RESUMEN
SARS-CoV-2 infection and vaccination generates enormous host-response heterogeneity and an age-dependent loss of immune-response quality. How the pre-exposure T cell repertoire contributes to this heterogeneity is poorly understood. We combined analysis of SARS-CoV-2-specific CD4+ T cells pre- and post-vaccination with longitudinal T cell receptor tracking. We identified strong pre-exposure T cell variability that correlated with subsequent immune-response quality and age. High-quality responses, defined by strong expansion of high-avidity spike-specific T cells, high interleukin-21 production, and specific immunoglobulin G, depended on an intact naive repertoire and exclusion of pre-existing memory T cells. In the elderly, T cell expansion from both compartments was severely compromised. Our results reveal that an intrinsic defect of the CD4+ T cell repertoire causes the age-dependent decline of immune-response quality against SARS-CoV-2 and highlight the need for alternative strategies to induce high-quality T cell responses against newly arising pathogens in the elderly.
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COVID-19 , SARS-CoV-2 , Anciano , Anticuerpos Antivirales , Humanos , Inmunidad , Inmunoglobulina G , Receptores de Antígenos de Linfocitos T , VacunaciónRESUMEN
The adult intestinal stem cells (ISCs), their hierarchies, mechanisms of maintenance and differentiation have been extensively studied. However, when and how ISCs are established during embryogenesis remains unknown. We show here that the transcription regulator Id2 controls the specification of embryonic Lgr5+ progenitors in the developing murine small intestine. Cell fate mapping analysis revealed that Lgr5+ progenitors emerge at E13.5 in wild-type embryos and differ from the rest on the intestinal epithelium by a characteristic ISC signature. In the absence of Id2, the intestinal epithelium differentiates into Lgr5+ cells already at E9.5. Furthermore, the size of the Lgr5+ cell pool is significantly increased. We show that Id2 restricts the activity of the Wnt signalling pathway at early stages and prevents precocious differentiation of the embryonic intestinal epithelium. Id2-deficient embryonic epithelial cells cultured ex vivo strongly activate Wnt target genes as well as markers of neoplastic transformation and form fast growing undifferentiated spheroids. Furthermore, adult ISCs from Id2-deficient mice display a distinct transcriptional signature, supporting an essential role for Id2 in the correct specification of ISCs.
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Proteína 2 Inhibidora de la Diferenciación/metabolismo , Intestino Delgado/embriología , Receptores Acoplados a Proteínas G/análisis , Células Madre/química , Células Madre/fisiología , Animales , Ratones , Vía de Señalización WntRESUMEN
BACKGROUND & AIMS: Excess and unresolved endoplasmic reticulum (ER) stress in intestinal epithelial cells (IECs) promotes intestinal inflammation. Activating transcription factor 6 (ATF6) is one of the signaling mediators of ER stress. We studied the pathways that regulate ATF6 and its role for inflammation in IECs. METHODS: We performed an RNA interference screen, using 23,349 unique small interfering RNAs targeting 7783 genes and a luciferase reporter controlled by an ATF6-dependent ERSE (ER stress-response element) promoter, to identify proteins that activate or inhibit the ATF6 signaling pathway in HEK293 cells. To validate the screening results, intestinal epithelial cell lines (Caco-2 cells) were transfected with small interfering RNAs or with a plasmid overexpressing a constitutively active form of ATF6. Caco-2 cells with a CRISPR-mediated disruption of autophagy related 16 like 1 gene (ATG16L1) were used to study the effect of ATF6 on ER stress in autophagy-deficient cells. We also studied intestinal organoids derived from mice that overexpress constitutively active ATF6, from mice with deletion of the autophagy related 16 like 1 or X-Box binding protein 1 gene in IECs (Atg16l1ΔIEC or Xbp1ΔIEC, which both develop spontaneous ileitis), from