RESUMEN
Chronic hepatitis B virus (HBV) infection affects 300 million patients worldwide1,2, in whom virus-specific CD8 T cells by still ill-defined mechanisms lose their function and cannot eliminate HBV-infected hepatocytes3-7. Here we demonstrate that a liver immune rheostat renders virus-specific CD8 T cells refractory to activation and leads to their loss of effector functions. In preclinical models of persistent infection with hepatotropic viruses such as HBV, dysfunctional virus-specific CXCR6+ CD8 T cells accumulated in the liver and, as a characteristic hallmark, showed enhanced transcriptional activity of cAMP-responsive element modulator (CREM) distinct from T cell exhaustion. In patients with chronic hepatitis B, circulating and intrahepatic HBV-specific CXCR6+ CD8 T cells with enhanced CREM expression and transcriptional activity were detected at a frequency of 12-22% of HBV-specific CD8 T cells. Knocking out the inhibitory CREM/ICER isoform in T cells, however, failed to rescue T cell immunity. This indicates that CREM activity was a consequence, rather than the cause, of loss in T cell function, further supported by the observation of enhanced phosphorylation of protein kinase A (PKA) which is upstream of CREM. Indeed, we found that enhanced cAMP-PKA-signalling from increased T cell adenylyl cyclase activity augmented CREM activity and curbed T cell activation and effector function in persistent hepatic infection. Mechanistically, CD8 T cells recognizing their antigen on hepatocytes established close and extensive contact with liver sinusoidal endothelial cells, thereby enhancing adenylyl cyclase-cAMP-PKA signalling in T cells. In these hepatic CD8 T cells, which recognize their antigen on hepatocytes, phosphorylation of key signalling kinases of the T cell receptor signalling pathway was impaired, which rendered them refractory to activation. Thus, close contact with liver sinusoidal endothelial cells curbs the activation and effector function of HBV-specific CD8 T cells that target hepatocytes expressing viral antigens by means of the adenylyl cyclase-cAMP-PKA axis in an immune rheostat-like fashion.
Asunto(s)
Linfocitos T CD8-positivos , Hepatitis B Crónica , Hígado , Animales , Humanos , Masculino , Ratones , Linfocitos T CD8-positivos/enzimología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/patología , Modulador del Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Virus de la Hepatitis B/inmunología , Hepatitis B Crónica/inmunología , Hepatitis B Crónica/virología , Hepatocitos/inmunología , Hepatocitos/virología , Hígado/inmunología , Hígado/virología , Fosforilación , Transducción de Señal , Activación de LinfocitosRESUMEN
Members of the Protein kinases D (PKD) family are described as regulators of T cell responses. From the two T cell-expressed isoforms PKD2 and PKD3, so far mainly the former was thoroughly investigated and is well understood. Recently, we have investigated also PKD3 using conventional as well as conditional T cell-specific knockout models. These studies suggested PKD3 to be a T cell-extrinsic regulator of the cells' fate. However, these former model systems did not take into account possible redundancies with the highly homologous PKD2. To overcome this issue and thus properly unravel PKD3's T cell-intrinsic functions, here we additionally used a mouse model overexpressing a constitutively active isoform of PKD3 specifically in the T cell compartment. These transgenic mice showed a slightly higher proportion of central memory T cells in secondary lymphoid organs and blood. This effect could not be explained via differences upon polyclonal stimulation in vitro, however, may be connected to the observed developmental aberrances in the CD8 single positive compartment during thymic development. Lastly, the observed alterations in the CD8+ T cell compartment did not impact proper immune response upon immunization with ovalbumin or in a subcutaneous tumour model suggesting only a small to absent biological relevance. Taking together the knowledge of all our published studies on PKD3 in the T cell compartment, we now conclude that T cell-intrinsic PKD3 is a fine-tuner of central memory T cell as well as CD8 single positive thymocyte development.
Asunto(s)
Linfocitos T CD8-positivos , Diferenciación Celular , Células T de Memoria , Ratones Transgénicos , Proteína Quinasa C , Timocitos , Animales , Timocitos/inmunología , Timocitos/metabolismo , Ratones , Linfocitos T CD8-positivos/inmunología , Proteína Quinasa C/metabolismo , Proteína Quinasa C/genética , Células T de Memoria/inmunología , Células T de Memoria/metabolismo , Ratones Endogámicos C57BL , Memoria Inmunológica , Ratones NoqueadosRESUMEN
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.
