RESUMEN
Necrotizing enterocolitis (NEC) is a devastating disease affecting premature infants with intestinal inflammation and necrosis. The neonatal intestinal inflammatory response is rich in macrophages, and blood monocyte count is low in human NEC. We previously found that NF-κB mediates the intestinal injury in experimental NEC. However, the role of NF-κB in myeloid cells during NEC remains unclear. Herein, inhibitor of kappaB kinase ß (IKKß), a critical kinase mediating NF-κB activation, was deleted in lysozyme M (Lysm)-expressing cells, which were found to be Cd11b+Ly6c+ monocytes but not Cd11b+Ly6c- macrophages in the dam-fed neonatal mouse intestine. NEC induced differentiation of monocytes into intestinal macrophages and up-regulation of monocyte recruitment genes (eg, L-selectin) in the macrophage compartment in wild-type mice, but not in pups with IKKß deletion in Lysm+ cells. Thus, NF-κB is required for NEC-induced monocyte activation, recruitment, and differentiation in neonatal intestines. Furthermore, pups with Lysm-IKKß deletion had improved survival and decreased incidence of severe NEC compared with littermate controls. Decreased NEC severity was not associated with an improved intestinal barrier. In contrast, NEC was unabated in mice with IKKß deletion in intestinal epithelial cells. Together, these data suggest that recruitment of Ly6c+ monocytes into the intestine, NF-κB activation in these cells, and differentiation of Ly6c+ monocytes into macrophages are critical cellular and molecular events in NEC development to promote disease.
Asunto(s)
Antígenos Ly/metabolismo , Enterocolitis Necrotizante/metabolismo , Células Epiteliales/metabolismo , Macrófagos/metabolismo , Monocitos/metabolismo , FN-kappa B/metabolismo , Animales , Antígenos Ly/genética , Enterocolitis Necrotizante/genética , Enterocolitis Necrotizante/patología , Células Epiteliales/patología , Eliminación de Gen , Quinasa I-kappa B/genética , Quinasa I-kappa B/metabolismo , Selectina L/genética , Selectina L/metabolismo , Macrófagos/patología , Ratones , Ratones Transgénicos , FN-kappa B/antagonistas & inhibidores , FN-kappa B/genética , Regulación hacia ArribaRESUMEN
BackgroundNecrotizing enterocolitis (NEC) is a devastating neonatal disease characterized by intestinal necrosis. Hypoxia-inducible factor-1α (HIF-1α) has a critical role in cellular oxygen homeostasis. Here, we hypothesized that prolyl hydroxylase (PHD) inhibition, which stabilizes HIF-1α, protects against NEC by promoting intestinal endothelial cell proliferation and improving intestinal microvascular integrity via vascular endothelial growth factor (VEGF) signaling.MethodsTo assess the role of PHD inhibition in a neonatal mouse NEC model, we administered dimethyloxalylglycine (DMOG) or vehicle to pups before or during the NEC protocol, and determined mortality and incidence of severe intestinal injury. We assessed intestinal VEGF by western blot analysis and quantified endothelial cell and epithelial cell proliferation following immunofluorescence.ResultsDMOG decreased mortality and incidence of severe NEC, increased intestinal VEGF expression, and increased intestinal villus endothelial and epithelial cell proliferation in experimental NEC. Inhibiting VEGFR2 signaling eliminated DMOG's protective effect on intestinal injury severity, survival, and endothelial cell proliferation while sparing DMOG's protective effect on intestinal epithelial cell proliferation.ConclusionDMOG upregulates intestinal VEGF, promotes endothelial cell proliferation, and protects against intestinal injury and mortality in experimental NEC in a VEGFR2 dependent manner. DMOG's protective effect on the neonatal intestinal mucosa may be mediated via VEGFR2 dependent improvement of the intestinal microvasculature.
