RESUMEN
Gastrointestinal (GI) discomfort is a hallmark of most gut disorders and represents an important component of chronic visceral pain1. For the growing population afflicted by irritable bowel syndrome, GI hypersensitivity and pain persist long after tissue injury has resolved2. Irritable bowel syndrome also exhibits a strong sex bias, afflicting women three times more than men1. Here, we focus on enterochromaffin (EC) cells, which are rare excitable, serotonergic neuroendocrine cells in the gut epithelium3-5. EC cells detect and transduce noxious stimuli to nearby mucosal nerve endings3,6 but involvement of this signalling pathway in visceral pain and attendant sex differences has not been assessed. By enhancing or suppressing EC cell function in vivo, we show that these cells are sufficient to elicit hypersensitivity to gut distension and necessary for the sensitizing actions of isovalerate, a bacterial short-chain fatty acid associated with GI inflammation7,8. Remarkably, prolonged EC cell activation produced persistent visceral hypersensitivity, even in the absence of an instigating inflammatory episode. Furthermore, perturbing EC cell activity promoted anxiety-like behaviours which normalized after blockade of serotonergic signalling. Sex differences were noted across a range of paradigms, indicating that the EC cell-mucosal afferent circuit is tonically engaged in females. Our findings validate a critical role for EC cell-mucosal afferent signalling in acute and persistent GI pain, in addition to highlighting genetic models for studying visceral hypersensitivity and the sex bias of gut pain.
Asunto(s)
Ansiedad , Células Enterocromafines , Dolor Visceral , Femenino , Humanos , Masculino , Ansiedad/complicaciones , Ansiedad/fisiopatología , Sistema Digestivo/inervación , Sistema Digestivo/fisiopatología , Células Enterocromafines/metabolismo , Síndrome del Colon Irritable/complicaciones , Síndrome del Colon Irritable/fisiopatología , Síndrome del Colon Irritable/psicología , Caracteres Sexuales , Dolor Visceral/complicaciones , Dolor Visceral/fisiopatología , Dolor Visceral/psicología , Inflamación/complicaciones , Inflamación/fisiopatología , Serotonina/metabolismo , Reproducibilidad de los ResultadosRESUMEN
Disease tolerance, the capacity of tissues to withstand damage caused by a stimulus without a decline in host fitness, varies across tissues, environmental conditions, and physiologic states. While disease tolerance is a known strategy of host defense, its role in noninfectious diseases has been understudied. Here, we provide evidence that a thermogenic fat-epithelial cell axis regulates intestinal disease tolerance during experimental colitis. We find that intestinal disease tolerance is a metabolically expensive trait, whose expression is restricted to thermoneutral mice and is not transferable by the microbiota. Instead, disease tolerance is dependent on the adrenergic state of thermogenic adipocytes, which indirectly regulate tolerogenic responses in intestinal epithelial cells. Our work has identified an unexpected mechanism that controls intestinal disease tolerance with implications for colitogenic diseases.
Asunto(s)
Tejido Adiposo Pardo/metabolismo , Colitis/inmunología , Neoplasias del Colon/inmunología , Resistencia a la Enfermedad , Infecciones por Enterobacteriaceae/inmunología , Adipocitos/metabolismo , Tejido Adiposo Pardo/citología , Animales , Azoximetano/administración & dosificación , Comunicación Celular , Citrobacter rodentium/patogenicidad , Colitis/inducido químicamente , Colitis/microbiología , Colitis/patología , Neoplasias del Colon/inducido químicamente , Neoplasias del Colon/patología , Sulfato de Dextran/toxicidad , Infecciones por Enterobacteriaceae/inducido químicamente , Infecciones por Enterobacteriaceae/microbiología , Infecciones por Enterobacteriaceae/patología , Células Epiteliales/metabolismo , Femenino , Humanos , Mucosa Intestinal/citología , Mucosa Intestinal/inmunología , Mucosa Intestinal/patología , Masculino , Ratones , Neoplasias Experimentales/inducido químicamente , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/patología , Termogénesis/inmunologíaRESUMEN
Central estrogen signaling coordinates energy expenditure, reproduction, and in concert with peripheral estrogen impacts skeletal homeostasis in females. Here, we ablate estrogen receptor alpha (ERα) in the medial basal hypothalamus and find a robust bone phenotype only in female mice that results in exceptionally strong trabecular and cortical bones, whose density surpasses other reported mouse models. Stereotaxic guided deletion of ERα in the arcuate nucleus increases bone mass in intact and ovariectomized females, confirming the central role of estrogen signaling in this sex-dependent bone phenotype. Loss of ERα in kisspeptin (Kiss1)-expressing cells is sufficient to recapitulate the bone phenotype, identifying Kiss1 neurons as a critical node in this powerful neuroskeletal circuit. We propose that this newly-identified female brain-to-bone pathway exists as a homeostatic regulator diverting calcium and energy stores from bone building when energetic demands are high. Our work reveals a previously unknown target for treatment of age-related bone disease.
