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 TransepitelialRESUMEN
Intestinal epithelial cells form a selectively permeable barrier to protect colon tissues from luminal microbiota and antigens and to mediate nutrient, fluid, and waste flux in the intestinal tract. Dysregulation of the epithelial cell barrier coincides with profound shifts in metabolic energy, especially in the colon, which exists in an energetically depleting state of physiological hypoxia. However, studies that systematically examine energy flux and adenylate metabolism during intestinal epithelial barrier development and restoration after disruption are lacking. Here, to delineate barrier-related energy flux, we developed an HPLC-based profiling method to track changes in energy flux and adenylate metabolites during barrier development and restoration. Cultured epithelia exhibited pooling of phosphocreatine and maintained ATP during barrier development. EDTA-induced epithelial barrier disruption revealed that hypoxanthine levels correlated with barrier resistance. Further studies uncovered that hypoxanthine supplementation improves barrier function and wound healing and that hypoxanthine appears to do so by increasing intracellular ATP, which improved cytoskeletal G- to F-actin polymerization. Hypoxanthine supplementation increased the adenylate energy charge in the murine colon, indicating potential to regulate adenylate energy charge-mediated metabolism in intestinal epithelial cells. Moreover, experiments in a murine colitis model disclosed that hypoxanthine loss during active inflammation correlates with markers of disease severity. In summary, our results indicate that hypoxanthine modulates energy metabolism in intestinal epithelial cells and is critical for intestinal barrier function.
Asunto(s)
Colitis/metabolismo , Colon/metabolismo , Metabolismo Energético , Hipoxantina/metabolismo , Mucosa Intestinal/metabolismo , Animales , Colitis/patología , Colon/patología , Femenino , Mucosa Intestinal/patología , Metaboloma , Ratones , Ratones Endogámicos C57BL , Consumo de Oxígeno , Permeabilidad , Uniones Estrechas/metabolismo , Uniones Estrechas/patologíaRESUMEN
There is interest in understanding post-translational modifications of proteins in inflammatory disease. Neddylation is the conjugation of the molecule neural precursor cell expressed, developmentally down-regulated 8 (NEDD8) to promote protein stabilization. Cullins are a family of NEDD8 targets important in the stabilization and degradation of proteins, such as hypoxia-inducible factor (HIF; via Cullin-2). Here, we elucidate the role of human deneddylase-1 (DEN-1, also called SENP8) in inflammatory responses in vitro and in vivo and define conditions for targeting neddylation in models of mucosal inflammation. HIF provides protection in inflammatory models, so we examined the contribution of DEN-1 to HIF stabilization. Pharmacologic targeting of neddylation activity with MLN4924 (IC50, 4.7 nM) stabilized HIF-1α, activated HIF promoter activity by 2.5-fold, and induced HIF-target genes in human epithelial cells up to 5-fold. Knockdown of DEN-1 in human intestinal epithelial cells resulted in increased kinetics in barrier formation, decreased permeability, and enhanced barrier restitution by 2 ± 0.5-fold. Parallel studies in vivo revealed that MLN4924 abrogated disease severity in murine dextran sulfate sodium colitis, including weight loss, colon length, and histologic severity. We conclude that DEN-1 is a regulator of cullin neddylation and fine-tunes the inflammatory response in vitro and in vivo. Pharmacologic inhibition of cullin neddylation may provide a therapeutic opportunity in mucosal inflammatory disease.
