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Cholesterol efflux capacity (CEC) is of interest given its potential relationship with several important clinical conditions including Alzheimer's disease. The inactivation of the APOE locus in mouse models supports the idea that it is involved in determining the CEC. With that in mind, we examine the impact of the plasma metabolome profile and the APOE genotype on the CEC in cognitively healthy elderly subjects. The study subjects were 144 unrelated healthy individuals. The plasma CEC was determined by exposing cultured mouse macrophages treated with BODIPY-cholesterol to human plasma. The metabolome profile was determined using NMR techniques. Multiple regression was performed to identify the most important predictors of CEC, as well as the NMR features most strongly associated with the APOE genotype. Plasma 3-hydroxybutyrate was the variable most strongly correlated with the CEC (r = 0.365; p = 7.3 × 10-6). Male sex was associated with a stronger CEC (r = -0.326, p = 6.8 × 10-5). Most of the NMR particles associated with the CEC did not correlate with the APOE genotype. The NMR metabolomics results confirmed the APOE genotype to have a huge effect on the concentration of plasma lipoprotein particles as well as those of other molecules including omega-3 fatty acids. In conclusion, the CEC of human plasma was associated with ketone body concentration, sex, and (to a lesser extent) the other features of the plasma lipoprotein profile. The APOE genotype exerted only a weak effect on the CEC via the modulation of the lipoprotein profile. The APOE locus was associated with omega-3 fatty acid levels independent of the plasma cholesterol level.
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Colesterol , Jejum , Animais , Camundongos , Humanos , Masculino , Adulto , Idoso , Espectroscopia de Ressonância Magnética , Genótipo , Apolipoproteínas E/genética , HDL-ColesterolRESUMO
Colorectal cancer consensus molecular subtypes (CMSs) are widely accepted and constitutes the basis for patient stratification to improve clinical practice. We aimed to find whether miRNAs could reproduce molecular subtypes, and to identify miRNA targets associated to the High-stroma/CMS4 subtype. The expression of 939 miRNAs was analyzed in tumors classified in CMS. TALASSO was used to find gene-miRNA interactions. A miR-mRNA regulatory network was constructed using Cytoscape. Candidate gene-miR interactions were validated in 293T cells. Hierarchical-Clustering identified three miRNA tumor subtypes (miR-LS; miR-MI; and miR-HS) which were significantly associated (p < 0.001) to the reported mRNA subtypes. miR-LS correlated with the low-stroma/CMS2; miR-MI with the mucinous-MSI/CMS1 and miR-HS with high-stroma/CMS4. MicroRNA tumor subtypes and association to CMSs were validated with TCGA datasets. TALASSO identified 1462 interactions (p < 0.05) out of 21,615 found between 176 miRs and 788 genes. Based on the regulatory network, 88 miR-mRNA interactions were selected as candidates. This network was functionally validated for the pair miR-30b/SLC6A6. We found that miR-30b overexpression silenced 3'-UTR-SLC6A6-driven luciferase expression in 293T-cells; mutation of the target sequence in the 3'-UTR-SLC6A6 prevented the miR-30b inhibitory effect. In conclusion CRC subtype classification using a miR-signature might facilitate a real-time analysis of the disease course and treatment response.
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The balance between laminin isoforms containing the α5 or the α4 chain in the endothelial basement membrane determines the site of leukocyte diapedesis under inflammatory conditions. Extracellular superoxide dismutase (SOD3) induces laminin α4 expression in tumor blood vessels, which is associated with enhanced intratumor T cell infiltration in primary human cancers. We show now that SOD3 overexpression in neoplastic and endothelial cells (ECs) reduces laminin α5 in tumor blood vessels. SOD3 represses the laminin α5 gene (LAMA5), but LAMA5 expression is not changed in SOD1-overexpressing cells. Transcriptomic analyses revealed SOD3 overexpression to change the transcription of 1682 genes in ECs, with the canonical and non-canonical NF-κB pathways as the major SOD3 targets. Indeed, SOD3 reduced the transcription of well-known NF-κB target genes as well as NF-κB-driven promoter activity in ECs stimulated with tumor necrosis factor (TNF)-α, an NF-κB signaling inducer. SOD3 inhibited the phosphorylation and degradation of IκBα (nuclear factor of the kappa light polypeptide gene enhancer in B-cells inhibitor alpha), an NF-κB inhibitor. Finally, TNF-α was found to be a transcriptional activator of LAMA5 but not of LAMA4; LAMA5 induction was prevented by SOD3. In conclusion, SOD3 is a major regulator of laminin balance in the basement membrane of tumor ECs, with potential implications for immune cell infiltration into tumors.
