RESUMEN
The gut microbiota has been implicated as a driver of irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD). Recently we described, mucosal biofilms, signifying alterations in microbiota composition and bile acid (BA) metabolism in IBS and ulcerative colitis (UC). Luminal oxygen concentration is a key factor in the gastrointestinal (GI) ecosystem and might be increased in IBS and UC. Here we analyzed the role of archaea as a marker for hypoxia in mucosal biofilms and GI homeostasis. The effects of archaea on microbiome composition and metabolites were analyzed via amplicon sequencing and untargeted metabolomics in 154 stool samples of IBS-, UC-patients and controls. Mucosal biofilms were collected in a subset of patients and examined for their bacterial, fungal and archaeal composition. Absence of archaea, specifically Methanobrevibacter, correlated with disrupted GI homeostasis including decreased microbial diversity, overgrowth of facultative anaerobes and conjugated secondary BA. IBS-D/-M was associated with absence of archaea. Presence of Methanobrevibacter correlated with Oscillospiraceae and epithelial short chain fatty acid metabolism and decreased levels of Ruminococcus gnavus. Absence of fecal Methanobrevibacter may indicate a less hypoxic GI environment, reduced fatty acid oxidation, overgrowth of facultative anaerobes and disrupted BA deconjugation. Archaea and Ruminococcus gnavus could distinguish distinct subtypes of mucosal biofilms. Further research on the connection between archaea, mucosal biofilms and small intestinal bacterial overgrowth should be performed.
Asunto(s)
Archaea , Bacterias , Biopelículas , Heces , Microbioma Gastrointestinal , Humanos , Biopelículas/crecimiento & desarrollo , Archaea/clasificación , Archaea/metabolismo , Archaea/genética , Archaea/aislamiento & purificación , Adulto , Persona de Mediana Edad , Femenino , Masculino , Bacterias/clasificación , Bacterias/genética , Bacterias/metabolismo , Bacterias/aislamiento & purificación , Heces/microbiología , Colon/microbiología , Methanobrevibacter/metabolismo , Methanobrevibacter/genética , Methanobrevibacter/crecimiento & desarrollo , Methanobrevibacter/aislamiento & purificación , Colitis Ulcerosa/microbiología , Colitis Ulcerosa/metabolismo , Síndrome del Colon Irritable/microbiología , Síndrome del Colon Irritable/metabolismo , Anciano , Mucosa Intestinal/microbiología , Mucosa Intestinal/metabolismo , Íleon/microbiología , Ácidos Grasos Volátiles/metabolismo , Adulto Joven , Ácidos y Sales Biliares/metabolismoRESUMEN
Lynch syndrome (LS) is the most prevalent heritable form of colorectal cancer. Its early onset and high lifetime risk for colorectal cancer emphasize the necessity for effective chemoprevention. NFE2L2 (NRF2) is often considered a potential druggable target, and many chemopreventive compounds induce NRF2. However, although NRF2 counteracts oxidative stress, it is also overexpressed in colorectal cancer and may promote tumorigenesis. In this study, we evaluated the role of NRF2 in the prevention of LS-associated neoplasia. We found increased levels of NRF2 in intestinal epithelia of mice with intestinal epithelium-specific Msh2 deletion (MSH2ΔIEC) compared with C57BL/6 (wild-type) mice, as well as an increase in downstream NRF2 targets NAD(P)H dehydrogenase (quinone 1) and glutamate-cysteine ligase catalytic subunit. Likewise, NRF2 levels were increased in human MSH2-deficient LS tumors compared with healthy human controls. In silico analysis of a publicly accessible RNA sequencing LS dataset also found an increase in downstream NRF2 targets. Upon crossing MSH2ΔIEC with Nrf2null (MSH2ΔIECNrf2null) mice, we unexpectedly found reduced tumorigenesis in MSH2ΔIECNrf2null mice compared with MSH2ΔIEC mice after 40 weeks, which occurred despite an increase in oxidative damage in MSH2ΔIECNrf2null mice. The loss of NRF2 impaired proliferation as seen by Ki67 intestinal staining and in organoid cultures. This was accompanied by diminished WNT/ß-catenin signaling, but apoptosis was unaffected. Microbial α-diversity increased over time with the loss of NRF2 based upon 16S rRNA gene amplicon sequencing of murine fecal samples. Altogether, we show that NRF2 protein levels are increased in MSH2 deficiency and associated neoplasia, but the loss of NRF2 attenuates tumorigenesis. Activation of NRF2 may not be a feasible strategy for chemoprevention in LS. Prevention Relevance: Patients with LS have an early onset and high lifetime risk for colorectal cancer. In this study, we show that NRF2 protein levels are increased in MSH2 deficiency and associated neoplasia, but the loss of NRF2 attenuates tumorigenesis. This suggests that NRF2 may not be a tumor suppressor in this specific context.
