RESUMO
Transglutaminase 2 (TG2) plays a pivotal role in the pathogenesis of celiac disease (CeD) by deamidating dietary gluten peptides, which facilitates antigenic presentation and a strong anti-gluten T cell response. Here, we elucidate the molecular mechanisms underlying the efficacy of the TG2 inhibitor ZED1227 by performing transcriptional analysis of duodenal biopsies from individuals with CeD on a long-term gluten-free diet before and after a 6-week gluten challenge combined with 100 mg per day ZED1227 or placebo. At the transcriptome level, orally administered ZED1227 effectively prevented gluten-induced intestinal damage and inflammation, providing molecular-level evidence that TG2 inhibition is an effective strategy for treating CeD. ZED1227 treatment preserved transcriptome signatures associated with mucosal morphology, inflammation, cell differentiation and nutrient absorption to the level of the gluten-free diet group. Nearly half of the gluten-induced gene expression changes in CeD were associated with the epithelial interferon-γ response. Moreover, data suggest that deamidated gluten-induced adaptive immunity is a sufficient step to set the stage for CeD pathogenesis. Our results, with the limited sample size, also suggest that individuals with CeD might benefit from an HLA-DQ2/HLA-DQ8 stratification based on gene doses to maximally eliminate the interferon-γ-induced mucosal damage triggered by gluten.
Assuntos
Doença Celíaca , Dieta Livre de Glúten , Proteínas de Ligação ao GTP , Perfilação da Expressão Gênica , Glutens , Mucosa Intestinal , Proteína 2 Glutamina gama-Glutamiltransferase , Transglutaminases , Doença Celíaca/imunologia , Humanos , Glutens/imunologia , Transglutaminases/metabolismo , Transglutaminases/antagonistas & inibidores , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/antagonistas & inibidores , Proteínas de Ligação ao GTP/genética , Mucosa Intestinal/patologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/imunologia , Mucosa Intestinal/efeitos dos fármacos , Feminino , Masculino , Adulto , Transcriptoma , Duodeno/patologia , Duodeno/imunologia , Duodeno/metabolismo , Interferon gama/metabolismo , Pessoa de Meia-Idade , Antígenos HLA-DQ/genética , Antígenos HLA-DQ/imunologia , Adulto Jovem , Imunidade Adaptativa/efeitos dos fármacosRESUMO
In vitro models of autoimmunity are constrained by an inability to culture affected epithelium alongside the complex tissue-resident immune microenvironment. Coeliac disease (CeD) is an autoimmune disease in which dietary gluten-derived peptides bind to the major histocompatibility complex (MHC) class II human leukocyte antigen molecules (HLA)-DQ2 or HLA-DQ8 to initiate immune-mediated duodenal mucosal injury1-4. Here, we generated air-liquid interface (ALI) duodenal organoids from intact fragments of endoscopic biopsies that preserve epithelium alongside native mesenchyme and tissue-resident immune cells as a unit without requiring reconstitution. The immune diversity of ALI organoids spanned T cells, B and plasma cells, natural killer (NK) cells and myeloid cells, with extensive T-cell and B-cell receptor repertoires. HLA-DQ2.5-restricted gluten peptides selectively instigated epithelial destruction in HLA-DQ2.5-expressing organoids derived from CeD patients, and this was antagonized by blocking MHC-II or NKG2C/D. Gluten epitopes stimulated a CeD organoid immune network response in lymphoid and myeloid subsets alongside anti-transglutaminase 2 (TG2) autoantibody production. Functional studies in CeD organoids revealed that interleukin-7 (IL-7) is a gluten-inducible pathogenic modulator that regulates CD8+ T-cell NKG2C/D expression and is necessary and sufficient for epithelial destruction. Furthermore, endogenous IL-7 was markedly upregulated in patient biopsies from active CeD compared with remission disease from gluten-free diets, predominantly in lamina propria mesenchyme. By preserving the epithelium alongside diverse immune populations, this human in vitro CeD model recapitulates gluten-dependent pathology, enables mechanistic investigation and establishes a proof of principle for the organoid modelling of autoimmunity.
