RESUMEN
BACKGROUND: Patients suffering from inflammatory bowel diseases (IBDs) express increased mucosal levels of pH-sensing receptors compared with non-IBD controls. Acidification leads to angiogenesis and extracellular matrix remodeling. We aimed to determine the expression of pH-sensing G protein-coupled receptor 4 (GPR4) in fibrotic lesions in Crohn's disease (CD) patients. We further evaluated the effect of deficiency in Gpr4 or its pharmacologic inhibition. METHODS: Paired samples from fibrotic and nonfibrotic terminal ileum were obtained from CD patients undergoing ileocaecal resection. The effects of Gpr4 deficiency were assessed in the spontaneous Il-10-/- and the chronic dextran sodium sulfate (DSS) murine colitis model. The effects of Gpr4 deficiency and a GPR4 antagonist (39c) were assessed in the heterotopic intestinal transplantation model. RESULTS: In human terminal ileum, increased expression of fibrosis markers was accompanied by an increase in GPR4 expression. A positive correlation between the expression of procollagens and GPR4 was observed. In murine disease models, Gpr4 deficiency was associated with a decrease in angiogenesis and fibrogenesis evidenced by decreased vessel length and expression of Edn, Vegfα, and procollagens. The heterotopic animal model for intestinal fibrosis, transplanted with terminal ileum from Gpr4-/- mice, revealed a decrease in mRNA expression of fibrosis markers and a decrease in collagen content and layer thickness compared with grafts from wild type mice. The GPR4 antagonist decreased collagen deposition. The GPR4 expression was also observed in human and murine intestinal fibroblasts. The GPR4 inhibition reduced markers of fibroblast activation stimulated by low pH, notably Acta2 and cTgf. CONCLUSIONS: Expression of GPR4 positively correlates with the expression of profibrotic genes and collagen. Deficiency of Gpr4 is associated with a decrease in angiogenesis and fibrogenesis. The GPR4 antagonist decreases collagen deposition. Targeting GPR4 with specific inhibitors may constitute a new treatment option for IBD-associated fibrosis.
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Colitis , Animales , Colitis/patología , Fibrosis , Humanos , Concentración de Iones de Hidrógeno , Intestinos/patología , Ratones , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
BACKGROUND: Genetic variations within the regulatory region of the gene encoding NOD-like receptor pyrin domain containing 3 (NLRP3) have been associated with Crohn's Disease (CD). NLRP3 is part of the NLRP3-inflammasome that mediates the maturation of IL-1ß and IL-18. Carrying the major allele of the single nucleotide polymorphisms (SNPs) rs10733113, rs4353135 and rs55646866 is associated with an increased risk for CD. We here studied the impact of these polymorphisms on clinical characteristics in patients of the Swiss IBD Cohort Study (SIBDCS). METHODS: We included 981 Crohn's disease (CD) patients and 690 ulcerative colitis (UC) patients of the SIBDCS. We analyzed whether three CD-associated NLRP3 polymorphisms have an impact on the clinical disease course in these patients. RESULTS: In CD patients presence of the major allele (G) of rs10733113 was associated with less surgeries and lower maximal CDAI and a similar trend was observed for rs55646866 and rs4353135. Presence of the major allele of all three SNPs was negatively correlated to maximal CDAI. In UC patients homozygous genotype for the major allele (CC) for rs55646866 was associated with a higher age at diagnosis and a higher MTWAI index. Homozygous genotype for the major allele of all three polymorphisms was associated with a higher number of ambulatory visits and longer hospital stays. CONCLUSIONS: In CD patients presence of the major allele of all three polymorphisms was associated with markers of a less severe disease course, while in UC the homozygous genotype for all major alleles suggested a more severe disease activity.
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Colitis Ulcerosa , Enfermedades Inflamatorias del Intestino , Estudios de Cohortes , Colitis Ulcerosa/genética , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Proteína con Dominio Pirina 3 de la Familia NLR , Proteínas NLR , Polimorfismo de Nucleótido Simple , Dominio Pirina , SuizaRESUMEN
INTRODUCTION: Intestinal fibrosis, characterized by excessive deposition of extracellular matrix proteins, is a common and severe clinical complication of inflammatory bowel disease (IBD). However, the mechanisms underlying fibrosis remain elusive, and currently, there are limited effective pharmacologic treatments that target the development of fibrosis. Hypoxia is one of the key microenvironmental factors influencing intestinal inflammation and has been linked to fibrosis. OBJECTIVE: In the present study, we sought to elucidate the impact of hypoxia on fibrotic gene expression in the intestinal mucosa. METHODS: Human volunteers, IBD patients, and dextran sulphate sodium-treated mice were exposed to hypoxia, and colonic biopsies were collected. The human intestinal epithelial cell line Caco-2, human THP-1 macrophages, and primary human gut fibroblasts were subjected to hypoxia, and changes in fibrotic gene expression were assessed. RESULTS: Human volunteers subjected to hypoxia presented reduced transcriptional levels of fibrotic and epithelial-mesenchymal transition markers in the intestinal mucosa. IBD patients showed a trend towards a decrease in tissue inhibitor of metalloproteinase 1 protein expression. In mice, hypoxic conditions reduced the colonic expression of several collagens and matrix metalloproteinases. Hypoxic Caco-2 cells, THP-1 cells, and primary gut fibroblasts showed a significant downregulation in the expression of fibrotic and tissue remodelling factors. CONCLUSIONS: Stabilization of hypoxia-inducible factors might represent a novel therapeutic approach for the treatment of IBD-associated fibrosis.
