RESUMEN
Neutrophils (polymorphonuclear leukocytes, PMNs) comprise a major component of the immune cell infiltrate during acute mucosal inflammation and have an important role in molding the inflammatory tissue environment. While PMNs are essential to clearance of invading microbes, the major PMN antimicrobial enzyme myeloperoxidase (MPO) can also promote bystander tissue damage. We hypothesized that blocking MPO would attenuate acute colitis and prevent the development of chronic colitis by limiting bystander tissue damage. Using the acute and chronic dextran sodium sulfate model of murine colitis, we demonstrated that MPO-deficient mice experienced less inflammation and more rapidly resolved colitis relative to wild-type controls. Mechanistic studies demonstrated that activated MPO disrupted intestinal epithelial barrier function through the dysregulation of the epithelial tight junction proteins. Our findings revealed that activated MPO chlorinates tyrosine within several tight junction proteins, thereby promoting tight junction mislocalization and dysfunction. These observations in cell models and in murine colitis were validated in human intestinal biopsies from individuals with ulcerative colitis and revealed a strong correlation between disease severity (Mayo score) and tissue chlorinated tyrosine levels. In summary, these findings implicate MPO as a viable therapeutic target to limit bystander tissue damage and preserve mucosal barrier function during inflammation.
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Modelos Animales de Enfermedad , Mucosa Intestinal , Neutrófilos , Peroxidasa , Proteínas de Uniones Estrechas , Peroxidasa/metabolismo , Animales , Ratones , Humanos , Mucosa Intestinal/patología , Mucosa Intestinal/metabolismo , Neutrófilos/metabolismo , Neutrófilos/inmunología , Proteínas de Uniones Estrechas/metabolismo , Colitis/patología , Colitis/metabolismo , Colitis/inducido químicamente , Halogenación , Inflamación/metabolismo , Inflamación/patología , Masculino , Ratones Noqueados , Sulfato de Dextran/toxicidad , Uniones Estrechas/metabolismo , Femenino , Ratones Endogámicos C57BL , Colitis Ulcerosa/patología , Colitis Ulcerosa/metabolismoRESUMEN
Colorectal cancer has been linked to chronic colitis and red meat consumption, which can increase colonic iron and heme. Heme oxygenase-1 ( Hmox1 ) metabolizes heme and releases ferrous iron, but its role in colonic tumorigenesis is not well-described. Recent studies suggest that ferroptosis, the iron-dependent form of cell death, protects against colonic tumorigenesis. Ferroptosis culminates in excessive lipid peroxidation that is constrained by the antioxidative glutathione pathway. We observed increased mucosal markers of ferroptosis and glutathione metabolism in the setting of murine and human colitis, as well as murine colonic neoplasia. We obtained similar results in murine and human colonic epithelial organoids exposed to heme and the ferroptosis activator erastin, especially induction of Hmox1 . RNA sequencing of colonic organoids from mice with deletion of intestinal epithelial Hmox1 (Hmox1 ΔIEC ) revealed increased ferroptosis and activated glutathione metabolism after heme exposure. In a colitis-associated cancer model we observed significantly fewer and smaller tumors in Hmox1 ΔIEC mice compared to littermate controls. Transcriptional profiling of Hmox1 ΔIEC tumors and tumor organoids revealed increased ferroptosis and oxidative stress markers in tumor epithelial cells. In total, our findings reveal ferroptosis as an important colitis-associated cancer signature pathway, and Hmox1 as a key regulator in the tumor microenvironment.