Assuntos
Doença Celíaca , Duodeno , Interleucina-7 , Mucosa Intestinal , Modelos Biológicos , Organoides , Humanos , Autoanticorpos/imunologia , Autoimunidade , Linfócitos B/imunologia , Linfócitos B/metabolismo , Biópsia , Doença Celíaca/imunologia , Doença Celíaca/patologia , Doença Celíaca/metabolismo , Duodeno/imunologia , Duodeno/patologia , Duodeno/metabolismo , Epitopos/imunologia , Glutens/imunologia , Glutens/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/imunologia , Antígenos HLA-DQ/imunologia , Antígenos HLA-DQ/metabolismo , Interleucina-7/metabolismo , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Células Matadoras Naturais/imunologia , Células Mieloides/imunologia , Organoides/imunologia , Organoides/metabolismo , Organoides/patologia , Proteína 2 Glutamina gama-Glutamiltransferase/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
The gastrointestinal (GI) tract is complex and consists of multiple organs with unique functions. Rare gene variants can cause congenital malformations of the human GI tract, although the molecular basis of these has been poorly studied. We identified a patient with compound-heterozygous variants in RFX6 presenting with duodenal malrotation and atresia, implicating RFX6 in development of the proximal intestine. To identify how mutations in RFX6 impact intestinal patterning and function, we derived induced pluripotent stem cells from this patient to generate human intestinal organoids (HIOs). We identified that the duodenal HIOs and human tissues had mixed regional identity, with gastric and ileal features. CRISPR-mediated correction of RFX6 restored duodenal identity. We then used gain- and loss-of-function and transcriptomic approaches in HIOs and Xenopus embryos to identify that PDX1 is a downstream transcriptional target of RFX6 required for duodenal development. However, RFX6 had additional PDX1-independent transcriptional targets involving multiple components of signaling pathways that are required for establishing early regional identity in the GI tract. In summary, we have identified RFX6 as a key regulator in intestinal patterning that acts by regulating transcriptional and signaling pathways.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio , Organoides , Fatores de Transcrição de Fator Regulador X , Transativadores , Humanos , Fatores de Transcrição de Fator Regulador X/genética , Fatores de Transcrição de Fator Regulador X/metabolismo , Animais , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/genética , Transativadores/metabolismo , Transativadores/genética , Organoides/metabolismo , Organoides/embriologia , Duodeno/metabolismo , Duodeno/embriologia , Intestinos/embriologia , Atresia Intestinal/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Padronização Corporal/genética , Transdução de Sinais/genética , Mutação/genéticaRESUMO
Iron is an essential cellular metal that is important for many physiological functions including erythropoiesis and host defense. It is absorbed from the diet in the duodenum and loaded onto transferrin (Tf), the main iron transport protein. Inefficient dietary iron uptake promotes many diseases, but mechanisms regulating iron absorption remain poorly understood. By assessing mice that harbor a macrophage-specific deletion of the tuberous sclerosis complex 2 (Tsc2), a negative regulator of mechanistic target of rapamycin complex 1 (mTORC1), we found that these mice possessed various defects in iron metabolism, including defective steady-state erythropoiesis and a reduced saturation of Tf with iron. This iron deficiency phenotype was associated with an iron import block from the duodenal epithelial cells into the circulation. Activation of mTORC1 in villous duodenal CD68+ macrophages induced serine protease expression and promoted local degradation of Tf, whereas the depletion of macrophages in mice increased Tf levels. Inhibition of mTORC1 with everolimus or serine protease activity with nafamostat restored Tf levels and Tf saturation in the Tsc2-deficient mice. Physiologically, Tf levels were regulated in the duodenum during the prandial process and Citrobacter rodentium infection. These data suggest that duodenal macrophages determine iron transfer to the circulation by controlling Tf availability in the lamina propria villi.
Assuntos
Ferro da Dieta , Transferrina , Camundongos , Animais , Transferrina/metabolismo , Ferro da Dieta/metabolismo , Ferro/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Dieta , Duodeno/metabolismo , Receptores da Transferrina/metabolismoRESUMO
Small organic molecules in the intestinal lumen, particularly short-chain fatty acids (SCFAs) and glucose, have long been postulated to enhance calcium absorption. Here, we used 45Ca radioactive tracer to determine calcium fluxes across the rat intestine after exposure to glucose and SCFAs. Confirming previous reports, glucose was found to increase the apical-to-basolateral calcium flux in the cecum. Under apical glucose-free conditions, SCFAs (e.g., butyrate) stimulated the cecal calcium fluxes by approximately twofold, while having no effect on proximal colon. Since SCFAs could be absorbed into the circulation, we further determined whether basolateral SCFA exposure rendered some positive actions. It was found that exposure of duodenum and cecum on the basolateral side to acetate or butyrate increased calcium fluxes. Under butyrate-rich conditions, cecal calcium transport was partially diminished by Na+/H+ exchanger 3 (NHE3) inhibitor (tenapanor) and nonselective transient receptor potential vanilloid subfamily 6 (TRPV6) inhibitor (miconazole). To confirm the contribution of TRPV6 to SCFA-stimulated calcium transport, we synthesized another TRPV6 inhibitor that was demonstrated by in silico molecular docking and molecular dynamics to occlude TRPV6 pore and diminish the glucose- and butyrate-induced calcium fluxes. Therefore, besides corroborating the importance of luminal molecules in calcium absorption, our findings provided foundation for development of more effective calcium-rich nutraceuticals in combination with various absorptive enhancers, e.g., glucose and SCFAs.NEW & NOTEWORTHY Organic molecules in the intestinal lumen, e.g., glucose and short-chain fatty acids (SCFAs), the latter of which are normally produced by microfloral fermentation, can stimulate calcium absorption dependent on transient receptor potential vanilloid subfamily 6 (TRPV6) and Na+/H+ exchanger 3 (NHE3). A selective TRPV6 inhibitor synthesized and demonstrated by in silico docking and molecular dynamics to specifically bind to the pore domain of TRPV6 was used to confirm a significant contribution of this channel. Our findings corroborate physiological significance of nutrients and SCFAs in enhancing calcium absorption.
