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1.
Nat Commun ; 12(1): 1368, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33649334

RESUMO

The homeostasis of the gut epithelium relies upon continuous renewal and proliferation of crypt-resident intestinal epithelial stem cells (IESCs). Wnt/ß-catenin signaling is required for IESC maintenance, however, it remains unclear how this pathway selectively governs the identity and proliferative decisions of IESCs. Here, we took advantage of knock-in mice harboring transgenic ß-catenin alleles with mutations that specifically impair the recruitment of N- or C-terminal transcriptional co-factors. We show that C-terminally-recruited transcriptional co-factors of ß-catenin act as all-or-nothing regulators of Wnt-target gene expression. Blocking their interactions with ß-catenin rapidly induces loss of IESCs and intestinal homeostasis. Conversely, N-terminally recruited co-factors fine-tune ß-catenin's transcriptional output to ensure proper self-renewal and proliferative behaviour of IESCs. Impairment of N-terminal interactions triggers transient hyperproliferation of IESCs, eventually resulting in exhaustion of the self-renewing stem cell pool. IESC mis-differentiation, accompanied by unfolded protein response stress and immune infiltration, results in a process resembling aberrant "villisation" of intestinal crypts. Our data suggest that IESC-specific Wnt/ß-catenin output requires selective modulation of gene expression by transcriptional co-factors.


Assuntos
Mucosa Intestinal/citologia , Células-Tronco/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética , beta Catenina/química , beta Catenina/metabolismo , Algoritmos , Animais , Sequência de Bases , Diferenciação Celular , Proliferação de Células , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Homeostase , Hiperplasia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , Proteínas Mutantes/metabolismo , Mutação/genética , Organoides/metabolismo , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais
2.
Science ; 371(6535)2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33737460

RESUMO

The intestine is a site of direct encounter with the external environment and must consequently balance barrier defense with nutrient uptake. To investigate how nutrient uptake is regulated in the small intestine, we tested the effect of diets with different macronutrient compositions on epithelial gene expression. We found that enzymes and transporters required for carbohydrate digestion and absorption were regulated by carbohydrate availability. The "on-demand" induction of this machinery required γδ T cells, which regulated this program through the suppression of interleukin-22 production by type 3 innate lymphoid cells. Nutrient availability altered the tissue localization and transcriptome of γδ T cells. Additionally, transcriptional responses to diet involved cellular remodeling of the epithelial compartment. Thus, this work identifies a role for γδ T cells in nutrient sensing.


Assuntos
Carboidratos da Dieta/administração & dosagem , Carboidratos da Dieta/metabolismo , Enterócitos/fisiologia , Interleucinas/metabolismo , Mucosa Intestinal/fisiologia , Receptores de Antígenos de Linfócitos T gama-delta , Subpopulações de Linfócitos T/fisiologia , Adaptação Fisiológica , Animais , Comunicação Celular , Proteínas na Dieta/administração & dosagem , Digestão , Regulação da Expressão Gênica , Interleucinas/genética , Absorção Intestinal , Mucosa Intestinal/citologia , Intestino Delgado/citologia , Intestino Delgado/metabolismo , Camundongos Endogâmicos C57BL , Nutrientes/administração & dosagem , Nutrientes/metabolismo , Subpopulações de Linfócitos T/imunologia , Transcrição Genética , Transcriptoma
3.
J Enzyme Inhib Med Chem ; 36(1): 659-668, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33641565

RESUMO

Human intestinal epithelial cell line-6 (HIEC-6) cells and primary human hepatocytes (PHHs) were treated with 3-amidinophenylalanine-derived inhibitors of trypsin-like serine proteases for 24 hours. It was proven that treatment with MI-1900 and MI-1907 was tolerated up to 50 µM in HIEC-6. These inhibitors did not cause elevations in extracellular H2O2 levels and in the concentrations of interleukin (IL)-6 and IL-8 and did not alter occludin distribution in HIEC-6. It was also found that MI-1900 and MI-1907 up to 50 µM did not affect cell viability, IL-6 and IL-8 and occludin levels of PHH. Based on our findings, these inhibitors could be safely applicable at 50 µM in HIEC-6 and in PHH; however, redox status was disturbed in case of PHH. Moreover, it has recently been demonstrated that MI-1900 prevents the replication and spread of the new SARS-CoV-2 in infected Calu-3 cells, most-likely via an inhibition of the membrane-bound host protease TMPRSS2.


