RESUMO
CD73-derived adenosine suppresses anti-cancer immunity, and CD73 inhibitors are currently evaluated in several clinical trials. Here, we have assessed enzyme kinetics of all key purinergic ectoenzymes in five cancer cell lines (Hodgkin lymphoma, multiple myeloma, pancreas adenocarcinoma, urinary bladder carcinoma, and glioblastoma) under normoxia and hypoxia. We found that adenosine metabolism varied considerably between individual cancer types. All cell lines investigated exhibited high ecto-adenosine deaminase (ADA) activity, which critically influenced the kinetics of adenosine accumulation. Combining kinetics data with single-cell RNA sequencing data on myeloma and glioblastoma cancerous tissue revealed that purine metabolism is not homogeneously organized, but it differs in a cancer type-specific fashion between malignant cells, stromal cells, and immune cells. Since purine metabolism in cancerous tissue is most likely spatially heterogeneous and differs between the various cell types, diffusion distances in the microenvironment as well as ADA activity may be important variables that influence the level of bioactive adenosine.
Assuntos
Glioblastoma , Mieloma Múltiplo , Humanos , Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo , 5'-Nucleotidase/metabolismo , Transdução de Sinais , Adenosina Desaminase/metabolismo , Microambiente TumoralRESUMO
Vascular inflammation plays a key role in the development of aortic diseases. A potential novel target for treatment might be CD73, an ecto-5'-nucleotidase that generates anti-inflammatory adenosine in the extracellular space. Here, we investigated whether a lack of CD73 results in enhanced aortic inflammation. To this end, angiotensin II was infused into wildtype and CD73-/- mice over 10 days. Before and after infusion, mice were analyzed using magnetic resonance imaging, ultrasound, flow cytometry, and histology. The impact of age and gender was investigated using female and male mice of three and six months of age, respectively. Angiotensin II infusion led to increased immune cell infiltration in both genotypes' aortae, but depletion of CD73 had no impact on immune cell recruitment. These findings were not modified by age or sex. No substantial difference in morphological or functional characteristics could be detected between wildtype and CD73-/- mice. Interestingly, the expression of CD73 on neutrophils decreased significantly in wildtype mice during treatment. In summary, we have found no evidence that CD73 deficiency affects the onset of aortic inflammation. However, as CD73 expression decreased during disease induction, an increase in CD73 by pharmaceutical intervention might result in lower vascular inflammation and less vascular disease.
Assuntos
5'-Nucleotidase , Angiotensina II , Animais , Feminino , Masculino , Camundongos , 5'-Nucleotidase/genética , Adenosina/metabolismo , Inflamação/genética , Camundongos KnockoutRESUMO
Type 2 diabetes is characterized by insulin hypersecretion followed by reduced glucose-stimulated insulin secretion (GSIS). Here we show that acute stimulation of pancreatic islets with the insulin secretagogue dextrorphan (DXO) or glibenclamide enhances GSIS, whereas chronic treatment with high concentrations of these drugs reduce GSIS but protect islets from cell death. Bulk RNA sequencing of islets shows increased expression of genes for serine-linked mitochondrial one-carbon metabolism (OCM) after chronic, but not acute, stimulation. In chronically stimulated islets, more glucose is metabolized to serine than to citrate, and the mitochondrial ATP/ADP ratio decreases, whereas the NADPH/NADP+ ratio increases. Activating transcription factor-4 (Atf4) is required and sufficient to activate serine-linked mitochondrial OCM genes in islets, with gain- and loss-of-function experiments showing that Atf4 reduces GSIS and is required, but not sufficient, for full DXO-mediated islet protection. In sum, we identify a reversible metabolic pathway that provides islet protection at the expense of secretory function.
Assuntos
Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Ilhotas Pancreáticas , Humanos , Diabetes Mellitus Tipo 2/metabolismo , Ilhotas Pancreáticas/metabolismo , Insulina/metabolismo , Glucose/metabolismo , Carbono/metabolismo , Células Secretoras de Insulina/metabolismoRESUMO
Neutrophils play a complex role during onset of tissue injury and subsequent resolution and healing. To assess neutrophil dynamics upon cardiovascular injury, here we develop a non-invasive, background-free approach for specific mapping of neutrophil dynamics by whole-body magnetic resonance imaging using targeted multimodal fluorine-loaded nanotracers engineered with binding peptides specifically directed against murine or human neutrophils. Intravenous tracer application before injury allowed non-invasive three-dimensional visualization of neutrophils within their different hematopoietic niches over the entire body and subsequent monitoring of their egress into affected tissues. Stimulated murine and human neutrophils exhibited enhanced labeling due to upregulation of their target receptors, which could be exploited as an in vivo readout for their activation state in both sterile and nonsterile cardiovascular inflammation. This non-invasive approach will allow us to identify hidden origins of bacterial or sterile inflammation in patients and also to unravel cardiovascular disease states on the verge of severe aggravation due to enhanced neutrophil infiltration or activation.