Asunto(s)
Neoplasias Colorrectales Hereditarias sin Poliposis , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , Proteína 2 Homóloga a MutS , Factor 2 Relacionado con NF-E2 , Animales , Factor 2 Relacionado con NF-E2/metabolismo , Factor 2 Relacionado con NF-E2/genética , Ratones , Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Neoplasias Colorrectales Hereditarias sin Poliposis/patología , Neoplasias Colorrectales Hereditarias sin Poliposis/metabolismo , Proteína 2 Homóloga a MutS/genética , Proteína 2 Homóloga a MutS/metabolismo , Humanos , Carcinogénesis/genética , Carcinogénesis/patología , Ratones Noqueados , Femenino , Mucosa Intestinal/patología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , MasculinoRESUMEN
Vitamin D (VD) is the most discussed antioxidant supplement for multiple sclerosis (MS) patients and many studies suggest correlations between a low VD serum level and onset and progression of the disease. While many studies in animals as well as clinical studies focused on the role of VD in the relapsing-remitting MS, knowledge is rather sparse for the progressive phase of the disease and the development of cortical pathology. In this study, we used our established rat model of cortical inflammatory demyelination, resembling features seen in late progressive MS, to address the question about whether VD could have positive effects on reducing cortical pathology, oxidative stress, and neurofilament light chain (NfL) serum levels. For this purpose, we used male Dark Agouti (DA) rats, with one group being supplemented with VD (400 IE per week; VD+) from the weaning on at age three weeks; the other group received standard rodent food. The rat brains were assessed using immunohistochemical markers against demyelination, microglial activation, apoptosis, neurons, neurofilament, and reactive astrocytes. To evaluate the effect of VD on oxidative stress and the antioxidant capacity, we used two different oxidized lipid markers (anti- Cu++ and HOCl oxidized LDL antibodies) along with colorimetric methods for protective polyphenols (PP) and total antioxidative capacity (TAC). NfL serum levels of VD+ and VD- animals were analyzed by fourth generation single-molecule array (SIMOA) analysis. We found significant differences between the VD+ and VD- animals both in histopathology as well as in all serum markers. Myelin loss and microglial activation is lower in VD+ animals and the number of apoptotic cells is significantly reduced with a higher neuronal survival. VD+ animals show significantly lower NfL serum levels, a higher TAC, and more PP. Additionally, there is a significant reduction of oxidized lipid markers in animals under VD supplementation. Our data thus show a positive effect of VD on cellular features of cortical pathology in our animal model, presumably due to protection against reactive oxygen species. In this study, VD enhanced remyelination and prevented neuroaxonal and oxidative damage, such as demyelination and neurodegeneration. However, more studies on VD dose relations are required to establish an optimal response while avoiding overdosing.
Asunto(s)
Esclerosis Múltiple Crónica Progresiva , Esclerosis Múltiple , Masculino , Ratas , Animales , Vitamina D , Antioxidantes/farmacología , Esclerosis Múltiple/tratamiento farmacológico , Vitaminas/farmacología , Vitaminas/uso terapéutico , Esclerosis Múltiple Crónica Progresiva/tratamiento farmacológico , Modelos AnimalesRESUMEN
With increasing urbanization and industrialization, the prevalence of inflammatory bowel diseases (IBDs) has steadily been rising over the past two decades. IBD involves flares of gastrointestinal (GI) inflammation accompanied by microbiota perturbations. However, microbial mechanisms that trigger such flares remain elusive. Here, we analyzed the association of the emerging pathogen atypical enteropathogenic E. coli (aEPEC) with IBD disease activity. The presence of diarrheagenic E. coli was assessed in stool samples from 630 IBD patients and 234 age- and sex-matched controls without GI symptoms. Microbiota was analyzed with 16S ribosomal RNA gene amplicon sequencing, and 57 clinical aEPEC isolates were subjected to whole-genome sequencing and in vitro pathogenicity experiments including biofilm formation, epithelial barrier function and the ability to induce pro-inflammatory signaling. The presence of aEPEC correlated with laboratory, clinical and endoscopic disease activity in ulcerative colitis (UC), as well as microbiota dysbiosis. In vitro, aEPEC strains induce epithelial p21-activated kinases, disrupt the epithelial barrier and display potent biofilm formation. The effector proteins espV and espG2 distinguish aEPEC cultured from UC and Crohn's disease patients, respectively. EspV-positive aEPEC harbor more virulence factors and have a higher pro-inflammatory potential, which is counteracted by 5-ASA. aEPEC may tip a fragile immune-microbiota homeostasis and thereby contribute to flares in UC. aEPEC isolates from UC patients display properties to disrupt the epithelial barrier and to induce pro-inflammatory signaling in vitro.
