INTRODUCTION:Intestinal fibrosis, characterized by excessive deposition of extracellular matrix proteins, is a common and severe clinical complication of inflammatory bowel disease (IBD). However, the mechanisms underlying fibrosis remain elusive, and currently, there are limited effective pharmacologic treatments that target the development of fibrosis. Hypoxia is one of the key microenvironmental factors influencing intestinal inflammation and has been linked to fibrosis.OBJECTIVE:In the present study, we sought to elucidate the impact of hypoxia on fibrotic gene expression in the intestinal mucosa.METHODS:Human volunteers, IBD patients, and dextran sulphate sodium-treated mice were exposed to hypoxia, and colonic biopsies were collected. The human intestinal epithelial cell line Caco-2, human THP-1 macrophages, and primary human gut fibroblasts were subjected to hypoxia, and changes in fibrotic gene expression were assessed.RESULTS:Human volunteers subjected to hypoxia presented reduced transcriptional levels of fibrotic and epithelial-mesenchymal transition markers in the intestinal mucosa. IBD patients showed a trend towards a decrease in tissue inhibitor of metalloproteinase 1 protein expression. In mice, hypoxic conditions reduced the colonic expression of several collagens and matrix metalloproteinases. Hypoxic Caco-2 cells, THP-1 cells, and primary gut fibroblasts showed a significant downregulation in the expression of fibrotic and tissue remodelling factors.CONCLUSIONS:Stabilization of hypoxia-inducible factors might represent a novel therapeutic approach for the treatment of IBD-associated fibrosis.

Environmental hypoxia influences the development of inflammatory bowel diseases. Adaptive responses to hypoxia are mediated through hypoxia-inducible factors, which are tightly regulated by oxygen- and iron-dependent hydroxylases. Regulation of uptake, storage and export of iron is mediated by signals reflecting oxygen and intracellular iron levels in enterocytes. Conversely, iron modulates responses to hypoxia. We sought to elucidate the effects of iron levels on hypoxia-associated responses in the intestinal epithelium. Human subjects were exposed to hypoxia, and colonic biopsies and serum samples were collected. The human intestinal epithelial cells HT-29, Caco-2 and T84 were subjected to hypoxia in the presence of iron or the iron chelator deferoxamine. Changes in inflammatory gene expression and signalling were assessed by qPCR and western blot. Chromatin immunoprecipitation was performed using antibodies against NF-κB and primers for promoter binding regions of TNF and IL-1β Human subjects presented reduced levels of ferritin and iron in the intestinal epithelium following hypoxia. Hypoxia reduced iron deprivation-associated TNF and IL-1β expression in HT-29 cells through the induction of autophagy. Contrarily, hypoxia triggered TNF and IL-1β expression, and NF-κB activation in Caco-2 and T84 cells. In Caco-2 cells, iron blocked early and late-stage autophagy while reducing hypoxia-associated TNF and IL-1β expression, and the binding of NF-κB to the promoter of TNF and IL-1β. Hypoxia-induced autophagy reduces inflammation in HT-29 cells. In Caco-2 cells, iron uptake is essential to prevent hypoxia-induced inflammatory processes. Iron mobilisation plays a crucial role in the maintenance of homeostasis in the hypoxic intestinal epithelium.

BACKGROUND & AIMS: Hypoxia-associated pathways influence the development of inflammatory bowel disease. Adaptive responses to hypoxia are mediated through hypoxia-inducible factors, which are regulated by iron-dependent hydroxylases. Signals reflecting oxygen tension and iron levels in enterocytes regulate iron metabolism. Conversely, iron availability modulates responses to hypoxia. In the present study we sought to elucidate how iron influences the responses to hypoxia in the intestinal epithelium. METHODS: Human subjects were exposed to hypoxia, and colonic biopsy specimens and serum samples were collected. HT-29, Caco-2, and T84 cells were subjected to normoxia or hypoxia in the presence of iron or the iron chelator deferoxamine. Changes in inflammatory gene expression and signaling were assessed by quantitative polymerase chain reaction and Western blot. Chromatin immunoprecipitation was performed using antibodies against nuclear factor (NF)-kappa B and primers for the promoter of tumor necrosis factor (TNF) and interleukin (IL)1 beta. RESULTS: Human subjects presented reduced levels of ferritin in the intestinal epithelium after hypoxia. Hypoxia reduced iron deprivation-associated TNF and IL1 beta expression in HT-29 cells through the induction of autophagy. Contrarily, hypoxia triggered TNF and IL1 beta expression, and NF-kappa B activation in Caco-2 and T84 cells. Iron blocked autophagy in Caco-2 cells, while reducing hypoxia-associated TNF and IL1 beta expression through the inhibition of NF-kappa B binding to the promoter of TNF and IL1 beta. CONCLUSIONS: Hypoxia promotes iron mobilization from the intestinal epithelium. Hypoxia-associated autophagy reduces inflammatory processes in HT-29 cells. In Caco-2 cells, iron uptake is essential to counteract hypoxia-induced inflammation. Iron mobilization into enterocytes may be a vital protective mechanism in the hypoxic inflamed mucosa.