Asunto(s)
Enfermedades Asintomáticas , Fenómenos Fisiológicos Bacterianos , Proliferación Celular , Proteínas de Unión al ADN/deficiencia , Leucemia/microbiología , Leucemia/patología , Proteínas Proto-Oncogénicas/deficiencia , Animales , Infecciones Bacterianas/inmunología , Infecciones Bacterianas/microbiología , Fenómenos Fisiológicos Bacterianos/inmunología , Proteínas de Unión al ADN/genética , Dioxigenasas , Femenino , Vida Libre de Gérmenes , Inflamación/microbiología , Interleucina-6/inmunología , Mucosa Intestinal/metabolismo , Lactobacillus/química , Lactobacillus/citología , Lactobacillus/inmunología , Masculino , Ratones , Penetrancia , Permeabilidad , Proteínas Proto-Oncogénicas/genética , Receptor Toll-Like 2/agonistasRESUMEN
BACKGROUND: The Protein kinase D3 (PKD3) has been implicated in signal transduction downstream of the T cell receptor (TCR). However, its role for the activation of primary T lymphocytes has not been elucidated so far. METHODS: Expression of PKD isoforms in primary murine T cells was determined by RT-PCR and SDS-Page. A germline PKD3-knockout mouse line was analyzed for its immune response to OVA/alum intraperitoneal immunization. Phenotyping of the T cell compartment ex vivo as well as upon stimulation in vitro was performed by flow cytometry. Additionally, cytokine expression was assessed by flow cytometry, RT-PCR and Luminex technology. RESULTS: PKD expression in T cells is modulated by TCR stimulation, leading to a rapid down-regulation on mRNA and on protein level. PKD3-deficient mice respond to immunization with enhanced T follicular helper cell generation. Furthermore, peripheral PKD3-deficient CD4+ T cells express more interleukin-2 than wild type CD4+ T cells upon TCR stimulation ex vivo. However, purified naïve CD4+ T cells do not differ in their phenotype upon differentiation in vitro from wild type T cells. Moreover, we observed a shift towards an effector/memory phenotype of splenic T cells at steady state, which might explain the contradictory results obtained with pan-T cells ex vivo and naïve-sorted T cells. CONCLUSION: While PKD3-deficiency in vivo in mice leads to a skewing of the T cell compartment towards a more activated phenotype, this kinase seems to be dispensable for naïve CD4+ T cell differentiation in vitro. Video Abstract.
Asunto(s)
Proteína Quinasa Activada por ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Linfocitos T , Animales , Linfocitos T CD4-Positivos , Ratones , Ratones Noqueados , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/metabolismoRESUMEN
Transforming growth-factor ß (TGFß) has been implicated in T helper 17 (Th17) cell biology and in triggering expression of interleukin-17A (IL-17A), which is a key Th17 cell cytokine. Deregulated TGFß receptor (TGFßR) signaling has been implicated in Th17-cell-mediated autoimmune pathogenesis. Nevertheless, the full molecular mechanisms involved in the activation of the TGFßR pathway in driving IL-17A expression remain unknown. Here, we identified protein kinase C α (PKCα) as a signaling intermediate specific to the Th17 cell subset in the activation of TGFßRI. We have shown that PKCα physically interacts and functionally cooperates with TGFßRI to promote robust SMAD2-3 activation. Furthermore, PKCα-deficient (Prkca(-/-)) cells demonstrated a defect in SMAD-dependent IL-2 suppression, as well as decreased STAT3 DNA binding within the Il17a promoter. Consistently, Prkca(-/-) cells failed to mount appropriate IL-17A, but not IL-17F, responses in vitro and were resistant to induction of Th17-cell-dependent experimental autoimmune encephalomyelitis in vivo.
Asunto(s)
Interleucina-17/metabolismo , Proteína Quinasa C-alfa/metabolismo , Células Th17/inmunología , Células Th17/metabolismo , Animales , Encefalomielitis Autoinmune Experimental/inducido químicamente , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/inmunología , Regulación de la Expresión Génica , Interleucina-17/inmunología , Ratones , Ratones Noqueados , Glicoproteína Mielina-Oligodendrócito/efectos adversos , Fragmentos de Péptidos/efectos adversos , Proteína Quinasa C-alfa/genética , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal , Proteínas Smad/metabolismo , Especificidad por SustratoRESUMEN
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.
