RESUMO
Angioimmunoblastic T cell lymphoma (AITL) is a peripheral T cell lymphoma that originates from T follicular helper (Tfh) cells and exhibits a prominent tumor microenvironment (TME). IDH2 and TET2 mutations co-occur frequently in AITL, but their contribution to tumorigenesis is poorly understood. We developed an AITL mouse model that is driven by Idh2 and Tet2 mutations. Malignant Tfh cells display aberrant transcriptomic and epigenetic programs that impair TCR signaling. Neoplastic Tfh cells bearing combined Idh2 and Tet2 mutations show altered cross-talk with germinal center B cells that promotes B cell clonal expansion while decreasing Fas-FasL interaction and reducing B cell apoptosis. The plasma cell count and angiogenesis are also increased in the Idh2-mutated tumors, implying a major relationship between Idh2 mutation and the characteristic AITL TME. Our mouse model recapitulates several features of human IDH2-mutated AITL and provides a rationale for exploring therapeutic targeting of Tfh-TME cross-talk for AITL patients.
Assuntos
Dioxigenases , Linfadenopatia Imunoblástica , Linfoma de Células T , Animais , Humanos , Camundongos , Dioxigenases/genética , Proteínas de Ligação a DNA/genética , Linfadenopatia Imunoblástica/genética , Isocitrato Desidrogenase/genética , Linfoma de Células T/genética , Mutação , Células T Auxiliares Foliculares/patologia , Linfócitos T Auxiliares-Indutores , Microambiente Tumoral/genéticaRESUMO
Regulatory T cells (Tregs) maintain immune homeostasis and prevent autoimmunity. Serine stimulates glutathione (GSH) synthesis and feeds into the one-carbon metabolic network (1CMet) essential for effector T cell (Teff) responses. However, serine's functions, linkage to GSH, and role in stress responses in Tregs are unknown. Here, we show, using mice with Treg-specific ablation of the catalytic subunit of glutamate cysteine ligase (Gclc), that GSH loss in Tregs alters serine import and synthesis and that the integrity of this feedback loop is critical for Treg suppressive capacity. Although Gclc ablation does not impair Treg differentiation, mutant mice exhibit severe autoimmunity and enhanced anti-tumor responses. Gclc-deficient Tregs show increased serine metabolism, mTOR activation, and proliferation but downregulated FoxP3. Limitation of cellular serine in vitro and in vivo restores FoxP3 expression and suppressive capacity of Gclc-deficient Tregs. Our work reveals an unexpected role for GSH in restricting serine availability to preserve Treg functionality.
Assuntos
Glutationa/metabolismo , Serina/metabolismo , Linfócitos T Reguladores/metabolismo , Animais , CamundongosRESUMO
Activated T cells produce reactive oxygen species (ROS), which trigger the antioxidative glutathione (GSH) response necessary to buffer rising ROS and prevent cellular damage. We report that GSH is essential for T cell effector functions through its regulation of metabolic activity. Conditional gene targeting of the catalytic subunit of glutamate cysteine ligase (Gclc) blocked GSH production specifically in murine T cells. Gclc-deficient T cells initially underwent normal activation but could not meet their increased energy and biosynthetic requirements. GSH deficiency compromised the activation of mammalian target of rapamycin-1 (mTOR) and expression of NFAT and Myc transcription factors, abrogating the energy utilization and Myc-dependent metabolic reprogramming that allows activated T cells to switch to glycolysis and glutaminolysis. In vivo, T-cell-specific ablation of murine Gclc prevented autoimmune disease but blocked antiviral defense. The antioxidative GSH pathway thus plays an unexpected role in metabolic integration and reprogramming during inflammatory T cell responses.
