RESUMEN
Intestinal chronic inflammation is associated with microbial dysbiosis and accumulation of various immune cells including myeloid-derived suppressor cells (MDSC), which profoundly impact the immune microenvironment, perturb homeostasis and increase the risk to develop colitis-associated colorectal cancer (CAC). However, the specific MDSCs-dysbiotic microbiota interactions and their collective impact on CAC development remain poorly understood. In this study, using a murine model of CAC, we demonstrate that CAC-bearing mice exhibit significantly elevated levels of highly immunosuppressive MDSCs, accompanied by microbiota alterations. Both MDSCs and bacteria that infiltrate the colon tissue and developing tumors can be found in close proximity, suggesting intricate MDSC-microbiota cross-talk within the tumor microenvironment. To investigate this phenomenon, we employed antibiotic treatment to disrupt MDSC-microbiota interactions. This intervention yielded a remarkable reduction in intestinal inflammation, decreased MDSC levels, and alleviated immunosuppression, all of which were associated with a significant reduction in tumor burden. Furthermore, we underscore the causative role of dysbiotic microbiota in the predisposition toward tumor development, highlighting their potential as biomarkers for predicting tumor load. We shed light on the intimate MDSCs-microbiota cross-talk, revealing how bacteria enhance MDSC suppressive features and activities, inhibit their differentiation into mature beneficial myeloid cells, and redirect some toward M2 macrophage phenotype. Collectively, this study uncovers the role of MDSC-bacteria cross-talk in impairing immune responses and promoting tumor growth, providing new insights into potential therapeutic strategies for CAC. SIGNIFICANCE: MDSCs-dysbiotic bacteria interactions in the intestine play a crucial role in intensifying immunosuppression within the CAC microenvironment, ultimately facilitating tumor growth, highlighting potential therapeutic targets for improving the treatment outcomes of CAC.
Asunto(s)
Neoplasias Asociadas a Colitis , Microbioma Gastrointestinal , Células Supresoras de Origen Mieloide , Neoplasias , Animales , Ratones , Inflamación , Microambiente TumoralRESUMEN
We have recently shown that neutrophil antitumor cytotoxicity is Ca2+ dependent and is mediated by TRPM2, an H2O2-dependent Ca2+ channel. However, neutrophil antitumor activity is dependent on context and is manifested in the premetastatic niche, but not at the primary site. We therefore hypothesized that expression of TRPM2 and the consequent susceptibility to neutrophil cytotoxicity may be associated with the epithelial/mesenchymal cellular state. We found that TRPM2 expression was upregulated during epithelial-to-mesenchymal transition (EMT), and mesenchymal cells were more susceptible to neutrophil cytotoxicity. Conversely, cells undergoing mesenchymal-to-epithelial transition (MET) expressed reduced levels of TRPM2, rendering them resistant to neutrophil cytotoxicity. Cells expressing reduced levels of TRPM2 were protected from neutrophil cytotoxicity and seeded more efficiently in the premetastatic lung. These data identify TRPM2 as the link between environmental cues at the primary tumor site, tumor cell susceptibility to neutrophil cytotoxicity, and disease progression. Furthermore, these data identify EMT as a process enhancing tumor-cell immune susceptibility and, by contrast, MET as a novel mode of immune evasion.Significance: EMT is required for metastatic spread and concomitantly enhances tumor cell susceptibility to neutrophil cytotoxicity. Cancer Res; 78(17); 5050-9. ©2018 AACR.
Asunto(s)
Neoplasias Pulmonares/genética , Neutrófilos/metabolismo , Canales Catiónicos TRPM/genética , Microambiente Tumoral/genética , Animales , Línea Celular Tumoral , Proliferación Celular/genética , Modelos Animales de Enfermedad , Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Peróxido de Hidrógeno/metabolismo , Neoplasias Pulmonares/patología , Ratones , Metástasis de la Neoplasia , Activación Neutrófila/genética , Activación Neutrófila/inmunología , Neutrófilos/patologíaRESUMEN
Mammalian DNA replication is a highly organized and regulated process. Large, Mb-sized regions are replicated at defined times along S-phase. Replication Timing (RT) is thought to play a role in shaping the mammalian genome by affecting mutation rates. Previous analyses relied on somatic RT profiles. However, only germline mutations are passed on to offspring and affect genomic composition. Therefore, germ cell RT information is necessary to evaluate the influences of RT on the mammalian genome. We adapted the RT mapping technique for limited amounts of cells, and measured RT from two stages in the mouse germline - primordial germ cells (PGCs) and spermatogonial stem cells (SSCs). RT in germline cells exhibited stronger correlations to both mutation rate and recombination hotspots density than those of RT in somatic tissues, emphasizing the importance of using correct tissues-of-origin for RT profiling. Germline RT maps exhibited stronger correlations to additional genetic features including GC-content, transposable elements (SINEs and LINEs), and gene density. GC content stratification and multiple regression analysis revealed independent contributions of RT to SINE, gene, mutation, and recombination hotspot densities. Together, our results establish a central role for RT in shaping multiple levels of mammalian genome composition.
