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1.
Nat Immunol ; 23(1): 50-61, 2022 01.
Artículo en Inglés | MEDLINE | ID: mdl-34853448

RESUMEN

NP105-113-B*07:02-specific CD8+ T cell responses are considered among the most dominant in SARS-CoV-2-infected individuals. We found strong association of this response with mild disease. Analysis of NP105-113-B*07:02-specific T cell clones and single-cell sequencing were performed concurrently, with functional avidity and antiviral efficacy assessed using an in vitro SARS-CoV-2 infection system, and were correlated with T cell receptor usage, transcriptome signature and disease severity (acute n = 77, convalescent n = 52). We demonstrated a beneficial association of NP105-113-B*07:02-specific T cells in COVID-19 disease progression, linked with expansion of T cell precursors, high functional avidity and antiviral effector function. Broad immune memory pools were narrowed postinfection but NP105-113-B*07:02-specific T cells were maintained 6 months after infection with preserved antiviral efficacy to the SARS-CoV-2 Victoria strain, as well as Alpha, Beta, Gamma and Delta variants. Our data show that NP105-113-B*07:02-specific T cell responses associate with mild disease and high antiviral efficacy, pointing to inclusion for future vaccine design.


Asunto(s)
Antígeno HLA-B7/inmunología , Epítopos Inmunodominantes/inmunología , Proteínas de la Nucleocápside/inmunología , SARS-CoV-2/inmunología , Linfocitos T Citotóxicos/inmunología , Anciano , Secuencia de Aminoácidos , Anticuerpos Antivirales/inmunología , Afinidad de Anticuerpos/inmunología , COVID-19/inmunología , COVID-19/patología , Línea Celular Transformada , Femenino , Perfilación de la Expresión Génica , Humanos , Memoria Inmunológica/inmunología , Masculino , Persona de Mediana Edad , Receptores de Antígenos de Linfocitos T/inmunología , Índice de Severidad de la Enfermedad , Virus Vaccinia/genética , Virus Vaccinia/inmunología , Virus Vaccinia/metabolismo
2.
Mol Cell ; 76(1): 110-125.e9, 2019 10 03.
Artículo en Inglés | MEDLINE | ID: mdl-31474573

RESUMEN

Failure to make adaptive immune responses is a hallmark of aging. Reduced B cell function leads to poor vaccination efficacy and a high prevalence of infections in the elderly. Here we show that reduced autophagy is a central molecular mechanism underlying immune senescence. Autophagy levels are specifically reduced in mature lymphocytes, leading to compromised memory B cell responses in old individuals. Spermidine, an endogenous polyamine metabolite, induces autophagy in vivo and rejuvenates memory B cell responses. Mechanistically, spermidine post-translationally modifies the translation factor eIF5A, which is essential for the synthesis of the autophagy transcription factor TFEB. Spermidine is depleted in the elderly, leading to reduced TFEB expression and autophagy. Spermidine supplementation restored this pathway and improved the responses of old human B cells. Taken together, our results reveal an unexpected autophagy regulatory mechanism mediated by eIF5A at the translational level, which can be harnessed to reverse immune senescence in humans.


Asunto(s)
Autofagia/efectos de los fármacos , Linfocitos B/efectos de los fármacos , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Senescencia Celular/efectos de los fármacos , Inmunosenescencia/efectos de los fármacos , Factores de Iniciación de Péptidos/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas de Unión al ARN/metabolismo , Espermidina/farmacología , Inmunidad Adaptativa/efectos de los fármacos , Factores de Edad , Envejecimiento , Animales , Linfocitos B/inmunología , Linfocitos B/metabolismo , Linfocitos B/patología , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/deficiencia , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Células HEK293 , Humanos , Memoria Inmunológica/efectos de los fármacos , Células Jurkat , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células 3T3 NIH , Factores de Iniciación de Péptidos/genética , Proteínas de Unión al ARN/genética , Transducción de Señal , Factor 5A Eucariótico de Iniciación de Traducción
3.
Immunity ; 47(3): 466-480.e5, 2017 09 19.
Artículo en Inglés | MEDLINE | ID: mdl-28916263