RESUMEN
Crohn disease (CD) is a highly morbid chronic inflammatory disease. Although many patients with CD also develop fibrostenosing complications, there are no medical therapies for intestinal fibrosis. This is due, in part, to a lack of high-fidelity biomimetic models to enhance understanding and drug development, which highlights the need for developing in vivo models of inflammatory bowel disease-related intestinal fibrosis. This study investigates whether the TNFΔARE mouse, a model of ileal inflammation, also develops intestinal fibrosis. Several clinically relevant outcomes were studied, including features of structural fibrosis, histologic fibrosis, and gene expression. These include the use of a new luminal casting technique, traditional histologic outcomes, use of second harmonic imaging, and quantitative PCR. These features were studied in aged TNFΔARE mice as well as in cohorts of numerous ages. At >24 weeks of age, TNFΔARE mice developed structural, histologic, and transcriptional changes of ileal fibrosis. Protein and RNA expression profiles showed changes as early as 6 weeks, coinciding with histologic changes as early as 14 to 15 weeks. Overt structural fibrosis was delayed until at least 16 weeks and was most developed after 24 weeks. This study found that the TNFΔARE mouse is a viable and highly tractable model of ileal fibrosis. This model and the techniques used herein can be leveraged for both mechanistic studies and therapeutic development for the treatment of intestinal fibrosis.
Asunto(s)
Enfermedad de Crohn , Intestinos , Ratones , Animales , Intestinos/patología , Enfermedad de Crohn/patología , Inflamación/patología , Íleon/metabolismo , FibrosisRESUMEN
On the backdrop of all acute inflammatory processes lies the activation of the resolution response. Recent years have witnessed an emerging interest in defining molecular factors that influence the resolution of inflammation. A keystone feature of the mucosal inflammatory microenvironment is hypoxia. The gastrointestinal tract, particularly the colon, exists in a state of physiological hypoxia and during active inflammation, this hypoxic state is enhanced as a result of infiltrating leukocyte oxygen consumption and the activation of oxygen consuming enzymes. Most evidence suggests that mucosal hypoxia promotes the active resolution of inflammation through a variety of mechanisms, including extracellular acidification, purine biosynthesis/salvage, the generation of specialized pro-resolving lipid mediators (ie. resolvins) and altered chemokine/cytokine expression. It is now appreciated that infiltrating innate immune cells (neutrophils, eosinophils, macrophages) have an important role in molding the tissue microenvironment to program an active resolution response. Structural or functional dysregulation of this inflammatory microenvironment can result in the loss of tissue homeostasis and ultimately progression toward chronicity. In this review, we will discuss how inflammatory hypoxia drives mucosal inflammatory resolution and its impact on other microenvironmental factors that influence resolution.
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Inflamación , Mucositis , Humanos , Hipoxia , Membrana Mucosa/metabolismo , NeutrófilosRESUMEN
Background & Aims: Crohn's disease (CD) is a highly morbid chronic inflammatory disease. The majority of CD patients also develop fibrostenosing complications. Despite this, there are no medical therapies for intestinal fibrosis. This is in part due to lack of high-fidelity biomimetic models to enhance understanding and drug development. There is a need to develop in vivo models of inflammatory bowel disease-related intestinal fibrosis. We sought to determine if the TNF ΔARE mouse, a model of ileal inflammation, may also develop intestinal fibrosis. Methods: Several clinically relevant outcomes were studied including features of structural fibrosis, histological fibrosis, and gene expression. These include the use of a luminal casting technique we developed, traditional histological outcomes, use of second harmonic imaging, and quantitative PCR. These features were studied in aged TNF ΔARE mice as well as in cohorts of numerous ages. Results: At ages of 24+ weeks, TNF ΔARE mice develop structural, histological, and genetic changes of ileal fibrosis. Genetic expression profiles have changes as early as six weeks, followed by histological changes occurring as early as 14-15 weeks, and overt structural fibrosis delayed until after 24 weeks. Discussion: The TNF ΔARE mouse is a viable and highly tractable model of intestinal fibrosis. This model and the techniques employed can be leveraged for both mechanistic studies and therapeutic development for the treatment of intestinal fibrosis.
RESUMEN
The intestinal mucosa exists in a state of "physiologic hypoxia," where oxygen tensions are markedly lower than those in other tissues. Intestinal epithelial cells (IECs) have evolved to maintain homeostasis in this austere environment through oxygen-sensitive transcription factors, including hypoxia-inducible factors (HIFs). Using an unbiased chromatin immunoprecipitation (ChIP) screen for HIF-1 targets, we identify autophagy as a major pathway induced by hypoxia in IECs. One important function of autophagy is to defend against intracellular pathogens, termed "xenophagy." Analysis reveals that HIF is a central regulator of autophagy and that in vitro infection of IECs with Salmonella Typhimurium results in induction of HIF transcriptional activity that tracks with the clearance of intracellular Salmonella. Work in vivo demonstrates that IEC-specific deletion of HIF compromises xenophagy and exacerbates bacterial dissemination. These results reveal that the interaction between hypoxia, HIF, and xenophagy is an essential innate immune component for the control of intracellular pathogens.