Asunto(s)
Activación de Linfocitos , Linfocitos T , Animales , Prueba de Cultivo Mixto de Linfocitos , Ratones , BazoRESUMEN
C-type lectin receptors (CLRs) that couple with the kinase Syk are major pattern recognition receptors for the activation of innate immunity and host defense. CLRs recognize fungi and other forms of microbial or sterile danger, and they induce inflammatory responses through the adaptor protein Card9. The mechanisms relaying CLR proximal signals to the core Card9 module are unknown. Here we demonstrated that protein kinase C-δ (PKCδ) was activated upon Dectin-1-Syk signaling, mediated phosphorylation of Card9 at Thr231, and was responsible for Card9-Bcl10 complex assembly and canonical NF-κB control. Prkcd(-/-) dendritic cells, but not those lacking PKCα, PKCß, or PKCθ, were defective in innate responses to Dectin-1, Dectin-2, or Mincle stimulation. Moreover, Candida albicans-induced cytokine production was blocked in Prkcd(-/-) cells, and Prkcd(-/-) mice were highly susceptible to fungal infection. Thus, PKCδ is an essential link between Syk activation and Card9 signaling for CLR-mediated innate immunity and host protection.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Células Dendríticas/enzimología , Células Dendríticas/inmunología , Inmunidad Innata , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lectinas Tipo C/metabolismo , Proteína Quinasa C/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Animales , Proteínas Adaptadoras de Señalización CARD , Candida albicans/inmunología , Ratones , Ratones Noqueados , Proteína Quinasa C-delta/metabolismo , Transducción de Señal , Quinasa SykRESUMEN
BACKGROUND: NR2F6 has been proposed as an alternative cancer immune checkpoint in the effector T cell compartment. However, a realistic assessment of the in vivo therapeutic potential of NR2F6 requires acute depletion. METHODS: Employing primary T cells isolated from Cas9-transgenic mice for electroporation of chemically synthesized sgRNA, we established a CRISPR/Cas9-mediated acute knockout protocol of Nr2f6 in primary mouse T cells. RESULTS: Analyzing these Nr2f6CRISPR/Cas9 knockout T cells, we reproducibly observed a hyper-reactive effector phenotype upon CD3/CD28 stimulation in vitro, highly reminiscent to Nr2f6-/- T cells. Importantly, CRISPR/Cas9-mediated Nr2f6 ablation prior to adoptive cell therapy (ACT) of autologous polyclonal T cells into wild-type tumor-bearing recipient mice in combination with PD-L1 or CTLA-4 tumor immune checkpoint blockade significantly delayed MC38 tumor progression and induced superior survival, thus further validating a T cell-inhibitory function of NR2F6 during tumor progression. CONCLUSIONS: These findings indicate that Nr2f6CRISPR/Cas9 knockout T cells are comparable to germline Nr2f6-/- T cells, a result providing an independent confirmation of the immune checkpoint function of lymphatic NR2F6. Taken together, CRISPR/Cas9-mediated acute Nr2f6 gene ablation in primary mouse T cells prior to ACT appeared feasible for potentiating established PD-L1 and CTLA-4 blockade therapies, thereby pioneering NR2F6 inhibition as a sensitizing target for augmented tumor regression. Video abstract.