RESUMEN

Neutrophils are critical and short-lived mediators of innate immunity that require constant replenishment. Their differentiation in the bone marrow requires extensive cytoplasmic and nuclear remodeling, but the processes governing these energy-consuming changes are unknown. While previous studies show that autophagy is required for differentiation of other blood cell lineages, its function during granulopoiesis has remained elusive. Here, we have shown that metabolism and autophagy are developmentally programmed and essential for neutrophil differentiation in vivo. Atg7-deficient neutrophil precursors had increased glycolytic activity but impaired mitochondrial respiration, decreased ATP production, and accumulated lipid droplets. Inhibiting autophagy-mediated lipid degradation or fatty acid oxidation alone was sufficient to cause defective differentiation, while administration of fatty acids or pyruvate for mitochondrial respiration rescued differentiation in autophagy-deficient neutrophil precursors. Together, we show that autophagy-mediated lipolysis provides free fatty acids to support a mitochondrial respiration pathway essential to neutrophil differentiation.


Asunto(s)
Autofagia , Diferenciación Celular , Ácidos Grasos no Esterificados/metabolismo , Neutrófilos/citología , Neutrófilos/metabolismo , Adaptación Biológica , Animales , Análisis por Conglomerados , Metabolismo Energético , Perfilación de la Expresión Génica , Técnicas de Inactivación de Genes , Glucosa/metabolismo , Metabolismo de los Lípidos , Lipólisis , Mielopoyesis , Neutrófilos/ultraestructura , Oxidación-Reducción , Ácido Pirúvico/metabolismo
4.
Nature ; 567(7746): 49-55, 2019 03.
Artículo en Inglés | MEDLINE | ID: mdl-30814735

RESUMEN

The colonic epithelium facilitates host-microorganism interactions to control mucosal immunity, coordinate nutrient recycling and form a mucus barrier. Breakdown of the epithelial barrier underpins inflammatory bowel disease (IBD). However, the specific contributions of each epithelial-cell subtype to this process are unknown. Here we profile single colonic epithelial cells from patients with IBD and unaffected controls. We identify previously unknown cellular subtypes, including gradients of progenitor cells, colonocytes and goblet cells within intestinal crypts. At the top of the crypts, we find a previously unknown absorptive cell, expressing the proton channel OTOP2 and the satiety peptide uroguanylin, that senses pH and is dysregulated in inflammation and cancer. In IBD, we observe a positional remodelling of goblet cells that coincides with downregulation of WFDC2-an antiprotease molecule that we find to be expressed by goblet cells and that inhibits bacterial growth. In vivo, WFDC2 preserves the integrity of tight junctions between epithelial cells and prevents invasion by commensal bacteria and mucosal inflammation. We delineate markers and transcriptional states, identify a colonic epithelial cell and uncover fundamental determinants of barrier breakdown in IBD.


Asunto(s)
Colon/citología , Colon/patología , Células Epiteliales/clasificación , Células Epiteliales/citología , Salud , Enfermedades Inflamatorias del Intestino/patología , Canales Iónicos/metabolismo , Animales , Biomarcadores/análisis , Colitis Ulcerosa/genética , Colitis Ulcerosa/microbiología , Colitis Ulcerosa/patología , Colon/microbiología , Células Epiteliales/microbiología , Células Epiteliales/patología , Predisposición Genética a la Enfermedad/genética , Células Caliciformes/citología , Células Caliciformes/metabolismo , Células Caliciformes/patología , Humanos , Concentración de Iones de Hidrógeno , Enfermedades Inflamatorias del Intestino/genética , Enfermedades Inflamatorias del Intestino/microbiología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Masculino , Ratones , Péptidos Natriuréticos/metabolismo , Proteínas/metabolismo , Análisis de la Célula Individual , Células Madre/citología , Células Madre/metabolismo , Células Madre/patología , Uniones Estrechas/metabolismo , Transcripción Genética , Proteína 2 de Dominio del Núcleo de Cuatro Disulfuros WAP
5.
Nucleic Acids Res ; 47(14): 7402-7417, 2019 08 22.
Artículo en Inglés | MEDLINE | ID: mdl-31127293

