardiac defects. Single Id knockout studies have not reported cardiomyopathies. To bypass embryonic lethality we used Tie2CRE-mediated recombination to conditionally delete Id1 against global Id3 ablation (Id cDKOs), which develops adult-onset dilated cardiomyopathy. We confirm upregulation of thrombospondin-1 (TSP1) in Id cDKO hearts. Colocalization studies reveal increased TSP1 expression in the vicinity of endothelial cells and near regions of endocardial fibrosis/disruption. Downstream fibrotic molecules were upregulated. Endocardial capillary density was reduced with evidence of vascular distention. Treatment of Id cDKO cardiac explants with LSKL, a peptide antagonist of TSP1 activation of TGFß, reversed the increased expression of fibrotic molecules. We conducted bone marrow transplant experiments in which we transferred bone marrow cells from Id cDKO mice into lethally irradiated WT mice. The majority of WT recipients of Id cDKO bone marrow cells phenocopied Id cDKO cardiac fibrosis 4 months post-transplantation. Injection of LSKL into adult Id cDKO mice led to downregulation of fibrotic molecules. The results prompt caution when bone marrow transfers from individuals potentially carrying mutations in the Id axis are applied in clinical settings.


Subject(s)
Endomyocardial Fibrosis/genetics , Hematopoiesis/genetics , Inhibitor of Differentiation Proteins/deficiency , Vascular Diseases/genetics , Animals , Apoptosis/genetics , Bone Marrow Cells/metabolism , Collagen/metabolism , Connective Tissue Growth Factor/metabolism , Disease Models, Animal , Endomyocardial Fibrosis/metabolism , Endomyocardial Fibrosis/pathology , Endothelial Cells/metabolism , Endothelium/metabolism , Gene Expression Regulation , Genotype , Inhibitor of Differentiation Proteins/genetics , Mice , Mice, Knockout , Thrombospondin 1/metabolism , Vascular Diseases/metabolism , Vascular Diseases/pathology
4.
PLoS One ; 11(4): e0154480, 2016.
Article in English | MEDLINE | ID: mdl-27128622

ABSTRACT

The Inhibitor of DNA Binding (Id) proteins play a crucial role in regulating hematopoiesis and are known to interact with E proteins and the bHLH family of transcription factors. Current efforts seek to elucidate the individual roles of Id members in regulating hematopoietic development and specification. However, the nature of their functional redundancies remains elusive since ablation of multiple Id genes is embryonically lethal. We developed a model to test this compensation in the adult. We report that global Id3 ablation with Tie2Cre-mediated conditional ablation of Id1 in both hematopoietic and endothelial cells (Id cDKO) extends viability to 1 year but leads to multi-lineage hematopoietic defects including the emergence of anemia associated with defective erythroid development, a novel phenotype unreported in prior single Id knockout studies. We observe decreased cell counts in the bone marrow and splenomegaly to dimensions beyond what is seen in single Id knockout models. Transcriptional dysregulation of hematopoietic regulators observed in bone marrow cells is also magnified in the spleen. E47 protein levels were elevated in Id cDKO bone marrow cell isolates, but decreased in the erythroid lineage. Chromatin immunoprecipitation (ChIP) studies reveal increased occupancy of E47 and GATA1 at the promoter regions of ß-globin and E2A. Bone marrow transplantation studies highlight the importance of intrinsic Id signals in maintaining hematopoietic homeostasis while revealing a strong extrinsic influence in the development of anemia. Together, these findings demonstrate that loss of Id compensation leads to dysregulation of the hematopoietic transcriptional network and multiple defects in erythropoietic development in adult mice.


Subject(s)
Anemia , Erythropoiesis/genetics , Gene Deletion , Hematopoietic Stem Cells/metabolism , Inhibitor of Differentiation Protein 1/deficiency , Inhibitor of Differentiation Proteins/deficiency , Anemia/genetics , Anemia/metabolism , Anemia/pathology , Animals , GATA1 Transcription Factor/genetics , GATA1 Transcription Factor/metabolism , Hematopoietic Stem Cells/pathology , Human Umbilical Vein Endothelial Cells , Humans , Inhibitor of Differentiation Protein 1/metabolism , Inhibitor of Differentiation Proteins/metabolism , Mice , Mice, Knockout , Transcription Factor 3/genetics , Transcription Factor 3/metabolism
5.
J Leukoc Biol ; 99(2): 321-31, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26382297

ABSTRACT

This study tested the hypothesis that B cells from salivary tissue are distinct in terms of proliferative capacity, immunoglobulin M secretion, repertoire, and autoantibody enrichment in Sjögren's syndrome. We sorted purified B cells from the spleen, cervical lymph nodes, and submandibular glands of a primary Sjögren's syndrome mouse model (Id3(-/-)). Enzyme-linked immunospot and proliferation assays were performed with stimulated B cells. We single-cell sorted B cells from the spleen, cervical lymph nodes, and submandibular gland tissue from Sjögren's syndrome mice and sequenced immunoglobulin M heavy-chain variable regions. Finally, autoantigen arrays were performed using immunoglobulin M derived from sera, cervical lymph nodes, spleens, and submandibular gland tissue of Id3(-/-) animals. Results suggest B cells from salivary tissue of Sjögren's syndrome mice are similar to those from secondary immune sites in terms of proliferative and secretory capacity. However, differences in repertoire usage, heavy chain complementarity-determining region 3 length, mutational frequency, and N region addition were observed among B cells derived from submandibular gland, cervical lymph node, and spleen tissue. Moreover, autoantigen array data show immunoglobulin M from salivary B cells have enriched specificity for Ro (Sjögren's syndrome A) and La (Sjögren's syndrome B). All together, these data suggest salivary B cells have unique repertoire characteristics that likely influence autoantigen binding and contribute to Sjögren's syndrome disease in a tissue-specific manner.


