RESUMO
The microbiological environment and their corresponding secreted metabolite spectrum are an essential modulator of the enterocyte function, effecting the whole organism. Intestinal porcine jejunal epithelial cell line (IPEC-J2) is an established in vitro model for differentiation of enterocytes in different cell culture models. An improved oxygen supply seems to be the main reason for differentiation in an air-liquid-interface culture, but this has not yet been conclusively clarified. In this context, the nutrition of the cell and its influence on the metabolism is also of crucial importance. The interest in short-chain fatty acids (SCFAs) has grown steadily in recent years due to their clinical relevance in certain diseases such as multiple sclerosis and other inflammatory diseases, but not much is known of FFAR2 and FFAR3 (free fatty acid receptor 2 and 3) in pigs. We want to address the questions: 1. about the distribution of FFAR2 and FFAR3 in vivo and in vitro in sus scrofa 2. whether there is an influence of propionic acid, glucose content and cultivation on metabolism of enterocytes? The morphological analysis of FFAR2 and FFAR3 in vivo was investigated through immunostaining of frozen sections of the porcine gut segments jejunum, ileum and colon. Both receptors are expressed along the gut and were found in the smooth muscle cells of the tunica muscularis and lamina muscularis mucosae. Furthermore, a high expression of FFAR2 and a low expression of FFAR3 in the enteric nerve system was also observed in jejunum, ileum and colon of sus scrofa. In addition, FFAR2 and FFAR3 within the vessels was investigated. FFAR3 showed a strong expression on endothelial cells of veins and lymphatic vessels but was not detectable on arteries. Furthermore, we demonstrate for the first time, FFAR2 and FFAR3 in IPEC-J2 cells on RNA- and protein level, as well as with confocal microscopy. In addition, ENO1 and NDUFA4 were investigated on RNA-level in IPEC-J2 cells as 2 important genes, which play an essential role in metabolism. Here, NDUFA4 is detected in the model animal sus scrofa as well as in the porcine cell line IPEC-J2. A potential impact of propionic acid and/or glucose and/or cultivation method on the metabolism of the cells was tested with the Seahorse analyzer. Here, a significant higher ECAR was observed in the SMC than in the OCR. In summary, we were able to show that the cultivation system appears to have a greater influence than the medium composition or nutrition of the cells. However, this can be modulated by incubation time or combination of different SCFAs.
Assuntos
Glucose , Propionatos , Animais , Propionatos/metabolismo , Glucose/metabolismo , Suínos , Linhagem Celular , Técnicas de Cultura de Células/métodos , Metaboloma , Receptores Acoplados a Proteínas G/metabolismo , Jejuno/metabolismo , Jejuno/citologia , Enterócitos/metabolismo , Mucosa Intestinal/metabolismoRESUMO
Human intestinal enteroids (HIEs) are gaining recognition as physiologically relevant models of the intestinal epithelium. While HIEs from adults are used extensively in biomedical research, few studies have used HIEs from infants. Considering the dramatic developmental changes that occur during infancy, it is important to establish models that represent infant intestinal characteristics and physiological responses. We established jejunal HIEs from infant surgical samples and performed comparisons to jejunal HIEs from adults using RNA sequencing (RNA-Seq) and morphologic analyses. We then validated differences in key pathways through functional studies and determined whether these cultures recapitulate known features of the infant intestinal epithelium. RNA-Seq analysis showed significant differences in the transcriptome of infant and adult HIEs, including differences in genes and pathways associated with cell differentiation and proliferation, tissue development, lipid metabolism, innate immunity, and biological adhesion. Validating these results, we observed a higher abundance of cells expressing specific enterocyte, goblet cell, and enteroendocrine cell markers in differentiated infant HIE monolayers, and greater numbers of proliferative cells in undifferentiated 3D cultures. Compared to adult HIEs, infant HIEs portray characteristics of an immature gastrointestinal epithelium including significantly shorter cell height, lower epithelial barrier integrity, and lower innate immune responses to infection with an oral poliovirus vaccine. HIEs established from infant intestinal tissues reflect characteristics of the infant gut and are distinct from adult cultures. Our data support the use of infant HIEs as an ex vivo model to advance studies of infant-specific diseases and drug discovery for this population. IMPORTANCE: Tissue or biopsy stem cell-derived human intestinal enteroids are increasingly recognized as physiologically relevant models of the human gastrointestinal epithelium. While enteroids from adults and fetal tissues have been extensively used for studying many infectious and non-infectious diseases, there are few reports on enteroids from infants. We show that infant enteroids exhibit both transcriptomic and morphological differences compared to adult cultures. They also differ in functional responses to barrier disruption and innate immune responses to infection, suggesting that infant and adult enteroids are distinct model systems. Considering the dramatic changes in body composition and physiology that begin during infancy, tools that appropriately reflect intestinal development and diseases are critical. Infant enteroids exhibit key features of the infant gastrointestinal epithelium. This study is significant in establishing infant enteroids as age-appropriate models for infant intestinal physiology, infant-specific diseases, and responses to pathogens.