Assuntos
Cálcio , Ácidos Graxos Voláteis , Ratos , Animais , Trocador 3 de Sódio-Hidrogênio/metabolismo , Cálcio/metabolismo , Simulação de Acoplamento Molecular , Ácidos Graxos Voláteis/farmacologia , Ácidos Graxos Voláteis/metabolismo , Butiratos/farmacologia , Proteínas de Transporte/metabolismo , Duodeno/metabolismo , Glucose/metabolismo , Absorção IntestinalRESUMO
AIMS/HYPOTHESIS: Metformin lowers postprandial glycaemic excursions in individuals with type 2 diabetes by modulating gastrointestinal function, including the stimulation of glucagon-like peptide-1 (GLP-1). The impact of varying the timing of metformin administration on postprandial glucose metabolism is poorly defined. We evaluated the effects of metformin, administered at different intervals before an intraduodenal glucose infusion, on the subsequent glycaemic, insulinaemic and GLP-1 responses in metformin-treated type 2 diabetes. METHODS: Sixteen participants with type 2 diabetes that was relatively well-controlled by metformin monotherapy were studied on four separate days in a crossover design. On each day, participants were randomised to receive a bolus infusion of metformin (1000 mg in 50 ml 0.9% saline) via a nasoduodenal catheter at t = -60, -30 or 0 min (and saline at the other timepoints) or saline at all timepoints (control), followed by an intraduodenal glucose infusion of 12.56 kJ/min (3 kcal/min) at t = 0-60 min. The treatments were blinded to both participants and investigators involved in the study procedures. Plasma glucose, insulin and total GLP-1 levels were measured every 30 min between t = -60 min and t = 120 min. RESULTS: There was a treatment-by-time interaction for metformin in reducing plasma glucose levels and increasing plasma GLP-1 and insulin levels (p<0.05 for each). The reduction in plasma glucose levels was greater when metformin was administered at t = -60 or -30 min vs t = 0 min (p<0.05 for each), and the increases in plasma GLP-1 levels were evident only when metformin was administered at t = -60 or -30 min (p<0.05 for each). Although metformin did not influence insulin sensitivity, it enhanced glucose-induced insulin secretion (p<0.05), and the increases in plasma insulin levels were comparable on the 3 days when metformin was given. CONCLUSIONS/INTERPRETATION: In well-controlled metformin-treated type 2 diabetes, glucose-lowering by metformin is greater when it is given before, rather than with, enteral glucose, and this is associated with a greater GLP-1 response. These observations suggest that administration of metformin before meals may optimise its effect in improving postprandial glycaemic control. TRIAL REGISTRATION: www.anzctr.org.au ACTRN12621000878875 FUNDING: The study was not funded by a specific research grant.
Assuntos
Glicemia , Estudos Cross-Over , Diabetes Mellitus Tipo 2 , Peptídeo 1 Semelhante ao Glucagon , Glucose , Hipoglicemiantes , Metformina , Humanos , Metformina/uso terapêutico , Metformina/administração & dosagem , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/sangue , Masculino , Peptídeo 1 Semelhante ao Glucagon/sangue , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Feminino , Pessoa de Meia-Idade , Método Duplo-Cego , Hipoglicemiantes/administração & dosagem , Hipoglicemiantes/uso terapêutico , Glucose/metabolismo , Insulina/sangue , Idoso , Adulto , Período Pós-Prandial , Duodeno/metabolismo , Duodeno/efeitos dos fármacosRESUMO
Beclin1 (Becn1) is a multifunctional protein involved in autophagy regulation, membrane trafficking, and tumor suppression. In this study, we examined the roles of Becn1 in the pancreas development by generating mice with conditional deletion of Becn1 in the pancreas using pancreatic transcriptional factor 1a (Ptf1a)-Cre mice (Becn1f/f; Ptf1aCre/+). Surprisingly, loss of Becn1 in the pancreas resulted in severe pancreatic developmental defects, leading to insufficient exocrine and endocrine pancreatic function. Approximately half of Becn1f/f; Ptf1aCre/+ mice died immediately after birth. However, duodenum and neural tissue development were almost normal, indicating that pancreatic insufficiency was the cause of death. These findings demonstrated a novel role for Becn1 in pancreas morphogenesis, differentiation, and growth, and suggested that loss of this factor leaded to pancreatic agenesis at birth.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Pâncreas , Animais , Camundongos , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Duodeno/metabolismo , Pâncreas/metabolismo , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: Gastrointestinal dysfunction has emerged as a prominent early feature of Parkinson's Disease, shedding new light on the pivotal role of the enteric nervous system in its pathophysiology. However, the role of immune-cell clusters and inflammatory and glial markers in the gut pathogenetic process needs further elucidation. OBJECTIVES: We aimed to study duodenum tissue samples to characterize PD's enteric nervous system pathology further. Twenty patients with advanced PD, six with early PD, and 18 matched controls were included in the PADUA-CESNE cohort. METHODS: Duodenal biopsies from 26 patients with early to advanced stage PD and 18 age-matched HCs were evaluated for the presence of surface markers (CD3+, CD4+, CD8+, CD20+, CD68+, HLA-DR), presence of misfolded alpha-synuclein and enteric glial alteration (GFAP). Correlation of immulogic pattern and clinical characteristic were analyzed. RESULTS: The findings validate that in patients with Parkinson's Disease, the activation and reactive gliosis are linked to the neurodegeneration triggered by the presence of misfolded alpha-synuclein in the enteric nervous system. This process intensifies from the initial to the advanced stages of the disease. The clusters of T- and B-lymphocytes in the enteric system, along with the overall expression of HLA-DR in antigen-presenting cells, exceeded those in the control group. Conversely, no differences in terms of macrophage populations were found. CONCLUSIONS: These findings broaden our understanding of the mechanisms underlying the enteric nervous system's involvement in PD and point to the gastrointestinal system as a potential therapeutic target, especially in the early stages of the disease. Moreover, our results propose a role of T- and B-lymphocytes in maintaining inflammation and ultimately influencing alpha-synuclein misfolding and aggregation.