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Macroautofagia , Infecciones por Salmonella , Humanos , Hipoxia/metabolismo , Mucosa Intestinal/metabolismo , Oxígeno/metabolismo , Infecciones por Salmonella/metabolismo , Factores de Transcripción/metabolismoRESUMEN
The healthy mammalian intestine is lined by a single layer of epithelial cells. These cells provide a selectively permeable barrier to luminal contents and normally do so in an efficient and effective manner. Barrier function in the healthy mucosa is provided via several mechanisms including epithelial junctional complexes, mucus production, as well as mucosal-derived antimicrobial proteins. As tissue metabolism is central to the maintenance of homeostasis in the mucosa, intestinal [Formula: see text] levels are uniquely low due to counter-current blood flow and the presence of the microbiota, resulting in the stabilization of the transcription factor hypoxia-inducible factor (HIF). Ongoing studies have revealed that HIF molds normal intestinal metabolism and is central to the coordination of barrier regulation during both homeostasis and active disease. During acute inflammation, HIF is central to controlling the rapid restitution of the epithelium consistent with normal wound healing responses. In contrast, HIF may also contribute to the fibrostenotic response associated with chronic, nonresolving inflammation. As such, HIF may function as a double-edged sword in the overall course of the inflammatory response. Here, we review recent literature on the contribution of HIF to mucosal barrier function, wound healing, and fibrosis.
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Mucosa Intestinal , Cicatrización de Heridas , Animales , Fibrosis , Humanos , Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Inflamación/metabolismo , Mucosa Intestinal/metabolismo , MamíferosRESUMEN
Neutrophil (PMN) infiltration during active inflammation imprints changes in the local tissue environment. Such responses are often accompanied by significant extracellular acidosis that result in predictable transcriptional responses. In this study, we explore the mechanisms involved in inflammatory acidification as a result of PMN-intestinal epithelial cell (IEC) interactions. Using recently developed tools, we revealed that PMN transepithelial migration (TEM)-associated inflammatory acidosis is dependent on the total number of PMNs present during TEM and is polarized toward the apical surface. Extending these studies, we demonstrate that physical separation of the PMNs and IECs prevented acidification, whereas inhibition of PMN TEM using neutralizing antibodies enhanced extracellular acidification. Utilizing pharmaceutical inhibitors, we demonstrate that the acidification response is independent of myeloperoxidase and dependent on reactive oxygen species generated during PMN TEM. In conclusion, inflammatory acidosis represents a polarized PMN-IEC-dependent response by an as yet to be fully determined mechanism.
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Mucosa Intestinal , Neutrófilos , Adhesión Celular , Células Cultivadas , Concentración de Iones de HidrógenoRESUMEN
Intestinal eosinophils are implicated in the inflammatory pathology of eosinophilic gastrointestinal diseases and inflammatory bowel diseases. Eosinophils also contribute to intestinal immunologic and tissue homeostasis and host defense. Recent studies in allergic airway disease suggest functional subphenotypes of eosinophils may underly their pathogenic versus protective roles. However, subphenotypes of intestinal eosinophils have not been defined and are complicated by their constitutive expression of the putative eosinophil inflammatory marker CD11c. Here, we propose a framework for subphenotype characterization of intestinal eosinophils based on relative intensity of surface CD11c expression. Using this flow cytometry framework in parallel with histology and BrdU tracing, we characterize intestinal eosinophil subphenotypes and monitor their plasticity at baseline and within the context of acute allergic and chronic systemic inflammation. Data reveal a conserved continuum of CD11c expression amongst intestinal eosinophils in health and acute disease states that overall tracked with other markers of activation. Oral allergen challenge induced recruitment of eosinophils into small intestinal lamina propria surrounding crypts, followed by in situ induction of CD11c expression in parallel with eosinophil redistribution into intestinal villi. Allergen challenge also elicited eosinophil transepithelial migration and the appearance of CD11clo CD11bhi eosinophils in the intestinal lumen. Chronic inflammation driven by overexpression of TNFα led to a qualitative shift in the relative abundance of CD11c-defined eosinophil subphenotypes favoring CD11chi -expressing eosinophils. These findings provide new insights into heterogeneity of intestinal tissue eosinophils and offer a framework for measuring and tracking eosinophil subphenotype versatility in situ in health and disease.
