RESUMO
Coenzyme A (CoA) serves as a vital cofactor in numerous enzymatic reactions involved in energy production, lipid metabolism, and synthesis of essential molecules. Dysregulation of CoA-dependent metabolic pathways can contribute to chronic diseases, such as inflammatory diseases, obesity, diabetes, cancer, and cardiovascular disorders. Additionally, CoA influences immune cell activation by modulating the metabolism of these cells, thereby affecting their proliferation, differentiation, and effector functions. Targeting CoA metabolism presents a promising avenue for therapeutic intervention, as it can potentially restore metabolic balance, mitigate chronic inflammation, and enhance immune cell function. This might ultimately improve the management and outcomes for these diseases. This review will more specifically focus on the contribution of pathways regulating the availability of the CoA precursor Vitamin B5/pantothenate in vivo and modulating the development of Th17-mediated inflammation, CD8-dependent anti-tumor immunity but also tissue repair processes in chronic inflammatory or degenerative diseases.
Assuntos
Coenzima A , Ácido Pantotênico , Humanos , Ácido Pantotênico/metabolismo , Coenzima A/metabolismo , Inflamação , ImunomodulaçãoRESUMO
OBJECTIVE: In the management of patients with IBD, there is a need to identify prognostic markers and druggable biological pathways to improve mucosal repair and probe the efficacy of tumour necrosis factor alpha biologics. Vnn1 is a pantetheinase that degrades pantetheine to pantothenate (vitamin B5, a precursor of coenzyme A (CoA) biosynthesis) and cysteamine. Vnn1 is overexpressed by inflamed colonocytes. We investigated its contribution to the tolerance of the intestinal mucosa to colitis-induced injury. DESIGN: We performed an RNA sequencing study on colon biopsy samples from patients with IBD stratified according to clinical severity and modalities of treatment. We generated the VIVA mouse transgenic model, which specifically overexpresses Vnn1 on intestinal epithelial cells and explored its susceptibility to colitis. We developed a pharmacological mimicry of Vnn1 overexpression by administration of Vnn1 derivatives. RESULTS: VNN1 overexpression on colonocytes correlates with IBD severity. VIVA mice are resistant to experimentally induced colitis. The pantetheinase activity of Vnn1 is cytoprotective in colon: it enhances CoA regeneration and metabolic adaptation of colonocytes; it favours microbiota-dependent production of short chain fatty acids and mostly butyrate, shown to regulate mucosal energetics and to be reduced in patients with IBD. This prohealing phenotype is recapitulated by treating control mice with the substrate (pantethine) or the products of pantetheinase activity prior to induction of colitis. In severe IBD, the protection conferred by the high induction of VNN1 might be compromised because its enzymatic activity may be limited by lack of available substrates. In addition, we identify the elevation of indoxyl sulfate in urine as a biomarker of Vnn1 overexpression, also detected in patients with IBD. CONCLUSION: The induction of Vnn1/VNN1 during colitis in mouse and human is a compensatory mechanism to reinforce the mucosal barrier. Therefore, enhancement of vitamin B5-driven metabolism should improve mucosal healing and might increase the efficacy of anti-inflammatory therapy.
Assuntos
Colite , Doenças Inflamatórias Intestinais , Humanos , Camundongos , Animais , Colite/metabolismo , Colo/patologia , Mucosa Intestinal/metabolismo , Doenças Inflamatórias Intestinais/genética , Ácidos Graxos Voláteis/metabolismo , Vitaminas , Sulfato de Dextrana , Modelos Animais de DoençasRESUMO
Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis of the skin and visceral organs and vascular alterations. SSc pathophysiology involves systemic inflammation and oxidative stress. Because the vanin-1 gene (vnn1) encodes an enzyme with pantetheinase activity that converts vasculoprotective pantethine into profibrotic pantothenic acid and pro-oxidant cystamine, we tested this pathway in the pathophysiology of SSc. Activation of the vanin-1/pantetheinase pathway was investigated in wild-type BALB/c mice with hypochlorous acid (HOCl)-induced SSc by ELISA and Western blotting. We then evaluated the effects of the inactivation of vnn1 on the development of fibrosis, endothelial alterations, and immunological activation in mice with HOCl- and bleomycin-induced SSc. We then explored the vanin-1/pantetheinase pathway in a cohort of patients with SSc and in controls. In wild-type mice with HOCl-induced SSc, the vanin-1/pantetheinase pathway was dysregulated, with elevation of vanin-1 activity in skin and high levels of serum pantothenic acid. Inactivation of the vnn1 gene in vnn1-/- mice with HOCl-induced SSc prevented the development of characteristic features of the disease, including fibrosis, immunologic abnormalities, and endothelial dysfunction. Remarkably, patients with diffuse SSc also had increased expression of vanin-1 in skin and blood and elevated levels of serum pantothenic acid that correlated with the severity of the disease. Our data demonstrate that vanin-1/pantetheinase controls fibrosis, vasculopathy, autoimmunity, and oxidative stress in SSc. The levels of vanin-1 expression and pantothenic acid determine SSc severity and can be used as markers of disease severity. More importantly, inhibition of vanin-1 can open new therapeutic approaches in SSc.