Asunto(s)
Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , Proteínas Represoras/metabolismo , Linfocitos T/inmunología , Animales , Secuencia de Bases , Sistemas CRISPR-Cas/genética , Antígeno CTLA-4/metabolismo , Células Cultivadas , Eliminación de Gen , Inhibidores de Puntos de Control Inmunológico/farmacología , Inmunidad/efectos de los fármacos , Ratones Endogámicos C57BL , Mutagénesis/genética , Neoplasias/patología , Receptor de Muerte Celular Programada 1/metabolismo , ARN Guía de Kinetoplastida/metabolismo , Proteínas Represoras/deficiencia , Reproducibilidad de los Resultados , Linfocitos T/efectos de los fármacosRESUMEN
Tumour metastasis is the primary cause of mortality in cancer patients and remains the key challenge for cancer therapy. New therapeutic approaches to block inhibitory pathways of the immune system have renewed hopes for the utility of such therapies. Here we show that genetic deletion of the E3 ubiquitin ligase Cbl-b (casitas B-lineage lymphoma-b) or targeted inactivation of its E3 ligase activity licenses natural killer (NK) cells to spontaneously reject metastatic tumours. The TAM tyrosine kinase receptors Tyro3, Axl and Mer (also known as Mertk) were identified as ubiquitylation substrates for Cbl-b. Treatment of wild-type NK cells with a newly developed small molecule TAM kinase inhibitor conferred therapeutic potential, efficiently enhancing anti-metastatic NK cell activity in vivo. Oral or intraperitoneal administration using this TAM inhibitor markedly reduced murine mammary cancer and melanoma metastases dependent on NK cells. We further report that the anticoagulant warfarin exerts anti-metastatic activity in mice via Cbl-b/TAM receptors in NK cells, providing a molecular explanation for a 50-year-old puzzle in cancer biology. This novel TAM/Cbl-b inhibitory pathway shows that it might be possible to develop a 'pill' that awakens the innate immune system to kill cancer metastases.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Células Asesinas Naturales/inmunología , Neoplasias Mamarias Experimentales/patología , Melanoma Experimental/patología , Metástasis de la Neoplasia/inmunología , Proteínas Proto-Oncogénicas c-cbl/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Anticoagulantes/farmacología , Anticoagulantes/uso terapéutico , Femenino , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/metabolismo , Masculino , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/inmunología , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/genética , Melanoma Experimental/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Metástasis de la Neoplasia/tratamiento farmacológico , Metástasis de la Neoplasia/prevención & control , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-cbl/deficiencia , Proteínas Proto-Oncogénicas c-cbl/genética , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/deficiencia , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación , Warfarina/farmacología , Warfarina/uso terapéutico , Tirosina Quinasa c-Mer , Tirosina Quinasa del Receptor AxlRESUMEN
BACKGROUND: The protein kinase C theta (PKCθ) has an important and non-redundant function downstream of the antigen receptor and co-receptor complex in T lymphocytes. PKCθ is not only essential for activation of NF-κB, AP-1 and NFAT and subsequent interleukin-2 expression, but also critical for positive selection and development of regulatory T lymphocytes in the thymus. Several domains regulate its activity, such as a pseudosubstrate sequence mediating an auto-inhibitory intramolecular interaction, the tandem C1 domains binding diacylglycerol, and phosphorylation at conserved tyrosine, threonine as well as serine residues throughout the whole length of the protein. To address the importance of the variable domain V1 at the very N-terminus, which is encoded by exon 2, a mutated version of PKCθ was analyzed for its ability to stimulate T lymphocyte activation. METHODS: T cell responses were analyzed with promoter luciferase reporter assays in Jurkat T cells transfected with PKCθ expression constructs. A mouse line expressing mutated instead of wild type PKCθ was analyzed in comparison to PKCθ-deficient and wild type mice for thymic development and T cell subsets by flow cytometry and T cell activation by quantitative RT-PCR, luminex analysis and flow cytometry. RESULTS: In cell lines, the exon 2-replacing mutation impaired the transactivation of interleukin-2 expression by constitutively active mutant form of PKCθ. Moreover, analysis of a newly generated exon 2-mutant mouse line (PKCθ-E2mut) revealed that the N-terminal replacement mutation results in an hypomorph mutant of PKCθ combined with reduced PKCθ protein levels in CD4+ T lymphocytes. Thus, PKCθ-dependent functions in T lymphocytes were affected resulting in impaired thymic development of single positive T lymphocytes in vivo. In particular, there was diminished generation of regulatory T lymphocytes. Furthermore, early activation responses such as interleukin-2 expression of CD4+ T lymphocytes were significantly reduced even though cell viability was not affected. Thus, PKCθ-E2mut mice show a phenotype similar to conventional PKCθ-deficient mice. CONCLUSION: Taken together, PKCθ-E2mut mice show a phenotype similar to conventional PKCθ-deficient mice. Both our in vitro T cell culture experiments and ex vivo analyses of a PKCθ-E2-mutant mouse line independently validate the importance of PKCθ downstream of the antigen-receptor complex for activation of CD4+ T lymphocytes.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Activación de Linfocitos , Mutación , Proteína Quinasa C-theta/genética , Animales , Células HEK293 , Humanos , Células Jurkat , Ratones , Ratones Endogámicos C57BL , Fenotipo , Proteína Quinasa C-theta/metabolismoRESUMEN
BACKGROUND: Protein kinase C θ has been established as an important signaling intermediate in T-effector-cell activation and survival pathways by controlling activity of the key transcription factors NF-κB and NFAT. Previous studies identified an activation-induced auto-phosphorylation site at Thr-219, located between the tandem C1 domains of the regulatory fragment in PKCθ, as a structural requirement for its correct membrane translocation and the subsequent transactivation of downstream signals leading to IL-2 production in a human T cell line. METHODS: The present work aimed to define the role of this phosphorylation switch on PKCθ in a physiological context through a homozygous T219A knockin mouse strain. T cell activation was analyzed by H3-thymidine uptake (proliferative response), qRT-PCR and luminex measurements (cytokine production). NFAT and NF-κB transactivation responses were estimated by Gel mobility shift and Alpha Screen assays. Frequencies of T cell subsets were analyzed by flow cytometry. RESULTS: Despite a normal T cell development, in vitro activated effector T cells clearly revealed a requirement of Thr-219 phosphorylation site on PKCθ for a transactivation of NF-κB and NFAT transcription factors and, subsequently, robust IL-2 and IFN-γ expression. CONCLUSION: This phenotype is reminiscent of the PKCθ knockout T cells, physiologically validating that this (p) Thr-219 auto-phosphorylation site indeed critically regulates PKCθ function in primary mouse T cells.
Asunto(s)
Técnicas de Sustitución del Gen , Fenotipo , Proteína Quinasa C-theta/genética , Proteína Quinasa C-theta/metabolismo , Animales , Citocinas/metabolismo , Ratones , Linfocitos T/citología , Linfocitos T/metabolismoRESUMEN
OBJECTIVE: Nuclear receptors are known to regulate both immune and barrier functions in the GI tract. The nuclear orphan receptor NR2F6 has been shown to suppress the expression of proinflammatory cytokines in T lymphocytes. NR2F6 gene expression is reduced in patients with IBS or UC, but its functional role and tissue dependency in healthy and inflamed gut have not yet been investigated. DESIGN: Intestinal inflammation was induced in wild-type, Nr2f6-deficient, Rag1-deficient or bone marrow-reconstituted mice by administration of chemical (dextran sodium sulfate (DSS)) and immunogenic (T cell transfer) triggers. Disease phenotypes were investigated by survival, body weight, colon length and analysis of immune cell infiltrates. Additionally, histology, intestinal permeability, tight junction proteins, bacterial fluorescence in situ hybridisation, apoptosis, cell proliferation and mucus production were investigated. RESULTS: Nr2f6-deficient mice were highly susceptible to DSS-induced colitis characterised by enhanced weight loss, increased colonic tissue destruction and immune cell infiltration together with enhanced intestinal permeability and reduced Muc2 expression. T cell transfer colitis and bone marrow reconstitution experiments demonstrated that disease susceptibility was not dependent on the expression of Nr2f6 in the immune compartment but on the protective role of NR2F6 in the intestinal epithelium. Mechanistically, we show that NR2F6 binds to a consensus sequence at -2 kb of the Muc2 promoter and transactivates Muc2 expression. Loss of NR2F6 alters intestinal permeability and results in spontaneous late-onset colitis in Nr2f6-deficient mice. CONCLUSION: We have for the first time identified a fundamental and non-redundant role of NR2F6 in protecting gut barrier homeostasis.