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Antígenos CD11/metabolismo , Eosinófilos , Hipersensibilidad , Alérgenos , Animales , Biomarcadores/metabolismo , Antígeno CD11c/metabolismo , Eosinófilos/metabolismo , Inflamación/patología , RatonesRESUMEN
Heme metabolism is a key regulator of inflammatory responses. Cobalt protoporphyrin IX (CoPP) is a heme analog and mimic that potently activates the NRF2/heme oxygenase-1 (HO-1) pathway, especially in monocytes and macrophages. We investigated the influence of CoPP on inflammatory responses using a murine model of colitis. Surprisingly, conditional deletion of myeloid HO-1 did not impact the colonic inflammatory response or the protective influence of CoPP in the setting of dextran sodium sulfate-induced colitis. Rather, we reveal that CoPP elicits a contradictory shift in blood myeloid populations relative to the colon during active intestinal inflammation. Major population changes include markedly diminished trafficking of CCR2+Ly6Chi monocytes to the inflamed colon, despite significant mobilization of this population into circulation. This resulted in significantly diminished colonic expansion of monocyte-derived macrophages and inflammatory cytokine expression. These findings were linked with significant induction of systemic CCL2 leading to a disrupted CCL2 chemoattractant gradient toward the colon and concentration-dependent suppression of circulating monocyte CCR2 expression. Administration of CoPP also induced macrophage differentiation toward a MarcohiHmox1hi anti-inflammatory erythrophagocytic phenotype, contributing to an overall decreased inflammatory profile. Such findings redefine protective influences of heme metabolism during inflammation, and highlight previously unreported immunosuppressive mechanisms of endogenous CCL2 induction.
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Colitis , Monocitos , Animales , Colitis/inducido químicamente , Colitis/metabolismo , Hemo/efectos adversos , Hemo-Oxigenasa 1/genética , Inflamación , Macrófagos/metabolismo , Ratones , Monocitos/metabolismoRESUMEN
Introduction: People living with HIV infection (PLWH) exhibit elevated levels of gastrointestinal inflammation. Potential causes of this inflammation include HIV infection and associated immune dysfunction, sexual behaviors among men who have sex with men (MSM) and gut microbiome composition. Methods: To better understand the etiology of gastrointestinal inflammation we examined levels of 28 fecal soluble immune factors (sIFs) and the fecal microbiome in well-defined cohorts of HIV seronegative MSM (MSM-SN), MSM with untreated HIV infection (MSM-HIV) and MSM with HIV on anti-retroviral treatment (MSMART). Additionally, fecal solutes from these participants were used to stimulate T-84 colonic epithelial cells to assess barrier function. Results: Both MSM cohorts with HIV had elevated levels of fecal calprotectin, a clinically relevant marker of GI inflammation, and nine inflammatory fecal sIFs (GM-CSF, ICAM-1, IL-1ß, IL-12/23, IL-15, IL-16, TNF-ß, VCAM-1, and VEGF). Interestingly, four sIFs (GM-CSF, ICAM-1, IL-7 and IL-12/23) were significantly elevated in MSM-SN compared to seronegative male non-MSM. Conversely, IL-22 and IL-13, cytokines beneficial to gut health, were decreased in all MSM with HIV and MSM-SN respectively. Importantly, all of these sIFs significantly correlated with calprotectin, suggesting they play a role in GI inflammation. Principal coordinate analysis revealed clustering of fecal sIFs by MSM status and significant associations with microbiome composition. Additionally, fecal solutes from participants in the MSM-HIV cohort significantly decreased colonic transcellular fluid transport in vitro, compared to non-MSM-SN, and this decrease associated with overall sIF composition and increased concentrations of eight inflammatory sIFs in participants with HIV. Lastly, elevated levels of plasma, sCD14 and sCD163, directly correlated with decreased transcellular transport and microbiome composition respectively, indicating that sIFs and the gut microbiome are associated with, and potentially contribute to, bacterial translocation. Conclusion: Taken together, these data demonstrate that inflammatory sIFs are elevated in MSM, regardless of HIV infection status, and are associated with the gut microbiome and intestinal barrier function.