RESUMEN
Fibroblast growth factor 23 (FGF23) is a main regulator of mineral homeostasis. Low and high circulating FGF23 levels are associated with bone, renal, cardiovascular diseases, and increased mortality. Understanding the factors and signaling pathways affecting FGF23 levels is crucial for the management of these diseases and their complications. Here, we show that activation of the Jak1/Stat3 signaling pathway leads to inflammation in liver and to an increase in hepatic FGF23 synthesis, a key hormone in mineral metabolism. This increased synthesis leads to massive C-terminal FGF23 circulating levels, the inactive C-terminal fragment, and increased intact FGF23 levels, the active form, resulting in imbalanced production and cleavage. Liver inflammation does not lead to activation of the calcineurin-NFAT pathway, and no signs of systemic inflammation could be observed. Despite the increase of active intact FGF23, excessive C-terminal FGF23 levels block the phosphaturic activity of FGF23. Therefore, kidney function and renal αKlotho expression are normal and no activation of the MAPK pathway was detected. In addition, activation of the Jak1/Stat3 signaling pathway leads to high calcitriol levels and low parathyroid hormone production. Thus, JAK1 is a central regulator of mineral homeostasis. Moreover, this study also shows that in order to assess the impact of high FGF23 levels on disease and kidney function, the source and the balance in FGF23 production and cleavage are critical.
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Factores de Crecimiento de Fibroblastos/metabolismo , Inflamación/metabolismo , Janus Quinasa 1/metabolismo , Hígado/inmunología , Hígado/metabolismo , Animales , Huesos/metabolismo , Línea Celular , Factor-23 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos/genética , Células HEK293 , Humanos , Inmunoprecipitación , Inflamación/genética , Janus Quinasa 1/genética , Riñón/metabolismo , Ratones , Factor de Transcripción STAT3/metabolismoRESUMEN
Fibroblast growth factor 23 (FGF23) regulates phosphate homeostasis, and its early rise in patients with chronic kidney disease is independently associated with all-cause mortality. Since inflammation is characteristic of chronic kidney disease and associates with increased plasma FGF23 we examined whether inflammation directly stimulates FGF23. In a population-based cohort, plasma tumor necrosis factor (TNF) was the only inflammatory cytokine that independently and positively correlated with plasma FGF23. Mouse models of chronic kidney disease showed signs of renal inflammation, renal FGF23 expression and elevated systemic FGF23 levels. Renal FGF23 expression coincided with expression of the orphan nuclear receptor Nurr1 regulating FGF23 in other organs. Antibody-mediated neutralization of TNF normalized plasma FGF23 and suppressed ectopic renal Fgf23 expression. Conversely, TNF administration to control mice increased plasma FGF23 without altering plasma phosphate. Moreover, in Il10-deficient mice with inflammatory bowel disease and normal kidney function, plasma FGF23 was elevated and normalized upon TNF neutralization. Thus, the inflammatory cytokine TNF contributes to elevated systemic FGF23 levels and also triggers ectopic renal Fgf23 expression in animal models of chronic kidney disease.