patients with Crohn's disease (CD) and healthy individuals (controls). Cells and organoids were incubated with tunicamycin to induce ER stress and/or chemical inhibitors of newly identified activator proteins of ATF6 signaling, and analyzed by real-time polymerase chain reaction and immunoblots. Atg16l1ΔIEC and control (Atg16l1fl/fl) mice were given intraperitoneal injections of tunicamycin and were treated with chemical inhibitors of ATF6 activating proteins. RESULTS: We identified and validated 15 suppressors and 7 activators of the ATF6 signaling pathway; activators included the regulatory subunit of casein kinase 2 (CSNK2B) and acyl-CoA synthetase long chain family member 1 (ACSL1). Knockdown or chemical inhibition of CSNK2B and ACSL1 in Caco-2 cells reduced activity of the ATF6-dependent ERSE reporter gene, diminished transcription of the ATF6 target genes HSP90B1 and HSPA5 and reduced NF-κB reporter gene activation on tunicamycin stimulation. Atg16l1ΔIEC and or Xbp1ΔIEC organoids showed increased expression of ATF6 and its target genes. Inhibitors of ACSL1 or CSNK2B prevented activation of ATF6 and reduced CXCL1 and tumor necrosis factor (TNF) expression in these organoids on induction of ER stress with tunicamycin. Injection of mice with inhibitors of ACSL1 or CSNK2B significantly reduced tunicamycin-mediated intestinal inflammation and IEC death and expression of CXCL1 and TNF in Atg16l1ΔIEC mice. Purified ileal IECs from patients with CD had higher levels of ATF6, CSNK2B, and HSPA5 messenger RNAs than controls; early-passage organoids from patients with active CD show increased levels of activated ATF6 protein, incubation of these organoids with inhibitors of ACSL1 or CSNK2B reduced transcription of ATF6 target genes, including TNF. CONCLUSIONS: Ileal IECs from patients with CD have higher levels of activated ATF6, which is regulated by CSNK2B and HSPA5. ATF6 increases expression of TNF and other inflammatory cytokines in response to ER stress in these cells and in organoids from Atg16l1ΔIEC and Xbp1ΔIEC mice. Strategies to inhibit the ATF6 signaling pathway might be developed for treatment of inflammatory bowel diseases.
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Factor de Transcripción Activador 6/metabolismo , Estrés del Retículo Endoplásmico/fisiología , Células Epiteliales/patología , Íleon/metabolismo , Íleon/patología , Enfermedades Inflamatorias del Intestino/metabolismo , Animales , Autofagia , Células CACO-2 , Técnicas de Cultivo de Célula , Chaperón BiP del Retículo Endoplásmico , Células HEK293 , Humanos , Enfermedades Inflamatorias del Intestino/etiología , Enfermedades Inflamatorias del Intestino/patología , Mucosa Intestinal/patología , Ratones , Transducción de SeñalRESUMEN
Epigenetic mechanisms, including chromatin structure, chromatin dynamics and histone modifications play an important role for maintenance and differentiation of pluripotent embryonic stem cells. However, little is known about the molecular mechanisms of adult stem cell specification and differentiation. Here, we used intestinal stem cells (ISCs) as a model system to reveal the epigenetic changes coordinating gene expression programs during these processes. We found that two distinct epigenetic mechanisms participate in establishing the transcriptional program promoting ISC specification from embryonic progenitors. A large number of adult ISC signature genes are targets of repressive DNA methylation in embryonic intestinal epithelial progenitors. On the other hand, genes essential for embryonic development acquire H3K27me3 and are silenced during ISC specification. We also show that the repression of ISC signature genes as well as the activation of enterocyte specific genes is accompanied by a global loss of H2A.Z during ISCs differentiation. Our results reveal that, already during ISC specification, an extensive remodeling of chromatin both at promoters and distal regulatory elements organizes transcriptional landscapes operating in differentiated enterocytes, thus explaining similar chromatin modification patterns in the adult gut epithelium.