Asunto(s)
Núcleo Arqueado del Hipotálamo/fisiología , Densidad Ósea , Receptor alfa de Estrógeno/fisiología , Kisspeptinas/metabolismo , Animales , Metabolismo Energético , Femenino , Homeostasis , Masculino , Ratones Transgénicos , Osteogénesis , Fenotipo , Caracteres SexualesRESUMEN
The enteric nervous system (ENS) is a large, complex division of the peripheral nervous system that regulates many digestive, immune, hormonal, and metabolic functions. Recent advances have elucidated the dynamic nature of the mature ENS, as well as the complex, bidirectional interactions among enteric neurons, glia, and the many other cell types that are important for mediating gut behaviors. Here, we provide an overview of ENS development and maintenance, and focus on the latest insights gained from the use of novel model systems and live-imaging techniques. We discuss major advances in the understanding of enteric glia, and the functional interactions among enteric neurons, glia, and enteroendocrine cells, a large class of sensory epithelial cells. We conclude by highlighting recent work on muscularis macrophages, a group of immune cells that closely interact with the ENS in the gut wall, and the importance of neurological-immune system communication in digestive health and disease.
Asunto(s)
Encéfalo/metabolismo , Sistema Nervioso Entérico/metabolismo , Enfermedades Gastrointestinales/metabolismo , Tracto Gastrointestinal/metabolismo , Animales , Encéfalo/inmunología , Encéfalo/patología , Sistema Nervioso Entérico/inmunología , Sistema Nervioso Entérico/patología , Enfermedades Gastrointestinales/inmunología , Enfermedades Gastrointestinales/patología , Tracto Gastrointestinal/inmunología , Tracto Gastrointestinal/patología , Humanos , NeurobiologíaRESUMEN
Epithelial dysfunction and crypt destruction are defining features of inflammatory bowel disease (IBD). However, current IBD therapies targeting epithelial dysfunction are lacking. The nuclear receptor LRH-1 (NR5A2) is expressed in intestinal epithelium and thought to contribute to epithelial renewal. Here we show that LRH-1 maintains intestinal epithelial health and protects against inflammatory damage. Knocking out LRH-1 in murine intestinal organoids reduces Notch signaling, increases crypt cell death, distorts the cellular composition of the epithelium, and weakens the epithelial barrier. Human LRH-1 (hLRH-1) rescues epithelial integrity and when overexpressed, mitigates inflammatory damage in murine and human intestinal organoids, including those derived from IBD patients. Finally, hLRH-1 greatly reduces disease severity in T-cell-mediated murine colitis. Together with the failure of a ligand-incompetent hLRH-1 mutant to protect against TNFα-damage, these findings provide compelling evidence that hLRH-1 mediates epithelial homeostasis and is an attractive target for intestinal disease.
Asunto(s)
Epitelio/patología , Homeostasis , Enfermedades Inflamatorias del Intestino/metabolismo , Enfermedades Inflamatorias del Intestino/patología , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Diferenciación Celular , Supervivencia Celular , Colitis/metabolismo , Colitis/patología , Modelos Animales de Enfermedad , Humanos , Ratones , Organoides/metabolismo , Receptores Notch/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Dietary, microbial, and inflammatory factors modulate the gut-brain axis and influence physiological processes ranging from metabolism to cognition. The gut epithelium is a principal site for detecting such agents, but precisely how it communicates with neural elements is poorly understood. Serotonergic enterochromaffin (EC) cells are proposed to fulfill this role by acting as chemosensors, but understanding how these rare and unique cell types transduce chemosensory information to the nervous system has been hampered by their paucity and inaccessibility to single-cell measurements. Here, we circumvent this limitation by exploiting cultured intestinal organoids together with single-cell measurements to elucidate intrinsic biophysical, pharmacological, and genetic properties of EC cells. We show that EC cells express specific chemosensory receptors, are electrically excitable, and modulate serotonin-sensitive primary afferent nerve fibers via synaptic connections, enabling them to detect and transduce environmental, metabolic, and homeostatic information from the gut directly to the nervous system.
