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1.
Immunity ; 49(3): 545-559.e5, 2018 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-30193848

RESUMO

Although the mammalian microbiota is well contained within the intestine, it profoundly shapes development and metabolism of almost every host organ. We questioned the range and depth of microbial metabolite penetration into the host, and how this is modulated by intestinal immunity. Chemically identical microbial and host metabolites were distinguished by stable isotope tracing from 13C-labeled live non-replicating Escherichia coli, differentiating 12C host isotopes with high-resolution mass spectrometry. Hundreds of endogenous microbial compounds penetrated 23 host tissues and fluids after intestinal exposure: subsequent 12C host metabolome signatures included lipidemia, reduced glycolysis, and inflammation. Penetrant bacterial metabolites from the small intestine were rapidly cleared into the urine, whereas induced antibodies curtailed microbial metabolite exposure by accelerating intestinal bacterial transit into the colon where metabolite transport mechanisms are limiting. Pervasive penetration of microbial molecules can cause extensive host tissue responses: these are limited by immune and non-immune intestinal mucosal adaptations to the microbiota.


Assuntos
Anticorpos/metabolismo , Microbioma Gastrointestinal/fisiologia , Glicólise/imunologia , Hiperlipidemias/imunologia , Inflamação/imunologia , Mamíferos/imunologia , Animais , Anticorpos/imunologia , Radioisótopos de Carbono/análise , Interações Hospedeiro-Patógeno , Imunidade , Cadeias Pesadas de Imunoglobulinas/genética , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
2.
J Hepatol ; 73(3): 628-639, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32205193

RESUMO

BACKGROUND & AIMS: Paneth cells (PCs) synthesize and secrete antimicrobial peptides that are key mediators of host-microbe interactions, establishing a balance between intestinal microflora and enteric pathogens. We observed that their number increases in experimental portal hypertension and aimed to investigate the mechanisms by which these cells can contribute to the regulation of portal pressure. METHODS: We first treated Math1Lox/LoxVilcreERT2 mice with tamoxifen to induce the complete depletion of intestinal PCs. Subsequently, we performed partial portal vein or bile duct ligation. We then studied the effects of these interventions on hemodynamic parameters, proliferation of blood vessels and the expression of genes regulating angiogenesis. Intestinal organoids were cultured and exposed to different microbial products to study the composition of their secreted products (by proteomics) and their effects on the proliferation and tube formation of endothelial cells (ECs). In vivo confocal laser endomicroscopy was used to confirm the findings on blood vessel proliferation. RESULTS: Portal hypertension was significantly attenuated in PC-depleted mice compared to control mice and was associated with a decrease in portosystemic shunts. Depletion of PCs also resulted in a significantly decreased density of blood vessels in the intestinal wall and mesentery. Furthermore, we observed reduced expression of intestinal genes regulating angiogenesis in Paneth cell depleted mice using arrays and next generation sequencing. Tube formation and wound healing responses were significantly decreased in ECs treated with conditioned media from PC-depleted intestinal organoids exposed to intestinal microbiota-derived products. Proteomic analysis of conditioned media in the presence of PCs revealed an increase in factors regulating angiogenesis and additional metabolic processes. In vivo endomicroscopy showed decreased vascular proliferation in the absence of PCs. CONCLUSIONS: These results suggest that in response to intestinal flora and microbiota-derived factors, PCs secrete not only antimicrobial peptides, but also pro-angiogenic signaling molecules, thereby promoting intestinal and mesenteric angiogenesis and regulating portal hypertension. LAY SUMMARY: Paneth cells are present in the lining of the small intestine. They prevent the passage of bacteria from the intestine into the blood circulation by secreting substances to fight bacteria. In this paper, we discovered that these substances not only act against bacteria, but also increase the quantity of blood vessels in the intestine and blood pressure in the portal vein. This is important, because high blood pressure in the portal vein may result in several complications which could be targeted with novel approaches.


