RESUMEN
In vitro models of autoimmunity are constrained by an inability to culture affected epithelium alongside the complex tissue-resident immune microenvironment. Coeliac disease (CeD) is an autoimmune disease in which dietary gluten-derived peptides bind to the major histocompatibility complex (MHC) class II human leukocyte antigen molecules (HLA)-DQ2 or HLA-DQ8 to initiate immune-mediated duodenal mucosal injury1-4. Here, we generated air-liquid interface (ALI) duodenal organoids from intact fragments of endoscopic biopsies that preserve epithelium alongside native mesenchyme and tissue-resident immune cells as a unit without requiring reconstitution. The immune diversity of ALI organoids spanned T cells, B and plasma cells, natural killer (NK) cells and myeloid cells, with extensive T-cell and B-cell receptor repertoires. HLA-DQ2.5-restricted gluten peptides selectively instigated epithelial destruction in HLA-DQ2.5-expressing organoids derived from CeD patients, and this was antagonized by blocking MHC-II or NKG2C/D. Gluten epitopes stimulated a CeD organoid immune network response in lymphoid and myeloid subsets alongside anti-transglutaminase 2 (TG2) autoantibody production. Functional studies in CeD organoids revealed that interleukin-7 (IL-7) is a gluten-inducible pathogenic modulator that regulates CD8+ T-cell NKG2C/D expression and is necessary and sufficient for epithelial destruction. Furthermore, endogenous IL-7 was markedly upregulated in patient biopsies from active CeD compared with remission disease from gluten-free diets, predominantly in lamina propria mesenchyme. By preserving the epithelium alongside diverse immune populations, this human in vitro CeD model recapitulates gluten-dependent pathology, enables mechanistic investigation and establishes a proof of principle for the organoid modelling of autoimmunity.
Asunto(s)
Enfermedad Celíaca , Duodeno , Interleucina-7 , Mucosa Intestinal , Modelos Biológicos , Organoides , Humanos , Autoanticuerpos/inmunología , Autoinmunidad , Linfocitos B/inmunología , Linfocitos B/metabolismo , Biopsia , Enfermedad Celíaca/inmunología , Enfermedad Celíaca/patología , Enfermedad Celíaca/metabolismo , Duodeno/inmunología , Duodeno/patología , Duodeno/metabolismo , Epítopos/inmunología , Glútenes/inmunología , Glútenes/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP/inmunología , Antígenos HLA-DQ/inmunología , Antígenos HLA-DQ/metabolismo , Interleucina-7/metabolismo , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Células Asesinas Naturales/inmunología , Células Mieloides/inmunología , Organoides/inmunología , Organoides/metabolismo , Organoides/patología , Proteína Glutamina Gamma Glutamiltransferasa 2/inmunología , Receptores de Antígenos de Linfocitos B/inmunología , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
Preeclampsia is a multi-organ complication of pregnancy characterized by sudden hypertension and proteinuria that is among the leading causes of preterm delivery and maternal morbidity and mortality worldwide. The heterogeneity of preeclampsia poses a challenge for understanding its etiology and molecular basis. Intriguingly, risk for the condition increases in high-altitude regions such as the Peruvian Andes. To investigate the genetic basis of preeclampsia in a population living at high altitude, we characterized genome-wide variation in a cohort of preeclamptic and healthy Andean families (n = 883) from Puno, Peru, a city located above 3,800 meters of altitude. Our study collected genomic DNA and medical records from case-control trios and duos in local hospital settings. We generated genotype data for 439,314 SNPs, determined global ancestry patterns, and mapped associations between genetic variants and preeclampsia phenotypes. A transmission disequilibrium test (TDT) revealed variants near genes of biological importance for placental and blood vessel function. The top candidate region was found on chromosome 13 of the fetal genome and contains clotting factor genes PROZ, F7, and F10. These findings provide supporting evidence that common genetic variants within coagulation genes play an important role in preeclampsia. A selection scan revealed a potential adaptive signal around the ADAM12 locus on chromosome 10, implicated in pregnancy disorders. Our discovery of an association in a functional pathway relevant to pregnancy physiology in an understudied population of Native American origin demonstrates the increased power of family-based study design and underscores the importance of conducting genetic research in diverse populations.
Asunto(s)
Preeclampsia , Altitud , Factores de Coagulación Sanguínea , Proteínas Sanguíneas/genética , Estudios de Casos y Controles , Factor VII/genética , Factor X/genética , Femenino , Humanos , Perú/epidemiología , Placenta , Preeclampsia/epidemiología , Preeclampsia/genética , EmbarazoRESUMEN
Uterine injury from procedures such as Cesarean sections (C-sections) often have severe consequences on subsequent pregnancy outcomes, leading to disorders such as placenta previa, placenta accreta, and infertility. With rates of C-section at ~30% of deliveries in the USA and projected to continue to climb, a deeper understanding of the mechanisms by which these pregnancy disorders arise and opportunities for intervention are needed. Here we describe a rodent model of uterine injury on subsequent in utero outcomes. We observed three distinct phenotypes: increased rates of resorption and death, embryo spacing defects, and placenta accreta-like features of reduced decidua and expansion of invasive trophoblasts. We show that the appearance of embryo spacing defects depends entirely on the phase of estrous cycle at the time of injury. Using RNA-seq, we identified perturbations in the expression of components of the COX/prostaglandin pathway after recovery from injury, a pathway that has previously been demonstrated to play an important role in embryo spacing. Therefore, we demonstrate that uterine damage in this mouse model causes morphological and molecular changes that ultimately lead to placental and embryonic developmental defects.