Subject(s)
B-Lymphocytes/immunology , Genes, Immunoglobulin , Immunoglobulin M/biosynthesis , Lymph Nodes/immunology , Sjogren's Syndrome/immunology , Spleen/immunology , Submandibular Gland/immunology , Animals , Autoantibodies/biosynthesis , Autoantibodies/immunology , Autoantigens/immunology , Complementarity Determining Regions/genetics , Disease Models, Animal , Female , Gene Rearrangement, B-Lymphocyte, Heavy Chain , Immunoglobulin Heavy Chains/genetics , Immunoglobulin M/immunology , Immunoglobulin Variable Region/genetics , Inhibitor of Differentiation Proteins/deficiency , Lymph Nodes/pathology , Lymphocyte Activation , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Organ Specificity , Sequence Analysis, DNA , Single-Cell Analysis , Sjogren's Syndrome/pathology , Spleen/pathology , Submandibular Gland/pathology
6.
Circ Res ; 117(3): e28-39, 2015 Jul 17.
Article in English | MEDLINE | ID: mdl-26082558

ABSTRACT

RATIONALE: B cells contribute to atherosclerosis through subset-specific mechanisms. Whereas some controversy exists about the role of B-2 cells, B-1a cells are atheroprotective because of secretion of atheroprotective IgM antibodies independent of antigen. B-1b cells, a unique subset of B-1 cells that respond specifically to T-cell-independent antigens, have not been studied within the context of atherosclerosis. OBJECTIVE: To determine whether B-1b cells produce atheroprotective IgM antibodies and function to protect against diet-induced atherosclerosis. METHODS AND RESULTS: We demonstrate that B-1b cells are sufficient to produce IgM antibodies against oxidation-specific epitopes on low-density lipoprotein both in vitro and in vivo. In addition, we demonstrate that B-1b cells provide atheroprotection after adoptive transfer into B- and T-cell deficient (Rag1(-/-)Apoe(-/-)) hosts. We implicate inhibitor of differentiation 3 (Id3) in the regulation of B-1b cells as B-cell-specific Id3 knockout mice (Id3(BKO)Apoe(-/-)) have increased numbers of B-1b cells systemically, increased titers of oxidation-specific epitope-reactive IgM antibodies, and significantly reduced diet-induced atherosclerosis when compared with Id3(WT)Apoe(-/-) controls. Finally, we report that the presence of a homozygous single nucleotide polymorphism in ID3 in humans that attenuates Id3 function is associated with an increased percentage of circulating B-1 cells and anti-malondialdehyde-low-density lipoprotein IgM suggesting clinical relevance. CONCLUSIONS: These results provide novel evidence that B-1b cells produce atheroprotective oxidation-specific epitope-reactive IgM antibodies and protect against atherosclerosis in mice and suggest that similar mechanisms may occur in humans.


Subject(s)
Atherosclerosis/immunology , B-Lymphocyte Subsets/immunology , Immunoglobulin M/immunology , Lipoproteins, LDL/immunology , Malondialdehyde/analogs & derivatives , Adoptive Transfer , Animals , Antibody Specificity , Aorta/pathology , Apolipoproteins E/deficiency , Atherosclerosis/blood , Atherosclerosis/genetics , Atherosclerosis/pathology , Atherosclerosis/prevention & control , B-Lymphocyte Subsets/transplantation , Cells, Cultured , Cholesterol/blood , Copper/immunology , Diet, Western/adverse effects , Epitopes/immunology , Homeodomain Proteins/genetics , Humans , Inhibitor of Differentiation Proteins/deficiency , Inhibitor of Differentiation Proteins/genetics , Inhibitor of Differentiation Proteins/physiology , Lipoproteins, LDL/chemistry , Lymphocyte Count , Male , Malondialdehyde/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Neoplasm Proteins/genetics , Neoplasm Proteins/physiology , Oxidation-Reduction , Plaque, Atherosclerotic/pathology , Polymorphism, Single Nucleotide , Toll-Like Receptor 4/immunology
7.
PLoS One ; 10(2): e0116839, 2015.
Article in English | MEDLINE | ID: mdl-25642713