Assuntos
Sistema Nervoso Entérico , Doença de Parkinson , Humanos , Doença de Parkinson/imunologia , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Sistema Nervoso Entérico/imunologia , Sistema Nervoso Entérico/patologia , Sistema Nervoso Entérico/metabolismo , Feminino , Masculino , Idoso , Pessoa de Meia-Idade , Estudos de Coortes , alfa-Sinucleína/metabolismo , alfa-Sinucleína/imunologia , Duodeno/imunologia , Duodeno/patologia , Duodeno/metabolismoRESUMO
Celiac disease (CD) is an immune-driven disease characterized by tissue damage in the small intestine of genetically-susceptible individuals. We evaluated here a crucial immune regulatory pathway involving TYRO3, AXL, and MERTK (TAM) receptors and their ligands PROS1 and GAS6 in duodenal biopsies of controls and CD patients. We found increased GAS6 expression associated with downregulation of PROS1 and variable TAM receptors levels in duodenum tissue of CD patients. Interestingly, CD3+ lymphocytes, CD68+, CD11c+ myeloid and epithelial cells, showed differential expressions of TAM components comparing CD vs controls. Principal component analysis revealed a clear segregation of two groups of CD patients based on TAM components and IFN signaling. In vitro validation demonstrated that monocytes, T lymphocytes and epithelial cells upregulated TAM components in response to IFN stimulation. Our findings highlight a dysregulated TAM axis in CD related to IFN signaling and contribute to a deeper understanding of the pathophysiology of CD.
Assuntos
Receptor Tirosina Quinase Axl , Doença Celíaca , Duodeno , Peptídeos e Proteínas de Sinalização Intercelular , Mucosa Intestinal , Proteína S , Receptores Proteína Tirosina Quinases , c-Mer Tirosina Quinase , Feminino , Humanos , Masculino , c-Mer Tirosina Quinase/genética , c-Mer Tirosina Quinase/metabolismo , Doença Celíaca/imunologia , Doença Celíaca/metabolismo , Doença Celíaca/genética , Duodeno/metabolismo , Duodeno/imunologia , Duodeno/patologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Interferons/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/imunologia , Proteína S/metabolismo , Proteína S/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/imunologia , Transdução de Sinais , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Tryptophan is an essential amino acid transformed by host and gut microbial enzymes into metabolites that regulate mucosal homeostasis through aryl hydrocarbon receptor (AhR) activation. Alteration of tryptophan metabolism has been associated with chronic inflammation; however, whether tryptophan supplementation affects the metabolite repertoire and AhR activation under physiological conditions in humans is unknown. We performed a randomized, double blind, placebo-controlled, crossover study in 20 healthy volunteers. Subjects on a low tryptophan background diet were randomly assigned to a 3-wk l-tryptophan supplementation (3 g/day) or placebo, and after a 2-wk washout switched to opposite interventions. We assessed gastrointestinal and psychological symptoms by validated questionnaires, AhR activation by cell reporter assay, tryptophan metabolites by liquid chromatography and high-resolution mass spectrometry, cytokine production in isolated monocytes by ELISA, and microbiota profile by 16S rRNA Illumina technique. Oral tryptophan supplementation was well tolerated, with no changes in gastrointestinal or psychological scores. Compared with placebo, tryptophan increased AhR activation capacity by duodenal contents, but not by feces. This was paralleled by higher urinary and plasma kynurenine metabolites and indoles. Tryptophan had a modest impact on fecal microbiome profiles and no significant effect on cytokine production. At the doses used in this study, oral tryptophan supplementation in humans induces microbial indole and host kynurenine metabolic pathways in the small intestine, known to be immunomodulatory. The results should prompt tryptophan intervention strategies in inflammatory conditions of the small intestine where the AhR pathway is impaired.NEW & NOTEWORTHY We demonstrate that in healthy subjects, orally administered tryptophan activates microbial indole and host kynurenine pathways in the small intestine, the primary metabolic site for dietary components, and the richest source of immune cells along the gut. This study provides novel insights in how to optimally activate immunomodulatory AhR pathways and indole metabolism in the small intestine, serving as basis for future therapeutic trials using l-tryptophan supplementation in chronic inflammatory conditions affecting the small intestine.