Asunto(s)
Momento de Replicación del ADN/genética , Replicación del ADN/genética , Genoma/genética , Células Germinativas/metabolismo , Células Madre/metabolismo , Animales , Composición de Base/genética , Línea Celular Tumoral , Células Cultivadas , Elementos Transponibles de ADN/genética , Femenino , Células Germinativas/citología , Mutación de Línea Germinal , Masculino , Mamíferos/genética , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Endogámicos NOD , Ratones SCID , Ratones Transgénicos , Elementos de Nucleótido Esparcido Corto/genética , Células Madre/citologíaRESUMEN
Neutrophils play a critical role in cancer, with both protumor and antitumor neutrophil subpopulations reported. The antitumor neutrophil subpopulation has the capacity to kill tumor cells and limit metastatic spread, yet not all tumor cells are equally susceptible to neutrophil cytotoxicity. Because cells that evade neutrophils have greater chances of forming metastases, we explored the mechanism neutrophils use to kill tumor cells. Neutrophil cytotoxicity was previously shown to be mediated by secretion of H2O2 We report here that neutrophil cytotoxicity is Ca2+ dependent and is mediated by TRPM2, a ubiquitously expressed H2O2-dependent Ca2+ channel. Perturbing TRPM2 expression limited tumor cell proliferation, leading to attenuated tumor growth. Concomitantly, cells expressing reduced levels of TRPM2 were protected from neutrophil cytotoxicity and seeded more efficiently in the premetastatic lung.Significance: These findings identify the mechanism utilized by neutrophils to kill disseminated tumor cells and to limit metastatic spread. Cancer Res; 78(10); 2680-90. ©2018 AACR.
Asunto(s)
Neoplasias de la Mama/patología , Canales de Calcio/metabolismo , Peróxido de Hidrógeno/metabolismo , Células Neoplásicas Circulantes/inmunología , Neutrófilos/inmunología , Canales Catiónicos TRPM/metabolismo , Animales , Sistemas CRISPR-Cas/genética , Calcio/metabolismo , Línea Celular Tumoral , Proliferación Celular/genética , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Células Neoplásicas Circulantes/patología , Neutrófilos/metabolismo , Canales Catiónicos TRPM/genéticaRESUMEN
The majority of mammalian genes contain one or more alternative polyadenylation sites. Choice of polyadenylation sites was suggested as one of the underlying mechanisms for generating longer/shorter transcript isoforms. Here, we demonstrate that mature mRNA transcripts can undergo additional cleavage and polyadenylation at a proximal internal site in the 3'-UTR, resulting in two stable, autonomous, RNA fragments: a coding sequence with a shorter 3'-UTR (body) and an uncapped 3'-UTR sequence downstream of the cleavage point (tail). Analyses of the human transcriptome has revealed thousands of such cleavage positions, suggesting a widespread post-transcriptional phenomenon producing thousands of stable 3'-UTR RNA tails that exist alongside their transcripts of origin. By analyzing the impact of microRNAs, we observed a significantly stronger effect for microRNA regulation at the body compared to the tail fragments. Our findings open a variety of future research prospects and call for a new perspective on 3'-UTR-dependent gene regulation.
Asunto(s)
Regiones no Traducidas 3'/genética , Isoformas de ARN/genética , Procesamiento Postranscripcional del ARN , ARN Mensajero/genética , Animales , Línea Celular Tumoral , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Células HEK293 , Humanos , Ratones Endogámicos C57BL , MicroARNs/genética , Sistemas de Lectura Abierta/genética , Poliadenilación , Caperuzas de ARNRESUMEN
Acquisition of a 'quiescence programme' by naive T cells is important to provide a stress-free environment and resistance to apoptosis while preserving their responsiveness to activating stimuli. Therefore, the survival and proper function of naive T cells depends on their ability to maintain quiescence. Recently we demonstrated that by preventing chronic unresolved endoplasmic reticulum (ER) stress, Schlafen2 (Slfn2) maintains a stress-free environment to conserve a pool of naive T cells ready to respond to a microbial invasion. These findings strongly suggest an intimate association between quiescence and stress signalling. However, the connection between ER stress conditions and loss of T-cell quiescence is unknown. Here we demonstrate that homeostasis of cholesterol and lipids, is disrupted in T cells and monocytes from Slfn2-mutant, elektra, mice with higher levels of lipid rafts and lipid droplets found in these cells. Moreover, elektra T cells had elevated levels of free cholesterol and cholesteryl ester due to increased de novo synthesis and higher levels of the enzyme HMG-CoA reductase. As cholesterol plays an important role in the transition of T cells from resting to active state, and ER regulates cholesterol and lipid synthesis, we suggest that regulation of cholesterol levels through the prevention of ER stress is an essential component of the mechanism by which Slfn2 regulates quiescence.