RESUMEN

The CRISPR system is widely used in genome editing for biomedical research. Here, using either dual paired Cas9D10A nickases or paired Cas9 nuclease we characterize unintended larger deletions at on-target sites that frequently evade common genotyping practices. We found that unintended larger deletions are prevalent at multiple distinct loci on different chromosomes, in cultured cells and mouse embryos alike. We observed a high frequency of microhomologies at larger deletion breakpoint junctions, suggesting the involvement of microhomology-mediated end joining in their generation. In populations of edited cells, the distribution of larger deletion sizes is dependent on proximity to sgRNAs and cannot be predicted by microhomology sequences alone.


Asunto(s)
Sistemas CRISPR-Cas , Deleción Cromosómica , Cromosomas de los Mamíferos/genética , Edición Génica/métodos , Eliminación de Secuencia , Animales , Línea Celular , Puntos de Rotura del Cromosoma , Cromosomas de los Mamíferos/metabolismo , Reparación del ADN por Unión de Extremidades , Desoxirribonucleasa I/genética , Desoxirribonucleasa I/metabolismo , Endonucleasas/genética , Endonucleasas/metabolismo , Ratones , Modelos Genéticos , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismo
6.
Eur J Immunol ; 48(7): 1120-1136, 2018 07.
Artículo en Inglés | MEDLINE | ID: mdl-29572905

RESUMEN

Zika virus (ZIKV) is a major public health concern in the Americas. We report that ZIKV infection and RNA extracted from ZIKV infected cells potently activated the induction of type I interferons (IFNs). This effect was fully dependent on the mitochondrial antiviral signaling protein (MAVS), implicating RIG-I-like receptors (RLRs) as upstream sensors of viral RNA. Indeed, RIG-I and the related RNA sensor MDA5 contributed to type I IFN induction in response to RNA from infected cells. We found that ZIKV NS5 from a recent Brazilian isolate blocked type I IFN induction downstream of RLRs and also inhibited type I IFN receptor (IFNAR) signaling. We defined the ZIKV NS5 nuclear localization signal and report that NS5 nuclear localization was not required for inhibition of signaling downstream of IFNAR. Mechanistically, NS5 blocked IFNAR signaling by both leading to reduced levels of STAT2 and by blocking phosphorylation of STAT1, two transcription factors activated by type I IFNs. Taken together, our observations suggest that ZIKV infection induces a type I IFN response via RLRs and that ZIKV interferes with this response by blocking signaling downstream of RLRs and IFNAR.


Asunto(s)
Proteína 58 DEAD Box/inmunología , Interferón Tipo I/metabolismo , ARN/inmunología , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT2/metabolismo , Proteínas no Estructurales Virales/metabolismo , Transporte Activo de Núcleo Celular , Brasil , Proteína 58 DEAD Box/genética , Regulación hacia Abajo , Células HEK293 , Humanos , Interferón Tipo I/genética , Fosforilación , Receptores Inmunológicos , Transducción de Señal , Replicación Viral , Virus Zika , Infección por el Virus Zika
7.
Mamm Genome ; 30(11-12): 319-328, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31667540