Asunto(s)
Células Quimiorreceptoras/metabolismo , Células Enterocromafines/metabolismo , Tracto Gastrointestinal/citología , Vías Nerviosas , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Canales de Calcio/metabolismo , Catecolaminas/metabolismo , Perfilación de la Expresión Génica , Humanos , Síndrome del Colon Irritable/patología , Ratones , Fibras Nerviosas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptores Odorantes/metabolismo , Receptores de Serotonina 5-HT3/metabolismo , Serotonina/metabolismo , Transducción de Señal , Sinapsis/metabolismo , Canal Catiónico TRPA1 , Canales de Potencial de Receptor Transitorio/metabolismoRESUMEN
The development of sustainable intestinal organoid cell culture has emerged as a new modality for the study of intestinal function and cellular processes. Organoid culture is providing a new testbed for therapeutic research and development. Intestinal organoids, self-renewing 3-dimensional structures comprised intestinal stem cells and their differentiated epithelial progeny allow for more facile and robust exploration of cellular activity, cell organization and structure, genetic manipulation, and vastly more physiologic modeling of intestinal response to stimuli as compared to traditional 2-dimensional cell line cultures. Intestinal organoids are affecting a wide variety of research into gastrointestinal pathology. The purpose of this review is to discuss the current state-of-the-art and future effect of research using enteroids and colonoids (organoids grown from the small and large intestines, respectively).
Asunto(s)
Investigación Biomédica/métodos , Enfermedades Intestinales , Organoides , Animales , Fibrosis Quística/genética , Fibrosis Quística/microbiología , Fibrosis Quística/fisiopatología , Fibrosis Quística/terapia , Humanos , Infecciones/genética , Infecciones/microbiología , Infecciones/fisiopatología , Infecciones/terapia , Enfermedades Intestinales/genética , Enfermedades Intestinales/microbiología , Enfermedades Intestinales/fisiopatología , Enfermedades Intestinales/terapia , Neoplasias Intestinales/genética , Neoplasias Intestinales/microbiología , Neoplasias Intestinales/fisiopatología , Neoplasias Intestinales/terapia , Modelos Biológicos , Organoides/microbiología , Organoides/fisiología , Organoides/fisiopatología , Medicina de Precisión/métodos , Ingeniería de Tejidos/métodosRESUMEN
Colorectal cancers (CRCs) account for nearly 10% of all cancer deaths in industrialized countries. Recent evidence points to a central role for the nuclear receptor liver receptor homolog-1 (LRH-1) in intestinal tumorigenesis. Interaction of LRH-1 with the Wnt/ß-catenin pathway, highly active in a critical subpopulation of CRC cells, underscores the importance of elucidating LRH-1's role in this disease. Reduction of LRH-1 diminishes tumor burden in murine models of CRC; however, it is not known whether LRH-1 is required for tumorigenesis, for proliferation, or for both. In this work, we address this question through shRNA-mediated silencing of LRH-1 in established CRC cell lines. LRH-1 mRNA knockdown results in significantly impaired proliferation in a cell line highly expressing the receptor and more modest impairment in a cell line with moderate LRH-1 expression. Cell-cycle analysis shows prolongation of G0/G1 with LRH-1 silencing, consistent with LRH-1 cell-cycle influences in other tissues. Cluster analysis of microarray gene expression demonstrates significant genome wide alterations with major effects in cell-cycle regulation, signal transduction, bile acid and cholesterol metabolism, and control of apoptosis. This study demonstrates a critical proproliferative role for LRH-1 in established colon cancer cell lines. LRH-1 exerts its effects via multiple signaling networks. Our results suggest that selected CRC patients could benefit from LRH-1 inhibitors.