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The inhibition of anabolic pathways, such as aerobic glycolysis, is a metabolic cornerstone of memory T cell differentiation and function. However, the signals that hamper these anabolic pathways are not completely known. Recent evidence pinpoints the chemokine receptor CCR5 as an important player in CD4+ T cell memory responses by regulating T cell antigen receptor (TCR) nanoclustering in an antigen-independent manner. This paper reports that CCR5 specifically restrains aerobic glycolysis in memory-like CD4+ T cells, but not in effector CD4+ T cells. CCR5-deficient memory CD4+ T cells thus show an abnormally high glycolytic/oxidative metabolism ratio. No CCR5-dependent change in glucose uptake nor in the expression of the main glucose transporters was detected in any of the examined cell types, although CCR5-deficient memory cells did show increased expression of the hexokinase 2 and pyruvate kinase M2 isoforms, plus the concomitant downregulation of Bcl-6, a transcriptional repressor of these key glycolytic enzymes. Further, the TCR nanoclustering defects observed in CCR5-deficient antigen-experienced CD4+ T cells were partially reversed by incubation with 2-deoxyglucose (2-DG), suggesting a link between inhibition of the glycolytic pathway and TCR nanoscopic organization. Indeed, the treatment of CCR5-deficient lymphoblasts with 2-DG enhanced IL-2 production after antigen re-stimulation. These results identify CCR5 as an important regulator of the metabolic fitness of memory CD4+ T cells, and reveal an unexpected link between T cell metabolism and TCR organization with potential influence on the response of memory T cells upon antigen re-encounter.
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Linfócitos T CD4-Positivos/metabolismo , Memória Imunológica , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores CCR5/fisiologia , Animais , Antígenos/imunologia , Células Cultivadas , Regulação da Expressão Gênica , Glucose/metabolismo , Glicólise/genética , Ligantes , Camundongos , Camundongos Transgênicos , Mitocôndrias/metabolismo , Nanoestruturas , Ovalbumina/imunologia , Consumo de Oxigênio , Fragmentos de Peptídeos/imunologia , Proteínas Proto-Oncogênicas c-bcl-6/biossíntese , Proteínas Proto-Oncogênicas c-bcl-6/genética , Receptores CCR5/deficiência , Organismos Livres de Patógenos EspecíficosRESUMO
The study of cancer biology should be based around a comprehensive vision of the entire tumor ecosystem, considering the functional, bioenergetic and metabolic state of tumor cells and those of their microenvironment, and placing particular importance on immune system cells. Enhanced understanding of the molecular bases that give rise to alterations of pathways related to tumor development can open up new therapeutic intervention opportunities, such as metabolic regulation applied to immunotherapy. This review outlines the role of various oncometabolites and immunometabolites, such as TCA intermediates, in shaping pro/anti-inflammatory activity of immune cells such as MDSCs, T lymphocytes, TAMs and DCs in cancer. We also discuss the extraordinary plasticity of the immune response and its implication in immunotherapy efficacy, and highlight different therapeutic intervention possibilities based on controlling the balanced systems of specific metabolites with antagonistic functions.
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BACKGROUND: Conventional type 1 dendritic cells (cDC1s) are central to antitumor immunity and their presence in the tumor microenvironment associates with improved outcomes in patients with cancer. DNGR-1 (CLEC9A) is a dead cell-sensing receptor highly restricted to cDC1s. DNGR-1 has been involved in both cross-presentation of dead cell-associated antigens and processes of disease tolerance, but its role in antitumor immunity has not been clarified yet. METHODS: B16 and MC38 tumor cell lines were inoculated subcutaneously into wild-type (WT) and DNGR-1-deficient mice. To overexpress Flt3L systemically, we performed gene therapy through the hydrodynamic injection of an Flt3L-encoding plasmid. To characterize the immune response, we performed flow cytometry and RNA-Seq of tumor-infiltrating cDC1s. RESULTS: Here, we found that cross-presentation of tumor antigens in the steady state was DNGR-1-independent. However, on Flt3L systemic overexpression, tumor growth was delayed in DNGR-1-deficient mice compared with WT mice. Of note, this protection was recapitulated by anti-DNGR-1-blocking antibodies in mice following Flt3L gene therapy. This improved antitumor immunity was associated with Batf3-dependent enhanced accumulation of CD8+ T cells and cDC1s within tumors. Mechanistically, the deficiency in DNGR-1 boosted an Flt3L-induced specific inflammatory gene signature in cDC1s, including Ccl5 expression. Indeed, the increased infiltration of cDC1s within tumors and their protective effect rely on CCL5/CCR5 chemoattraction. Moreover, FLT3LG and CCL5 or CCR5 gene expression signatures correlate with an enhanced cDC1 signature and a favorable overall survival in patients with cancer. Notably, cyclophosphamide elevated serum Flt3L levels and, in combination with the absence of DNGR-1, synergized against tumor growth. CONCLUSION: DNGR-1 limits the accumulation of tumor-infiltrating cDC1s promoted by Flt3L. Thus, DNGR-1 blockade may improve antitumor immunity in tumor therapy settings associated to high Flt3L expression.