ABSTRACT

Melanoma tissues and cell lines are heterogeneous, and include cells with invasive, proliferative, stem cell-like, and differentiated properties. Such heterogeneity likely contributes to the aggressiveness of the disease and resistance to therapy. One model suggests that heterogeneity arises from rare cancer stem cells (CSCs) that produce distinct cancer cell lineages. Another model suggests that heterogeneity arises through reversible cellular plasticity, or phenotype-switching. Recent work indicates that phenotype-switching may include the ability of cancer cells to dedifferentiate to a stem cell-like state. We set out to investigate the phenotype-switching capabilities of melanoma cells, and used unbiased methods to identify genes that may control such switching. We developed a system to reversibly synchronize melanoma cells between 2D-monolayer and 3D-stem cell-like growth states. Melanoma cells maintained in the stem cell-like state showed a striking upregulation of a gene set related to development and neural stem cell biology, which included SRY-box 2 (SOX2) and Inhibitor of DNA Binding 4 (ID4). A gene set related to cancer cell motility and invasiveness was concomitantly downregulated. Intense and pervasive ID4 protein expression was detected in human melanoma tissue samples, suggesting disease relevance for this protein. SiRNA knockdown of ID4 inhibited switching from monolayer to 3D-stem cell-like growth, and instead promoted switching to a highly differentiated, neuronal-like morphology. We suggest that ID4 is upregulated in melanoma as part of a stem cell-like program that facilitates further adaptive plasticity. ID4 may contribute to disease by preventing stem cell-like melanoma cells from progressing to a normal differentiated state. This interpretation is guided by the known role of ID4 as a differentiation inhibitor during normal development. The melanoma stem cell-like state may be protected by factors such as ID4, thereby potentially identifying a new therapeutic vulnerability to drive differentiation to the normal cell phenotype.


Subject(s)
Cell Differentiation , Gene Expression Regulation, Neoplastic , Inhibitor of Differentiation Proteins/metabolism , Melanoma/metabolism , Melanoma/pathology , Cell Adhesion , Cell Line, Tumor , Gene Knockdown Techniques , Humans , Inhibitor of Differentiation Proteins/deficiency , Inhibitor of Differentiation Proteins/genetics , Melanoma/genetics , Neoplasm Invasiveness , Neoplastic Stem Cells/pathology , Phenotype , RNA, Small Interfering/genetics , SOXB1 Transcription Factors/deficiency , SOXB1 Transcription Factors/genetics , Transcription, Genetic
8.
Diabetologia ; 58(4): 758-70, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25636209

ABSTRACT

AIMS/HYPOTHESIS: Oxidative stress is implicated in beta cell glucotoxicity in type 2 diabetes. Inhibitor of differentiation (ID) proteins are transcriptional regulators induced by hyperglycaemia in islets, but the mechanisms involved and their role in beta cells are not clear. Here we investigated whether or not oxidative stress regulates ID levels in beta cells and the role of ID proteins in beta cells during oxidative stress. METHODS: MIN6 cells were cultured in H2O2 or ribose to induce oxidative stress. ID1, ID3 and small MAF proteins (MAFF, MAFG and MAFK) were inhibited using small interfering RNA. Isolated islets from Id1(-/-), Id3(-/-) and diabetic db/db mice were used. RESULTS: ID1-4 expression was upregulated in vivo in the islets of diabetic db/db mice and stimulated in vitro by ribose and H2O2. Id1/3 inhibition reduced the expression of multiple antioxidant genes and potentiated oxidative stress-induced apoptosis. This finding was associated with increased levels of intracellular reactive oxygen species, altered mitochondrial morphology and reduced expression of Tfam, which encodes a mitochondrial transcription factor, and respiratory chain components. Id1/3 inhibition also reduced the expression of small MAF transcription factors (MafF, MafG and MafK), interacting partners of nuclear factor, erythroid 2-like 2 (NFE2L2), master regulator of the antioxidant response. Inhibition of small MAFs reduced the expression of antioxidant genes and potentiated oxidative stress-induced apoptosis, thus recapitulating the effects of Id1/3 inhibition. CONCLUSIONS/INTERPRETATION: Our study identifies IDs as a novel family of oxidative stress-responsive proteins in beta cells. IDs are crucial regulators of the adaptive antioxidant-mitochondrial response that promotes beta cell survival during oxidative stress through a novel link to the NFE2L2-small MAF pathway.


Subject(s)
Antioxidants/metabolism , Diabetes Mellitus/metabolism , Inhibitor of Differentiation Protein 1/metabolism , Inhibitor of Differentiation Proteins/metabolism , Insulin-Secreting Cells/metabolism , Mitochondria/metabolism , Oxidative Stress , Animals , Apoptosis , Cell Line , Diabetes Mellitus/genetics , Disease Models, Animal , Gene Expression Regulation , Inhibitor of Differentiation Protein 1/deficiency , Inhibitor of Differentiation Protein 1/genetics , Inhibitor of Differentiation Proteins/deficiency , Inhibitor of Differentiation Proteins/genetics , Maf Transcription Factors, Small/genetics , Maf Transcription Factors, Small/metabolism , Mice, Inbred C57BL , Mice, Knockout , NF-E2-Related Factor 2/genetics , NF-E2-Related Factor 2/metabolism , RNA Interference , Signal Transduction , Time Factors , Tissue Culture Techniques , Transfection
9.
Cytokine ; 69(2): 226-33, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25010390

ABSTRACT

Elevated levels of the cytokine IL-13 has been found to be associated with autoimmune diseases, including Sjögren's Syndrome. However, whether IL-13 plays a causative role in disease development is not known and cannot be easily studied in humans. Our previous work has shown that levels of IL-13 are elevated in Id3 knockout mice, which has been established as a model for primary Sjögren's Syndrome. Here, we utilized an IL-13 reporter to determine the source of the elevated IL-13 levels observed in Id3 knockout mice and assess its contribution to SS pathology. Our results indicate that T cells, notably CD4 and γδ T cells, in Id3 knockout mice acquire IL-13 competency at an elevated rate well before disease symptoms become apparent. We also show that T cells developing early in life are more predisposed to produce IL-13. Finally, analysis of Id3 and IL-13 double deficient mice demonstrated that IL-13 plays an essential role in the deterioration of gland function. Our study provides crucial genetic evidence that enhanced IL-13 production by T cells can play a causative role in the exocrinopathy observed in Id3 knockout mice.