Asunto(s)
Proteínas Cullin/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Enfermedades Inflamatorias del Intestino/metabolismo , Enfermedades Inflamatorias del Intestino/prevención & control , Animales , Línea Celular , Proteínas Cullin/antagonistas & inhibidores , Ciclopentanos/farmacología , Modelos Animales de Enfermedad , Endopeptidasas/genética , Endopeptidasas/metabolismo , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Enfermedades Inflamatorias del Intestino/patología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Redes y Vías Metabólicas , Ratones Endogámicos C57BL , Proteína NEDD8 , Inhibidores de Proteasas/farmacología , Estabilidad Proteica , Pirimidinas/farmacología , Ubiquitinas/metabolismoRESUMEN
Although acute lung injury (ALI) contributes significantly to critical illness, resolution often occurs spontaneously through endogenous pathways. We recently found that mechanical ventilation increases levels of pulmonary adenosine, a signaling molecule known to attenuate lung inflammation. In this study, we hypothesized a contribution of transcriptionally controlled pathways to pulmonary adenosine receptor (ADOR) signaling during ALI. We gained initial insight from microarray analysis of pulmonary epithelia exposed to conditions of cyclic mechanical stretch, a mimic for ventilation-induced lung disease. Surprisingly, these studies revealed a selective induction of the ADORA2B. Using real-time RT-PCR and Western blotting, we confirmed an up to 9-fold induction of the ADORA2B following cyclic mechanical stretch (A549, Calu-3, or human primary alveolar epithelial cells). Studies using ADORA2B promoter constructs identified a prominent region within the ADORA2B promoter conveying stretch responsiveness. This region of the promoter contained a binding site for the transcription factor hypoxia-inducible factor (HIF)-1. Additional studies using site-directed mutagenesis or transcription factor binding assays demonstrated a functional role for HIF-1 in stretch-induced increases of ADORA2B expression. Moreover, studies of ventilator-induced lung injury revealed induction of the ADORA2B during ALI in vivo that was abolished following HIF inhibition or genetic deletion of Hif1a. Together, these studies implicate HIF in the transcriptional control of pulmonary adenosine signaling during ALI.
Asunto(s)
Lesión Pulmonar Aguda/fisiopatología , Subunidad alfa del Factor 1 Inducible por Hipoxia/fisiología , Receptor de Adenosina A2B/genética , Estrés Mecánico , Lesión Pulmonar Inducida por Ventilación Mecánica/fisiopatología , Lesión Pulmonar Aguda/metabolismo , Adenosina/fisiología , Animales , Sitios de Unión , Células Cultivadas , Células Epiteliales/fisiología , Femenino , Genes Reporteros , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/deficiencia , Pulmón/metabolismo , Pulmón/fisiopatología , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Regiones Promotoras Genéticas/genética , Receptor de Adenosina A2B/biosíntesis , Receptor de Adenosina A2B/fisiología , Transcripción GenéticaRESUMEN
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/genéticaRESUMEN
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 PolimerasaRESUMEN
Hypoxia has been widely implicated in many pathological conditions, including those associated with inflammation and tumorigenesis. A number of recent studies have implicated hypoxia in the control of vasculogenesis and permeability, the basis for which is not fully understood. Here we examine the transcriptional regulation of angiogenesis and permeability by hypoxia in endothelial cells. Guided by a global profiling approach in cultured endothelial cells, these studies revealed the selective induction of human gravin (protein kinase A anchoring protein 12) by hypoxia. Analysis of the cloned gravin promoter identified a functional hypoxia-responsive region including 2 binding sites for hypoxia-inducible factor (HIF). Site-directed mutagenesis identified the most distal HIF-binding site as essential for the induction of gravin by hypoxia. Further studies examining gravin gain and loss of function confirmed strong dependence of gravin in control of microvascular endothelial tube formation, wherein gravin functions as a "braking" system for angiogenesis. Additional studies in confluent endothelia revealed that gravin functionally couples to control endothelial barrier function in response to protein kinase A (PKA) agonists. Taken together, these results demonstrate transcriptional coordination of gravin by HIF-1α and amplified PKA-dependent endothelial responses. These findings provide an important link between hypoxia and metabolic conditions associated with inflammation and angiogenesis.
Asunto(s)
Proteínas de Anclaje a la Quinasa A/metabolismo , Proteínas de Ciclo Celular/metabolismo , Factor 1 Inducible por Hipoxia/metabolismo , Proteínas de Anclaje a la Quinasa A/genética , Proteínas de Ciclo Celular/genética , Hipoxia de la Célula/genética , Hipoxia de la Célula/fisiología , Línea Celular , Humanos , Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Mutagénesis Sitio-DirigidaRESUMEN
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/metabolismoRESUMEN
Recent studies have demonstrated dramatic shifts in metabolic supply-and-demand ratios during inflammation, a process resulting in localized tissue hypoxia within inflammatory lesions ("inflammatory hypoxia"). As part of the adaptive immune response, T cells are recruited to sites of inflammatory hypoxia. Given the profound effects of hypoxia on gene regulation, we hypothesized that T-cell differentiation is controlled by hypoxia. To pursue this hypothesis, we analyzed the transcriptional consequences of ambient hypoxia (1% oxygen) on a broad panel of T-cell differentiation factors. Surprisingly, these studies revealed selective, robust induction of FoxP3, a key transcriptional regulator for regulatory T cells (Tregs). Studies of promoter binding or loss- and gain-of-function implicated hypoxia-inducible factor (HIF)-1α in inducing FoxP3. Similarly, hypoxia enhanced Treg abundance in vitro and in vivo. Finally, Treg-intrinsic HIF-1α was required for optimal Treg function and Hif1a-deficient Tregs failed to control T-cell-mediated colitis. These studies demonstrate that hypoxia is an intrinsic molecular cue that promotes FoxP3 expression, in turn eliciting potent anti-inflammatory mechanisms to limit tissue damage in conditions of reduced oxygen availability.