Assuntos
Glutamato-Cisteína Ligase/deficiência , Glutationa/metabolismo , Inflamação/metabolismo , Linfócitos T/metabolismo , Animais , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/metabolismo , Metabolismo Energético/genética , Glutamato-Cisteína Ligase/genética , Glutamina/metabolismo , Glicólise , Immunoblotting , Inflamação/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fatores de Transcrição NFATC/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
T-cell proliferation is regulated by ubiquitination but the underlying molecular mechanism remains obscure. Here we report that Lys-48-linked ubiquitination of the transcription factor KLF4 mediated by the E3 ligase Mule promotes T-cell entry into S phase. Mule is elevated in T cells upon TCR engagement, and Mule deficiency in T cells blocks proliferation because KLF4 accumulates and drives upregulation of its transcriptional targets E2F2 and the cyclin-dependent kinase inhibitors p21 and p27. T-cell-specific Mule knockout (TMKO) mice develop exacerbated experimental autoimmune encephalomyelitis (EAE), show impaired generation of antigen-specific CD8+ T cells with reduced cytokine production, and fail to clear LCMV infections. Thus, Mule-mediated ubiquitination of the novel substrate KLF4 regulates T-cell proliferation, autoimmunity and antiviral immune responses in vivo.
Assuntos
Proliferação de Células , Fatores de Transcrição Kruppel-Like/química , Fatores de Transcrição Kruppel-Like/metabolismo , Linfócitos T/citologia , Linfócitos T/enzimologia , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Motivos de Aminoácidos , Animais , Feminino , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/imunologia , Coriomeningite Linfocítica/genética , Coriomeningite Linfocítica/imunologia , Coriomeningite Linfocítica/fisiopatologia , Coriomeningite Linfocítica/virologia , Vírus da Coriomeningite Linfocítica/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Linfócitos T/imunologia , Linfócitos T/metabolismo , Proteínas Supressoras de Tumor/genética , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
The regulation of toll-like receptor (TLR) signaling in a tumor microenvironment is poorly understood despite its importance in cancer biology. To address this problem, TLR7-responses of chronic lymphocytic leukemia (CLL) cells were studied in the presence and absence of a human stromal cell-line derived from a leukemic spleen. CLL cells alone produced high levels of tumor necrosis factor (TNF)-α and proliferated in response to TLR7-agonists. A signal transducer and activator of transcription 3 -activating stromal factor, identified as interleukin (IL)-6, was found to upregulate microRNA (miR)-17 and miR-19a, target TLR7 and TNFA messenger RNA, and induce a state of tolerance to TLR7-agonists in CLL cells. Overexpression of the miR-17-92 cluster tolerized CLL cells directly and miR-17 and miR-19a antagomiRs restored TLR7-signaling. Inhibition of IL-6 signaling with antibodies or small-molecule Janus kinase inhibitors reversed tolerization and increased TLR7-stimulated CLL cell numbers in vitro and in NOD-SCIDγc (null) mice. These results suggest IL-6 can act as tumor suppressor in CLL by inhibiting TLR-signaling.
Assuntos
Linfócitos B/imunologia , Regulação Leucêmica da Expressão Gênica , Interleucina-6/imunologia , MicroRNAs/imunologia , Células Estromais/imunologia , Animais , Anticorpos Neutralizantes/farmacologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/patologia , Linhagem Celular Tumoral , Técnicas de Cocultura , Humanos , Tolerância Imunológica , Interleucina-6/genética , Janus Quinase 1/genética , Janus Quinase 1/imunologia , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , MicroRNAs/genética , Oligonucleotídeos/farmacologia , RNA Longo não Codificante , RNA Mensageiro/genética , RNA Mensageiro/imunologia , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/imunologia , Transdução de Sinais , Células Estromais/efeitos dos fármacos , Células Estromais/patologia , Receptor 7 Toll-Like/antagonistas & inibidores , Receptor 7 Toll-Like/genética , Receptor 7 Toll-Like/imunologia , Microambiente Tumoral , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Appropriate control of immune responses is a critical determinant of health. Here, we show that choline acetyltransferase (ChAT) is expressed and ACh is produced by B cells and other immune cells that have an impact on innate immunity. ChAT expression occurs in mucosal-associated lymph tissue, subsequent to microbial colonization, and is reduced by antibiotic treatment. MyD88-dependent Toll-like receptor up-regulates ChAT in a transient manner. Unlike the previously described CD4(+) T-cell population that is stimulated by norepinephrine to release ACh, ChAT(+) B cells release ACh after stimulation with sulfated cholecystokinin but not norepinephrine. ACh-producing B-cells reduce peritoneal neutrophil recruitment during sterile endotoxemia independent of the vagus nerve, without affecting innate immune cell activation. Endothelial cells treated with ACh in vitro reduced endothelial cell adhesion molecule expression in a muscarinic receptor-dependent manner. Despite this ability, ChAT(+) B cells were unable to suppress effector T-cell function in vivo. Therefore, ACh produced by lymphocytes has specific functions, with ChAT(+) B cells controlling the local recruitment of neutrophils.
