ABSTRACT
Memory T cells are critical for the immune response to recurring infections. Their instantaneous reactivity to pathogens is empowered by the persistent expression of cytokine-encoding mRNAs. How the translation of proteins from pre-formed cytokine-encoding mRNAs is prevented in the absence of infection has remained unclear. Here we found that protein production in memory T cells was blocked via a 3' untranslated region (3' UTR)-mediated process. Germline deletion of AU-rich elements (AREs) in the Ifng-3' UTR led to chronic cytokine production in memory T cells. This aberrant protein production did not result from increased expression and/or half-life of the mRNA. Instead, AREs blocked the recruitment of cytokine-encoding mRNA to ribosomes; this block depended on the ARE-binding protein ZFP36L2. Thus, AREs mediate repression of translation in mouse and human memory T cells by preventing undesirable protein production from pre-formed cytokine-encoding mRNAs in the absence of infection.
Subject(s)
3' Untranslated Regions/genetics , AU Rich Elements/genetics , Interferon-gamma/genetics , RNA, Messenger/genetics , T-Lymphocytes/immunology , Animals , Cells, Cultured , Epigenetic Repression , Immunologic Memory , Lymphocyte Activation , Mice , Mice, Inbred C57BL , Mice, Transgenic , Peptide Chain Elongation, Translational , Ribosomes/metabolism , Tristetraprolin/genetics , Tristetraprolin/metabolismABSTRACT
Innate CD8+ T cells express a memory-like phenotype and demonstrate a strong cytotoxic capacity that is critical during the early phase of the host response to certain bacterial and viral infections. These cells arise in the thymus and depend on IL-4 and IL-15 for their development. Even though innate CD8+ T cells exist in the thymus of WT mice in low numbers, they are highly enriched in KO mice that lack certain kinases, leading to an increase in IL-4 production by thymic NKT cells. Our work describes that in C57BL/6 WT mice undergoing a Th1 biased infectious disease, the thymus experiences an enrichment of single positive CD8 (SP8) thymocytes that share all the established phenotypical and functional characteristics of innate CD8+ T cells. Moreover, through in vivo experiments, we demonstrate a significant increase in survival and a lower parasitemia in mice adoptively transferred with SP8 thymocytes from OT I-T. cruzi-infected mice, demonstrating that innate CD8+ thymocytes are able to protect against a lethal T. cruzi infection in an Ag-independent manner. Interestingly, we obtained similar results when using thymocytes from systemic IL-12 + IL-18-treated mice. This data indicates that cytokines triggered during the acute stage of a Th1 infectious process induce thymic production of IL-4 along with IL-15 expression resulting in an adequate niche for development of innate CD8+ T cells as early as the double positive (DP) stage. Our data demonstrate that the thymus can sense systemic inflammatory situations and alter its conventional CD8 developmental pathway when a rapid innate immune response is required to control different types of pathogens.
Subject(s)
Interleukin-15/metabolism , Interleukin-4/metabolism , Thymus Gland/immunology , Animals , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Cell Differentiation/immunology , Cytokines/metabolism , Female , Immunity, Innate/genetics , Interleukin-12/metabolism , Interleukin-15/genetics , Interleukin-18/metabolism , Interleukin-4/genetics , Male , Mice , Mice, Inbred C57BL , Natural Killer T-Cells/immunology , Signal Transduction , Th1 Cells/immunology , Thymocytes/metabolism , Thymus Gland/metabolism , Thymus Gland/pathologyABSTRACT
Low grade, chronic inflammation is a critical risk factor for immunologic dysfunction including autoimmune diseases. However, the multiplicity of complex mechanisms and lack of relevant murine models limit our understanding of the precise role of chronic inflammation. To address these hurdles, we took advantage of multi-omics data and a unique murine model with a low but chronic expression of IFN-γ, generated by replacement of the AU-rich element (ARE) in the 3' UTR region of IFN-γ mRNA with random nucleotides. Herein, we demonstrate that low but differential expression of IFN-γ in mice by homozygous or heterozygous ARE replacement triggers distinctive gut microbial alterations, of which alteration is female-biased with autoimmune-associated microbiota. Metabolomics data indicates that gut microbiota-dependent metabolites have more robust sex-differences than microbiome profiling, particularly those involved in fatty acid oxidation and nuclear receptor signaling. More importantly, homozygous ARE-Del mice have dramatic changes in tryptophan metabolism, bile acid and long-chain lipid metabolism, which interact with gut microbiota and nuclear receptor signaling similarly with sex-dependent metabolites. Consistent with these findings, nuclear receptor signaling, encompassing molecules such as PPARs, FXR, and LXRs, was detectable as a top canonical pathway in comparison of blood and tissue-specific gene expression between female homozygous vs heterozygous ARE-Del mice. Further analysis implies that dysregulated autophagy in macrophages is critical for breaking self-tolerance and gut homeostasis, while pathways interact with nuclear receptor signaling to regulate inflammatory responses. Overall, pathway-based integration of multi-omics data provides systemic and cellular insights about how chronic inflammation driven by IFN-γ results in the development of autoimmune diseases with specific etiopathological features.