Asunto(s)
Aminoácidos Dicarboxílicos/farmacología , Enterocolitis Necrotizante/patología , Intestinos/patología , Microcirculación , Animales , Animales Recién Nacidos , Proliferación Celular , Modelos Animales de Enfermedad , Células Endoteliales/citología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Intestinos/irrigación sanguínea , Intestinos/lesiones , Ratones , Ratones Endogámicos C57BL , Prolil Hidroxilasas/metabolismo , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Intestinal adaptation to small-bowel resection (SBR) after necrotizing enterocolitis expands absorptive surface areas and promotes enteral autonomy. Survivin increases proliferation and blunts apoptosis. The current study examines survivin in intestinal epithelial cells after ileocecal resection. Wild-type and epithelial Pik3r1 (p85α)-deficient mice underwent sham surgery or 30% resection. RNA and protein were isolated from small bowel to determine levels of ß-catenin target gene expression, activated caspase-3, survivin, p85α, and Trp53. Healthy and post-resection human infant small-bowel sections were analyzed for survivin, Ki-67, and TP53 by immunohistochemistry. Five days after ileocecal resection, epithelial levels of survivin increased relative to sham-operated on mice, which correlated with reduced cleaved caspase-3, p85α, and Trp53. At baseline, p85α-deficient intestinal epithelial cells had less Trp53 and more survivin, and relative responses to resection were blunted compared with wild-type. In infant small bowel, survivin in transit amplifying cells increased 71% after SBR. Resection increased proliferation and decreased numbers of TP53-positive epithelial cells. Data suggest that ileocecal resection reduces p85α, which lowers TP53 activation and releases survivin promoter repression. The subsequent increase in survivin among transit amplifying cells promotes epithelial cell proliferation and lengthens crypts. These findings suggest that SBR reduces p85α and TP53, which increases survivin and intestinal epithelial cell expansion during therapeutic adaptation in patients with short bowel syndrome.
Asunto(s)
Adaptación Fisiológica/fisiología , Proteínas Inhibidoras de la Apoptosis/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Síndrome del Intestino Corto/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Western Blotting , Fosfatidilinositol 3-Quinasa Clase Ia , Procedimientos Quirúrgicos del Sistema Digestivo/efectos adversos , Modelos Animales de Enfermedad , Enterocolitis Necrotizante/cirugía , Proteínas de la Matriz Extracelular/metabolismo , Regulación de la Expresión Génica , Humanos , Inmunohistoquímica , Lactante , Recién Nacido , Proteínas Inhibidoras de la Apoptosis/biosíntesis , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Represoras/biosíntesis , Síndrome del Intestino Corto/etiología , SurvivinRESUMEN
BACKGROUND: Decreased intestinal perfusion may contribute to the development of necrotizing enterocolitis (NEC). Vascular endothelial growth factor (VEGF) is an angiogenic protein necessary for the development and maintenance of capillary networks. Whether VEGF is dysregulated in NEC remains unknown. OBJECTIVES: The objective of this study was to determine whether intestinal VEGF expression is altered in a neonatal mouse model of NEC and in human NEC patients. METHODS: We first assessed changes of intestinal VEGF mRNA and protein in a neonatal mouse NEC model before significant injury occurs. We then examined whether exposure to formula feeding, bacterial inoculation, cold stress and/or intermittent hypoxia affected intestinal VEGF expression. Last, we visualized VEGF protein in intestinal tissues of murine and human NEC and control cases by immunohistochemistry. RESULTS: Intestinal VEGF protein and mRNA were significantly decreased in pups exposed to the NEC protocol compared to controls. Hypoxia, cold stress and commensal bacteria, when administered together, significantly downregulated intestinal VEGF expression, while they had no significant effect when given alone. VEGF was localized to a few single intestinal epithelial cells and some cells of the lamina propria and myenteric plexus. VEGF staining was decreased in murine and human NEC intestines when compared to control tissues. CONCLUSION: Intestinal VEGF protein is reduced in human and experimental NEC. Decreased VEGF production might contribute to NEC pathogenesis.