RESUMO
CD73-derived adenosine plays a major role in damage-induced tissue responses by inhibiting inflammation. Damage-associated stimuli, such as hypoxia and mechanical stress, induce the cellular release of ATP and NAD+ and upregulate the expression of the nucleotide-degrading purinergic ectoenzyme cascade, including adenosine-generating CD73. Extracellular NAD+ also serves as substrate for mono-ADP-ribosylation of cell surface proteins, which in human cells is mediated by ecto-ADP-ribosyltransferase 1 (ARTC1). Here we explored, whether human CD73 enzymatic activity is regulated by mono-ADP-ribosylation, using recombinant human CD73 in the presence of ARTC1 with etheno-labelled NAD+ as substrate. Multi-colour immunoblotting with an anti-etheno-adenosine antibody showed ARTC1-mediated transfer of ADP-ribose together with the etheno label to CD73. HPLC analysis of the enzymatic activity of in vitro-ribosylated CD73 revealed strong inhibition of adenosine generation in comparison to non-ribosylated CD73. Mass spectrometry of in vitro-ribosylated CD73 identified six ribosylation sites. 3D model analysis indicated that three of them (R328, R354, R545) can interfere with CD73 enzymatic activity. Our study identifies human CD73 as target for ARTC1-mediated mono-ADP-ribosylation, which can profoundly modulate its adenosine-generating activity. Thus, in settings with enhanced release of NAD+ as substrate for ARTC1, assessment of CD73 protein expression in human tissues may not be predictive of adenosine formation resulting in anti-inflammatory activity.
Assuntos
5'-Nucleotidase/metabolismo , Adenosina , ADP-Ribosilação , Adenosina/metabolismo , Adenosina Difosfato Ribose/metabolismo , Proteínas Ligadas por GPI/metabolismo , Humanos , Proteínas de Membrana , NADRESUMO
BACKGROUND: Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that leads to a breakdown of tolerance to self-antigens resulting in inflammation and organ damage. The anti-inflammatory activity of CD73-derived adenosine is well documented, however, its role in SLE pathogenesis is unknown. METHODS: Human peripheral blood immune cells were obtained from adult SLE patients (SLE) and healthy controls (HC). Expression and activity of purinergic ectoenzymes were assessed by qRT-PCR, flow cytometry and HPLC. Genes encoding purinergic ectoenzymes in SLE patients were analysed with targeted DNA sequencing. FINDINGS: Among circulating immune cells (both in HC and SLE), CD73 was most highly expressed on B cells, which was mirrored by high enzymatic activity only in HC. CD73 protein molecular weight was unchanged in SLE, however, the enzymatic activity of CD73 on SLE B cells was almost fully abolished. Accordingly, AMP accumulated in cultured SLE B cells. A similar discrepancy between protein expression and enzymatic activity was observed for NAD-degrading CD38 on SLE B cells. No differences were found in the rate of extracellular ATP degradation and expression of CD39, CD203a/c, and CD157. DNA sequencing identified no coding variants in CD73 in SLE patients. INTERPRETATION: We describe a new pathomechanism for SLE, by which inactivation of CD73 on B cells produces less anti-inflammatory adenosine, resulting in immune cell activation. CD73 inactivation was not due to genetic variation but may be related to posttranslational modification. FUNDING: The German Research Council, Medical Faculty of the Heinrich-Heine-University Duesseldorf, Hiller Research Foundation, and Cardiovascular Research Institute Duesseldorf.