Subject(s)
Autoimmune Diseases/immunology , Dysbiosis/immunology , Inflammation/immunology , Interferon-gamma/metabolism , Macrophages/immunology , 3' Untranslated Regions/genetics , AU Rich Elements/genetics , Animals , Autophagy , Chronic Disease , Female , Gastrointestinal Microbiome/immunology , Interferon-gamma/genetics , Male , Mice , Mice, Knockout , Receptors, Cytoplasmic and Nuclear/metabolism , Sexism , Signal TransductionABSTRACT
We have reported on a murine model of autoimmune cholangitis, generated by altering the AU-rich element (ARE) by deletion of the interferon gamma (IFN-γ) 3' untranslated region (coined ARE-Del-/- ), that has striking similarities to human primary biliary cholangitis (PBC) with female predominance. Previously, we suggested that the sex bias of autoimmune cholangitis was secondary to intense and sustained type I and II IFN signaling. Based on this thesis, and to define the mechanisms that lead to portal inflammation, we specifically addressed the hypothesis that type I IFNs are the driver of this disease. To accomplish these goals, we crossed ARE-Del-/- mice with IFN type I receptor alpha chain (Ifnar1) knockout mice. We report herein that loss of type I IFN receptor signaling in the double construct of ARE-Del-/- Ifnar1-/- mice dramatically reduces liver pathology and abrogated sex bias. More importantly, female ARE-Del-/- mice have an increased number of germinal center (GC) B cells as well as abnormal follicular formation, sites which have been implicated in loss of tolerance. Deletion of type I IFN signaling in ARE-Del-/- Ifnar1-/- mice corrects these GC abnormalities, including abnormal follicular structure. CONCLUSION: Our data implicate type I IFN signaling as a necessary component of the sex bias of this murine model of autoimmune cholangitis. Importantly these data suggest that drugs that target the type I IFN signaling pathway would have potential benefit in the earlier stages of PBC. (Hepatology 2018;67:1408-1419).
Subject(s)
Autoimmune Diseases/immunology , Cholangitis/immunology , Interferon Type I/genetics , Liver/pathology , Animals , B-Lymphocytes/immunology , Disease Models, Animal , Female , Flow Cytometry , Liver/immunology , Liver/metabolism , Male , Mice , Mice, Knockout , Sex Factors , Signal Transduction/immunologyABSTRACT
UNLABELLED: In most autoimmune diseases the serologic hallmarks of disease precede clinical pathology by years. Therefore, the use of animal models in defining early disease events becomes critical. We took advantage of a "designer" mouse with dysregulation of interferon gamma (IFNγ) characterized by prolonged and chronic expression of IFNγ through deletion of the IFNγ 3'-untranslated region adenylate uridylate-rich element (ARE). The ARE-Del(-/-) mice develop primary biliary cholangitis (PBC) with a female predominance that mimics human PBC that is characterized by up-regulation of total bile acids, spontaneous production of anti-mitochondrial antibodies, and portal duct inflammation. Transfer of CD4 T cells from ARE-Del(-/-) to B6/Rag1(-/-) mice induced moderate portal inflammation and parenchymal inflammation, and RNA sequencing of liver gene expression revealed that up-regulated genes potentially define early stages of cholangitis. Interestingly, up-regulated genes specifically overlap with the gene expression signature of biliary epithelial cells in PBC, implying that IFNγ may play a pathogenic role in biliary epithelial cells in the initiation stage of PBC. Moreover, differentially expressed genes in female mice have stronger type 1 and type 2 IFN signaling and lymphocyte-mediated immune responses and thus may drive the female bias of the disease. CONCLUSION: Changes in IFNγ expression are critical for the pathogenesis of PBC. (Hepatology 2016;64:1189-1201).