Assuntos
Amidoidrolases/metabolismo , Escleroderma Sistêmico/metabolismo , Animais , Feminino , Proteínas Ligadas por GPI/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Ácido Pantotênico/metabolismoRESUMO
BACKGROUND AND AIMS: Pretreatment with clofibrate, a peroxisome proliferator-activated receptor alpha (PPARa) agonist, protects mice from acetaminophen (APAP) injury. Protection is not due to alterations in APAP metabolism and is dependent on PPARa expression. Gene array analysis revealed that mice receiving clofibrate have enhanced hepatic Vanin-1 (Vnn1) gene expression, a response that is also PPARa dependent. METHODS: We examined the role of Vnn1 by comparing the responses of Vnn1 knockout and wild-type mice following APAP hepatotoxicity. APAP metabolism, hepatotoxicity, and compensatory hepatocyte proliferation and immune responses were assessed. RESULTS: Vnn1 knockout mice are more susceptible to APAP hepatotoxicity despite no differences in hepatic glutathione content, gene expression of APAP metabolizing enzymes, or hepatic capacity to bioactivate or detoxify APAP ex vivo. Together, these data strongly suggest that the susceptibility of Vnn1 knockout mice is not due to differences in APAP metabolism. Immunochemistry revealed a lack of proliferating cell nuclear antigen-positive hepatocytes and F4/80-positive macrophages in and around areas of centrilobular necrosis in APAP-treated Vnn1 knockouts. Hepatic gene induction of pro-inflammatory cytokines was either significantly reduced or completely blunted in these mice. This was correlated with a reduction in early recruitment of cells positive for granulocyte differentiation antigen 1 or integrin alpha M. Heightened toxicity was also observed in CCl4 and ConA hepatitis models in the absence of Vnn1. CONCLUSIONS: These results indicate that mice lacking Vnn1 have deficiencies in compensatory repair and immune responses following toxic APAP exposure and that these mechanisms may contribute to the enhanced hepatotoxicity seen.
Assuntos
Acetaminofen/efeitos adversos , Amidoidrolases/deficiência , Proliferação de Células/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/imunologia , Fígado/imunologia , Acetaminofen/farmacologia , Amidoidrolases/imunologia , Animais , Doença Hepática Induzida por Substâncias e Drogas/genética , Doença Hepática Induzida por Substâncias e Drogas/patologia , Clofibrato/farmacologia , Proteínas Ligadas por GPI/deficiência , Proteínas Ligadas por GPI/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Hepatócitos/imunologia , Hepatócitos/patologia , Fígado/patologia , Macrófagos/imunologia , Macrófagos/patologia , Camundongos , Camundongos Knockout , PPAR alfa/genética , PPAR alfa/imunologiaRESUMO
Tissue pantetheinase, encoded by the VNN1 gene, regulates response to stress, and previous studies have shown that VNN genes contribute to the susceptibility to malaria. Herein, we evaluated the role of pantetheinase on erythrocyte homeostasis and on the development of malaria in patients and in a new mouse model of pantetheinase insufficiency. Patients with cerebral malaria have significantly reduced levels of serum pantetheinase activity (PA). In mouse, we show that a reduction in serum PA predisposes to severe malaria, including cerebral malaria and severe anemia. Therefore, scoring pantetheinase in serum may serve as a severity marker in malaria infection. This disease triggers an acute stress in erythrocytes, which enhances cytoadherence and hemolysis. We speculated that serum pantetheinase might contribute to erythrocyte resistance to stress under homeostatic conditions. We show that mutant mice with a reduced serum PA are anemic and prone to phenylhydrazine-induced anemia. A cytofluorometric and spectroscopic analysis documented an increased frequency of erythrocytes with an autofluorescent aging phenotype. This is associated with an enhanced oxidative stress and shear stress-induced hemolysis. Red blood cell transfer and bone marrow chimera experiments show that the aging phenotype is not cell intrinsic but conferred by the environment, leading to a shortening of red blood cell half-life. Therefore, serum pantetheinase level regulates erythrocyte life span and modulates the risk of developing complicated malaria.