Asunto(s)
Enterocolitis Necrotizante/genética , Mucosa Intestinal/patología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Animales Recién Nacidos , Respuesta al Choque por Frío , Modelos Animales de Enfermedad , Regulación hacia Abajo , Humanos , Hipoxia/fisiopatología , Inmunohistoquímica , Lactante , Recién Nacido , Mucosa Intestinal/metabolismo , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/genética , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
Milk fat globule-EGF factor 8 (MFG-E8) is expressed by macrophages and plays an important role in attenuating inflammation and maintaining tissue homeostasis. Previously, we and others found that lipopolysaccharide (LPS) inhibits MFG-E8 gene expression in macrophages. Here, we characterized the 5'-flanking region of the mouse MFG-E8 gene. To functionally analyze the upstream regulatory region of the MFG-E8 gene, a series of luciferase reporter gene constructs containing deleted or mutated regulatory elements were prepared. Using the luciferase assay, we revealed that Sp1 binding motifs within the proximal promoter region were necessary for full activity of the MFG-E8 promoter, whereas AP-1 like binding sequence at -372 played a role in governing the promoter activity at a homeostatic level. With chromatin immunoprecipitation assay, we showed that Sp1 and c-Jun physically interact with the MFG-E8 promoter region in vivo. In addition, Sp1 was found to regulate the MFG-E8 promoter activity positively and c-Jun negatively. Furthermore, we demonstrated that LPS inhibited MFG-E8 promoter activity via targeting Sp1 and AP-1-like motifs in the 5'-flanking region. Collectively, our data indicate that Sp1 and AP-1-related factors are involved in the regulation of MFG-E8 gene transcription by targeting their binding sites in the 5'-flanking region under physiological and inflammatory states.
Asunto(s)
Antígenos de Superficie/genética , Inflamación/inducido químicamente , Proteínas de la Leche/genética , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-jun/metabolismo , Factor de Transcripción Sp1/metabolismo , Animales , Antígenos de Superficie/química , Antígenos de Superficie/metabolismo , Sitios de Unión/efectos de los fármacos , Células Cultivadas , Regulación de la Expresión Génica/efectos de los fármacos , Inflamación/genética , Inflamación/metabolismo , Lipopolisacáridos/farmacología , Macrófagos Peritoneales/citología , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Ratones , Proteínas de la Leche/química , Proteínas de la Leche/metabolismo , Regiones Promotoras Genéticas/efectos de los fármacosRESUMEN
Whether intestinal barrier disruption precedes or is the consequence of intestinal injury in necrotizing enterocolitis (NEC) remains unknown. Using a neonatal mouse NEC model, we examined the changes in intestinal permeability and specific tight-junction (TJ) proteins preceding NEC and asked whether these changes are prevented by administration of Bifidobacterium infantis, a probiotic known to decrease NEC incidence in humans. Compared with dam-fed controls, pups submitted to the NEC protocol developed i) significantly increased intestinal permeability at 12 and 24 hours (as assessed by 70-kDa fluorescein isothiocyanate-dextran transmucosal flux); ii) occludin and claudin 4 internalization at 12 hours (as assessed by immunofluorescence and low-density membrane fraction immunoblotting); iii) increased claudin 2 expression at 6 hours and decreased claudin 4 and 7 expression at 24 hours; and iv) increased claudin 2 protein at 48 hours. Similar results were seen in human NEC, with claudin 2 protein increased. In mice, administration of B. infantis micro-organisms attenuated increases in intestinal permeability, preserved claudin 4 and occludin localization at TJs, and decreased NEC incidence. Thus, an increase in intestinal permeability precedes NEC and is associated with internalization of claudin 4 and occludin. Administration of B. infantis prevents these changes and reduces NEC incidence. The beneficial effect of B. infantis is, at least in part, due to its TJ and barrier-preserving properties.