Subject(s)
Autoimmune Diseases/etiology , Cholangitis/immunology , Interferon-gamma/biosynthesis , Animals , Autoimmune Diseases/metabolism , Cholangitis/metabolism , Female , Male , Mice , Sex FactorsABSTRACT
Aplastic anemia (AA) is characterized by hypocellular marrow and peripheral pancytopenia. Because interferon gamma (IFN-γ) can be detected in peripheral blood mononuclear cells of AA patients, it has been hypothesized that autoreactive T lymphocytes may be involved in destroying the hematopoietic stem cells. We have observed AA-like symptoms in our IFN-γ adenylate-uridylate-rich element (ARE)-deleted (del) mice, which constitutively express a low level of IFN-γ under normal physiologic conditions. Because no T-cell autoimmunity was observed, we hypothesized that IFN-γ may be directly involved in the pathophysiology of AA. In these mice, we did not detect infiltration of T cells in bone marrow (BM), and the existing T cells seemed to be hyporesponsive. We observed inhibition in myeloid progenitor differentiation despite an increase in serum levels of cytokines involved in hematopoietic differentiation and maturation. Furthermore, there was a disruption in erythropoiesis and B-cell differentiation. The same phenomena were also observed in wild-type recipients of IFN-γ ARE-del BM. The data suggest that AA occurs when IFN-γ inhibits the generation of myeloid progenitors and prevents lineage differentiation, as opposed to infiltration of activated T cells. These results may be useful in improving treatment as well as maintaining a disease-free status.
Subject(s)
Anemia, Aplastic/immunology , Cell Differentiation/immunology , Cell Lineage/immunology , Hematopoietic Stem Cells/immunology , Interferon-gamma/immunology , Anemia, Aplastic/genetics , Anemia, Aplastic/metabolism , Animals , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/pharmacology , Bone Marrow Cells/immunology , Bone Marrow Cells/metabolism , Bone Marrow Transplantation/methods , Cell Differentiation/genetics , Cell Lineage/genetics , Erythropoiesis/drug effects , Erythropoiesis/genetics , Erythropoiesis/immunology , Flow Cytometry , Hematopoietic Stem Cells/metabolism , Humans , Interferon-gamma/genetics , Interferon-gamma/metabolism , Mice, Inbred BALB C , Mice, Knockout , T-Lymphocytes/immunology , T-Lymphocytes/metabolismABSTRACT
We generated a mouse model with a 162 nt AU-rich element (ARE) region deletion in the 3' untranslated region (3'UTR) of the interferon-gamma (IFN-γ) gene that results in chronic circulating serum IFN-γ levels. Mice homozygous for the ARE deletion (ARE-Del) (-/-) present both serologic and cellular abnormalities typical of patients with systemic lupus erythematosus (SLE). ARE-Del(-/-) mice display increased numbers of pDCs in bone marrow and spleen. Addition of IFN-γ to Flt3-ligand (Flt3L) treated in vitro bone marrow cultures results in a 2-fold increase in pDCs with concurrent increases in IRF8 expression. Marginal zone B (MZB) cells and marginal zone macrophages (MZMs) are absent in ARE-Del(-/-) mice. ARE-Del(+/-) mice retain both MZB cells and MZMs and develop no or mild autoimmunity. However, low dose clodronate treatment in ARE-Del(+/-) mice specifically eliminates MZMs and promotes anti-DNA antibody development and glomerulonephritis. Our findings demonstrate the consequences of a chronic IFN-γ milieu on B220(+) cell types and in particular the impact of MZB cell loss on MZM function in autoimmunity. Furthermore, similarities between disease states in ARE-Del(-/-) mice and SLE patients suggest that IFN-γ may not only be a product of SLE but may be critical for disease onset and progression.
Subject(s)
AU Rich Elements/genetics , Base Sequence , Interferon-gamma , Lupus Nephritis/immunology , Sequence Deletion , Animals , Antibodies, Antinuclear/immunology , B-Lymphocytes/immunology , B-Lymphocytes/pathology , Gene Expression Regulation/genetics , Gene Expression Regulation/immunology , Humans , Interferon Regulatory Factors/genetics , Interferon Regulatory Factors/immunology , Interferon-gamma/genetics , Interferon-gamma/immunology , Lupus Nephritis/genetics , Macrophages/immunology , Macrophages/pathology , Membrane Proteins/genetics , Membrane Proteins/immunology , Mice , Mice, KnockoutABSTRACT
The thymus plays a crucial role in T cell differentiation, a complex process influenced by various factors such as antigens, the microenvironment and thymic architecture. The way the thymus resolves infections is critical, as chronic persistence of microbes or inflammatory mediators can obstruct the differentiation. Here, we illustrate that following inflammatory T helper 1 infectious processes like those caused by Candida albicans or Trypanosoma cruzi, single positive thymocytes adopt a mature phenotype. Further investigations focused on T. cruzi infection, reveal a substantial existence of CD44+ cells in both the cortical and medullary areas of the thymus at the onset of infection. This disturbance coincides with heightened interferon gamma (IFNγ) production by thymocytes and an increased cytotoxic capacity against T. cruzi-infected macrophages. Additionally, we observe a reduced exportation capacity in T. cruzi-infected mice. Some alterations can be reversed in IFNγ knockout mice (KO). Notably, the majority of these effects can be replicated by systemic expression of interleukin (IL)-12+IL-18, underlining the predominantly inflammatory rather than pathogen-specific nature of these phenomena. Understanding the mechanisms through which systemic inflammation disrupts normal T cell development, as well as subsequent T cell exportation to secondary lymphoid organs (SLO) is pivotal for comprehending susceptibility to diseases in different pathological scenarios.