Assuntos
Antivirais/farmacologia , Células Epiteliais/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Fenilalanina/farmacologia , Inibidores de Proteases/farmacologia , Serina Endopeptidases/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/enzimologia , Regulação da Expressão Gênica/efeitos dos fármacos , Hepatócitos/citologia , Hepatócitos/enzimologia , Humanos , Peróxido de Hidrogênio/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/enzimologia , Ocludina/genética , Ocludina/metabolismo , Oxirredução/efeitos dos fármacos , Fenilalanina/análogos & derivados , Cultura Primária de Células , Serina Endopeptidases/genética
4.
J Vis Exp ; (168)2021 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-33645571

RESUMO

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water while preventing passage of luminal bacteria and exogenous substances. A breach of this layer results in increased permeability to luminal contents and recruitment of immune cells, both of which are hallmarks of pathologic states in the gut including inflammatory bowel disease (IBD). Mechanisms regulating epithelial barrier function and transepithelial migration (TEpM) of polymorphonuclear neutrophils (PMN) are incompletely understood due to the lack of experimental in vivo methods allowing quantitative analyses. Here, we describe a robust murine experimental model that employs an exteriorized intestinal segment of either ileum or proximal colon. The exteriorized intestinal loop (iLoop) is fully vascularized and offers physiological advantages over ex vivo chamber-based approaches commonly used to study permeability and PMN migration across epithelial cell monolayers. We demonstrate two applications of this model in detail: (1) quantitative measurement of intestinal permeability through detection of fluorescence-labeled dextrans in serum after intraluminal injection, (2) quantitative assessment of migrated PMN across the intestinal epithelium into the gut lumen after intraluminal introduction of chemoattractants. We demonstrate feasibility of this model and provide results utilizing the iLoop in mice lacking the epithelial tight junction-associated protein JAM-A compared to controls. JAM-A has been shown to regulate epithelial barrier function as well as PMN TEpM during inflammatory responses. Our results using the iLoop confirm previous studies and highlight the importance of JAM-A in regulation of intestinal permeability and PMN TEpM in vivo during homeostasis and disease. The iLoop model provides a highly standardized method for reproducible in vivo studies of intestinal homeostasis and inflammation and will significantly enhance understanding of intestinal barrier function and mucosal inflammation in diseases such as IBD.


Assuntos
Mucosa Intestinal/citologia , Mucosa Intestinal/fisiologia , Modelos Biológicos , Migração Transendotelial e Transepitelial , Animais , Linhagem Celular , Quimiocinas/farmacologia , Citometria de Fluxo , Mucosa Intestinal/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Neutrófilos/citologia , Permeabilidade , Padrões de Referência , Migração Transendotelial e Transepitelial/efeitos dos fármacos
5.
Methods Mol Biol ; 2273: 263-278, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33604860

RESUMO

Tissue engineering is an elegant tool to create organs in vitro, that can help obviate the lack of organ donors in transplantation medicine and provide the opportunity of studying complex biological systems in vitro, thereby reducing the need for animal experiments. Artificial intestine models are at the core of Fish-AI, an EU FET-Open research project dedicated to the development of a 3D in vitro platform that is intended to enable the aquaculture feed industry to predict the nutritional and health value of alternative feed sources accurately and efficiently.At present, it is impossible to infer the health and nutrition value through the chemical characterization of any given feed. Therefore, each new feed must be tested through in vivo growth trials. The procedure is lengthy, expensive and requires the use of many animals. Furthermore, although this process allows for a precise evaluation of the final effect of each feed, it does not improve our basic knowledge of the cellular and molecular mechanisms determining such end-results. In turn, this lack of mechanistic knowledge severely limits the capacity to understand and predict the biological value of a single raw material and of their different combinations.The protocol described herein allows to develop the two main components essential to produce a functional platform for the efficient and reliable screening of feeds that the feed industry is currently developing for improving their health and nutritional value. It is here applied to the Rainbow Trout, but it can be fruitfully used to many other fish species.