Asunto(s)
Factores de Transcripción COUP/metabolismo , Colitis/metabolismo , Colitis/patología , Animales , Colitis/etiología , Sulfato de Dextran , Modelos Animales de Enfermedad , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Ratones , Mucina 2/metabolismo , Proteínas Represoras , Proteínas de Uniones Estrechas/metabolismoRESUMEN
OBJECTIVE: Nicotinamide phosphoribosyltransferase (NAMPT, also referred to as pre-B cell colony-enhancing factor or visfatin) is critically required for the maintenance of cellular nicotinamide adenine dinucleotide (NAD) supply catalysing the rate-limiting step of the NAD salvage pathway. NAMPT is strongly upregulated in inflammation including IBD and counteracts an increased cellular NAD turnover mediated by NAD-depleting enzymes. These constitute an important mechanistic link between inflammatory, metabolic and transcriptional pathways and NAD metabolism. DESIGN: We investigated the impact of NAMPT inhibition by the small-molecule inhibitor FK866 in the dextran sulfate sodium (DSS) model of colitis and the azoxymethane/DSS model of colitis-associated cancer. The impact of NAD depletion on differentiation of mouse and human primary monocytes/macrophages was studied in vitro. Finally, we tested the efficacy of FK866 compared with dexamethasone and infliximab in lamina propria mononuclear cells (LPMNC) isolated from patients with IBD. RESULTS: FK866 ameliorated DSS-induced colitis and suppressed inflammation-associated tumorigenesis in mice. FK866 potently inhibited NAMPT activity as demonstrated by reduced mucosal NAD, resulting in reduced abundances and activities of NAD-dependent enzymes including PARP1, Sirt6 and CD38, reduced nuclear factor kappa B activation, and decreased cellular infiltration by inflammatory monocytes, macrophages and activated T cells. Remarkably, FK866 effectively supressed cytokine release from LPMNCs of patients with IBD. As FK866 was also effective in Rag1-/- mice, we mechanistically linked FK866 treatment with altered monocyte/macrophage biology and skewed macrophage polarisation by reducing CD86, CD38, MHC-II and interleukin (IL)-6 and promoting CD206, Egr2 and IL-10. CONCLUSION: Our data emphasise the importance of NAD immunometabolism for mucosal immunity and highlight FK866-mediated NAMPT blockade as a promising therapeutic approach in acute intestinal inflammation.
Asunto(s)
Acrilamidas/farmacología , Diferenciación Celular/efectos de los fármacos , Colitis Ulcerosa , Neoplasias del Colon , Dexametasona/farmacología , Infliximab/farmacología , NAD/metabolismo , Nicotinamida Fosforribosiltransferasa/metabolismo , Piperidinas/farmacología , Animales , Colitis Ulcerosa/inmunología , Colitis Ulcerosa/metabolismo , Neoplasias del Colon/inmunología , Neoplasias del Colon/metabolismo , Metabolismo Energético , Fármacos Gastrointestinales/farmacología , Humanos , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Macrófagos/metabolismo , Macrófagos/patología , Ratones , Monocitos/metabolismo , Monocitos/patologíaRESUMEN
Coronins are evolutionarily conserved proteins that were originally identified as modulators of actin-dependent processes. Studies analyzing complete Coronin 1a knock-out mice have shown that this molecule is an important regulator of naive T cell homeostasis and it has been linked to immune deficiencies as well as autoimmune disorders. Nevertheless, because Coronin 1A is strongly expressed in all leukocyte subsets, it is not conclusive whether or not this phenotype is attributed to a T cell-intrinsic function of Coronin 1A. To address this research question, we have generated a T cell-specific Coronin 1a knock-out mouse (Coro1afl/fl × Cd4[Cre]). Deletion of Coronin 1A specifically in T cells led to a strong reduction in T cell number and a shift toward the effector/memory phenotype in peripheral lymphoid organs when compared with Cd4[Cre] mice expressing wild-type Coronin 1A. In contrast to peripheral lymphoid tissue, thymocyte number and subsets were not affected by the deletion of Coronin 1a Furthermore, T cell-specific Coronin 1a knock-out mice were largely resistant to the induction of autoimmunity when tested in the myelin oligoglycoprotein-induced EAE mouse model of multiple sclerosis. Thus, the phenotype of T cell-specific Coronin 1a deletion resembles the phenotype observed with conventional (whole body) Coronin 1a knock-out mice. In summary, our findings provide formal proof of the predominant T cell-intrinsic role of Coronin 1A.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Memoria Inmunológica , Proteínas de Microfilamentos/inmunología , Esclerosis Múltiple/inmunología , Animales , Linfocitos T CD4-Positivos/patología , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/patología , Ratones , Ratones Noqueados , Proteínas de Microfilamentos/genética , Esclerosis Múltiple/genética , Esclerosis Múltiple/patologíaRESUMEN
Dysfunctional mitochondria contribute to the development of many diseases and pathological conditions through the excessive production of reactive oxygen species (ROS), and, where studied, ablation of p66Shc (p66) was beneficial. p66 translocates to the mitochondria and oxidizes cytochrome c to yield H2O2, which in turn initiates cell death. PKCß-mediated phosphorylation of serine 36 in p66 has been implicated as a key regulatory step preceding mitochondrial translocation, ROS production, and cell death, and PKCß thus may provide a target for therapeutic intervention. We performed a reassessment of PKCß regulation of the oxidoreductase activity of p66. Although our experiments did not substantiate Ser36 phosphorylation by PKCß, they instead provided evidence for Ser139 and Ser213 as PKCß phosphorylation sites regulating the pro-oxidant and pro-apoptotic function of p66. Mutation of another predicted PKCß phosphorylation site also located in the phosphotyrosine binding domain, threonine 206, had no phenotype. Intriguingly, p66 with Thr206 and Ser213 mutated to glutamic acid showed a gain-of-function phenotype with significantly increased ROS production and cell death induction. Taken together, these data argue for a complex mechanism of PKCß-dependent regulation of p66 activation involving Ser139 and a motif surrounding Ser213.