Assuntos
5'-Nucleotidase/metabolismo , Adenosina/metabolismo , Linfócitos B/imunologia , Linfócitos B/metabolismo , Lúpus Eritematoso Sistêmico/etiologia , Lúpus Eritematoso Sistêmico/metabolismo , Trifosfato de Adenosina/metabolismo , Biomarcadores , Vias Biossintéticas , Estudos de Casos e Controles , Cromatografia Líquida de Alta Pressão , Suscetibilidade a Doenças , Feminino , Proteínas Ligadas por GPI/metabolismo , Humanos , Imunofenotipagem , Leucócitos/imunologia , Leucócitos/metabolismo , Lúpus Eritematoso Sistêmico/diagnóstico , Lúpus Eritematoso Sistêmico/terapia , Masculino , Índice de Gravidade de DoençaRESUMO
Myocardial infarction (MI) activates the epicardium to form epicardial stromal cells (EpiSC) that reside in the epicardial hypoxic microenvironment. Paracrine factors secreted by EpiSC were shown to modulate the injury response of the post-MI heart and improve cardiac function. We have previously reported that the expression of the angiogenic cytokines vascular endothelial growth factor A (VEGFA) and IL-6 is strongly upregulated in EpiSC by adenosine acting via the A2B receptor (A2B R). Since tissue hypoxia is well known to be a potent stimulus for the generation of extracellular adenosine, the present study explored the crosstalk of A2B R activation and hypoxia-hypoxia-inducible factor 1 alpha (HIF-1α) signaling in cultured EpiSC, isolated from rat hearts 5 days after MI. We found substantial nuclear accumulation of HIF-1α after A2B R activation even in the absence of hypoxia. This normoxic HIF-1α induction was PKC-dependent and involved upregulation of HIF-1α mRNA expression. While the influence of hypoxia on adenosine generation and A2B R signaling was only minor, hypoxia and A2B R activation cumulatively increased VEGFA expression. Normoxic A2B R activation triggered an HIF-1α-associated cell-protective metabolic switch and reduced oxygen consumption. HIF-1α targets and negative regulators PHD2 and PHD3 were only weakly induced by A2B R signaling, which may result in a sustained HIF-1α activity. The A2B R-mediated normoxic HIF-1α induction was also observed in cardiac fibroblasts from healthy mouse hearts, suggesting that this mechanism is also functional in other A2B R-expressing cell types. Altogether, we identified A2B R-mediated HIF-1α induction as novel aspect in the HIF-1α-adenosine crosstalk, which modulates EpiSC activity and can amplify HIF-1α-mediated cardioprotection.
Assuntos
Cardiotônicos/metabolismo , Hipóxia Celular , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Infarto do Miocárdio/prevenção & controle , Pericárdio/metabolismo , Receptor A2B de Adenosina/metabolismo , Células Estromais/metabolismo , Animais , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Masculino , Infarto do Miocárdio/etiologia , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Pericárdio/patologia , Ratos , Ratos Wistar , Receptor A2B de Adenosina/genética , Células Estromais/patologiaRESUMO
Cerium (Ce) oxide nanoparticles (CNP; nanoceria) are reported to have cytotoxic effects on certain cancerous cell lines, while at the same concentration they show no cytotoxicity on normal (healthy) cells. Redox-active CNP exhibit both selective prooxidative as well as antioxidative properties. The former is proposed to be responsible for impairment of tumor growth and invasion and the latter for rescuing normal cells from reactive oxygen species (ROS)-induced damage. Here we address possible underlying mechanisms of prooxidative effects of CNP in a metastatic human melanoma cell line. Malignant melanoma is the most aggressive form of skin cancer, and once it becomes metastatic the prognosis is very poor. We have shown earlier that CNP selectively kill A375 melanoma cells by increasing intracellular ROS levels, whose basic amount is significantly higher than in the normal (healthy) counterpart, the melanocytes. Here we show that CNP initiate a mitochondrial increase of ROS levels accompanied by an increase in mitochondrial thiol oxidation. Furthermore, we observed CNP-induced changes in mitochondrial bioenergetics, dynamics, and cristae morphology demonstrating mitochondrial dysfunction which finally led to tumor cell death. CNP-induced cell death is abolished by administration of PEG-conjugated catalase. Overall, we propose that cerium oxide nanoparticles mediate cell death via hydrogen peroxide production linked to mitochondrial dysfunction.