Assuntos
Mucosa Intestinal , Humanos , Lactente , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Adulto , Diferenciação Celular , Jejuno/citologia , Jejuno/imunologia , Transcriptoma , Organoides , Imunidade Inata , Feminino , Masculino , Recém-Nascido , EnterócitosRESUMO
Acute intestinal mucositis is a common off-target effect of chemotherapy, leading to co-morbidities such as vomiting, diarrhea, sepsis, and death. We previously demonstrated that the presence of enteric bacteria modulates the extent of jejunal epithelial damage induced by doxorubicin (DXR) in mice. Despite conventional thinking of the crypt as a sterile environment, recent evidence suggests that bacterial signaling influences aISC function. In this study, we labeled aISCs using transgenic Lgr5-driven fluorescence or with immunostaining for OLFM4. We examined the effect of DXR in both germ free (GF) mice and mice depleted of microbiota using an established antimicrobial treatment protocol (AMBx). We found differences in DXR-induced loss of aISCs between GF mice and mice treated with AMBx. aISCs were decreased after DXR in GF mice, whereas AMBx mice retained aISC expression after DXR. Neither group of mice exhibited an inflammatory response to DXR, suggesting the difference in aISC retention was not due to differences in local tissue inflammation. Therefore, we suspected that there was a protective microbial signal present in the AMBx mice that was not present in the GF mice. 16S rRNA sequencing of jejunal luminal contents demonstrated that AMBx altered the fecal and jejunal microbiota. In the jejunal contents, AMBx mice had increased abundance of Ureaplasma and Burkholderia. These results suggest pro-survival signaling from microbiota in AMBx-treated mice to the aISCs, and that this signaling maintains aISCs in the face of chemotherapeutic injury. Manipulation of the enteric microbiota presents a therapeutic target for reducing the severity of chemotherapy-associated mucositis.
Assuntos
Antineoplásicos/efeitos adversos , Doxorrubicina/efeitos adversos , Jejuno/efeitos dos fármacos , Mucosite/prevenção & controle , Células-Tronco/efeitos dos fármacos , Administração Oral , Animais , Antibacterianos/administração & dosagem , Antibacterianos/farmacologia , Antineoplásicos/administração & dosagem , Bactérias/classificação , Bactérias/efeitos dos fármacos , Bactérias/genética , Bactérias/isolamento & purificação , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/administração & dosagem , Microbioma Gastrointestinal/efeitos dos fármacos , Vida Livre de Germes , Humanos , Jejuno/citologia , Jejuno/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Mucosite/microbiologia , Células-Tronco/citologia , Fatores de TempoRESUMO
Porcine epidemic diarrhea virus (PEDV) infection leads to diarrhea and subsequently to decreased feed efficiency and growth in weaned pigs. Given that few studies have addressed the host-virus interaction in vivo, this study focused on endoplasmic reticulum (ER) stress and unfolded protein response (UPR) in jejunal epithelial cells during PEDV infection. Eight-week-old pigs (n = 64) were orally inoculated with PEDV IN19338 strain (n = 40) or sham-inoculated (n = 24) and analyzed for PEDV viral RNA shedding using reverse transcription-quantitative polymerase chain reaction and for viral antigen within enterocytes using immunohistochemistry (IHC). ER stress was analyzed in a subset of 9 PEDV-inoculated pigs with diarrhea, detectable viral RNA, and viral antigen (PEDV-immunopositive pigs). Compared with control pigs, PEDV-immunopositive pigs had a reduced ratio of villus height to crypt depth in the jejunum (P = .002, n = 9 per group), consistent with intestinal injury. The protein levels of ATF6, IRE1, PERK, XBP1u, ATF4, GRP78, and caspase-3 were assessed in jejunal epithelial cells at the villus tips via IHC. Both ER stress and UPR were demonstrated in PEDV-immunopositive pigs by the increased expression of ATF6 (P = .047), IRE1 (P = .007), and ATF4 (P = .001). The expression of GRP78 (P = .024) and caspase-3 (P = .004) were also increased, indicating an accompanying increase in ER protein folding capacity and apoptosis. Overall, these results reveal that PEDV infection induces ER stress and UPR in intestinal epithelial cells of weaned pigs.
Assuntos
Infecções por Coronavirus/veterinária , Estresse do Retículo Endoplasmático , Células Epiteliais/virologia , Vírus da Diarreia Epidêmica Suína , Resposta a Proteínas não Dobradas , Animais , Chaperona BiP do Retículo Endoplasmático , Jejuno/citologia , SuínosRESUMO
Circadian rhythms regulate diverse aspects of gastrointestinal physiology ranging from the composition of microbiota to motility. However, development of the intestinal circadian clock and detailed mechanisms regulating circadian physiology of the intestine remain largely unknown. In this report, we show that both pluripotent stem cell-derived human intestinal organoids engrafted into mice and patient-derived human intestinal enteroids possess circadian rhythms and demonstrate circadian phase-dependent necrotic cell death responses to Clostridium difficile toxin B (TcdB). Intriguingly, mouse and human enteroids demonstrate anti-phasic necrotic cell death responses to TcdB. RNA-Seq analysis shows that ~3-10% of the detectable transcripts are rhythmically expressed in mouse and human enteroids. Remarkably, we observe anti-phasic gene expression of Rac1, a small GTPase directly inactivated by TcdB, between mouse and human enteroids, and disruption of Rac1 abolishes clock-dependent necrotic cell death responses. Our findings uncover robust functions of circadian rhythms regulating clock-controlled genes in both mouse and human enteroids governing organism-specific, circadian phase-dependent necrotic cell death responses, and lay a foundation for human organ- and disease-specific investigation of clock functions using human organoids for translational applications.