Subject(s)
Exocrine Glands/pathology , Inhibitor of Differentiation Proteins/deficiency , Interleukin-13/biosynthesis , T-Lymphocytes/metabolism , Aging/immunology , Animals , Animals, Newborn , Exocrine Glands/metabolism , Inhibitor of Differentiation Proteins/metabolism , Mice, Knockout , Receptors, Antigen, T-Cell, gamma-delta/metabolism , Salivary Glands/immunology , Salivary Glands/pathology , Thymocytes/metabolism
10.
J Immunol ; 192(3): 1055-1063, 2014 Feb 01.
Article in English | MEDLINE | ID: mdl-24379125

ABSTRACT

The innate-like T cells expressing Vγ1.1 and Vδ6.3 represent a unique T cell lineage sharing features with both the γδ T and the invariant NKT cells. The population size of Vγ1.1(+)Vδ6.3(+) T cells is tightly controlled and usually contributes to a very small proportion of thymic output, but the underlying mechanism remains enigmatic. Deletion of Id3, an inhibitor of E protein transcription factors, can induce an expansion of the Vγ1.1(+)Vδ6.3(+) T cell population. This phenotype is much stronger on the C57BL/6 background than on the 129/sv background. Using quantitative trait linkage analysis, we identified Id2, a homolog of Id3, to be the major modifier of Id3 in limiting Vγ1.1(+)Vδ6.3(+) T cell expansion. The Vγ1.1(+)Vδ6.3(+) phenotype is attributed to an intrinsic weakness of Id2 transcription from Id2 C57BL/6 allele, leading to an overall reduced dosage of Id proteins. However, complete removal of both Id2 and Id3 genes in developing T cells suppressed the expansion of Vγ1.1(+)Vδ6.3(+) T cells because of decreased proliferation and increased cell death. We showed that conditional knockout of Id2 alone is sufficient to promote a moderate expansion of γδ T cells. These regulatory effects of Id2 and Id3 on Vγ1.1(+)Vδ6.3(+) T cells are mediated by titration of E protein activity, because removing one or more copies of E protein genes can restore Vγ1.1(+)Vδ6.3(+) T cell expansion in Id2 and Id3 double conditional knockout mice. Our data indicated that Id2 and Id3 collaboratively control survival and expansion of the γδ lineage through modulating a proper threshold of E proteins.


Subject(s)
Inhibitor of Differentiation Protein 2/physiology , Inhibitor of Differentiation Proteins/physiology , Receptors, Antigen, T-Cell, gamma-delta/analysis , T-Lymphocyte Subsets/immunology , Alleles , Animals , Basic Helix-Loop-Helix Transcription Factors/deficiency , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/physiology , Cell Lineage , Crosses, Genetic , Gene Rearrangement, delta-Chain T-Cell Antigen Receptor , Gene Rearrangement, gamma-Chain T-Cell Antigen Receptor , Inhibitor of Differentiation Protein 2/deficiency , Inhibitor of Differentiation Protein 2/genetics , Inhibitor of Differentiation Proteins/deficiency , Inhibitor of Differentiation Proteins/genetics , Lymphocyte Count , Mice , Mice, Inbred C57BL , Mice, Knockout , Specific Pathogen-Free Organisms , T-Lymphocyte Subsets/chemistry , T-Lymphocyte Subsets/cytology , Thymus Gland/cytology , Thymus Gland/immunology
11.
J Immunol ; 191(12): 5973-83, 2013 Dec 15.
Article in English | MEDLINE | ID: mdl-24244015

ABSTRACT

Invariant NKT (iNKT) cells display characteristics of both adaptive and innate lymphoid cells (ILCs). Like other ILCs, iNKT cells constitutively express ID proteins, which antagonize the E protein transcription factors that are essential for adaptive lymphocyte development. However, unlike ILCs, ID2 is not essential for thymic iNKT cell development. In this study, we demonstrated that ID2 and ID3 redundantly promoted iNKT cell lineage specification involving the induction of the signature transcription factor PLZF and that ID3 was critical for development of TBET-dependent NKT1 cells. In contrast, both ID2 and ID3 limited iNKT cell numbers by enforcing the postselection checkpoint in conventional thymocytes. Therefore, iNKT cells show both adaptive and innate-like requirements for ID proteins at distinct checkpoints during iNKT cell development.