Assuntos
Cério/farmacologia , Citotoxinas/farmacologia , Melanoma/tratamento farmacológico , Mitocôndrias/efeitos dos fármacos , Antioxidantes/química , Antioxidantes/farmacologia , Catalase/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cério/química , Citotoxinas/química , Humanos , Melanoma/metabolismo , Melanoma/patologia , Mitocôndrias/patologia , Nanopartículas/química , Metástase Neoplásica , Compostos de Sulfidrila/metabolismoRESUMO
Epicardium-derived cells (EPDC) and atrial stromal cells (ASC) display cardio-regenerative potential, but the molecular details are still unexplored. Signals which induce activation, migration and differentiation of these cells are largely unknown. Here we have isolated rat ventricular EPDC and rat/human ASC and performed genetic and proteomic profiling. EPDC and ASC expressed epicardial/mesenchymal markers (WT-1, Tbx18, CD73, CD90, CD44, CD105), cardiac markers (Gata4, Tbx5, troponin T) and also contained phosphocreatine. We used cell surface biotinylation to isolate plasma membrane proteins of rEPDC and hASC, Nano-liquid chromatography with subsequent mass spectrometry and bioinformatics analysis identified 396 rat and 239 human plasma membrane proteins with 149 overlapping proteins. Functional GO-term analysis revealed several significantly enriched categories related to extracellular matrix (ECM), cell migration/differentiation, immunology or angiogenesis. We identified receptors for ephrin and growth factors (IGF, PDGF, EGF, anthrax toxin) known to be involved in cardiac repair and regeneration. Functional category enrichment identified clusters around integrins, PI3K/Akt-signaling and various cardiomyopathies. Our study indicates that EPDC and ASC have a similar molecular phenotype related to cardiac healing/regeneration. The cell surface proteome repository will help to further unravel the molecular details of their cardio-regenerative potential and their role in cardiac diseases.
Assuntos
Átrios do Coração/citologia , Ventrículos do Coração/citologia , Pericárdio/citologia , Proteoma/genética , Células Estromais/citologia , Animais , Diferenciação Celular , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/metabolismo , Átrios do Coração/metabolismo , Ventrículos do Coração/metabolismo , Humanos , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Pericárdio/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fator de Crescimento Derivado de Plaquetas/genética , Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteoma/metabolismo , Proteômica , Ratos , Ratos Wistar , Transdução de Sinais , Células Estromais/metabolismoRESUMO
The present protocol describes a unique approach that enables the collection of cardiac transudate (CT) from the isolated, saline-perfused rat heart. After isolation and retrograde perfusion of the heart according to the Langendorff technique, the heart is inverted into an upside-down position and is mechanically stabilized by a balloon catheter inserted into the left ventricle. Then, a thin latex cap - previously cast to match the average size of the rat heart - is placed over the epicardial surface. The outlet of the latex cap is connected to silicon tubing, with the distal opening 10 cm below the base level of the heart, creating slight suction. CT continuously produced on the epicardial surface is collected in ice-cooled vials for further analysis. The rate of CT formation ranged from 17 to 147 µL/min (n = 14) in control and infarcted hearts, which represents 0.1-1% of the coronary venous effluent perfusate. Proteomic analysis and high performance liquid chromatography (HPLC) revealed that the collected CT contains a wide spectrum of proteins and purinergic metabolites.
Assuntos
Cateterismo Cardíaco/métodos , Exsudatos e Transudatos/metabolismo , Coração/fisiologia , Modelos Biológicos , Miocárdio/metabolismo , Animais , Cateteres Cardíacos , Cromatografia Líquida de Alta Pressão , Vasos Coronários , Ventrículos do Coração , Técnicas In Vitro , Masculino , Perfusão , Proteômica , RatosRESUMO
