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2.
Biomed Res Int ; 2021: 1086206, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34423029

RESUMO

As a new type of noncoding RNA, circular RNA (circRNA) is stable in cells and not easily degraded. This type of RNA can also competitively bind miRNAs to regulate the expression of their target genes. The role of circRNA in the mechanism of intestinal oxidative stress (OS) in weaned piglets is still unclear. In our research, diquat (DQ) was used to induce OS in small intestinal epithelial cells (IPEC-J2) to construct an OS cell model. Mechanistically, dual luciferase reporter assays, fluorescence in situ hybridization (FISH), and western blotting were performed to confirm that circGLI3 directly sponged miR-339-5p and regulated the expression of VEGFA. Overexpression of circGLI3 promoted IPEC-J2 cell proliferation, increased the proportion of S-phase cells (P < 0.01), and reduced reactive oxygen species (ROS) generation when IPEC-J2 cells were subjected to OS. circGLI3 can increase the activity of glutathione peroxidase (GSH-Px) and the total antioxidant capacity (T-AOC) in IPEC-J2 cells and reduce the malondialdehyde (MDA) content and levels of inflammatory factors. Therefore, overexpression of circGLI3 reduced oxidative damage, whereas miR-339-5p mimic counteracted these effects. We identified a regulatory network composed of circGLI3, miR-339-5p, and VEGFA and verified that circGLI3 regulates VEGFA by directly binding miR-339-5p. The expression of VEGFA affects IPEC-J2 cell proliferation, cell cycle progression, and ROS content and changes the levels of antioxidant enzymes and inflammatory factors. This study reveals the molecular mechanism by which circGLI3 inhibits OS in the intestine of piglets and provides a theoretical basis for further research on the effect of OS on intestinal function.


Assuntos
Diquat/efeitos adversos , Intestino Delgado/citologia , MicroRNAs/genética , RNA Circular/genética , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Células Epiteliais/química , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Glutationa Peroxidase/metabolismo , Intestino Delgado/química , Intestino Delgado/efeitos dos fármacos , Malondialdeído/metabolismo , Modelos Biológicos , Estresse Oxidativo , Suínos , Fator A de Crescimento do Endotélio Vascular/metabolismo
3.
Nat Commun ; 12(1): 4462, 2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34294718

RESUMO

RORγt+ lymphocytes, including interleukin 17 (IL-17)-producing gamma delta T (γδT17) cells, T helper 17 (Th17) cells, and group 3 innate lymphoid cells (ILC3s), are important immune regulators. Compared to Th17 cells and ILC3s, γδT17 cell metabolism and its role in tissue homeostasis remains poorly understood. Here, we report that the tissue milieu shapes splenic and intestinal γδT17 cell gene signatures. Conditional deletion of mitochondrial transcription factor A (Tfam) in RORγt+ lymphocytes significantly affects systemic γδT17 cell maintenance and reduces ILC3s without affecting Th17 cells in the gut. In vivo deletion of Tfam in RORγt+ lymphocytes, especially in γδT17 cells, results in small intestine tissue remodeling and increases small intestine length by enhancing the type 2 immune responses in mice. Moreover, these mice show dysregulation of the small intestine transcriptome and metabolism with less body weight but enhanced anti-helminth immunity. IL-22, a cytokine produced by RORγt+ lymphocytes inhibits IL-13-induced tuft cell differentiation in vitro, and suppresses the tuft cell-type 2 immune circuit and small intestine lengthening in vivo, highlighting its key role in gut tissue remodeling.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Grupo de Alta Mobilidade/metabolismo , Intestino Delgado/imunologia , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Animais , Diferenciação Celular , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Feminino , Perfilação da Expressão Gênica , Proteínas de Grupo de Alta Mobilidade/deficiência , Proteínas de Grupo de Alta Mobilidade/genética , Homeostase/imunologia , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Intestino Delgado/citologia , Intestino Delgado/metabolismo , Masculino , Metaboloma , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Organoides , Proteína com Dedos de Zinco da Leucemia Promielocítica/genética , Proteína com Dedos de Zinco da Leucemia Promielocítica/metabolismo , Subpopulações de Linfócitos T/citologia , Células Th17/citologia , Células Th17/imunologia , Células Th17/metabolismo
4.
Int J Radiat Biol ; 97(9): 1206-1216, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34264173