Assuntos
Relógios Circadianos , Jejuno/citologia , Organoides/metabolismo , Animais , Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/toxicidade , Morte Celular , Células Cultivadas , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Organoides/efeitos dos fármacos , Organoides/fisiologia , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Epigenetic mechanisms are gatekeepers for the gene expression patterns that establish and maintain cellular identity in mammalian development, stem cells and adult homeostasis. Amongst many epigenetic marks, methylation of histone 3 lysine 4 (H3K4) is one of the most widely conserved and occupies a central position in gene expression. Mixed lineage leukemia 1 (MLL1/KMT2A) is the founding mammalian H3K4 methyltransferase. It was discovered as the causative mutation in early onset leukemia and subsequently found to be required for the establishment of definitive hematopoiesis and the maintenance of adult hematopoietic stem cells. Despite wide expression, the roles of MLL1 in non-hematopoietic tissues remain largely unexplored. To bypass hematopoietic lethality, we used bone marrow transplantation and conditional mutagenesis to discover that the most overt phenotype in adult Mll1-mutant mice is intestinal failure. MLL1 is expressed in intestinal stem cells (ISCs) and transit amplifying (TA) cells but not in the villus. Loss of MLL1 is accompanied by loss of ISCs and a differentiation bias towards the secretory lineage with increased numbers and enlargement of goblet cells. Expression profiling of sorted ISCs revealed that MLL1 is required to promote expression of several definitive intestinal transcription factors including Pitx1, Pitx2, Foxa1, Gata4, Zfp503 and Onecut2, as well as the H3K27me3 binder, Bahcc1. These results were recapitulated using conditional mutagenesis in intestinal organoids. The stem cell niche in the crypt includes ISCs in close association with Paneth cells. Loss of MLL1 from ISCs promoted transcriptional changes in Paneth cells involving metabolic and stress responses. Here we add ISCs to the MLL1 repertoire and observe that all known functions of MLL1 relate to the properties of somatic stem cells, thereby highlighting the suggestion that MLL1 is a master somatic stem cell regulator.
Assuntos
Células-Tronco Adultas/fisiologia , Diferenciação Celular/genética , Histona-Lisina N-Metiltransferase/genética , Insuficiência Intestinal/genética , Mucosa Intestinal/patologia , Proteína de Leucina Linfoide-Mieloide/genética , Animais , Transplante de Medula Óssea , Metilação de DNA , Modelos Animais de Doenças , Epigênese Genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Insuficiência Intestinal/patologia , Mucosa Intestinal/citologia , Jejuno/citologia , Jejuno/patologia , Camundongos , Camundongos Transgênicos , Mutagênese , Mutação , Proteína de Leucina Linfoide-Mieloide/metabolismo , Nicho de Células-TroncoRESUMO
The human intestine contains numerous mononuclear phagocytes (MNP), including subsets of conventional dendritic cells (cDC), macrophages (Mf) and monocytes, each playing their own unique role within the intestinal immune system and homeostasis. The ability to isolate and interrogate MNPs from fresh human tissue is crucial if we are to understand the role of these cells in homeostasis, disease settings and immunotherapies. However, liberating these cells from tissue is problematic as many of the key surface identification markers they express are susceptible to enzymatic cleavage and they are highly susceptible to cell death. In addition, the extraction process triggers immunological activation/maturation which alters their functional phenotype. Identifying the evolving, complex and highly heterogenous repertoire of MNPs by flow cytometry therefore requires careful selection of digestive enzyme blends that liberate viable cells and preserve recognition epitopes involving careful selection of antibody clones to enable analysis and sorting for functional assays. Here we describe a method for the anatomical separation of mucosa and submucosa as well as isolating lymphoid follicles from human jejunum, ileum and colon. We also describe in detail the optimised enzyme digestion methods needed to acquire functionally immature and biologically functional intestinal MNPs. A comprehensive list of screened antibody clones is also presented which allows for the development of high parameter flow cytometry panels to discriminate all currently identified human tissue MNP subsets including pDCs, cDC1, cDC2 (langerin+ and langerin-), newly described DC3, monocytes, Mf1, Mf2, Mf3 and Mf4. We also present a novel method to account for autofluorescent signal from tissue macrophages. Finally, we demonstrate that these methods can successfully be used to sort functional, immature intestinal DCs that can be used for functional assays such as cytokine production assays.
