RESUMO
Depending on the context, robust and durable T lymphocyte activation is either desirable, as in the case of anti-tumor responses, or unwanted, in cases of autoimmunity when chronic stimulation leads to self-tissue damage. Therefore, reliable in vivo models are of great importance to identify and validate regulatory pathways of T lymphocyte activation. Here, we describe an in vivo mixed-lymphocyte-reaction (MLR) approach, which is based on the so-called parent-into-F1 (P â F1) mouse model in combination with the congenic marker CD45.1/2 and cell proliferation dye-labeling. This setup allows us to track adoptively transferred allogenic CD4+ and CD8+ T lymphocytes and analyze their phenotype as well as the proliferation by flow cytometry in the blood and spleen. We could show hypo-reactive responses of T lymphocytes isolated from knockout mice with a known defect in T lymphocyte activation. Thus, this MLR-based in vivo model provides the opportunity to analyze positive regulators of T cell responses under physiological conditions of polyclonal T lymphocyte activation in vivo.
Assuntos
Ativação Linfocitária , Linfócitos T , Animais , Teste de Cultura Mista de Linfócitos , Camundongos , BaçoRESUMO
Somatic mutations in tet methylcytosine dioxygenase 2 (TET2), which encodes an epigenetic modifier enzyme, drive the development of haematopoietic malignancies1-7. In both humans and mice, TET2 deficiency leads to increased self-renewal of haematopoietic stem cells with a net developmental bias towards the myeloid lineage1,4,8,9. However, pre-leukaemic myeloproliferation (PMP) occurs in only a fraction of Tet2-/- mice8,9 and humans with TET2 mutations1,3,5-7, suggesting that extrinsic non-cell-autonomous factors are required for disease onset. Here we show that bacterial translocation and increased interleukin-6 production, resulting from dysfunction of the small-intestinal barrier, are critical for the development of PMP in mice that lack Tet2 expression in haematopoietic cells. Furthermore, in symptom-free Tet2-/- mice, PMP can be induced by disrupting intestinal barrier integrity, or in response to systemic bacterial stimuli such as the toll-like receptor 2 agonist. PMP was reversed by antibiotic treatment and failed to develop in germ-free Tet2-/- mice, which illustrates the importance of microbial signals in the development of this condition. Our findings demonstrate the requirement for microbial-dependent inflammation in the development of PMP and provide a mechanistic basis for the variation in PMP penetrance observed in Tet2-/- mice. This study will prompt new lines of investigation that may profoundly affect the prevention and management of haematopoietic malignancies.
Assuntos
Doenças Assintomáticas , Fenômenos Fisiológicos Bacterianos , Proliferação de Células , Proteínas de Ligação a DNA/deficiência , Leucemia/microbiologia , Leucemia/patologia , Proteínas Proto-Oncogênicas/deficiência , Animais , Infecções Bacterianas/imunologia , Infecções Bacterianas/microbiologia , Fenômenos Fisiológicos Bacterianos/imunologia , Proteínas de Ligação a DNA/genética , Dioxigenases , Feminino , Vida Livre de Germes , Inflamação/microbiologia , Interleucina-6/imunologia , Mucosa Intestinal/metabolismo , Lactobacillus/química , Lactobacillus/citologia , Lactobacillus/imunologia , Masculino , Camundongos , Penetrância , Permeabilidade , Proteínas Proto-Oncogênicas/genética , Receptor 2 Toll-Like/agonistasRESUMO
BACKGROUND: The serine/threonine protein kinase C (PKC) theta has been firmly implicated in T cell-mediated immunity. Because its role in macrophages has remained undefined, we employed PKCtheta-deficient (PKCtheta (-/-)) mice in order to investigate if PKCtheta plays a role in macrophage-mediated immune responses during bacterial infections. RESULTS: Our results demonstrate that PKCtheta plays an important role in host defense against the Gram-negative, intracellular bacterium Salmonella typhimurium, as reflected both by markedly decreased survival and a significantly enhanced number of bacteria in spleen and liver of PKCtheta (-/-) mice, when compared to wild-type mice. Of note, albeit macrophages do not express detectable PKCtheta, PKCtheta mRNA expression was found to be profoundly upregulated during the first hours of lipopolysaccharide (LPS)/interferon-gamma (IFNgamma)-, but not IL-4-mediated cell polarization conditions in vitro. Mechanistically, despite expressing normal levels of classically activated macrophage (CAM) markers, PKCtheta-deficient CAMs expressed significantly higher levels of the anti-inflammatory cytokine IL-10 in vivo and in vitro when challenged with S. typhimurium or LPS/IFNgamma. Neutralization of IL-10 recovered immune control to S. typhimurium infection in PKCtheta-deficient macrophages. CONCLUSIONS: Taken together, our data provide genetic evidence that PKCtheta promotes a potent pro-inflammatory CAM phenotype that is instrumental to mounting protective anti-bacterial immunity. Mechanistically, PKCtheta exerts a host-protective role against S. typhimurium infection, and acts as an essential link between TLR4/IFNgammaR signaling and selective suppression of the anti-inflammatory cytokine IL-10 at the onset of CAM differentiation in the course of a bacterial infection.
