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1.
Immunity ; 40(1): 66-77, 2014 Jan 16.
Artículo en Inglés | MEDLINE | ID: mdl-24412613

RESUMEN

Acute intestinal inflammation involves early accumulation of neutrophils (PMNs) followed by either resolution or progression to chronic inflammation. Based on recent evidence that mucosal metabolism influences disease outcomes, we hypothesized that transmigrating PMNs influence the transcriptional profile of the surrounding mucosa. Microarray studies revealed a cohort of hypoxia-responsive genes regulated by PMN-epithelial crosstalk. Transmigrating PMNs rapidly depleted microenvironmental O2 sufficiently to stabilize intestinal epithelial cell hypoxia-inducible factor (HIF). By utilizing HIF reporter mice in an acute colitis model, we investigated the relative contribution of PMNs and the respiratory burst to "inflammatory hypoxia" in vivo. CGD mice, lacking a respiratory burst, developed accentuated colitis compared to control, with exaggerated PMN infiltration and diminished inflammatory hypoxia. Finally, pharmacological HIF stabilization within the mucosa protected CGD mice from severe colitis. In conclusion, transcriptional imprinting by infiltrating neutrophils modulates the host response to inflammation, via localized O2 depletion, resulting in microenvironmental hypoxia and effective inflammatory resolution.


Asunto(s)
Colitis/inmunología , Hipoxia/inmunología , Membrana Mucosa/metabolismo , Neutrófilos/patología , Animales , Comunicación Celular , Movimiento Celular , Células Cultivadas , Microambiente Celular , Colitis/inducido químicamente , Colon/patología , Modelos Animales de Enfermedad , Hipoxia/inducido químicamente , Factor 1 Inducible por Hipoxia/metabolismo , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Análisis por Micromatrices , Membrana Mucosa/patología , NADPH Oxidasa 2 , NADPH Oxidasas/genética , Estrés Oxidativo , Oxígeno/metabolismo , Estabilidad Proteica/efectos de los fármacos , Migración Transendotelial y Transepitelial
2.
J Immunol ; 192(3): 1267-76, 2014 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-24367025

RESUMEN

Cytokines secreted at sites of inflammation impact the onset, progression, and resolution of inflammation. In this article, we investigated potential proresolving mechanisms of IFN-γ in models of inflammatory bowel disease. Guided by initial microarray analysis, in vitro studies revealed that IFN-γ selectively induced the expression of IL-10R1 on intestinal epithelia. Further analysis revealed that IL-10R1 was expressed predominantly on the apical membrane of polarized epithelial cells. Receptor activation functionally induced canonical IL-10 target gene expression in epithelia, concomitant with enhanced barrier restitution. Furthermore, knockdown of IL-10R1 in intestinal epithelial cells results in impaired barrier function in vitro. Colonic tissue isolated from murine colitis revealed that levels of IL-10R1 and suppressor of cytokine signaling 3 were increased in the epithelium and coincided with increased tissue IFN-γ and IL-10 cytokines. In parallel, studies showed that treatment of mice with rIFN-γ was sufficient to drive expression of IL-10R1 in the colonic epithelium. Studies of dextran sodium sulfate colitis in intestinal epithelial-specific IL-10R1-null mice revealed a remarkable increase in disease susceptibility associated with increased intestinal permeability. Together, these results provide novel insight into the crucial and underappreciated role of epithelial IL-10 signaling in the maintenance and restitution of epithelial barrier and of the temporal regulation of these pathways by IFN-γ.


Asunto(s)
Células Epiteliales/metabolismo , Interferón gamma/farmacología , Subunidad alfa del Receptor de Interleucina-10/biosíntesis , Interleucina-10/fisiología , Mucosa Intestinal/metabolismo , Animales , Línea Celular , Polaridad Celular , Colitis/inducido químicamente , Colitis/metabolismo , Citocinas/biosíntesis , Citocinas/genética , Sulfato de Dextran/toxicidad , Dextranos/farmacocinética , Células Epiteliales/efectos de los fármacos , Células Epiteliales/ultraestructura , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/farmacocinética , Regulación de la Expresión Génica , Humanos , Interferón gamma/biosíntesis , Interferón gamma/genética , Interferón gamma/fisiología , Subunidad alfa del Receptor de Interleucina-10/genética , Ratones , Ratones Endogámicos C57BL , Permeabilidad , Proteínas Recombinantes/farmacología , Factor de Transcripción STAT3/metabolismo , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas/biosíntesis , Proteínas Supresoras de la Señalización de Citocinas/genética
3.
Proc Natl Acad Sci U S A ; 110(49): 19820-5, 2013 Dec 03.
Artículo en Inglés | MEDLINE | ID: mdl-24248342