Assuntos
Separação Celular , Colo/citologia , Citometria de Fluxo , Íleo/citologia , Mucosa Intestinal/citologia , Jejuno/citologia , Fagócitos/metabolismo , Biomarcadores/metabolismo , Células Cultivadas , Citocinas/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Monócitos/imunologia , Monócitos/metabolismo , Fagócitos/imunologia , FenótipoRESUMO
The cellular and tissue damage induced by oxidative stress (OS) contribute to a variety of human diseases, which include gastrointestinal diseases. Salvianolic acid B (Sal B), which is a natural polyphenolic acid in Salvia miltiorrhiza, exhibits prominent antioxidant properties. However, its precise function and molecular mechanisms in protecting normal intestine epithelium from OS-induced damage are still poorly defined. In this study, we tried to clarify this relationship. Here, we found Sal B addiction in the rat intestinal epithelial cell, IEC-6, prevented H2O2-induced cell viability decrease and apoptosis induction, ameliorated H2O2-induced intestinal epithelial barrier dysfunction and mitochondrial dysfunction, and suppressed H2O2-induced production of ROS to varying degrees, ranging from 10% to 30%. Moreover, by employing an ischemia reperfusion model of rats, we also discovered that Sal B treatment reversed ischemia and a reperfusion-caused decrease in villus height and crypt depth, decreased proliferation of enterocytes, and increased the apoptotic index in the jejunum and ileum. Mechanistically, Sal B treatment up-regulated the phosphorylated level of Akt and GSK3ß in enterocytes in vitro and in vivo, and PI3K inhibitor LY294002 treatment abrogated the protective effects of Sal B. Meanwhile, the inactivation of GSK3ß reversed the oxidative stress-induced apoptosis and mitochondrial dysfunction in IEC-6 cells. Together, our results demonstrated that the damage of intestinal epithelial cells in in vitro and in vivo models were both attenuated by Sal B treatment, and such antioxidant activity might very possibly be attributed to the activation of Akt/GSK3ß signaling.
Assuntos
Antioxidantes/farmacologia , Benzofuranos/farmacologia , Enteropatias/tratamento farmacológico , Animais , Antioxidantes/uso terapêutico , Apoptose/efeitos dos fármacos , Benzofuranos/uso terapêutico , Linhagem Celular , Modelos Animais de Doenças , Enterócitos/efeitos dos fármacos , Enterócitos/patologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Íleo/citologia , Íleo/efeitos dos fármacos , Íleo/patologia , Enteropatias/patologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/patologia , Jejuno/citologia , Jejuno/efeitos dos fármacos , Jejuno/patologia , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Estresse Oxidativo/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Salvia miltiorrhiza/química , Transdução de Sinais/efeitos dos fármacosRESUMO
Functional loss of myosin Vb (MYO5B) induces a variety of deficits in intestinal epithelial cell function and causes a congenital diarrheal disorder, microvillus inclusion disease (MVID). The impact of MYO5B loss on differentiated cell lineage choice has not been investigated. We quantified the populations of differentiated epithelial cells in tamoxifen-induced, epithelial cell-specific MYO5B-knockout (VilCreERT2 Myo5bfl/fl) mice utilizing digital image analysis. Consistent with our RNA-sequencing data, MYO5B loss induced a reduction in tuft cells in vivo and in organoid cultures. Paneth cells were significantly increased by MYO5B deficiency along with expansion of the progenitor cell zone. We further investigated the effect of lysophosphatidic acid (LPA) signaling on epithelial cell differentiation. Intraperitoneal LPA significantly increased tuft cell populations in both control and MYO5B-knockout mice. Transcripts for Wnt ligands were significantly downregulated by MYO5B loss in intestinal epithelial cells, whereas Notch signaling molecules were unchanged. Additionally, treatment with the Notch inhibitor dibenzazepine (DBZ) restored the populations of secretory cells, suggesting that the Notch pathway is maintained in MYO5B-deficient intestine. MYO5B loss likely impairs progenitor cell differentiation in the small intestine in vivo and in vitro, partially mediated by Wnt/Notch imbalance. Notch inhibition and/or LPA treatment may represent an effective therapeutic approach for treatment of MVID.