Subject(s)
Chagas Disease , Cytokines , Mice, Knockout , Th1 Cells , Thymus Gland , Trypanosoma cruzi , Animals , Chagas Disease/immunology , Chagas Disease/parasitology , Chagas Disease/pathology , Chagas Disease/metabolism , Trypanosoma cruzi/immunology , Mice , Thymus Gland/immunology , Thymus Gland/pathology , Th1 Cells/immunology , Cytokines/metabolism , Interferon-gamma/metabolism , Interferon-gamma/immunology , Mice, Inbred C57BL , Inflammation/immunology , Cell DifferentiationABSTRACT
Mature lymphocyte immigration into the thymus has been documented in mouse, rat, and pig models, and highly increases when cells acquire an activated phenotype. Entrance of peripheral B and T cells into the thymus has been described in healthy and pathological situations. However, it has not been proposed that leukocyte recirculation to the thymus could be a common feature occurring during the early phase of a Th1 inflammatory/infectious process when a large number of peripheral cells acquire an activated phenotype and the cellularity of the thymus is seriously compromised. The data we present here demonstrate that in well-established Th1 models triggered by different types of immunogens, for example, LPS treatment (a bacterial product), Candida albicans infection (a fungus), and after Trypanosoma cruzi infection (a parasite), a large number of mature peripheral B and T cells enter the thymus. This effect is dependent on, but not exclusive of, the available space in the thymus. Our data also demonstrate that MCP-1/CCR2 (where MCP-1 is monocyte chemoattractant protein-1) interaction is responsible for the infiltration of peripheral cells to the thymus in these Th1-inflammatory/infectious situations. Finally, systemic expression of IL-12 and IL-18 produced during the inflammatory process is ultimately responsible for these migratory events.
Subject(s)
B-Lymphocytes/immunology , Candida albicans/immunology , Candidiasis/immunology , Chagas Disease/immunology , Chemokine CCL2/metabolism , Receptors, CCR2/metabolism , Th1 Cells/immunology , Trypanosoma cruzi/immunology , Animals , B-Lymphocytes/microbiology , B-Lymphocytes/parasitology , Cell Movement , Cells, Cultured , Female , Interleukin-12/immunology , Interleukin-18/immunology , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Protein Binding , Th1 Cells/microbiology , Th1 Cells/parasitology , Thymus Gland/immunology , Thymus Gland/pathologyABSTRACT
The fate of invariant NKT (iNKT) cells following activation remains controversial and unclear. We systemically examined how iNKT cells are regulated following TCR-dependent and -independent activation with α-galactosylceramide (αGC) or IL-18 plus IL-12, respectively. Our studies reveal activation by αGC or IL-18 plus IL-12 induced transient depletion of iNKT cells exclusively in the liver that was independent of caspase 3-mediated apoptosis. The loss of iNKT cells was followed by repopulation and expansion of phenotypically distinct cells via different mechanisms. Liver iNKT cell expansion following αGC, but not IL-18 plus IL-12, treatment required an intact spleen and IFN-γ. Additionally, IL-18 plus IL-12 induced a more prolonged expansion of liver iNKT cells compared with αGC. iNKT cells that repopulate the liver following αGC had higher levels of suppressive receptors PD-1 and Lag3, whereas those that repopulate the liver following IL-18 plus IL-12 had increased levels of TCR and ICOS. In contrast to acute treatment that caused a transient loss of iNKT cells, chronic αGC or IL-18 plus IL-12 treatment caused long-term systemic loss requiring an intact thymus for repopulation of the liver. This report reveals a previously undefined role for the liver in the depletion of activated iNKT cells. Additionally, TCR-dependent and -independent activation differentially regulate iNKT cell distribution and phenotype. These results provide new insights for understanding how iNKT cells are systemically regulated following activation.