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Factores de Crecimiento de Fibroblastos/sangre , Enfermedades Inflamatorias del Intestino/inmunología , Insuficiencia Renal Crónica/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Adulto , Animales , Línea Celular , Estudios de Cohortes , Modelos Animales de Enfermedad , Femenino , Factor-23 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos/inmunología , Factores de Crecimiento de Fibroblastos/metabolismo , Humanos , Enfermedades Inflamatorias del Intestino/sangre , Interleucina-10/deficiencia , Interleucina-10/genética , Riñón/inmunología , Riñón/patología , Masculino , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Cultivo Primario de Células , Insuficiencia Renal Crónica/sangre , Insuficiencia Renal Crónica/patología , Factor de Necrosis Tumoral alfa/sangre , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Nonalcoholic steatohepatitis (NASH), a primary cause of liver disease, leads to complications such as fibrosis, cirrhosis, and carcinoma, but the pathophysiology of NASH is incompletely understood. Epstein-Barr virus-induced G protein-coupled receptor 2 (EBI2) and its oxysterol ligand 7α,25-dihydroxycholesterol (7α,25-diHC) are recently discovered immune regulators. Several lines of evidence suggest a role of oxysterols in NASH pathogenesis, but rigorous testing has not been performed. We measured oxysterol levels in the livers of NASH patients by LC-MS and tested the role of the EBI2-7α,25-diHC system in a murine feeding model of NASH. Free oxysterol profiling in livers from NASH patients revealed a pronounced increase in 24- and 7-hydroxylated oxysterols in NASH compared with controls. Levels of 24- and 7-hydroxylated oxysterols correlated with histological NASH activity. Histological analysis of murine liver samples demonstrated ballooning and liver inflammation. No significant genotype-related differences were observed in Ebi2-/- mice and mice with defects in the 7α,25-diHC synthesizing enzymes CH25H and CYP7B1 compared with wild-type littermate controls, arguing against an essential role of these genes in NASH pathogenesis. Elevated 24- and 7-hydroxylated oxysterol levels were confirmed in murine NASH liver samples. Our results suggest increased bile acid synthesis in NASH samples, as judged by the enhanced level of 7α-hydroxycholest-4-en-3-one and impaired 24S-hydroxycholesterol metabolism as characteristic biochemical changes in livers affected by NASH.
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Hígado/metabolismo , Hígado/patología , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/patología , Oxiesteroles/metabolismo , Adulto , Alanina Transaminasa/sangre , Animales , Aspartato Aminotransferasas/sangre , Colesterol/sangre , Cromatografía Liquida , Citometría de Flujo , Humanos , Hidroxicolesteroles/sangre , Hidroxicolesteroles/metabolismo , Masculino , Espectrometría de Masas , Ratones , Ratones Noqueados , Persona de Mediana Edad , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Enfermedad del Hígado Graso no Alcohólico/sangre , Oxiesteroles/sangre , Esteroide Hidroxilasas/genética , Esteroide Hidroxilasas/metabolismoRESUMEN
The gene encoding for Epstein-Barr virus-induced G-protein-coupled receptor 2 (EBI2) is a risk gene for inflammatory bowel disease (IBD). Together with its oxysterol ligand 7α,25-dihydroxycholesterol, EBI2 mediates migration and differentiation of immune cells. However, the role of EBI2 in the colonic immune system remains insufficiently studied. We found increased mRNA expression of EBI2 and oxysterol-synthesizing enzymes (CH25H, CYP7B1) in the inflamed colon of patients with ulcerative colitis and mice with acute or chronic dextran sulfate sodium (DSS) colitis. Accordingly, we detected elevated levels of 25-hydroxylated oxysterols, including 7α,25-dihydroxycholesterol in mice with acute colonic inflammation. Knockout of EBI2 or CH25H did not affect severity of DSS colitis; however, inflammation was decreased in male EBI2-/- mice in the IL-10 colitis model. The colonic immune system comprises mucosal lymphoid structures, which accumulate upon chronic inflammation in IL-10-deficient mice and in chronic DSS colitis. However, EBI2-/- mice formed significantly less colonic lymphoid structures at baseline and showed defects in inflammation-induced accumulation of lymphoid structures. In summary, we report induction of the EBI2-7α,25-dihydroxycholesterol axis in colitis and a role of EBI2 for the accumulation of lymphoid tissue during homeostasis and inflammation. These data implicate the EBI2-7α,25-dihydroxycholesterol axis in IBD pathogenesis.
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Colitis/metabolismo , Colon/patología , Receptores Acoplados a Proteínas G/metabolismo , Estructuras Linfoides Terciarias/patología , Animales , Movimiento Celular , Células Cultivadas , Colitis/inducido químicamente , Colitis/inmunología , Sulfato de Dextran , Modelos Animales de Enfermedad , Femenino , Humanos , Enfermedades Inflamatorias del Intestino/inmunología , Enfermedades Inflamatorias del Intestino/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oxiesteroles/metabolismo , Receptores Acoplados a Proteínas G/genética , Factores Sexuales , Transducción de SeñalRESUMEN
BACKGROUND AND AIMS: Inflammatory bowel disease [IBD] is accompanied by lesions in the epithelial barrier, which allow translocation of bacterial products from the gut lumen to the host's circulation. IMM-124E is a colostrum-based product containing high levels of anti-E.coli-LPS IgG, and might limit exposure to bacterial endotoxins. Here, we investigated whether IMM-124E can ameliorate intestinal inflammation. METHODS: Acute colitis was induced in WT C57Bl/6J mice by administration of 2.5% dextran sodium sulphate [DSS] for 7 days. T cell transfer colitis was induced via transfer of 0.5 x 106 naïve T cells into RAG2-/- C57Bl/6J mice. IMM-124E was administered daily by oral gavage, either preventively or therapeutically. RESULTS: Treatment with IMM-124E significantly ameliorated colitis in acute DSS colitis and in T cell transfer colitis. Maximum anti-inflammatory effects were detected at an IMM-124E concentration of 100 mg/kg body weight, whereas 25 mg/kg and 500 mg/kg were less effective. Histology revealed reduced levels of infiltrating immune cells and less pronounced mucosal damage. Flow cytometry revealed reduced numbers of effector T helper cells in the intestine, whereas levels of regulatory T cells were enhanced. IMM-124E treatment reduced the DSS-induced increase of serum levels of lipopolysaccharide [LPS]-binding protein, indicating reduced systemic LPS exposure. CONCLUSIONS: Our results demonstrate that oral treatment with IMM-124E significantly reduces intestinal inflammation, via decreasing the accumulation of pathogenic T cells and concomitantly increasing the induction of regulatory T cells. Our study confirms the therapeutic efficacy of IMM-124E in acute colitis and suggests that administration of IMM-124E might represent a novel therapeutic strategy to induce or maintain remission in chronic colitis.