Asunto(s)
Placenta Accreta , Placenta , Humanos , Embarazo , Femenino , Animales , Ratones , Diestro , Útero , Cesárea/efectos adversos , Estudios RetrospectivosRESUMEN
The obstetrical conditions placenta accreta spectrum (PAS) and placenta previa are a significant source of pregnancy-associated morbidity and mortality, yet the specific molecular and cellular underpinnings of these conditions are not known. In this study, we identified misregulated gene expression patterns in tissues from placenta previa and percreta (the most extreme form of PAS) compared with control cases. By comparing this gene set with existing placental single-cell and bulk RNA-Seq datasets, we show that the upregulated genes predominantly mark extravillous trophoblasts. We performed immunofluorescence on several candidate molecules and found that PRG2 and AQPEP protein levels are upregulated in both the fetal membranes and the placental disk in both conditions. While this increased AQPEP expression remains restricted to trophoblasts, PRG2 is mislocalized and is found throughout the fetal membranes. Using a larger patient cohort with a diverse set of gestationally aged-matched controls, we validated PRG2 as a marker for both previa and PAS and AQPEP as a marker for only previa in the fetal membranes. Our findings suggest that the extraembryonic tissues surrounding the conceptus, including both the fetal membranes and the placental disk, harbor a signature of previa and PAS that is characteristic of EVTs and that may reflect increased trophoblast invasiveness.
Asunto(s)
Proteína Mayor Básica del Eosinófilo/genética , Membranas Extraembrionarias/metabolismo , Regulación de la Expresión Génica , Metaloproteasas/genética , Placenta Accreta/metabolismo , Placenta Previa/metabolismo , Proteoglicanos/genética , Proteína Mayor Básica del Eosinófilo/metabolismo , Femenino , Humanos , Metaloproteasas/metabolismo , Embarazo , Proteoglicanos/metabolismoRESUMEN
Facilitates chromatin transcription (FACT) is a conserved histone chaperone that reorganizes nucleosomes and ensures chromatin integrity during DNA transcription, replication and repair. Key to the broad functions of FACT is its recognition of histones H2A-H2B (ref. 2). However, the structural basis for how histones H2A-H2B are recognized and how this integrates with the other functions of FACT, including the recognition of histones H3-H4 and other nuclear factors, is unknown. Here we reveal the crystal structure of the evolutionarily conserved FACT chaperone domain Spt16M from Chaetomium thermophilum, in complex with the H2A-H2B heterodimer. A novel 'U-turn' motif scaffolded onto a Rtt106-like module embraces the α1 helix of H2B. Biochemical and in vivo assays validate the structure and dissect the contribution of histone tails and H3-H4 towards Spt16M binding. Furthermore, we report the structure of the FACT heterodimerization domain that connects FACT to replicative polymerases. Our results show that Spt16M makes several interactions with histones, which we suggest allow the module to invade the nucleosome gradually and block the strongest interaction of H2B with DNA. FACT would thus enhance 'nucleosome breathing' by re-organizing the first 30 base pairs of nucleosomal histone-DNA contacts. Our snapshot of the engagement of the chaperone with H2A-H2B and the structures of all globular FACT domains enable the high-resolution analysis of the vital chaperoning functions of FACT, shedding light on how the complex promotes the activity of enzymes that require nucleosome reorganization.
Asunto(s)
Chaetomium/química , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Histonas/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Secuencias de Aminoácidos , Secuencia Conservada , Cristalografía por Rayos X , ADN/química , ADN/metabolismo , Replicación del ADN , Histonas/química , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Nucleosomas/química , Nucleosomas/metabolismo , Unión Proteica , Multimerización de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Especificidad por SustratoRESUMEN
The Xeroderma pigmentosum complementation group C (XPC) complex is a versatile factor involved in both nucleotide excision repair and transcriptional coactivation as a critical component of the NANOG, OCT4, and SOX2 pluripotency gene regulatory network. Here we present the structure of the human holo-XPC complex determined by single-particle electron microscopy to reveal a flexible, ear-shaped structure that undergoes localized loss of order upon DNA binding. We also determined the structure of the complete yeast homolog Rad4 holo-complex to find a similar overall architecture to the human complex, consistent with their shared DNA repair functions. Localized differences between these structures reflect an intriguing phylogenetic divergence in transcriptional capabilities that we present here. Having positioned the constituent subunits by tagging and deletion, we propose a model of key interaction interfaces that reveals the structural basis for this difference in functional conservation. Together, our findings establish a framework for understanding the structure-function relationships of the XPC complex in the interplay between transcription and DNA repair.