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Neoplasias do Colo/terapia , Células Dendríticas/metabolismo , Terapia Genética , Lectinas Tipo C/metabolismo , Melanoma Experimental/terapia , Proteínas de Membrana/metabolismo , Receptores Imunológicos/metabolismo , Neoplasias Cutâneas/terapia , Animais , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Quimiocina CCL5/genética , Quimiocina CCL5/metabolismo , Técnicas de Cocultura , Neoplasias do Colo/genética , Neoplasias do Colo/imunologia , Neoplasias do Colo/metabolismo , Células Dendríticas/imunologia , Regulação Neoplásica da Expressão Gênica , Lectinas Tipo C/genética , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Melanoma Experimental/metabolismo , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Receptores CCR5/genética , Receptores CCR5/metabolismo , Receptores Imunológicos/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/metabolismo , Carga Tumoral , Evasão Tumoral , Microambiente TumoralRESUMO
Pyruvate is a key molecule in the metabolic fate of mammalian cells; it is the crossroads from where metabolism proceeds either oxidatively or ends with the production of lactic acid. Pyruvate metabolism is regulated by many enzymes that together control carbon flux. Mitochondrial pyruvate carrier (MPC) is responsible for importing pyruvate from the cytosol to the mitochondrial matrix, where it is oxidatively phosphorylated to produce adenosine triphosphate (ATP) and to generate intermediates used in multiple biosynthetic pathways. MPC activity has an important role in glucose homeostasis, and its alteration is associated with diabetes, heart failure, and neurodegeneration. In cancer, however, controversy surrounds MPC function. In some cancers, MPC upregulation appears to be associated with a poor prognosis. However, most transformed cells undergo a switch from oxidative to glycolytic metabolism, the so-called Warburg effect, which, amongst other possibilities, is induced by MPC malfunction or downregulation. Consequently, impaired MPC function might induce tumors with strong proliferative, migratory, and invasive capabilities. Moreover, glycolytic cancer cells secrete lactate, acidifying the microenvironment, which in turn induces angiogenesis, immunosuppression, and the expansion of stromal cell populations supporting tumor growth. This review examines the latest findings regarding the tumorigenic processes affected by MPC.
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BACKGROUND: Preclinical research suggests that the efficacy of immune checkpoint inhibitors in breast cancer can be enhanced by combining them with antiangiogenics, particularly in a sequential fashion. We sought to explore the efficacy and biomarkers of combining the anti-PD-L1 durvalumab plus the antiangiogenic bevacizumab after bevacizumab monotherapy for advanced HER2-negative breast cancer. METHODS: Patients had advanced HER2-negative disease that progressed while receiving single-agent bevacizumab maintenance as a part of a previous chemotherapy plus bevacizumab regimen. Treatment consisted of bi-weekly durvalumab plus bevacizumab (10 mg/kg each i.v.). Peripheral-blood mononuclear cells (PBMCs) were obtained before the first durvalumab dose and every 4 weeks and immunophenotyped by flow-cytometry. A fresh pre-durvalumab tumor biopsy was obtained; gene-expression studies and immunohistochemical staining to assess vascular normalization and characterize the immune infiltrate were conducted. Patients were classified as "non-progressors" if they had clinical benefit (SD/PR/CR) at 4 months. The co-primary endpoints were the changes in the percentage T cell subpopulations in PBMCs in progressors versus non-progressors, and PFS/OS time. RESULTS: Twenty-six patients were accrued. Median PFS and OS were 3.5 and 11 months; a trend for a longer OS was detected for the hormone-positive subset (19.8 versus 7.4 months in triple-negatives; P = 0.11). Clinical benefit rate at 2 and 4 months was 60% and 44%, respectively, without significant differences between hormone-positive and triple-negative (P = 0.73). Non-progressors' tumors displayed vascular normalization features as a result of previous bevacizumab, compared with generally abnormal patterns observed in progressors. Non-progressors also showed increased T-effector and T-memory signatures and decreased TREG signatures in gene expression studies in baseline-post-bevacizumab-tumors compared with progressors. Notably, analysis of PBMC populations before durvalumab treatment was concordant with the findings in tumor samples and showed a decreased percentage of circulating TREGs in non-progressors. CONCLUSIONS: This study reporting on sequential bevacizumab+durvalumab in breast cancer showed encouraging activity in a heavily pre-treated cohort. The correlative studies agree with the preclinical rationale supporting an immunopriming effect exerted by antiangiogenic treatment, probably by reducing TREGs cells both systemically and in tumor tissue. The magnitude of this benefit should be addressed in a randomized setting. TRIAL REGISTRATION: (www.clinicaltrials.gov): NCT02802098 . Registered on June 16, 2020.