Assuntos
Técnicas de Cultura de Células/métodos , Gelatina/química , Mucosa Intestinal/citologia , Oncorhynchus mykiss , Tecidos Suporte/química , Acrilamidas/química , Ração Animal/análise , Animais , Aquicultura/métodos , Materiais Biocompatíveis/química , Separação Celular/métodos , Células Cultivadas , Norbornanos/química , Oncorhynchus mykiss/crescimento & desenvolvimento
6.
Nature ; 592(7852): 99-104, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33627870

RESUMO

The small intestine is the main organ for nutrient absorption, and its extensive resection leads to malabsorption and wasting conditions referred to as short bowel syndrome (SBS). Organoid technology enables an efficient expansion of intestinal epithelium tissue in vitro1, but reconstruction of the whole small intestine, including the complex lymphovascular system, has remained challenging2. Here we generate a functional small intestinalized colon (SIC) by replacing the native colonic epithelium with ileum-derived organoids. We first find that xenotransplanted human ileum organoids maintain their regional identity and form nascent villus structures in the mouse colon. In vitro culture of an organoid monolayer further reveals an essential role for luminal mechanistic flow in the formation of villi. We then develop a rat SIC model by repositioning the SIC at the ileocaecal junction, where the epithelium is exposed to a constant luminal stream of intestinal juice. This anatomical relocation provides the SIC with organ structures of the small intestine, including intact vasculature and innervation, villous structures, and the lacteal (a fat-absorbing lymphatic structure specific to the small intestine). The SIC has absorptive functions and markedly ameliorates intestinal failure in a rat model of SBS, whereas transplantation of colon organoids instead of ileum organoids invariably leads to mortality. These data provide a proof of principle for the use of intestinal organoids for regenerative purposes, and offer a feasible strategy for SBS treatment.


Assuntos
Colo/citologia , Íleo/transplante , Mucosa Intestinal/citologia , Organoides/transplante , Regeneração , Medicina Regenerativa/métodos , Síndrome do Intestino Curto/terapia , Animais , Colo/irrigação sanguínea , Colo/inervação , Colo/cirurgia , Modelos Animais de Doenças , Xenoenxertos , Humanos , Íleo/citologia , Mucosa Intestinal/irrigação sanguínea , Mucosa Intestinal/inervação , Mucosa Intestinal/cirurgia , Masculino , Técnicas de Cultura de Órgãos , Organoides/citologia , Ratos , Ratos Endogâmicos Lew , Síndrome do Intestino Curto/patologia , Síndrome do Intestino Curto/cirurgia
8.
Life Sci ; 271: 119195, 2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33581125

RESUMO

AIMS: Ulcerative colitis and Crohn's disease, collectively known as inflammatory bowel disease (IBD), are chronic inflammatory disorders of the intestine for which key elements in disease initiation and perpetuation are defects in epithelial barrier integrity. Achieving mucosal healing is essential to ameliorate disease outcome and so new therapies leading to epithelial homeostasis and repair are under investigation. This study was designed to determine the mechanisms by which IL-22 regulates intestinal epithelial cell function. MAIN METHODS: Human intestinal organoids and resections, as well as mice were used to evaluate the effect of IL-22 on stem cell expansion, proliferation and expression of mucus components. IL-22 effect on barrier function was assessed in polarized T-84 cell monolayers. Butyrate co-treatments and organoid co-cultures with immune cells were performed to monitor the impact of microbial-derived metabolites and inflammatory environments on IL-22 responses. KEY FINDINGS: IL-22 led to epithelial stem cell expansion, proliferation, barrier dysfunction and anti-microbial peptide production in human and mouse models evaluated. IL-22 also altered the mucus layer by inducing an increase in membrane mucus but a decrease in secreted mucus and goblet cell content. IL-22 had the same effect on anti-microbial peptides and membrane mucus in both healthy and IBD human samples. In contrast, this IL-22-associated epithelial phenotype was different when treatments were performed in presence of butyrate and organoids co-cultured with immune cells. SIGNIFICANCE: Our data indicate that IL-22 promotes epithelial regeneration, innate defense and membrane mucus production, strongly supporting the potential clinical utility of IL-22 as a mucosal healing therapy in IBD.