Assuntos
Síndromes de Malabsorção/genética , Microvilosidades/patologia , Mucolipidoses/genética , Miosina Tipo V/deficiência , Receptores Notch/metabolismo , Via de Sinalização Wnt/genética , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células Cultivadas , Dibenzazepinas/farmacologia , Modelos Animais de Doenças , Enterócitos/efeitos dos fármacos , Enterócitos/metabolismo , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/patologia , Jejuno/citologia , Jejuno/efeitos dos fármacos , Jejuno/patologia , Lisofosfolipídeos/farmacologia , Lisofosfolipídeos/uso terapêutico , Síndromes de Malabsorção/tratamento farmacológico , Síndromes de Malabsorção/patologia , Camundongos , Camundongos Knockout , Microvilosidades/genética , Mucolipidoses/tratamento farmacológico , Mucolipidoses/patologia , Miosina Tipo V/genética , Organoides , Cultura Primária de Células , Receptores Notch/antagonistas & inibidores , Células-Tronco/fisiologia , Via de Sinalização Wnt/efeitos dos fármacosRESUMO
Although CD8+ T cell tolerance to tissue-specific antigen (TSA) is essential for host homeostasis, the mechanisms underlying peripheral cross-tolerance and whether they may differ between tissue sites remain to be fully elucidated. Here, we demonstrate that peripheral cross-tolerance to intestinal epithelial cell (IEC)-derived antigen involves the generation and suppressive function of FoxP3+CD8+ T cells. FoxP3+CD8+ Treg generation was dependent on intestinal cDC1, whose absence led to a break of tolerance and epithelial destruction. Mechanistically, intestinal cDC1-derived PD-L1, TGFß, and retinoic acid contributed to the generation of gut-tropic CCR9+CD103+FoxP3+CD8+ Tregs Last, CD103-deficient CD8+ T cells lacked tolerogenic activity in vivo, indicating a role for CD103 in FoxP3+CD8+ Treg function. Our results describe a role for FoxP3+CD8+ Tregs in cross-tolerance in the intestine for which development requires intestinal cDC1.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Tolerância Periférica , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Transferência Adotiva , Animais , Apresentação de Antígeno , Autoantígenos/imunologia , Autoantígenos/metabolismo , Autoimunidade , Linfócitos T CD8-Positivos/metabolismo , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/metabolismo , Feminino , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Jejuno/citologia , Jejuno/imunologia , Camundongos , Modelos Animais , Cultura Primária de Células , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Reguladores/metabolismo , Quimeras de TransplanteRESUMO
The present study aims to investigate the roles of water intake in serotonin production and release in rat jejunum. We evaluated the changes in concentrations of serotonin in the portal vein and mesenteric lymph vessel induced by the intragastric administration of distilled water. The density of granules in enterochromaffin cells and the immunoreactivity of serotonin in the jejunal villi were investigated before and after water intake. The effects of intravenous administration of serotonin and/or ketanserin on mesenteric lymph flow and concentrations of albumin and IL-22 in the lymph were also addressed. Water intake increased serotonin concentration in the portal vein, but not in the mesenteric lymph vessel. The flux of serotonin through the portal vein was significantly larger than that through the mesenteric lymph vessel. Water intake decreased the density of granules in the enterochromaffin cells and increased the immunoreactivity of serotonin in the jejunal villi. The intravenous administration of serotonin increased significantly mesenteric lymph flow and the concentrations of albumin and IL-22; both were significantly reduced by the intravenous pretreatment with ketanserin. We showed that serotonin released from enterochromaffin cells by water intake was mainly transported through the portal vein. Additionally, serotonin in blood was found to increase mesenteric lymph formation with permeant albumin in the jejunal villi via the activation of 5-HT2 receptor.
Assuntos
Ingestão de Líquidos , Células Enterocromafins/metabolismo , Jejuno/metabolismo , Serotonina/metabolismo , Albuminas/metabolismo , Animais , Grânulos Citoplasmáticos/metabolismo , Interleucinas/sangue , Jejuno/citologia , Jejuno/fisiologia , Masculino , Veia Porta/fisiologia , Ratos , Ratos Sprague-Dawley , Serotonina/sangue , Interleucina 22RESUMO
Mycotoxin contamination in foods is a major risk factor for human and animal health due to its prevalence in cereals and their by-products. Deoxynivalenol (DON), mainly produced by Fusarium genera, is the most common mycotoxin detected in cereal products. Deoxynivalenol disrupts intestinal barrier function and decreases protein levels of tight junction proteins (TJP). However, the overall mechanism by which DON regulates specific TJP turnover and epithelial cell integrity remains unclear. Herein, we show that DON (2 µM) decreases the protein stability and accelerates the degradation of TJP in the lysosome. Interestingly, pretreatment of cells with dynasore (a dynamin-dependent endocytosis inhibitor) protected against DON-induced degradation of claudin-3 and 4. Immunofluorescence analysis also shows that the decreased membrane presence of claudin-4 and ZO-1 induced by DON is reversible with dynamin inhibition, whereas the pretreatment with cytochalasin D (an actin-dependent endocytosis inhibitor) reverses the degradation of claudin-1 and 4 induced by DON. We also show that the endocytosis and degradation of claudin-1 is regulated by p38 mitogen-activated protein kinase (MAPK), whereas the endocytosis of claudin-4 and ZO-1 is mediated by c-Jun-N-terminal kinase (JNK). Resveratrol, with JNK inhibitory activity, also prevents the endocytosis and degradation of claudin-4 and ZO-1 and protects against DON-induced decrease in transepithelial electrical resistance (TEER) and increase in FITC-dextran permeability. Collectively, this study, for the first time, shows that DON accelerates the endocytosis and degradation of TJP and this is regulated by the activation of p38 MAPK and JNK signaling pathways. Therefore, natural bioactive compounds with p38 MAPK and JNK inhibitory activities may be effective in preventing the DON-induced TJP disruption and preserve gut barrier function in vivo.