Assuntos
Infecções por Escherichia coli/metabolismo , Escherichia coli/metabolismo , Microbioma Gastrointestinal/genética , Hipertensão Portal/metabolismo , Hipertensão Portal/microbiologia , Neovascularização Patológica/metabolismo , Celulas de Paneth/metabolismo , Animais , Meios de Cultivo Condicionados , Modelos Animais de Doenças , Infecções por Escherichia coli/microbiologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Intestino Delgado/metabolismo , Intestino Delgado/microbiologia , Masculino , Camundongos , Camundongos Transgênicos , Organoides/metabolismo , Organoides/microbiologia , Celulas de Paneth/efeitos dos fármacos , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Proteoma , Proteômica/métodos , Tamoxifeno/farmacologia
3.
Digestion ; 101(6): 717-729, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31550710

RESUMO

BACKGROUND: The gut-liver-axis presents the pathophysiological hallmark for multiple liver diseases and has been proposed to be modulated during stress and shock. Access to the gut-liver-axis needs crossing of the mucus and gut-vascular barrier. The role of ß-adrenoreceptor-activation for both barriers has not been defined and is characterized here. METHODS: Splanchnic ß-adrenergic stimulation was achieved by chronic intraperitoneal application of isoproterenol via alzet-pump in vivo. The intestinal permeability and gut-vascular barrier function was assessed in ileal loop experiments. The extravasation of predefined sizes of fluorescence isothiocyanate (FITC)-dextran molecules in ileal microcirculation was evaluated by intravital confocal laser endomicroscopy in vivo. Mucus parameters thickness, goblet cell count and mucin-expression were assessed by stereomicroscopy, immunostaining and RNA-sequencing respectively. Ileal lamina propria (LP) as well as mesenteric lymph node mononuclear cells was assessed by FACS. RESULTS: Healthy mice lack translocation of 4 kDa-FITC-dextran from the small intestine to the liver, whereas isoproterenol-treated mice demonstrate pathological translocation (PBT). Mucus layer is reduced in thickness with loss of goblet-cells and mucin-2-staining and -expression in isoproterenol-treated animals under standardized gnotobiotic conditions. Isoproterenol disrupts the gut vascular barrier displaying Ileal extravasation of large-sized 70- and 150 kDa-FITC-dextran. This pathological endothelial permeability and accessibility induced by isoproterenol associates with an augmented expression of plasmalemmal-vesicle-associated-protein-1 in intestinal vessel. Ileal LP after isoproterenol treatment contains more CD11c+-dendritic cells (DC) with increased appearance of CCR7+ DC in mesenteric lymph nodes. CONCLUSIONS: Isoproterenol impairs the intestinal muco-epithelial and endothelial-vascular barrier promoting PBT to the liver. This barrier dysfunction on multiple levels potentially can contribute to liver injury induced by catecholamines during states of increased ß-adrenergic drive.


Assuntos
Mucosa Intestinal , Isoproterenol , Muco , Animais , Isoproterenol/farmacologia , Fígado , Camundongos , Permeabilidade
4.
J Hepatol ; 71(6): 1216-1228, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31419514

RESUMO

BACKGROUND & AIMS: Fatty liver disease, including non-alcoholic fatty liver (NAFLD) and steatohepatitis (NASH), has been associated with increased intestinal barrier permeability and translocation of bacteria or bacterial products into the blood circulation. In this study, we aimed to unravel the role of both intestinal barrier integrity and microbiota in NAFLD/NASH development. METHODS: C57BL/6J mice were fed with high-fat diet (HFD) or methionine-choline-deficient diet for 1 week or longer to recapitulate aspects of NASH (steatosis, inflammation, insulin resistance). Genetic and pharmacological strategies were then used to modulate intestinal barrier integrity. RESULTS: We show that disruption of the intestinal epithelial barrier and gut vascular barrier (GVB) are early events in NASH pathogenesis. Mice fed HFD for only 1 week undergo a diet-induced dysbiosis that drives GVB damage and bacterial translocation into the liver. Fecal microbiota transplantation from HFD-fed mice into specific pathogen-free recipients induces GVB damage and epididymal adipose tissue enlargement. GVB disruption depends on interference with the WNT/ß-catenin signaling pathway, as shown by genetic intervention driving ß-catenin activation only in endothelial cells, preventing GVB disruption and NASH development. The bile acid analogue and farnesoid X receptor agonist obeticholic acid (OCA) drives ß-catenin activation in endothelial cells. Accordingly, pharmacologic intervention with OCA protects against GVB disruption, both as a preventive and therapeutic agent. Importantly, we found upregulation of the GVB leakage marker in the colon of patients with NASH. CONCLUSIONS: We have identified a new player in NASH development, the GVB, whose damage leads to bacteria or bacterial product translocation into the blood circulation. Treatment aimed at restoring ß-catenin activation in endothelial cells, such as administration of OCA, protects against GVB damage and NASH development. LAY SUMMARY: The incidence of fatty liver disease is reaching epidemic levels in the USA, with more than 30% of adults having NAFLD (non-alcoholic fatty liver disease), which can progress to more severe non-alcoholic steatohepatitis (NASH). Herein, we show that disruption of the intestinal epithelial barrier and gut vascular barrier are early events in the development of NASH. We show that the drug obeticholic acid protects against barrier disruption and thereby prevents the development of NASH, providing further evidence for its use in the prevention or treatment of NASH.