Assuntos
Amidoidrolases/sangue , Eritrócitos/fisiologia , Malária/fisiopatologia , Adolescente , Adulto , Amidoidrolases/metabolismo , Anemia , Animais , Criança , Pré-Escolar , Modelos Animais de Doenças , Suscetibilidade a Doenças , Feminino , Proteínas Ligadas por GPI/sangue , Proteínas Ligadas por GPI/metabolismo , Homeostase , Humanos , Lactente , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo , Adulto JovemRESUMO
BACKGROUND & AIMS: Peroxisome proliferator-activated receptor alpha (PPARα) is a key regulator of hepatic fat oxidation that serves as an energy source during starvation. Vanin-1 has been described as a putative PPARα target gene in liver, but its function in hepatic lipid metabolism is unknown. METHODS: We investigated the regulation of vanin-1, and total vanin activity, by PPARα in mice and humans. Furthermore, the function of vanin-1 in the development of hepatic steatosis in response to starvation was examined in Vnn1 deficient mice, and in rats treated with an inhibitor of vanin activity. RESULTS: Liver microarray analyses reveals that Vnn1 is the most prominently regulated gene after modulation of PPARα activity. In addition, activation of mouse PPARα regulates hepatic- and plasma vanin activity. In humans, consistent with regulation by PPARα, plasma vanin activity increases in all subjects after prolonged fasting, as well as after treatment with the PPARα agonist fenofibrate. In mice, absence of vanin-1 exacerbates the fasting-induced increase in hepatic triglyceride levels. Similarly, inhibition of vanin activity in rats induces accumulation of hepatic triglycerides upon fasting. Microarray analysis reveal that the absence of vanin-1 associates with gene sets involved in liver steatosis, and reduces pathways involved in oxidative stress and inflammation. CONCLUSIONS: We show that hepatic vanin-1 is under extremely sensitive regulation by PPARα and that plasma vanin activity could serve as a readout of changes in PPARα activity in human subjects. In addition, our data propose a role for vanin-1 in regulation of hepatic TG levels during fasting.
Assuntos
Amidoidrolases/fisiologia , Metabolismo dos Lipídeos , Fígado/metabolismo , PPAR alfa/fisiologia , Animais , Fígado Gorduroso/etiologia , Proteínas Ligadas por GPI/fisiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Wistar , Inanição/metabolismoRESUMO
Pantetheinase is an ubiquitous enzyme which hydrolyses D-pantetheine into cysteamine and pantothenate (vitamin B5) on the dissimilative pathway of CoA. Pantetheinase isoforms are encoded by the Vnn (vanin) genes and Vnn1 is the predominant tissue isoform in mice and humans. In the present article, we review the results showing the regulation of Vnn1 expression during developmental, repair and inflammatory situations and the impact of a Vnn1 deficiency in mouse models of pathologies. We document the involvement of the Vnn1 pantetheinase in situations of increased tissue needs and propose that Vnn1 through recycling of pantothenate and release of cysteamine in tissues participates in the adaptive response of the tissue to stress.
Assuntos
Amidoidrolases/metabolismo , Animais , Cisteamina/metabolismo , Proteínas Ligadas por GPI/metabolismo , Humanos , Ácido Pantotênico/metabolismo , Estresse Fisiológico/fisiologiaRESUMO
The tumor microenvironment is a dynamic network of stromal, cancer, and immune cells that interact and compete for resources. We have previously identified the Vanin1 pathway as a tumor suppressor of sarcoma development via vitamin B5 and coenzyme A regeneration. Using an aggressive sarcoma cell line that lacks Vnn1 expression, we showed that the administration of pantethine, a vitamin B5 precursor, attenuates tumor growth in immunocompetent but not nude mice. Pantethine boosts antitumor immunity, including the polarization of myeloid and dendritic cells towards enhanced IFNγ-driven antigen presentation pathways and improved the development of hypermetabolic effector CD8+ T cells endowed with potential antitumor activity. At later stages of treatment, the effect of pantethine was limited by the development of immune cell exhaustion. Nevertheless, its activity was comparable with that of anti-PD1 treatment in sensitive tumors. In humans, VNN1 expression correlates with improved survival and immune cell infiltration in soft-tissue sarcomas, but not in osteosarcomas. Pantethine could be a potential therapeutic immunoadjuvant for the development of antitumor immunity.