Assuntos
Células Epiteliais/fisiologia , Homeostase/fisiologia , Interleucinas/fisiologia , Interleucinas/uso terapêutico , Mucosa Intestinal/fisiologia , Animais , Linhagem Celular , Técnicas de Cocultura , Colite Ulcerativa/tratamento farmacológico , Colite Ulcerativa/patologia , Células Epiteliais/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Humanos , Interleucinas/farmacologia , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Organoides/efeitos dos fármacos , Organoides/fisiologia
9.
J Vis Exp ; (168)2021 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-33616118

RESUMO

The intestinal epithelium is comprised of a single layer of cells that act as a barrier between the gut lumen and the interior of the body. Disruption in the continuity of this barrier can result in inflammatory disorders such as inflammatory bowel disease. One of the limitations in the study of intestinal epithelial biology has been the lack of primary cell culture models, which has obliged researchers to use model cell lines derived from carcinomas. The advent of three dimensional (3D) enteroids has given epithelial biologists a powerful tool to generate primary cell cultures, nevertheless, these structures are embedded in extracellular matrix and lack the maturity characteristic of differentiated intestinal epithelial cells. Several techniques to generate intestinal epithelial monolayers have been published, but most are derived from established 3D enteroids making the process laborious and expensive. Here we describe a protocol to generate primary epithelial colon monolayers directly from murine intestinal crypts. We also detail experimental approaches that can be used with this model such as the generation of confluent cultures on permeable filters, confluent monolayer for scratch wound healing studies and sparse and confluent monolayers for immunofluorescence analysis.


Assuntos
Diferenciação Celular , Colo/citologia , Células Epiteliais/citologia , Mucosa Intestinal/citologia , Cultura Primária de Células/métodos , Animais , Técnicas de Cultura de Células/métodos , Células Cultivadas , Camundongos , Camundongos Endogâmicos C57BL
10.
Methods Mol Biol ; 2241: 243-255, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33486741

RESUMO

Eosinophils are primarily tissue-dwelling leukocytes. Utilization of flow cytometry techniques applied to digested tissues is expanding the scope of organs within which eosinophils are identified at baseline and is providing deeper insights into categorizing phenotypically and functionally distinct tissue-resident eosinophil subpopulations in health and disease. Here we describe a tissue digestion protocol and flow cytometry gating strategy for identification and isolation of tissue eosinophils from the small intestine of mice. This protocol is also amenable to the isolation and characterization of colonic eosinophils, and of intestinal eosinophils from human resected tissues.


Assuntos
Eosinófilos/citologia , Citometria de Fluxo/métodos , Intestino Delgado/citologia , Animais , Separação Celular/métodos , Mucosa Intestinal/citologia , Intestinos/citologia , Leucócitos/citologia , Camundongos
11.
Nucleic Acids Res ; 49(2): 791-804, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33398338

RESUMO

The proteolytic cleavage of histone tails, also termed histone clipping, has been described as a mechanism for permanent removal of post-translational modifications (PTMs) from histone proteins. Such activity has been ascribed to ensure regulatory function in key cellular processes such as differentiation, senescence and transcriptional control, for which different histone-specific proteases have been described. However, all these studies were exclusively performed using cell lines cultured in vitro and no clear evidence that histone clipping is regulated in vivo has been reported. Here we show that histone H3 N-terminal tails undergo extensive cleavage in the differentiated cells of the villi in mouse intestinal epithelium. Combining biochemical methods, 3D organoid cultures and in vivo approaches, we demonstrate that intestinal H3 clipping is the result of multiple proteolytic activities. We identified Trypsins and Cathepsin L as specific H3 tail proteases active in small intestinal differentiated cells and showed that their proteolytic activity is differentially affected by the PTM pattern of histone H3 tails. Together, our findings provide in vivo evidence of H3 tail proteolysis in mammalian tissues, directly linking H3 clipping to cell differentiation.


Assuntos
Enterócitos/metabolismo , Histonas/metabolismo , Intestino Delgado/citologia , Celulas de Paneth/metabolismo , Peptídeo Hidrolases/metabolismo , Processamento de Proteína Pós-Traducional , Células-Tronco/metabolismo , Animais , Catepsina L/metabolismo , Diferenciação Celular , Homeostase , Mucosa Intestinal/citologia , Camundongos , Microvilosidades/ultraestrutura , Nucleossomos/metabolismo , Nucleossomos/ultraestrutura , Organoides , Domínios Proteicos , Tripsina/metabolismo
12.
Nat Cell Biol ; 23(1): 23-31, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33398177

