Necrotizing enterocolitis (NEC) is an inflammatory bowel necrosis of premature infants. In tissue samples of NEC, we identified numerous macrophages and a few neutrophils but not many lymphocytes. We hypothesized that these pathoanatomic characteristics of NEC represent a common tissue injury response of the gastrointestinal tract to a variety of insults at a specific stage of gut development. To evaluate developmental changes in mucosal inflammatory response, we used trinitrobenzene sulfonic acid (TNBS)-induced inflammation as a nonspecific insult and compared mucosal injury in newborn vs. adult mice. Enterocolitis was induced in 10-day-old pups and adult mice (n = 25 animals per group) by administering TNBS by gavage and enema. Leukocyte populations were enumerated in human NEC and in murine TNBS-enterocolitis using quantitative immunofluorescence. Chemokine expression was measured using quantitative polymerase chain reaction, immunoblots, and immunohistochemistry. Macrophage recruitment was investigated ex vivo using intestinal tissue-conditioned media and bone marrow-derived macrophages in a microchemotaxis assay. Similar to human NEC, TNBS enterocolitis in pups was marked by a macrophage-rich leukocyte infiltrate in affected tissue. In contrast, TNBS-enterocolitis in adult mice was associated with pleomorphic leukocyte infiltrates. Macrophage precursors were recruited to murine neonatal gastrointestinal tract by the chemokine CXCL5, a known chemoattractant for myeloid cells. We also demonstrated increased expression of CXCL5 in surgically resected tissue samples of human NEC, indicating that a similar pathway was active in NEC. We concluded that gut mucosal injury in the murine neonate is marked by a macrophage-rich leukocyte infiltrate, which contrasts with the pleomorphic leukocyte infiltrates in adult mice. In murine neonatal enterocolitis, macrophages were recruited to the inflamed gut mucosa by the chemokine CXCL5, indicating that CXCL5 and its cognate receptor CXCR2 merit further investigation as potential therapeutic targets in NEC.
Background Necrotizing enterocolitis (NEC) is an inflammatory bowel necrosis of premature infants. Based on our recent findings of increased Smad7 expression in surgically-resected bowel affected by NEC, we hypothesized that NEC macrophages undergo inflammatory activation because increased Smad7 expression renders these cells resistant to normal, gut-specific, transforming growth factor (TGF)-β-mediated suppression of inflammatory pathways. Methods We used surgically-resected human NEC tissue, murine models of NEC-like injury, bone marrow-derived and intestinal macrophages, and RAW264.7 cells. Smad7 and IκB kinase-beta (IKK-β) were measured by quantitative polymerase chain reaction (qPCR), Western blots, and immunohistochemistry. Promoter activation was confirmed in luciferase reporter and chromatin immunoprecipitation assays. Results NEC macrophages showed increased Smad7 expression, particularly in areas with severe tissue damage and high bacterial load. LPS-induced Smad7 expression suppressed TGF-β signaling and augmented NF-κB activation and cytokine production in macrophages. Smad7-mediated NF-κB activated was likely mediated via increased expression of IKK-β, which, further increased Smad7 expression in a feed-forward loop. We show that Smad7 induced IKK-β expression through direct binding to the IKK-β promoter and its transcriptional activation. Conclusions Smad7 expression in NEC macrophages interrupts TGF-β signaling and promotes NF-κB-mediated inflammatory signaling in these cells through increased expression of IKK-β.
Necrotizing enterocolitis (NEC) is an idiopathic, inflammatory bowel necrosis of premature infants. Clinical studies have linked NEC with antecedent red blood cell (RBC) transfusions, but the underlying mechanisms are unclear. Here we report a neonatal murine model to investigate this association. C57BL/6 mouse pups rendered anemic by timed phlebotomy and then given RBC transfusions develop NEC-like intestinal injury with prominent necrosis, inflammation, and submucosal edema/separation of the lamina propria in the ileocecal region and colon within 12–24 h. The anemic intestine is infiltrated by inflammatory macrophages, which are activated in situ by RBC transfusions via a Toll-like receptor (TLR)-4-mediated mechanism and cause bowel injury. Chelation of RBC degradation products with haptoglobin, absence of TLR4, macrophage depletion, and inhibition of macrophage activation is protective. Intestinal injury worsens with increasing severity and the duration of anemia prior to transfusion, indicating a need for the re-evaluation of current transfusion guidelines for premature infants.