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Colitis Ulcerosa , Escherichia coli Enteropatógena , Infecciones por Escherichia coli , Microbioma Gastrointestinal , Enfermedades Inflamatorias del Intestino , Humanos , Escherichia coli Enteropatógena/genéticaRESUMEN
The gut mucosal environment is key in host health; protecting against pathogens and providing a niche for beneficial bacteria, thereby facilitating a mutualistic balance between host and microbiome. Lack of dietary fiber results in erosion of the mucosal layer, suggested to be a result of increased mucus-degrading gut bacteria. This study aimed to use quantitative analyses to investigate the diet-induced imbalance of mucosal homeostasis. Seven days of fiber-deficiency affected intestinal anatomy and physiology, seen by reduced intestinal length and loss of the colonic crypt-structure. Moreover, the mucus layer was diminished, muc2 expression decreased, and impaired mucus secretion was detected by stable isotope probing. Quantitative microbiome profiling of the gut microbiota showed a diet-induced reduction in bacterial load and decreased diversity across the intestinal tract, including taxa with fiber-degrading and butyrate-producing capabilities. Most importantly, there was little change in the absolute abundance of known mucus-degrading bacteria, although, due to the general loss of taxa, relative abundance would erroneously indicate an increase in mucus degraders. These findings underscore the importance of using quantitative methods in microbiome research, suggesting erosion of the mucus layer during fiber deprivation is due to diminished mucus production rather than overgrowth of mucus degraders.
Asunto(s)
Fibras de la Dieta , Moco , Bacterias , Butiratos/metabolismo , Fibras de la Dieta/metabolismo , Homeostasis , Mucosa Intestinal/metabolismo , Moco/metabolismoRESUMEN
Aberrant innate immune responses to the gut microbiota are causally involved in the pathogenesis of inflammatory bowel diseases (IBD). The exact triggers and main signaling pathways activating innate immune cells and how they modulate adaptive immunity in IBD is still not completely understood. Here, we report that the PI3K/PTEN signaling pathway in dendritic cells enhances IL-6 production in a model of DSS-induced colitis. This results in exacerbated Th1 cell responses and increased mortality in DC-specific PTEN knockout (PTENΔDC) animals. Depletion of the gut microbiota using antibiotics as well as blocking IL-6R signaling rescued mortality in PTENΔDC mice, whereas adoptive transfer of Flt3L-derived PTEN-/- DCs into WT recipients exacerbated DSS-induced colitis and increased mortality. Taken together, we show that the PI3K signaling pathway in dendritic cells contributes to disease pathology by promoting IL-6 mediated Th1 responses.
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Colitis , Enfermedades Inflamatorias del Intestino , Animales , Células Dendríticas , Sulfato de Dextran/efectos adversos , Modelos Animales de Enfermedad , Interleucina-6/metabolismo , Ratones , Ratones Endogámicos C57BL , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de SeñalRESUMEN
BACKGROUND & AIMS: Irritable bowel syndrome (IBS) and inflammatory bowel diseases result in a substantial reduction in quality of life and a considerable socioeconomic impact. In IBS, diagnosis and treatment options are limited, but evidence for involvement of the gut microbiome in disease pathophysiology is emerging. Here we analyzed the prevalence of endoscopically visible mucosal biofilms in gastrointestinal disease and associated changes in microbiome composition and metabolism. METHODS: The presence of mucosal biofilms was assessed in 1426 patients at 2 European university-based endoscopy centers. One-hundred and seventeen patients were selected for in-depth molecular and microscopic analysis using 16S ribosomal RNA gene amplicon-sequencing of colonic biopsies and fecal samples, confocal microscopy with deep learning-based image analysis, scanning electron microscopy, metabolomics, and in vitro biofilm formation assays. RESULTS: Biofilms were present in 57% of patients with IBS and 34% of patients with ulcerative colitis compared with 6% of controls (P < .001). These yellow-green adherent layers of the ileum and right-sided colon were microscopically confirmed to be dense bacterial biofilms. 16S-sequencing links the presence of biofilms to a dysbiotic gut microbiome, including overgrowth of Escherichia coli and Ruminococcus gnavus. R. gnavus isolates cultivated from patient biofilms also formed biofilms in vitro. Metabolomic analysis found an accumulation of bile acids within biofilms that correlated with fecal bile acid excretion, linking this phenotype with a mechanism of diarrhea. CONCLUSIONS: The presence of mucosal biofilms is an endoscopic feature in a subgroup of IBS and ulcerative colitis with disrupted bile acid metabolism and bacterial dysbiosis. They provide novel insight into the pathophysiology of IBS and ulcerative colitis, illustrating that biofilm can be seen as a tipping point in the development of dysbiosis and disease.