Assuntos
Jejuno/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas de Junções Íntimas/metabolismo , Tricotecenos/toxicidade , Animais , Linhagem Celular , Endocitose/efeitos dos fármacos , Jejuno/citologia , Jejuno/patologia , Permeabilidade , Estabilidade Proteica/efeitos dos fármacos , Suínos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Intestinal epithelial cells are tightly bound by tight junction proteins (TJP) which are dynamic and sensitive to environmental stress. However, the role of the endocytic pathway in the regulation of TJP abundance and tight junction integrity during nutrient stress is poorly understood. Therefore, this study was conducted to investigate the regulation of TJP abundance during nutrient starvation and the role of the endocytic mechanism in this process. IPEC-J2 cells were subjected to nutrient starvation in Krebs-Ringer bicarbonate buffer (KRB) and abundance of TJP, an indication of tight junction remodeling, was characterized with RT-PCR, western blotting and immunofluorescence. Abundance of TJP was dynamically regulated by nutrient starvation. The protein levels of claudin-1, 3, and 4 were initially downregulated within the first 6 hours of starvation, and then, increased thereafter (P < .01). However, there was no change in occludin and ZO-1. Lysosome and proteasome inhibitors were used to determine the contribution of these protein degradation pathways to the TJP remodeling. Short-term starvation-induced degradation of claudin-1, 3, and 4 was found to be lysosome dependent. Specifically, the downregulation of claudin-3 and 4 was via a dynamin-dependent, but clathrin and caveolae independent, endocytic pathway and this downregulation was partly reversed by amino acids supplementation. Interestingly, the re-synthesis of TJP with prolonged starvation partly depended on proteasome function. Collectively, this study, for the first time, elucidated a major role for dynamin-dependent endocytosis of claudin-3 and 4 during nutrient stress in intestinal epithelial cells. Therefore, transient endocytosis inhibition may be a potential mechanism for preserving tight junction integrity and function in metabolic or pathological states such as inflammatory bowel disease that involves destruction of intestinal epithelial TJP.
Assuntos
Endocitose , Enterócitos/metabolismo , Nutrientes/deficiência , Inanição/metabolismo , Junções Íntimas/metabolismo , Animais , Linhagem Celular , Dinaminas/metabolismo , Jejuno/citologia , Ocludina/metabolismo , Suínos , Proteína da Zônula de Oclusão-1/metabolismoRESUMO
OBJECTIVES: Altered enteroendocrine cell (EEC) function in obesity and type 2 diabetes is not fully understood. Understanding the transcriptional program that controls EEC differentiation is important because some EEC types harbor significant therapeutic potential for type 2 diabetes. METHODS: EEC isolation from jejunum of obese individuals with (ObD) or without (Ob) type 2 diabetes was obtained with a new method of cell sorting. EEC transcriptional profiles were established by RNA-sequencing in a first group of 14 Ob and 13 ObD individuals. EEC lineage and densities were studied in the jejunum of a second independent group of 37 Ob, 21 ObD and 22 non obese (NOb) individuals. RESULTS: The RNA seq analysis revealed a distinctive transcriptomic signature and a decreased differentiation program in isolated EEC from ObD compared to Ob individuals. In the second independent group of ObD, Ob and NOb individuals a decreased GLP-1 cell lineage and GLP-1 maturation from proglucagon, were observed in ObD compared to Ob individuals. Furthermore, jejunal density of GLP-1-positive cells was significantly reduced in ObD compared to Ob individuals. CONCLUSIONS: These results highlight that the transcriptomic signature of EEC discriminate obese subjects according to their diabetic status. Furthermore, type 2 diabetes is associated with reduced GLP-1 cell differentiation and proglucagon maturation leading to low GLP-1-cell density in human obesity. These mechanisms could account for the decrease plasma GLP-1 observed in metabolic diseases.
Assuntos
Diabetes Mellitus Tipo 2 , Células Enteroendócrinas/metabolismo , Jejuno/citologia , Obesidade , Adulto , Células Cultivadas , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/epidemiologia , Diabetes Mellitus Tipo 2/metabolismo , Células Enteroendócrinas/citologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Obesidade/complicações , Obesidade/epidemiologia , Obesidade/metabolismoRESUMO
There is increasing evidence that the study of normal human enteroids duplicates many known aspects of human intestinal physiology. However, this epithelial cell-only model lacks the many nonepithelial intestinal cells present in the gastrointestinal tract and exposure to the mechanical forces to which the intestine is exposed. We tested the hypothesis that physical shear forces produced by luminal and blood flow would provide an intestinal model more closely resembling normal human jejunum. Jejunal enteroid monolayers were studied in the Emulate, Inc. Intestine-Chip under conditions of constant luminal and basolateral flow that was designed to mimic normal intestinal fluid flow, with human umbilical vein endothelial cells (HUVECs) on the basolateral surface and with Wnt3A, R-spondin, and Noggin only on the luminal surface. The jejunal enteroids formed monolayers that remained confluent for 6-8 days, began differentiating at least as early as day 2 post plating, and demonstrated continuing differentiation over the entire time of the study, as shown by quantitative real-time polymerase chain reaction and Western blot analysis. Differentiation impacted villus genes and proteins differently with early expression of regenerating family member 1α (REG1A), early reduction to a low but constant level of expression of Na+-K+-2Cl- cotransporter 1 (NKCC1), and increasing expression of sucrase-isomaltase (SI) and downregulated in adenoma (DRA). These results were consistent with continual differentiation, as was shown to occur in mouse villus enterocytes. Compared with differentiated enteroid monolayers grown on Transwell inserts, enteroids exposed to flow were more differentiated but exhibited increased apoptosis and reduced carbohydrate metabolism, as shown by proteomic analysis. This study of human jejunal enteroids-on-chip suggests that luminal and basolateral flow produce a model of continual differentiation over time and NaCl absorption that mimics normal intestine and should provide new insights in intestinal physiology.NEW & NOTEWORTHY This study showed that polarized enteroid models in which there is no basolateral Wnt3a, are differentiated, regardless of the Wnt3a status of the apical media. The study supports the concept that in the human intestine villus differentiation is not an all or none phenomenon, demonstrating that at different days after lack of basolateral Wnt exposure, clusters of genes and proteins exist geographically along the villus with different domains having different functions.