Asunto(s)
Neoplasias del Colon/genética , Neoplasias del Colon/patología , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Receptores Citoplasmáticos y Nucleares/genética , Células CACO-2 , Ciclo Celular/genética , Proliferación Celular , Técnicas de Silenciamiento del Gen , Células HT29 , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Receptores Citoplasmáticos y Nucleares/metabolismo , Reproducibilidad de los ResultadosRESUMEN
The DSX (Doublesex) transcription factor regulates somatic sexual differentiation in Drosophila. Female and male isoforms (DSX F and DSX M) are formed due to sex-specific RNA splicing. DNA recognition, mediated by a shared N-terminal zinc module (the DM domain), is enhanced by a C-terminal dimerization element. Sex-specific extension of this element in DSX F and DSX M leads to assembly of distinct transcriptional preinitiation complexes. Here, we describe the structure of the extended C-terminal dimerization domain of DSX F as determined by multidimensional NMR spectroscopy. The core dimerization element is well ordered, giving rise to a dense network of interresidue nuclear Overhauser enhancements. The structure contains dimer-related UBA folds similar to those defined by x-ray crystallographic studies of a truncated domain. Whereas the proximal portion of the female tail extends helix 3 of the UBA fold, the distal tail is disordered. Ala substitutions in the proximal tail disrupt the sex-specific binding of IX (Intersex), an obligatory partner protein and putative transcriptional coactivator; IX-DSX F interaction is, by contrast, not disrupted by truncation of the distal tail. Mutagenesis of the UBA-like dimer of DSX F highlights the importance of steric and electrostatic complementarity across the interface. Two temperature-sensitive mutations at this interface have been characterized in yeast model systems. One weakens a network of solvated salt bridges, whereas the other perturbs the underlying nonpolar interface. These mutations confer graded gene-regulatory activity in yeast within a physiological temperature range and so may provide novel probes for genetic analysis of a sex-specific transcriptional program in Drosophila development.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Caracteres Sexuales , Animales , Cristalografía por Rayos X/métodos , Drosophila melanogaster , Femenino , Espectroscopía de Resonancia Magnética , Masculino , Modelos Genéticos , Conformación Molecular , Mutagénesis Sitio-Dirigida , Mutación , Estructura Terciaria de Proteína , Factores Sexuales , Activación TranscripcionalRESUMEN
Male- and female-specific isoforms of the Doublesex (DSX) transcription factor regulate somatic sexual differentiation in Drosophila. The isoforms (DSX(M) and DSX(F)) share an N-terminal DNA binding domain (the DM motif), broadly conserved among metazoan sex-determining pathways. DM-DNA recognition is enhanced by a C-terminal dimerization domain. The crystal structure of this domain, determined at a resolution of 1.6 A, reveals a novel dimeric arrangement of ubiquitin-associated (UBA) folds. Although this alpha-helical motif is well characterized in pathways of DNA repair and subcellular trafficking, to our knowledge this is its first report in a transcription factor. Dimerization is mediated by a non-canonical hydrophobic interface extrinsic to the putative ubiquitin binding surface. Key side chains at this interface, identified by alanine scanning mutagenesis, are conserved among DSX homologs. The mechanism of dimerization is thus unrelated to the low affinity domain swapping observed among ubiquitin-associated CUE domains. The unexpected observation of a ubiquitin-associated fold in DSX extends the repertoire of alpha-helical dimerization elements in transcription factors. The possibility that the ubiquitination machinery participates in the regulation of sexual dimorphism is discussed.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Drosophila/química , Regulación de la Expresión Génica , Ubiquitina/química , Alanina/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X , ADN/química , Dimerización , Drosophila melanogaster , Electrones , Femenino , Enlace de Hidrógeno , Masculino , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación Proteica , Pliegue de Proteína , Isoformas de Proteínas , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Factores Sexuales , Factores de Transcripción/química , Técnicas del Sistema de Dos HíbridosRESUMEN
Doublesex (DSX) is a transcription factor in Drosophila melanogaster that regulates somatic sexual differentiation. Homologues have been found in diverse metazoans, suggesting a conserved role in development. Here, the expression, purification and crystallization of a novel 63-amino-acid dimerization domain and selenomethionine analogue are described. Native crystals belong to space group P2(1)2(1)2(1) and diffract to a resolution of 1.6 A with synchrotron radiation. Selenomethionine-containing crystals provide anomalous dispersion for phasing.