Preterm infants may be at risk of necrotizing enterocolitis (NEC) due to deficiency of transforming growth factor-β 2 (TGF-β2) in the developing intestine. We hypothesized that low epithelial TGF-β2expression in preterm intestine and during NEC results from diminished autocrine induction of TGF-β2in these cells. Premature baboons delivered at 67% gestation were treated per current norms for human preterm infants. NEC was diagnosed by clinical and radiological findings. Inflammatory cytokines, TGF-β2, Smad7, Ski, and strawberry notch N (SnoN)/Ski-like oncoprotein (SKIL) was measured using quantitative reverse transcriptase-polymerase chain reaction, immunoblots, and immunohistochemistry. Smad7 effects were examined in transfected IEC6 intestinal epithelial cells in vitro. Findings were validated in archived human tissue samples of NEC. NEC was recorded in seven premature baboons. Consistent with existing human data, premature baboon intestine expressed less TGF-β2than term intestine. TGF-β2expression was regulated in epithelial cells in an autocrine fashion, which was interrupted in the premature intestine and during NEC due to increased expression of Smad7. LPS increased Smad7 binding to the TGF-β2promoter and was associated with dimethylation of the lysine H3K9, a marker of transcriptional silencing, on the nucleosome of TGF-β2. Increased Smad7 expression in preterm intestine was correlated with the deficiency of SnoN/SKIL, a repressor of the Smad7 promoter. Smad7 inhibits autocrine expression of TGF-β2in intestinal epithelial cells in the normal premature intestine and during NEC. Increased Smad7 expression in the developing intestine may be due to a developmental deficiency of the SnoN/SKIL oncoprotein.
Chlorine gas (Cl 2 ) exposure during accidents or in the military setting results primarily in injury to the lungs. However, the potential for Cl 2 exposure to promote injury to the systemic vasculature leading to compromised vascular function has not been studied. We hypothesized that Cl 2 promotes extrapulmonary endothelial dysfunction characterized by a loss of endothelial nitric oxide synthase (eNOS)-derived signaling. Male Sprague Dawley rats were exposed to Cl 2 for 30 minutes, and eNOS-dependent vasodilation of aorta as a function of Cl 2 dose (0-400 ppm) and time after exposure (0-48 h) were determined. Exposure to Cl 2 (250-400 ppm) significantly inhibited eNOS-dependent vasodilation (stimulated by acetycholine) at 24 to 48 hours after exposure without affecting constriction responses to phenylephrine or vasodilation responses to an NO donor, suggesting decreased NO formation. Consistent with this hypothesis, eNOS protein expression was significantly decreased (z 60%) in aorta isolated from Cl 2 -exposed versus airexposed rats. Moreover, inducible nitric oxide synthase (iNOS) mRNA was up-regulated in circulating leukocytes and aorta isolated 24 hours after Cl 2 exposure, suggesting stimulation of inflammation in the systemic vasculature. Despite decreased eNOS expression and activity, no changes in mean arterial blood pressure were observed. However, injection of 1400W, a selective inhibitor of iNOS, increased mean arterial blood pressure only in Cl 2 -exposed animals, suggesting that iNOS-derived NO compensates for decreased eNOS-derived NO. These results highlight the potential for Cl 2 exposure to promote postexposure systemic endothelial dysfunction via disruption of vascular NO homeostasis mechanisms.
Cytokines and growth factors play diverse roles in the uninflamed fetal/neonatal intestinal mucosa and in the development of inflammatory bowel injury during necrotizing enterocolitis (NEC). During gestational development and the early neonatal period, the fetal/premature intestine is exposed to high levels of many “inflammatory” cytokines and growth factors, first via swallowed amniotic fluid in utero and then, after birth, in colostrum and mother’s milk. This article reviews the dual, seemingly counter-intuitive roles of cytokines, where these agents play a “trophic” role and promote maturation of the uninflamed mucosa, but can also cause inflammation and promote intestinal injury during NEC.