RESUMEN

Genetic factors play a significant role in risk for mood and anxiety disorders. Polymorphisms in genes that regulate the brain monoamine systems, such as catabolic enzymes and transporters, are attractive candidates for being risk factors for emotional disorders given the weight of evidence implicating monoamines involvement in these conditions. Several common genetic variants have been identified in the human serotonin transporter (5-HTT) gene, including a repetitive sequence located in the promoter region of the locus called the serotonin transporter-linked polymorphic region (5-HTT-LPR). This polymorphism has been associated with a number of mental traits in both humans and primates, including depression, neuroticism, and harm avoidance. Some, but not all, studies found a link between the polymorphism and 5-HTT levels, leaving open the question of whether the polymorphism affects risk for mental traits via changes in 5-HTT expression. To investigate the impact of the polymorphism on gene expression, serotonin homeostasis, and behavioral traits, we set out to develop a mouse model of the human 5-HTT-LPR. Here we describe the creation and characterization of a set of mouse lines with single-copy human transgenes carrying the short and long 5-HTT-LPR variants. Although we were not able to detect differences in expression between the short and long variants, we encountered several technical issues concerning the design of our humanized mice that are likely to have influenced our findings. Our study serves as a cautionary note for future studies aimed at studying human transgene regulation in the context of the living mouse.


Asunto(s)
Polimorfismo Genético , Proteínas de Transporte de Serotonina en la Membrana Plasmática/genética , Animales , Línea Celular , Expresión Génica , Humanos , Ratones Transgénicos , Transgenes
8.
J Infect Dis ; 217(2): 257-262, 2018 01 04.
Artículo en Inglés | MEDLINE | ID: mdl-29202190

RESUMEN

Interferon-induced transmembrane 3 (IFITM3) is known to restrict the entry of a range of enveloped viruses. The single nucleotide polymorphism rs12252-C within IFITM3 has been shown to be associated with severe influenza A virus infection. It has been suggested that rs12252-C results in expression of a truncated IFITM3 protein lacking the first 21 amino acids. By performing high-throughput RNA sequencing on primary dendritic cells and peripheral blood mononuclear cells isolated from pandemic H1N1 influenza and human immunodeficiency virus-1 (HIV-1) infected patients we show that full-length IFITM3 mRNA is dominantly expressed (>99%) across all rs12252 genotypes. Full-length IFITM3 protein can be detected in all genotypes.


Asunto(s)
Gripe Humana/genética , Gripe Humana/patología , Proteínas de la Membrana/genética , Polimorfismo de Nucleótido Simple , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Células Dendríticas/inmunología , Humanos , Subtipo H1N1 del Virus de la Influenza A/aislamiento & purificación , Gripe Humana/virología , Leucocitos Mononucleares , Análisis de Secuencia de ARN , Reino Unido
9.
Methods Protoc ; 6(6)2023 Nov 17.
Artículo en Inglés | MEDLINE | ID: mdl-37987358

RESUMEN

As our readers know, Methods and Protocols is a multidisciplinary peer-reviewed scientific journal that provides a forum to the publication of novel approaches in the fields of Life Sciences, Chemistry, and Biomedical Sciences and their intersection with other related scientific fields such as Physics, Earth Sciences, and Environmental Research [...].

10.
Cancer Res ; 83(20): 3400-3413, 2023 Oct 13.
Artículo en Inglés | MEDLINE | ID: mdl-37463466

RESUMEN

GTP cyclohydrolase (GCH1) is the rate-limiting enzyme for tetrahydrobiopterin (BH4) biosynthesis. The catalysis of BH4 biosynthesis is tightly regulated for physiological neurotransmission, inflammation, and vascular tone. Paradoxically, BH4 has emerged as an oncometabolite regulating tumor growth, but the effects on tumor development remain controversial. Here, we found that GCH1 potentiated the growth of triple-negative breast cancer (TNBC) and HER2+ breast cancer and transformed nontumor breast epithelial cells. Independent of BH4 production, GCH1 protein induced epithelial-to-mesenchymal transition by binding to vimentin (Vim), which was mediated by HSP90. Conversely, GCH1 ablation impaired tumor growth, suppressed Vim in TNBC, and inhibited EGFR/ERK signaling while activating the p53 pathway in estrogen receptor-positive tumor cells. GCH1 deficiency increases tumor cell sensitivity to HSP90 inhibition and endocrine treatments. In addition, high GCH1 correlated with poor breast cancer survival. Together, this study reveals an enzyme-independent oncogenic role of GCH1, presenting it as a potential target for therapeutic development. SIGNIFICANCE: GTP cyclohydrolase functions as an oncogene in breast cancer and binds vimentin to induce epithelial-to-mesenchymal transition independently of its enzyme activity, which confers targetable vulnerabilities for developing breast cancer treatment strategies.

