RESUMEN
The B cell response to different pathogens uses tailored effector mechanisms and results in functionally specialized memory B (Bm) cell subsets, including CD21+ resting, CD21-CD27+ activated and CD21-CD27- Bm cells. The interrelatedness between these Bm cell subsets remains unknown. Here we showed that single severe acute respiratory syndrome coronavirus 2-specific Bm cell clones showed plasticity upon antigen rechallenge in previously exposed individuals. CD21- Bm cells were the predominant subsets during acute infection and early after severe acute respiratory syndrome coronavirus 2-specific immunization. At months 6 and 12 post-infection, CD21+ resting Bm cells were the major Bm cell subset in the circulation and were also detected in peripheral lymphoid organs, where they carried tissue residency markers. Tracking of individual B cell clones by B cell receptor sequencing revealed that previously fated Bm cell clones could redifferentiate upon antigen rechallenge into other Bm cell subsets, including CD21-CD27- Bm cells, demonstrating that single Bm cell clones can adopt functionally different trajectories.
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Subgrupos de Linfocitos B , COVID-19 , Humanos , SARS-CoV-2 , Células B de Memoria , Linfocitos BRESUMEN
Mucosal barrier immunity is essential for the maintenance of the commensal microflora and combating invasive bacterial infection. Although immune and epithelial cells are thought to be the canonical orchestrators of this complex equilibrium, here, we show that the enteric nervous system (ENS) plays an essential and non-redundant role in governing the antimicrobial protein (AMP) response. Using confocal microscopy and single-molecule fluorescence in situ mRNA hybridization (smFISH) studies, we observed that intestinal neurons produce the pleiotropic cytokine IL-18. Strikingly, deletion of IL-18 from the enteric neurons alone, but not immune or epithelial cells, rendered mice susceptible to invasive Salmonella typhimurium (S.t.) infection. Mechanistically, unbiased RNA sequencing and single-cell sequencing revealed that enteric neuronal IL-18 is specifically required for homeostatic goblet cell AMP production. Together, we show that neuron-derived IL-18 signaling controls tissue-wide intestinal immunity and has profound consequences on the mucosal barrier and invasive bacterial killing.
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Inmunidad Mucosa/inmunología , Interleucina-18/inmunología , Mucosa Intestinal/inmunología , Animales , Citocinas/inmunología , Sistema Nervioso Entérico/inmunología , Sistema Nervioso Entérico/metabolismo , Células Epiteliales/inmunología , Femenino , Células Caliciformes/inmunología , Interleucina-18/biosíntesis , Mucosa Intestinal/metabolismo , Intestino Delgado/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/inmunología , Ratas , Ratas Sprague-Dawley , Infecciones por Salmonella/inmunología , Salmonella typhimurium/inmunología , Transducción de Señal/inmunologíaRESUMEN
Due to its stimulatory potential for immunomodulatory CD4+ regulatory T (Treg) cells, low-dose interleukin-2 (IL-2) immunotherapy has gained considerable attention for the treatment of autoimmune diseases. In this investigator-initiated single-arm non-placebo-controlled phase-2 clinical trial of low-dose IL-2 immunotherapy in systemic lupus erythematosus (SLE) patients, we generated a comprehensive atlas of in vivo human immune responses to low-dose IL-2. We performed an in-depth study of circulating and cutaneous immune cells by imaging mass cytometry, high-parameter flow cytometry, transcriptomics, and targeted serum proteomics. Low-dose IL-2 stimulated various circulating immune cells, including Treg cells with a skin-homing phenotype that appeared in the skin of SLE patients in close interaction with endothelial cells. Analysis of surface proteins and transcriptomes revealed different IL-2-driven Treg cell activation programs, including gut-homing CD38+, skin-homing HLA-DR+, and highly proliferative inflammation-homing CD38+ HLA-DR+ Treg cells. Collectively, these data define the distinct human Treg cell subsets that are responsive to IL-2 immunotherapy.