Subject(s)
Cell Differentiation/immunology , Liver/immunology , Liver/metabolism , Lymphocyte Activation/immunology , Lymphocyte Depletion , Natural Killer T-Cells/cytology , Natural Killer T-Cells/immunology , Receptors, Antigen, T-Cell/physiology , Animals , Caspase 3/metabolism , Galactosylceramides/physiology , Immunophenotyping , Interleukin-12/physiology , Interleukin-18/physiology , Liver/cytology , Lymphocyte Depletion/methods , Lymphoid Tissue/cytology , Lymphoid Tissue/immunology , Lymphoid Tissue/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Natural Killer T-Cells/metabolism , Receptors, Antigen, T-Cell/metabolism , Signal Transduction/immunology , Spleen/cytology , Spleen/immunology , Spleen/metabolismABSTRACT
The presence of CD8+ T cells with a memory phenotype in nonimmunized mice has been noted for decades, but it was not until about 2 decades ago that they began to be studied in greater depth. Currently called virtual memory CD8+ T cells, they consist of a heterogeneous group of cells with memory characteristics, without any previous contact with their specific antigens. These cells were identified in mice, but a few years ago, a cell type with characteristics equivalent to the murine ones was described in healthy humans. In this review, we address the different aspects of its biology mainly developed in murine models and what is currently known about its cellular equivalent in humans.
Subject(s)
CD8-Positive T-Lymphocytes , Immunologic Memory , Humans , Mice , Animals , Mice, Inbred C57BLABSTRACT
The linker for activation of T cells (LAT) and the linker for activation of B cells (LAB/NTAL/LAT2) are integral proteins in receptor coupling to downstream events. Both proteins are expressed in natural killer (NK) cells and LAT is phosphorylated during target cell interactions or ligation of the immunoreceptor tyrosine-based activation motif (ITAM)-coupled CD16. Regardless, Lat(-/-) mice exhibit normal natural and antibody-mediated killing. Here we place both LAT and LAB in the DAP12 pathway of NK cells. Moreover, we unveil a LAT-independent pathway that requires expression of Syk. Mice lacking either LAT or LAB have a skewed Ly49 repertoire, and activated NK cells from Lat(-/-) mice have reduced responses to the ITAM-coupled receptor NK1.1. In contrast, resting Lat(-/-) NK cells show intact NK1.1 responses, whereas NK cells without LAB are hyperactive. Elimination of both adaptors severely reduces NK1.1 signaling under both conditions. Together these data show that NK ITAMs preferentially use a signaling cassette regulated by interplay between LAT and LAB. Activation by interleukin-2 causes a shift to greater dependency on LAT due to suppression of Syk signaling. The overlapping use of multiple adaptors permits fine-tuning of NK-cell ITAM responses over the course of an immune response.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Antigens, Ly/immunology , Killer Cells, Natural/immunology , Lectins, C-Type/immunology , Lymphocyte Activation/immunology , Signal Transduction/immunology , Adaptor Proteins, Vesicular Transport/metabolism , Amino Acid Motifs , Animals , Antibodies/pharmacology , Calcium Signaling/drug effects , Cell Line , Cytokines/biosynthesis , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Killer Cells, Natural/cytology , Killer Cells, Natural/drug effects , Lymphocyte Activation/drug effects , Membrane Proteins/metabolism , Mice , Mice, Inbred C57BL , Phosphoproteins/metabolism , Phosphorylation/drug effects , Protein-Tyrosine Kinases/metabolism , Receptors, IgG/metabolism , Receptors, NK Cell Lectin-Like , Signal Transduction/drug effects , Syk KinaseABSTRACT
Cell cycle regulation is essential for proper homeostasis of hematopoietic cells. Cdk2 is a major regulator of S phase entry, is activated by mitogenic cytokines, and has been suggested to be involved in antigen-induced apoptosis of T lymphocytes. The role of Cdk2 in hematopoietic cells and apoptosis in vivo has not yet been addressed. To determine whether Cdk2 plays a role in these cells, we performed multiple analyses of bone marrow cells, thymocytes, and splenocytes from Cdk2 knockout mice. We found that Cdk2 is not required in vivo to induce apoptosis in lymphocytes, a result that differs from previous pharmacological in vitro studies. Furthermore, thymocyte maturation was not affected by the lack of Cdk2. We then analyzed the hematopoietic stem cell compartment and found similar proportions of stem cells and progenitors in Cdk2(-)(/)(-) and wild-type animals. Knockouts of Cdk2 inhibitors (p21, p27) affect stem cell renewal, but a competitive graft experiment indicated that renewal and multilineage differentiation are normal in the absence of Cdk2. Finally, we stimulated T lymphocytes or macrophages to induce proliferation and observed normal reactivation of Cdk2(-)(/)(-) quiescent cells. Our results indicate that Cdk2 is not required for proliferation and differentiation of hematopoietic cells in vivo, although in vitro analyses consider Cdk2 to be a major player in proliferation and apoptosis in these cells and a potential target for therapy.