11.
Heliyon ; 9(10): e20076, 2023 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-37842619

RESUMEN

Mutations within viral epitopes can result in escape from T cells, but the contribution of mutations in flanking regions of epitopes in SARS-CoV-2 has not been investigated. Focusing on two SARS-CoV-2 nucleoprotein CD8+ epitopes, we investigated the contribution of these flanking mutations to proteasomal processing and T cell activation. We found decreased NP9-17-B*27:05 CD8+ T cell responses to the NP-Q7K mutation, likely due to a lack of efficient epitope production by the proteasome, suggesting immune escape caused by this mutation. In contrast, NP-P6L and NP-D103 N/Y mutations flanking the NP9-17-B*27:05 and NP105-113-B*07:02 epitopes, respectively, increased CD8+ T cell responses associated with enhanced epitope production by the proteasome. Our results provide evidence that SARS-CoV-2 mutations outside the epitope could have a significant impact on proteasomal processing, either contributing to T cell escape or enhancement that may be exploited for future vaccine design.

12.
Cell Rep ; 42(5): 112470, 2023 05 30.
Artículo en Inglés | MEDLINE | ID: mdl-37141092

RESUMEN

Most existing studies characterizing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cell responses are peptide based. This does not allow evaluation of whether tested peptides are processed and presented canonically. In this study, we use recombinant vaccinia virus (rVACV)-mediated expression of SARS-CoV-2 spike protein and SARS-CoV-2 infection of angiotensin-converting enzyme (ACE)-2-transduced B cell lines to evaluate overall T cell responses in a small cohort of recovered COVID-19 patients and uninfected donors vaccinated with ChAdOx1 nCoV-19. We show that rVACV expression of SARS-CoV-2 antigen can be used as an alternative to SARS-CoV-2 infection to evaluate T cell responses to naturally processed spike antigens. In addition, the rVACV system can be used to evaluate the cross-reactivity of memory T cells to variants of concern (VOCs) and to identify epitope escape mutants. Finally, our data show that both natural infection and vaccination could induce multi-functional T cell responses with overall T cell responses remaining despite the identification of escape mutations.


Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , ChAdOx1 nCoV-19 , Vacunación , Anticuerpos Antivirales
13.
Sci Rep ; 12(1): 18639, 2022 11 03.
Artículo en Inglés | MEDLINE | ID: mdl-36329114