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Bacterias/crecimiento & desarrollo , Biopelículas/crecimiento & desarrollo , Colitis Ulcerosa/microbiología , Colon/microbiología , Colonoscopía , Microbioma Gastrointestinal , Mucosa Intestinal/microbiología , Síndrome del Colon Irritable/microbiología , Austria , Bacterias/metabolismo , Bacterias/ultraestructura , Estudios de Casos y Controles , Colitis Ulcerosa/metabolismo , Colitis Ulcerosa/patología , Colon/metabolismo , Colon/patología , Aprendizaje Profundo , Alemania , Humanos , Interpretación de Imagen Asistida por Computador , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Síndrome del Colon Irritable/metabolismo , Síndrome del Colon Irritable/patología , Metabolómica , Microscopía Confocal , Microscopía Electrónica de Rastreo , Valor Predictivo de las Pruebas , RibotipificaciónRESUMEN
Inflammatory bowel disease is a group of conditions with rising incidence caused by genetic and environmental factors including diet. The chelator ethylenediaminetetraacetate (EDTA) is widely used by the food and pharmaceutical industry among numerous other applications, leading to a considerable environmental exposure. Numerous safety studies in healthy animals have revealed no relevant toxicity by EDTA. Here we show that, in the presence of intestinal inflammation, EDTA is surprisingly capable of massively exacerbating inflammation and even inducing colorectal carcinogenesis at doses that are presumed to be safe. This toxicity is evident in two biologically different mouse models of inflammatory bowel disease, the AOM/DSS and the IL10-/- model. The mechanism of this effect may be attributed to disruption of intercellular contacts as demonstrated by in vivo confocal endomicroscopy, electron microscopy and cell culture studies. Our findings add EDTA to the list of food additives that might be detrimental in the presence of intestinal inflammation, but the toxicity of which may have been missed by regulatory safety testing procedures that utilize only healthy models. We conclude that the current use of EDTA especially in food and pharmaceuticals should be reconsidered. Moreover, we suggest that intestinal inflammatory models should be implemented in the testing of food additives to account for the exposure of this primary organ to environmental and dietary stress.
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Carcinogénesis/genética , Colitis/patología , Neoplasias del Colon/patología , Ácido Edético/efectos adversos , Animales , Carcinogénesis/efectos de los fármacos , Colitis/inducido químicamente , Colitis/genética , Neoplasias del Colon/inducido químicamente , Neoplasias del Colon/genética , Modelos Animales de Enfermedad , Aditivos Alimentarios/efectos adversos , Humanos , Enfermedades Inflamatorias del Intestino/inducido químicamente , Enfermedades Inflamatorias del Intestino/genética , Enfermedades Inflamatorias del Intestino/patología , Interleucina-10/genética , Ratones , Ratones NoqueadosRESUMEN
BACKGROUND & AIMS: p21-activated kinase-1 (PAK1) belongs to a family of serine-threonine kinases and contributes to cellular pathways such as nuclear factor-κB (NF-κB), mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT), and Wingless-related integration site(Wnt)/ß-catenin, all of which are involved in intestinal homeostasis. Overexpression of PAK1 is linked to inflammatory bowel disease as well as colitis-associated cancer (CAC), and similarly was observed in interleukin (IL)10 knockout (KO) mice, a model of colitis and CAC. Here, we tested the effects of PAK1 deletion on intestinal inflammation and carcinogenesis in IL10 KO mice. METHODS: IL10/PAK1 double-knockout (DKO) mice were generated and development of colitis and CAC was analyzed. Large intestines were measured and prepared for histology or RNA isolation. Swiss rolls were stained with H&E and periodic acid-Schiff. Co-immunoprecipitation and immunofluorescence were performed using intestinal organoids, SW480, and normal human colon epithelial cells 1CT. RESULTS: When compared with IL10 KO mice, DKOs showed longer colons and prolonged crypts, despite having higher inflammation and numbers of dysplasia. Crypt hyperproliferation was associated with Notch1 activation and diminished crypt differentiation, indicated by a reduction of goblet cells. Gene expression analysis indicated up-regulation of the Notch1 target hairy and enhancer of split-1 and the stem cell receptor leucin-rich repeat-containing G-protein-coupled receptor 5 in DKO mice. Interestingly, the stem cell marker olfactomedin-4 was present in colonic tissue. Increased ß-catenin messenger RNA and cytoplasmic accumulation indicated aberrant Wnt signaling. Co-localization and direct interaction of Notch1 and PAK1 was found in colon epithelial cells. Notch1 activation abrogated this effect whereas silencing of PAK1 led to Notch1 activation. CONCLUSIONS: PAK1 contributes to the regulation of crypt homeostasis under inflammatory conditions by controlling Notch1. This identifies a novel PAK1-Notch1 axis in intestinal pathophysiology of inflammatory bowel disease and CAC.