BACKGROUND: T cells are required for proper healing after myocardial infarction. The mechanism of their beneficial action, however, is unknown. The proinflammatory danger signal ATP, released from damaged cells, is degraded by the ectonucleotidases CD39 and CD73 to the anti-inflammatory mediator adenosine. Here, we investigate the contribution of CD73-derived adenosine produced by T cells to cardiac remodeling after ischemia/reperfusion and define its mechanism of action. METHODS: Myocardial ischemia (50 minutes followed by reperfusion) was induced in global CD73-/- and CD4-CD73-/- mice. Tissue injury, T-cell purinergic signaling, cytokines, and cardiac function (magnetic resonance tomography at 9.4 T over 4 weeks) were analyzed. RESULTS: Changes in functional parameters of CD4-CD73-/- mice were identical to those in global CD73 knockouts (KOs). T cells infiltrating the injured heart significantly upregulated at the gene (quantitative polymerase chain reaction) and protein (enzymatic activity) levels critical transporters and enzymes (connexin43, connexin37, pannexin-1, equilibrative nucleoside transporter 1, CD39, CD73, ecto-nucleotide pyrophosphatase/phosphodiesterases 1 and 3, CD157, CD38) for the accelerated release and hydrolysis of ATP, cAMP, AMP, and NAD to adenosine. It is surprising that a lack of CD39 on T cells (from CD39-/- mice) did not alter ATP hydrolysis and very likely involves pyrophosphatases (ecto-nucleotide pyrophosphatase/phosphodiesterases 1 and 3). Circulating T cells predominantly expressed A2a receptor (A2aR) transcripts. After myocardial infarction, A2b receptor (A2bR) transcription was induced in both T cells and myeloid cells in the heart. Thus, A2aR and A2bR signaling may contribute to myocardial responses after myocardial infarction. In the case of T cells, this was associated with an accelerated secretion of proinflammatory and profibrotic cytokines (interleukin-2, interferon-γ, and interleukin-17) when CD73 was lacking. Cytokine production by T cells from peripheral lymph nodes was inhibited by A2aR activation (CGS-21680). The A2bR agonist BAY 60-6583 showed off-target effects. The adenosine receptor agonist NECA inhibited interferon-γ and stimulated interleukin-6 production, each of which was antagonized by a specific A2bR antagonist (PSB-603). CONCLUSIONS: This work demonstrates that CD73 on T cells plays a crucial role in the cardiac wound healing process after myocardial infarction. The underlying mechanism involves a profound increase in the hydrolysis of ATP/NAD and AMP, resulting primarily from the upregulation of pyrophosphatases and CD73. We also define A2bR/A2aR-mediated autacoid feedback inhibition of proinflammatory/profibrotic cytokines by T cell-derived CD73.
Assuntos
5'-Nucleotidase/metabolismo , Infarto do Miocárdio/metabolismo , Receptor A2A de Adenosina/metabolismo , Receptor A2B de Adenosina/metabolismo , Linfócitos T/metabolismo , Cicatrização/fisiologia , 5'-Nucleotidase/imunologia , Animais , Movimento Celular/fisiologia , Reprogramação Celular/fisiologia , Feminino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Infarto do Miocárdio/imunologia , Receptor A2A de Adenosina/imunologia , Receptor A2B de Adenosina/imunologia , Linfócitos T/imunologiaRESUMO
Extracellular adenosine formation from ATP is controlled by ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase/CD39) and ecto-5'-nucleotidase (e-5NT/CD73); the latter converts AMP to adenosine and inorganic phosphate, representing the rate limiting step controlling the ratio between extracellular ATP and adenosine. Evidence that cellular expression and activity of CD39 and CD73 may be subject to changes under pathophysiological conditions has identified this pathway as an endogenous modulator in several diseases and was shown to be involved in the molecular mechanism of drugs, such as methotrexate, salicylates , interferon-ß. We evaluated whether CD73/adenosine/A2A signalling pathway is involved in nimesulide anti-inflammatory effect, in vivo and in vitro. We found that the adenosine A2A agonist, 4-[2-[[6-amino-9-(N-ethyl-ß-d-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid hydrochloride (CGS21680, 2mg/kg ip.), inhibited carrageenan-induced rat paw oedema and the effect was reversed by co-administration of the A2A antagonist -(2-[7-amino-2-[2-furyl][1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl-amino]ethyl)phenol (ZM241385; 3mg/kg i.p.). Nimesulide (5mg/kg i.p.) anti-inflammatory effect was inhibited by pre-treatment with ZM241385 (3mg/kg i.p.) and by local administration of the CD73 inhibitor, adenosine 5'-(α,ß-methylene)diphosphate (APCP; 400µg/paw). Furthermore, we found increased activity of 5'-nucleotidase/CD73 in paws and plasma of nimesulide treated rats, 4h following oedema induction. In vitro, the inhibitory effect of nimesulide on nitrite and prostaglandin E2 production by lipopolysaccharide-activated J774 cell line was reversed by ZM241385 and APCP. Furthermore, nimesulide increased CD73 activity in J774 macrophages while it did not inhibit nitrite accumulation by lipopolysaccharide-activated SiRNA CD73 silenced J774 macrophages. Our data demonstrate that the anti-inflammatory effect of nimesulide in part is mediated by CD73-derived adenosine acting on A2A receptors.