Assuntos
Linfócitos T CD8-Positivos , Sarcoma , Humanos , Camundongos , Animais , Coenzima A/farmacologia , Ácido Pantotênico/farmacologia , Sarcoma/tratamento farmacológico , Microambiente TumoralRESUMO
Aggressive tumors often display mitochondrial dysfunction. Upon oxidative stress, mitochondria undergo fission through OMA1-mediated cleavage of the fusion effector OPA1. In yeast, a redox-sensing switch participates in OMA1 activation. 3D modeling of OMA1 comforted the notion that cysteine 403 might participate in a similar sensor in mammalian cells. Using prime editing, we developed a mouse sarcoma cell line in which OMA1 cysteine 403 was mutated in alanine. Mutant cells showed impaired mitochondrial responses to stress including ATP production, reduced fission, resistance to apoptosis, and enhanced mitochondrial DNA release. This mutation prevented tumor development in immunocompetent, but not nude or cDC1 dendritic cell-deficient, mice. These cells prime CD8+ lymphocytes that accumulate in mutant tumors, whereas their depletion delays tumor control. Thus, OMA1 inactivation increased the development of anti-tumor immunity. Patients with complex genomic soft tissue sarcoma showed variations in the level of OMA1 and OPA1 transcripts. High expression of OPA1 in primary tumors was associated with shorter metastasis-free survival after surgery, and low expression of OPA1, with anti-tumor immune signatures. Targeting OMA1 activity may enhance sarcoma immunogenicity.
Assuntos
GTP Fosfo-Hidrolases , Sarcoma , Camundongos , Animais , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Cisteína/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Mitocôndrias/metabolismo , Sarcoma/genética , Sarcoma/metabolismo , Mamíferos/metabolismo , Metaloproteases/genética , Metaloproteases/metabolismoRESUMO
Colitis involves immune cell-mediated tissue injuries, but the contribution of epithelial cells remains largely unclear. Vanin-1 is an epithelial ectoenzyme with a pantetheinase activity that provides cysteamine/cystamine to tissue. Using the 2,4,6-trinitrobenzene sulfonic acid (TNBS)-colitis model we show here that Vanin-1 deficiency protects from colitis. This protection is reversible by administration of cystamine or bisphenol A diglycidyl ether, a peroxisome proliferator-activated receptor (PPAR)gamma antagonist. We further demonstrate that Vanin-1, by antagonizing PPARgamma, licenses the production of inflammatory mediators by intestinal epithelial cells. We propose that Vanin-1 is an epithelial sensor of stress that exerts a dominant control over innate immune responses in tissue. Thus, the Vanin-1/pantetheinase activity might be a new target for therapeutic intervention in inflammatory bowel disease.
Assuntos
Moléculas de Adesão Celular/fisiologia , Colite/metabolismo , Células Epiteliais/metabolismo , PPAR gama/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/fisiologia , Amidoidrolases , Animais , Compostos Benzidrílicos , Peso Corporal , Moléculas de Adesão Celular/deficiência , Moléculas de Adesão Celular/genética , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Quimiocina CXCL2 , Quimiocinas/genética , Quimiocinas/metabolismo , Colite/induzido quimicamente , Colite/patologia , Colo/efeitos dos fármacos , Colo/metabolismo , Colo/patologia , Ciclo-Oxigenase 2/metabolismo , Cistamina/farmacologia , Citocinas/genética , Citocinas/metabolismo , Células Epiteliais/efeitos dos fármacos , Compostos de Epóxi/farmacologia , Proteínas Ligadas por GPI , Regulação da Expressão Gênica/efeitos dos fármacos , Interleucina-1beta/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos SCID , PPAR gama/antagonistas & inibidores , PPAR gama/genética , Prostaglandina D2/análogos & derivados , Prostaglandina D2/farmacologia , Análise de Sobrevida , Ácido TrinitrobenzenossulfônicoRESUMO
A strong bias related to age is observed in COVID-19 patients with pediatric subjects developing a milder disease than adults. We hypothesized that a specific SARS-CoV-2 effect conjugated with preexisting differences in the immune systems may explain this. Using flow cytometry, we investigated basal immune differences in a cohort consisting of 16 non-infected young and 16 aged individuals and further leveraged an in vitro whole blood model of SARS-CoV-2 infection so that functional differences could be mined as well. In short, blood diluted in culture media was incubated 5 or 24 h with the trimeric spike protein or controls. Following unsupervised analysis, we first confirmed that the immune lymphoid and myeloid systems in adults are less efficient and prone to develop higher inflammation than those in children. We notably identified in adults a higher CD43 lymphocyte expression, known for its potentially inhibitory role. The spike protein induced different responses between adults and children, notably a higher increase of inflammatory markers together with lower monocyte and B cell activation in adults. Interestingly, CD169, a CD43 ligand overexpressed in COVID-19 patients, was confirmed to be strongly modulated by the spike protein. In conclusion, the spike protein exacerbated the preexisting lower immune responsiveness and higher inflammatory potential in adults. Altogether, some of the markers identified may explain the marked age bias and be predictive of severity.