RESUMEN
The intestinal epithelium is a highly structured tissue composed of repeating crypt-villus units. Enterocytes perform the diverse tasks of absorbing a wide range of nutrients while protecting the body from the harsh bacterium-rich environment. It is unknown whether these tasks are spatially zonated along the villus axis. Here, we extracted a large panel of landmark genes characterized by transcriptomics of laser capture microdissected villus segments and utilized it for single-cell spatial reconstruction, uncovering broad zonation of enterocyte function along the villus. We found that enterocytes at villus bottoms express an anti-bacterial gene program in a microbiome-dependent manner. They next shift to sequential expression of carbohydrates, peptides, and fat absorption machineries in distinct villus compartments. Finally, they induce a Cd73 immune-modulatory program at the villus tips. Our approach can be used to uncover zonation patterns in other organs when prior knowledge of landmark genes is lacking.
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Enterocitos/metabolismo , Transcriptoma , Animales , Diferenciación Celular , Movimiento Celular , Enterocitos/citología , Enterocitos/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Análisis de la Célula IndividualRESUMEN
Empiric probiotics are commonly consumed by healthy individuals as means of life quality improvement and disease prevention. However, evidence of probiotic gut mucosal colonization efficacy remains sparse and controversial. We metagenomically characterized the murine and human mucosal-associated gastrointestinal microbiome and found it to only partially correlate with stool microbiome. A sequential invasive multi-omics measurement at baseline and during consumption of an 11-strain probiotic combination or placebo demonstrated that probiotics remain viable upon gastrointestinal passage. In colonized, but not germ-free mice, probiotics encountered a marked mucosal colonization resistance. In contrast, humans featured person-, region- and strain-specific mucosal colonization patterns, hallmarked by predictive baseline host and microbiome features, but indistinguishable by probiotics presence in stool. Consequently, probiotics induced a transient, individualized impact on mucosal community structure and gut transcriptome. Collectively, empiric probiotics supplementation may be limited in universally and persistently impacting the gut mucosa, meriting development of new personalized probiotic approaches.
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Microbioma Gastrointestinal , Probióticos/administración & dosificación , Adolescente , Adulto , Anciano , Animales , Bacterias/genética , Bacterias/aislamiento & purificación , Heces/microbiología , Femenino , Mucosa Gástrica/microbiología , Humanos , Mucosa Intestinal/microbiología , Masculino , Metagenómica , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Efecto Placebo , Análisis de Componente Principal , ARN Ribosómico 16S/genética , ARN Ribosómico 16S/metabolismo , Transcriptoma , Adulto JovenRESUMEN
Probiotics are widely prescribed for prevention of antibiotics-associated dysbiosis and related adverse effects. However, probiotic impact on post-antibiotic reconstitution of the gut mucosal host-microbiome niche remains elusive. We invasively examined the effects of multi-strain probiotics or autologous fecal microbiome transplantation (aFMT) on post-antibiotic reconstitution of the murine and human mucosal microbiome niche. Contrary to homeostasis, antibiotic perturbation enhanced probiotics colonization in the human mucosa but only mildly improved colonization in mice. Compared to spontaneous post-antibiotic recovery, probiotics induced a markedly delayed and persistently incomplete indigenous stool/mucosal microbiome reconstitution and host transcriptome recovery toward homeostatic configuration, while aFMT induced a rapid and near-complete recovery within days of administration. In vitro, Lactobacillus-secreted soluble factors contributed to probiotics-induced microbiome inhibition. Collectively, potential post-antibiotic probiotic benefits may be offset by a compromised gut mucosal recovery, highlighting a need of developing aFMT or personalized probiotic approaches achieving mucosal protection without compromising microbiome recolonization in the antibiotics-perturbed host.