RESUMO

PURPOSE: Intestinal damage induced by radiation exposure is a major clinic concern of radiotherapy for patients with abdominal or pelvic tumor. Melatonin (N-acetyl-5-methoxytryptamine) is likely be an ideal radioprotector to protect individuals from radiation exposure. The study aimed to define the role of melatonin in small intestinal damage caused by abdominal irradiation (ABI). MATERIALS AND METHODS: 30-day survival rate and pathological histology of the intestines from melatonin-treated mice after 13 Gy ABI exposure was first detected. Next, quantitative proteomics analysis of the small intestines tissue was examined and GO term and KEGG pathways analysis were performed. RESULTS: Melatonin treatment before ABI exposure significantly increased 30-day survival rate to 83% and ameliorated damage to the intestinal epithelial cells. Melatonin significantly altered the proteins profile of the small intestines following irradiation. For the irradiated mice treated with melatonin in comparison with the irradiated mice, the enriched GO terms were mainly involved in defense response to other organism (BP, GO: 0098542), response to other organism (BP, GO: 0051707), anion transmembrane transporter activity (MF, GO: 0008509), and secondary active transmembrane transporter activity (MF, GO: 0015291). In the process of antioxidant activity (MF, GO: 0016209), melatonin treatment prior to radiation exhibited high protein levels of Sod3 and Gpx3. The markedly KEGG pathways for melatonin treatment prior to radiation mainly included protein digestion and absorption (ko 04974) and mineral absorption (ko 04978). p53 signaling pathway and DNA repair pathways were enriched in melatonin treated mice. The amount of radiation-induced DNA damage and the cell apoptosis of the small intestines was decreased in the melatonin-treated mice. CONCLUSIONS: Melatonin may protect small intestines from radiation damage through increasing DNA repair and decreasing cell apoptosis of the small intestines. Our data provided perspective for the study of melatonin in mitigating ABI-caused intestinal damage.


Assuntos
Raios gama/efeitos adversos , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/metabolismo , Melatonina/farmacologia , Proteômica , Protetores contra Radiação/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Dano ao DNA , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/efeitos da radiação , Intestino Delgado/citologia , Intestino Delgado/efeitos da radiação , Masculino , Camundongos
5.
IEEE Trans Image Process ; 30: 7025-7037, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34329165

RESUMO

Quantitative phase imaging (QPI) is an emerging label-free technique that produces images containing morphological and dynamical information without contrast agents. Unfortunately, the phase is wrapped in most imaging system. Phase unwrapping is the computational process that recovers a more informative image. It is particularly challenging with thick and complex samples such as organoids. Recent works that rely on supervised training show that deep learning is a powerful method to unwrap the phase; however, supervised approaches require large and representative datasets which are difficult to obtain for complex biological samples. Inspired by the concept of deep image priors, we propose a deep-learning-based method that does not need any training set. Our framework relies on an untrained convolutional neural network to accurately unwrap the phase while ensuring the consistency of the measurements. We experimentally demonstrate that the proposed method faithfully recovers the phase of complex samples on both real and simulated data. Our work paves the way to reliable phase imaging of thick and complex samples with QPI.