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Colitis/tratamiento farmacológico , Calostro/química , Animales , Western Blotting , Bovinos , Colitis/patología , Colon/patología , Calostro/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Inmunoglobulina G/inmunología , Inmunoglobulina G/uso terapéutico , Lipopolisacáridos/inmunología , Ratones , Ratones Endogámicos C57BL , Peroxidasa/metabolismoRESUMEN
BACKGROUND: Tissue inflammation in inflammatory bowel diseases [IBD] is associated with local acidification. Genetic variants in the pH-sensing G protein-coupled receptor 65, also known as T cell death-associated gene 8 [TDAG8], have been implicated in IBD and other autoimmune diseases. Since the role of TDAG8 in intestinal inflammation remains unclear, we investigated the function of TDAG8 using murine colitis models. METHODS: The effects of TDAG8 deficiency were assessed in dextran sodium sulphate [DSS], IL-10-/-, and T cell transfer colitis murine models. RNA sequencing of acidosis-activated TDAG8-/- and wild-type [WT] peritoneal macrophages [MΦs] was performed. RESULTS: mRNA expression of IFN-γ, TNF, IL-6, and iNOS in TDAG8-/- mice increased significantly in colonic lymphoid patches and in colonic tissue in acute and chronic DSS colitis, respectively. In transfer colitis, there was a trend towards increased IFN-γ, iNOS, and IL-6 expression in mice receiving TDAG8-/- T cells. However, absence of TDAG8 did not lead to changes in clinical scores in the models tested. Increased numbers of infiltrating MΦs and neutrophils, but not CD3+ T cells, were observed in DSS-treated TDAG8-/- mice. No differences in infiltrating CD3+ T cells were observed between mice receiving TDAG8-/- or WT naïve T cells in transfer colitis. RNA sequencing showed that acidosis activation of TDAG8 in MΦs modulated the expression of immune response genes. CONCLUSIONS: TDAG8 deficiency triggers colonic MΦ and neutrophil infiltration, and expression of pro-inflammatory mediators in DSS colitis models. In transfer colitis, mice receiving TDAG8-/- T cells presented a significantly higher spleen weight and a tendency towards increased expression of pro-inflammatory markers of monocyte/MΦ activity.
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Enfermedades Inflamatorias del Intestino/metabolismo , Macrófagos/metabolismo , Animales , Modelos Animales de Enfermedad , Enfermedades Inflamatorias del Intestino/patología , Interferón gamma/metabolismo , Interleucina-6 , Macrófagos/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Óxido Nítrico Sintasa de Tipo II/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
BACKGROUND AND AIMS: pH-sensing ovarian cancer G-protein coupled receptor-1 [OGR1/GPR68] is regulated by key inflammatory cytokines. Patients suffering from inflammatory bowel diseases [IBDs] express increased mucosal levels of OGR1 compared with non-IBD controls. pH-sensing may be relevant for progression of fibrosis, as extracellular acidification leads to fibroblast activation and extracellular matrix remodelling. We aimed to determine OGR1 expression in fibrotic lesions in the intestine of Crohn's disease [CD] patients, and the effect of Ogr1 deficiency in fibrogenesis. METHODS: Human fibrotic and non-fibrotic terminal ileum was obtained from CD patients undergoing ileocaecal resection due to stenosis. Gene expression of fibrosis markers and pH-sensing receptors was analysed. For the initiation of fibrosis in vivo, spontaneous colitis by Il10-/-, dextran sodium sulfate [DSS]-induced chronic colitis and the heterotopic intestinal transplantation model were used. RESULTS: Increased expression of fibrosis markers was accompanied by an increase in OGR1 [2.71 ± 0.69 vs 1.18 ± 0.03, p = 0.016] in fibrosis-affected human terminal ileum, compared with the non-fibrotic resection margin. Positive correlation between OGR1 expression and pro-fibrotic cytokines [TGFB1 and CTGF] and pro-collagens was observed. The heterotopic animal model for intestinal fibrosis transplanted with terminal ileum from Ogr1-/- mice showed a decrease in mRNA expression of fibrosis markers as well as a decrease in collagen layer thickness and hydroxyproline compared with grafts from wild-type mice. CONCLUSIONS: OGR1 expression was correlated with increased expression levels of pro-fibrotic genes and collagen deposition. Ogr1 deficiency was associated with a decrease in fibrosis formation. Targeting OGR1 may be a potential new treatment option for IBD-associated fibrosis.