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Proteínas de Ciclo Celular/metabolismo , Proliferación Celular , Senescencia Celular , Colesterol/biosíntesis , Activación de Linfocitos , Mutación , Linfocitos T/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Ésteres del Colesterol/biosíntesis , Estrés del Retículo Endoplásmico , Genotipo , Hidroximetilglutaril-CoA Reductasas/metabolismo , Gotas Lipídicas/metabolismo , Microdominios de Membrana/metabolismo , Ratones Endogámicos C57BL , Ratones Mutantes , Monocitos/inmunología , Monocitos/metabolismo , Fenotipo , Linfocitos T/inmunología , Regulación hacia ArribaRESUMEN
BACKGROUND: Generalized verrucosis (GV) is a chronic and progressive cutaneous human papillomavirus (HPV) infection resulting in multiple warts and associated with acquired or genetic immune defects. We identified a consanguineous Arab family manifesting GV and recurrent bacterial and viral infections, in association with inflammatory bowel disease (IBD). OBJECTIVE: To identify the mutated gene responsible for GV, recurrent infections and IBD, in this family. METHODS: Flow cytometry of peripheral blood mononuclear cells was performed, as well as proliferation and cell cycle assays of T cells. Whole exome sequencing was utilized to detect candidate mutated genes, assuming an autosomal recessive mode of inheritance. Skin fibroblasts from a patient, the mother and control were incubated with sorbitol to detect the phosphorylation ability of TAOK2, and a clonogenic assay was performed to assess the survival and proliferative capacity of fibroblasts' colonies. RESULTS: Despite normal immunophenotyping of T and B cells, T cell proliferation upon activation was impaired in a patient compared to a heterozygous family member and a control. Genetic analyses identified a rare homozygous missense variant, c.2098C>T (p.R700C) in the TAOK2 gene, segregating with the disease phenotype in the family. TAOK2 encodes the TAO2 kinase, a mitogen activated protein kinase kinase kinase (MAP3K) in the p38-MAPK cascade. The mutation is predicted to disrupt its normal folding and molecular interaction; however, no impairment was observed in TAOK2 kinase activity toward its downstream target, MEK3/6, in patient's fibroblasts. Despite this normal kinase activity, a noticeably higher survival/proliferation of patient's skin fibroblasts was found. CONCLUSIONS: A mutation in TAOK2 appears to cause a novel form of primary immunodeficiency, characterized by an impaired T cell proliferation upon activation. This novel cause of GV gives further support to the importance of the p38-MAPK pathway in the immune response against HPV, and possibly also in the pathogenesis of IBD.
Asunto(s)
Síndromes de Inmunodeficiencia/genética , Activación de Linfocitos/genética , Infecciones por Papillomavirus/genética , Proteínas Serina-Treonina Quinasas/genética , Linfocitos T/inmunología , Verrugas/genética , Biopsia , Proliferación Celular/genética , Niño , Preescolar , Enfermedad Crónica , Consanguinidad , Femenino , Pruebas Genéticas , Homocigoto , Humanos , Síndromes de Inmunodeficiencia/inmunología , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Activación de Linfocitos/inmunología , Sistema de Señalización de MAP Quinasas/inmunología , Mutación , Infecciones por Papillomavirus/inmunología , Linaje , Fenotipo , Enfermedades de Inmunodeficiencia Primaria , Recurrencia , Piel/inmunología , Piel/patología , Linfocitos T/metabolismo , Verrugas/inmunología , Secuenciación del ExomaRESUMEN
Immunologically naïve lymphocytes are kept in a quiescent state until antigen engagement. These quiescent immune cells are characterized by small cell size, lack of spontaneous proliferation and low metabolic rate. Lymphocyte quiescence is actively enforced condition which ensures the preservation of proper differentiation and proliferation capabilities of naïve and memory lymphocytes. Previously we described a chemically induced mutation in Schlafen2 (Slfn2), termed elektra, which breaks quiescence and compromises immunity. However, the mechanism by which Slfn2 maintains quiescence remains unknown. Here we demonstrate that elektra T cells display chronic ER stress under steady state conditions. Modulation of ER stress response by depletion of either UPR mediators XBP1 or CHOP, improved viability and partially corrected the developmental abnormalities and proliferation capabilities of elektra T cells. Altogether, our results demonstrate a functional connection between Slfn2 induced quiescence in T cells and ER homeostasis, clarifying a novel mechanism by which immune cell quiescence is maintained.