Assuntos
Aprendizado Profundo , Holografia/métodos , Processamento de Imagem Assistida por Computador/métodos , Microscopia/métodos , Algoritmos , Animais , Células Cultivadas , Intestino Delgado/citologia , Camundongos , Organoides/citologia , Organoides/diagnóstico por imagem , Técnicas de Cultura de Tecidos
6.
Nutrients ; 13(6)2021 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-34204790

RESUMO

The beneficial effects of human milk suppressing the development of intestinal pathologies such as necrotizing enterocolitis in preterm infants are widely known. Human milk (HM) is rich in a multitude of bioactive factors that play major roles in promoting postnatal maturation, differentiation, and the development of the microbiome. Previous studies showed that HM is rich in hyaluronan (HA) especially in colostrum and early milk. This study aims to determine the role of HA 35 KDa, a HM HA mimic, on intestinal proliferation, differentiation, and the development of the intestinal microbiome. We show that oral HA 35 KDa supplementation for 7 days in mouse pups leads to increased villus length and crypt depth, and increased goblet and Paneth cells, compared to controls. We also show that HA 35 KDa leads to an increased predominance of Clostridiales Ruminococcaceae, Lactobacillales Lactobacillaceae, and Clostridiales Lachnospiraceae. In seeking the mechanisms involved in the changes, bulk RNA seq was performed on samples from the terminal ileum and identified upregulation in several genes essential for cellular growth, proliferation, and survival. Taken together, this study shows that HA 35 KDa supplemented to mouse pups promotes intestinal epithelial cell proliferation, as well as the development of Paneth cells and goblet cell subsets. HA 35 KDa also impacted the intestinal microbiota; the implications of these responses need to be determined.


Assuntos
Suplementos Nutricionais , Microbioma Gastrointestinal/efeitos dos fármacos , Ácido Hialurônico/farmacologia , Intestino Delgado/crescimento & desenvolvimento , Animais , Animais Recém-Nascidos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Caliciformes/citologia , Mucosa Intestinal/efeitos dos fármacos , Intestino Delgado/citologia , Intestinos/citologia , Intestinos/crescimento & desenvolvimento , Camundongos , Celulas de Paneth/citologia
7.
Front Immunol ; 12: 653560, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34149694

RESUMO

Gut-associated lymphoid tissue (GALT) is crucial for the maintenance of the intestinal homeostasis, but it is also the potential site of the activation of autoreactive cells and initiation/propagation of autoimmune diseases in the gut and in the distant organs. Type 3 innate lymphoid cells (ILC3) residing in the GALT integrate signals from food ingredients and gut microbiota metabolites in order to control local immunoreactivity. Notably, ILC3 secrete IL-17 and GM-CSF that activate immune cells in combating potentially pathogenic microorganisms. ILC3 also produce IL-22 that potentiates the strength and integrity of epithelial tight junctions, production of mucus and antimicrobial peptides thus enabling the proper function of the intestinal barrier. The newly discovered function of small intestine ILC3 is the secretion of IL-2 and the promotion of regulatory T cell (Treg) generation and function. Since the intestinal barrier dysfunction, together with the reduction in small intestine ILC3 and Treg numbers are associated with the pathogenesis of type 1 diabetes (T1D), the focus of this article is intestinal ILC3 modulation for the therapy of T1D. Of particular interest is free fatty acids receptor 2 (FFAR2), predominantly expressed on intestinal ILC3, that can be stimulated by available selective synthetic agonists. Thus, we propose that FFAR2-based interventions by boosting ILC3 beneficial functions may attenuate autoimmune response against pancreatic ß cells during T1D. Also, it is our opinion that treatments based on ILC3 stimulation by functional foods can be used as prophylaxis in individuals that are genetically predisposed to develop T1D.