RESUMEN

Mucosal surfaces of the lower gastrointestinal tract are subject to frequent, pronounced fluctuations in oxygen tension, particularly during inflammation. Adaptive responses to hypoxia are orchestrated largely by the hypoxia-inducible transcription factors (HIFs). As HIF-1α and HIF-2α are coexpressed in mucosal epithelia that constitute the barrier between the lumen and the underlying immune milieu, we sought to define the discrete contribution of HIF-1 and HIF-2 transactivation pathways to intestinal epithelial cell homeostasis. The present study identifies creatine kinases (CKs), key metabolic enzymes for rapid ATP generation via the phosphocreatine-creatine kinase (PCr/CK) system, as a unique gene family that is coordinately regulated by HIF. Cytosolic CKs are expressed in a HIF-2-dependent manner in vitro and localize to apical intestinal epithelial cell adherens junctions, where they are critical for junction assembly and epithelial integrity. Supplementation with dietary creatine markedly ameliorated both disease severity and inflammatory responses in colitis models. Further, enzymes of the PCr/CK metabolic shuttle demonstrate dysregulated mucosal expression in a subset of ulcerative colitis and Crohn disease patients. These findings establish a role for HIF-regulated CK in epithelial homeostasis and reveal a fundamental link between cellular bioenergetics and mucosal barrier.


Asunto(s)
Translocador Nuclear del Receptor de Aril Hidrocarburo/metabolismo , Hipoxia de la Célula/fisiología , Colitis/metabolismo , Creatina Quinasa/metabolismo , Creatina/metabolismo , Regulación Enzimológica de la Expresión Génica/fisiología , Transducción de Señal/fisiología , Análisis de Varianza , Western Blotting , Cromatografía Líquida de Alta Presión , Cartilla de ADN/genética , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Regulación Enzimológica de la Expresión Génica/genética , Técnicas de Silenciamiento del Gen , Humanos , Inmunoprecipitación , Reacción en Cadena de la Polimerasa
4.
J Immunol ; 190(1): 392-400, 2013 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-23209320

RESUMEN

A deeper understanding of the mechanisms that control responses to inflammation is critical to the development of effective therapies. We sought to define the most proximal regulators of the Cullin (Cul)-RING ligases, which play a central role in the stabilization of NF-κB and hypoxia-inducible factor (HIF). In these studies, we identify the human deneddylase-1 (SENP8) as a key regulator of Cul neddylation response in vitro and in vivo. Using human microvascular endothelial cells (HMECs), we examined inflammatory responses to LPS or TNF-α by assessing Cul neddylation status, NF-κB and HIF-1α stabilization, and inflammatory cytokine secretion. HMECs with an intact neddylation pathway showed a time-dependent induction of Cul-1 neddylation, nuclear translocation of NF-κB, stabilization of HIF-1α, and increased NF-κB/HIF-α promoter activity in response to LPS. HMECs lacking SENP8 were unable to neddylate Cul-1 and subsequently were unable to activate NF-κB or HIF-1α. Pharmacological targeting of neddylation (MLN4924) significantly abrogated NF-κB responses, induced HIF-1α promoter activity, and reduced secretion of TNF-α-elicited proinflammatory cytokines. MLN4924 stabilized HIF and abrogated proinflammatory responses while maintaining anti-inflammatory IL-10 responses in vivo following LPS administration. These studies identify SENP8 as a proximal regulator of Cul neddylation and provide an important role for SENP8 in fine-tuning the inflammatory response. Moreover, our findings provide feasibility for therapeutic targeting of the Culs during inflammation.


Asunto(s)
Proteínas Cullin/fisiología , Endopeptidasas/fisiología , Endotelio Vascular/enzimología , Endotelio Vascular/inmunología , Mediadores de Inflamación/fisiología , Ubiquitinas/fisiología , Células Cultivadas , Proteínas Cullin/metabolismo , Endopeptidasas/deficiencia , Endopeptidasas/genética , Endotelio Vascular/citología , Precursores Enzimáticos/metabolismo , Precursores Enzimáticos/fisiología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Microcirculación/inmunología , Proteína NEDD8 , Ubiquitinas/metabolismo
5.
J Immunol ; 186(11): 6505-14, 2011 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-21515785