Asunto(s)
Proteína Quinasa C beta/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src/metabolismo , Células 3T3 , Animales , Muerte Celular , Eliminación de Gen , Células HEK293 , Humanos , Peróxido de Hidrógeno/metabolismo , Ratones , Mitocondrias/genética , Mitocondrias/metabolismo , Modelos Moleculares , Estrés Oxidativo , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Fosforilación , Mutación Puntual , Proteína Quinasa C beta/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , Serina/genética , Serina/metabolismo , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src/genéticaRESUMEN
The protein kinase C (PKC) family of serine-threonine kinases plays a central role in T lymphocyte activation. Here, we identify NR2F6, a nuclear zinc-finger orphan receptor, as a critical PKC substrate and essential regulator of CD4(+) T cell activation responses. NR2F6 potently antagonized the ability of T helper 0 (Th0) and Th17 CD4(+) T cells to induce expression of key cytokine genes such as interleukin-2 (IL-2) and IL-17. Mechanistically, NR2F6 directly interfered with the DNA binding of nuclear factor of activated T cells (NF-AT):activator protein 1 (AP-1) but not nuclear factor kappaB (NF-kappa B) and, subsequently, transcriptional activity of the NF-AT-dependent IL-17A cytokine promoter. Consistent with our model, Nr2f6-deficient mice had hyperreactive lymphocytes, developed a late-onset immunopathology, and were hypersusceptible to Th17-dependent experimental autoimmune encephalomyelitis. Our study establishes NR2F6 as a transcriptional repressor of IL-17 expression in Th17-differentiated CD4(+) T cells in vitro and in vivo.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Linfocitos T CD4-Positivos/inmunología , Proteínas de Unión al ADN/metabolismo , Interleucina-17/metabolismo , Activación de Linfocitos , Proteína Quinasa C/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Enfermedades Autoinmunes/metabolismo , Autoinmunidad/inmunología , Linfocitos T CD4-Positivos/metabolismo , Factores de Transcripción COUP , Proteínas de Unión al ADN/deficiencia , Interleucina-17/inmunología , Interleucina-2/inmunología , Interleucina-2/metabolismo , Ratones , Ratones Noqueados , Receptores Citoplasmáticos y Nucleares/deficiencia , Proteínas Represoras , Linfocitos T Colaboradores-Inductores/metabolismo , Factor de Transcripción AP-1/metabolismoRESUMEN
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.
Asunto(s)
Isoenzimas/metabolismo , Macrófagos/inmunología , Proteína Quinasa C/metabolismo , Infecciones por Salmonella/inmunología , Animales , Células Cultivadas , Interferón gamma/genética , Interferón gamma/metabolismo , Interleucina-10/genética , Interleucina-10/metabolismo , Interleucina-4/genética , Interleucina-4/metabolismo , Isoenzimas/genética , Activación de Macrófagos , Ratones , Ratones Endogámicos C57BL , Proteína Quinasa C/genética , Proteína Quinasa C-theta , Salmonella typhimurium/inmunologíaRESUMEN
Rab GTPases control membrane traffic and receptor-mediated endocytosis. Within this context, Rab5a plays an important role in the spatial regulation of intracellular transport and signal transduction processes. Here, we report a previously uncharacterized role for Rab5a in the regulation of T-cell motility. We show that Rab5a physically associates with protein kinase Cϵ (PKCϵ) in migrating T-cells. After stimulation of T-cells through the integrin LFA-1 or the chemokine receptor CXCR4, Rab5a is phosphorylated on an N-terminal Thr-7 site by PKCϵ. Both Rab5a and PKCϵ dynamically interact at the centrosomal region of migrating cells, and PKCϵ-mediated phosphorylation on Thr-7 regulates Rab5a trafficking to the cell leading edge. Furthermore, we demonstrate that Rab5a Thr-7 phosphorylation is functionally necessary for Rac1 activation, actin rearrangement, and T-cell motility. We present a novel mechanism by which a PKCϵ-Rab5a-Rac1 axis regulates cytoskeleton remodeling and T-cell migration, both of which are central for the adaptive immune response.