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Antibacterianos/farmacología , Microbioma Gastrointestinal/efectos de los fármacos , Probióticos/administración & dosificación , Adolescente , Adulto , Anciano , Animales , Trasplante de Microbiota Fecal , Heces/microbiología , Femenino , Humanos , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/microbiología , Lactobacillus/efectos de los fármacos , Lactobacillus/genética , Lactobacillus/aislamiento & purificación , Lactococcus/genética , Lactococcus/aislamiento & purificación , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , ARN Ribosómico 16S/análisis , ARN Ribosómico 16S/genética , ARN Ribosómico 16S/metabolismo , Adulto JovenRESUMEN
The tumour evolution model posits that malignant transformation is preceded by randomly distributed driver mutations in cancer genes, which cause clonal expansions in phenotypically normal tissues. Although clonal expansions can remodel entire tissues1-3, the mechanisms that result in only a small number of clones transforming into malignant tumours remain unknown. Here we develop an in vivo single-cell CRISPR strategy to systematically investigate tissue-wide clonal dynamics of the 150 most frequently mutated squamous cell carcinoma genes. We couple ultrasound-guided in utero lentiviral microinjections, single-cell RNA sequencing and guide capture to longitudinally monitor clonal expansions and document their underlying gene programmes at single-cell transcriptomic resolution. We uncover a tumour necrosis factor (TNF) signalling module, which is dependent on TNF receptor 1 and involving macrophages, that acts as a generalizable driver of clonal expansions in epithelial tissues. Conversely, during tumorigenesis, the TNF signalling module is downregulated. Instead, we identify a subpopulation of invasive cancer cells that switch to an autocrine TNF gene programme associated with epithelial-mesenchymal transition. Finally, we provide in vivo evidence that the autocrine TNF gene programme is sufficient to mediate invasive properties and show that the TNF signature correlates with shorter overall survival of patients with squamous cell carcinoma. Collectively, our study demonstrates the power of applying in vivo single-cell CRISPR screening to mammalian tissues, unveils distinct TNF programmes in tumour evolution and highlights the importance of understanding the relationship between clonal expansions in epithelia and tumorigenesis.
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Sistemas CRISPR-Cas , Carcinoma de Células Escamosas , Transformación Celular Neoplásica , Evolución Clonal , Células Clonales , Análisis de la Célula Individual , Factores de Necrosis Tumoral , Animales , Femenino , Humanos , Masculino , Ratones , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Evolución Clonal/genética , Células Clonales/citología , Células Clonales/metabolismo , Células Clonales/patología , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Sistemas CRISPR-Cas/genética , Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión Génica , Macrófagos/metabolismo , Mutación , Invasividad Neoplásica/genética , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Transducción de Señal/genética , Análisis de la Célula Individual/métodos , Transcriptoma/genética , Factores de Necrosis Tumoral/genética , Factores de Necrosis Tumoral/metabolismo , Comunicación Autocrina , Análisis de SupervivenciaRESUMEN
It is estimated that only 0.02% of disseminated tumour cells are able to seed overt metastases1. While this suggests the presence of environmental constraints to metastatic seeding, the landscape of host factors controlling this process remains largely unclear. Here, combining transposon technology2 and fluorescence niche labelling3, we developed an in vivo CRISPR activation screen to systematically investigate the interactions between hepatocytes and metastatic cells. We identify plexin B2 as a critical host-derived regulator of liver colonization in colorectal and pancreatic cancer and melanoma syngeneic mouse models. We dissect a mechanism through which plexin B2 interacts with class IV semaphorins on tumour cells, leading to KLF4 upregulation and thereby promoting the acquisition of epithelial traits. Our results highlight the essential role of signals from the liver parenchyma for the seeding of disseminated tumour cells before the establishment of a growth-promoting niche. Our findings further suggest that epithelialization is required for the adaptation of CRC metastases to their new tissue environment. Blocking the plexin-B2-semaphorin axis abolishes metastatic colonization of the liver and therefore represents a therapeutic strategy for the prevention of hepatic metastases. Finally, our screening approach, which evaluates host-derived extrinsic signals rather than tumour-intrinsic factors for their ability to promote metastatic seeding, is broadly applicable and lays a framework for the screening of environmental constraints to metastasis in other organs and cancer types.