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Anticorpos Monoclonais/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Bevacizumab/administração & dosagem , Neoplasias da Mama/tratamento farmacológico , Adulto , Idoso , Inibidores da Angiogênese/administração & dosagem , Inibidores da Angiogênese/efeitos adversos , Anticorpos Monoclonais/efeitos adversos , Antineoplásicos Imunológicos/administração & dosagem , Antineoplásicos Imunológicos/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/imunologia , Antígeno B7-H1/metabolismo , Bevacizumab/efeitos adversos , Mama/patologia , Neoplasias da Mama/sangue , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Progressão da Doença , Feminino , Humanos , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/imunologia , Pessoa de Meia-Idade , Projetos Piloto , Intervalo Livre de Progressão , Estudo de Prova de Conceito , Receptor ErbB-2/análise , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologiaRESUMO
Tumor-infiltrating lymphocytes (TILs) are major players in the immune-mediated control of cancer and the response to immunotherapy. In primary cancers, however, TILs are commonly absent, suggesting T-cell entry into the tumor microenvironment (TME) to be selectively restricted. Blood and lymph vessels are the first barriers that circulating T-cells must cross to reach the tumor parenchyma. Certainly, the crossing of the endothelial cell (EC) basement membrane (EC-BM)-an extracellular matrix underlying EC-is a limiting step in T-cell diapedesis. This review highlights new data suggesting the antioxidant enzyme superoxide dismutase-3 (SOD3) to be a regulator of EC-BM composition in the tumor vasculature. In the EC, SOD3 induces vascular normalization and endows the EC-BM with the capacity for the extravasation of effector T-cells into the TME, which it achieves via the WNT signaling pathway. However, when activated in tumor cells, this same pathway is reported to exclude TILs. SOD3 also regulates TIL density in primary human colorectal cancers (CRC), thus affecting the relapse rate and patient survival.
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Membrana Basal/patologia , Neoplasias Colorretais/metabolismo , Células Endoteliais/patologia , Linfócitos do Interstício Tumoral/imunologia , Superóxido Dismutase/metabolismo , Linfócitos T/imunologia , Animais , Movimento Celular , Neoplasias Colorretais/patologia , Espaço Extracelular , Humanos , Microambiente Tumoral , Via de Sinalização WntRESUMO
The conversion of a non-T cell-inflamed into a T cell-inflamed tumor microenvironment (TME) is a key to sensitizing tumors to T-cell-based immunotherapies. Recent data show that the extracellular superoxide dismutase (SOD3) alters endothelial basement membrane (EC-BM) composition, providing permissive signals that enhance tumor infiltration by effector T cells. Abbreviations: AJ, adherens junction; EC, endothelial cell; EC-BM, endothelial basement membrane; HIF, hypoxia-inducible factor; ICAM-1, intercellular adhesion molecule-1; LAMA4, laminin-α4; SOD3, superoxide dismutase-3; TME, tumor microenvironment; VCAM-1, vascular cell adhesion molecule-1; VEGF, vascular-endothelial growth factor.