Asunto(s)
Linfocitos T CD4-Positivos/citología , Linfocitos T CD8-positivos/citología , Proteínas de Ciclo Celular/genética , Estrés del Retículo Endoplásmico , Animales , Antígenos/química , Apoptosis , Proteínas de Ciclo Celular/metabolismo , Proliferación Celular , Supervivencia Celular , Retículo Endoplásmico/metabolismo , Femenino , Homeostasis , Memoria Inmunológica , Inflamación , Sistema de Señalización de MAP Quinasas , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Transducción de Señal , Bazo/citología , Factores de Transcripción/metabolismo , Proteína 1 de Unión a la X-Box/metabolismoRESUMEN
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of thymocytes. Despite significant improvement in the treatment of T-ALL, approximately 20% of children and most adults undergo relapse. Previous findings demonstrated that loss of T-cell quiescence due to a mutation in the Slfn2 gene (elektra) leads to acquisition of an aberrant developmental program by which T-cells lose their renewal capabilities and undergo apoptosis. Here we show that the elektra mutation in Slfn2 completely prevents a severe lymphoproliferative disease caused by overexpression of BCL2 in combination with Fas deficiency in mice. Moreover, Slfn2 impaired-function protects mice from experimental disease similar to human T-ALL by severely impairing the proliferation potential and survival of leukemic T-cells, partially by activation of the p53 tumor suppressor protein. Our study suggest that in certain malignancies, such as T-ALL, a novel therapeutic strategy may be applied by imposing aberrant development of leukemic cells. Furthermore, as the elektra mutation in Slfn2 seems to impair only T-cells and monocytes, targeting Slfn2 is expected to be harmless to other cell types, and thereby could be a promising target for treating malignancies. Together our results demonstrate the potential of targeting Slfn2 and its human paralog for T-ALL treatment.
Asunto(s)
Proteínas de Ciclo Celular/genética , Recurrencia Local de Neoplasia/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Fase de Descanso del Ciclo Celular , Timocitos/metabolismo , Traslado Adoptivo , Animales , Apoptosis , Proteínas de Ciclo Celular/metabolismo , Supervivencia Celular , Progresión de la Enfermedad , Regulación hacia Abajo , Células HEK293 , Humanos , Leucemia Experimental/metabolismo , Leucemia Experimental/patología , Leucemia Experimental/terapia , Mutación con Pérdida de Función , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Terapia Molecular Dirigida/métodos , Monocitos/metabolismo , Monocitos/patología , Recurrencia Local de Neoplasia/patología , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/terapia , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Receptor Notch1/metabolismo , Timocitos/patología , Transducción Genética , Proteína p53 Supresora de Tumor/metabolismo , Receptor fas/genéticaRESUMEN
BACKGROUND: Persistent influenza virus replication during antiviral therapy in patients undergoing hematopoietic stem cell transplantation (HSCT) could promote the emergence of antiviral drug resistance. OBJECTIVES: To follow the viral genotypic and drug susceptibility changes in a patient who developed progressive influenza A/H3N2 pneumonia despite oseltamivir therapy after haploidentical HSCT. STUDY DESIGN: Direct genotypic analysis of the neuraminidase (NA) and hemagglutinin (HA) genes in successive bronchoalveolar lavage specimens was employed in combination with hemagglutination and NA enzymatic activity assays of the corresponding viral isolates. RESULTS: The emergence of NA oseltamivir-resistance mutation R292K was detected by 12 days of oseltamivir treatment with 44,286-fold increase in oseltamivir IC50. Resurgence of wild type viral population was identified by 7 days after cessation of oseltamivir. Sequential HA mutations R228S and A138S were identified and associated with a shift in the HA receptor binding pattern reflected by loss of the ability to agglutinate chicken erythrocytes. CONCLUSIONS: These rapid evolutionary changes warrant close virologic monitoring of immunocompromised patients treated for influenza infection, and raise concern about the efficacy of mono-drug therapy for influenza-associated disease in HSCT recipients.