RESUMEN

Clusterin (CLU) is one of the most significant genetic risk factors for late onset Alzheimer's disease (AD). However, the mechanisms by which CLU contributes to AD development and pathogenesis remain unclear. Studies have demonstrated that the trafficking and localisation of glycosylated CLU proteins is altered by CLU-AD mutations and amyloid-ß (Aß), which may contribute to AD pathogenesis. However, the roles of non-glycosylated and glycosylated CLU proteins in mediating Aß toxicity have not been studied in human neurons. iPSCs with altered CLU trafficking were generated following the removal of CLU exon 2 by CRISPR/Cas9 gene editing. Neurons were generated from control (CTR) and exon 2 -/- edited iPSCs and were incubated with aggregated Aß peptides. Aß induced changes in cell death and neurite length were quantified to determine if altered CLU protein trafficking influenced neuronal sensitivity to Aß. Finally, RNA-Seq analysis was performed to identify key transcriptomic differences between CLU exon 2 -/- and CTR neurons. The removal of CLU exon 2, and the endoplasmic reticulum (ER)-signal peptide located within, abolished the presence of glycosylated CLU and increased the abundance of intracellular, non-glycosylated CLU. While non-glycosylated CLU levels were unaltered by Aß25-35 treatment, the trafficking of glycosylated CLU was altered in control but not exon 2 -/- neurons. The latter also displayed partial protection against Aß-induced cell death and neurite retraction. Transcriptome analysis identified downregulation of multiple extracellular matrix (ECM) related genes in exon 2 -/- neurons, potentially contributing to their reduced sensitivity to Aß toxicity. This study identifies a crucial role of glycosylated CLU in facilitating Aß toxicity in human neurons. The loss of these proteins reduced both, cell death and neurite damage, two key consequences of Aß toxicity identified in the AD brain. Strikingly, transcriptomic differences between exon 2 -/- and control neurons were small, but a significant and consistent downregulation of ECM genes and pathways was identified in exon 2 -/- neurons. This may contribute to the reduced sensitivity of these neurons to Aß, providing new mechanistic insights into Aß pathologies and therapeutic targets for AD.


Asunto(s)
Enfermedad de Alzheimer , Clusterina , Humanos , Clusterina/genética , Clusterina/metabolismo , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/toxicidad , Péptidos beta-Amiloides/metabolismo , Neuronas/metabolismo , Encéfalo/metabolismo
14.
Commun Biol ; 5(1): 111, 2022 02 04.
Artículo en Inglés | MEDLINE | ID: mdl-35121793

RESUMEN

Salmonella enterica represent a major disease burden worldwide. S. enterica serovar Typhi (S. Typhi) is responsible for potentially life-threatening Typhoid fever affecting 10.9 million people annually. While non-typhoidal Salmonella (NTS) serovars usually trigger self-limiting diarrhoea, invasive NTS bacteraemia is a growing public health challenge. Dendritic cells (DCs) are key professional antigen presenting cells of the human immune system. The ability of pathogenic bacteria to subvert DC functions and prevent T cell recognition contributes to their survival and dissemination within the host. Here, we adapted dual RNA-sequencing to define how different Salmonella pathovariants remodel their gene expression in tandem with that of infected DCs. We find DCs harness iron handling pathways to defend against invading Salmonellas, which S. Typhi is able to circumvent by mounting a robust response to nitrosative stress. In parallel, we uncover the alternative strategies invasive NTS employ to impair DC functions.


Asunto(s)
Reprogramación Celular/fisiología , Células Dendríticas/metabolismo , Salmonella enterica/clasificación , Células Dendríticas/inmunología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Mutación
15.
Mol Pain ; 7: 66, 2011 Sep 11.
Artículo en Inglés | MEDLINE | ID: mdl-21906401

RESUMEN

Progress in the somatosensory field has been restricted by the limited number of genetic tools available to study gene function in peripheral sensory neurons. Here we generated a Cre-driver mouse line that expresses Cre-recombinase from the locus of the sensory neuron specific gene Advillin. These mice displayed almost exclusive Cre-mediated recombination in all peripheral sensory neurons. As such, the Advillin-Cre-driver line will be a powerful tool for targeting peripheral neurons in future investigations.