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Infecciones por VIH , Microbiota , Minorías Sexuales y de Género , Humanos , Masculino , Factor Estimulante de Colonias de Granulocitos y Macrófagos , Molécula 1 de Adhesión Intercelular , Homosexualidad Masculina , Factores Inmunológicos , Inflamación , Interleucina-12 , Complejo de Antígeno L1 de LeucocitoRESUMEN
Early accumulation of neutrophils (PMN) is a hallmark of acute intestinal inflammation. This acute inflammation is either resolved or progresses to chronic inflammation. Without efficient PMN clearance at sites of infiltration, PMN can accumulate and contribute to chronic inflammatory conditions, including the intestinal diseases ulcerative colitis (UC) and Crohn's Disease (CD). The pH in the distal colon in individuals with active UC can range between a pH of 5 and 6, whereas healthy individuals maintain colonic pH in the range of 6.8-7.4. Extracellular pH has been shown to influence both intestinal epithelial cells and the infiltrating immune cells. More specifically, extracellular acidosis significantly impacts PMN. At pH below 6.5, there are increases in the production of H2O2, inhibition of apoptosis, and increases in the functional lifespan of PMN. Given the significant presence of PMN and extracellular acidification at sites of inflammation, we developed a novel model that allows for the monitoring of extracellular pH during PMN transepithelial migration in real time. Here, we describe this model and how it can be utilized to measure both the apical and basal pH during PMN trafficking. This model can be utilized to monitor extracellular pH under a wide range of conditions; including, hypoxia, PMN transepithelial migration, and for extended periods of time.
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Peróxido de Hidrógeno , Mucosa Intestinal , Colon , Humanos , Concentración de Iones de Hidrógeno , NeutrófilosRESUMEN
Sites of acute inflammation become austere environments for the procurement of energy. The combination of oxygen depletion (hypoxia) and decreased glucose availability requires surprising metabolic adaptability. In this issue of the JCI, Watts et al. examined the metabolic adaptability of murine neutrophils to the setting of acute pulmonary inflammation elicited by exposure to nebulized endotoxin. While neutrophils are generally considered a primarily glycolytic cell type, Watts et al. used a combination of labeled amino acids and high-resolution proteomics to reveal that the harsh environment of the inflammatory lesion drives neutrophils toward de novo protein synthesis and extracellular protein scavenging as a primary fuel. This study provides compelling evidence that tissue neutrophils scavenge extracellular proteins to fuel carbon metabolism, which aids in de novo protein synthesis and the promotion of an inflammatory phenotype. These observations reveal the surprisingly creative extent to which cells and tissues might adapt to energy-deficient inflammatory environments.
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Dieta Paleolítica , Neutrófilos , Animales , Endotoxinas , Glucólisis , Pulmón , RatonesRESUMEN
Metabolic changes associated with tissue inflammation result in significant extracellular acidosis (EA). Within mucosal tissues, intestinal epithelial cells (IEC) have evolved adaptive strategies to cope with EA through the up-regulation of SLC26A3 to promote pH homeostasis. We hypothesized that EA significantly alters IEC gene expression as an adaptive mechanism to counteract inflammation. Using an unbiased RNA sequencing approach, we defined the impact of EA on IEC gene expression to define molecular mechanisms by which IEC respond to EA. This approach identified a unique gene signature enriched in cyclic AMP response element-binding protein (CREB)-regulated gene targets. Utilizing loss- and gain-of-function approaches in cultured epithelia and murine colonoids, we demonstrate that EA elicits prominent CREB phosphorylation through cyclic AMP-independent mechanisms that requires elements of the mitogen-activated protein kinase signaling pathway. Further analysis revealed that EA signals through the G protein-coupled receptor GPR31 to promote induction of FosB, NR4A1, and DUSP1. These studies were extended to an in vivo murine model in conjunction with colonization of a pH reporter Escherichia coli strain that demonstrated significant mucosal acidification in the TNFΔARE model of murine ileitis. Herein, we observed a strong correlation between the expression of acidosis-associated genes with bacterial reporter sfGFP intensity in the distal ileum. Finally, the expression of this unique EA-associated gene signature was increased during active inflammation in patients with Crohn's disease but not in the patient control samples. These findings establish a mechanism for EA-induced signals during inflammation-associated acidosis in both murine and human ileitis.