fluid-borne hepatocyte growth factor protects rat pups against experimental necrotizing enterocolitis. Am J Physiol Gastrointest Liver Physiol 306: G361-G369, 2014. First published January 9, 2014; doi:10.1152/ajpgi.00272.2013.-Fetal swallowing of amniotic fluid, which contains numerous cytokines and growth factors, plays a key role in gut mucosal development. Preterm birth interrupts this exposure to amniotic fluid-borne growth factors, possibly contributing to the increased risk of necrotizing enterocolitis (NEC) in premature infants. We hypothesized that supplementation of formula feeds with amniotic fluid can provide amniotic fluid-borne growth factors and prevent experimental NEC in rat pups. We compared NEC-like injury in rat pups fed with infant formula vs. formula supplemented either with 30% amniotic fluid or recombinant hepatocyte growth factor (HGF). Cytokines/growth factors in amniotic fluid were measured by immunoassays. Amniotic fluid and HGF effects on enterocyte migration, proliferation, and survival were measured in cultured IEC6 intestinal epithelial cells. Finally, we used an antibody array to investigate receptor tyrosine kinase (RTK) activation and immunoblots to measure phosphoinositide 3-kinase (PI3K) signaling. Amniotic fluid supplementation in oral feeds protected rat pups against NEC-like injury. HGF was the most abundant growth factor in rat amniotic fluid in our panel of analytes. Amniotic fluid increased cell migration, proliferation, and cell survival in vitro. These effects were reproduced by HGF and blocked by anti-HGF antibody or a PI3K inhibitor. HGF transactivated several RTKs in IEC6 cells, indicating that its effects extended to multiple signaling pathways. Finally, similar to amniotic fluid, recombinant HGF also reduced the frequency and severity of NEC-like injury in rat pups. Amniotic fluid supplementation protects rat pups against experimental NEC, which is mediated, at least in part, by HGF. amniotic fluid; NEC; HGF; inflammation; phosphoinositide 3-kinase NECROTIZING ENTEROCOLITIS (NEC), an inflammatory bowel necrosis of preterm infants, is a leading cause of death among neonates born before 32 wk of gestation or with a birth weight Ͻ1,500 g (20, 27). Although the etiology of NEC remains unclear, epidemiological studies show an association with diverse risk factors such as maternal chorioamnionitis, perinatal asphyxia, indomethacin therapy, viral infections, and blood transfusions (20). Current pathophysiological models suggest that NEC occurs when altered/disrupted epithelial barrier in the preterm intestine allows luminal bacteria to translocate across the epithelial barrier into the lamina propria, triggering a severe mucosal inflammatory response and tissue damage (21).During intrauterine development, the fetus ingests progressively large volumes of amniotic fluid, which contains several cytokines and growth factors known to promote gut mucosal development (19). In the third trimester, the human fetus swallows nearly 550 ml/day amniotic fluid (29, 40). We hypothesiz...
BackgroundWe have shown previously that enteral administration of 2, 4, 6-trinitrobenzene sulfonic acid in 10-day-old C57BL/6 pups produces an acute necrotizing enterocolitis with histopathological and inflammatory changes similar to human necrotizing enterocolitis (NEC). To determine whether murine neonatal TNBS-mediated intestinal injury could be used as a NEC model, we compared gene expression profiles of TNBS-mediated intestinal injury and NEC.MethodsWhole genome microarray analysis was performed on proximal colon from control and TNBS-treated pups (n=8/group). For comparison, we downloaded human microarray data of NEC (n=5) and surgical control (n=4) from a public database. Data were analyzed using the software programs Partek Genomics Suite and Ingenuity Pathway Analysis.ResultsWe detected extensive changes in gene expression in murine TNBS-mediated intestinal injury and human NEC. Using fold-change cut-offs of ±1.5, we identified 4440 differentially-expressed genes (DEGs) in murine TNBS-mediated injury and 1377 in NEC. Murine TNBS-mediated injury and NEC produced similar changes in expression of orthologous genes (r = 0.611, p<0.001), and also activated nearly-identical biological processes and pathways. Lipopolysaccharide was top predicted upstream regulator in both the murine and human datasets.ConclusionsMurine neonatal TNBS-mediated enterocolitis and human NEC activate nearly-identical biological processes, signaling pathways, and transcriptional networks.
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