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Sistemas CRISPR-Cas , Hepatocitos , Neoplasias Hepáticas , Hígado , Metástasis de la Neoplasia , Proteínas del Tejido Nervioso , Animales , Femenino , Humanos , Masculino , Ratones , Línea Celular Tumoral , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/metabolismo , Sistemas CRISPR-Cas/genética , Modelos Animales de Enfermedad , Elementos Transponibles de ADN , Fluorescencia , Hepatocitos/metabolismo , Hepatocitos/citología , Hepatocitos/patología , Factor 4 Similar a Kruppel/metabolismo , Hígado/citología , Hígado/metabolismo , Hígado/patología , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/prevención & control , Neoplasias Hepáticas/secundario , Melanoma/metabolismo , Melanoma/patología , Metástasis de la Neoplasia/tratamiento farmacológico , Metástasis de la Neoplasia/patología , Metástasis de la Neoplasia/prevención & control , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Semaforinas/antagonistas & inhibidores , Semaforinas/metabolismoRESUMEN
In the past decade, single-cell transcriptomics has helped to uncover new cell types and states and led to the construction of a cellular compendium of health and disease. Despite this progress, some difficult-to-sequence cells remain absent from tissue atlases. Eosinophils-elusive granulocytes that are implicated in a plethora of human pathologies1-5-are among these uncharted cell types. The heterogeneity of eosinophils and the gene programs that underpin their pleiotropic functions remain poorly understood. Here we provide a comprehensive single-cell transcriptomic profiling of mouse eosinophils. We identify an active and a basal population of intestinal eosinophils, which differ in their transcriptome, surface proteome and spatial localization. By means of a genome-wide CRISPR inhibition screen and functional assays, we reveal a mechanism by which interleukin-33 (IL-33) and interferon-γ (IFNγ) induce the accumulation of active eosinophils in the inflamed colon. Active eosinophils are endowed with bactericidal and T cell regulatory activity, and express the co-stimulatory molecules CD80 and PD-L1. Notably, active eosinophils are enriched in the lamina propria of a small cohort of patients with inflammatory bowel disease, and are closely associated with CD4+ T cells. Our findings provide insights into the biology of eosinophils and highlight the crucial contribution of this cell type to intestinal homeostasis, immune regulation and host defence. Furthermore, we lay a framework for the characterization of eosinophils in human gastrointestinal diseases.
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Colitis , Eosinófilos , Inmunidad , Intestinos , Animales , Humanos , Ratones , Colitis/inmunología , Colitis/patología , Eosinófilos/clasificación , Eosinófilos/citología , Eosinófilos/inmunología , Eosinófilos/metabolismo , Enfermedades Inflamatorias del Intestino/inmunología , Análisis de Expresión Génica de una Sola Célula , Transcriptoma , Proteoma , Interleucina-33 , Interferón gamma , Linfocitos T , Antígeno B7-1/metabolismo , Intestinos/inmunología , Intestinos/patologíaRESUMEN
Immunological memory is a hallmark of adaptive immunity and facilitates an accelerated and enhanced immune response upon re-infection with the same pathogen1,2. Since the outbreak of the ongoing COVID-19 pandemic, a key question has focused on which SARS-CoV-2-specific T cells stimulated during acute infection give rise to long-lived memory T cells3. Here, using spectral flow cytometry combined with cellular indexing of transcriptomes and T cell receptor sequencing, we longitudinally characterized individual SARS-CoV-2-specific CD8+ T cells of patients with COVID-19 from acute infection to 1 year into recovery and found a distinct signature identifying long-lived memory CD8+ T cells. SARS-CoV-2-specific memory CD8+ T cells persisting 1 year after acute infection express CD45RA, IL-7 receptor-α and T cell factor 1, but they maintain low expression of CCR7, thus resembling CD45RA+ effector memory T cells. Tracking individual clones of SARS-CoV-2-specific CD8+ T cells, we reveal that an interferon signature marks clones that give rise to long-lived cells, whereas prolonged proliferation and mechanistic target of rapamycin signalling are associated with clonal disappearance from the blood. Collectively, we describe a transcriptional signature that marks long-lived, circulating human memory CD8+ T cells following an acute viral infection.