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Laminina , Neoplasias , Endotélio Vascular , Humanos , Superóxido Dismutase/genética , Linfócitos T , Microambiente TumoralRESUMO
CCR5 is not only a coreceptor for HIV-1 infection in CD4+ T cells, but also contributes to their functional fitness. Here, we show that by limiting transcription of specific ceramide synthases, CCR5 signaling reduces ceramide levels and thereby increases T-cell antigen receptor (TCR) nanoclustering in antigen-experienced mouse and human CD4+ T cells. This activity is CCR5-specific and independent of CCR5 co-stimulatory activity. CCR5-deficient mice showed reduced production of high-affinity class-switched antibodies, but only after antigen rechallenge, which implies an impaired memory CD4+ T-cell response. This study identifies a CCR5 function in the generation of CD4+ T-cell memory responses and establishes an antigen-independent mechanism that regulates TCR nanoclustering by altering specific lipid species.
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Antígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Ceramidas/imunologia , Memória Imunológica , Receptores CCR5/deficiência , Animais , Antígenos/genética , Linfócitos T CD4-Positivos/citologia , Ceramidas/genética , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Receptores CCR5/imunologiaRESUMO
BACKGROUND: Tumor-infiltrating lymphocytes (TILs), mainly CD8+ cytotoxic T lymphocytes (CTL), are linked to immune-mediated control of human cancers and response to immunotherapy. Tumors have nonetheless developed specific mechanisms that selectively restrict T cell entry into the tumor microenvironment. The extracellular superoxide dismutase (SOD3) is an anti-oxidant enzyme usually downregulated in tumors. We hypothesize that upregulation of SOD3 in the tumor microenvironment might be a mechanism to boost T cell infiltration by normalizing the tumor-associated endothelium. RESULTS: Here we show that SOD3 overexpression in endothelial cells increased in vitro transmigration of naïve and activated CD4+ and CD8+ T cells, but not of myeloid cells. Perivascular expression of SOD3 also specifically increased CD4+ and CD8+ effector T cell infiltration into tumors and improved the effectiveness of adoptively transferred tumor-specific CD8+ T cells. SOD3-induced enhanced transmigration in vitro and tumor infiltration in vivo were not associated to upregulation of T cell chemokines such as CXCL9 or CXCL10, nor to changes in the levels of endothelial adhesion receptors such as intercellular adhesion molecule-1 (ICAM-1) or vascular cell adhesion molecule-1 (VCAM-1). Instead, SOD3 enhanced T cell infiltration via HIF-2α-dependent induction of specific WNT ligands in endothelial cells; this led to WNT signaling pathway activation in the endothelium, FOXM1 stabilization, and transcriptional induction of laminin-α4 (LAMA4), an endothelial basement membrane component permissive for T cell infiltration. In patients with stage II colorectal cancer, SOD3 was associated with increased CD8+ TIL density and disease-free survival. SOD3 expression was also linked to a T cell-inflamed gene signature using the COAD cohort from The Cancer Genome Atlas program. CONCLUSION: Our findings suggest that SOD3-induced upregulation of LAMA4 in endothelial cells boosts selective tumor infiltration by T lymphocytes, thus transforming immunologically "cold" into "hot" tumors. High SOD3 levels are associated with human colon cancer infiltration by CD8+ T cells, with potential consequences for the clinical outcome of these patients. Our results also uncover a cell type-specific, distinct activity of the WNT pathway for the regulation of T cell infiltration into tumors.
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Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Células Endoteliais/imunologia , Linfócitos do Interstício Tumoral/imunologia , Neoplasias/imunologia , Superóxido Dismutase/metabolismo , Superóxido Dismutase/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Feminino , Humanos , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias/metabolismo , Neoplasias/patologia , Transdução de Sinais , Superóxido Dismutase/genética , Células Tumorais Cultivadas , Microambiente TumoralRESUMO
Cancer immunotherapy relies on the ability of immune cells to kill malignant cells. The cytotoxic T lymphocyte (CTL) response is perhaps the functional measure that best reflects cell-mediated immunity against cancer. Straightforward methods that facilitate quantitative evaluation of the potency of compounds that can modulate T cell-mediated, tumor antigen-specific immune responses are central in the screening cascade when searching for new immunotherapeutic agents for cancer. Here we describe a simple, sensitive method, based on flow cytometry analyses, to quantitatively measure cytotoxicity in vitro. We provide examples that validate its feasibility and specificity using CD8+ T lymphocytes specific for a surrogate tumor antigen, and a blocking antibody for the inhibitory PD-1/PD-L1 axis. This method can nonetheless be applied to the screening of virtually any cytotoxicity modulatory compound, including antibodies and small molecules or T cell-based therapies, and can be scaled up for high-throughput workflow and automation.