Assuntos
Estudos Cross-Over , Duodeno , Voluntários Saudáveis , Receptores de Hidrocarboneto Arílico , Triptofano , Humanos , Triptofano/metabolismo , Triptofano/administração & dosagem , Receptores de Hidrocarboneto Arílico/metabolismo , Masculino , Adulto , Feminino , Duodeno/metabolismo , Duodeno/efeitos dos fármacos , Método Duplo-Cego , Suplementos Nutricionais , Microbioma Gastrointestinal/efeitos dos fármacos , Adulto Jovem , Administração Oral , Cinurenina/metabolismo , Citocinas/metabolismo , Fezes/microbiologia , Fezes/química , Indóis/farmacologia , Indóis/administração & dosagem , Fatores de Transcrição Hélice-Alça-Hélice BásicosRESUMO
PURPOSE: A large proportion of Common variable immunodeficiency (CVID) patients has duodenal inflammation with increased intraepithelial lymphocytes (IEL) of unknown aetiology. The histologic similarities to celiac disease, lead to confusion regarding treatment (gluten-free diet) of these patients. We aimed to elucidate the role of epigenetic DNA methylation in the aetiology of duodenal inflammation in CVID and differentiate it from true celiac disease. METHODS: DNA was isolated from snap-frozen pieces of duodenal biopsies and analysed for differences in genome-wide epigenetic DNA methylation between CVID patients with increased IEL (CVID_IEL; n = 5) without IEL (CVID_N; n = 3), celiac disease (n = 3) and healthy controls (n = 3). RESULTS: The DNA methylation data of 5-methylcytosine in CpG sites separated CVID and celiac diseases from healthy controls. Differential methylation in promoters of genes were identified as potential novel mediators in CVID and celiac disease. There was limited overlap of methylation associated genes between CVID_IEL and Celiac disease. High frequency of differentially methylated CpG sites was detected in over 100 genes nearby transcription start site (TSS) in both CVID_IEL and celiac disease, compared to healthy controls. Differential methylation of genes involved in regulation of TNF/cytokine production were enriched in CVID_IEL, compared to healthy controls. CONCLUSION: This is the first study to reveal a role of epigenetic DNA methylation in the etiology of duodenal inflammation of CVID patients, distinguishing CVID_IEL from celiac disease. We identified potential biomarkers and therapeutic targets within gene promotors and in high-frequency differentially methylated CpG regions proximal to TSS in both CVID_IEL and celiac disease.
Assuntos
Doença Celíaca , Imunodeficiência de Variável Comum , Ilhas de CpG , Metilação de DNA , Duodeno , Epigênese Genética , Humanos , Imunodeficiência de Variável Comum/genética , Duodeno/metabolismo , Duodeno/patologia , Doença Celíaca/genética , Feminino , Masculino , Adulto , Pessoa de Meia-Idade , Ilhas de CpG/genética , Regiões Promotoras Genéticas/genética , Linfócitos Intraepiteliais/imunologia , Adulto Jovem , Estudo de Associação Genômica Ampla , 5-Metilcitosina/metabolismoRESUMO
Growing empirical evidence suggests that nutrition and bacterial metabolites might impact the systemic immune response in the context of disease and autoimmunity. We report that long-chain fatty acids (LCFAs) enhanced differentiation and proliferation of T helper 1 (Th1) and/or Th17 cells and impaired their intestinal sequestration via p38-MAPK pathway. Alternatively, dietary short-chain FAs (SCFAs) expanded gut T regulatory (Treg) cells by suppression of the JNK1 and p38 pathway. We used experimental autoimmune encephalomyelitis (EAE) as a model of T cell-mediated autoimmunity to show that LCFAs consistently decreased SCFAs in the gut and exacerbated disease by expanding pathogenic Th1 and/or Th17 cell populations in the small intestine. Treatment with SCFAs ameliorated EAE and reduced axonal damage via long-lasting imprinting on lamina-propria-derived Treg cells. These data demonstrate a direct dietary impact on intestinal-specific, and subsequently central nervous system-specific, Th cell responses in autoimmunity, and thus might have therapeutic implications for autoimmune diseases such as multiple sclerosis.
Assuntos
Autoimunidade/efeitos dos fármacos , Sistema Nervoso Central/imunologia , Gorduras na Dieta/farmacologia , Duodeno/imunologia , Encefalomielite Autoimune Experimental/etiologia , Ácidos Graxos/farmacologia , Linfopoese/efeitos dos fármacos , Subpopulações de Linfócitos T/efeitos dos fármacos , Animais , Gorduras na Dieta/toxicidade , Duodeno/metabolismo , Duodeno/microbiologia , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/metabolismo , Ácidos Graxos/química , Ácidos Graxos/toxicidade , Transplante de Microbiota Fecal , Microbioma Gastrointestinal/fisiologia , Regulação da Expressão Gênica/imunologia , Ácidos Láuricos/toxicidade , Receptores X do Fígado , Sistema de Sinalização das MAP Quinases , Camundongos , Peso Molecular , Receptores Nucleares Órfãos/biossíntese , Receptores Nucleares Órfãos/genética , Receptores Acoplados a Proteínas G/biossíntese , Receptores Acoplados a Proteínas G/genética , Baço/imunologia , Baço/patologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Reguladores/imunologia , Células Th1/imunologia , Células Th17/imunologia , TranscriptomaRESUMO
BACKGROUND AND AIMS: The duodenum has been shown to play a key role in glucose homeostasis. Duodenal mucosal resurfacing (DMR) is an endoscopic procedure for patients with type 2 diabetes (T2D) in which the duodenal mucosa is hydrothermally ablated. DMR improves glycemic control, but the underlying mechanisms remain unclear. Here, we report changes in glucoregulatory hormones and indices of insulin sensitivity and beta cell function after DMR. METHODS: We included 28 patients on noninsulin glucose-lowering medications who underwent open-label DMR and a mixed meal test (MMT) in Revita-1 or Revita-2 studies. Inclusion criteria were a hemoglobin A1c from 7.6% to 10.4% and a body mass index of 24 to 40 kg/m2. Baseline and 3-month MMT data included plasma glucose, insulin, C-peptide, glucagon-like peptide-1 (GLP-1), and gastric inhibitory polypeptide (GIP) concentrations. Glucoregulatory hormones, insulin sensitivity indices (Homeostatic Model Assessment for Insulin Resistance [HOMA-IR], Matsuda index [MI], and hepatic insulin resistance) and beta cell function (insulinogenic index, disposition index [DI], and insulin secretion rate [ISR]) were assessed. RESULTS: Fasting insulin, glucagon, and C-peptide decreased significantly. Insulin sensitivity (HOMA-IR, MI, and hepatic insulin resistance) and beta cell function (DI and ISR) all improved significantly. Declines in postprandial glucose, mainly driven by a decrease in fasting levels, and in postprandial glucagon were observed, whereas GLP-1 and GIP did not change. CONCLUSIONS: Insulin sensitivity and insulin secretion improved 3 months after DMR. It is unlikely that incretin changes are responsible for improved glucose control after DMR. These data add to the growing evidence validating the duodenum as a therapeutic target for patients with T2D. (Clinical trial registration numbers: NCT02413567 and NCT03653091.).