BACKGROUND:The impact of environmental hypoxia on inflammatory bowel disease (IBD) is controversial with studies showing detrimental and protective effects.Hypoxia regulates autophagy and nucleotide-binding oligomerization domain receptor, pyrin domain containing (NLRP)3, two innate immune mechanisms linked by mutual regulation that have been associated to the development of IBD.We investigated the functional impact of hypoxia on the development of colitis with special emphasis on autophagy and NLRP3 regulation.METHODS: Healthy volunteers and patients with IBD, as well as wild-type, interleukin (IL)-10 -/- , NLRP3 -/- and IL-10 -/- NLRP3 -/- double knockout mice were subjected to hypoxia and changes in inflammatory signaling and gene expression were analyzed in colon biopsies using RT-qPCR and Western blotting.The effects of hypoxia on autophagy, NLRP3 regulation and inflammation were further assessed in vitro using the intestinal epithelial cell line HT-29.RESULTS: Hypoxia significantly reduced tumor necrosis factor α, IL-6 and NLRP3 expression and increased autophagy gene expression in colon biopsies of patients with Crohn's disease.In normoxia, IL-10 -/- , but not IL-10 -/- Nlrp3 -/- mice presented an accumulation of autophagy proteins and an increase in NF-κB activation and inflammatory cytokine expression, which was significantly reduced under hypoxia.In vitro, hypoxia-induced autophagy downregulated NF-κB signaling in intestinal epithelial cells.Hypoxia also reduced NLRP3 expression, and silencing of NLRP3 activated autophagy following the dephosphorylation of mammalian target of rapamycin (mTOR).Co-immunoprecipitation experiments identified NLRP3 as a novel binding partner of mTOR.CONCLUSION: Our results show that hypoxia counteracts inflammation through a novel mechanism involving the downregulation of the binding partner of mTOR NLRP3 and subsequent activation of autophagy.

Background: The impact of hypoxia on inflammatory bowel disease (IBD) is controversial with studies showing detrimental and protective effects. Hypoxia regulates autophagy and nucleotide-binding oligomerization domain receptor, pyrin domain containing (NLRP)3, two innate immune mechanisms linked by mutual regulation that have been associated to the development of IBD. We investigated the functional impact of hypoxia on the development of colitis with special emphasis on autophagy and NLRP3 regulation. Methods: Healthy volunteers and patients with IBD, as well as wild-type, interleukin (IL)-10−/−, NLRP3−/− and IL-10−/− NLRP3−/− double knockout mice were subjected to hypoxia and changes in inflammatory signaling and gene expression were analyzed in colon biopsies using RT-qPCR and Western blotting. The effects of hypoxia on autophagy, NLRP3 regulation and inflammation were further assessed in vitro using the intestinal epithelial cell line HT-29. Results: Hypoxia significantly reduced tumor necrosis factor α, IL-6 and NLRP3 expression and increased autophagy gene expression in colon biopsies of patients with Crohn's disease. In normoxia, IL-10−/−, but not IL-10−/− Nlrp3−/− mice presented an accumulation of autophagy proteins and an increase in NF-κB activation and inflammatory cytokine expression, which was significantly reduced under hypoxia. In vitro, hypoxia-induced autophagy downregulated NF-κB signaling. Hypoxia also reduced NLRP3 expression, and silencing of NLRP3 activated autophagy following the dephosphorylation of mammalian target of rapamycin (mTOR). Co-immunoprecipitation experiments identified NLRP3 as a novel binding partner of mTOR. Conclusions: Our results show that hypoxia counteracts inflammation through a novel mechanism involving the downregulation of the binding partner of mTOR NLRP3 and subsequent activation of autophagy.