RESUMO

A detailed understanding of intestinal stem cell (ISC) self-renewal and differentiation is required to treat chronic intestinal diseases. However, the different models of ISC lineage hierarchy1-6 and segregation7-12 are subject to debate. Here, we have discovered non-canonical Wnt/planar cell polarity (PCP)-activated ISCs that are primed towards the enteroendocrine or Paneth cell lineage. Strikingly, integration of time-resolved lineage labelling with single-cell gene expression analysis revealed that both lineages are directly recruited from ISCs via unipotent transition states, challenging the existence of formerly predicted bi- or multipotent secretory progenitors7-12. Transitory cells that mature into Paneth cells are quiescent and express both stem cell and secretory lineage genes, indicating that these cells are the previously described Lgr5+ label-retaining cells7. Finally, Wnt/PCP-activated Lgr5+ ISCs are molecularly indistinguishable from Wnt/ß-catenin-activated Lgr5+ ISCs, suggesting that lineage priming and cell-cycle exit is triggered at the post-transcriptional level by polarity cues and a switch from canonical to non-canonical Wnt/PCP signalling. Taken together, we redefine the mechanisms underlying ISC lineage hierarchy and identify the Wnt/PCP pathway as a new niche signal preceding lateral inhibition in ISC lineage priming and segregation.


Assuntos
Linhagem da Célula , Polaridade Celular , Células Enteroendócrinas/citologia , Mucosa Intestinal/citologia , Celulas de Paneth/citologia , Células-Tronco/citologia , Proteínas Wnt/metabolismo , Animais , Autorrenovação Celular , Células Enteroendócrinas/metabolismo , Feminino , Perfilação da Expressão Gênica , Mucosa Intestinal/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Celulas de Paneth/metabolismo , Receptores Acoplados a Proteínas-G/fisiologia , Análise de Célula Única , Células-Tronco/metabolismo , beta Catenina/metabolismo
13.
Food Chem ; 339: 127985, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-32920305

RESUMO

There is limited research focusing on the effects of human gut microbiota on the oral bioaccessibility and intestinal absorption of pesticide residues in food. In the present study, we use a modified setup of the Simulator of the Human Intestinal Microbial Ecosystem for the determination of pesticide residue bioaccessibility in Chaenomeles speciosa, and a Caco-2 cell model of human intestinal absorption. Results showed that gut microbiota played a dual role based their effects on contaminant release and metabolism in the bioaccessibility assay, and Lactobacillus plantarum was one of key bacterial species in the gut microbiota that influenced pesticide stability significantly. The addition of L. plantarum to the system reduced the relative amounts (by 11.40-86.51%) of six pesticides. The interaction between the food matrix and human gut microbiota led to different absorption rates, and the barrier effects increased with an increase in incubation time.


Assuntos
Microbioma Gastrointestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Praguicidas/farmacologia , Rosaceae/química , Bactérias/metabolismo , Células CACO-2 , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/microbiologia , Lactobacillus plantarum/efeitos dos fármacos , Lactobacillus plantarum/isolamento & purificação , Neonicotinoides/metabolismo , Neonicotinoides/farmacologia , Nitrocompostos/metabolismo , Nitrocompostos/farmacologia , Compostos Organotiofosforados/metabolismo , Compostos Organotiofosforados/farmacologia , Praguicidas/química , Praguicidas/metabolismo , Rosaceae/metabolismo , Tiametoxam/metabolismo , Tiametoxam/farmacologia
14.
Methods Mol Biol ; 2237: 247-256, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33237424

RESUMO

Gut mucosal immune cells play an essential role in health due to their ability to orchestrate host signaling events in response to exogenous antigens. These antigens may originate from microorganisms including viruses, commensal or pathogenic bacteria, or single-celled eukaryotes, as well as from dietary foodstuff-derived proteins or products. A critical technological capacity to understand host responses to antigens is the ability to efficiently isolate and functionally characterize immune cells from intestinal tissues. Additionally, after characterization, it is of paramount importance to understand the exact functions of these immune cells under different disease states or genetic variables. Here, we outline methods for immune cell isolation from murine small and large intestines with the goal of undertaking a functional analysis of isolated cell types using antibody array platforms.


Assuntos
Citometria de Fluxo/métodos , Mucosa Intestinal/citologia , Linfócitos/imunologia , Análise Serial de Proteínas/métodos , Animais , Imunoensaio/métodos , Linfócitos/citologia , Camundongos
15.
Life Sci ; 260: 118428, 2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-32931798