Assuntos
Glicemia , Diabetes Mellitus Tipo 2 , Duodeno , Polipeptídeo Inibidor Gástrico , Peptídeo 1 Semelhante ao Glucagon , Resistência à Insulina , Células Secretoras de Insulina , Insulina , Mucosa Intestinal , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Glicemia/metabolismo , Peptídeo C/sangue , Peptídeo C/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Duodeno/cirurgia , Duodeno/metabolismo , Ressecção Endoscópica de Mucosa/métodos , Polipeptídeo Inibidor Gástrico/metabolismo , Glucagon/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Hemoglobinas Glicadas/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/cirurgia , Projetos PilotoRESUMO
BACKGROUND: Spinal cord injury (SCI) disrupts intestinal barrier function, thereby increasing antigen permeation and leading to poor outcomes. Despite the intestinal tract's anatomic and physiologic heterogeneity, studies following SCI have not comprehensively addressed intestinal pathophysiology with regional specificity. AIMS AND METHODS: We used an experimental model of high thoracic SCI to investigate (1) regional mucosal oxidative stress using dihydroethidium labeling; (2) regional paracellular permeability to small- and large-molecular probes via Ussing chamber; (3) regional intestinal tight junction (TJ) protein expression; and (4) hindgut perfusion via the caudal mesenteric artery. RESULTS: Dihydroethidium staining was significantly elevated within duodenal mucosa at 3-day post-SCI. Molar flux of [14C]-urea was significantly elevated in duodenum and proximal colon at 3-day post-SCI, while molar flux of [3H]-inulin was significantly elevated only in duodenum at 3-day post-SCI. Barrier permeability was mirrored by a significant increase in the expression of pore-forming TJ protein claudin-2 in duodenum and proximal colon at 3-day post-SCI. Claudin-2 expression remained significantly elevated in proximal colon at 3-week post-SCI. Expression of the barrier-forming TJ protein occludin was significantly reduced in duodenum at 3-day post-SCI. Caudal mesenteric artery flow was unchanged by SCI at 3 days or 3 weeks despite significant reductions in mean arterial pressure. CONCLUSION: These data show that T3-SCI provokes elevated mucosal oxidative stress, altered expression of TJ proteins, and elevated intestinal barrier permeability in the proximal intestine. In contrast, mucosal oxidative stress and intestinal barrier permeability were unchanged in the hindgut after SCI. This regional heterogeneity may result from differential sensitivity to reduced mesenteric perfusion, though further studies are required to establish a causal link. Understanding regional differences in intestinal pathophysiology is essential for developing effective treatments and standards of care for individuals with SCI.