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Antígenos Virales/inmunología , Biomarcadores/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , COVID-19/inmunología , Células T de Memoria/inmunología , Células T de Memoria/metabolismo , SARS-CoV-2/inmunología , Enfermedad Aguda , COVID-19/virología , Proliferación Celular , Células Clonales/citología , Células Clonales/inmunología , Humanos , Interferones/inmunología , Subunidad alfa del Receptor de Interleucina-7/metabolismo , Antígenos Comunes de Leucocito/metabolismo , Estudios Longitudinales , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores CCR7/metabolismo , Factor 1 de Transcripción de Linfocitos T/metabolismo , Factores de Tiempo , TranscriptomaRESUMEN
Asymmetric subcellular mRNA localization allows spatial regulation of gene expression and functional compartmentalization. In neurons, localization of specific mRNAs to neurites is essential for cellular functioning. However, it is largely unknown how transcript sorting works in a sequence-specific manner. Here, we combined subcellular transcriptomics and massively parallel reporter assays and tested â¼50 000 sequences for their ability to localize to neurites. Mapping the localization potential of >300 genes revealed two ways neurite targeting can be achieved: focused localization motifs and broadly encoded localization potential. We characterized the interplay between RNA stability and localization and identified motifs able to bias localization towards neurite or soma as well as the trans-acting factors required for their action. Based on our data, we devised machine learning models that were able to predict the localization behavior of novel reporter sequences. Testing this predictor on native mRNA sequencing data showed good agreement between predicted and observed localization potential, suggesting that the rules uncovered by our MPRA also apply to the localization of native full-length transcripts.
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Neuronas , Estabilidad del ARN , Neuritas/metabolismo , Neuronas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transactivadores/metabolismoRESUMEN
Hundreds of RNAs are enriched in the projections of neuronal cells. For the vast majority of them, though, the sequence elements that regulate their localization are unknown. To identify RNA elements capable of directing transcripts to neurites, we deployed a massively parallel reporter assay that tested the localization regulatory ability of thousands of sequence fragments drawn from endogenous mouse 3' UTRs. We identified peaks of regulatory activity within several 3' UTRs and found that sequences derived from these peaks were both necessary and sufficient for RNA localization to neurites in mouse and human neuronal cells. The localization elements were enriched in adenosine and guanosine residues. They were at least tens to hundreds of nucleotides long as shortening of two identified elements led to significantly reduced activity. Using RNA affinity purification and mass spectrometry, we found that the RNA-binding protein Unk was associated with the localization elements. Depletion of Unk in cells reduced the ability of the elements to drive RNAs to neurites, indicating a functional requirement for Unk in their trafficking. These results provide a framework for the unbiased, high-throughput identification of RNA elements and mechanisms that govern transcript localization in neurons.
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Neuronas , Secuencias Reguladoras de Ácido Ribonucleico , Regiones no Traducidas 3'/genética , Animales , Humanos , Ratones , Neuronas/metabolismo , Nucleótidos/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Análisis de Secuencia de ARNRESUMEN
The liver is a major metastatic target organ, and little is known about the role of immunity in controlling hepatic metastases. Here, we discovered that the concerted and nonredundant action of two innate lymphocyte subpopulations, conventional natural killer cells (cNKs) and tissue-resident type I innate lymphoid cells (trILC1s), is essential for antimetastatic defense. Using different preclinical models for liver metastasis, we found that trILC1 controls metastatic seeding, whereas cNKs restrain outgrowth. Whereas the killing capacity of trILC1s was not affected by the metastatic microenvironment, the phenotype and function of cNK cells were affected in a cancer type-specific fashion. Thus, individual cancer cell lines orchestrate the emergence of unique cNK subsets, which respond differently to tumor-derived factors. Our findings will contribute to the development of therapies for liver metastasis involving hepatic innate cells.