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Colitis/genética , Colágeno/genética , Enfermedad de Crohn/genética , Enfermedad de Crohn/patología , Mucosa Intestinal/patología , ARN Mensajero/metabolismo , Receptores Acoplados a Proteínas G/genética , Actinas/genética , Animales , Biomarcadores , Colitis/inducido químicamente , Colágeno/metabolismo , Colágeno Tipo I/genética , Cadena alfa 1 del Colágeno Tipo I , Colágeno Tipo III/genética , Factor de Crecimiento del Tejido Conjuntivo/genética , Sulfato de Dextran , Femenino , Fibrosis , Expresión Génica , Humanos , Íleon/metabolismo , Íleon/patología , Íleon/trasplante , Mucosa Intestinal/metabolismo , Masculino , Ratones , Ratones Noqueados , Factor de Crecimiento Transformador beta1/genética , Trasplante Heterotópico , Vimentina/genéticaRESUMEN
Gp96 is an endoplasmic reticulum chaperone for multiple protein substrates. Its lack in intestinal macrophages of Crohn's disease (CD) patients is correlated with loss of tolerance against the host gut flora. Gp96 has been stablished to be an essential chaperone for Toll-like receptors (TLRs). We studied the impact of gp96-knockdown on TLR-function in macrophages. TLR2 and TLR4 expression was only decreased but not abolished when gp96 was knocked-down in cell lines, whereas in a monocyte/macrophage specific knock-out mouse model (LysMCre) TLR4 was abolished, while TLR2 was still present. Lipopolysaccharide (LPS)-induced NF-κB activation was still observed in the absence of gp96, and gp96-deficient macrophages were able to up-regulate surface TLR4 upon LPS treatment, suggesting that there is another chaperone involved in the folding of TLR4 upon stress responses. Moreover, LPS-dependent pro-inflammatory cytokines were still expressed, although to a lesser extent in the absence of gp96, which reinforces the fact that gp96 is involved in regulating signaling cascades downstream of TLR4 are impaired upon loss of gp96. In addition, we have also found a reduced phosphorylation of ERK and p38 kinases and an impaired response upon CSF1R activation in gp96 deficient macrophages. Our findings indicate that the loss of gp96 not only impairs TLR4 signaling, but is also associated with a diminished phosphorylation of ERK and mitogen-activated stress kinases resulting in an impaired signalling through several receptors, including CSF1R.
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Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Glicoproteínas de Membrana/deficiencia , Receptor Toll-Like 4/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Línea Celular , Células HEK293 , Humanos , Interleucina-8/metabolismo , Glicoproteínas de Membrana/genética , Ratones Endogámicos C57BL , Ratones Transgénicos , Inhibidor NF-kappaB alfa/metabolismo , FN-kappa B/metabolismo , Fosforilación/fisiología , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/genéticaRESUMEN
BACKGROUND AND AIMS: During active inflammation, intraluminal intestinal pH is decreased in patients with inflammatory bowel disease [IBD]. Acidic pH may play a role in IBD pathophysiology. Recently, proton-sensing G-protein coupled receptors were identified, including GPR4, OGR1 [GPR68], and TDAG8 [GPR65]. We investigated whether GPR4 is involved in intestinal inflammation. METHODS: The role of GPR4 was assessed in murine colitis models by chronic dextran sulphate sodium [DSS] administration and by cross-breeding into an IL-10 deficient background for development of spontaneous colitis. Colitis severity was assessed by body weight, colonoscopy, colon length, histological score, cytokine mRNA expression, and myeloperoxidase [MPO] activity. In the spontaneous Il-10-/- colitis model, the incidence of rectal prolapse and characteristics of lamina propria leukocytes [LPLs] were analysed. RESULTS: Gpr4-/- mice showed reduced body weight loss and histology score after induction of chronic DSS colitis. In Gpr4-/-/Il-10-/- double knock-outs, the onset and progression of rectal prolapse were significantly delayed and mitigated compared with Gpr4+/+/Il-10-/- mice. Double knock-out mice showed lower histology scores, MPO activity, CD4+ T helper cell infiltration, IFN-γ, iNOS, MCP-1 [CCL2], CXCL1, and CXCL2 expression compared with controls. In colon, GPR4 mRNA was detected in endothelial cells, some smooth muscle cells, and some macrophages. CONCLUSIONS: Absence of GPR4 ameliorates colitis in IBD animal models, indicating an important regulatory role in mucosal inflammation, thus providing a new link between tissue pH and the immune system. Therapeutic inhibition of GPR4 may be beneficial for the treatment of IBD.