Assuntos
Mucosa Intestinal , Estresse Oxidativo , Permeabilidade , Traumatismos da Medula Espinal , Animais , Mucosa Intestinal/metabolismo , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/fisiopatologia , Masculino , Estresse Oxidativo/fisiologia , Ratos , Ratos Sprague-Dawley , Duodeno/metabolismo , Vértebras Torácicas , Junções Íntimas/metabolismo , Ocludina/metabolismo , Colo/metabolismo , Colo/irrigação sanguínea , Modelos Animais de DoençasRESUMO
BACKGROUND: Calprotectin, a damage-associated molecular pattern protein of the S100/calgranulin family, is a potential marker of gastrointestinal inflammation in dogs and mainly originates from activated macrophages and granulocytes. Increased calprotectin concentrations are reported in feces and serum samples from dogs with chronic inflammatory enteropathy (CIE), but mucosal calprotectin expression has not been extensively investigated in canine CIE. Thus, we aimed to evaluate gastrointestinal mucosal concentrations of calprotectin in 62 dogs (44 dogs with CIE compared to 18 healthy Beagles) using a particle-enhanced turbidimetric immunoassay method. Additionally, we assessed the relationship of gastric, duodenal, jejunal, ileal, and colonic mucosal calprotectin levels with the clinical disease severity (canine clinical inflammatory bowel disease activity index, CIBDAI), histopathologic findings, clinical outcome, and serum albumin concentrations to further evaluate the potential of calprotectin as a biomarker for CIE. RESULTS: Mucosal calprotectin concentrations in dogs with CIE were significantly higher in the duodenum (median: 276.2 µg/g) and colon (median: 298.2 µg/g) compared to healthy controls (median: 94.3 µg/g, P = 0.0039; and median: 112.0 µg/g, P = 0.0061). Similar numerical differences in the ileum and cecum were not statistically significant, and mucosal calprotectin concentrations correlated significantly among the different gastrointestinal segments. Histologic lesion severity was linked to mucosal calprotectin concentrations for inflammatory and structural histology criteria in the duodenum and colon (all P < 0.05). Higher mucosal calprotectin levels in the duodenum and across all segments correlated with lower serum albumin concentrations (both P < 0.05); duodenal mucosal calprotectin concentrations were more than sixfold higher in hypoalbuminemic dogs (median: 1441 µg/g, n = 4) than normoalbuminemic dogs (median: 227 µg/g, n = 40). There was no significant association of mucosal calprotectin levels with CIBDAI scores or individual clinical outcomes. CONCLUSIONS: These results show that duodenal and colonic mucosal calprotectin concentrations are increased in dogs with CIE, providing further supporting evidence for the diagnostic potential of fecal calprotectin (presumably reflecting mucosal) concentrations and in dogs with CIE. Further longitudinal research is needed to assess changes in mucosal calprotectin concentrations with clinical response to treatment vs. mucosal disease remission and to determine the clinical utility of fecal calprotectin concentrations to diagnose and monitor dogs with CIE in clinical practice.
Assuntos
Colo , Doenças do Cão , Mucosa Intestinal , Complexo Antígeno L1 Leucocitário , Animais , Cães , Doenças do Cão/metabolismo , Doenças do Cão/patologia , Complexo Antígeno L1 Leucocitário/análise , Mucosa Intestinal/patologia , Mucosa Intestinal/metabolismo , Masculino , Feminino , Colo/patologia , Colo/metabolismo , Duodeno/patologia , Duodeno/metabolismo , Biomarcadores/metabolismo , Doenças Inflamatórias Intestinais/veterinária , Doenças Inflamatórias Intestinais/patologia , Doenças Inflamatórias Intestinais/metabolismo , Índice de Gravidade de DoençaRESUMO
BACKGROUND: Currently, society and industry generate huge amounts of plastics worldwide. The ubiquity of microplastics is obvious, but its impact on the animal and human organism remains not fully understood. The digestive tract is one of the first barriers between pathogens and xenobiotics and a living organism. Its proper functioning is extremely important in order to maintain homeostasis. The aim of this study was to determine the effect of microplastic on enteric nervous system and histological structure of swine duodenum. The experiment was carried out on 15 sexually immature gilts, approximately 8 weeks old. The animals were randomly divided into 3 study groups (n = 5/group). The control group received empty gelatin capsules once a day for 28 days, the first research group received daily gelatin capsules with polyethylene terephthalate (PET) particles as a mixture of particles of various sizes (maximum particle size 300 µm) at a dose of 0.1 g/animal/day. The second study group received a dose ten times higher-1 g/animal/day. RESULTS: A dose of 1 g/day/animal causes more changes in the enteric nervous system and in the histological structure of duodenum. Statistically significant differences in the expression of cocaine and amphetamine regulated transcript, galanin, neuronal nitric oxide synthase, substance P, vesicular acetylcholine transporter and vasoactive intestinal peptide between control and high dose group was noted. The histopathological changes were more frequently observed in the pigs receiving higher dose of PET. CONCLUSION: Based on this study it may be assumed, that oral intake of microplastic might have potential negative influence on digestive tract, but it is dose-dependent.
Assuntos
Microplásticos , Plásticos , Humanos , Suínos , Animais , Feminino , Polietilenotereftalatos/metabolismo , Polietilenotereftalatos/farmacologia , Gelatina/metabolismo , Gelatina/farmacologia , Duodeno/metabolismo , NeurôniosRESUMO
2-ethylhexyl diphenyl phosphate (EHDPHP) is a widely used organophosphorus flame retardant and plasticizer, which is commonly found in the environment. EHDPHP not only potentially harms the environment but also causes different degrees of damage to the organism. In this study, the duodenum of chicks was selected as the potential toxic target organ to explore the mechanism of duodenal injury induced by EHDPHP exposure. Ninety one-day-old healthy male chicks were selected and randomly divided into C1(control group), C2(solvent control group), L(800â¯mg/kg), M(1600â¯mg/kg), H(3200â¯mg/kg) according to different doses of EHDPHP after one week of environmental adaptation. The chicks were given continuous gavage for 14 d, 28 d, and 42 d. It was found that constant exposure to EHDPHP caused an increase in duodenal MDA content, a decrease in P-gp, SOD, GSH-Px activities, and a decrease in duodenal mucosal immune factor (sIgA, GSH-Px). The expression of sIgM and mucosal link proteins (CLDN, OCLN, ZO-1, JAM) decreased, and the expression of the inflammatory protein (NF-κB, COX2) in duodenal tissues was up-regulated. The results showed that continuous exposure to EHDPHP could cause duodenal oxidative stress, inflammation, and mucosal barrier damage in chicks, which provided a basis for studying the mechanism of toxic damage caused by EHDPHP in poultry.