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Neoplasias Asociadas a Colitis/inmunología , Colitis/inmunología , Receptor Notch1/metabolismo , Quinasas p21 Activadas/metabolismo , Animales , Línea Celular , Colitis/inducido químicamente , Colitis/complicaciones , Colitis/patología , Neoplasias Asociadas a Colitis/patología , Colon/efectos de los fármacos , Colon/inmunología , Colon/patología , Modelos Animales de Enfermedad , Femenino , Silenciador del Gen , Humanos , Interleucina-10/genética , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/inmunología , Mucosa Intestinal/patología , Masculino , Ratones , Ratones Noqueados , Organoides , Piroxicam/administración & dosificación , Piroxicam/toxicidad , Cultivo Primario de Células , Regulación hacia Arriba , Vía de Señalización Wnt/inmunología , Quinasas p21 Activadas/genéticaRESUMEN
Colorectal cancer is a multifactorial disease involving inherited DNA mutations, environmental factors, gut inflammation and intestinal microbiota. Certain germline mutations within the DNA mismatch repair system are associated with Lynch syndrome tumors including right-sided colorectal cancer with mucinous phenotype and presence of an inflammatory infiltrate. Such tumors are more often associated with bacterial biofilms, which may contribute to disease onset and progression. Inflammatory bowel diseases are also associated with colorectal cancer and intestinal dysbiosis. Herein we addressed the question, whether inflammation can aggravate colorectal cancer development under mismatch repair deficiency. MSH2loxP/loxP Vill-cre mice were crossed into the IL-10-/- background to study the importance of inflammation and mucosal bacteria as a driver of tumorigenesis in a Lynch syndrome mouse model. An increase in large bowel tumorigenesis was found in double knockout mice both under conventional housing and under specific pathogen-free conditions. This increase was mostly due to the development of proximal tumors, a hotspot for tumorigenesis in Lynch syndrome, and was associated with a higher degree of inflammation. Additionally, bacterial invasion into the mucus of tumor crypts was observed in the proximal tumors. Inflammation shifted fecal and mucosal microbiota composition and was associated with enrichment in Escherichia-Shigella as well as Akkermansia, Bacteroides and Parabacteroides genera in fecal samples. Tumor-bearing double knockout mice showed a similar enrichment for Escherichia-Shigella and Parabacteroides. Lactobacilli, Lachnospiraceae and Muribaculaceae family members were depleted upon inflammation. In summary, chronic inflammation aggravates colonic tumorigenesis under mismatch repair deficiency and is associated with a shift in microbiota composition.
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Carcinogénesis/patología , Neoplasias Colorrectales Hereditarias sin Poliposis/microbiología , Neoplasias Colorrectales Hereditarias sin Poliposis/parasitología , Neoplasias Colorrectales/microbiología , Neoplasias Colorrectales/patología , Animales , Bacterias/patogenicidad , Biopelículas/crecimiento & desarrollo , Carcinogénesis/genética , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Neoplasias Colorrectales/genética , Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Reparación de la Incompatibilidad de ADN/genética , Modelos Animales de Enfermedad , Disbiosis/genética , Disbiosis/microbiología , Disbiosis/patología , Microbioma Gastrointestinal/genética , Mutación de Línea Germinal/genética , Inflamación/genética , Inflamación/microbiología , Inflamación/patología , Interleucina-10/genética , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Inflammatory bowel disease increases the odds of developing colitis-associated cancer. We hypothesized that Western-style diet (WD) aggravates azoxymethane (AOM)/dextran sulfate sodium salt (DSS)-induced colitis-associated tumorigenesis and that switching to the standard AIN93G diet will ameliorate disease symptoms even after cancer initiation. Female BALB/c mice received either WD (WD group) or standard AIN93G diet (AIN group) for the whole experimental period. After five weeks, the mice received 12.5 mg/kg AOM intraperitoneally, followed by three DSS cycles. In one group of mice, the WD was switched to AIN93G the day before starting the first DSS cycle (WD/AIN group). Feeding the WD during the whole experimental period aggravated colitis symptoms, shortened the colon (p < 0.05), changed microbiota composition and increased tumor promotion. On molecular level, the WD reduced proliferation (p < 0.05) and increased expression of the vitamin D catabolizing enzyme Cyp24a1 (p < 0.001). The switch to the AIN93G diet ameliorated this effect, reflected by longer colons, fewer (p < 0.05) and smaller (p < 0.01) aberrant colonic crypt foci, comparable with the AIN group. Our results show that switching to a healthy diet, even after cancer initiation is able to revert the deleterious effect of the WD and could be an effective preventive strategy to reduce colitis symptoms and prevent tumorigenesis.
Asunto(s)
Colitis/inducido químicamente , Colitis/complicaciones , Neoplasias Colorrectales/prevención & control , Dieta Saludable , Dieta Occidental/efectos adversos , Focos de Criptas Aberrantes/patología , Animales , Azoximetano/administración & dosificación , Colon/patología , Neoplasias Colorrectales/etiología , Neoplasias Colorrectales/patología , Sulfato de Dextran/administración & dosificación , Modelos Animales de Enfermedad , Femenino , Microbioma Gastrointestinal/fisiología , Hígado/enzimología , Ratones , Ratones Endogámicos BALB C , Vitamina D/metabolismoRESUMEN
The mechanistic target of rapamycin complex 2 (mTORC2) is a potentially novel and promising anticancer target due to its critical roles in proliferation, apoptosis, and metabolic reprogramming of cancer cells. However, the activity and function of mTORC2 in distinct cells within malignant tissue in vivo is insufficiently explored. Surprisingly, in primary human and mouse colorectal cancer (CRC) samples, mTORC2 signaling could not be detected in tumor cells. In contrast, only macrophages in tumor-adjacent areas showed mTORC2 activity, which was downregulated in stromal macrophages residing within human and mouse tumor tissues. Functionally, inhibition of mTORC2 by specific deletion of Rictor in macrophages stimulated tumorigenesis in a colitis-associated CRC mouse model. This phenotype was driven by a proinflammatory reprogramming of mTORC2-deficient macrophages that promoted colitis via the cytokine SPP1/osteopontin to stimulate tumor growth. In human CRC patients, high SPP1 levels and low mTORC2 activity in tumor-associated macrophages correlated with a worsened clinical prognosis. Treatment of mice with a second-generation mTOR inhibitor that inhibits mTORC2 and mTORC1 exacerbated experimental colorectal tumorigenesis in vivo. In conclusion, mTORC2 activity is confined to macrophages in CRC and limits tumorigenesis. These results suggest activation but not inhibition of mTORC2 as a therapeutic strategy for colitis-associated CRC.