Subject(s)
Clonal Selection, Antigen-Mediated , Inhibitor of Differentiation Protein 2/physiology , Inhibitor of Differentiation Proteins/physiology , Lymphopoiesis/physiology , Natural Killer T-Cells/cytology , T-Lymphocyte Subsets/cytology , Adoptive Transfer , Animals , Antigens, Differentiation, T-Lymphocyte/biosynthesis , Antigens, Differentiation, T-Lymphocyte/genetics , Cells, Cultured , Gene Rearrangement, alpha-Chain T-Cell Antigen Receptor , Immunoglobulin Joining Region/genetics , Immunoglobulin Variable Region/genetics , Inhibitor of Differentiation Protein 2/genetics , Inhibitor of Differentiation Proteins/deficiency , Interferon-gamma/biosynthesis , Interleukin-4/biosynthesis , Kruppel-Like Transcription Factors/biosynthesis , Kruppel-Like Transcription Factors/genetics , Lymphocyte Count , Mice , Mice, Knockout , Natural Killer T-Cells/metabolism , Promyelocytic Leukemia Zinc Finger Protein , Radiation Chimera , T-Box Domain Proteins/biosynthesis , T-Box Domain Proteins/deficiency , T-Box Domain Proteins/genetics , T-Lymphocyte Subsets/metabolism , Thymus Gland/cytology , Thymus Gland/growth & development
12.
Arterioscler Thromb Vasc Biol ; 33(12): 2771-9, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24115031

ABSTRACT

OBJECTIVE: Natural immunity is emerging as an important mediator of protection from atherogenesis. Natural IgM antibodies that recognize oxidation-specific epitopes on low-density lipoprotein or phospholipids and the B-1a B cells that produce them attenuate atherosclerosis. We previously demonstrated that Apoe(-/-) mice globally deficient in the helix-loop-helix protein inhibitor of differentiation 3 (Id3) develop early diet-induced atherosclerosis. Furthermore, B cell-mediated attenuation of atherosclerosis in B cell-deficient mice was dependent on Id3. Here, we sought to determine whether Id3 regulates B-1a B cells and the natural antibodies that they produce and identify mechanisms mediating these effects. APPROACH AND RESULTS: Mice lacking Id3 had significantly fewer B-1a B cells in the spleen and peritoneal cavity and reduced serum levels of the natural antibody E06. B cell-specific deletion of Id3 revealed that this effect was not because of the loss of Id3 in B cells. Interleukin (IL)-33 induced abundant, Id3-dependent IL-5 production in the recently identified innate lymphoid cell, the natural helper (NH) cell, but not Th2 or mast cells. In addition, delivery of IL-5 to Id3-deficient mice restored B-1a B cell proliferation. B-1a B cells were present in aortic samples also containing NH cells. Aortic NH cells produced IL-5, a B-1a B cell mitogen in response to IL-33 stimulation. CONCLUSIONS: These studies are the first to identify NH and B-1a B cells in the aorta and provide evidence that Id3 is a key regulator of NH cell IL-5 production and B-1a B cell homeostasis.


Subject(s)
Aorta/immunology , Aortic Diseases/immunology , Atherosclerosis/immunology , B-Lymphocyte Subsets/immunology , Cell Proliferation , Immunity, Innate , Inhibitor of Differentiation Proteins/metabolism , Interleukin-5/metabolism , Animals , Antigens, CD19/genetics , Antigens, CD19/metabolism , Aortic Diseases/genetics , Apolipoproteins E/genetics , Apolipoproteins E/metabolism , Atherosclerosis/genetics , Cells, Cultured , Disease Models, Animal , Inhibitor of Differentiation Proteins/deficiency , Inhibitor of Differentiation Proteins/genetics , Interleukin-33 , Interleukin-5/genetics , Interleukins/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , T-Lymphocytes, Helper-Inducer/metabolism , Transfection
13.
Mol Cancer ; 12: 67, 2013 Jun 21.
Article in English | MEDLINE | ID: mdl-23786676

ABSTRACT

BACKGROUND: Inhibitor of differentiation 4 (Id4), a member of the helix-loop-helix family of transcriptional regulators has emerged as a tumor suppressor in prostate cancer. Id4 is expressed in the normal prostate where its expression is also regulated by androgens. In this study we investigated the effect of loss of Id4 (Id4-/-) on adult prostate morphology. METHODS: Histological analysis was performed on prostates from 6-8 weeks old Id4-/-, Id4+/- and Id4+/+ mice. Expression of Id1, Sox9, Myc, androgen receptor, Akt, p-Akt, Pten and Nkx3.1 was investigated by immunohistochemistry. Androgen receptor binding on NKX3.1 promoter was studied by chromatin immuno-precipitation. Id4 was either over-expressed or silenced in prostate cancer cell lines DU145 and LNCaP respectively followed by analysis of PTEN, NKX3.1 and Sox9 expression. RESULTS: Id4-/- mice had smaller prostates with fewer tubules, smaller tubule diameters and subtle mPIN like lesions. Levels of androgen receptor were similar between wild type and Id4-/- prostate. Decreased NKX3.1 expression was in part due to decreased androgen receptor binding on NKX3.1 promoter in Id4-/- mice. The increase in the expression of Myc, Sox9, Id1, Ki67 and decrease in the expression of PTEN, Akt and phospho-AKT was associated with subtle mPIN like lesions in Id4-/- prostates. Finally, prostate cancer cell line models in which Id4 was either silenced or over-expressed confirmed that Id4 regulates NKX3.1, Sox9 and PTEN. CONCLUSIONS: Our results suggest that loss of Id4 attenuates normal prostate development and promotes hyperplasia/dysplasia with subtle mPIN like lesions characterized by gain of Myc and Id1 and loss of Nkx3.1 and Pten expression. One of the mechanisms by which Id4 may regulate normal prostate development is through regulating androgen receptor binding to respective response elements such as those on NKX3.1 promoter. In spite of these complex alterations, large neoplastic lesions in Id4-/- prostates were not observed suggesting the possibility of mechanisms/pathways such as loss of Akt that could restrain the formation of significant pre-cancerous lesions.