Assuntos
Isoenzimas/metabolismo , Macrófagos/imunologia , Proteína Quinase C/metabolismo , Infecções por Salmonella/imunologia , Animais , Células Cultivadas , Interferon gama/genética , Interferon gama/metabolismo , Interleucina-10/genética , Interleucina-10/metabolismo , Interleucina-4/genética , Interleucina-4/metabolismo , Isoenzimas/genética , Ativação de Macrófagos , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase C/genética , Proteína Quinase C-theta , Salmonella typhimurium/imunologiaRESUMO
LAMTOR2 (p14), a part of the larger LAMTOR/Ragulator complex, plays a crucial role in EGF-dependent activation of p42/44 mitogen-activated protein kinases (MAPK, ERK1/2). In this study, we investigated the role of LAMTOR2 in nerve growth factor (NGF)-mediated neuronal differentiation. Stimulation of PC12 (rat adrenal pheochromocytoma) cells with NGF is known to activate the MAPK. Pharmacological inhibition of MEK1 as well as siRNA-mediated knockdown of both p42 and p44 MAPK resulted in inhibition of neurite outgrowth. Contrary to expectations, siRNA-mediated knockdown of LAMTOR2 effectively augmented neurite formation and neurite length of PC12 cells. Ectopic expression of a siRNA-resistant LAMTOR2 ortholog reversed this phenotype back to wildtype levels, ruling out nonspecific off-target effects of this LAMTOR2 siRNA approach. Mechanistically, LAMTOR2 siRNA treatment significantly enhanced NGF-dependent MAPK activity, and this effect again was reversed upon expression of the siRNA-resistant LAMTOR2 ortholog. Studies of intracellular trafficking of the NGF receptor TrkA revealed a rapid colocalization with early endosomes, which was modulated by LAMTOR2 siRNA. Inhibition of LAMTOR2 and concomitant destabilization of the remaining members of the LAMTOR complex apparently leads to a faster release of the TrkA/MAPK signaling module and nuclear increase of activated MAPK. These results suggest a modulatory role of the MEK1 adapter protein LAMTOR2 in NGF-mediated MAPK activation required for induction of neurite outgrowth in PC12 cells.
Assuntos
Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fator de Crescimento Neural/metabolismo , Proteínas/metabolismo , Animais , Diferenciação Celular/fisiologia , Endossomos/metabolismo , Cinética , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Fator de Crescimento Neural/genética , Células PC12 , Proteínas/genética , Ratos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
In T cells PKCθ mediates the activation of critical signals downstream of TCR/CD28 stimulation. We investigated the molecular mechanisms by which PKCθ regulates NFκB transactivation by examining PKCθ/ß single and double knockout mice and observed a redundant involvement of PKCθ and PKCß in this signaling pathway. Mechanistically, we define a PKCθ-CYLD protein complex and an interaction between the positive PKCθ/ß and the negative CYLD signaling pathways that both converge at the level of TAK1/IKK/I-κBα/NFκB and NFAT transactivation. In Jurkat leukemic T cells, CYLD is endoproteolytically processed in the initial minutes of stimulation by the paracaspase MALT1 in a PKC-dependent fashion, which is required for robust IL-2 transcription. However, in primary T cells, CYLD processing occurs with different kinetics and an altered dependence on PKC. The formation of a direct PKCθ/CYLD complex appears to regulate the short-term spatial distribution of CYLD, subsequently affecting NFκB and NFAT repressional activity of CYLD prior to its MALT1-dependent inactivation. Taken together, our study establishes CYLD as a new and critical PKCθ interactor in T cells and reveals that antagonistic PKCθ/ß-CYLD crosstalk is crucial for the adjustment of immune thresholds in primary mouse CD3(+) T cells.