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Carcinogénesis/inmunología , Colitis Ulcerosa/patología , Neoplasias Colorrectales/inmunología , Macrófagos/inmunología , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Animales , Carcinogénesis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Colitis Ulcerosa/sangre , Colitis Ulcerosa/inducido químicamente , Colitis Ulcerosa/inmunología , Colon/citología , Colon/efectos de los fármacos , Colon/inmunología , Colon/patología , Neoplasias Colorrectales/mortalidad , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/prevención & control , Sulfato de Dextran/toxicidad , Modelos Animales de Enfermedad , Femenino , Humanos , Mucosa Intestinal/citología , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/inmunología , Mucosa Intestinal/patología , Estimación de Kaplan-Meier , Macrófagos/metabolismo , Masculino , Diana Mecanicista del Complejo 2 de la Rapamicina/antagonistas & inhibidores , Ratones , Ratones Transgénicos , Morfolinas/farmacología , Osteopontina/sangre , Osteopontina/metabolismo , Cultivo Primario de Células , Pronóstico , Tasa de SupervivenciaRESUMEN
Disruption of mucosal structure and barrier function contribute to the pathogenesis of inflammatory bowel disease (IBD). Efficacy of therapy in IBD is based on endoscopic mucosal healing, which occurs by a dynamic interplay of epithelial cell regeneration, migration and differentiation. Both mesalamine (5-ASA) and azathioprine (AZTP) promote this process through mechanisms not clearly understood. We examined molecular pathways implicated in epithelial barrier function that were altered by 5-ASA and AZTP. Paracellular permeability induced by inflammatory mediators was mitigated by both compounds through restoration of cellular anchoring complexes. 5-ASA and AZTP induced rearrangement and membranous localization of junctional proteins and modulated genes involved in tight junctions. Intestinal organoids from wildtype-mice treated with TNF-α and IL-10- deficient-mice displayed impaired epithelial barrier with loss of membranous E-cadherin and reduced Desmoglein-2 expression. These effects were counteracted by 5-ASA and AZTP. Unlike AZTP that exhibited antiproliferative effects, 5-ASA promoted wound healing in colon epithelial cells. Both affected cellular senescence, cell cycle distribution and restricted cells in G1 or S phase without inducing apoptosis. This study provides mechanistic evidence that molecular actions of 5-ASA and AZTP on intestinal epithelia are fundamental in the resolution of barrier dysfunction.
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Azatioprina/farmacología , Células Epiteliales/efectos de los fármacos , Inflamación , Enfermedades Inflamatorias del Intestino/fisiopatología , Intestinos/efectos de los fármacos , Mesalamina/farmacología , Animales , Antiinflamatorios no Esteroideos , Azatioprina/uso terapéutico , Colitis , Células Epiteliales/fisiología , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Intestinos/fisiopatología , Mesalamina/uso terapéutico , Ratones , Cicatrización de HeridasRESUMEN
Patients with inflammatory bowel disease (IBD) have a higher risk of developing colitis-associated-cancer (CAC); however, the underlying processes of disease progression are not completely understood. Here, the molecular processes of inflammation-driven colon carcinogenesis were investigated using IL10-deficient mice (IL10 KO). IL10 KO mice were euthanized after development of colitis and dysplasia. IHC was performed for markers of colitis-induced DNA damage (CIDD): oxidative DNA lesions (8-oxoG), double-strand breaks (DSB; γH2AX). and DSB repair. MSI, LOH (Trp53, Apc), and global methylation (CIMP) were assessed on microdissected tissue. Comet assay for DNA damage, immunofluorescence, and immunoblotting were performed on intestinal organoids from wild-type (WT) and IL10 KO mice. Sequential biopsies and surgical specimens from IBD and CAC patients were used for IHC analysis. Severity of inflammation correlated with number of dysplasia. 8-oxoG and γH2AX-positive cells were significantly increased in inflamed and dysplastic areas along with activation of DSB repair. The amount of positively stained cells strongly correlated with degree of inflammation (8-oxoG: R = 0.923; γH2AX: R = 0.858). Neither CIMP, MSI nor LOH was observed. Enhanced DSBs in IL10 KO organoids were confirmed by comet assay and increased expression of γH2AX. Human clinical specimens exhibited significantly higher γH2AX and 8-oxoG in IBD, dysplasia, and CAC compared with normal mucosa. These data indicate that inflammation-driven colon carcinogenesis in IL10 KO mice and IBD patients is associated with oxidative DNA damage and overt presence of DSB. Mol Cancer Res; 16(4); 634-42. ©2018 AACR.