Assuntos
Adenosina/metabolismo , Anti-Inflamatórios/farmacologia , Inibidores de Ciclo-Oxigenase 2/farmacologia , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia , 5'-Nucleotidase/antagonistas & inibidores , 5'-Nucleotidase/sangue , 5'-Nucleotidase/metabolismo , Animais , Anti-Inflamatórios/uso terapêutico , Linhagem Celular , Inibidores de Ciclo-Oxigenase 2/administração & dosagem , Dinoprostona/sangue , Edema/tratamento farmacológico , Edema/imunologia , Edema/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Masculino , Ratos Wistar , Receptor A2A de Adenosina/metabolismo , Sulfonamidas/administração & dosagemRESUMO
IL-17 production defines Th17 cells, which orchestrate immune responses and autoimmune diseases. Human Th17 cells can be efficiently generated with appropriate cytokines from precommitted precursors, but the requirements of uncommitted T cells are still ill defined. In standard human Th17 cultures, IL-17 production was restricted to CCR6(+)CD45RA(+) T cells, which expressed CD95 and produced IL-17 ex vivo, identifying them as Th17 memory stem cells. Uncommitted naive CD4(+) T cells upregulated CCR6, RORC2, and IL-23R expression with Th17-promoting cytokines but in addition required sustained TCR stimulation, late mammalian target of rapamycin (mTOR) activity, and HIF-1α to produce IL-17. However, in standard high-density cultures, nutrients like glucose and amino acids became progressively limiting, and mTOR activity was consequently not sustained, despite ongoing TCR stimulation and T cell proliferation. Sustained, nutrient-dependent mTOR activity also induced spontaneous IL-22 and IFN-γ production, but these cytokines had also unique metabolic requirements. Thus, glucose promoted IL-12-independent Th1 differentiation, whereas aromatic amino acid-derived AHR ligands were selectively required for IL-22 production. The identification of Th17 memory stem cells and the stimulation requirements for induced human Th17/22 differentiation have important implications for T cell biology and for therapies targeting the mTOR pathway.
Assuntos
Diferenciação Celular/imunologia , Memória Imunológica/fisiologia , Interferon gama/imunologia , Interleucinas/imunologia , Transdução de Sinais/imunologia , Células Th17/imunologia , Feminino , Humanos , Masculino , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/imunologia , Receptores CCR6/imunologia , Receptores de Interleucina/imunologia , Células Th17/citologia , Interleucina 22RESUMO
IL-21 promotes Th17 differentiation, and Th17 cells that upregulate T-bet, IFN-γ, and GM-CSF drive experimental autoimmune diseases in mice. Anti-IL-21 treatment of autoimmune patients is therefore a therapeutic option, but the role of IL-21 in human T cell differentiation is incompletely understood. IL-21 was produced at high levels by human CD4(+) central memory T cells, suggesting that it is associated with early T cell differentiation. Consistently, it was inhibited by forced expression of T-bet or RORC2, the lineage-defining transcription factors of Th1 and Th17 effector cells, respectively. Although IL-21 was efficiently induced by IL-12 in naive CD4(+) T cells, it inhibited the generation of Th1 effector cells in a negative feedback loop. IL-21 was also induced by IL-6 and promoted Th17 differentiation, but it was not absolutely required. Importantly, however, IL-21 promoted IL-10 secretion but inhibited IFN-γ and GM-CSF production in developing Th17 cells, and consequently prevented the generation of polyfunctional Th1/17 effector cells. Moreover, in Th17 memory cells, IL-21 selectively inhibited T-bet upregulation and GM-CSF production. In summary, IL-21 is a central memory T cell-associated cytokine that promotes Th17 differentiation and IL-10 production, but inhibits the generation of potentially pathogenic Th1/17 effector cells. These findings shed new light on the role of IL-21 in T cell differentiation, and have relevant implications for anti-IL-21 therapy of autoimmune diseases.