Assuntos
Complexo CD3 , Cisteína Endopeptidases/metabolismo , NF-kappa B/metabolismo , Fatores de Transcrição NFATC/metabolismo , Proteína Quinase C/metabolismo , Transdução de Sinais , Linfócitos T/metabolismo , Animais , Complexo CD3/metabolismo , Caspases/metabolismo , Linhagem Celular , Cisteína Endopeptidases/genética , Enzima Desubiquitinante CYLD , Ativação Enzimática , Regulação da Expressão Gênica , Humanos , Células Jurkat , Ativação Linfocitária , MAP Quinase Quinase 4/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Camundongos , Camundongos Knockout , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa , Proteínas de Neoplasias/metabolismo , Fenótipo , Ligação Proteica , Proteína Quinase C/genética , Proteólise , Linfócitos T/imunologia , Ativação TranscricionalRESUMO
The Bcl-2 regulated apoptosis pathway is critical for the elimination of autoreactive lymphocytes, thereby precluding autoimmunity. T cells escaping this process can be kept in check by regulatory T (Treg) cells expressing the transcription and lineage commitment factor Foxp3. Despite the well-established role of Bcl-2 family proteins in shaping the immune system and their frequent deregulation in autoimmune pathologies, it is poorly understood how these proteins affect Treg cell development and function. Here we compared the relative expression of a panel of 40 apoptosis-associated genes in Treg vs. conventional CD4(+) T cells. Physiological significance of key-changes was validated using gene-modified mice lacking or overexpressing pro- or anti-apoptotic Bcl-2 family members. We define a key role for the Bim/Bcl-2 axis in Treg cell development, homeostasis and function but exclude a role for apoptosis induction in responder T cells as relevant suppression mechanism. Notably, only lack of the pro-apoptotic BH3-only protein Bim or Bcl-2 overexpression led to accumulation of Treg cells while loss of pro-apoptotic Bad, Bmf, Puma or Noxa had no effect. Remarkably, apoptosis resistant Treg cells showed reduced suppressive capacity in a model of T cell-driven colitis, posing a caveat for the use of such long-lived cells in possible therapeutic settings.
Assuntos
Apoptose/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Transdução de Sinais/genética , Linfócitos T Reguladores/metabolismo , Animais , Antineoplásicos/farmacologia , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 11 Semelhante a Bcl-2 , Linfócitos T CD4-Positivos/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Células Cultivadas , Etoposídeo/farmacologia , Feminino , Citometria de Fluxo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Perfilação da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/metabolismo , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Timo/citologia , Timo/metabolismoRESUMO
Using model tumor T cell lines, protein kinase C (PKC) alpha has been implicated in IL-2 cytokine promoter activation in response to Ag receptor stimulation. In this study, for the first time, PKCalpha null mutant mice are analyzed and display normal T and B lymphocyte development. Peripheral CD3(+) PKCalpha-deficient T cells show unimpaired activation-induced IL-2 cytokine secretion, surface expression of CD25, CD44, and CD69, as well as transactivation of the critical transcription factors NF-AT, NF-kappaB, AP-1, and STAT5 in vitro. Nevertheless, CD3/CD28 Ab- and MHC alloantigen-induced T cell proliferation and IFN-gamma production are severely impaired in PKCalpha(-/-) CD3(+) T cells. Consistently, PKCalpha-deficient CD3(+) T cells from OVA-immunized PKCalpha-deficient mice exhibit markedly reduced recall proliferation to OVA in in vitro cultures. In vivo, PKCalpha-deficient mice give diminished OVA-specific IgG2a and IgG2b responses following OVA immunization experiments. In contrast, OVA-specific IgM and IgG1 responses and splenic PKCalpha(-/-) B cell proliferation are unimpaired. Our genetic data, thus, define PKCalpha as the physiological and nonredundant PKC isotype in signaling pathways that are necessary for T cell-dependent IFN-gamma production and IgG2a/2b Ab responses.