RESUMO

AIMS: The benefits of utilizing laboratory mice include low cost, ease of maintenance, and accessibility of molecular tools. However, the ages of experimental mice in the literature vary drastically. We hypothesized that there exists age-related variation in the murine small intestine across developmental stages. MATERIALS AND METHODS: Segments of small intestine were harvested from C57BL/6J mice of varying ages (E17 to 24 weeks; n = 3-4/group). Slides were analyzed for morphometric parameters, cell types, and crypt proliferation index (CPI). Secondary analysis comparing age-matched males and females (n = 4/group) was performed. Means were compared with Student's t-test and variance of proportions with the Chi-squared test to a significance of p < 0.05. KEY FINDINGS: There were small but significant differences including regional variation in villus height, which abolished when examining the small intestine as a whole. Sexually immature mice had increased CPI compared to mature animals. The most dramatic differences were seen in mice at weaning, which demonstrated shallower crypts, increased CPI, fewer Paneth and goblet cells, and more enterochromaffin cells. Examination of embryonic intestine revealed an underdeveloped mucosa lacking differentiated cells. There were minimal differences when comparing age-matched males and females. SIGNIFICANCE: Small, but statistically significant differences in villus height, crypt depth, and crypt proliferation are present in mice across early developmental stages. Mice at weaning exhibit variation in crypt-villus cell composition compared to older animals, which may explain the propensity for certain intestinal conditions in the very young. Investigators studying the GI mucosa should employ consistent age-matching in order to allow direct comparison between studies.


Assuntos
Mucosa Intestinal/citologia , Mucosa Intestinal/crescimento & desenvolvimento , Intestino Delgado/crescimento & desenvolvimento , Fatores Etários , Animais , Feminino , Mucosa Intestinal/embriologia , Intestino Delgado/citologia , Intestino Delgado/embriologia , Masculino , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Varredura
16.
Adv Exp Med Biol ; 1265: 133-151, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32761574

RESUMO

The intestine interacts with a diverse community of antigens and bacteria. To keep its homeostasis, the gut has evolved with a complex defense system, including intestinal microbiota, epithelial layer and lamina propria. Various factors (e.g., nutrients) affect the intestinal defensive system and progression of intestinal diseases. This review highlights the current understanding about the role of amino acids (AAs) in protecting the intestine from harm. Amino acids (e.g., arginine, glutamine and tryptophan) are essential for the function of intestinal microbiota, epithelial cells, tight junction, goblet cells, Paneth cells and immune cells (e.g., macrophages, B cells and T cells). Through the modulation of the intestinal defensive system, AAs maintain the integrity and function of the intestinal mucosa and inhibit the progression of various intestinal diseases (e.g., intestinal infection and intestinal colitis). Thus, adequate intake of functional AAs is crucial for intestinal and whole-body health in humans and other animals.


Assuntos
Aminoácidos/metabolismo , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Animais , Colite , Microbioma Gastrointestinal , Humanos , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Junções Íntimas
17.
Nat Med ; 26(9): 1480-1490, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32747828

RESUMO

Colonic antigen-experienced lymphocytes such as tissue-resident memory CD8+ T cells can respond rapidly to repeated antigen exposure. However, their cellular phenotypes and the mechanisms by which they drive immune regulation and inflammation remain unclear. Here we compiled an unbiased atlas of human colonic CD8+ T cells in health and ulcerative colitis (UC) using single-cell transcriptomics with T-cell receptor repertoire analysis and mass cytometry. We reveal extensive heterogeneity in CD8+ T-cell composition, including expanded effector and post-effector terminally differentiated CD8+ T cells. While UC-associated CD8+ effector T cells can trigger tissue destruction and produce tumor necrosis factor (TNF)-α, post-effector cells acquire innate signatures to adopt regulatory functions that may mitigate excessive inflammation. Thus, we identify colonic CD8+ T-cell phenotypes in health and UC, define their clonal relationships and characterize terminally differentiated dysfunctional UC CD8+ T cells expressing IL-26, which attenuate acute colitis in a humanized IL-26 transgenic mouse model.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Colite Ulcerativa/patologia , Interleucinas/metabolismo , Mucosa Intestinal/patologia , Fator de Necrose Tumoral alfa/metabolismo , Animais , Colo/patologia , Feminino , Perfilação da Expressão Gênica , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transcriptoma/genética
18.
Nat Commun ; 11(1): 3769, 2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32724083

RESUMO

Butyrophilin-like (Btnl) genes are emerging as major epithelial determinants of tissue-associated γδ T cell compartments. Thus, the development of signature, murine TCRγδ+ intraepithelial lymphocytes (IEL) in gut and skin depends on Btnl family members, Btnl1 and Skint1, respectively. In seeking mechanisms underlying these profound effects, we now show that normal gut and skin γδ IEL development additionally requires Btnl6 and Skint2, respectively, and furthermore that different Btnl heteromers can seemingly shape different intestinal γδ+ IEL repertoires. This formal genetic evidence for the importance of Btnl heteromers also applied to the steady-state, since sustained Btnl expression is required to maintain the signature TCR.Vγ7+ IEL phenotype, including specific responsiveness to Btnl proteins. In sum, Btnl proteins are required to select and to maintain the phenotypes of tissue-protective γδ IEL compartments, with combinatorially diverse heteromers having differential impacts on different IEL subsets.