Assuntos
Acetilcolina/biossíntese , Imunidade Adaptativa/fisiologia , Imunidade Inata/fisiologia , Linfócitos/imunologia , Linfócitos/metabolismo , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Colina O-Acetiltransferase/genética , Colina O-Acetiltransferase/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Feminino , Tecido Linfoide/imunologia , Tecido Linfoide/metabolismo , Tecido Linfoide/microbiologia , Macrófagos/imunologia , Macrófagos/metabolismo , Metagenoma/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Fator 88 de Diferenciação Mieloide/deficiência , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Neuroimunomodulação , Gravidez , Receptores de Neurotransmissores/imunologia , Receptores de Neurotransmissores/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Receptores Toll-Like/metabolismoRESUMO
The endogenous metabolite of estradiol, 2-Methoxyestradiol (2ME2), is an antimitotic and antiangiogenic cancer drug candidate that also exhibits disease-modifying activity in animal models of rheumatoid arthritis (RA). We found that 2ME2 dramatically suppresses development of mouse experimental autoimmune encephalomyelitis (EAE), a rodent model of multiple sclerosis (MS). 2ME2 inhibits in vitro lymphocyte activation, cytokine production, and proliferation in a dose-dependent fashion. 2ME2 treatment of lymphocytes specifically reduced the nuclear translocation and transcriptional activity of nuclear factor of activated T-cells (NFAT) c1, whereas NF-κB and activator protein 1 (AP-1) activation were not adversely affected. We therefore propose that 2ME2 attenuates EAE through disruption of the NFAT pathway and subsequent lymphocyte activation. By extension, our findings provide a molecular rationale for the use of 2ME2 as a tolerable oral immunomodulatory agent for the treatment of autoimmune disorders such as MS in humans.
Assuntos
Encefalomielite Autoimune Experimental/tratamento farmacológico , Estradiol/análogos & derivados , 2-Metoxiestradiol , Animais , Autoimunidade , Linfócitos T CD4-Positivos/citologia , Citocinas/biossíntese , Estradiol/farmacologia , Humanos , Ativação Linfocitária , Linfócitos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Esclerose Múltipla/imunologia , NF-kappa B/metabolismo , Fatores de Transcrição NFATC/metabolismo , Transdução de Sinais , Fator de Transcrição AP-1/metabolismo , Moduladores de Tubulina/farmacologiaRESUMO
Cellular homeostasis is controlled by pathways that balance cell death with survival. Mcl-1 ubiquitin ligase E3 (Mule) is an E3 ubiquitin ligase that targets the proapoptotic molecule p53 for polyubiquitination and degradation. To elucidate the role of Mule in B lymphocyte homeostasis, B cell-specific Mule knockout (BMKO) mice were generated using the Cre-LoxP recombination system. Analysis of BMKO mice showed that Mule was essential for B cell development, proliferation, homeostasis, and humoral immune responses. p53 transactivation was increased by two- to fourfold in Mule-deficient B cells at steady state. Genetic ablation of p53 in BMKO mice restored B cell development, proliferation, and homeostasis. p53 protein was increased in resting Mule-deficient mouse embryonic fibroblasts (MEFs) and embryonic stem (ES) cells. Loss of Mule in both MEFs and B cells at steady state resulted in increased levels of phospho-ataxia telangiectasia mutated (ATM) and the ATM substrate p53. Under genotoxic stress, BMKO B cells were resistant to apoptosis, and control MEFs exhibited evidence of a physical interaction between Mule and phospho-ATM. Phospho-ATM, phospho-p53, and Brca1 levels were reduced in Mule-deficient B cells and MEFs subjected to genotoxic stress. Thus, Mule regulates the ATM-p53 axis to maintain B cell homeostasis under both steady-state and stress conditions.