Asunto(s)
Factores de Transcripción Forkhead/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Hipoxia , Inflamación/genética , Mucosa Intestinal/metabolismo , Linfocitos T Reguladores/metabolismo , Animales , Hipoxia de la Célula , Proliferación Celular , Células Cultivadas , Colitis/genética , Colitis/metabolismo , Femenino , Citometría de Flujo , Factores de Transcripción Forkhead/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Inflamación/metabolismo , Interleucina-1/farmacología , Mucosa Intestinal/patología , Células Jurkat , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T Reguladores/efectos de los fármacos , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
PROBLEM: The interleukin-17 (IL-17) family includes pro-inflammatory cytokines IL-17A-F with important roles in mucosal defence, barrier integrity and tissue regeneration. IL-17A can be dysregulated in fertility complications, including pre-eclampsia, endometriosis and miscarriage. Because mammalian subclasses (eutherian, metatherian, and prototherian) have different related reproductive strategies, IL-17 genes and proteins were investigated in the three mammalian classes to explore their involvement in female fertility. METHOD OF STUDY: Gene and protein sequences for IL-17s are found in eutherian, metatherian and prototherian mammals. Through synteny and multiple sequence protein alignment, the relationships among mammalian IL-17s were inferred. Publicly available datasets of early pregnancy stages and female fertility in therian mammals were collected and analysed to retrieve information on IL-17 expression. RESULTS: Synteny mapping and phylogenetic analyses allowed the classification of mammalian IL-17 family orthologs of human IL-17. Despite differences in their primary amino acid sequence, metatherian and prototherian IL-17s share the same tertiary structure as human IL-17s, suggesting similar functions. The analysis of available datasets for female fertility in therian mammals shows up-regulation of IL-17A and IL-17D during placentation. IL-17B and IL-17D are also found to be over-expressed in human fertility complication datasets, such as endometriosis or recurrent implantation failure. CONCLUSIONS: The conservation of the IL-17 gene and protein across mammals suggests similar functions in all the analysed species. Despite significant differences, the upregulation of IL-17 expression is associated with the establishment of pregnancy in eutherian and metatherian mammals. The dysregulation of IL-17s in human reproductive disorders suggests them as a potential therapeutic target.
Asunto(s)
Fertilidad , Interleucina-17 , Mamíferos , Filogenia , Femenino , Interleucina-17/metabolismo , Interleucina-17/genética , Animales , Humanos , Fertilidad/genética , Embarazo , Mamíferos/genética , Evolución Molecular , SinteníaRESUMEN
BACKGROUND/AIMS: Stabilization of the hypoxia-inducible factor (HIF-1α) is proposed to provide a protective host-response to C. difficile intoxication. Here, we aimed to elucidate whether nitric oxide and/or reactive oxygen species produced during C. difficile toxin exposure could influence HIF-1α stability and initiate protection against epithelial cell damage. METHODS/RESULTS: HIF-1α and inducible nitric oxide synthase (iNOS) proteins were up-regulated whereas factor-inhibiting HIF-1 (FIH-1) protein was down-regulated in Caco-2 epithelial cell monolayers with in vitro toxin exposure. We demonstrate using the biotin-switch assay that the stabilization of HIF-1α protein occurred via iNOS-dependent nitrosylation. Inhibition of iNOS activity by selective inhibitor (1400W) attenuated HIF-1α stabilization and exacerbated toxin-dependent disruptions in Caco-2 monolayer morphology and tight junctional integrity in vitro. Treatment of Caco-2 cell monolayers with N-actylcysteine (NAC), a scavenger of reactive oxygen species (ROS), attenuated toxin-dependent increases in iNOS and HIF-1α protein levels but had no effect on FIH-1 responses. In addition, mice that were exposed to C. difficile toxin in vivo also demonstrated a significant increase in HIF-1α protein and nitrosylation levels. CONCLUSION: Taken together, these data suggest that important synergistic actions exist between nitric oxide and ROS to stabilize HIF-1α and its innate, protective actions in the context of C. difficile toxin-mediated epithelial injury.