Assuntos
Switching de Imunoglobulina/genética , Imunoglobulina G/classificação , Imunoglobulina G/genética , Isotipos de Imunoglobulinas/genética , Proteína Quinase C-alfa/deficiência , Proteína Quinase C-alfa/genética , Animais , Complexo CD3/biossíntese , Proliferação de Células , Células Cultivadas , Interferon gama/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase C-alfa/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
We here investigate the crosstalk of PKC and PKA signaling during primary CD3(+) T-lymphocyte activation using pharmacologic inhibitors and activators in combination with our established panel of PKC isotype-deficient mouse T cells in vitro. PKCtheta and PKA inversely affect the CD3/CD28-induced IL-2 expression, whereas other PKC isotypes are dispensable in this signaling pathway. Gene ablation of PKCtheta selectively results in a profound reduction of IL-2 production; however, complete abrogation of IL-2 production in these PKCtheta(-/-) T cells was achieved only by simultaneous coactivation of the cAMP/PKA pathway in CD3(+) T cells. Conversely, the reduced IL-2 production in PKC inhibitor-treated T cells can be rescued by inhibition of the cAMP/PKA pathway in wild-type but not in PKCtheta(-/-) T cells. Mechanistically, the cAMP/PKA and PKCtheta pathways converge at the level of NF-AT, as shown by DNA binding analysis. The combined increase in PKA and decrease in PKCtheta activity leads to an enhanced inhibition of nuclear NF-AT translocation. This PKCtheta/PKA crosstalk significantly affects neither the NF-kappaB, the AP-1, nor the CREB pathways. Taken together, this opposite effect between the positive PKCtheta and the negative cAMP/PKA signaling pathways appears rate limiting for NF-AT transactivation and IL-2 secretion responses of CD3(+) T lymphocytes.
Assuntos
Complexo CD3/imunologia , Fatores de Transcrição NFATC/imunologia , Proteína Quinase C/imunologia , Proteínas Serina-Treonina Quinases/imunologia , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/genética , Transporte Ativo do Núcleo Celular/imunologia , Animais , Antígenos CD28/imunologia , Proteína de Ligação a CREB/imunologia , Núcleo Celular/imunologia , Células Cultivadas , AMP Cíclico/imunologia , Proteínas Quinases Dependentes de AMP Cíclico , Deleção de Genes , Interleucina-2/imunologia , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos , NF-kappa B/imunologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/deficiência , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Linfócitos T/citologia , Fator de Transcrição AP-1/imunologiaRESUMO
Phosphopeptide mapping identified a major autophosphorylation site, phospho (p)Thr-219, between the tandem C1 domains of the regulatory fragment in protein kinase C (PKC)theta. Confirmation of this identification was derived using (p)Thr-219 antisera that reacted with endogenous PKCtheta in primary CD3+ T cells after stimulation with phorbol ester, anti-CD3 or vanadate. The T219A mutation abrogated the capacity of PKCtheta to mediate NF-kappaB, NF-AT and interleukin-2 promoter transactivation, and reduced PKCtheta's ability in Jurkat T cells to phosphorylate endogenous cellular substrates. In particular, the T219A mutation impaired crosstalk of PKCtheta with Akt/PKBalpha in NF-kappaB activation. Yet, this novel (p)Thr-219 site did not affect catalytic activity or second-messenger lipid-binding activity in vitro. Instead, the T219A mutation prevented proper recruitment of PKCtheta in activated T cells. The PKCthetaT219A mutant defects were largely rescued by addition of a myristoylation signal to force its proper membrane localization. We conclude that autophosphorylation of PKCtheta at Thr-219 plays an important role in the correct targeting and cellular function of PKCtheta upon antigen receptor ligation.