Assuntos
Diferenciação Celular , Jejuno/citologia , Microfluídica/métodos , Cultura Primária de Células/métodos , Estresse Mecânico , Adulto , Apoptose , Proteínas de Transporte/metabolismo , Células Cultivadas , Enterócitos/citologia , Enterócitos/metabolismo , Feminino , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Jejuno/metabolismo , Litostatina/metabolismo , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Trombospondinas/metabolismo , Proteína Wnt3A/metabolismoRESUMO
The intestine is often examined histologically in connection with allergies and in search for pathological changes. To be able to examine the intestine histologically with a microscope, it must be sampled and processed correctly. For microscopic analysis, the samples have to be cut into thin sections, stained, and mounted on slides. Since it is not possible to cut fresh samples without damaging them, they must first be fixed. The most common method, which is described herein, is the fixation in formalin with subsequent embedding in paraffin and staining of the slides with hematoxylin and eosin (H&E). Hematoxylin solutions (in this case Mayer's hemalum solution) stain the acidic components of the cell, i.e., cell nuclei, blue. The staining with eosin gives a pink staining of cytoplasm. This chapter describes the method of processing intestinal tissue for paraffin-embedding, sectioning, and staining with H&E. Tissue processing can be done in tissue processing machines or manually. We describe the manual processing that is often used for smaller batches of samples.
Assuntos
Íleo/patologia , Jejuno/anatomia & histologia , Inclusão em Parafina/métodos , Coloração e Rotulagem/métodos , Fixação de Tecidos/métodos , Animais , Galinhas , Amarelo de Eosina-(YS)/química , Formaldeído/química , Hematoxilina/química , Imuno-Histoquímica/métodos , Jejuno/citologia , Microtomia/métodos , Inclusão em Parafina/instrumentação , Suínos , Fixação de Tecidos/instrumentaçãoRESUMO
Flammulina velutipes polysaccharides (FVP) can improve gut health through gut microbiota and metabolism regulation. In this study, the 28-days fed experiment was used to investigate gut microbime and metabolic profiling induced by FVP. After treatment, intestinal tissue section showed the higher villus height and villus height/crypt depth (V/C) value in FVP-treated group. The 16 s rRNA gene sequencing revealed microbiota composition alteration caused by FVP, as the Firmicutes phylum increased while Bacteroidetes phylum slightly decreased. The metabolic profiling was detected by LC/MS and results showed 56 and 99 compounds were dramatically changed after FVP treatment in positive and negative ion mode, respectively. Annotation in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways displayed the adjustment of energy metabolism, amino acid metabolism, nucleotide metabolism and other related basic pathways after FVP treatment. Our study suggested that FVP can be developed as a dietary supplement for intestine health promotion.
Assuntos
Carboidratos da Dieta/farmacologia , Flammulina/química , Microbioma Gastrointestinal/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Redes e Vias Metabólicas/efeitos dos fármacos , Polissacarídeos/farmacologia , Aminoácidos/metabolismo , Animais , Bacteroidetes/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Cromatografia Líquida , Metabolismo Energético/efeitos dos fármacos , Firmicutes/efeitos dos fármacos , Mucosa Intestinal/anatomia & histologia , Mucosa Intestinal/citologia , Jejuno/citologia , Jejuno/efeitos dos fármacos , Masculino , Espectrometria de Massas , Metabolômica , Camundongos , Camundongos Endogâmicos C57BL , Nucleotídeos/metabolismo , Polissacarídeos/química , RNA Ribossômico 16S/genéticaRESUMO
BACKGROUND: Age-dependent changes in the intestinal gene expression of enzymes involved in the metabolism of citrulline and arginine are well characterized. Enteroids, a novel ex-vivo model that recreates the three-dimensional structure of the intestinal crypt-villus unit, have shown to replicate molecular and physiological profiles of the intestinal segment from where they originated ("location memory"). OBJECTIVE: The present study tested the hypothesis that enteroids recapitulate the developmental changes observed in vivo regarding citrulline production in pigs ("developmental memory"). METHODS: Preterm (10- and 5-d preterm) and term pigs at birth, together with 7- and 35-d-old pigs were studied. Gene expression was measured in jejunal samples and in enteroids derived from this segment. Whole body citrulline production was measured by isotope dilution and enteroid citrulline production by accumulation in the media. RESULTS: With the exception of arginase I and inducible nitric oxide synthase, all the genes investigated expressed in jejunum were expressed by enteroids. In the jejunum, established markers of development (lactase and sucrase-isomaltase), as well as genes that code for enzymes involved in the production and utilization of citrulline and arginine, underwent the ontogenic changes described in the literature. However, enteroid expression of these genes, as well as citrulline production, failed to recapitulate the changes observed in vivo. CONCLUSIONS: Under culture conditions used in our study, enteroids derived from jejunal crypts of pigs at different ages failed to replicate the gene expression observed in whole tissue and whole body citrulline production. Additional extracellular cues may be needed to reproduce the age-dependent phenotype.