Assuntos
Galinhas , Duodeno , Retardadores de Chama , Estresse Oxidativo , Animais , Estresse Oxidativo/efeitos dos fármacos , Duodeno/efeitos dos fármacos , Duodeno/patologia , Duodeno/metabolismo , Masculino , Retardadores de Chama/toxicidade , Inflamação/induzido quimicamente , Inflamação/patologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/patologia , Mucosa Intestinal/metabolismo , Compostos Organofosforados/toxicidade , Organofosfatos/toxicidadeRESUMO
Diabetes mellitus, as a chronic metabolic disorder, significantly impacts the pancreas and among other organs, affects duodenal function. Emerging evidence suggests that probiotics can exert beneficial effects on gut health and metabolism. In our previous research, we evaluated the probiotic Lactobacillus paraplantarum BGCG11 primarily for its protective properties against diabetic rats' damaged liver and kidneys. In this work, we further examined the effects of probiotic strain BGCG11 on the function of the duodenum and pancreas in diabetic rats. We explored the potential mechanisms underlying the probiotic's effects, focusing on general indicators of diabetes, the architecture and morphology of pancreatic islets, duodenal integrity (measuring the transfer of fluid and serum zonulin level), and the modulation of gut microbiota composition. Our findings reveal the protective and regulatory roles of L. paraplantarum BGCG11 in mitigating diabetes-induced pancreatic and duodenal dysfunction regardless of its application time (pre- or post-treatment), highlighting its therapeutic potential in managing diabetes-related gastrointestinal complications.
Assuntos
Diabetes Mellitus Experimental , Duodeno , Microbioma Gastrointestinal , Lactobacillus , Pâncreas , Probióticos , Animais , Probióticos/farmacologia , Duodeno/microbiologia , Duodeno/metabolismo , Ratos , Diabetes Mellitus Experimental/terapia , Masculino , Microbioma Gastrointestinal/efeitos dos fármacos , Pâncreas/patologia , Pâncreas/metabolismo , Pâncreas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacosRESUMO
Diagnosis of biliopancreatic cancers by the available serum tumor markers, imaging, and histopathological tissue specimen examination remains a challenge. Circulating cell-free DNA derived from matched pairs of secretin-stimulated duodenal fluid (DF) and plasma from 10 patients with biliopancreatic diseases and 8 control subjects was analyzed using AmpliSeq™ HD technology for Ion Torrent Next-Generation Sequencing to evaluate the potential of liquid biopsy with DF in biliopancreatic cancers. The median cfDNA concentration was greater in DF-derived than in plasma-derived samples. A total of 13 variants were detected: 11 vs. 1 were exclusive for DF relative to the plasma source, and 1 was shared between the two body fluids. According to the four-tier systems, 10 clinical tier-I-II (76.9%), 1 tier-III (7.7%), and 2 tier-IV (15.4%) variants were identified. Notably, the 11 tier-I-III variants were exclusively found in DF-derived cfDNA from five patients with biliopancreatic cancers, and were detected in seven genes (KRAS, TP53, BRAF, CDKN2A, RNF43, GNAS, and PIK3CA); 82% of the tier-I-III variants had a low abundance, with a VAF < 6%. The mutational profiling of DF seems to be a reliable and promising tool for identifying cancer-associated alterations in malignant cancers of the biliopancreatic tract.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Mutação , Neoplasias Pancreáticas , Humanos , Masculino , Feminino , Pessoa de Meia-Idade , Idoso , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/patologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Duodeno/metabolismo , Duodeno/patologia , Biomarcadores Tumorais/genética , Biópsia Líquida/métodos , Adulto , Ácidos Nucleicos Livres/genética , Neoplasias do Sistema Biliar/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , CromograninasRESUMO
Background and Objectives: Hyperprolactinemia, as a potential side-effect of some antipsychotic medications, is associated with decreased bone density and an increased risk of fractures. This study investigates whether calcium and vitamin D supplementation affects prolactin receptor (Prlr) gene expression in the duodenum, vertebrae, and kidneys of female rats with sulpiride-induced hyperprolactinemia. Materials and Methods: Twenty-one-week-old female Wistar rats were assigned to three groups: Group S consisted of ten rats who received sulpiride injections (10 mg/kg) twice daily for 6 weeks; Group D (10 rats) received daily supplementation of 50 mg calcium and 500 IU vitamin D along with sulpiride for the last 3 weeks; and Group C consisting of seven age-matched nulliparous rats serving as a control group. Real-time PCR was used to assess Prlr gene expression in the duodenum, vertebrae, and kidneys. Results: In Group S, Prlr gene expression was notably decreased in the duodenum (p < 0.01) but elevated in the vertebrae and kidneys compared to Group C. Conversely, Group D exhibited significantly increased Prlr expression in the duodenum (p < 0.01) alongside elevated expression in the vertebrae and kidneys. Conclusions: In sulpiride-induced hyperprolactinemia, decreased Prlr gene expression in the duodenum may lead to reduced intestinal calcium absorption. Consequently, prolactin may draw calcium from the skeletal system to maintain calcium balance, facilitated by increased Prlr gene expression in the vertebrae. However, vitamin D supplementation in sulpiride-induced hyperprolactinemia notably enhances Prlr gene expression in the duodenum, potentially ameliorating intestinal calcium absorption and mitigating adverse effects on bone health.