Assuntos
COVID-19 , Monócitos , Glicoproteína da Espícula de Coronavírus , Adulto , Idoso , Criança , Humanos , COVID-19/imunologia , Monócitos/imunologia , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/metabolismoRESUMO
To identify COVID-19-associated immunophenotyping patterns at hospital admission and to determine if some patterns could predict the need for mechanical ventilation (MV). DESIGN: Prospective observational monocentric cohort study. SETTING: A university-affiliated hospital in Marseille, France. PATIENTS: Thirty patients presenting with laboratory-confirmed COVID-19 pneumonia were enrolled within the first 48 hours of hospital admission and compared with 18 healthy controls. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Whole-blood leukocytes were immunophenotyped with a rapid and simplified one-step flow cytometry method. Thirty-eight immune and five laboratory parameters were compared first between COVID-19 patients and controls and then between the COVID-19 patients who received or not MV during their stays. The variables that significantly discriminated MV from non-MV patients in univariate analysis were entered into a multiple stepwise logistic regression analysis. The COVID-19 patients were predominantly male (87%), aged 61 years (50-71 yr), and 93% received early corticosteroid therapy. Sixteen patients (53%) were managed with noninvasive respiratory support, and 14 (47%) required MV. Compared with controls, COVID-19 patients were characterized by an immune signature featuring: 1) decreased HLA-DR expression on monocytes; 2) reduced basophils, eosinophils, T-cells, NK cells, and nonclassical monocyte count; and 3) up regulation of CD169 on monocytes, CD64 on neutrophils, the adhesion/migration markers (CD62L and CD11b), and the checkpoint inhibitor CD274 on myeloid cells. Among the COVID-19 patients, those who received MV had lower level of CD4 and HLA-DR on monocytes, lower CD8+ T-cell count, and higher lactate dehydrogenase at hospital admission. In multivariate analysis, only CD4 on monocytes (p = 0.032) and CD8+ T-cell count (p = 0.026) were associated with MV requirement. The model combining these two variables provided an area under curve of 0.97 (95% CI, 0.83-0.99). CONCLUSIONS: The association of low CD4 on monocytes and low CD8+ T-cell count at hospital admission was highly predictive of the need for MV in hospitalized patients with COVID-19 pneumonia.
RESUMO
Metabolic rewiring offers novel therapeutic opportunities in cancer. Until recently, there was scant information regarding soft tissue sarcomas, due to their heterogeneous tissue origin, histological definition and underlying genetic history. Novel large-scale genomic and metabolomics approaches are now helping stratify their physiopathology. In this review, we show how various genetic alterations skew activation pathways and orient metabolic rewiring in sarcomas. We provide an update on the contribution of newly described mechanisms of metabolic regulation. We underscore mechanisms that are relevant to sarcomagenesis or shared with other cancers. We then discuss how diverse metabolic landscapes condition the tumor microenvironment, anti-sarcoma immune responses and prognosis. Finally, we review current attempts to control sarcoma growth using metabolite-targeting drugs.