Assuntos
Linfócitos B/enzimologia , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Linfócitos B/citologia , Linfócitos B/imunologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Linhagem Celular , Proliferação de Células , Dano ao DNA/imunologia , Feminino , Ordem dos Genes , Marcação de Genes , Homeostase/genética , Homeostase/imunologia , Imunidade Humoral/genética , Switching de Imunoglobulina/genética , Isotipos de Imunoglobulinas/sangue , Ativação Linfocitária/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Supressora de Tumor p53/genética , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/genéticaRESUMO
Mice deficient for interferon regulatory factor (Irf)2 (Irf2(-/-) mice) exhibit immunological abnormalities and cannot survive lymphocytic choriomeningitis virus infection. The pancreas of these animals is highly inflamed, a phenotype replicated by treatment with poly(I:C), a synthetic double-stranded RNA. Trypsinogen5 mRNA was constitutively up-regulated about 1,000-fold in Irf2(-/-) mice compared with controls as assessed by quantitative RT-PCR. Further knockout of IFNα/ß receptor 1(Ifnar1) abolished poly(I:C)-induced pancreatitis but had no effect on the constitutive up-regulation of trypsinogen5 gene, indicating crucial type I IFN signaling to elicit the inflammation. Analysis of Ifnar1(-/-) mice confirmed type I IFN-dependent transcriptional activation of dsRNA-sensing pattern recognition receptor genes MDA5, RIG-I, and TLR3, which induced poly(I:C)-dependent cell death in acinar cells in the absence of IRF2. We speculate that Trypsin5, the trypsinogen5 gene product, leaking from dead acinar cells triggers a chain reaction leading to lethal pancreatitis in Irf2(-/-) mice because it is resistant to a major endogenous trypsin inhibitor, Spink3.
Assuntos
Fator Regulador 2 de Interferon/metabolismo , Pancreatite/metabolismo , RNA de Cadeia Dupla/metabolismo , Transcrição Gênica , Tripsinogênio/genética , Células Acinares/metabolismo , Animais , Catepsina B/metabolismo , Glicoproteínas/metabolismo , Células HEK293 , Células HeLa , Humanos , Camundongos , Camundongos Transgênicos , Pancreatite/genética , Poli I-C/genética , Proteínas Secretadas pela Próstata/metabolismo , Inibidor da Tripsina Pancreática de Kazal , Inibidores da Tripsina/farmacologiaRESUMO
14-3-3σ regulates cytokinesis and cell cycle arrest induced by DNA damage but its role in the immune system is unknown. Using gene-targeted 14-3-3σ-deficient (i.e., KO) mice, we studied the role of 14-3-3σ in B-cell functions. Total numbers of B cells were reduced by spontaneous apoptosis of peripheral B cells. Upon B-cell antigen receptor engagement in vitro, KO B cells did not proliferate properly or up-regulate CD86. In response to T cell-independent antigens, KO B cells showed poor secretion of antigen-specific IgM. This deficit led to increased lethality of KO mice after vesicular stomatitis virus infection. KO B cells showed elevated total FOXO transcriptional activity but also increased FOXO1 degradation. Coimmunoprecipitation revealed that endogenous 14-3-3σ protein formed a complex with FOXO1 protein. Our results suggest that 14-3-3σ maintains FOXO1 at a consistent level critical for normal B-cell antigen receptor signaling and B-cell survival.