Asunto(s)
Inmunidad Adaptativa/fisiología , Movimiento Celular/fisiología , Proteína Quinasa C-epsilon/metabolismo , Linfocitos T/metabolismo , Proteínas de Unión al GTP rab5/metabolismo , Línea Celular , Citoesqueleto/genética , Citoesqueleto/metabolismo , Femenino , Humanos , Antígeno-1 Asociado a Función de Linfocito/genética , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Masculino , Fosforilación/fisiología , Proteína Quinasa C-epsilon/genética , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Linfocitos T/citología , Proteínas de Unión al GTP rab5/genéticaRESUMEN
BACKGROUND: Protein kinase C-θ (PKCθ) plays an important role in signal transduction down-stream of the T cell receptor and T cells deficient of PKCθ show impaired NF-κB as well as NFAT/AP-1 activation resulting in strongly decreased IL-2 expression and proliferation. However, it is not yet entirely clear, how the function of PKCθ - upon T cell activation - is regulated on a molecular level. FINDINGS: Employing a yeast two-hybrid screen and co-immunoprecipitation analyses, we here identify coronin 1A (Coro1A) as a novel PKCθ-interacting protein. We show that the NH2-terminal WD40 domains of Coro1A and the C2-like domain of PKCθ are sufficient for the interaction. Furthermore, we confirm a physical interaction by GST-Coro1A mediated pull-down of endogenous PKCθ protein. Functionally, wild-type but not Coro1A lacking its actin-binding domain negatively interferes with PKCθ-dependent NF-κB, Cyclin D1 and IL-2 transactivation when analysed with luciferase promoter activation assays in Jurkat T cells. This could be phenocopied by pharmacological inhibitors of actin polymerization and PKC, respectively. Mechanistically, Coro1A overexpression attenuates both lipid raft and plasma membrane recruitment of PKCθ in CD3/CD28-activated T cells. Using primary CD3(+) T cells, we observed that (opposite to PKCθ) Coro1A does not localize preferentially to the immunological synapse. In addition, we show that CD3(+) T cells isolated from Coro1A-deficient mice show impaired IKK/NF-κB transactivation. CONCLUSIONS: Together, these findings both in Jurkat T cells as well as in primary T cells indicate a regulatory role of Coro1A on PKCθ recruitment and function downstream of the TCR leading to NF-κB transactivation.
Asunto(s)
Isoenzimas/metabolismo , Proteínas de Microfilamentos/metabolismo , Complejos Multiproteicos/metabolismo , Proteína Quinasa C/metabolismo , Linfocitos T/metabolismo , Animales , Humanos , Isoenzimas/genética , Células Jurkat , Ratones , Proteínas de Microfilamentos/genética , Complejos Multiproteicos/genética , FN-kappa B/genética , FN-kappa B/metabolismo , Proteína Quinasa C/genética , Proteína Quinasa C-theta , Transporte de Proteínas/fisiología , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/citología , Activación Transcripcional/fisiologíaRESUMEN
PKC-θ plays a central role in TCR-induced IL-2 production and T-cell proliferation. The aim of the present study was to analyse how PKC-θ is regulated in human T cells during T-cell activation and differentiation. We show that PKC-θ is found in a high-molecular disulfide-linked complex in naïve T cells, and that PKC-θ most likely is inactive in this form. In parallel with the accumulation of the major redox regulators, glutathione and thioredoxin, PKC-θ is gradually reduced to the 82 kDa active form during T-cell activation. We demonstrate that PKC-θ is recruited to the plasma membrane in the disulfide-linked form in naïve T cells, and that activation of PKC-θ is redox dependent and requires de novo synthesis of glutathione. This is the first study that shows that the activity of PKC-θ is regulated by the intracellular redox state, and that PKC-θ is recruited to the plasma membrane in an inactive form in naïve T cells. Our observations underscore the existence of major differences in TCR signaling in naïve versus primed T cells.