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Testes Imunológicos de Citotoxicidade/métodos , Citometria de Fluxo/métodos , Fatores Imunológicos/farmacologia , Neoplasias/imunologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Animais , Antígenos de Neoplasias , Humanos , Fatores Imunológicos/uso terapêutico , Imunoterapia , Neoplasias/terapia , Linfócitos T Citotóxicos/imunologiaRESUMO
BACKGROUND: Binding of the programmed death-1 (PD-1) receptor to its ligands (PD-L1/2) transduces inhibitory signals that promote exhaustion of activated T cells. Blockade of the PD-1 pathway is widely used for cancer treatment, yet the inhibitory signals transduced by PD-1 in T cells remain elusive. METHODS: Expression profiles of human CD8+ T cells in resting, activated (CD3 + CD28) and PD-1-stimulated cells (CD3 + CD28 + PD-L1-Fc) conditions were evaluated by RNA-seq. Bioinformatic analyses were used to identify signaling pathways differentially regulated in PD-1-stimulated cells. Metabolic analyses were performed with SeaHorse technology, and mitochondrial ultrastructure was determined by transmission electron microscopy. PD-1-regulated mitochondrial genes were silenced using short-hairpin RNA in primary cells. Blue native gel electrophoresis was used to determine respiratory supercomplex assembly. RESULTS: PD-1 engagement in human CD8+ T cells triggers a specific, progressive genetic program different from that found in resting cells. Gene ontology identified metabolic processes, including glycolysis and oxidative phosphorylation (OXPHOS), as the main pathways targeted by PD-1. We observed severe functional and structural alterations in the mitochondria of PD-1-stimulated cells, including a reduction in the number and length of mitochondrial cristae. These cristae alterations were associated with reduced expression of CHCHD3 and CHCHD10, two proteins that form part of the mitochondrial contact site and cristae organizing system (MICOS). Although PD-1-stimulated cells showed severe cristae alterations, assembly of respiratory supercomplexes was unexpectedly greater in these cells than in activated T cells. CHCHD3 silencing in primary CD8+ T cells recapitulated some effects induced by PD-1 stimulation, including reduced mitochondrial polarization and interferon-γ production following T cell activation with anti-CD3 and -CD28 activating antibodies. CONCLUSIONS: Our results suggest that mitochondria are the main targets of PD-1 inhibitory activity. PD-1 reprograms CD8+ T cell metabolism for efficient use of fatty acid oxidation; this mitochondrial phenotype might explain the long-lived phenotype of PD-1-engaged T cells.
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Linfócitos T CD8-Positivos/imunologia , Mitocôndrias/imunologia , Antígeno B7-H1/imunologia , Antígeno B7-H1/metabolismo , Células Cultivadas , Expressão Gênica/imunologia , Glicólise , Células HEK293 , Humanos , Leucócitos Mononucleares , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/imunologia , Fosforilação Oxidativa , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/imunologia , Receptor de Morte Celular Programada 1/metabolismo , Transdução de Sinais/imunologiaRESUMO
Adult progenitor cells reside in specialized microenvironments which maintain their undifferentiated cell state and trigger regenerative responses following injury. Although these environments are well described in several tissues, the cellular components that comprise the cardiac environment where progenitor cells are located remain unknown. Here we use Bmi1CreERT and Bmi1GFP mice as genetic tools to trace cardiac damage-responsive cells throughout the mouse lifespan. In adolescent mice, Bmi1+ damage-responsive cells are broadly distributed throughout the myocardium. In adult mice, however, Bmi1+ cells are confined predominately in perivascular areas with low levels of reactive oxygen species (ROS) and their number decline in an age-dependent manner. In vitro co-culture experiments with endothelial cells supported a regulatory role of the endothelium in damage-responsive cell behavior. Accordingly, in vivo genetic decrease of ROS levels in adult heart disengaged Bmi1+ cells from the cardiovascular network, recapitulating an adolescent-like Bmi1 expression profile. Thus, we identify cardiac perivascular regions as low-stress microenvironments that favor the maintenance of adult damage-responsive cells.