Assuntos
Proteínas 14-3-3/imunologia , Linfócitos B/imunologia , Fatores de Transcrição Forkhead/imunologia , Homeostase/imunologia , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Transferência Adotiva , Animais , Antígenos/imunologia , Apoptose/imunologia , Linfócitos B/citologia , Linfócitos B/metabolismo , Western Blotting , Proliferação de Células , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Feminino , Ficoll/análogos & derivados , Ficoll/imunologia , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ligação Proteica , Receptores de Antígenos de Linfócitos B/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Trinitrobenzenos/imunologiaRESUMO
Fas is highly expressed in activated and germinal center (GC) B cells but can potentially be inactivated by misguided somatic hypermutation. We employed conditional Fas-deficient mice to investigate the physiological functions of Fas in various B cell subsets. B cell-specific Fas-deficient mice developed fatal lymphoproliferation due to activation of B cells and T cells. Ablation of Fas specifically in GC B cells reproduced the phenotype, indicating that the lymphoproliferation initiates in the GC environment. B cell-specific Fas-deficient mice also showed an accumulation of IgG1(+) memory B cells expressing high amounts of CD80 and the expansion of CD28-expressing CD4(+) Th cells. Blocking T cell-B cell interaction and GC formation completely prevented the fatal lymphoproliferation. Thus, Fas-mediated selection of GC B cells and the resulting memory B cell compartment is essential for maintaining the homeostasis of both T and B lymphocytes.
Assuntos
Linfócitos B/imunologia , Centro Germinativo/imunologia , Linfócitos T/imunologia , Receptor fas/metabolismo , Animais , Antígenos CD/imunologia , Antígenos CD/metabolismo , Linfócitos B/metabolismo , Antígeno B7-1/imunologia , Antígeno B7-1/metabolismo , Antígenos CD28/imunologia , Antígenos CD28/metabolismo , Antígenos CD40/imunologia , Antígenos CD40/metabolismo , Antígeno CTLA-4 , Comunicação Celular , Diferenciação Celular , Proliferação de Células , Citocinas/sangue , Centro Germinativo/metabolismo , Homeostase , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes , Linfócitos T/metabolismo , Receptor fas/deficiência , Receptor fas/imunologiaRESUMO
We have previously reported the cloning and characterization of CARD6, a caspase recruitment domain (CARD)-containing protein that is structurally related to the interferon (IFN)-inducible GTPases. CARD6 associates with microtubules and with receptor-interacting protein 2 (RIP2). RIP2 mediates NF-kappaB activation induced by the intracellular nucleotide-binding oligomerization domain (NOD) receptors that sense bacterial peptidoglycan. Here we report that the expression of CARD6 and RIP2 in bone marrow-derived macrophages is rapidly induced by beta IFN and gamma IFN. This IFN-induced upregulation of CARD6 is suppressed by lipopolysaccharide (LPS), in contrast to LPS's enhancement of IFN-induced RIP2 upregulation. We generated CARD6-deficient (CARD6(-/-)) mice and carried out extensive analyses of signaling pathways mediating innate and adaptive immune responses, including the NOD pathways, but did not detect any abnormalities. Moreover, CARD6(-/-) mice were just as susceptible as wild-type mice to infection by Salmonella enterica serovar Typhimurium, Listeria monocytogenes, Candida albicans, lymphocytic choriomeningitis virus, or mouse adenovirus type 1. Thus, although structural and in vitro analyses strongly suggest an important role for CARD6 in immune defense, the physiological function of CARD6 remains obscure.