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Colitis/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Prolapso Rectal/etiología , Animales , Quimiocina CCL2/metabolismo , Quimiocina CXCL1/metabolismo , Quimiocina CXCL2/metabolismo , Colitis/inducido químicamente , Colitis/complicaciones , Colitis/patología , Sulfato de Dextran , Células Endoteliales/metabolismo , Femenino , Concentración de Iones de Hidrógeno , Interferón gamma/metabolismo , Interleucina-10/genética , Mucosa Intestinal/patología , Macrófagos/metabolismo , Masculino , Ratones , Ratones Noqueados , Miocitos del Músculo Liso/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Peroxidasa/metabolismo , Protones , ARN Mensajero/metabolismo , Prolapso Rectal/genética , Linfocitos T Colaboradores-Inductores/patologíaRESUMEN
Trichuris suis ova (TSO) have been tested for therapeutic application in inflammatory bowel diseases (IBD) yet understanding of the underlying mechanisms and safety in an immunocompromised host is limited due to lack of a suitable animal model. We used a recently established rabbit model of dextran sodium sulphate (DSS) induced colitis to study the efficacy, mechanisms and safety of TSO therapy in immunocompetent and immunosuppressed animals. TSO treatment prevented the DSS induced weight loss, delayed the onset of DSS induced symptoms by 2 days and significantly reduced the disease activity (DAI). TSO treatment protected caecal histology and prevented the colitis-associated loss in faecal microbiota diversity. Mainly the transcriptome of lamina propria mononuclear cells (LPMC) was affected by TSO treatment, showing dampened innate and adaptive inflammatory responses. The protective effect of TSO was lost in immunosuppressed rabbits, where TSO exacerbated colitis. Our data show that preventive TSO treatment ameliorates colitis severity in immunocompetent rabbits, modulates LPMC immune responses and reduces faecal dysbiosis. In contrast, the same TSO treatment exacerbates colitis in immunosuppressed animals. Our data provide further evidence for a therapeutic effect of TSO in IBD, yet caution is required with regard to TSO treatment in immunosuppressed patients.
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Terapia Biológica , Colitis/etiología , Colitis/prevención & control , Terapia de Inmunosupresión , Trichuris/inmunología , Animales , Terapia Biológica/métodos , Colitis/tratamiento farmacológico , Colitis/patología , Sulfato de Dextran/efectos adversos , Modelos Animales de Enfermedad , Microbioma Gastrointestinal , Perfilación de la Expresión Génica , Humanos , Huésped Inmunocomprometido , Enfermedades Inflamatorias del Intestino/etiología , Enfermedades Inflamatorias del Intestino/patología , Enfermedades Inflamatorias del Intestino/terapia , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Macrófagos/inmunología , Macrófagos/metabolismo , Conejos , TranscriptomaRESUMEN
The use of molecular markers for inflammation in the gastrointestinal tract could empower optical imaging modalities for early diagnosis and eventually personalized timely treatments. A major hurdle to the widespread use of functional fluorescence imaging is the absence of suitable contrast agents, in particular to be administered via the oral route due to the usual proteolytic susceptibility of the biomarkers. By designing a retro-inverso peptide, starting from a previously described sequence specific for N-cadherin, we achieved resistance to gastrointestinal degradation and even slightly improved specificity towards the target, both in ex vivo and in vivo experimental colitis. Simulations at fundamental molecular level suggested that the introduced retro-inverso modifications did not affect the folding of the peptide, leaving its ability to interact with the binding pocket of the monomeric N-cadherin unaltered, even when fluorescently labeled. Possible further derivatization of this sequence could be envisaged to further extend the potential of the designed retro-inverso peptide as diagnostic or theranostic agent for the oral route.