Assuntos
Diabetes Mellitus Tipo 1/tratamento farmacológico , Fatores Imunológicos/farmacologia , Mucosa Intestinal/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Receptores de Superfície Celular/agonistas , Animais , Autoimunidade/efeitos dos fármacos , Diabetes Mellitus Tipo 1/dietoterapia , Diabetes Mellitus Tipo 1/imunologia , Alimento Funcional , Humanos , Imunidade Inata/efeitos dos fármacos , Imunidade nas Mucosas/efeitos dos fármacos , Fatores Imunológicos/uso terapêutico , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Intestino Delgado/citologia , Intestino Delgado/imunologia , Intestino Delgado/metabolismo , Linfócitos/imunologia , Linfócitos/metabolismo , Camundongos , Modelos Animais , Receptores de Superfície Celular/metabolismo
8.
Nat Commun ; 12(1): 3318, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34083536

RESUMO

Dormancy, a reversible quiescent cellular state characterized by greatly reduced metabolic activity, protects from genetic damage, prolongs survival and is crucial for tissue homeostasis and cellular response to injury or transplantation. Dormant cells have been characterized in many tissues, but their identification, isolation and characterization irrespective of tissue of origin remains elusive. Here, we develop a live cell ratiometric fluorescent Optical Stem Cell Activity Reporter (OSCAR) based on the observation that phosphorylation of RNA Polymerase II (RNApII), a hallmark of active mRNA transcription elongation, is largely absent in dormant stem cells from multiple lineages. Using the small intestinal crypt as a model, OSCAR reveals in real time the dynamics of dormancy induction and cellular differentiation in vitro, and allows the identification and isolation of several populations of transcriptionally diverse OSCARhigh and OSCARlow intestinal epithelial cell states in vivo. In particular, this reporter is able to identify a dormant OSCARhigh cell population in the small intestine. OSCAR therefore provides a tool for a better understanding of dormant stem cell biology.


Assuntos
RNA Polimerase II/metabolismo , Fase de Repouso do Ciclo Celular/fisiologia , Animais , Separação Celular , Quinase 9 Dependente de Ciclina/metabolismo , Citometria de Fluxo , Corantes Fluorescentes/metabolismo , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Intestino Delgado/citologia , Intestino Delgado/metabolismo , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Transgênicos , RNA Mensageiro/metabolismo , Transcrição Genética
9.
Biochem Biophys Res Commun ; 559: 135-140, 2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-33940384

RESUMO

Dedicator of cytokinesis 8 (DOCK8) is a guanine nucleotide exchange factor (GEF) for Cdc42. In humans, homozygous or compound heterozygous deletions in DOCK8 cause a combined immunodeficiency characterized by various allergic diseases including food allergies. Although group 2 innate lymphoid cells (ILC2s) contribute to the development of allergic inflammation by producing interleukin (IL)-5 and IL-13, the role of ILC2s in DOCK8 deficiency has not been fully explored. With the use of cytometry by time-of-flight (CyTOF), we performed high-dimensional phenotyping of intestinal immune cells and found that DOCK8-deficient (Dock8-/-) mice exhibited expansion of ILC2s and other leukocytes associated with type 2 immunity in the small intestine. Moreover, IL-5- and IL-13-producing cells markedly increased in Dock8-/- mice, and the majority of them were lineage-negative cells, most likely ILC2s. Intestinal ILC2s expanded when DOCK8 expression was selectively deleted in hematopoietic cells. Importantly, intestinal ILC2 expansion was also observed in Dock8VAGR mice having mutations in the catalytic center of DOCK8, thereby failing to activate Cdc42. Our findings indicate that DOCK8 is a negative regulator of intestinal ILC2s to inhibit their expansion via Cdc42 activation, and that deletion of DOCK8 causes a skewing to type 2 immunity in the gut.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/imunologia , Imunidade Inata , Intestino Delgado/imunologia , Linfócitos/imunologia , Animais , Deleção de Genes , Fatores de Troca do Nucleotídeo Guanina/genética , Intestino Delgado/citologia , Intestino Delgado/metabolismo , Linfócitos/citologia , Camundongos Endogâmicos C57BL
10.
Science ; 372(6539)2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33859001

RESUMO

The intestinal mucus layer, an important element of epithelial protection, is produced by goblet cells. Intestinal goblet cells are assumed to be a homogeneous cell type. In this study, however, we delineated their specific gene and protein expression profiles and identified several distinct goblet cell populations that form two differentiation trajectories. One distinct subtype, the intercrypt goblet cells (icGCs), located at the colonic luminal surface, produced mucus with properties that differed from the mucus secreted by crypt-residing goblet cells. Mice with defective icGCs had increased sensitivity to chemically induced colitis and manifested spontaneous colitis with age. Furthermore, alterations in mucus and reduced numbers of icGCs were observed in patients with both active and remissive ulcerative colitis, which highlights the importance of icGCs in maintaining functional protection of the epithelium.