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Interferons/farmacologia , NF-kappa B/metabolismo , Proteínas Adaptadoras de Sinalização NOD/química , Transdução de Sinais/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Alelos , Animais , Blastocisto/metabolismo , Proteínas Adaptadoras de Sinalização CARD/genética , Células Cultivadas , Células Clonais , Cruzamentos Genéticos , Eletroporação , Embrião de Mamíferos , Células-Tronco Embrionárias/citologia , Ativação Enzimática , Fibroblastos/metabolismo , Heterozigoto , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microinjeções , Mutação , Proteínas Adaptadoras de Sinalização NOD/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes/metabolismo , Recombinação GenéticaRESUMO
Diverse functions have been reported for lipocalin 2. To investigate these functions in vivo, we generated gene-targeted lipocalin 2-deficient mice (Lcn2-/- mice). In vitro studies have suggested that lipocalin 2 is important for cellular apoptosis induced by IL-3 withdrawal, and for the induction of kidney differentiation during embryogenesis. Analysis of Lcn2-/- mice showed normal cell death upon IL-3 withdrawal and normal kidney development. However, we found that Lcn2-/- mice exhibited an increased susceptibility to bacterial infections, in keeping with the proposed function of lipocalin 2 in iron sequestration. Neutrophils isolated from Lcn2-/- mice showed significantly less bacteriostatic activity compared with WT controls. The bacteriostatic property of the WT neutrophils was abolished by the addition of exogenous iron, indicating that the main function of lipocalin 2 in the antibacterial innate immune response is to limit this essential element. Another important function ascribed to lipocalin 2 has been its protective role against kidney ischemia-reperfusion injury. We analyzed Lcn2-/- mice using a mouse model for severe renal failure and could not detect any significant differences compared with their WT littermates.
Assuntos
Proteínas de Fase Aguda/deficiência , Proteínas de Fase Aguda/metabolismo , Infecções por Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Escherichia coli/fisiologia , Proteínas Oncogênicas/deficiência , Proteínas Oncogênicas/metabolismo , Traumatismo por Reperfusão/metabolismo , Proteínas de Fase Aguda/genética , Animais , Apoptose , Células Cultivadas , Infecções por Escherichia coli/genética , Infecções por Escherichia coli/imunologia , Imunidade Inata/imunologia , Rim/metabolismo , Rim/patologia , Lipocalina-2 , Lipocalinas , Camundongos , Camundongos Knockout , Proteínas Oncogênicas/genética , Traumatismo por Reperfusão/imunologiaRESUMO
Host response to infectious agents must be rapid and powerful. One mechanism is the release of presynthesized membrane-bound TNF. TNF shedding is mediated by TNF-alpha converting enzyme, which is selectively inhibited by the tissue inhibitor of metalloproteinase 3 (TIMP3). We show that loss of TIMP3 impacts innate immunity by dysregulating cleavage of TNF and its receptors. Cultured timp3-/- macrophages release more TNF in response to LPS than wild-type macrophages. In timp3-/- mice, LPS causes serum levels of TNF and its receptors to rise more rapidly and remain higher compared with wild-type mice. The altered kinetics of ligand and receptor shedding enhances TNF signaling in timp3-/- mice, indicated by elevated serum IL-6. Physiologically, timp3-/- mice are more susceptible to LPS-induced mortality. Ablation of the TNF receptor gene p55 (Tnfrsf1a) or treatment with a synthetic metalloproteinase inhibitor rescues timp3-/- mice. Thus, TIMP3 is essential for normal innate immune function.