Asunto(s)
Acidosis/genética , Antiportadores/genética , Enfermedad de Crohn/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Ileítis/genética , Receptores Acoplados a Proteínas G/genética , Transportadores de Sulfato/genética , Acidosis/metabolismo , Acidosis/patología , Animales , Antiportadores/metabolismo , Enfermedad de Crohn/metabolismo , Enfermedad de Crohn/patología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Modelos Animales de Enfermedad , Fosfatasa 1 de Especificidad Dual/genética , Fosfatasa 1 de Especificidad Dual/metabolismo , Regulación de la Expresión Génica , Humanos , Ileítis/metabolismo , Ileítis/patología , Íleon/metabolismo , Íleon/patología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Ratones , Ratones Endogámicos C57BL , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Organoides/metabolismo , Organoides/patología , Fosforilación , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Análisis de Secuencia de ARN , Transducción de Señal , Transportadores de Sulfato/metabolismoRESUMEN
During episodes of acute inflammation, polymorphonuclear leukocytes (PMNs) are actively recruited to sites of inflammation or injury where they provide anti-microbial and wound-healing functions. One enzyme crucial for fulfilling these functions is myeloperoxidase (MPO), which generates hypochlorous acid from Cl- and hydrogen peroxide. The potential exists, however, that uncontrolled the extracellular generation of hypochlorous acid by MPO can cause bystander tissue damage and inhibit the healing response. Previous work suggests that the microbiota-derived tryptophan metabolites 1H-indole and related molecules ("indoles") are protective during intestinal inflammation, although their precise mechanism of action is unclear. In the present work, we serendipitously discovered that indoles are potent and selective inhibitors of MPO. Using both primary human PMNs and recombinant human MPO in a cell-free system, we revealed that indoles inhibit MPO at physiologic concentrations. Particularly, indoles block the chlorinating activity of MPO, a reliable marker for MPO-associated tissue damage, as measured by coulometric-coupled HPLC. Further, we observed direct interaction between indoles and MPO using the established biochemical techniques microscale thermophoresis and STD-NMR. Utilizing a murine colitis model, we demonstrate that indoles inhibit bystander tissue damage, reflected in decreased colon 3-chlorotyrosine and pro-inflammatory chemokine expression in vivo. Taken together, these results identify microbiota-derived indoles that acts as endogenous immunomodulatory compounds through their actions on MPO, suggesting a symbiotic association between the gut microbiota and host innate immune system. Such findings offer exciting new targets for future pharmacological intervention.