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Colitis/diagnóstico por imagen , Péptidos/administración & dosificación , Administración Oral , Animales , Cadherinas/metabolismo , Colitis/inducido químicamente , Colitis/metabolismo , Sulfato de Dextran , Células Epiteliales/metabolismo , Femenino , Ratones Endogámicos C57BL , Microscopía FluorescenteRESUMEN
Hypoxia regulates autophagy and nucleotide-binding oligomerization domain receptor, pyrin domain containing (NLRP)3, two innate immune mechanisms linked by mutual regulation and associated to IBD. Here we show that hypoxia ameliorates inflammation during the development of colitis by modulating autophagy and mammalian target of rapamycin (mTOR)/NLRP3 pathway. Hypoxia significantly reduces tumor necrosis factor α, interleukin (IL)-6 and NLRP3 expression, and increases the turnover of the autophagy protein p62 in colon biopsies of Crohn's disease patients, and in samples from dextran sulfate sodium-treated mice and Il-10 -/- mice. In vitro, NF-κB signaling and NLRP3 expression are reduced through hypoxia-induced autophagy. We also identify NLRP3 as a novel binding partner of mTOR. Dimethyloxalylglycine-mediated hydroxylase inhibition ameliorates colitis in mice, downregulates NLRP3 and promotes autophagy. We suggest that hypoxia counteracts inflammation through the downregulation of the binding of mTOR and NLRP3 and activation of autophagy.Hypoxia and HIF-1α activation are protective in mouse models of colitis, and the latter regulates autophagy. Here Cosin-Roger et al. show that hypoxia ameliorates intestinal inflammation in Crohn's patients and murine colitis models by inhibiting mTOR/NLRP3 pathway and promoting autophagy.
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Colitis Ulcerosa/metabolismo , Enfermedad de Crohn/metabolismo , Inflamación/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Animales , Autofagia/fisiología , Colitis/inducido químicamente , Sulfato de Dextran/toxicidad , Regulación hacia Abajo , Regulación de la Expresión Génica/fisiología , Humanos , Interleucina-10/genética , Interleucina-10/metabolismo , Ratones , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR/genética , ARN Interferente Pequeño , Serina-Treonina Quinasas TOR/genéticaRESUMEN
Inflammatory bowel disease (IBD) may develop due to an immunogenic response to commensal gut microbiota triggered by environmental factors in the genetically susceptible host. Isotretinoin, applied in the treatment of severe acne, has been variably associated with IBD, but prior treatment with antibiotics, also associated with IBD development, confounds confirmation of this association. This study investigated the effects of doxycycline, metronidazole (frequently used in the treatment of acne and IBD, respectively) and isotretinoin on murine gut (faecal) microbiota after 2 weeks of treatment and after a 4-week recovery period. Faecal microbiota composition was assessed by 16S rRNA gene sequencing on the GS-FLX 454 platform with primers directed against the variable regions V1-V2. Doxycycline had a modest effect on bacterial richness and evenness, but had pronounced persistent and significant effects on the abundance of certain operational taxonomic units compared with the control group. In contrast, metronidazole induced a pronounced reduction in diversity after treatment, but these effects did not persist after the recovery period. This study demonstrates differential effects of antibiotics on the gut microbiota with doxycycline, unlike metronidazole, mediating long-term changes in the murine gut microbiota. Isotretinoin had no significant effect on the faecal microbiota.
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Antibacterianos/administración & dosificación , Fármacos Dermatológicos/administración & dosificación , Doxiciclina/administración & dosificación , Microbioma Gastrointestinal/efectos de los fármacos , Isotretinoína/administración & dosificación , Metronidazol/administración & dosificación , Animales , Análisis por Conglomerados , ADN Ribosómico/química , ADN Ribosómico/genética , Femenino , Ratones Endogámicos BALB C , Filogenia , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADNRESUMEN
BACKGROUND AND AIMS: An imbalance between cellular antioxidant defence system[s] and reactive oxygen species [ROS]-driven oxidative stress has been implicated in the pathogenesis of inflammatory bowel disease. Peroxiredoxin [PRDX] 6 contributes to an appropriate redox balance by clearing ROS and reducing peroxidized membrane phospholipids. We here studied the role of PRDX6 in acute and chronic dextran sodium sulphate [DSS]-induced colitis. METHODS: To investigate the impact of PRDX6 on intestinal inflammation, we used wild type [WT], Prdx6 knock-out mice [Prdx6-/-] and transgenic mice [Prdx6tg/tg], overexpressing Prdx6. Acute and chronic colitis was induced by DSS in WT, Prdx6-/- and Prdx6tg/tg mice. Colitis was evaluated by endoscopy, colon length, histopathological assessment and myeloperoxidase [MPO] activity. Changes in mRNA and protein expression of pro-inflammatory cytokines and antioxidant enzymes were evaluated by real-time quantitative polymerase chain reaction [RT-qPCR] and western blot. Total glutathione [GSH] levels in colon samples were determined. RESULTS: Prdx6-/- mice exposed to acute and chronic DSS showed a significant decrease in the clinical parameters and in colonic expression of pro-inflammatory cytokines compared with WT mice. mRNA expression of antioxidant enzymes in colon samples was significantly increased in Prdx6-/- compared with WT mice exposed to acute and chronic DSS. In addition, total GSH levels were increased in Prdx6-/- mice treated with DSS in comparison with WT. Overexpression of Prdx6 did not significantly influence acute and chronic colitis. CONCLUSIONS: Our data indicate that a lack of the antioxidant enzyme PRDX6 protects against the development of acute and chronic experimental colitis and is associated with increased expression and function of other antioxidant enzymes, suggesting effective compensatory mechanisms.