Assuntos
Translocação Bacteriana/efeitos dos fármacos , Permeabilidade Capilar , Ácido Quenodesoxicólico/análogos & derivados , Microbioma Gastrointestinal/fisiologia , Mucosa Intestinal , Hepatopatia Gordurosa não Alcoólica , Animais , Permeabilidade Capilar/efeitos dos fármacos , Permeabilidade Capilar/fisiologia , Ácido Quenodesoxicólico/farmacologia , Dieta Hiperlipídica , Modelos Animais de Doenças , Disbiose/imunologia , Inflamação/metabolismo , Resistência à Insulina , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Fígado/patologia , Camundongos , Hepatopatia Gordurosa não Alcoólica/imunologia , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/terapia , Substâncias Protetoras/farmacologia
5.
J Hepatol ; 71(6): 1126-1140, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31295531

RESUMO

BACKGROUND & AIMS: Pathological bacterial translocation (PBT) in cirrhosis is the hallmark of spontaneous bacterial infections, increasing mortality several-fold. Increased intestinal permeability is known to contribute to PBT in cirrhosis, although the role of the mucus layer has not been addressed in detail. A clear route of translocation for luminal intestinal bacteria is yet to be defined, but we hypothesize that the recently described gut-vascular barrier (GVB) is impaired in experimental portal hypertension, leading to increased accessibility of the vascular compartment for translocating bacteria. MATERIALS: Cirrhosis was induced in mouse models using bile-duct ligation (BDL) and CCl4. Pre-hepatic portal-hypertension was induced by partial portal vein ligation (PPVL). Intestinal permeability was compared in these mice after GFP-Escherichia coli or different sized FITC-dextrans were injected into the intestine. RESULTS: Healthy and pre-hepatic portal-hypertensive (PPVL) mice lack translocation of FITC-dextran and GFP-E. coli from the small intestine to the liver, whereas BDL and CCl4-induced cirrhotic mice demonstrate pathological translocation, which is not altered by prior thoracic-duct ligation. The mucus layer is reduced in thickness, with loss of goblet cells and Muc2-staining and expression in cirrhotic but not PPVL mice. These changes are associated with bacterial overgrowth in the inner mucus layer and pathological translocation of GFP-E. coli through the ileal epithelium. GVB is profoundly altered in BDL and CCl4-mice with Ileal extravasation of large-sized 150 kDa-FITC-dextran, but only slightly altered in PPVL mice. This pathological endothelial permeability and accessibility in cirrhotic mice is associated with augmented expression of PV1 in intestinal vessels. OCA but not fexaramine stabilizes the GVB, whereas both FXR-agonists ameliorate gut to liver translocation of GFP-E. coli. CONCLUSIONS: Cirrhosis, but not portal hypertension per se, grossly impairs the endothelial and muco-epithelial barriers, promoting PBT to the portal-venous circulation. Both barriers appear to be FXR-modulated, with FXR-agonists reducing PBT via the portal-venous route. LAY SUMMARY: For intestinal bacteria to enter the systemic circulation, they must cross the mucus and epithelial layer, as well as the gut-vascular barrier. Cirrhosis disrupts all 3 of these barriers, giving bacteria access to the portal-venous circulation and thus, the gut-liver axis. Diminished luminal bile acid availability, cirrhosis and the associated reduction in farnesoid x receptor (FXR) signaling seem, at least partly, to mediate these changes, as FXR-agonists reduce bacterial translocation via the portal-venous route to the liver in cirrhosis.


Assuntos
Translocação Bacteriana/efeitos dos fármacos , Permeabilidade Capilar , Dextranos/farmacocinética , Escherichia coli , Microbioma Gastrointestinal/fisiologia , Mucosa Intestinal , Cirrose Hepática Experimental , Receptores Citoplasmáticos e Nucleares , Animais , Ácidos e Sais Biliares/metabolismo , Permeabilidade Capilar/efeitos dos fármacos , Permeabilidade Capilar/fisiologia , Modelos Animais de Doenças , Escherichia coli/isolamento & purificação , Escherichia coli/fisiologia , Hipertensão Portal/metabolismo , Hipertensão Portal/fisiopatologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Cirrose Hepática Experimental/metabolismo , Cirrose Hepática Experimental/microbiologia , Cirrose Hepática Experimental/fisiopatologia , Camundongos , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores Citoplasmáticos e Nucleares/metabolismo
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