Subject(s)
Homeodomain Proteins/metabolism , Inhibitor of Differentiation Proteins/deficiency , PTEN Phosphohydrolase/metabolism , Prostate/growth & development , Prostatic Intraepithelial Neoplasia/metabolism , Receptors, Androgen/metabolism , Transcription Factors/metabolism , Animals , Apoptosis , Humans , Inhibitor of Differentiation Proteins/metabolism , Male , Mice , Mice, Knockout , Phenotype , Phosphorylation , Prostate/pathology , Prostatic Intraepithelial Neoplasia/pathology , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Proto-Oncogene Proteins c-akt/metabolism
14.
Arterioscler Thromb Vasc Biol ; 32(12): 2855-61, 2012 Dec.
Article in English | MEDLINE | ID: mdl-23042815

ABSTRACT

OBJECTIVE: Inhibitor of differention-3 (Id3) promotes B cells homing to the aorta and atheroprotection in Apoe(-/-) mice. We sought to determine the impact of loss of Id3 in the Ldlr((-/-)) mouse model of diet-induced atherosclerosis and identify novel Id3 targets in the vessel wall. METHODS AND RESULTS: Ex vivo optical imaging confirmed that Id3((-/-)) Ldlr((-/-)) mice have significantly fewer aortic B cells than Id3((+/+)) Ldlr(-/-) mice. After 8 and 16 weeks of Western diet, Id3((-/-)) Ldlr((-/-)) mice developed significantly more atherosclerosis than Id3((+/+)) Ldlr((-/-)) mice, with Id3(+/-) Ldlr(-/-) mice demonstrating an intermediate phenotype. There were no differences in serum lipid levels between genotypes. Immunostaining demonstrated that aortas from Id3((-/-)) Ldlr((-/-)) mice had greater intimal macrophage density and C-C chemokine ligand 20 and vascular cell adhesion molecule 1 (VCAM-1) expression compared with Id3((+/+)) Ldlr(-/-) mice. Real-time polymerase chain reaction demonstrated increased VCAM-1 mRNA levels in the aortas of Id3(-/-) Ldlr(-/-) mice. Primary vascular smooth muscle cells from Id3((-/-)) mice expressed greater amounts of VCAM-1 protein compared with control. Gain and loss of function studies in primary vascular smooth muscle cells identified a role for Id3 in repressing VCAM-1 promoter activation. Chromatin immunoprecipitation demonstrated interaction of E12 with the VCAM-1 promoter, which is inhibited by Id3. CONCLUSIONS: Id3 is an atheroprotective transcription regulator with targets in both B cells and vessel wall cells leading to reduced macrophage accumulation and reduced atherosclerosis formation.


Subject(s)
Atherosclerosis/physiopathology , Cell Movement/physiology , Cell Proliferation , Inhibitor of Differentiation Proteins/deficiency , Macrophages/pathology , Receptors, LDL/deficiency , Vascular Cell Adhesion Molecule-1/metabolism , Animals , Aorta/metabolism , Aorta/pathology , Atherosclerosis/epidemiology , Atherosclerosis/metabolism , Atherosclerosis/pathology , B-Lymphocytes/metabolism , B-Lymphocytes/pathology , Chemokine CCL20/metabolism , Disease Models, Animal , Inhibitor of Differentiation Proteins/genetics , Inhibitor of Differentiation Proteins/metabolism , Macrophages/metabolism , Mice , Mice, Knockout , Prevalence , Receptors, LDL/genetics , Receptors, LDL/metabolism , Risk Factors
15.
J Immunol ; 189(8): 3822-30, 2012 Oct 15.
Article in English | MEDLINE | ID: mdl-22972921

ABSTRACT

Lineage commitment is regulated during hematopoiesis, with stepwise loss of differentiation potential ultimately resulting in lineage commitment. In this study we describe a novel population of B/NK bipotent precursors among common lymphoid progenitors in the fetal liver and the bone marrow. The absence of T cell precursor potential, both in vivo and in vitro, is due to low Notch1 expression and secondary to inhibition of E2A activity by members of the inhibitor of DNA binding (Id) protein family. Our results demonstrate a new, Id protein-dependent, molecular mechanism of Notch1 repression, operative in both fetal and adult common lymphoid progenitors, where T cell potential is selectively inhibited without affecting either the B or NK programs. This study identifies Id proteins as negative regulators of T cell specification, before B and NK commitment, and provides important insights into the transcriptional networks orchestrating hematopoiesis.