Assuntos
Inflamação/imunologia , Inibidor Tecidual de Metaloproteinase-3/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Animais , Células Cultivadas , Imunidade Inata , Cinética , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores do Fator de Necrose Tumoral/metabolismo , Inibidor Tecidual de Metaloproteinase-3/deficiência , Inibidor Tecidual de Metaloproteinase-3/genéticaRESUMO
Transcription factors of the interferon regulatory factor (IRF) family contribute to the regulation of cell proliferation and apoptosis. Here, we show that CD4(+) T helper (Th) cells lacking IRF4 (IRF4(-/-)) are highly sensitive to apoptosis. After infection of IRF4(-/-) mice with the protozoan parasite Leishmania major, the lesion-draining lymph nodes developed the prototypic lymphadenopathy of wild-type mice after 4 wk, but demonstrated almost total loss of cellularity and enhanced apoptosis after 7 wk. In vitro, activation of IRF4(-/-) CD4(+) Th cells led to greatly increased apoptosis compared with wild-type cells. Coculture of IRF4(-/-) and IRF4(+/+) CD4(+) cells did not increase survival of IRF4(-/-) CD4(+) cells, indicating that the enhanced rate of IRF4(-/-) Th cell apoptosis was neither transferable nor due to lack of a cytokine. Enhanced CD4(+) cell apoptosis was also observed after anti-CD95 mAb treatment, despite normal CD95 expression. Removal of endogenous cytokines, notably interleukin (IL)-4, led to increased and equally high levels of IRF4(-/-) and IRF4(+/+) cell apoptosis, whereas the protective activity of exogenous IL-4 was reduced in IRF4(-/-) CD4(+) cells despite normal expression of the IL-4 receptor. Therefore, IRF4 is central in protecting CD4(+) cells against proapoptotic stimuli.
Assuntos
Apoptose , Linfócitos T CD4-Positivos/metabolismo , Proteínas de Ligação a DNA/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Fatores de Transcrição/genética , Animais , Anexina A5/farmacologia , Antígenos CD4/biossíntese , Divisão Celular , Corantes/farmacologia , Citocinas/metabolismo , Proteínas de Ligação a DNA/fisiologia , Citometria de Fluxo , Marcação In Situ das Extremidades Cortadas , Fatores Reguladores de Interferon , Leishmania major/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Linfócitos T Auxiliares-Indutores/metabolismo , Fatores de Tempo , Fatores de Transcrição/fisiologia , Receptor fas/biossínteseRESUMO
Because survivin-null embryos die at an early embryonic stage, the role of survivin in thymocyte development is unknown. We have investigated the role by deleting the survivin gene only in the T lineage and show here that loss of survivin blocks the transition from CD4- CD8- double negative (DN) thymocytes to CD4+ CD8+ double positive cells. Although the pre-T cell receptor signaling pathway is intact in survivin-deficient thymocytes, the cells cannot respond to its signals. In response to proliferative stimuli, cycling survivin-deficient DN cells exhibit cell cycle arrest, a spindle formation defect, and increased cell death. Strikingly, loss of survivin activates the tumor suppressor p53. However, the developmental defects caused by survivin deficiency cannot be rescued by p53 inactivation or introduction of Bcl-2. These lines of evidence indicate that developing thymocytes depend on the cytoprotective function of survivin and that this function is tightly coupled to cell proliferation but independent of p53 and Bcl-2. Thus, survivin plays a critical role in early thymocyte development.
Assuntos
Ciclo Celular/fisiologia , Morte Celular/fisiologia , Divisão Celular/fisiologia , Proteínas Associadas aos Microtúbulos/fisiologia , Linfócitos T/fisiologia , Timo/imunologia , Proteína Supressora de Tumor p53/fisiologia , Animais , Citometria de Fluxo , Proteínas Inibidoras de Apoptose , Cinética , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/deficiência , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Neoplasias , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Survivina , Linfócitos T/citologia , Timo/citologiaRESUMO
The translocation t(11;18)(q21;q21) involving MALT1 is the most common chromosomal abnormality in lymphomas of mucosa-associated lymphoid tissue. Although the paracaspase MALT1 can bind to BCL10, the physiological function of MALT1 is unknown. Using mouse models, we show that Malt1 is essential for T cell activation, proliferation, and IL-2 production in response to TCR ligation and strictly required for signal-specific NF-kappaB activation induced by the TCR but not TNF-alpha or IL-1 signaling. Malt1 operates downstream of Bcl10, controls the catalytic activity of the canonical IKK complex, and regulates the signaling of Jnk and p38 MAP kinases. In contrast to Bcl10 disruption, however, inactivation of Malt1 has only mild effects on B cell activation and does not cause defects during neurodevelopment. Thus, Malt1 is an essential regulator of Bcl10 signaling that is differentially required depending on cellular context.