Asunto(s)
Antivirales/farmacología , Farmacorresistencia Viral , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Subtipo H3N2 del Virus de la Influenza A/efectos de los fármacos , Subtipo H3N2 del Virus de la Influenza A/aislamiento & purificación , Gripe Humana/virología , Sustitución de Aminoácidos/genética , Líquido del Lavado Bronquioalveolar/virología , Preescolar , Femenino , Genotipo , Pruebas de Inhibición de Hemaglutinación , Humanos , Concentración 50 Inhibidora , Mutación Missense , Oseltamivir/farmacología , ARN Viral/genéticaRESUMEN
T cell AgR zeta chain down-regulation associated with T cell dysfunction has been described in cancer, infectious, and autoimmune diseases. We have previously shown that chronic inflammation is mandatory for the induction of an immunosuppressive environment leading to this phenomenon. To identify the key immunosuppressive components, we used an in vivo mouse model exhibiting chronic inflammation-induced immunosuppression. Herein, we demonstrate that: 1) under chronic inflammation secondary lymphatic organs display various immunological milieus; zeta chain down-regulation and T cell dysfunction are induced in the spleen, peripheral blood, and bone marrow, but not in lymph nodes, correlating with elevated levels of Gr1(+)Mac-1(+) myeloid suppressor cells (MSC); 2) MSC are responsible for the induction of such an immunosuppression under both normal and inflammatory conditions; and 3) normal T cells administered into mice exhibiting an immunosuppressive environment down-regulate their zeta expression. Such an environment is anticipated to limit the success of immunotherapeutic strategies based on vaccination and T cell transfer, which are currently under investigation for immunotherapy of cancer.
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Inflamación/inmunología , Tejido Linfoide/citología , Células Mieloides/inmunología , Receptores de Antígenos de Linfocitos T/biosíntesis , Linfocitos T/inmunología , Animales , Enfermedad Crónica , Regulación hacia Abajo , Femenino , Citometría de Flujo , Tolerancia Inmunológica , Tejido Linfoide/inmunología , Tejido Linfoide/patología , Antígeno de Macrófago-1/metabolismo , Ratones , Ratones Endogámicos BALB CRESUMEN
Human CD56(bright) NK cells accumulate in the maternal decidua during pregnancy and are found in direct contact with fetal trophoblasts. Several mechanisms have been proposed to explain the inability of NK cells to kill the semiallogeneic fetal cells. However, the actual functions of decidual NK (dNK) cells during pregnancy are mostly unknown. Here we show that dNK cells, but not peripheral blood-derived NK subsets, regulate trophoblast invasion both in vitro and in vivo by production of the interleukin-8 and interferon-inducible protein-10 chemokines. Furthermore, dNK cells are potent secretors of an array of angiogenic factors and induce vascular growth in the decidua. Notably, such functions are regulated by specific interactions between dNK-activating and dNK-inhibitory receptors and their ligands, uniquely expressed at the fetal-maternal interface. The overall results support a 'peaceful' model for reproductive immunology, in which elements of innate immunity have been incorporated in a constructive manner to support reproductive tissue development.
Asunto(s)
Decidua/citología , Células Asesinas Naturales/fisiología , Intercambio Materno-Fetal/fisiología , Embarazo/inmunología , Trofoblastos/fisiología , Inductores de la Angiogénesis/metabolismo , Animales , Antígenos CD/fisiología , Antígeno CD56/inmunología , Femenino , Feto/citología , Humanos , Interleucina-8/biosíntesis , Receptor Leucocitario Tipo Inmunoglobulina B1 , Glicoproteínas de Membrana/fisiología , Ratones , Receptor 2 Gatillante de la Citotoxidad Natural , Receptor 3 Gatillante de la Citotoxidad Natural , Receptores de Quimiocina/biosíntesis , Receptores Inmunológicos/fisiología , Receptores KIR , Trofoblastos/metabolismoRESUMEN
T cell antigen receptor zeta chain down-regulation and impaired in vitro T cell function have been described in cancer and autoimmune and infectious diseases. However, the immunological basis for this phenomenon is unknown. Sustained exposure to antigen and chronic systemic inflammation, factors shared by the various pathologies, might account for this phenomenon. We developed an in vivo experimental system that mimics these conditions and show that sustained exposure of mice to bacterial antigens was sufficient to induce T cell antigen receptor zeta chain down-regulation and impair T cell function, provided an interferon-gamma-dependent T helper type 1 immune response developed. This indicates zeta chain down-regulation could be a physiological response that attenuates an exacerbated immune response. However, it can act as a 'double-edged sword', impairing immune responses to chronic diseases.