RESUMEN

Tissues of the mucosa are lined by an epithelium that provides barrier and transport functions. It is now appreciated that inflammatory responses in inflammatory bowel diseases are accompanied by striking shifts in tissue metabolism. In this paper, we examined global metabolic consequences of mucosal inflammation using both in vitro and in vivo models of disease. Initial analysis of the metabolic signature elicited by inflammation in epithelial models and in colonic tissue isolated from murine colitis demonstrated that levels of specific metabolites associated with cellular methylation reactions are significantly altered by model inflammatory systems. Furthermore, expression of enzymes central to all cellular methylation, S-adenosylmethionine synthetase and S-adenosylhomocysteine hydrolase, are increased in response to inflammation. Subsequent studies showed that DNA methylation is substantially increased during inflammation and that epithelial NF-κB activity is significantly inhibited following treatment with a reversible S-adenosylhomocysteine hydrolase inhibitor, DZ2002. Finally, these studies demonstrated that inhibition of cellular methylation in a murine model of colitis results in disease exacerbation while folate supplementation to promote methylation partially ameliorates the severity of murine colitis. Taken together, these results identify a global change in methylation, which during inflammation, translates to an overall protective role in mucosal epithelia.


Asunto(s)
Colitis/metabolismo , Inflamación/metabolismo , Mucosa Intestinal/metabolismo , Metabolómica/métodos , Adenina/análogos & derivados , Adenina/farmacología , Adenosilhomocisteinasa/genética , Adenosilhomocisteinasa/metabolismo , Animales , Western Blotting , Butiratos/farmacología , Línea Celular Tumoral , Colitis/genética , Colon/efectos de los fármacos , Colon/metabolismo , Colon/patología , Metilación de ADN/efectos de los fármacos , Sulfato de Dextran/farmacología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Perfilación de la Expresión Génica/métodos , Células HeLa , Humanos , Inflamación/genética , Interferón gamma/metabolismo , Interferón gamma/farmacología , Mucosa Intestinal/patología , Espectroscopía de Resonancia Magnética , Metionina Adenosiltransferasa/genética , Metionina Adenosiltransferasa/metabolismo , Metilación/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Mucositis/genética , Mucositis/metabolismo , FN-kappa B/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
6.
J Immunol ; 186(3): 1790-8, 2011 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-21199896

RESUMEN

Numerous studies have revealed that hypoxia and inflammation occur coincidentally in mucosal disorders, such as inflammatory bowel disease. During inflammation, epithelial-expressed hypoxia-inducible factor (HIF) serves an endogenously protective function. In this study, we sought to explore how mucosal immune responses influence HIF-dependent end points. Guided by a screen of relevant inflammatory mediators, we identified IFN-γ as a potent repressor of HIF-dependent transcription in human intestinal epithelial cells. Analysis of HIF levels revealed that HIF-1ß, but not HIF-1α, is selectively repressed by IFN-γ in a JAK-dependent manner. Cloning and functional analysis of the HIF-1ß promoter identified a prominent region for IFN-γ-dependent repression. Further studies revealed that colonic IFN-γ and HIF-1ß levels were inversely correlated in a murine colitis model. Taken together, these studies demonstrated that intestinal epithelial HIF is attenuated by IFN-γ through transcriptional repression of HIF-1ß. These observations are relevant to the pathophysiology of colitis (i.e., that loss of HIF signaling during active inflammation may exacerbate disease pathogenesis).


Asunto(s)
Translocador Nuclear del Receptor de Aril Hidrocarburo/antagonistas & inhibidores , Translocador Nuclear del Receptor de Aril Hidrocarburo/genética , Colitis/inmunología , Interferón gamma/fisiología , Mucosa Intestinal/inmunología , Mucosa Intestinal/patología , Proteínas Represoras/fisiología , Animales , Translocador Nuclear del Receptor de Aril Hidrocarburo/fisiología , Células CACO-2 , Línea Celular Tumoral , Células Cultivadas , Clonación Molecular , Colitis/enzimología , Colitis/patología , Sulfato de Dextran/toxicidad , Modelos Animales de Enfermedad , Femenino , Humanos , Mediadores de Inflamación/antagonistas & inhibidores , Mediadores de Inflamación/fisiología , Mucosa Intestinal/enzimología , Ratones , Ratones Endogámicos C57BL , Procolágeno-Prolina Dioxigenasa/fisiología , Transducción de Señal/inmunología
7.
Proc Natl Acad Sci U S A ; 107(32): 14298-303, 2010 Aug 10.
Artículo en Inglés | MEDLINE | ID: mdl-20660763