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Adenocarcinoma/patología , Efecto Espectador , Colitis/patología , Neoplasias Colorrectales/patología , Indoles/farmacología , Neutrófilos/enzimología , Peroxidasa/antagonistas & inhibidores , Adenocarcinoma/inmunología , Adenocarcinoma/metabolismo , Animales , Colitis/inmunología , Colitis/metabolismo , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/metabolismo , Halogenación , Humanos , Ratones , Ratones Endogámicos C57BL , Microbiota , Células Tumorales Cultivadas , Tirosina/metabolismoRESUMEN
Primary sclerosing cholangitis (PSC) is a progressive fibrosing cholestatic liver disease that is strongly associated with inflammatory bowel disease (IBD). PSC-associated IBD (PSC-IBD) displays a unique phenotype characterized by right-side predominant colon inflammation and increased risk of colorectal cancer compared to non-PSC-IBD. The frequent association and unique phenotype of PSC-IBD suggest distinctive underlying disease mechanisms from other chronic liver diseases or IBD alone. Multidrug resistance protein 2 knockout (Mdr2-/-) mice develop spontaneous cholestatic liver injury and fibrosis mirroring human PSC. As a novel model of PSC-IBD, we treated Mdr2-/- mice with dextran sulfate sodium (DSS) to chemically induce colitis (Mdr2-/-/DSS). Mdr2-/- mice demonstrate alterations in fecal bile acid composition and enhanced colitis susceptibility with increased colonic adhesion molecule expression, particularly mucosal addressin-cell adhesion molecule 1 (MAdCAM-1). In vitro, ursodeoxycholic acid (UDCA) co-treatment resulted in a dose dependent attenuation of TNF-α-induced endothelial MAdCAM-1 expression. In the combined Mdr2-/-/DSS model, UDCA supplementation attenuated colitis severity and downregulated intestinal MAdCAM-1 expression. These findings suggest a potential mechanistic role for alterations in bile acid signaling in modulating MAdCAM-1 expression and colitis susceptibility in cholestasis-associated colitis. Together, our findings provide a novel model and new insight into the pathogenesis and potential treatment of PSC-IBD.
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Ácidos y Sales Biliares/metabolismo , Moléculas de Adhesión Celular/metabolismo , Colangitis Esclerosante/metabolismo , Colestasis/metabolismo , Colitis/metabolismo , Colon/metabolismo , Enfermedades Inflamatorias del Intestino/metabolismo , Mucoproteínas/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Animales , Moléculas de Adhesión Celular/genética , Colon/patología , Sulfato de Dextran , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Humanos , Ratones , Ratones Noqueados , Mucoproteínas/genética , Factor de Necrosis Tumoral alfa/metabolismo , Ácido Ursodesoxicólico/metabolismo , Miembro 4 de la Subfamilia B de Casete de Unión a ATPRESUMEN
Intestinal epithelial cells (IECs) exist in a metabolic state of low oxygen tension termed "physiologic hypoxia." An important factor in maintaining intestinal homeostasis is the transcription factor hypoxia-inducible factor (HIF), which is stabilized under hypoxic conditions and mediates IEC homeostatic responses to low oxygen tension. To identify HIF transcriptional targets in IEC, chromatin immunoprecipitation (ChIP) was performed in Caco-2 IECs using HIF-1α- or HIF-2α-specific antibodies. ChIP-enriched DNA was hybridized to a custom promoter microarray (termed ChIP-chip). This unbiased approach identified autophagy as a major HIF-1-targeted pathway in IEC. Binding of HIF-1 to the ATG9A promoter, the only transmembrane component within the autophagy pathway, was particularly enriched by exposure of IEC to hypoxia. Validation of this ChIP-chip revealed prominent induction of ATG9A, and luciferase promoter assays identified a functional hypoxia response element upstream of the TSS. Hypoxia-mediated induction of ATG9A was lost in cells lacking HIF-1. Strikingly, we found that lentiviral-mediated knockdown (KD) of ATG9A in IECs prevents epithelial barrier formation by >95% and results in significant mislocalization of multiple tight junction (TJ) proteins. Extensions of these findings showed that ATG9A KD cells have intrinsic abnormalities in the actin cytoskeleton, including mislocalization of the TJ binding protein vasodilator-stimulated phosphoprotein. These results implicate ATG9A as essential for multiple steps of epithelial TJ biogenesis and actin cytoskeletal regulation. Our findings have novel applicability for disorders that involve a compromised epithelial barrier and suggest that targeting ATG9A may be a rational strategy for future therapeutic intervention.