Assuntos
Linfoma de Zona Marginal Tipo Células B/metabolismo , Proteínas de Neoplasias/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais/fisiologia , Animais , Caspases , Linfoma de Zona Marginal Tipo Células B/genética , Camundongos , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa , NF-kappa B/metabolismo , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/genética , Receptores de Antígenos de Linfócitos B/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/imunologiaRESUMO
The mitochondrial proapoptotic protein Smac/DIABLO has recently been shown to potentiate apoptosis by counteracting the antiapoptotic function of the inhibitor of apoptosis proteins (IAPs). In response to apoptotic stimuli, Smac is released into the cytosol and promotes caspase activation by binding to IAPs, thereby blocking their function. These observations have suggested that Smac is a new regulator of apoptosis. To better understand the physiological function of Smac in normal cells, we generated Smac-deficient (Smac(-/-)) mice by using homologous recombination in embryonic stem (ES) cells. Smac(-/-) mice were viable, grew, and matured normally and did not show any histological abnormalities. Although the cleavage in vitro of procaspase-3 was inhibited in lysates of Smac(-/-) cells, all types of cultured Smac(-/-) cells tested responded normally to all apoptotic stimuli applied. There were also no detectable differences in Fas-mediated apoptosis in the liver in vivo. Our data strongly suggest the existence of a redundant molecule or molecules capable of compensating for a loss of Smac function.
Assuntos
Apoptose/fisiologia , Proteínas de Transporte/metabolismo , Proteínas Mitocondriais/metabolismo , Animais , Anticorpos/metabolismo , Anticorpos/farmacologia , Proteínas Reguladoras de Apoptose , Linfócitos B/fisiologia , Proteínas de Transporte/genética , Caspase 3 , Caspase 8 , Caspase 9 , Caspases/metabolismo , Células Cultivadas , Embrião de Mamíferos/fisiologia , Ativação Enzimática , Marcação de Genes , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Knockout , Proteínas Mitocondriais/genética , Células-Tronco/citologia , Células-Tronco/metabolismo , Taxa de Sobrevida , Linfócitos T/fisiologia , Receptor fas/metabolismoRESUMO
Toll-like receptors (TLRs), which recognize pathogen-associated molecular patterns, and members of the pro-inflammatory interleukin-1 receptor (IL-1R) family, share homologies in their cytoplasmic domains called Toll/IL-1R/plant R gene homology (TIR) domains. Intracellular signalling mechanisms mediated by TIRs are similar, with MyD88 (refs 5-8) and TRAF6 (refs 9, 10) having critical roles. Signal transduction between MyD88 and TRAF6 is known to involve the serine-threonine kinase IL-1 receptor-associated kinase 1 (IRAK-1) and two homologous proteins, IRAK-2 (ref. 12) and IRAK-M. However, the physiological functions of the IRAK molecules remain unclear, and gene-targeting studies have shown that IRAK-1 is only partially required for IL-1R and TLR signalling. Here we show by gene-targeting that IRAK-4, an IRAK molecule closely related to the Drosophila Pelle protein, is indispensable for the responses of animals and cultured cells to IL-1 and ligands that stimulate various TLRs. IRAK-4-deficient animals are completely resistant to a lethal dose of lipopolysaccharide (LPS). In addition, animals lacking IRAK-4 are severely impaired in their responses to viral and bacterial challenges. Our results indicate that IRAK-4 has an essential role in innate immunity.