Subject(s)
Basic Helix-Loop-Helix Transcription Factors/antagonists & inhibitors , Cell Differentiation/immunology , DNA-Binding Proteins/antagonists & inhibitors , Down-Regulation/immunology , Receptor, Notch1/antagonists & inhibitors , Stem Cells/immunology , T-Lymphocyte Subsets/immunology , fms-Like Tyrosine Kinase 3/deficiency , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Cell Differentiation/genetics , Cell Lineage/genetics , Cell Lineage/immunology , DNA-Binding Proteins/genetics , DNA-Binding Proteins/physiology , Down-Regulation/genetics , Inhibitor of Differentiation Protein 2/deficiency , Inhibitor of Differentiation Protein 2/genetics , Inhibitor of Differentiation Proteins/deficiency , Inhibitor of Differentiation Proteins/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Multigene Family/genetics , Multigene Family/immunology , Receptor, Notch1/biosynthesis , Receptor, Notch1/genetics , Stem Cells/cytology , Stem Cells/metabolism , T-Lymphocyte Subsets/cytology , T-Lymphocyte Subsets/metabolism , fms-Like Tyrosine Kinase 3/biosynthesis , fms-Like Tyrosine Kinase 3/genetics
16.
Immunity ; 36(3): 348-61, 2012 Mar 23.
Article in English | MEDLINE | ID: mdl-22425249

ABSTRACT

The double-positive (DP) to single-positive (SP) transition during T cell development is initiated by downregulation of the E protein transcription factors HEB and E2A. Here, we have demonstrated that in addition to regulating the onset of this transition, HEB and E2A also play a separate role in CD4(+) lineage choice. Deletion of HEB and E2A in DP thymocytes specifically blocked the development of CD4(+) lineage T cells. Furthermore, deletion of the E protein inhibitors Id2 and Id3 allowed CD4(+) T cell development but blocked CD8(+) lineage development. Analysis of the CD4(+) lineage transcriptional regulators ThPOK and Gata3 placed HEB and E2A upstream of CD4(+) lineage specification. These studies identify an important role for E proteins in the activation of CD4(+) lineage differentiation as thymocytes undergo the DP to SP transition.


Subject(s)
Basic Helix-Loop-Helix Transcription Factors/immunology , CD4-Positive T-Lymphocytes/immunology , Animals , Basic Helix-Loop-Helix Transcription Factors/deficiency , Basic Helix-Loop-Helix Transcription Factors/genetics , CD4 Antigens/genetics , CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Cell Differentiation/immunology , Inhibitor of Differentiation Protein 2/deficiency , Inhibitor of Differentiation Protein 2/genetics , Inhibitor of Differentiation Protein 2/immunology , Inhibitor of Differentiation Proteins/deficiency , Inhibitor of Differentiation Proteins/genetics , Inhibitor of Differentiation Proteins/immunology , Interleukin-7 Receptor alpha Subunit/metabolism , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Receptors, CCR7/metabolism , Up-Regulation
17.
J Androl ; 33(4): 667-74, 2012.
Article in English | MEDLINE | ID: mdl-22016354

ABSTRACT

Inhibitor of DNA binding 3 (ID3) protein binds to and inhibits the function of basic helix-loop-helix transcription factors. The expression of ID3 protein in postproliferative Sertoli cells in the testis, as well as its unique, highly region-specific expression profile along the adult epididymis, implies novel functional roles for this regulator of transcription. The purpose of this study was to determine the effect of Id3 deficiency on male fertility and progeny outcome. We observed that fertility status of Id3(-/-) males is adversely affected. Males having this null mutation did not sire any litters when crossed with wild-type C57Bl/6 females; although pregnancies were induced in these females, these never resulted in the birth of live pups. Additionally, abnormal reproductive outcomes and developmental phenotypes were observed in gestational day 18 fetuses resulting from crosses with an Id3-deficient sire and wild-type CD-1 females; these were characterized by an elevated rate of fetal death, reduced body weights of live fetuses, a higher rate of external malformations, and aberrant skeletal development. Importantly, we observed that mutation of this gene differentially affects the phenotype of resulting offspring, depending on the genotype of the parent, not necessarily the genotype of the fetus itself. These results provide novel insight into the role of Id3, suggesting that Id3 might coordinate gene expression programs in the male reproductive system associated with the generation of healthy offspring.


Subject(s)
Fertility/genetics , Inhibitor of Differentiation Proteins/genetics , Reproduction/genetics , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Female , Fetal Development/genetics , Inhibitor of Differentiation Proteins/deficiency , Male , Mice , Mice, Inbred C57BL , Mutation , Pregnancy , Pregnancy Outcome
18.
Arterioscler Thromb Vasc Biol ; 32(2): 317-24, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22075252

ABSTRACT

OBJECTIVE: Inhibitor of differentiation-3 (Id3) has been implicated in promoting angiogenesis, a key determinant of high-fat diet (HFD)-induced visceral adiposity. Yet the role of Id3 in HFD-induced angiogenesis and visceral adipose expansion is unknown. METHODS AND RESULTS: Id3(-/-) mice demonstrated a significant attenuation of HFD-induced visceral fat depot expansion compared to wild type littermate controls. Importantly, unlike other Id proteins, loss of Id3 did not affect adipose depot size in young mice fed chow diet or differentiation of adipocytes in vitro or in vivo. Contrast enhanced ultrasound revealed a significant attenuation of visceral fat microvascular blood volume in HFD-fed mice null for Id3 compared to wild type controls. HFD induced Id3 and VEGFA expression in the visceral stromal vascular fraction and Id3(-/-) mice had significantly lower levels of VEGFA protein in visceral adipose tissue compared to wild type. Furthermore, HFD-induced VEGFA expression in visceral adipose tissue was completely abolished by loss of Id3. Consistent with this effect, Id3 abolished E12-mediated repression of VEGFA promoter activity. CONCLUSIONS: Results identify Id3 as an important regulator of HFD-induced visceral adipose VEGFA expression, microvascular blood volume, and depot expansion. Inhibition of Id3 may have potential as a therapeutic strategy to limit visceral adiposity.