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Proteínas Relacionadas con la Autofagia/metabolismo , Mucosa Intestinal/metabolismo , Proteínas de la Membrana/metabolismo , Uniones Estrechas/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas Relacionadas con la Autofagia/genética , Células CACO-2 , Hipoxia de la Célula/fisiología , Línea Celular , Células Epiteliales/metabolismo , Regulación de la Expresión Génica/genética , Células HeLa , Humanos , Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Proteínas de la Membrana/genética , Regiones Promotoras Genéticas/genética , Elementos de Respuesta/genética , Proteínas de Uniones Estrechas/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Transporte Vesicular/genéticaRESUMEN
Acute intestinal inflammation includes the early accumulation of neutrophils (PMN). Based on recent evidence that PMN infiltration "imprints" changes in the local tissue environment through local oxygen depletion and the release of adenine nucleotides, we hypothesized that the interaction between transmigrating PMN and intestinal epithelial cells (IECs) results in inflammatory acidification of the tissue. Using newly developed tools, we revealed that active PMN transepithelial migration (TEM) significantly acidifies the local microenvironment, a decrease of nearly 2 pH units. Using unbiased approaches, we sought to define acid-adaptive pathways elicited by PMN TEM. Given the significant amount of adenosine (Ado) generated during PMN TEM, we profiled the influence of Ado on IECs gene expression by microarray and identified the induction of SLC26A3, the major apical Cl-/HCO3- exchanger in IECs. Utilizing loss- and gain-of-function approaches, as well as murine and human colonoids, we demonstrate that Ado-induced SLC26A3 promotes an adaptive IECs phenotype that buffers local pH during active inflammation. Extending these studies, chronic murine colitis models were used to demonstrate that SLC26A3 expression rebounds during chronic DSS-induced inflammation. In conclusion, Ado signaling during PMN TEM induces an adaptive tissue response to inflammatory acidification through the induction of SLC26A3 expression, thereby promoting pH homeostasis.
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Acidosis/inmunología , Antiportadores/metabolismo , Colitis/inmunología , Inflamación/inmunología , Mucosa Intestinal/fisiología , Intestinos/inmunología , Neutrófilos/inmunología , Transportadores de Sulfato/metabolismo , Acidosis/inducido químicamente , Adaptación Fisiológica , Adenosina/metabolismo , Animales , Antiportadores/genética , Células Cultivadas , Colitis/inducido químicamente , Modelos Animales de Enfermedad , Humanos , Enfermedades del Sistema Inmune , Inflamación/inducido químicamente , Trastornos Leucocíticos , Ratones , Activación Neutrófila , Dodecil Sulfato de Sodio , Transportadores de Sulfato/genética , Migración Transendotelial y Transepitelial , Regulación hacia ArribaRESUMEN
Dicentric and centric ring chromosomes are used for radiation-induced damage analysis and biodosimetry after radiation exposure. However, Giemsa stain-based cytogenetic analysis is labor-intense and time-consuming. Moreover, the disadvantage of Giemsa based chromosome analysis is a potential poor reproducibility when researchers are not fully trained for analysis. These problems come from analysis of morphological abnormality of chromosomal aberrations. Locus-specific FISH probes were used to overcome this problem. Centromere probes can visualize centromere locations and help identify dicentric chromosomes and centric rings. Telomere probes help to identify terminal deletion and telomere fusions. Probes were originally designed with a DNA probe but Peptide nucleic acid (PNA) probes took the place of DNA probes. This chapter introduces PNA telomere and centromere FISH staining and accurate analysis of chromosomal aberrations.
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Centrómero/metabolismo , Aberraciones Cromosómicas/efectos de la radiación , Ácidos Nucleicos de Péptidos/química , Radiación , Coloración y Etiquetado , Telómero/metabolismo , Animales , Humanos , Hibridación Fluorescente in Situ , Metafase/efectos de la radiación , RatonesRESUMEN
Fluorescence in situ Hybridization (FISH) utilizes peptide nucleic acid (PNA) probes to identify specific DNA sequences. PNA probes have been effectively used to identify chromosome aberrations and have been shown to greatly aid in biodosimetery assays involved in identifying dicentrics. Traditional techniques have required the heat denaturing of the DNA in formamide followed by multiple hours at moderated temperatures to allow the probe to hybridize to its specific target. Over the past 30 years, advancements in both protocols and probes have made FISH a more reliable technique for both biological research and medical diagnostics, additionally the protocol has been shortened to several minutes. We will introduce two modified PNA FISH protocols, a rapid microwave-based approach and nonclassical hybridization protocol.