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Miocárdio/metabolismo , Estresse Oxidativo , Complexo Repressor Polycomb 1/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fatores Etários , Animais , Permeabilidade Capilar , Proliferação de Células , Células Endoteliais/metabolismo , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Estresse Oxidativo/genética , Complexo Repressor Polycomb 1/genética , Proteínas Proto-Oncogênicas/genética , Espécies Reativas de Oxigênio/metabolismoRESUMO
The arsenal of cancer therapies has evolved to target T lymphocytes and restore their capacity to destroy tumor cells. T cells rely on diacylglycerol (DAG) to carry out their functions. DAG availability and signaling are regulated by the enzymes diacylglycerol kinase (DGK) α and ζ, whose excess function drives T cells into hyporesponsive states. Targeting DGKα is a promising strategy for coping with cancer; its blockade could reinstate T-cell attack on tumors while limiting tumor growth, due to positive DGKα functions in several oncogenic pathways. Here, we made a side-by-side comparison of the effects of commercial pharmacological DGK inhibitors on T-cell responses with those promoted by DGKα and DGKζ genetic deletion or silencing. We show the specificity for DGKα of DGK inhibitors I and II and the structurally similar compound ritanserin. Inhibitor treatment promoted Ras/ERK (extracellular signal-regulated kinase) signaling and AP-1 (Activator protein-1) transcription, facilitated DGKα membrane localization, reduced the requirement for costimulation, and cooperated with enhanced activation following DGKζ silencing/deletion. DGKiII and ritanserin had similar effects on TCR proximal signaling, but ritanserin counteracted long-term T-cell activation, an effect that was potentiated in DGKα-/- cells. In contrast with enhanced activation triggered by pharmacological inhibition, DGKα silencing/genetic deletion led to impaired Lck (lymphocyte-specific protein tyrosine kinase) activation and limited costimulation responses. Our results demonstrate that pharmacological inhibition of DGKα downstream of the TCR provides a gain-of-function effect that amplifies the DAG-dependent signaling cascade, an ability that could be exploited therapeutically to reinvigorate T cells to attack tumors.
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Diacilglicerol Quinase/imunologia , Imunoterapia/métodos , Linfócitos T/imunologia , Animais , Humanos , Camundongos , Transdução de Sinais , TransfecçãoRESUMO
One drawback of chemotherapy is poor drug delivery to tumor cells, due in part to hyperpermeability of the tumor vasculature. Extracellular superoxide dismutase (SOD3) is an antioxidant enzyme usually repressed in the tumor milieu. Here we show that specific SOD3 re-expression in tumor-associated endothelial cells (ECs) increases doxorubicin (Doxo) delivery into and chemotherapeutic effect on tumors. Enhanced SOD3 activity fostered perivascular nitric oxide accumulation and reduced vessel leakage by inducing vascular endothelial cadherin (VEC) transcription. SOD3 reduced HIF prolyl hydroxylase domain protein activity, which increased hypoxia-inducible factor-2α (HIF-2α) stability and enhanced its binding to a specific VEC promoter region. EC-specific HIF-2α ablation prevented both the SOD3-mediated increase in VEC transcription and the enhanced Doxo effect. SOD3, VEC, and HIF-2α levels correlated positively in primary colorectal cancers, which suggests a similar interconnection of these proteins in human malignancy.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Doxorrubicina/administração & dosagem , Células Endoteliais/metabolismo , Neoplasias/tratamento farmacológico , Superóxido Dismutase/metabolismo , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Antineoplásicos/administração & dosagem , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Caderinas/genética , Caderinas/metabolismo , Dioxigenases/genética , Dioxigenases/metabolismo , Tratamento Farmacológico , Células Endoteliais/efeitos dos fármacos , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/genética , Neoplasias/metabolismo , Estabilidade Proteica , Superóxido Dismutase/genéticaRESUMO
M1 and M2 macrophage phenotypes, which mediate proinflammatory and antiinflammatory functions, respectively, represent the extremes of immunoregulatory plasticity in the macrophage population. This plasticity can also result in intermediate macrophage states that support a balance between these opposing functions. In sepsis, M1 macrophages can compensate for hyperinflammation by acquiring an M2-like immunosuppressed status that increases the risk of secondary infection and death. The M1 to M2 macrophage reprogramming that develops during LPS tolerance resembles the pathological antiinflammatory response to sepsis. Here, we determined that p21 regulates macrophage reprogramming by shifting the balance between active p65-p50 and inhibitory p50-p50 NF-κB pathways. p21 deficiency reduced the DNA-binding affinity of the p50-p50 homodimer in LPS-primed and -rechallenged macrophages, impairing their ability to attenuate IFN-ß production and acquire an M2-like hyporesponsive status. High p21 levels in sepsis patients correlated with low IFN-ß expression, and p21 knockdown in human monocytes corroborated its role in IFN-ß regulation. The data demonstrate that p21 adjusts the equilibrium between p65-p50 and p50-p50 NF-κB pathways to mediate macrophage plasticity in LPS tolerance. Identifying p21-related pathways involved in monocyte reprogramming may lead to potential targets for sepsis treatment.