Asunto(s)
Reparación del ADN , Proteínas de Unión al ADN/química , Complejos Multiproteicos/química , Animales , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Homeodominio/química , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Proteína Homeótica Nanog , Factor 3 de Transcripción de Unión a Octámeros/química , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Factores de Transcripción SOXB1/química , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Relación Estructura-ActividadRESUMEN
The embryonic stem cell (ESC) state is transcriptionally controlled by OCT4, SOX2, and NANOG with cofactors, chromatin regulators, noncoding RNAs, and other effectors of signaling pathways. Uncovering components of these regulatory circuits and their interplay provides the knowledge base to deploy ESCs and induced pluripotent stem cells. We recently identified the DNA-repair complex xeroderma pigmentosum C (XPC)-RAD23B-CETN2 as a stem cell coactivator (SCC) required for OCT4/SOX2 transcriptional activation. Here we investigate the role of SCC genome-wide in murine ESCs by mapping regions bound by RAD23B and analyzing transcriptional profiles of SCC-depleted ESCs. We establish OCT4 and SOX2 as the primary transcription factors recruiting SCC to regulatory regions of pluripotency genes and identify the XPC subunit as essential for interaction with the two proteins. The present study reveals new mechanistic and functional aspects of SCC transcriptional activity, and thus underscores the diversified functions of this regulatory complex.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Células Madre Embrionarias/citología , Regulación del Desarrollo de la Expresión Génica , Animales , Sitios de Unión , Diferenciación Celular , Linaje de la Célula , Reparación del ADN , Genoma , Células HEK293 , Humanos , Inmunoglobulina G/química , Lentivirus/metabolismo , Ratones , Ratones Noqueados , Células Madre Pluripotentes/citología , Regiones Promotoras Genéticas , Unión Proteica , Factores de Transcripción SOXB1/metabolismo , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Uterine injury from procedures such as Cesarean sections (C-sections) often have severe consequences on subsequent pregnancies, leading to disorders such as uterine placenta previa, placenta accreta spectrum (PAS), and Cesarean scar pregnancy. With rates of C-section at â¼30% of deliveries in the US and projected to continue to climb, an understanding of the mechanisms by which these pregnancy disorders arise and opportunities for intervention are sorely needed. However, there are currently very few animal models of uterine injury and its subsequent impacts on maternal as well as in utero and postnatal fetal outcomes. Here, we describe a procedure for a novel model of surgically induced uterine injury in the genetically tractable laboratory mouse (Mus musculus). We describe preparatory steps for surgery, the induction of uterine injury itself, and post-surgical recovery. We then provide supporting information regarding downstream dissection of pregnant mice. Lastly, we include additional information regarding estrous cycle staging in order to perform surgeries and dissections at the relevant phase in non-pregnant mice. This procedure for incurring uterine injury in a mouse model presents an important step forward in understanding uterine damage and its associated pregnancy disorders. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Preparation for surgery Basic Protocol 2: Surgery and induction of uterine injury Basic Protocol 3: Mating and dissection of pregnant mice as endpoint analyses Support Protocol: Estrous staging of animals.
Asunto(s)
Modelos Animales de Enfermedad , Útero , Animales , Femenino , Ratones , Útero/cirugía , Útero/patología , Embarazo , Cesárea/efectos adversosRESUMEN
The development of neoplasia involves a complex and continuous interplay between malignantly transformed cells and the tumour microenvironment (TME). Cancer immunotherapies targeting the immune TME have been increasingly validated in clinical trials but response rates vary substantially between tumour histologies and are often transient, idiosyncratic and confounded by resistance. Faithful experimental models of the patient-specific tumour immune microenvironment, capable of recapitulating tumour biology and immunotherapy effects, would greatly improve patient selection, target identification and definition of resistance mechanisms for immuno-oncology therapeutics. In this Review, we discuss currently available and rapidly evolving 3D tumour organoid models that capture important immune features of the TME. We highlight diverse opportunities for organoid-based investigations of tumour immunity, drug development and precision medicine.
Asunto(s)
Neoplasias , Organoides , Microambiente Tumoral , Microambiente Tumoral/inmunología , Humanos , Organoides/inmunología , Organoides/patología , Neoplasias/inmunología , Neoplasias/patología , Neoplasias/terapia , Medicina de Precisión , Inmunoterapia/métodos , Animales , Modelos BiológicosRESUMEN
The RNA-guided CRISPR-associated protein Cas9 is used for genome editing, transcriptional modulation, and live-cell imaging. Cas9-guide RNA complexes recognize and cleave double-stranded DNA sequences on the basis of 20-nucleotide RNA-DNA complementarity, but the mechanism of target searching in mammalian cells is unknown. Here, we use single-particle tracking to visualize diffusion and chromatin binding of Cas9 in living cells. We show that three-dimensional diffusion dominates Cas9 searching in vivo, and off-target binding events are, on average, short-lived (<1 second). Searching is dependent on the local chromatin environment, with less sampling and slower movement within heterochromatin. These results reveal how the bacterial Cas9 protein interrogates mammalian genomes and navigates eukaryotic chromatin structure.