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Células Madre Adultas/metabolismo , Cromatina/química , Células Madre Embrionarias/metabolismo , Enterocitos/metabolismo , Silenciador del Gen , Mucosa Intestinal/metabolismo , Células Madre Adultas/citología , Animales , Diferenciación Celular , Linaje de la Célula , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina , Metilación de ADN , Embrión de Mamíferos , Células Madre Embrionarias/citología , Enterocitos/citología , Histonas/genética , Histonas/metabolismo , Intestinos/citología , Masculino , Ratones , Ratones Transgénicos , Transcripción GenéticaRESUMEN
Endoplasmic reticulum (ER) stress and autophagy are tightly controlled cellular processes, which are responsible for maintaining protein homeostasis in a cell. Impairment of the interlinking pathways have been implicated in a number of human diseases, prominently in inflammatory bowel disease, where genetic variants in several independent autophagy and ER stress related loci have been associated to increased disease risk. Autophagy is a selective quality control process, which governs the integrity of the cell by removal of aged organelles and proteins via the lysosome, but recently has been shown to actively license the outcome of other signaling pathways by guiding the proteolytic removal of signaling protein complexes (adaptophagy). In this review, we summarize our knowledge on regulated proteolytic events involved in ER stress responses and autophagy, their interplay and potential regulatory effects with a particular focus on intestinal inflammation. This article is part of a Special Issue entitled: Proteolysis as a Regulatory Event in Pathophysiology edited by Stefan Rose-John.
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Autofagia/genética , Estrés del Retículo Endoplásmico/genética , Enfermedades Inflamatorias del Intestino/genética , Proteolisis , Humanos , Enfermedades Inflamatorias del Intestino/patología , Mucosa Intestinal/metabolismo , Intestinos/patología , Lisosomas/metabolismo , Transducción de SeñalRESUMEN
Alcohol-associated liver disease is accompanied by intestinal mycobiome dysbiosis, yet the impacts on liver disease are unclear. We demonstrate that Candida albicans-specific T helper 17 (Th17) cells are increased in circulation and present in the liver of patients with alcohol-associated liver disease. Chronic ethanol administration in mice causes migration of Candida albicans (C. albicans)-reactive Th17 cells from the intestine to the liver. The antifungal agent nystatin decreased C. albicans-specific Th17 cells in the liver and reduced ethanol-induced liver disease in mice. Transgenic mice expressing T cell receptors (TCRs) reactive to Candida antigens developed more severe ethanol-induced liver disease than transgene-negative littermates. Adoptively transferring Candida-specific TCR transgenic T cells or polyclonal C. albicans-primed T cells exacerbated ethanol-induced liver disease in wild-type mice. Interleukin-17 (IL-17) receptor A signaling in Kupffer cells was required for the effects of polyclonal C. albicans-primed T cells. Our findings indicate that ethanol increases C. albicans-specific Th17 cells, which contribute to alcohol-associated liver disease.
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Candida albicans , Células Th17 , Ratones , Animales , Candida , Ratones Transgénicos , Etanol/toxicidadRESUMEN
Genetic variants in the DNA methyltransferase 3 A (DNMT3A) locus have been associated with inflammatory bowel disease (IBD). DNMT3A is part of the epigenetic machinery physiologically involved in DNA methylation. We show that DNMT3A plays a critical role in maintaining intestinal homeostasis and gut barrier function. DNMT3A expression is downregulated in intestinal epithelial cells from IBD patients and upon tumor necrosis factor treatment in murine intestinal organoids. Ablation of DNMT3A in Caco-2 cells results in global DNA hypomethylation, which is linked to impaired regenerative capacity, transepithelial resistance and intercellular junction formation. Genetic deletion of Dnmt3a in intestinal epithelial cells (Dnmt3aΔIEC) in mice confirms the phenotype of an altered epithelial ultrastructure with shortened apical-junctional complexes, reduced Goblet cell numbers and increased intestinal permeability in the colon in vivo. Dnmt3aΔIEC mice suffer from increased susceptibility to experimental colitis, characterized by reduced epithelial regeneration. These data demonstrate a critical role for DNMT3A in orchestrating intestinal epithelial homeostasis and response to tissue damage and suggest an involvement of impaired epithelial DNMT3A function in the etiology of IBD.