Assuntos
Colo/citologia , Células Caliciformes/fisiologia , Mucosa Intestinal/citologia , Muco/fisiologia , Animais , Diferenciação Celular , Colite/induzido quimicamente , Colite/fisiopatologia , Colite Ulcerativa/patologia , Colite Ulcerativa/fisiopatologia , Colo/fisiologia , Células Caliciformes/citologia , Humanos , Mucosa Intestinal/fisiologia , Intestino Delgado/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-ets/genética , Transcriptoma
11.
Int J Mol Sci ; 22(7)2021 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-33805523

RESUMO

The intestinal epithelium serves as a dynamic barrier to protect the host tissue from exposure to a myriad of inflammatory stimuli in the luminal environment. Intestinal epithelial cells (IECs) encompass differentiated and specialized cell types that are equipped with regulatory genes, which allow for sensing of the luminal environment. Potential inflammatory cues can instruct IECs to undergo a diverse set of phenotypic alterations. Aging is a primary risk factor for a variety of diseases; it is now well-documented that aging itself reduces the barrier function and turnover of the intestinal epithelium, resulting in pathogen translocation and immune priming with increased systemic inflammation. In this study, we aimed to provide an effective epigenetic and regulatory outlook that examines age-associated alterations in the intestines through the profiling of microRNAs (miRNAs) on isolated mouse IECs. Our microarray analysis revealed that with aging, there is dysregulation of distinct clusters of miRNAs that was present to a greater degree in small IECs (22 miRNAs) compared to large IECs (three miRNAs). Further, miRNA-mRNA interaction network and pathway analyses indicated that aging differentially regulates key pathways between small IECs (e.g., toll-like receptor-related cascades) and large IECs (e.g., cell cycle, Notch signaling and small ubiquitin-related modifier pathway). Taken together, current findings suggest novel gene regulation pathways by epithelial miRNAs in aging within the gastrointestinal tissues.


Assuntos
Envelhecimento/fisiologia , Células Epiteliais/fisiologia , Mucosa Intestinal/citologia , MicroRNAs/fisiologia , Animais , Simulação por Computador , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Intestino Grosso/citologia , Intestino Delgado/citologia , Camundongos Endogâmicos C57BL , RNA Mensageiro
12.
Nature ; 593(7857): 147-151, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33828301

RESUMO

Bile acids are lipid-emulsifying metabolites synthesized in hepatocytes and maintained in vivo through enterohepatic circulation between the liver and small intestine1. As detergents, bile acids can cause toxicity and inflammation in enterohepatic tissues2. Nuclear receptors maintain bile acid homeostasis in hepatocytes and enterocytes3, but it is unclear how mucosal immune cells tolerate high concentrations of bile acids in the small intestine lamina propria (siLP). CD4+ T effector (Teff) cells upregulate expression of the xenobiotic transporter MDR1 (encoded by Abcb1a) in the siLP to prevent bile acid toxicity and suppress Crohn's disease-like small bowel inflammation4. Here we identify the nuclear xenobiotic receptor CAR (encoded by Nr1i3) as a regulator of MDR1 expression in T cells that can safeguard against bile acid toxicity and inflammation in the mouse small intestine. Activation of CAR induced large-scale transcriptional reprogramming in Teff cells that infiltrated the siLP, but not the colon. CAR induced the expression of not only detoxifying enzymes and transporters in siLP Teff cells, as in hepatocytes, but also the key anti-inflammatory cytokine IL-10. Accordingly, CAR deficiency in T cells exacerbated bile acid-driven ileitis in T cell-reconstituted Rag1-/- or Rag2-/- mice, whereas pharmacological activation of CAR suppressed it. These data suggest that CAR acts locally in T cells that infiltrate the small intestine to detoxify bile acids and resolve inflammation. Activation of this program offers an unexpected strategy to treat small bowel Crohn's disease and defines lymphocyte sub-specialization in the small intestine.