Asunto(s)
Técnicas Genéticas , Integrasas/metabolismo , Proteínas de Microfilamentos/metabolismo , Envejecimiento/metabolismo , Animales , Conducta Animal , Embrión de Mamíferos/metabolismo , Dosificación de Gen/genética , Hiperalgesia/patología , Hiperalgesia/fisiopatología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Nocicepción/fisiología , Células Receptoras Sensoriales/metabolismo , Coloración y Etiquetado , beta-Galactosidasa/metabolismo
16.
Sci Adv ; 7(49): eabj9247, 2021 Dec 03.
Artículo en Inglés | MEDLINE | ID: mdl-34860543

RESUMEN

The transcription factor FOXN1 is a master regulator of thymic epithelial cell (TEC) development and function. Here, we demonstrate that FOXN1 expression is differentially regulated during organogenesis and participates in multimolecular nuclear condensates essential for the factor's transcriptional activity. FOXN1's C-terminal sequence regulates the diffusion velocity within these aggregates and modulates the binding to proximal gene regulatory regions. These dynamics are altered in a patient with a mutant FOXN1 that is modified in its C-terminal sequence. This mutant is transcriptionally inactive and acts as a dominant negative factor displacing wild-type FOXN1 from condensates and causing athymia and severe lymphopenia in heterozygotes. Expression of the mutated mouse ortholog selectively impairs mouse TEC differentiation, revealing a gene dose dependency for individual TEC subtypes. We have therefore identified the cause for a primary immunodeficiency disease and determined the mechanism by which this FOXN1 gain-of-function mutant mediates its dominant negative effect.

17.
Cells ; 9(6)2020 06 16.
Artículo en Inglés | MEDLINE | ID: mdl-32560274

RESUMEN

The Zika virus (ZIKV) has received much attention due to an alarming increase in cases of neurological disorders including congenital Zika syndrome associated with infection. To date, there is no effective treatment available. An immediate response by the innate immune system is crucial for effective control of the virus. Using CRISPR/Cas9-mediated knockouts in A549 cells, we investigated the individual contributions of the RIG-I-like receptors MDA5 and RIG-I to ZIKV sensing and control of this virus by using a Brazilian ZIKV strain. We show that RIG-I is the main sensor for ZIKV in A549 cells. Surprisingly, we observed that loss of RIG-I and consecutive type I interferon (IFN) production led to virus-induced apoptosis. ZIKV non-structural protein NS5 was reported to interfere with type I IFN receptor signaling. Additionally, we show that ZIKV NS5 inhibits type I IFN induction. Overall, our study highlights the importance of RIG-I-dependent ZIKV sensing for the prevention of virus-induced cell death and shows that NS5 inhibits the production of type I IFN.


Asunto(s)
Muerte Celular/fisiología , Proteína 58 DEAD Box/metabolismo , Receptores Inmunológicos/metabolismo , Infección por el Virus Zika/virología , Animales , Chlorocebus aethiops/virología , Humanos , Inmunidad Innata/inmunología , Transducción de Señal/inmunología , Células Vero/virología , Proteínas no Estructurales Virales/metabolismo , Virus Zika/inmunología , Virus Zika/metabolismo , Infección por el Virus Zika/inmunología
18.
Methods Protoc ; 3(3)2020 07 29.
Artículo en Inglés | MEDLINE | ID: mdl-32751356

RESUMEN

Use of dual sgRNAs is a common CRISPR/Cas9-based strategy for the creation of genetic deletions. The ease of screening combined with a rather high rate of success makes this approach a reliable genome engineering procedure. Recently, a number of studies using CRISPR/Cas9 have revealed unwanted large-scale rearrangements, duplications, inversions or larger-than-expected deletions. Strict quality control measures are required to validate the model system, and this crucially depends on knowing which potential experimental outcomes to expect. Using the dual sgRNA deletion approach, our team discovered high levels of excision, inversion and re-insertion at the site of targeting. We detected those at a variety of genomic loci and in several immortalized cell lines, demonstrating that inverted re-insertions are a common by-product with an overall frequency between 3% and 20%. Our findings imply an inherent danger in the misinterpretation of screening data when using only a single PCR screening. While amplification of the region of interest might classify clones as wild type (WT) based on amplicon size, secondary analyses can discover heterozygous (HET) clones among presumptive WTs, and events deemed as HET clones could potentially be full KO. As such, screening for inverted re-insertions helps in decreasing the number of clones required to obtain a full KO. With this technical note, we want to raise awareness of this phenomenon and suggest implementing a standard secondary PCR while screening for deletions.