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Colitis/enzimología , Colitis/genética , Citocinas/metabolismo , Peroxiredoxina VI/genética , Enfermedad Aguda , Animales , Enfermedad Crónica , Colitis/inducido químicamente , Colitis/patología , Colon/metabolismo , Sulfato de Dextran , Endoscopía Gastrointestinal , Células Epiteliales/metabolismo , Femenino , Glutamato-Cisteína Ligasa/genética , Glutatión/metabolismo , Glutatión Sintasa/genética , Humanos , Enfermedades Inflamatorias del Intestino/genética , Interferón gamma/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Factor 2 Relacionado con NF-E2/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Peroxidasa/metabolismo , Peroxiredoxina III/genética , Peroxiredoxina VI/metabolismo , Peroxirredoxinas/genética , ARN Mensajero/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
OBJECTIVE: Western lifestyle and diet are major environmental factors playing a role in the development of IBD. Titanium dioxide (TiO2) nanoparticles are widely used as food additives or in pharmaceutical formulations and are consumed by millions of people on a daily basis. We investigated the effects of TiO2 in the development of colitis and the role of the nucleotide-binding oligomerisation domain receptor, pyrin domain containing (NLRP)3 inflammasome. DESIGN: Wild-type and NLRP3-deficient mice with dextran sodium sulfate-induced colitis were orally administered with TiO2 nanoparticles. The proinflammatory effects of TiO2 particles in cultured human intestinal epithelial cells (IECs) and macrophages were also studied, as well as the ability of TiO2 crystals to traverse IEC monolayers and accumulate in the blood of patients with IBD using inductively coupled plasma mass spectrometry. RESULTS: Oral administration of TiO2 nanoparticles worsened acute colitis through a mechanism involving the NLRP3 inflammasome. Importantly, crystals were found to accumulate in spleen of TiO2-administered mice. In vitro, TiO2 particles were taken up by IECs and macrophages and triggered NLRP3-ASC-caspase-1 assembly, caspase-1 cleavage and the release of NLRP3-associated interleukin (IL)-1ß and IL-18. TiO2 also induced reactive oxygen species generation and increased epithelial permeability in IEC monolayers. Increased levels of titanium were found in blood of patients with UC having active disease. CONCLUSION: These findings indicate that individuals with a defective intestinal barrier function and pre-existing inflammatory condition, such as IBD, might be negatively impacted by the use of TiO2 nanoparticles.
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Colitis/inmunología , Colorantes/efectos adversos , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR , Nanopartículas/efectos adversos , Titanio/efectos adversos , Animales , Caspasa 1/metabolismo , Colitis/inducido químicamente , Colitis/metabolismo , Colorantes/administración & dosificación , Sulfato de Dextran/efectos adversos , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Humanos , Interleucina-18/biosíntesis , Interleucina-1beta/metabolismo , Intestinos/citología , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Nanopartículas/administración & dosificación , Especies Reactivas de Oxígeno/metabolismo , Bazo/patología , Titanio/administración & dosificación , Titanio/sangreRESUMEN
BACKGROUND AND AIMS: The transcription factor Nrf2 is a major modulator of the cellular antioxidant response. Oxidative burst of infiltrating macrophages leads to a massive production of reactive oxygen species in inflamed tissue of inflammatory bowel disease patients. This oxidative burst contributes to tissue destruction and epithelial permeability, but it is also an essential part of the antibacterial defence. We therefore investigated the impact of the Nrf2 orchestrated antioxidant response in both acute and chronic intestinal inflammation. METHODS: To study the role of Nrf2 overexpression in mucosal inflammation, we used transgenic mice conditionally expressing a constitutively active form of Nrf2 [caNrf2] either in epithelial cells or in the myeloid cell lineage. Acute colitis was induced by dextran sulphate sodium [DSS] in transgenic and control animals, and changes in gene expression were evaluated by genome-wide expression studies. Long-term effects of Nrf2 activation were studied in mice with an IL-10-/- background. RESULTS: Expression of caNrf2 either in epithelial cells or myeloid cells resulted in aggravation of DSS-induced acute colitis. Aggravation of inflammation by caNrf2 was not observed in the IL-10-/- model of spontaneous chronic colitis, where even a trend towards reduced prolapse rate was observed. CONCLUSIONS: Our findings show that a well-balanced redox homeostasis is as important in epithelial cells as in myeloid cells during induction of colitis. Aggravation of acute DSS colitis in response to constitutive Nrf2 expression emphasises the importance of tight regulation of Nrf2 during the onset of intestinal inflammation.