RESUMEN

Resolvin-E1 (RvE1) has been demonstrated to promote inflammatory resolution in numerous disease models. Given the importance of epithelial cells to coordination of mucosal inflammation, we hypothesized that RvE1 elicits an epithelial resolution signature. Initial studies revealed that the RvE1-receptor (ChemR23) is expressed on intestinal epithelial cells (IECs) and that microarray profiling of cells exposed to RvE1 revealed regulation of inflammatory response gene expression. Notably, RvE1 induced intestinal alkaline phosphatase (ALPI) expression and significantly enhanced epithelial ALPI enzyme activity. One role recently attributed to ALPI is the detoxification of bacterial LPS. In our studies, RvE1-exposed epithelia detoxified LPS (assessed by attenuation of NF-kappaB signaling). Furthermore, in epithelial-bacterial interaction assays, we determined that ALPI retarded the growth of Escherichia coli. To define these features in vivo, we used a murine dextran sulfate sodium (DSS) model of colitis. Compared with vehicle controls, administration of RvE1 resulted in significant improvement of disease activity indices (e.g., body weight, colon length) concomitant with increased ALPI expression in the intestinal epithelium. Moreover, inhibition of ALPI activity resulted in increased severity of colitis in DSS-treated animals and partially abrogated the protective influence of RvE1. Together, these data implicate a previously unappreciated role for ALPI in RvE1-mediated inflammatory resolution.


Asunto(s)
Fosfatasa Alcalina/genética , Ácido Eicosapentaenoico/análogos & derivados , Inflamación/prevención & control , Mucosa Intestinal/enzimología , Lipopolisacáridos/antagonistas & inhibidores , Animales , Colitis/prevención & control , Ácido Eicosapentaenoico/farmacología , Células Epiteliales/química , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Receptores de Superficie Celular/análisis , Activación Transcripcional
8.
Gastroenterology ; 141(1): 208-16, 2011 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-21557945

RESUMEN

BACKGROUND & AIMS: Inflammatory bowel disease (IBD) refers to 2 chronic inflammatory diseases of the intestine, ie, ulcerative colitis and Crohn's disease. IBD results from environmental factors (eg, bacterial antigens) triggering a dysregulated immune response in genetically predisposed hosts. Although the basis of IBD is incompletely understood, a number of recent studies have implicated defective innate immune responses in the pathogenesis of IBD. In this regard, there is much interest in therapies that activate innate immunity (eg, recombinant granulocyte-macrophage colony-stimulating factor). METHODS: In this study, we screened expression and function of circulating leukocyte granulocyte-macrophage colony-stimulating factor receptor (CD116) messenger RNA and surface protein in 52 IBD patients and 52 healthy controls. RESULTS: Our results show that both granulocyte and monocyte CD116 levels, but not CD114 or interleukin-3Rα, were significantly decreased in IBD compared to control (P<.001) and disease controls (irritable bowel syndrome; P<.001; rheumatoid arthritis; P<.025). IBD-associated CD116 repression was more prominent in patients with ulcerative colitis compared to Crohn's disease (P<.05), was independent of disease activity (P>.05), and was not influenced by current medications (P>.05). Receiver operating characteristic curve analysis revealed that leukocyte CD116 expression is a sensitive (85%) and specific (92%) biomarker for IBD. Moreover, granulocyte CD116-mediated function (phosphorylation of signal transducers and activators of transcription 3) paralleled decreased expression of CD116 in IBD granulocytes compared to control (P<.001). CONCLUSIONS: These studies identify defective expression and function of CD116 as a distinguishing feature of IBD and implicate an associated defect in innate immune responses toward granulocyte-macrophage colony-stimulating factor.


Asunto(s)
Colitis Ulcerosa/inmunología , Enfermedad de Crohn/inmunología , Inmunidad Innata , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/sangre , Adulto , Biomarcadores/sangre , Estudios de Casos y Controles , Colitis Ulcerosa/sangre , Colitis Ulcerosa/genética , Colorado , Enfermedad de Crohn/sangre , Enfermedad de Crohn/genética , Regulación hacia Abajo , Femenino , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Granulocitos/inmunología , Humanos , Inmunidad Innata/genética , Masculino , Persona de Mediana Edad , Fosforilación , Valor Predictivo de las Pruebas , Pronóstico , ARN Mensajero/sangre , Curva ROC , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Factor de Transcripción STAT3/metabolismo , Índice de Severidad de la Enfermedad
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