Assuntos
Doenças Autoimunes/imunologia , Diferenciação Celular/imunologia , Memória Imunológica , Interleucinas/imunologia , Células Th1/imunologia , Células Th17/imunologia , Animais , Doenças Autoimunes/patologia , Doenças Autoimunes/terapia , Feminino , Humanos , Interleucina-10/imunologia , Interleucina-12/imunologia , Interleucina-6/imunologia , Masculino , Camundongos , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/imunologia , Proteínas com Domínio T/imunologia , Células Th1/patologia , Células Th17/patologia , Regulação para Cima/imunologiaRESUMO
Extracellular nucleotides and nucleosides have been implicated as important signaling molecules in the pathogenesis of acute lung injury (ALI). While adenosine is known to inhibit T cell activation, little information is available as to ATP and NAD degrading enzymes, the expression of ATP and adenosine receptors/transporters in different T cell subsets. ALI was induced by challenging mice with intra-tracheal instillation of 60 µl (3 µg/g) LPS. After 3 d and 7 d blood, lung tissue and bronchoalveolar lavage was collected and immune cells were analyzed using flow cytometry. The transcriptional phenotype of T helper cells, cytotoxic and regulatory T cells sorted by FACS was assessed by measuring the expression profile of 28 genes related to purinergic signaling using TaqMan Array Micro Fluidic Cards. Catabolism of ATP, NAD and cAMP by activated CD4+ T cells was evaluated by HPLC. CD73 was found to be highly abundant on lymphoid cells with little abundance on myeloid cells, while the opposite was true for CD39. After ALI, the abundance of CD39 and CD73 significantly increased on all T cell subsets derived from lung tissue and bronchoalveolar space. Expression analysis in T cell subsets of the lung revealed ATP (Cd39, Cd73) and NAD (Cd38, Cd157, Cd296, Pc-1) degrading enzymes. However, only transcription of Cd38, Cd39, Cd73, Ent1 and A2a receptor was significantly upregulated after ALI in T helper cells. CD4+ T cells from injured lung rapidly metabolized extracellular ATP to AMP and adenosine but not NAD or cAMP. These findings show that lung T cells--the dominant cell fraction in the later phase of ALI--exhibit a unique expression pattern of purinergic signaling molecules. Adenosine is formed by T cells at an enhanced rate from ATP but not from NAD and together with upregulated A2a receptor is likely to modulate the healing process after acute lung injury.
Assuntos
Leucócitos/patologia , Lipopolissacarídeos/toxicidade , Lesão Pulmonar/patologia , Receptores Purinérgicos/metabolismo , Transdução de Sinais , 5'-Nucleotidase/imunologia , Animais , Antígenos CD/imunologia , Apirase/imunologia , Cromatografia Líquida de Alta Pressão , Leucócitos/imunologia , Lesão Pulmonar/induzido quimicamente , Lesão Pulmonar/metabolismo , Camundongos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Dendritic cells (DC) have the unique capacities to induce primary T-cell responses. In mice, CD8α(+)DC are specialized to cross-prime CD8(+) T cells and produce interleukin-12 (IL-12) that promotes cytotoxicity. Human BDCA-3(+)DC share several relevant characteristics with CD8α(+)DC, but the capacities of human DC subsets to induce CD8(+) T-cell responses are incompletely understood. Here we compared CD1c(+) myeloid DC (mDC)1, BDCA-3(+)mDC2, and plasmacytoid DC (pDC) in peripheral blood and lymphoid tissues for phenotype, cytokine production, and their capacities to prime cytotoxic T cells. mDC1 were surprisingly the only human DC that secreted high amounts of IL-12p70, but they required combinational Toll-like receptor (TLR) stimulation. mDC2 and pDC produced interferon-λ and interferon-α, respectively. Importantly, mDC1 and mDC2 required different combinations of TLR ligands to cross-present protein antigens to CD8(+) T cells. pDC were inefficient and also expressed lower levels of major histocompatibility complex and co-stimulatory molecules. Nevertheless, all DC induced CD8(+) memory T-cell expansions upon licensing by CD4(+) T cells, and primed naive CD8(+) T cells following appropriate TLR stimulation. However, because mDC1 produced IL-12, they induced the highest levels of cytotoxic molecules. In conclusion, CD1c(+)mDC1 are the relevant source of IL-12 for naive T cells and are fully equipped to cross-prime cytotoxic T-cell responses.