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Inmunidad Innata/inmunología , Células Asesinas Naturales/inmunología , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/secundario , Linfocitos/inmunología , Animales , Femenino , Regulación Neoplásica de la Expresión Génica , Integrina alfa1/metabolismo , Interleucina-15/metabolismo , Hígado/inmunología , Hígado/patología , Neoplasias Hepáticas/genética , Ratones , Ratones Endogámicos C57BL , RNA-Seq , Análisis de la Célula Individual , Transcriptoma/genética , Factor de Crecimiento Transformador beta/metabolismo , Microambiente Tumoral/genética , Microambiente Tumoral/inmunologíaRESUMEN
OBJECTIVE: Inflammatory bowel disease (IBD) is a multifactorial condition driven by genetic and environmental risk factors. A genetic variation in the protein tyrosine phosphatase non-receptor type 22 (PTPN22) gene has been associated with autoimmune disorders while protecting from the IBD subtype Crohn's disease. Mice expressing the murine orthologous PTPN22-R619W variant are protected from intestinal inflammation in the model of acute dextran sodium sulfate (DSS)-induced colitis. We previously identified food-grade titanium dioxide (TiO2, E171) as a neglected IBD risk factor. Here, we investigate the interplay of the PTPN22 variant and TiO2-mediated effects during IBD pathogenesis. DESIGN: Acute DSS colitis was induced in wild-type and PTPN22 variant mice (PTPN22-R619W) and animals were treated with TiO2 nanoparticles during colitis induction. Disease-triggering mechanisms were investigated using bulk and single-cell RNA sequencing. RESULTS: In mice, administration of TiO2 nanoparticles abrogated the protective effect of the variant, rendering PTPN22-R619W mice susceptible to DSS colitis. In early disease, cytotoxic CD8+ T-cells were found to be reduced in the lamina propria of PTPN22-R619W mice, an effect reversed by TiO2 administration. Normalisation of T-cell populations correlated with increased Ifng expression and, at a later stage of disease, the promoted prevalence of proinflammatory macrophages that triggered severe intestinal inflammation. CONCLUSION: Our findings indicate that the consumption of TiO2 nanoparticles might have adverse effects on the gastrointestinal health of individuals carrying the PTPN22 variant. This demonstrates that environmental factors interact with genetic risk variants and can reverse a protective mechanism into a disease-promoting effect.
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Colitis , Enfermedad de Crohn , Enfermedades Inflamatorias del Intestino , Nanopartículas , Ratones , Animales , Enfermedad de Crohn/genética , Enfermedad de Crohn/complicaciones , Linfocitos T CD8-positivos/metabolismo , Colitis/inducido químicamente , Colitis/genética , Colitis/prevención & control , Inflamación/complicaciones , Sulfato de Dextran , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , Proteína Tirosina Fosfatasa no Receptora Tipo 22/genéticaRESUMEN
The molecular mechanisms of angiogenesis have been intensely studied, but many genes that control endothelial behavior and fate still need to be described. Here, we characterize the role of Apold1 (Apolipoprotein L domain containing 1) in angiogenesis in vivo and in vitro. Single-cell analyses reveal that - across tissues - the expression of Apold1 is restricted to the vasculature and that Apold1 expression in endothelial cells (ECs) is highly sensitive to environmental factors. Using Apold1-/- mice, we find that Apold1 is dispensable for development and does not affect postnatal retinal angiogenesis nor alters the vascular network in adult brain and muscle. However, when exposed to ischemic conditions following photothrombotic stroke as well as femoral artery ligation, Apold1-/- mice display dramatic impairments in recovery and revascularization. We also find that human tumor endothelial cells express strikingly higher levels of Apold1 and that Apold1 deletion in mice stunts the growth of subcutaneous B16 melanoma tumors, which have smaller and poorly perfused vessels. Mechanistically, Apold1 is activated in ECs upon growth factor stimulation as well as in hypoxia, and Apold1 intrinsically controls EC proliferation but not migration. Our data demonstrate that Apold1 is a key regulator of angiogenesis in pathological settings, whereas it does not affect developmental angiogenesis, thus making it a promising candidate for clinical investigation.
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Células Endoteliales , Neovascularización Fisiológica , Animales , Humanos , Ratones , Células Endoteliales/metabolismo , Miembro Posterior/irrigación sanguínea , Hipoxia/metabolismo , Isquemia/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Neovascularización Fisiológica/genética , Proteínas Inmediatas-Precoces/metabolismoRESUMEN
The mammalian liver consists of hexagon-shaped lobules that are radially polarized by blood flow and morphogens. Key liver genes have been shown to be differentially expressed along the lobule axis, a phenomenon termed zonation, but a detailed genome-wide reconstruction of this spatial division of labour has not been achieved. Here we measure the entire transcriptome of thousands of mouse liver cells and infer their lobule coordinates on the basis of a panel of zonated landmark genes, characterized with single-molecule fluorescence in situ hybridization. Using this approach, we obtain the zonation profiles of all liver genes with high spatial resolution. We find that around 50% of liver genes are significantly zonated and uncover abundant non-monotonic profiles that peak at the mid-lobule layers. These include a spatial order of bile acid biosynthesis enzymes that matches their position in the enzymatic cascade. Our approach can facilitate the reconstruction of similar spatial genomic blueprints for other mammalian organs.