19.
Sci Rep ; 8(1): 5549, 2018 04 03.
Artículo en Inglés | MEDLINE | ID: mdl-29615718

RESUMEN

T cell function can be compromised during chronic infections or through continuous exposure to tumor antigens by the action of immune checkpoint receptors, such as programmed cell death protein 1 (PD-1). Systemic administration of blocking antibodies against the PD-1 pathway can restore T cell function, and has been approved for the treatment of several malignancies, although there is a risk of adverse immune-related side-effects. We have developed a method for generating gene knockouts in human antigen (Ag)-specific cytotoxic T-Lymphocyte (CTLs) using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome editing. Using this method, we generated several transduced CD4+ or CD8+ antigen-specific polyclonal CTL lines and clones, and validated gene modifications of the PD-1 gene. We compared these T-cell lines and clones with control groups in the presence of programmed death-ligand 1 (PD-L1) and observed improved effector functions in the PD1-disrupted cell group. Overall, we have developed a versatile tool for functional genomics in human antigen-specific CTL studies. Furthermore, we provide an alternative strategy for current cell-based immunotherapy that will minimize the side effects caused by antibody blockade therapy.


Asunto(s)
Antígenos de Neoplasias/inmunología , Edición Génica , Inmunoterapia , Neoplasias/tratamiento farmacológico , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Linfocitos T Citotóxicos/inmunología , Sistemas CRISPR-Cas , Humanos , Activación de Linfocitos , Neoplasias/inmunología , Receptor de Muerte Celular Programada 1/genética , Receptor de Muerte Celular Programada 1/inmunología
20.
Nat Commun ; 9(1): 5375, 2018 12 18.
Artículo en Inglés | MEDLINE | ID: mdl-30560907

RESUMEN

During development, it is unclear if lineage-fated cells derive from multilineage-primed progenitors and whether active mechanisms operate to restrict cell fate. Here we investigate how mesoderm specifies into blood-fated cells. We document temporally restricted co-expression of blood (Scl/Tal1), cardiac (Mesp1) and paraxial (Tbx6) lineage-affiliated transcription factors in single cells, at the onset of blood specification, supporting the existence of common progenitors. At the same time-restricted stage, absence of SCL results in expansion of cardiac/paraxial cell populations and increased cardiac/paraxial gene expression, suggesting active suppression of alternative fates. Indeed, SCL normally activates expression of co-repressor ETO2 and Polycomb-PRC1 subunits (RYBP, PCGF5) and maintains levels of Polycomb-associated histone marks (H2AK119ub/H3K27me3). Genome-wide analyses reveal ETO2 and RYBP co-occupy most SCL target genes, including cardiac/paraxial loci. Reduction of Eto2 or Rybp expression mimics Scl-null cardiac phenotype. Therefore, SCL-mediated transcriptional repression prevents mis-specification of blood-fated cells, establishing active repression as central to fate determination processes.


Asunto(s)
Linaje de la Célula/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas Nucleares/metabolismo , Proteínas Represoras/metabolismo , Proteína 1 de la Leucemia Linfocítica T Aguda/metabolismo , Factores de Transcripción/metabolismo , Animales , Diferenciación Celular/fisiología , Línea Celular , Separación Celular/métodos , Embrión de Mamíferos , Citometría de Flujo/métodos , Código de Histonas/fisiología , Mesodermo/citología , Mesodermo/fisiología , Ratones , Células Madre Embrionarias de Ratones , Proteínas Nucleares/genética , Complejo Represivo Polycomb 1/metabolismo , Proteínas del Grupo Polycomb/metabolismo , ARN Interferente Pequeño/metabolismo , Proteínas Represoras/genética , Proteína 1 de la Leucemia Linfocítica T Aguda/genética , Factores de Transcripción/genética
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