Asunto(s)
Colitis Ulcerosa/genética , Histonas/metabolismo , Interleucina-10/genética , Neoplasias Gástricas/genética , Animales , Colitis Ulcerosa/complicaciones , Colitis Ulcerosa/metabolismo , Roturas del ADN de Doble Cadena , Modelos Animales de Enfermedad , Guanina/análogos & derivados , Guanina/metabolismo , Humanos , Ratones , Ratones Noqueados , Estrés Oxidativo , Neoplasias Gástricas/etiología , Neoplasias Gástricas/metabolismoRESUMEN
Microsatellite instability (MSI) is present in ulcerative colitis (UC) and colitis-associated colorectal cancers (CAC). Certain factors released by polymorphonuclear cells (PMNs) may drive mucosal frameshift mutations resulting in MSI and cancer. Here, we applied a co-culture system with PMNs and colon epithelial cells to identify such culprit factors. Subjecting HCT116 + chr3 and human colonic epithelial cells (HCEC)-1CT MSI-reporter cell lines harboring mono-, di- or tetranucleotide DNA repeats linked to enhanced green fluorescent protein (EGFP) to activated PMNs induced frameshift mutations within all repeats, as quantified by flow cytometry. Activated PMNs released superoxide and hydrogen peroxide (H2O2), as measured by lucigenin-amplified chemiluminescence and fluorometry, respectively. Catalase, which scavenges H2O2, reduced such PMN-induced MSI. The NADPH-oxidase inhibitor apocynin, which blocks the oxidative burst in PMNs, similarly inhibited PMN-induced MSI. A bead-based multiplex assay revealed that PMNs release a wide range of cytokines such as interleukin (IL)-8, IL-6 and tumor necrosis factor-α (TNF-α). In vitro, these cytokines increased MSI in colon epithelial cells, and the Janus kinase (JAK) inhibitor tofacitinib abolished IL-6-induced or PMN-induced MSI. Intracellular reactive oxygen species (ROS) formation, as measured by 2',7'-dichlorofluorescein diacetate (DCFDA) assay, was induced upon cytokine treatment. DNA oxidation upon IL-6 was present, as detected by formamidopyrimidine glycosylase (FPG)-modified comet assay. In conclusion, activated PMNs induce frameshift mutations in colon epithelial cells resulting in MSI. Both oxidative burst with release of ROS and PMN-secreted cytokines, such as IL-8, IL-6 or TNF-α, contribute to MSI. ROS scavengers and/or specific inhibitors of cytokine signaling may delay or prevent cancer development in the setting of colitis.
Asunto(s)
Colitis/complicaciones , Neoplasias Colorrectales/etiología , Inestabilidad de Microsatélites , Mutagénesis/fisiología , Neutrófilos/metabolismo , Línea Celular Tumoral , Técnicas de Cocultivo , Colitis/metabolismo , Citocinas/metabolismo , Mutación del Sistema de Lectura , Humanos , Estrés Oxidativo/fisiología , Especies Reactivas de Oxígeno/efectos adversos , Especies Reactivas de Oxígeno/metabolismoRESUMEN
HuR is an RNA-binding protein implicated in immune homeostasis and various cancers, including colorectal cancer. HuR binding to AU-rich elements within the 3' untranslated region of mRNAs encoding oncogenes, growth factors, and various cytokines leads message stability and translation. In this study, we evaluated HuR as a small-molecule target for preventing colorectal cancer in high-risk groups such as those with familial adenomatosis polyposis (FAP) or inflammatory bowel disease (IBD). In human specimens, levels of cytoplasmic HuR were increased in colonic epithelial cells from patients with IBD, IBD-cancer, FAP-adenoma, and colorectal cancer, but not in patients with IBD-dysplasia. Intraperitoneal injection of the HuR small-molecule inhibitor MS-444 in AOM/DSS mice, a model of IBD and inflammatory colon cancer, augmented DSS-induced weight loss and increased tumor multiplicity, size, and invasiveness. MS-444 treatment also abrogated tumor cell apoptosis and depleted tumor-associated eosinophils, accompanied by a decrease in IL18 and eotaxin-1. In contrast, HuR inhibition in APCMin mice, a model of FAP and colon cancer, diminished the number of small intestinal tumors generated. In this setting, fecal microbiota, evaluated by 16S rRNA gene amplicon sequencing, shifted to a state of reduced bacterial diversity, with an increased representation of Prevotella, Akkermansia, and Lachnospiraceae Taken together, our results indicate that HuR activation is an early event in FAP-adenoma but is not present in IBD-dysplasia. Furthermore, our results offer a preclinical proof of concept for HuR inhibition as an effective means of FAP chemoprevention, with caution advised in the setting of IBD. Cancer Res; 77(9); 2424-38. ©2017 AACR.