Assuntos
Redes e Vias Metabólicas , Metaboloma , Sarcoma/metabolismo , Animais , Regulação Neoplásica da Expressão Gênica , Humanos , Metabolômica , Sarcoma/genética , Transdução de Sinais , Transcriptoma , Microambiente TumoralRESUMO
Flow cytometry is a powerful analytical technique that is increasingly used in scientific investigations and healthcare; however, it requires time-consuming, multi-step sample procedures, which limits its use to specialized laboratories. In this study, we propose a new universal one-step method in which white blood cell staining and red blood cell lysis are carried out in a single step, using a gentle lysis solution mixed with fluorescent antibody conjugates or probes in a dry or liquid format. The blood sample may be obtained from a routine venipuncture or directly from a fingerprick, allowing for near-patient analysis. This procedure enables the analysis of common white blood cell markers as well as markers related to infections or sepsis. This simpler and faster protocol may help to democratize the use of flow cytometry in the research and medical fields. Graphic abstract: One-step White Blood Cell Extracellular Staining Method for Flow Cytometry.
RESUMO
Blood cell analysis is a major pillar of biomedical research and healthcare. These analyses are performed in central laboratories. Rapid shipment from collection site to the central laboratories is currently needed because cells and biomarkers degrade rapidly. The dried blood spot from a fingerstick allows the preservation of cellular molecules for months but entire cells are never recovered. Here leucocyte elution is optimized from dried blood spots. Flow cytometry and mRNA expression profiling are used to analyze the recovered cells. 50-70% of the leucocytes that are dried on a polyester solid support via elution after shaking the support with buffer are recovered. While red blood cells lyse upon drying, it is found that the majority of leucocytes are preserved. Leucocytes have an altered structure that is improved by adding fixative in the elution buffer. Leucocytes are permeabilized, allowing an easy staining of all cellular compartments. Common immunophenotyping and mRNAs are preserved. The ability of a new biomarker (CD169) to discriminate between patients with and without Severe Acute Respiratory Syndrome induced by Coronavirus 2 (SARS-CoV-2) infections is also preserved. Leucocytes from blood can be dried, shipped, and/or stored for at least 1 month, then recovered for a wide variety of analyses, potentially facilitating biomedical applications worldwide.
Assuntos
Doenças Transmissíveis/diagnóstico , Testes Diagnósticos de Rotina/métodos , Teste em Amostras de Sangue Seco/métodos , Hematologia/métodos , Imunofenotipagem/métodos , Anticorpos Antivirais/sangue , Biomarcadores/sangue , Coleta de Amostras Sanguíneas/métodos , COVID-19/diagnóstico , Separação Celular/métodos , Doenças Transmissíveis/virologia , Eritrócitos/virologia , Citometria de Fluxo/métodos , Humanos , Leucócitos/virologia , RNA Mensageiro/sangue , SARS-CoV-2/genéticaRESUMO
Inflammatory bowel diseases (IBD) are characterized during their active phase by polymorphonuclear leukocyte (PMNL) transepithelial migration. The efflux of PMNL into the mucosa is associated with the production of proinflammatory cytokines and the release of ATP from damaged and necrotic cells. The expression and function of purinergic P2X(7) receptor (P2X(7)R) in intestinal epithelial cells (IEC) and its potential role in the "cross talk" between IEC and PMNL have not been explored. The aims of the present study were 1) to examine P2X(7)R expression in IEC (T84 cells) and in human intestinal biopsies; 2) to detect any changes in P2X(7)R expression in T84 cells during PMNL transepithelial migration, and during the active and quiescent phases of IBD; and 3) to test whether P2X(7)R stimulation in T84 monolayers can induce caspase-1 activation and IL-1beta release by IEC. We found that a functional ATP-sensitive P2X(7)R is constitutively expressed at the apical surface of IEC T84 cells. PMNL transmigration regulates dynamically P2X(7)R expression and alters its distribution from the apical to basolateral surface of IEC during the early phase of PMNL transepithelial migration in vitro. P2X(7)R expression was weak in intestinal biopsies obtained during the active phase of IBD. We show that activation of epithelial P2X(7)R is mandatory for PMNL-induced caspase-1 activation and IL-1beta release by IEC. Overall, these changes in P2X(7)R function may serve to tailor the intensity of the inflammatory response and to prevent IL-1beta overproduction and inflammatory disease.