Assuntos
Ácidos e Sais Biliares/metabolismo , Regulação da Expressão Gênica , Intestino Delgado/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Linfócitos T/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Linfócitos T CD4-Positivos/metabolismo , Doença de Crohn/metabolismo , Feminino , Ileíte/metabolismo , Inflamação/metabolismo , Interleucina-10/biossíntese , Interleucina-10/genética , Intestino Delgado/citologia , Camundongos
13.
Parasit Vectors ; 14(1): 153, 2021 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-33712075

RESUMO

BACKGROUND: Toxoplasma gondii is a parasite that primarily infects through the oral route. Nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) play crucial roles in the immune responses generated during parasitic infection and also drive the inflammatory response against invading parasites. However, little is known about the regulation of NLRs and inflammasome activation in T. gondii-infected human small intestinal epithelial (FHs 74 Int) cells. METHODS: FHs 74 Int cells infected with T. gondii were subsequently evaluated for morphological changes, cytotoxicity, expression profiles of NLRs, inflammasome components, caspase-cleaved interleukins (ILs), and the mechanisms of NLRP3 and NLRP6 inflammasome activation. Immunocytochemistry, lactate dehydrogenase assay, reverse transcription polymerase chain reaction (RT-PCR), real-time quantitative RT-PCR, and western blotting techniques were utilized for analysis. RESULTS: Under normal and T. gondii-infected conditions, members of the NLRs, inflammasome components and caspase-cleaved ILs were expressed in the FHs Int 74 cells, except for NLRC3, NLRP5, and NLRP9. Among the NLRs, mRNA expression of NOD2, NLRP3, NLRP6, and NAIP1 was significantly increased in T. gondii-infected cells, whereas that of NLRP2, NLRP7, and CIITA mRNAs decreased significantly in a time-dependent manner. In addition, T. gondii infection induced NLRP3, NLRP6 and NLRC4 inflammasome activation and production of IL-1ß, IL-18, and IL-33 in FHs 74 Int cells. T. gondii-induced NLRP3 inflammasome activation was strongly associated with the phosphorylation of p38 MAPK; however, JNK1/2 had a weak effect. NLRP6 inflammasome activation was not related to the MAPK pathway in FHs 74 Int cells. CONCLUSIONS: This study highlighted the expression profiles of NLRs and unraveled the underlying mechanisms of NLRP3 inflammasome activation in T. gondii-infected FHs 74 Int cells. These findings may contribute to understanding of the mucosal and innate immune responses induced by the NLRs and inflammasomes during T. gondii infection in FHs 74 Int cells.


Assuntos
Células Epiteliais/parasitologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata , Inflamassomos/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Proteínas NLR/genética , Linhagem Celular , Humanos , Inflamassomos/imunologia , Intestino Delgado/citologia , Intestino Delgado/parasitologia , Proteínas NLR/classificação , Proteínas NLR/imunologia , RNA Mensageiro
14.
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
15.
Neurochem Res ; 46(6): 1410-1422, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33656693