Hypoxia regulates autophagy and nucleotide-binding oligomerization domain receptor, pyrin domain containing (NLRP)3, two innate immune mechanisms linked by mutual regulation and associated to IBD. Here we show that hypoxia ameliorates inflammation during the development of colitis by modulating autophagy and mammalian target of rapamycin (mTOR)/NLRP3 pathway. Hypoxia significantly reduces tumor necrosis factor α, interleukin (IL)-6 and NLRP3 expression, and increases the turnover of the autophagy protein p62 in colon biopsies of Crohn's disease patients, and in samples from dextran sulfate sodium-treated mice and Il-10 -/- mice. In vitro, NF-κB signaling and NLRP3 expression are reduced through hypoxia-induced autophagy. We also identify NLRP3 as a novel binding partner of mTOR. Dimethyloxalylglycine-mediated hydroxylase inhibition ameliorates colitis in mice, downregulates NLRP3 and promotes autophagy. We suggest that hypoxia counteracts inflammation through the downregulation of the binding of mTOR and NLRP3 and activation of autophagy.Hypoxia and HIF-1α activation are protective in mouse models of colitis, and the latter regulates autophagy. Here Cosin-Roger et al. show that hypoxia ameliorates intestinal inflammation in Crohn's patients and murine colitis models by inhibiting mTOR/NLRP3 pathway and promoting autophagy.

critical for accurate quantification of target genes using quantitative real time PCR (RT-qPCR) and is a crucial prerequisite for achieving reliable conclusions.However, no studies have addressed the performance of reference genes in that model.Therefore, this study aimed to determine the stability of reference genes in DSS-experimental murine colitis.Methods: Colonic inflammation was induced in male C57BL/6 mice using DSS 5% (n = 6) in drinking water for 5 days, and the control group (n = 6) received water.RNA was extracted from inflamed and non-inflamed colon.Using RT-qPCR, comparative analysis of 13 reference genes was performed by studying the effect of inflammation on their expression, and gene stability including geNorm, BestKeeper and comprehensive ranking analyses.Using the 13 reference genes, the relative colonic TNF-aand IL-1b gene expression was determined by calculating the difference in the threshold cycle.Results: Intestinal inflammation significantly altered the stability of mucosal reference gene expression.Commonly-used glyceraldehyde-3-phosphate dehydrogenase (GAPDH), b-actin (ACTB), or b2-microglobulin (B2M) showed the highest variability between the inflamed and control groups.Conversely, TATAbox-binding protein (TBP) and eukaryotic translation elongation factor 2 ( Eef2) were not affected by inflammation and were the most stable genes.Normalization of colonic TNF-a and IL-1b mRNA levels was dependent on the reference gene used.Depending on the genes used to normalize the data, statistical significance varied from significant when TBP and Eef2 were used to non-significant when GAPDH, ACTB or B2M were used.Conclusions: These findings support the importance of using stable reference genes when using the DSS model.Suboptimal reference genes may explain controversial results from published studies in the context of experimental colitis.We recommend using TBP and Eef2 instead of GAPDH, ACTB or B2M as reference genes.This study highlights the appropriate choice of reference genes to ensure adequate normalization of RT-qPCR data.

BACKGROUND & AIMS: A novel family of proton-sensing G-protein-coupled receptors, including ovarian cancer G-protein-coupled receptor 1 (OGR1) (GPR68) has been identified to play a role in pH homeostasis. Hypoxia is known to change tissue pH as a result of anaerobic glucose metabolism through the stabilization of hypoxia-inducible factor-1 alpha. We investigated how hypoxia regulates the expression of OGR1 in the intestinal mucosa and associated cells.METHODS: OGR1 expression in murine tumors, human colonic tissue, and myeloid cells was determined by quantitative reverse-transcription polymerase chain reaction. The influence of hypoxia on OGR1 expression was studied in monocytes/macrophages and intestinal mucosa of inflammatory bowel disease (IBD) patients. Changes in OGR1 expression in MonoMac6 (MM6) cells under hypoxia were determined upon stimulation with tumor necrosis factor (TNF), in the presence or absence of nuclear factor-kappa B (NF-kappa B) inhibitors. To study the molecular mechanisms involved, chromatin immunoprecipitation analysis of the OGR1 promoter was performed.RESULTS: OGR1 expression was significantly higher in tumor tissue compared with normal murine colon tissue. Hypoxia positively regulated the expression of OGR1 in MM6 cells, mouse peritoneal macrophages, primary human intestinal macrophages, and colonic tissue from IBD patients. In MM6 cells, hypoxia-enhanced TNF-induced OGR1 expression was reversed by inhibition of NF-kappa B. In addition to the effect of TNF and hypoxia, OGR1 expression was increased further at low pH. Chromatin immunoprecipitation analysis showed that HIF-1 alpha, but not NF-kappa B, binds to the promoter of OGR1 under hypoxia.CONCLUSIONS: The enhancement of TNF- and hypoxia-induced OGR1 expression under low pH points to a positive feed-forward regulation of OGR1 activity in acidic conditions, and supports a role for OGR1 in the pathogenesis of IBD.