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Perfilación de la Expresión Génica , Hepatocitos/metabolismo , Hígado/citología , Hígado/fisiología , Análisis de la Célula Individual , Animales , Ácidos y Sales Biliares/biosíntesis , Genoma/genética , Hibridación Fluorescente in Situ , Hígado/enzimología , Masculino , Ratones , Ratones Endogámicos C57BL , Análisis de Secuencia de ARN , Imagen Individual de Molécula , Transcriptoma/genéticaRESUMEN
BACKGROUND & AIMS: Intestinal epithelial homeostasis depends on a tightly regulated balance between intestinal epithelial cell (IEC) death and proliferation. While the disruption of several IEC death regulating factors result in intestinal inflammation, the loss of the anti-apoptotic BCL2 family members BCL2 and BCL2L1 has no effect on intestinal homeostasis in mice. We investigated the functions of the antiapoptotic protein MCL1, another member of the BCL2 family, in intestinal homeostasis in mice. METHODS: We generated mice with IEC-specific disruption of Mcl1 (Mcl1ΔIEC mice) or tamoxifen-inducible IEC-specific disruption of Mcl1 (i-Mcl1ΔIEC mice); these mice and mice with full-length Mcl1 (controls) were raised under normal or germ-free conditions. Mice were analyzed by endoscopy and for intestinal epithelial barrier permeability. Intestinal tissues were analyzed by histology, in situ hybridization, proliferation assays, and immunoblots. Levels of calprotectin, a marker of intestinal inflammation, were measured in intestinal tissues and feces. RESULTS: Mcl1ΔIEC mice spontaneously developed apoptotic enterocolopathy, characterized by increased IEC apoptosis, hyperproliferative crypts, epithelial barrier dysfunction, and chronic inflammation. Loss of MCL1 retained intestinal crypts in a hyperproliferated state and prevented the differentiation of intestinal stem cells. Proliferation of intestinal stem cells in MCL1-deficient mice required WNT signaling and was associated with DNA damage accumulation. By 1 year of age, Mcl1ΔIEC mice developed intestinal tumors with morphologic and genetic features of human adenomas and carcinomas. Germ-free housing of Mcl1ΔIEC mice reduced markers of microbiota-induced intestinal inflammation but not tumor development. CONCLUSION: The antiapoptotic protein MCL1, a member of the BCL2 family, is required for maintenance of intestinal homeostasis and prevention of carcinogenesis in mice. Loss of MCL1 results in development of intestinal carcinomas, even under germ-free conditions, and therefore does not involve microbe-induced chronic inflammation. Mcl1ΔIEC mice might be used to study apoptotic enterocolopathy and inflammatory bowel diseases.
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Carcinoma/patología , Mucosa Intestinal/patología , Neoplasias Intestinales/patología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Animales , Apoptosis/genética , Apoptosis/inmunología , Carcinogénesis/genética , Carcinogénesis/inmunología , Carcinogénesis/patología , Carcinoma/diagnóstico , Carcinoma/genética , Modelos Animales de Enfermedad , Endoscopía , Células Epiteliales/patología , Humanos , Enfermedades Inflamatorias del Intestino/inmunología , Enfermedades Inflamatorias del Intestino/patología , Mucosa Intestinal/diagnóstico por imagen , Neoplasias Intestinales/diagnóstico , Neoplasias Intestinales/genética , Ratones , Ratones Transgénicos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genéticaRESUMEN
Malignant cell growth is fueled by interactions between tumor cells and the stromal cells composing the tumor microenvironment. The human liver is a major site of tumors and metastases, but molecular identities and intercellular interactions of different cell types have not been resolved in these pathologies. Here, we apply single cell RNA-sequencing and spatial analysis of malignant and adjacent non-malignant liver tissues from five patients with cholangiocarcinoma or liver metastases. We find that stromal cells exhibit recurring, patient-independent expression programs, and reconstruct a ligand-receptor map that highlights recurring tumor-stroma interactions. By combining transcriptomics of laser-capture microdissected regions, we reconstruct a zonation atlas of hepatocytes in the non-malignant sites and characterize the spatial distribution of each cell type across the tumor microenvironment. Our analysis provides a resource for understanding human liver malignancies and may expose potential points of interventions.