Asunto(s)
Toxinas Bacterianas/toxicidad , Clostridioides difficile , Células Epiteliales/efectos de los fármacos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Óxido Nítrico/farmacología , Especies Reactivas de Oxígeno/farmacología , Animales , Células CACO-2 , Humanos , Immunoblotting , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Estabilidad Proteica/efectos de los fármacosRESUMEN
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/inmunologíaRESUMEN
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 InversaRESUMEN
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 TranscripcionalRESUMEN
The effects of COVID-19 on fertility services became evident in early 2020. Fertility treatments were initially suspended following advice from international fertility governing bodies. We performed a web-based study to investigate the attitudes of male and female fertility patients in Ireland, for risk mitigation strategies and pregnancy advice during the first wave of COVID-19. Despite international recommendations and uncertainty regarding COVID-19 and pregnancy, over two thirds of patients continued trying to conceive, while awaiting recommencement of fertility services. When services resumed, the majority were keen to continue fertility treatment. They were agreeable to telemedicine in place of face-to-face consultations. They felt that privacy was maintained and were comfortable signing consent forms via video link. Large numbers, however, strongly disagreed with the no-partner policy for embryo transfer and early pregnancy scanning, highlighting the importance of partner support. Patients felt strongly that fertility treatments should be classified as essential services and that every effort should be made to continue treatments in future pandemics. These results highlight the importance of maintaining fertility services, while adapting to new practices that may be required. The primary concern of the infertility population is the desire for pregnancy and parenthood. This innate human need trumps concerns regarding COVID-19 for the majority of those affected.
Asunto(s)
COVID-19 , Infertilidad , Embarazo , Humanos , Masculino , Femenino , COVID-19/prevención & control , Pandemias/prevención & control , SARS-CoV-2 , Fertilidad , Infertilidad/terapiaRESUMEN
The gastrointestinal epithelium is anatomically positioned to provide a selective barrier between the anaerobic lumen and lamina propria, which has a high rate of metabolism. Supported by a complex vasculature, this important barrier is affected by reduced blood flow and resultant tissue hypoxia, particularly during the severe metabolic shifts associated with active inflammation in individuals with inflammatory bowel disease. Activation of hypoxia-inducible factor (HIF) under these conditions promotes resolution of inflammation in mouse models of disease. Protective influences of HIF are attributed, in part, to the complex regulation of barrier protection with the intestinal mucosa. Reagents that activate HIF, via inhibition of the prolyl hydroxylase enzymes, might be developed to induce hypoxia-mediated resolution in patients with intestinal mucosal inflammatory disease.
Asunto(s)
Hipoxia/fisiopatología , Enfermedades Inflamatorias del Intestino/prevención & control , Mucosa Intestinal/metabolismo , Animales , Formación de Anticuerpos , Vasos Sanguíneos/metabolismo , Progresión de la Enfermedad , Metabolismo Energético , Humanos , Hipoxia/genética , Hipoxia/metabolismo , Enfermedades Inflamatorias del Intestino/fisiopatología , Enfermedades Inflamatorias del Intestino/terapia , Mucosa Intestinal/inmunología , Intestinos/irrigación sanguínea , Microcirculación , Oxígeno/metabolismo , Procolágeno-Prolina Dioxigenasa/antagonistas & inhibidores , Procolágeno-Prolina Dioxigenasa/metabolismo , Transcripción GenéticaRESUMEN
Traditionally, the assessment of endometrial receptivity at transvaginal ultrasound scan has been based on the thickness and the morphological appearance of the endometrium. The objective of this study was to prospectively evaluate endometrial thickness (ET), endometrial morphology and uterine artery Doppler parameters prior to assisted reproduction treatment (ART) in the prediction of pregnancy outcome. This was a prospective cohort study. ET, morphology and uterine artery Doppler (UtAD) pulsatility index (PI) and resistance index (RI) were measured in the mid-luteal stage of the menstrual cycle ultrasonographically, timed with urinary luteinizing hormone testing. A total of 50 women were included in the analysis. The clinical pregnancy rate (CPR) per embryo transfer was 42.0% (n = 21/50). Twenty nine women (58.0%) had an unsuccessful outcome. There were no differences in mean ± SD endometrial thickness (ET) (10.0 ± 1.8 mm vs. 10.5 ± 2.4; p = 0.43), or endometrial morphology (100% (n = 21) vs 100% (n = 29); p = 1.00) between the pregnant and not pregnant groups. Similarly, there were no differences in mean ± SD UtAD PI (2.17 ± 0.83 vs. 2.07 ± 0.81; p = 0.67 or mean ± SD UtAD RI (0.84 ± 0.10 vs. 0.81 ± 0.10; p = 0.30). Ultrasonographic endometrial assessment did not differentiate between those who would have a subsequent clinical pregnancy.