Asunto(s)
Bifidobacterium/fisiología , Claudinas/metabolismo , Enterocolitis Necrotizante/patología , Enterocolitis Necrotizante/fisiopatología , Intestinos/microbiología , Intestinos/patología , Uniones Estrechas/metabolismo , Animales , Animales Recién Nacidos , Caveolina 1/metabolismo , Claudinas/genética , Modelos Animales de Enfermedad , Regulación hacia Abajo/genética , Endocitosis , Enterocolitis Necrotizante/metabolismo , Enterocolitis Necrotizante/microbiología , Enterocitos/metabolismo , Enterocitos/patología , Humanos , Lactante , Intestinos/fisiopatología , Intestinos/ultraestructura , Ratones , Ratones Endogámicos C57BL , Ocludina/metabolismo , Permeabilidad , Transporte de Proteínas , Estrés Fisiológico , Uniones Estrechas/ultraestructura , Regulación hacia Arriba/genéticaRESUMEN
Platelet-activating factor (PAF), an endogenous proinflammatory phospholipid, when injected intravascularly to rats and mice, causes shock, acute bowel injury, and a rapid activation of NF-kappaB p50-p50 with upregulation of the chemokine CXCL2 in the intestine. In this study, we investigate the mechanism of NF-kappaB activation and the role of the NF-kappaB p50 subunit in PAF-induced shock and acute bowel injury. NF-kappaB p50-deficient mice and wild-type mice were anesthetized and tracheotomized, and their carotid artery was cannulated for blood pressure monitoring, blood sampling, and PAF administration. For determination of bowel injury, shock, and survival, PAF (2.2 microg/kg, intra-arterially, i.a.) was injected. Two hours later, animals were euthanized, and their small intestines were removed for histological examination. For biochemical studies, PAF (1.5 microg/kg i.a.) was administered and the small intestine removed after 15-60 min. We found that PAF induced an increase in p105 processing within 30 min, but there were no changes in the levels of the NF-kappaB inhibitory proteins IkappaBalpha and beta. NF-kappaB p50-deficient mice were protected against PAF-induced mortality, shock, intestinal hypoperfusion, and injury compared with wild-type animals. We also found that p50-deficient mice had decreased gene expression of CXCL2 and TNF and a decrease in CXCL2 protein production compared with wild-type mice. Our study suggests that PAF increases the processing of NF-kappaB p105 into p50, with upregulation of proinflammatory cytokines, which leads to PAF-induced systemic inflammatory response and acute bowel injury.
Asunto(s)
Íleon/metabolismo , Enfermedades Inflamatorias del Intestino/metabolismo , Subunidad p50 de NF-kappa B/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Quimiocina CXCL2/genética , Quimiocina CXCL2/metabolismo , Modelos Animales de Enfermedad , Hipotensión/inducido químicamente , Hipotensión/metabolismo , Proteínas I-kappa B/metabolismo , Íleon/irrigación sanguínea , Íleon/patología , Mediadores de Inflamación/metabolismo , Enfermedades Inflamatorias del Intestino/inducido químicamente , Enfermedades Inflamatorias del Intestino/patología , Enfermedades Inflamatorias del Intestino/fisiopatología , Enfermedades Inflamatorias del Intestino/prevención & control , Ratones , Ratones Noqueados , Inhibidor NF-kappaB alfa , Subunidad p50 de NF-kappa B/deficiencia , Subunidad p50 de NF-kappa B/genética , Factor de Activación Plaquetaria , Circulación Esplácnica , Factores de Tiempo , Factores de Necrosis Tumoral/genética , Factores de Necrosis Tumoral/metabolismo , Regulación hacia ArribaRESUMEN
Necrotizing enterocolitis (NEC) is a major cause of morbidity and death in premature infants. NEC is associated with increased levels of pro-inflammatory cytokines in plasma and tissues that are regulated by the transcription factor nuclear factor-kappaB (NF-kappaB). It remains unknown, however, whether NF-kappaB mediates injury in neonatal NEC. We therefore examined the activation status of NF-kappaB perinatally in the small intestine and in a neonatal rat model of NEC. We found that intestinal NF-kappaB is strongly activated at birth and, in dam-fed newborn rats, is down-regulated within a day. In contrast, NF-kappaB remains strongly activated at both d 1 and d 2 in stressed animals, and this is accompanied by a significant decrease in the levels of the endogenous NF-kappaB inhibitor protein IkappaBalpha and IkappaBbeta at d 2. To determine the importance of elevated NF-kappaB activity in intestinal injury in NEC, we administered the NEMO-binding domain (NBD) peptide that selectively inhibits the critical upstream IkappaB kinase (IKK). NBD but not a control peptide decreased mortality and bowel injury in this model, supporting the hypothesis that bowel injury in NEC results from elevated NF-kappaB activity. Our findings therefore lead us to conclude that selective NF-kappaB inhibition represents a promising therapeutic strategy for NEC.