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Regulação da Expressão Gênica , Interferon beta/metabolismo , Macrófagos/metabolismo , Subunidade p50 de NF-kappa B/metabolismo , Sepse/imunologia , Idoso , Animais , Citocinas/metabolismo , Feminino , Humanos , Inflamação , Lipopolissacarídeos , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Monócitos/metabolismo , Fenótipo , Ligação Proteica , Multimerização Proteica , Sepse/terapia , Fator de Transcrição RelA/metabolismoRESUMO
Evidence links aryl hydrocarbon receptor (AHR) activation to rheumatoid arthritis (RA) pathogenesis, although results are inconsistent. AHR agonists inhibit pro-inflammatory cytokine expression in macrophages, pivotal cells in RA aetiopathogenesis, which hints at specific circuits that regulate the AHR pathway in RA macrophages. We compared microRNA (miR) expression in CD14(+) cells from patients with active RA or with osteoarthritis (OA). Seven miR were downregulated and one (miR-223) upregulated in RA compared to OA cells. miR-223 upregulation correlated with reduced Notch3 and Notch effector expression in RA patients. Overexpression of the Notch-induced repressor HEY-1 and co-culture of healthy donor monocytes with Notch ligand-expressing cells showed direct Notch-mediated downregulation of miR-223. Bioinformatics predicted the AHR regulator ARNT (AHR nuclear translocator) as a miR-223 target. Pre-miR-223 overexpression silenced ARNT 3'UTR-driven reporter expression, reduced ARNT (but not AHR) protein levels and prevented AHR/ARNT-mediated inhibition of pro-inflammatory cytokine expression. miR-223 counteracted AHR/ARNT-induced Notch3 upregulation in monocytes. Levels of ARNT and of CYP1B1, an AHR/ARNT signalling effector, were reduced in RA compared to OA synovial tissue, which correlated with miR-223 levels. Our results associate Notch signalling to miR-223 downregulation in RA macrophages, and identify miR-223 as a negative regulator of the AHR/ARNT pathway through ARNT targeting.
Assuntos
Artrite Reumatoide/genética , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Citocinas/metabolismo , Macrófagos/metabolismo , MicroRNAs/genética , Receptores Notch/genética , Idoso , Artrite Reumatoide/patologia , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Técnicas de Cocultura , Citocinas/genética , Feminino , Perfilação da Expressão Gênica/métodos , Células HEK293 , Humanos , Masculino , Pessoa de Meia-Idade , Osteoartrite/genética , Osteoartrite/patologia , Transdução de SinaisRESUMO
APRIL (a proliferation-inducing ligand) is a cytokine of the tumor necrosis factor family associated mainly with hematologic malignancies. APRIL is also overexpressed in breast carcinoma tissue lesions, although neither its role in breast tumorigenesis nor the underlying molecular mechanism is known. Here, we show that several breast cancer cell lines express APRIL and both its receptors, B cell maturation antigen (BCMA) and transmembrane activator and CAML-interactor (TACI), independently of luminal or basal tumor cell phenotype, and that the mitogen-activated protein kinases p38, ERK1/2, and JNK1/2 are activated in response to APRIL. The inflammatory stimulus poly I:C, a toll-like receptor (TLR) 3 ligand, enhanced APRIL secretion. Silencing experiments decreased cell proliferation, demonstrating that APRIL is a critical autocrine factor for breast tumor growth. Studies of 4T1 orthotopic breast tumors in APRIL transgenic mice showed that an APRIL-enriched environment increased tumor growth and promoted lung metastasis associated with enhanced tumor cell proliferation; BCMA and TACI expression suggests that both participate in these processes. We detected APRIL, BCMA and TACI in human luminal, triple-negative breast carcinomas and HER2 breast carcinomas, with increased levels in more aggressive basal tumors. APRIL was observed near Ki67(+) nuclei and was distributed heterogeneously in the cancer cells, in the leukocyte infiltrate, and in the myoepithelial layer adjacent to the tumor area; these results imply that APRIL provides proliferation signals to tumor cells through paracrine and autocrine signaling. Our study identifies participation of APRIL signaling in breast cancer promotion; we propose impairment of this pathway as a potential therapeutic strategy.