Subject(s)
Apoptosis , Cyclin-Dependent Kinase 2/deficiency , Hematopoiesis/physiology , T-Lymphocytes/cytology , Animals , Bone Marrow Cells/cytology , Mice , Spleen/cytology , Stem Cells/cytologyABSTRACT
IL-7 is required for T cell differentiation and mature T cell homeostasis and promotes pro-B cell proliferation and survival. Tyrosine phosphorylation plays a central role in IL-7 signaling. We identified by two-dimensional electrophoresis followed by anti-phosphotyrosine immunoblotting and mass spectrometry sixteen tyrosine phosphorylated proteins from the IL-7-dependent cell line D1. IL-7 stimulation induced the phosphorylation of the proteins STI1, ATIC and hnRNPH, involved in pathways related to survival, proliferation and gene expression, respectively, and increased the phosphorylation of CrkL, a member of a family of adaptors including the highly homologous Crk isoforms CrkII and CrkI, important in multiple signaling pathways. We observed an increased phosphorylation of CrkL in murine pro-B cells and in murine and human T cells. In addition, IL-7 increased the association of CrkL with the transcription factor Stat5, essential for IL-7 pro-survival activity. The selective tyrosine kinase inhibitor Imatinib. counteracted the IL-7 pro-survival effect in D1 cells and decreased CrkL phosphorylation. These data suggested that CrkL could play a pro-survival role in IL-7-mediated signaling. We observed that pro-B cells also expressed, in addition to CrkL, the Crk isoforms CrkII and CrkI and therefore utilized pro-B cells conditionally deficient in all three to evaluate the role of these proteins. The observation that the IL-7 pro-survival effect was reduced in Crk/CrkL conditionally-deficient pro-B cells further pointed to a pro-survival role of these adaptors. To further evaluate the role of these proteins, gene expression studies were performed in Crk/CrkL conditionally-deficient pro-B cells. IL-7 decreased the transcription of the receptor LAIR1, which inhibits B cell proliferation, in a Crk/CrkL-dependent manner, suggesting that the Crk family of proteins may promote pro-B cell proliferation. Our data contribute to the understanding of IL-7 signaling and suggest the involvement of Crk family proteins in pathways promoting survival and proliferation.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Interleukin-7/pharmacology , Nuclear Proteins/metabolism , Adaptor Proteins, Signal Transducing/genetics , Animals , Apoptosis/drug effects , B-Lymphocytes/cytology , B-Lymphocytes/drug effects , B-Lymphocytes/metabolism , Gene Expression Regulation/drug effects , Humans , Imatinib Mesylate/pharmacology , Male , Mice , Mice, Inbred C57BL , Nuclear Proteins/genetics , Phosphorylation/drug effects , Protein Isoforms/genetics , Protein Isoforms/metabolism , STAT5 Transcription Factor/metabolism , Signal Transduction/drug effects , T-Lymphocytes/cytology , T-Lymphocytes/drug effects , T-Lymphocytes/metabolismABSTRACT
Posttranscriptional control of gamma interferon (IFN-gamma) gene expression has not been extensively studied and is poorly understood. Our work describes a posttranscriptional mechanism that modulates IFN-gamma mRNA expression in stimulated natural killer (NK) cells through nuclear retention of the IFN-gamma mRNA. This is evidenced by the elevated and sustained nuclear accumulation of both precursor and processed IFN-gamma mRNAs in NK cells stimulated with interleukin-12 (IL-12). The elevated nuclear mRNA accumulation persists long after transcriptional activity has subsided and the rate of cytoplasmic IFN-gamma mRNA accumulation has dropped. The IL-12-induced nuclear retention of the IFN-gamma mRNA prevails until a secondary cytokine stimulus is received. The secondary stimulus, which is initiated by IL-2, mediates transcription-independent movement of the nuclear IFN-gamma mRNA. Concurrent with the nucleocytoplasmic movement of the IFN-gamma mRNA, we have observed increases in the amount of processed nuclear IFN-gamma mRNA that are greater than that seen for the unprocessed IFN-gamma mRNA. The increase in processed IFN-gamma mRNA appears to be due to increased mRNA stability which then promotes increased nucleocytoplasmic shuttling of the mature IFN-gamma mRNA. These data support a model whereby mobilization of nuclear IFN-gamma mRNA stores allows NK cells to rapidly and robustly respond to secondary cytokine activators in a transcription-independent manner, thus shortening the time for overall cellular response to inflammatory signals.