RESUMO

Propofol and dexmedetomidine are popular used for sedation in ICU, however, inadequate attention has been paid to their effect on gastrointestinal tract (GIT) motility. Present study aimed to compare the effect of propofol and dexmedetomidine on GIT motility at parallel level of sedation and explore the possible mechanism. Male C57BL/6 mice (8-10 weeks) were randomly divided into control, propofol and dexmedetomidine group. After intraperitoneal injection of propofol or dexmedetomidine, comparable sedative level was confirmed by sedative score, physiological parameters and electroencephalogram (EEG). Different segments of GIT motility in vivo (gastric emptying, small intestine transit, distal colon bead expulsion, stool weight and number of fecal pellets, gastrointestinal transit and whole gut transit time) and colonic migrating motor complexes (CMMCs) pattern in vitro were evaluated. The Ca2+ response of primary enteric glia was examined under the treatment of propofol or dexmedetomidine. There is little difference in physiological parameters and composite permutation entropy index (CPEI) between administration of 50 mg/kg propofol and 40 µg/kg dexmedetomidine, indicated that parallel level of sedation was reached. Data showed that propofol and dexmedetomidine had significantly inhibitory effect on GIT motility while dexmedetomidine was stronger. Also, the amplitude (ΔF/F0) of Ca2+ response in primary enteric glia was attenuated after treated with the sedatives while the effect of dexmedetomidine was greater than propofol. These findings demonstrated that dexmedetomidine caused stronger inhibitory effects on GIT motility in sedative mice, which may involve impaired Ca2+ response in enteric glia. Hence, dexmedetomidine should be carefully applied especially for potential GIT dysmotility patient.


Assuntos
Cálcio/metabolismo , Dexmedetomidina/farmacologia , Motilidade Gastrointestinal/efeitos dos fármacos , Hipnóticos e Sedativos/farmacologia , Neuroglia/efeitos dos fármacos , Propofol/farmacologia , Animais , Células Cultivadas , Colo/efeitos dos fármacos , Defecação/efeitos dos fármacos , Esvaziamento Gástrico/efeitos dos fármacos , Trânsito Gastrointestinal/efeitos dos fármacos , Intestino Delgado/citologia , Intestino Delgado/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL
16.
Int J Mol Sci ; 22(4)2021 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-33672854

RESUMO

Enteric glial cells (EGCs) of the enteric nervous system are critically involved in the maintenance of intestinal epithelial barrier function (IEB). The underlying mechanisms remain undefined. Glial cell line-derived neurotrophic factor (GDNF) contributes to IEB maturation and may therefore be the predominant mediator of this process by EGCs. Using GFAPcre x Ai14floxed mice to isolate EGCs by Fluorescence-activated cell sorting (FACS), we confirmed that they synthesize GDNF in vivo as well as in primary cultures demonstrating that EGCs are a rich source of GDNF in vivo and in vitro. Co-culture of EGCs with Caco2 cells resulted in IEB maturation which was abrogated when GDNF was either depleted from EGC supernatants, or knocked down in EGCs or when the GDNF receptor RET was blocked. Further, TNFα-induced loss of IEB function in Caco2 cells and in organoids was attenuated by EGC supernatants or by recombinant GDNF. These barrier-protective effects were blunted when using supernatants from GDNF-deficient EGCs or by RET receptor blockade. Together, our data show that EGCs produce GDNF to maintain IEB function in vitro through the RET receptor.


Assuntos
Sistema Nervoso Entérico/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Mucosa Intestinal/metabolismo , Neuroglia/metabolismo , Animais , Células CACO-2 , Células Cultivadas , Técnicas de Cocultura , Meios de Cultivo Condicionados/farmacologia , Sistema Nervoso Entérico/efeitos dos fármacos , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Fator Neurotrófico Derivado de Linhagem de Célula Glial/farmacologia , Humanos , Mucosa Intestinal/efeitos dos fármacos , Intestino Delgado/citologia , Intestino Delgado/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Neuroglia/efeitos dos fármacos , Permeabilidade/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Fator de Necrose Tumoral alfa/farmacologia
17.
Vet Res ; 52(1): 33, 2021 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-33632315