Subject(s)
Adiposity/physiology , Dietary Fats/pharmacology , Inhibitor of Differentiation Proteins/metabolism , Intra-Abdominal Fat/metabolism , Adipocytes/pathology , Animals , Blood Volume/physiology , Inhibitor of Differentiation Proteins/deficiency , Inhibitor of Differentiation Proteins/genetics , Intra-Abdominal Fat/blood supply , Intra-Abdominal Fat/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Models, Animal , Neovascularization, Physiologic/physiology , Signal Transduction/physiology , Vascular Endothelial Growth Factor A/metabolism
19.
Circ Res ; 110(1): e1-12, 2012 Jan 06.
Article in English | MEDLINE | ID: mdl-22034493

ABSTRACT

RATIONALE: B cells are abundant in the adventitia of normal and diseased vessels. Yet, the molecular and cellular mechanisms mediating homing of B cells to the vessel wall and B-cell effects on atherosclerosis are poorly understood. Inhibitor of differentiation-3 (Id3) is important for atheroprotection in mice and polymorphism in the human ID3 gene has been implicated as a potential risk marker of atherosclerosis in humans. Yet, the role of Id3 in B-cell regulation of atherosclerosis is unknown. OBJECTIVE: To determine if Id3 regulates B-cell homing to the aorta and atheroprotection and identify molecular and cellular mechanisms mediating this effect. METHODS AND RESULTS: Loss of Id3 in Apoe(-/-) mice resulted in early and increased atherosclerosis. Flow cytometry revealed a defect in Id3(-/-) Apoe(-/-) mice in the number of B cells in the aorta but not the spleen, lymph nodes, and circulation. Similarly, B cells transferred from Id3(-/-) Apoe(-/-) mice into B-cell-deficient mice reconstituted spleen, lymph node, and blood similarly to B cells from Id3(+/+) Apoe(-/-) mice, but aortic reconstitution and B-cell-mediated inhibition of diet-induced atherosclerosis was significantly impaired. In addition to retarding initiation of atherosclerosis, B cells homed to regions of existing atherosclerosis, reduced macrophage content in plaque, and attenuated progression of disease. The chemokine receptor CCR6 was identified as an important Id3 target mediating aortic homing and atheroprotection. CONCLUSIONS: Together, these results are the first to identify the Id3-CCR6 pathway in B cells and demonstrate its role in aortic B-cell homing and B-cell-mediated protection from early atherosclerosis.


Subject(s)
Aorta/pathology , Atherosclerosis/prevention & control , Atherosclerosis/physiopathology , B-Lymphocytes/pathology , Cell Movement/physiology , Inhibitor of Differentiation Proteins/physiology , Animals , Aorta/physiopathology , Apolipoproteins E/deficiency , Apolipoproteins E/genetics , Atherosclerosis/etiology , B-Lymphocytes/physiology , Diet/adverse effects , Disease Models, Animal , Disease Progression , Incidence , Inhibitor of Differentiation Proteins/deficiency , Inhibitor of Differentiation Proteins/genetics , Mice , Mice, Knockout , Plaque, Atherosclerotic/pathology , Plaque, Atherosclerotic/physiopathology , Receptors, CCR6/physiology , Signal Transduction/physiology
20.
Mol Immunol ; 49(1-2): 227-33, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21924496

ABSTRACT

Id3-/- mice represent a model for T cell mediated primary Sjogren's syndrome (PSS). An intriguing feature of this disease model is the early appearance of impaired salivary function or exocrinopathy prior to lymphocytic infiltration of the salivary glands. This phenomenon prompted us to examine the role of cytokines produced by T cells in the systemic regulation of gland function. A comprehensive examination of serum cytokine profiles revealed elevated levels of IL-13 in Id3-/- mice. We found that the increase in serum IL-13 levels in Id3-/- mice was largely dependent on αß T cells. Removal of αß T cells in Id3-/- mice also eliminates disease symptoms, including lymphocytic infiltration in the gland tissues, and impaired saliva production. We further show that the number of mast cells in the salivary glands of Id3-/- mice is significantly increased, in a trend inversely related to the saliva production. This increase in the number of mast cells is also dependent on the presence of αß T cells. Treatment of young Id3-/- mice with anti-IL-13 antibodies over a two-month period resulted in a reduction of both serum IL-13 levels and the number of mast cells in the salivary gland tissues, as well as correspondingly improved saliva production. These findings indicate a potentially important role for IL-13 in gland regulation and disease pathology.


Subject(s)
CD4-Positive T-Lymphocytes/immunology , Inhibitor of Differentiation Proteins/immunology , Interleukin-13/immunology , Mast Cells/pathology , Receptors, Antigen, T-Cell, alpha-beta/immunology , Sjogren's Syndrome/immunology , Animals , Cell Separation , Disease Models, Animal , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Inhibitor of Differentiation Proteins/deficiency , Inhibitor of Differentiation Proteins/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Microscopy, Fluorescence , Reverse Transcriptase Polymerase Chain Reaction , Sjogren's Syndrome/genetics , Sjogren's Syndrome/pathology
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