Subject(s)
Gene Expression Regulation/physiology , Interferon-gamma/genetics , Interleukin-12/metabolism , Interleukin-2/metabolism , RNA Processing, Post-Transcriptional/physiology , Cell Line , Cell Nucleus/metabolism , Humans , Interferon-gamma/metabolism , Interleukin-12/pharmacology , Interleukin-2/pharmacology , Killer Cells, Natural/cytology , Killer Cells, Natural/drug effects , Killer Cells, Natural/metabolism , Polyadenylation/physiology , RNA Stability/drug effects , RNA Stability/physiology , RNA, Messenger/metabolismABSTRACT
The proinflammatory cytokine, interleukin-18 (IL-18), is a natural killer (NK) cell activator that induces NK cell cytotoxicity and interferon-gamma (IFN-gamma) expression. In this report, we define a novel role for IL-18 as an NK cell protective agent. Specifically, IL-18 prevents NK cell death initiated by different and distinct stress mechanisms. IL-18 reduces NK cell self-destruction during NK-targeted cell killing, and in the presence of staurosporin, a potent apoptotic inducer, IL-18 reduces caspase-3 activity. The critical regulatory step in this process is downstream of the mitochondrion and involves reduced cleavage and activation of caspase-9 and caspase-3. The ability of IL-18 to regulate cell survival is not limited to a caspase death pathway in that IL-18 augments tumor necrosis factor (TNF) signaling, resulting in increased and prolonged mRNA expression of c-apoptosis inhibitor 2 (cIAP2), a prosurvival factor and caspase-3 inhibitor, and TNF receptor-associated factor 1 (TRAF1), a prosurvival protein. The cumulative effects of IL-18 define a novel role for this cytokine as a molecular survival switch that functions to both decrease cell death through inhibition of the mitochondrial apoptotic pathway and enhance TNF induction of prosurvival factors.
Subject(s)
Apoptosis , Inhibitor of Apoptosis Proteins/biosynthesis , Interleukin-18/pharmacology , Killer Cells, Natural/immunology , Signal Transduction , Cells, Cultured , Humans , Inflammation Mediators/pharmacology , Inhibitor of Apoptosis Proteins/genetics , Killer Cells, Natural/drug effects , NF-kappa B/metabolism , RNA, Messenger/metabolism , Receptors, Tumor Necrosis Factor/metabolism , Signal Transduction/drug effects , TNF Receptor-Associated Factor 1/biosynthesis , TNF Receptor-Associated Factor 1/genetics , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
Large granular lymphocyte (LGL) leukemia is a clonal proliferative disease of T and natural killer (NK) cells. Interleukin (IL)-15 is important for the development and progression of LGL leukemia and is a survival factor for normal NK and T memory cells. IL-15 alters expression of Bcl-2 family members, Bcl-2, Bcl-XL, Bim, Noxa, and Mcl-1; however, effects on Bid have not been shown. Using an adoptive transfer model, we show that NK cells from Bid-deficient mice survive longer than cells from wild-type control mice when transferred into IL-15-null mice. In normal human NK cells, IL-15 significantly reduces Bid accumulation. Decreases in Bid are not due to alterations in RNA accumulation but result from increased proteasomal degradation. IL-15 up-regulates the E3 ligase HDM2 and we find that HDM2 directly interacts with Bid. HDM2 suppression by short hairpin RNA increases Bid accumulation lending further support for HDM2 involvement in Bid degradation. In primary leukemic LGLs, Bid levels are low but are reversed with bortezomib treatment with subsequent increases in LGL apoptosis. Overall, these data provide a novel molecular mechanism for IL-15 control of Bid that potentially links this cytokine to leukemogenesis through targeted proteasome degradation of Bid and offers the possibility that proteasome inhibitors may aid in the treatment of LGL leukemia.
Subject(s)
BH3 Interacting Domain Death Agonist Protein/immunology , Interleukin-15/immunology , Leukemia, Large Granular Lymphocytic/immunology , Lymphocytes/immunology , Proteasome Endopeptidase Complex/metabolism , Animals , BH3 Interacting Domain Death Agonist Protein/biosynthesis , BH3 Interacting Domain Death Agonist Protein/deficiency , BH3 Interacting Domain Death Agonist Protein/metabolism , Humans , Interleukin-15/deficiency , Interleukin-15/metabolism , Interleukin-15/pharmacology , Interleukin-2/immunology , Interleukin-2/metabolism , Killer Cells, Natural/immunology , Leukemia, Large Granular Lymphocytic/enzymology , Leukemia, Large Granular Lymphocytic/metabolism , Lymphocytes/enzymology , Lymphocytes/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Proteasome Inhibitors , Proto-Oncogene Proteins c-mdm2/immunology , Proto-Oncogene Proteins c-mdm2/metabolismABSTRACT
Our previous studies have identified mechanisms by which cytokine production, blocked by Ly49G2 receptor cross-linking, can be overridden. In this study we analyzed the regulation of other ITAM-positive receptor signaling on NK, NKT, and T cells and characterized the biochemical pathways involved in this signaling. Our studies demonstrate that cross-linking of NKG2D and NK1.1 results in a synergistic NK IFN-gamma response when combined with IL-12 or IL-18. Examination of NKT- and T-cell responses demonstrated that cross-linking of NKG2D and CD3 resulted in potent synergy when combined with IL-12 and, to a lesser degree, with IL-18. We have now found that both the p38 MAP kinase and the ERK-dependent signal transduction pathways are required for the synergistic response. Further mechanistic examination of the synergy indicated a potent up-regulation of total IFN-gamma mRNA in the nuclear and the cytoplasmic compartment, but mRNA half-life was not affected. Fifteen minutes of IL-12 pretreatment was sufficient to result in maximal synergistic activation, indicating that the response of the cells to the IL-12 signal was rapid and immediate. Thus, our data demonstrate that multiple convergent signals maximize the innate immune response by triggering complementary biochemical signaling pathways.