Assuntos
Antígenos CD1/metabolismo , Células Dendríticas/citologia , Glicoproteínas/metabolismo , Interleucina-12/metabolismo , Linfócitos T Citotóxicos/citologia , Animais , Apresentação de Antígeno , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Proliferação de Células , Separação Celular , Citocinas/metabolismo , Células Dendríticas/metabolismo , Humanos , Memória Imunológica , Interferon-alfa/metabolismo , Interferon gama/metabolismo , Ativação Linfocitária , Camundongos , Fenótipo , Receptores Toll-Like/metabolismoRESUMO
CCR6 is a chemokine receptor expressed on Th17 cells and regulatory T cells that is induced by T-cell priming with certain cytokines, but how its expression and stability are regulated at the molecular level is largely unknown. Here, we identified and characterized a noncoding region of the human CCR6 locus that displayed unmethylated CpG motifs (differentially methylated region [DMR]) selectively in CCR6(+) lymphocytes. CCR6 expression on circulating CD4(+) T cells was stable on cytokine-induced proliferation but partially down-regulated on T-cell receptor stimulation. However, CCR6 down-regulation was mostly transient, and the DMR within the CCR6 locus remained demethylated. Notably, in vitro induction of CCR6 expression with cytokines in T-cell receptor-activated naive CD4(+) T cells was not associated with a demethylated DMR and resulted in unstable CCR6 expression. Conversely, treatment with the DNA methylation inhibitor 5'-azacytidine induced demethylation of the DMR and led to increased and stable CCR6 expression. Finally, when cloned into a reporter gene plasmid, the DMR displayed transcriptional activity in memory T cells that was suppressed by DNA methylation. In summary, we have identified a noncoding region of the human CCR6 gene with methylation-sensitive transcriptional activity in CCR6(+) T cells that controls stable CCR6 expression via epigenetic mechanisms.
Assuntos
Metilação de DNA/genética , Epigênese Genética/genética , Regulação da Expressão Gênica/genética , Receptores CCR6/genética , Linfócitos T/metabolismo , Separação Celular , Citometria de Fluxo , Expressão Gênica , Humanos , Reação em Cadeia da Polimerase , TransfecçãoRESUMO
Interleukin (IL)-10 produced by regulatory T cell subsets is important for the prevention of autoimmunity and immunopathology, but little is known about the phenotype and function of IL-10-producing memory T cells. Human CD4(+)CCR6(+) memory T cells contained comparable numbers of IL-17- and IL-10-producing cells, and CCR6 was induced under both Th17-promoting conditions and upon tolerogenic T cell priming with transforming growth factor (TGF)-beta. In normal human spleens, the majority of CCR6(+) memory T cells were in the close vicinity of CCR6(+) myeloid dendritic cells (mDCs), and strikingly, some of them were secreting IL-10 in situ. Furthermore, CCR6(+) memory T cells produced suppressive IL-10 but not IL-2 upon stimulation with autologous immature mDCs ex vivo, and secreted IL-10 efficiently in response to suboptimal T cell receptor (TCR) stimulation with anti-CD3 antibodies. However, optimal TCR stimulation of CCR6(+) T cells induced expression of IL-2, interferon-gamma, CCL20, and CD40L, and autoreactive CCR6(+) T cell lines responded to various recall antigens. Notably, we isolated autoreactive CCR6(+) T cell clones with context-dependent behavior that produced IL-10 with autologous mDCs alone, but that secreted IL-2 and proliferated upon stimulation with tetanus toxoid. We propose the novel concept that a population of memory T cells, which is fully equipped to participate in secondary immune responses upon recognition of a relevant recall antigen, contributes to the maintenance of tolerance under steady-state conditions.
Assuntos
Memória Imunológica , Receptores CCR6/genética , Linfócitos T Reguladores/imunologia , Linfócitos T/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Regulação da Expressão Gênica , Homeostase/imunologia , Humanos , Terapia de Imunossupressão , Interferon gama/genética , Interleucina-10/biossíntese , Interleucina-2/biossíntese , Interleucina-2/genética , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T Reguladores/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Two subsets of natural and adaptive regulatory T (T reg) cells have been described, but the identity of adaptive type 1 regulatory (Tr1)-like cells in humans is unclear. We analyzed a subset of human blood CD4(+) T cells--CD45RA(-)CD25(-)interleukin (IL)-7 receptor (R)(-) cells--that rapidly secreted high levels of IL-10 together with interferon gamma, but produced little IL-2. These IL-7R(-) T cells were rare, anergic, and largely Foxp3(-). They expressed low levels of Bcl-2 but high levels of Ki-67 and ICOS, suggesting that they have been recently activated in vivo. Consistently, they responded selectively to persistent foreign and self-antigens under steady-state conditions. Unlike natural CD25(+) T reg cells, IL-7R(-) cells suppressed naive and memory T cell proliferation in an IL-10-dependent fashion, and they required strong T cell receptor stimulation for suppression. To our knowledge, this is the first report that identifies Tr1-like cells in human blood. These IL-10-secreting cells have characteristics of chronically activated Th1 effector cells and are distinct from CD25(+) T reg cells.