Assuntos
Trifosfato de Adenosina/metabolismo , Quimiotaxia de Leucócito , Células Epiteliais/metabolismo , Doenças Inflamatórias Intestinais/metabolismo , Mucosa Intestinal/metabolismo , Infiltração de Neutrófilos , Neutrófilos/metabolismo , Receptores Purinérgicos P2/metabolismo , Biópsia , Caspase 1/metabolismo , Linhagem Celular Tumoral , Ativação Enzimática , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Células Epiteliais/patologia , Humanos , Imuno-Histoquímica , Mediadores da Inflamação/metabolismo , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/patologia , Interleucina-1beta/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Neutrófilos/patologia , Interferência de RNA , RNA Mensageiro/metabolismo , Receptores Purinérgicos P2/efeitos dos fármacos , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X7 , Índice de Gravidade de Doença , Fatores de Tempo , Análise Serial de TecidosRESUMO
Free cysteamine levels in mouse tissues have been strictly correlated to the presence of membrane-bound pantetheinase activity encoded by Vanin-1. Vanin-1 is involved in many biological processes in mouse, from thymus homing to sexual development. Vanin-1 -/- mice are fertile and grow and develop normally; they better control inflammation and most of the knockout effects were rescued by cystamine treatment. Gene structure analysis showed the presence of an oxidative stimuli-responsive ARE-like sequence in the promoter. In this paper we investigate antioxidant-detoxifying enzymatic activities at the tissue level, comparing Vanin-1 -/- and wild-type mice. In Vanin-1 null animals we pointed out a decrease in the Se-independent glutathione peroxidase activity. The decrease in enzymatic activity appeared to be correlated to an impairment of GST isoenzyme levels. In particular a significant drop in GSTA3 together with a minor decrement in GSTM1 and an increase in GSTP1 levels was detected in Vanin-1 -/- livers. Cystamine administration to Vanin-1 -/- mice restored specifically GSTA3 levels and the corresponding enzymatic activity without influencing protein expression. A possible role of cystamine on protein stability/folding can be postulated.
Assuntos
Antioxidantes/metabolismo , Moléculas de Adesão Celular/genética , Cistamina/farmacologia , Glutationa Transferase/metabolismo , Protetores contra Radiação/farmacologia , Amidoidrolases , Animais , Western Blotting , Catalase/metabolismo , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Proteínas Ligadas por GPI , Glutationa Peroxidase/metabolismo , Glutationa Redutase/metabolismo , Isoenzimas/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Superóxido Dismutase/metabolismoRESUMO
Like other tumors, aggressive soft tissue sarcomas (STS) use glycolysis rather than mitochondrial oxidative phosphorylation (OXPHOS) for growth. Given the importance of the cofactor coenzyme A (CoA) in energy metabolism, we investigated the impact of Vnn1 pantetheinase-an enzyme that degrades pantetheine into pantothenate (vitamin B5, the CoA biosynthetic precursor) and cysyteamine-on tumor growth. Using two models, we show that Vnn1+ STS remain differentiated and grow slowly, and that in patients a detectable level of VNN1 expression in STS is associated with an improved prognosis. Increasing pantetheinase activity in aggressive tumors limits their growth. Using combined approaches, we demonstrate that Vnn1 permits restoration of CoA pools, thereby maintaining OXPHOS. The simultaneous production of cysteamine limits glycolysis and release of lactate, resulting in a partial inhibition of STS growth in vitro and in vivo. We propose that the Warburg effect observed in aggressive STS is reversed by induction of Vnn1 pantetheinase and the rewiring of cellular energy metabolism by its products.
RESUMO
Vanin-1 is a membrane-anchored pantetheinase highly expressed in the gut and liver. It hydrolyzes pantetheine to pantothenic acid (vitamin B5) and the low-molecular-weight thiol cysteamine. The latter is believed to be a key regulating factor of several essential metabolic pathways, acting through sulfhydryl-disulfide exchange reactions between sulfhydryl groups of the enzymes and the oxidized form, cystamine. Its physiological importance remains to be elucidated, however. To explore this point, we developed Vanin-1-deficient mice that lack free cysteamine. We examined the susceptibility of deficient mice to intestinal inflammation, either acute (NSAID administration) or chronic (Schistosoma infection). We found that Vanin-1(-/-) mice better controlled inflammatory reaction and intestinal injury in both experiments. This protection was associated with increased gamma-glutamylcysteine synthetase activity and increased stores of reduced glutathione, as well as reduced inflammatory cell activation in inflamed tissues. Oral administration of cystamine reversed all aspects of the deficient phenotype. These findings suggest that one cysteamine function is to upregulate inflammation. Consequently, the pantetheinase activity of Vanin-1 molecule could be a target for a new anti-inflammatory strategy.