Assuntos
Citrulina/metabolismo , Duodeno/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Mucosa Intestinal/metabolismo , Jejuno/metabolismo , Animais , Animais Recém-Nascidos , Duodeno/citologia , Feminino , Mucosa Intestinal/citologia , Jejuno/citologia , Masculino , SuínosRESUMO
Neutrophil infiltration to ischemic tissues following reperfusion worsens injury. A key driver of neutrophil recruitment and activation is the complement factor C5a, which signals through two receptors, C5aR1 and C5aR2. In this study, we used a neutrophil-dependent mouse model of intestinal ischemia-reperfusion (IR) injury to investigate the underexplored role of C5aR2 in neutrophil mobilization, recruitment, and disease outcomes. We show that intestinal IR induces rapid neutrophil mobilization along with a concomitant reduction in plasma C5a levels that is driven by both C5aR1 and C5aR2. Intestinal IR in C5aR2-/- mice led to worsened intestinal damage and increased neutrophil infiltration. Inhibition of C5aR1 signaling in C5aR2-/- mice with PMX53 prevented neutrophil accumulation and reduced IR pathology, suggesting a key requirement for enhanced neutrophil C5aR1 activation in the absence of C5aR2 signaling. Interestingly, C5aR2 deficiency also reduced circulating neutrophil numbers after IR, as well as following G-CSF-mediated bone marrow mobilization, which was independent of C5aR1, demonstrating that C5aR2 has unique and distinct functions from C5aR1 in neutrophil egress. Despite enhanced tissue injury in C5aR2-/- IR mice, there were significant reductions in intestinal proinflammatory cytokines, highlighting complicated dual protective/pathogenic roles for C5aR2 in pathophysiology. Collectively, we show that C5aR2 is protective in intestinal IR by inhibiting C5aR1-mediated neutrophil recruitment to the ischemic tissue. This is despite the potentially local pathogenic effects of C5aR2 in increasing intestinal proinflammatory cytokines and enhancing circulating neutrophil numbers in response to mobilizing signals. Our data therefore suggest that this balance between the dual pro- and anti-inflammatory roles of C5aR2 ultimately dictates disease outcomes.
Assuntos
Isquemia Mesentérica/imunologia , Infiltração de Neutrófilos , Receptor da Anafilatoxina C5a/metabolismo , Traumatismo por Reperfusão/imunologia , Animais , Complemento C5a/análise , Complemento C5a/metabolismo , Modelos Animais de Doenças , Humanos , Jejuno/citologia , Jejuno/imunologia , Jejuno/patologia , Masculino , Isquemia Mesentérica/sangue , Isquemia Mesentérica/complicações , Isquemia Mesentérica/patologia , Camundongos , Camundongos Knockout , Receptor da Anafilatoxina C5a/genética , Traumatismo por Reperfusão/sangue , Traumatismo por Reperfusão/patologiaRESUMO
BACKGROUND: The gut is the only organ system with intrinsic neural reflexes. Intrinsic primary afferent neurons (IPANs) of the enteric nervous system initiate intrinsic reflexes, form gut-brain connections, and undergo considerable neuroplasticity to cause digestive diseases. They remain inaccessible to study in mice in the absence of a selective marker. Advillin is used as a marker for primary afferent neurons in dorsal root ganglia. The aim of this study was to test the hypothesis that advillin is expressed in IPANs of the mouse jejunum. METHODS: Advillin expression was assessed with immunohistochemistry and using transgenic mice expressing an inducible Cre recombinase under the advillin promoter were used to drive tdTomato and the genetically encoded calcium indicator GCaMP5. These mice were used to characterize the morphology and physiology of advillin-expressing enteric neurons using confocal microscopy, calcium imaging, and whole-cell patch-clamp electrophysiology. KEY RESULTS: Advillin is expressed in about 25% of myenteric neurons of the mouse jejunum, and these neurons demonstrate the requisite properties of IPANs. Functionally, they demonstrate calcium responses following mechanical stimuli of the mucosa and during antidromic action potentials. They have Dogiel type II morphology with neural processes that mostly remain within the myenteric plexus, but also project to the mucosa and express NeuN and calcitonin gene-related peptide (CGRP), but not nNOS. CONCLUSIONS AND INFERENCES: Advillin marks jejunal IPANs providing accessibility to this important neuronal population to study and model digestive disease.