Asunto(s)
Poliposis Adenomatosa del Colon/genética , Neoplasias Colorrectales/genética , Proteína 1 Similar a ELAV/genética , Enfermedades Inflamatorias del Intestino/genética , Poliposis Adenomatosa del Colon/microbiología , Poliposis Adenomatosa del Colon/patología , Animales , Apoptosis/efectos de los fármacos , Carcinogénesis/genética , Proliferación Celular/efectos de los fármacos , Quimiocina CCL11/genética , Neoplasias Colorrectales/microbiología , Neoplasias Colorrectales/patología , Proteína 1 Similar a ELAV/antagonistas & inhibidores , Heces/microbiología , Furanos/administración & dosificación , Microbioma Gastrointestinal/efectos de los fármacos , Microbioma Gastrointestinal/genética , Células HCT116 , Humanos , Enfermedades Inflamatorias del Intestino/microbiología , Enfermedades Inflamatorias del Intestino/patología , Interleucina-18/genética , Ratones , Naftoles/administración & dosificación , Células RAW 264.7RESUMEN
Anemia affects a fourth of the global population, with iron deficiency remaining the primary cause. It is associated with diminished work capacity, fatigue, impaired cognitive function, and can negatively impact the course of diseases like chronic heart failure or chronic kidney disease. Treatment options include oral and intravenous iron; however, conditions such as inflammatory bowel disease, celiac disease, or autoimmune gastritis can diminish the efficacy of oral iron. Timely recognition of iron deficiency anemia and administration of appropriate therapy not only improves quality of life, but also reduces the need for blood transfusions. Proper selection of iron-deficient patients for whom further diagnostic testing is necessary facilitates identification of underlying diseases that require specific treatment, and avoids unnecessary invasive testing.
Asunto(s)
Anemia Ferropénica/diagnóstico , Adulto , Algoritmos , Anemia Ferropénica/sangre , Anemia Ferropénica/tratamiento farmacológico , Anemia Ferropénica/etiología , Femenino , Hemoglobinometría , Humanos , Hierro/sangre , Hierro/uso terapéutico , Masculino , Embarazo , Valores de ReferenciaRESUMEN
The HECT domain E3 ligase HACE1 has been identified as a tumor suppressor in multiple cancers. Here, we report that HACE1 is a central gatekeeper of TNFR1-induced cell fate. Genetic inactivation of HACE1 inhibits TNF-stimulated NF-κB activation and TNFR1-NF-κB-dependent pathogen clearance in vivo. Moreover, TNF-induced apoptosis was impaired in hace1 mutant cells and knockout mice in vivo. Mechanistically, HACE1 is essential for the ubiquitylation of the adaptor protein TRAF2 and formation of the apoptotic caspase-8 effector complex. Intriguingly, loss of HACE1 does not impair TNFR1-mediated necroptotic cell fate via RIP1 and RIP3 kinases. Loss of HACE1 predisposes animals to colonic inflammation and carcinogenesis in vivo, which is markedly alleviated by genetic inactivation of RIP3 kinase and TNFR1. Thus, HACE1 controls TNF-elicited cell fate decisions and exerts tumor suppressor and anti-inflammatory activities via a TNFR1-RIP3 kinase-necroptosis pathway.
Asunto(s)
Linaje de la Célula , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Caspasa 8/metabolismo , Linaje de la Célula/efectos de los fármacos , Colitis/metabolismo , Colitis/patología , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Sulfato de Dextran , Embrión de Mamíferos/citología , Activación Enzimática/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Eliminación de Gen , Ratones Endogámicos C57BL , Mutación/genética , FN-kappa B/metabolismo , Necrosis , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Factor 2 Asociado a Receptor de TNF/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Ubiquitinación/efectos de los fármacosRESUMEN
p21-activated kinase 1 (PAK1) is a serine/threonine kinase that is overexpressed in colorectal cancer. PAK1 is a target of mesalamine [5-aminosylicylic acid (5-ASA)], a common drug for the treatment of ulcerative colitis with prospective chemopreventive properties. Here, we investigated whether PAK1 deletion impedes tumorigenesis in murine intestinal cancer models. Ten-week-old APC(min) or APC(min)/PAK1(-/-) mice were monitored for 8 weeks, euthanized, and assessed for tumor number and size. Six- to 8-week-old PAK1(-/-) and wild-type (WT) mice received one 10 mg/kg intraperitoneal injection of azoxymethane (AOM) and four cycles of 1.7% dextran sodium sulfate (DSS) for 4 days followed by 14 days of regular water. Mice also received 5-ASA via diet. Tumor incidence and size was assessed via colonoscopy and pathology. Molecular targets of PAK1 and 5-ASA were evaluated via immunohistochemistry (IHC) in both models. PAK1 deletion reduced tumor multiplicity and tumor burden but did not alter average tumor size in APC(min) mice. IHC revealed that PAK1 deletion reduced p-AKT, ß-catenin, and c-Myc expression in APC(min) adenomas. Colonoscopy and pathologic analysis revealed that PAK1 deletion reduced tumor multiplicity without affecting tumor size in AOM/DSS-treated mice. 5-ASA treatment and PAK1 deletion impeded tumor multiplicity and dysplastic lesions in AOM/DSS mice. IHC further revealed that 5-ASA blocked ß-catenin signaling via inhibition of PAK1/p-AKT. These data indicate that PAK1 contributes to initiation of intestinal carcinogenesis.