Assuntos
Butirofilinas/metabolismo , Imunidade Celular , Linfócitos Intraepiteliais/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Animais , Butirofilinas/genética , Butirofilinas/imunologia , Perfilação da Expressão Gênica , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Linfócitos Intraepiteliais/metabolismo , Camundongos , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T gama-delta/imunologia
19.
Nature ; 584(7821): 415-419, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32641829

RESUMO

Sexual dimorphism arises from genetic differences between male and female cells, and from systemic hormonal differences1-3. How sex hormones affect non-reproductive organs is poorly understood, yet highly relevant to health given the sex-biased incidence of many diseases4. Here we report that steroid signalling in Drosophila from the ovaries to the gut promotes growth of the intestine specifically in mated females, and enhances their reproductive output. The active ovaries of the fly produce the steroid hormone ecdysone, which stimulates the division and expansion of intestinal stem cells in two distinct proliferative phases via the steroid receptors EcR and Usp and their downstream targets Broad, Eip75B and Hr3. Although ecdysone-dependent growth of the female gut augments fecundity, the more active and more numerous intestinal stem cells also increase female susceptibility to age-dependent gut dysplasia and tumorigenesis, thus potentially reducing lifespan. This work highlights the trade-offs in fitness traits that occur when inter-organ signalling alters stem-cell behaviour to optimize organ size.


Assuntos
Drosophila melanogaster/metabolismo , Fertilidade/fisiologia , Intestinos/crescimento & desenvolvimento , Longevidade/fisiologia , Tamanho do Órgão/fisiologia , Ovário/metabolismo , Esteroides/metabolismo , Envelhecimento , Animais , Carcinogênese , Proliferação de Células , Copulação/fisiologia , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/anatomia & histologia , Drosophila melanogaster/citologia , Drosophila melanogaster/fisiologia , Ecdisona/metabolismo , Feminino , Mucosa Intestinal/anatomia & histologia , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Intestinos/anatomia & histologia , Intestinos/citologia , Intestinos/patologia , Masculino , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Esteroides/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Fatores de Transcrição/metabolismo
20.
Cell Rep ; 32(1): 107863, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32610043

RESUMO

Severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) is an unprecedented worldwide health problem that requires concerted and global approaches to stop the coronavirus 2019 (COVID-19) pandemic. Although SARS-CoV-2 primarily targets lung epithelium cells, there is growing evidence that the intestinal epithelium is also infected. Here, using both colon-derived cell lines and primary non-transformed colon organoids, we engage in the first comprehensive analysis of the SARS-CoV-2 life cycle in human intestinal epithelial cells (hIECs). Our results demonstrate that hIECs fully support SARS-CoV-2 infection, replication, and production of infectious de novo virus particles. We found that viral infection elicits an extremely robust intrinsic immune response where interferon-mediated responses are efficient at controlling SARS-CoV-2 replication and de novo virus production. Taken together, our data demonstrate that hIECs are a productive site of SARS-CoV-2 replication and suggest that the enteric phase of SARS-CoV-2 may participate in the pathologies observed in COVID-19 patients by contributing to increasing patient viremia and fueling an exacerbated cytokine response.


Assuntos
Betacoronavirus/crescimento & desenvolvimento , Colo/virologia , Células Epiteliais/imunologia , Interferons/imunologia , Mucosa Intestinal/imunologia , Betacoronavirus/imunologia , Células CACO-2 , Linhagem Celular Tumoral , Colo/citologia , Colo/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/patologia , Síndrome da Liberação de Citocina/imunologia , Síndrome da Liberação de Citocina/virologia , Citocinas/sangue , Células Epiteliais/virologia , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/virologia , Pandemias , Pneumonia Viral/imunologia , Pneumonia Viral/patologia , Síndrome Respiratória Aguda Grave/imunologia , Síndrome Respiratória Aguda Grave/patologia , Replicação Viral/imunologia
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