RESUMO

In livestock species, the monolayer of epithelial cells covering the digestive mucosa plays an essential role for nutrition and gut barrier function. However, research on farm animal intestinal epithelium has been hampered by the lack of appropriate in vitro models. Over the past decade, methods to culture livestock intestinal organoids have been developed in pig, bovine, rabbit, horse, sheep and chicken. Gut organoids from farm animals are obtained by seeding tissue-derived intestinal epithelial stem cells in a 3-dimensional culture environment reproducing in vitro the stem cell niche. These organoids can be generated rapidly within days and are formed by a monolayer of polarized epithelial cells containing the diverse differentiated epithelial progeny, recapitulating the original structure and function of the native epithelium. The phenotype of intestinal organoids is stable in long-term culture and reflects characteristics of the digestive segment of origin. Farm animal intestinal organoids can be amplified in vitro, cryopreserved and used for multiple experiments, allowing an efficient reduction of the use of live animals for experimentation. Most of the studies using livestock intestinal organoids were used to investigate host-microbe interactions at the epithelial surface, mainly focused on enteric infections with viruses, bacteria or parasites. Numerous other applications of farm animal intestinal organoids include studies on nutrient absorption, genome editing and bioactive compounds screening relevant for agricultural, veterinary and biomedical sciences. Further improvements of the methods used to culture intestinal organoids from farm animals are required to replicate more closely the intestinal tissue complexity, including the presence of non-epithelial cell types and of the gut microbiota. Harmonization of the methods used to culture livestock intestinal organoids will also be required to increase the reproducibility of the results obtained in these models. In this review, we summarize the methods used to generate and cryopreserve intestinal organoids in farm animals, present their phenotypes and discuss current and future applications of this innovative culture system of the digestive epithelium.


Assuntos
Animais Domésticos/anatomia & histologia , Técnicas de Cultura de Células/veterinária , Criopreservação/veterinária , Intestino Grosso/citologia , Intestino Delgado/citologia , Organoides/citologia , Animais , Técnicas de Cultura de Células/métodos , Criopreservação/métodos , Células Epiteliais/citologia , Mucosa Intestinal/citologia
18.
J Exp Med ; 218(4)2021 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-33533917

RESUMO

Conventional CD4+ T cells are differentiated into CD4+CD8αα+ intraepithelial lymphocytes (IELs) in the intestine; however, the roles of intestinal epithelial cells (IECs) are poorly understood. Here, we showed that IECs expressed MHC class II (MHC II) and programmed death-ligand 1 (PD-L1) induced by the microbiota and IFN-γ in the distal part of the small intestine, where CD4+ T cells were transformed into CD4+CD8αα+ IELs. Therefore, IEC-specific deletion of MHC II and PD-L1 hindered the development of CD4+CD8αα+ IELs. Intracellularly, PD-1 signals supported the acquisition of CD8αα by down-regulating the CD4-lineage transcription factor, T helper-inducing POZ/Krüppel-like factor (ThPOK), via the Src homology 2 domain-containing tyrosine phosphatase (SHP) pathway. Our results demonstrate that noncanonical antigen presentation with cosignals from IECs constitutes niche adaptation signals to develop tissue-resident CD4+CD8αα+ IELs.


Assuntos
Antígeno B7-H1/deficiência , Linfócitos T CD4-Positivos/imunologia , Antígenos CD8/metabolismo , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular/genética , Células Epiteliais/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Mucosa Intestinal/imunologia , Linfócitos Intraepiteliais/imunologia , Transferência Adotiva/métodos , Animais , Apresentação do Antígeno/genética , Apresentação do Antígeno/imunologia , Antígeno B7-H1/genética , Diferenciação Celular/imunologia , Células Cultivadas , Microbioma Gastrointestinal/imunologia , Antígenos de Histocompatibilidade Classe II/genética , Mucosa Intestinal/citologia , Intestino Delgado/citologia , Intestino Delgado/imunologia , Espaço Intracelular/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
19.
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
20.
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
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