Subject(s)
Antigens, Ly/immunology , Interleukin-12/pharmacology , Interleukin-18/immunology , Lectins, C-Type/immunology , Receptors, Immunologic/immunology , Amino Acid Substitution , Animals , Antigens, Ly/genetics , Cell Line , Cytokines/analysis , Flow Cytometry , Interferon-gamma/genetics , Interleukin-18/pharmacology , Killer Cells, Natural/drug effects , Killer Cells, Natural/immunology , Lectins, C-Type/genetics , Mice , Mice, Inbred BALB C , NK Cell Lectin-Like Receptor Subfamily K , Polymerase Chain Reaction , Receptors, Immunologic/genetics , Receptors, NK Cell Lectin-Like , Receptors, Natural Killer Cell , Ribonucleases , T-Lymphocytes/drug effects , T-Lymphocytes/immunologyABSTRACT
The analysis of several Ly49 genes has identified a tissue-specific promoter adjacent to the previously defined first exon. The current study reveals the presence of an additional Ly49 promoter (Pro-1) and two noncoding exons upstream of the previously defined promoter (Pro-2). DNA sequences homologous to Pro-1 are present 4-10 kb upstream of Pro-2 in all Ly49 genes examined, and Pro-1 transcripts were detected from the Ly49a, e, g, o, and v genes. Pro-1 activity can be detected in bone marrow, embryonic thymus, freshly isolated liver NK cells, and the murine LNK cell line, but it does not function in adult thymus, sorted NK-T cells, spleen NK cells, or the EL-4 T cell line, even though these cells express Ly49 proteins. Luciferase reporter assays identified a Pro-1 core promoter region that functions in the LNK cell line but not EL-4 cells. The novel promoter is not active in mature NK cells, suggesting that Pro-1 represents an early Ly49 promoter.
Subject(s)
Antigens, Ly/genetics , Bone Marrow Cells/immunology , Bone Marrow Cells/metabolism , Gene Expression Regulation/immunology , Membrane Glycoproteins/genetics , Promoter Regions, Genetic/immunology , Thymus Gland/immunology , Thymus Gland/metabolism , 5' Untranslated Regions/immunology , Alternative Splicing/immunology , Animals , Base Sequence , Cell Line , Exons/immunology , Fetus , Killer Cells, Natural , Lectins, C-Type , Liver/cytology , Liver/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Molecular Sequence Data , Multigene Family/immunology , NK Cell Lectin-Like Receptor Subfamily A , RNA, Messenger/isolation & purification , Receptors, NK Cell Lectin-Like , Transcription Initiation Site , Transcription, Genetic/immunology , Tumor Cells, CulturedABSTRACT
The engagement of antigen receptor can initiate apoptosis of T lymphocytes through the induced expression of Fas ligand (FasL). Forskolin, an activator of the cAMP/PKA pathway, results in antagonism of Fas-dependent, activation-induced cell death (AICD) by suppressed expression of the FasL. We report that forskolin-mediated induction of inducible cAMP early repressor (ICER) correlates with transcriptional attenuation of FasL expression in the AICD model 2B4 T cell hybridoma. ICER is inducible in human peripheral blood CD3(+) T cells, but in CD19(+) B cells, its induction is less responsive to forskolin treatment. Increased expression of ICER correlates with decreased FasL expression in both T and NK cells. ICER binds specifically to the proximal DNA binding site of the nuclear factor of activated T cells (NFAT) in the FasL promoter and in the presence of the minimal NFAT DNA-binding domain, the proximal NFAT motif allows ICER and NFAT to form an NFAT/ICER ternary complex in vitro. Moreover, in the activated 2B4 T cell hybridoma, the proximal NFAT motif participates in the down-regulation of the FasL promoter mediated by ICER. These findings provide further insight into the mechanism involved in cAMP-mediated transcriptional attenuation of FasL expression in T and NK lymphocytes.