Asunto(s)
Resultado del Embarazo , Arteria Uterina , Embarazo , Femenino , Humanos , Arteria Uterina/diagnóstico por imagen , Estudios Prospectivos , Transferencia de Embrión , Índice de Embarazo , Endometrio/diagnóstico por imagenRESUMEN
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.
Asunto(s)
Macroautofagia , Infecciones por Salmonella , Humanos , Hipoxia/metabolismo , Mucosa Intestinal/metabolismo , Oxígeno/metabolismo , Infecciones por Salmonella/metabolismo , Factores de Transcripción/metabolismoRESUMEN
OBJECTIVE: The dose-response effects of dysferlin transgenesis were analyzed to determine if the dysferlin-deficient myopathies are good candidates for gene replacement therapy. METHODS: We have generated 3 lines of transgenic mice, expressing low, mid, and high levels of full-length human dysferlin from a muscle-specific promoter. Transgenic skeletal muscle was analyzed and scored for morphological and functional deficits. RESULTS: Overexpression of dysferlin in mice resulted in a striking phenotype of kyphosis, irregular gait, and reduced muscle mass and strength. Moreover, protein dosage correlated with phenotype severity. In contrast to dysferlin-null skeletal muscle, no evidence of sarcolemmal impairment was revealed. Rather, increased levels of Ca(2+)-regulated, dysferlin-binding proteins and endoplasmic reticulum stress chaperone proteins were observed in muscle lysates from transgenic mice as compared with controls. INTERPRETATION: Expression levels of dysferlin are important for appropriate function without deleterious or cytotoxic effects. As a corollary, we propose that future endeavors in gene replacement for correction of dysferlinopathy should be tailored to take account of this.
Asunto(s)
Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Enfermedades Musculares/genética , Enfermedades Musculares/metabolismo , Animales , Proteínas de Unión al Calcio/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Disferlina , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/patología , Dosificación de Gen , Regulación de la Expresión Génica/genética , Predisposición Genética a la Enfermedad/genética , Humanos , Cifosis/genética , Cifosis/patología , Cifosis/fisiopatología , Cojera Animal/genética , Cojera Animal/patología , Cojera Animal/fisiopatología , Ratones , Ratones Transgénicos , Chaperonas Moleculares/metabolismo , Debilidad Muscular/genética , Debilidad Muscular/patología , Debilidad Muscular/fisiopatología , Músculo Esquelético/fisiopatología , Enfermedades Musculares/fisiopatología , Fenotipo , Regiones Promotoras Genéticas/genética , Estrés Fisiológico/genéticaRESUMEN
Inflammatory diseases influence tissue metabolism, significantly altering the profile of extracellular adenine nucleotides. A number of studies have suggested that adenosine (Ado) may function as an endogenously generated anti-inflammatory molecule. Given the central role of intestinal epithelial cells to the development of colitis, we hypothesized that specific Ado receptors would contribute to disease resolution in mucosal inflammation as modeled by dextran sodium sulfate (DSS) colitis. Initial profiling studies revealed that murine intestinal epithelial cells express predominantly the Ado A2B receptor (AA2BR) and to a lesser extent AA2AR. Guided by these results, we examined the contribution of AA2BR to colitis. Initial studies indicated that the severity of colitis was increased in Aa2br(-/-) mice relative to Aa2br(+/+) controls, as reflected by increased weight loss, colonic shortening, and disease activity indices. Likewise, enteral administration of the selective AA2BR inhibitor PSB1115 to Aa2br(+/+) mice resulted in a similar increase in severity of DSS colitis. Cytokine profiling of colonic tissue revealed specific deficiencies in IL-10 in Aa2br(-/-) mice relative to controls. Extensions of these findings in cultured human intestinal epithelial cells revealed that stable Ado analogs induce IL-10 mRNA and protein and that such increases can be blocked with PSB1115. Taken together, these studies indicate a central regulatory role for AA2BR-modulated IL-10 in the acute inflammatory phase of DSS colitis, thereby implicating AA2BR as an endogenously protective molecule expressed on intestinal epithelial cells.