Intestinal Epithelial Apoptosis Research Articles

Intestinal Conditioning After Cardiac Arrest: The Use of Normothermic Extracorporeal Membrane Oxygenation in the Non-Heart-Beating Animal Model.

The effect of normothermic extracorporeal membrane oxygenation (NECMO) on small bowel preservation in a clinically relevant large animal model of expected donation after cardiac death (eDCD) was evaluated. Thirty domestic crossbred donor pigs were divided into five groups. The first group served as the live donation (LD) group, the second group served as the donation after cardiac death (DCD) group, and the remaining were further assigned into three subgroups: E1 group (1 h NECMO support), E3 group (3 h NECMO support), and E5 group (5 h NECMO support). Pathology, electron microscopy, energy metabolism, cell apoptosis, and tight junction (TJ) protein expression level of intestinal mucosa and the level of plasma d-lactic acid were evaluated in normal, cardiac death and at the end of extracorporeal support, respectively. The mean arterial pressure and PaO2 were maintained over 60 and 267 mmHg during NECMO support, respectively. One hour of extracorporeal support could improve the energy status in intestines of the DCD group. Although the histologic damage and apoptosis of the E1 group had no significant difference with those of the LD and DCD groups (P > 0.05), the levels of intestinal mucosa TJ protein decreased (P < 0.05), and plasma d-lactic acid increased progressively (P < 0.05). With the extension of extracorporeal support, the degree of intestinal mucosa damage and intestinal permeability gradually increased, as well as the content of adenosine triphosphate in intestinal mucosa. The normothermic extracorporeal support for 1 h in DCD is beneficial for improving the energy status and viability of the bowel. However, the integrity of intestinal mucosa was destroyed gradually as extracorporeal support time went by. And the activation of intestinal epithelial apoptosis and hyperoxia might be the factors that lead to intestinal mucosa injury.

Cyclic AMP and Protein Kinase A Mediated Signaling Plays a Crucial Role in Cronobacter sakazakii-induced Intestinal Epithelial Apoptosis, Exposing a New Potential Therapy for Necrotizing Enterocolitis

Cyclic AMP and Protein Kinase A Mediated Signaling Plays a Crucial Role in Cronobacter sakazakii-induced Intestinal Epithelial Apoptosis, Exposing a New Potential Therapy for Necrotizing Enterocolitis

The preventive effects of dexmedetomidine against intestinal ischemia-reperfusion injury in Wistar rats.

Intestinal ischemia-reperfusion is a major problem, which may lead to multiorgan failure and death. The aim of this study was to evaluate the protective effects of dexmedetomidine on cell proliferation, antioxidant system, cell death, and structural integrity in intestinal injury induced by ischemia-reperfusion in rats. Animals were randomized into three groups: group A, sham-operated or control; group B, intestinal ischemia/reperfusion (IR); and group C, intestinal IR pretreated with 50 μg of dexmedetomidine. Intestine tissue was collected from all rats 30 min after desufflation, and fresh frozen for histological and biochemical evaluation. The intestinal tissue of group B rats showed a significant decrease in the antioxidant enzyme activities. However, these enzyme activities were improved by the administration of dexmedetomidine. Inhibiting the protein expression of MCP7, PAR2, P-JAK, P-STAT1, and P-STAT3 proved the protective effect of dexmedetomidine. The immunohistochemical staining revealed its protective effect by maintaining the normal structural integrity, less caspase-3 immuno reactivity, and increased cell proliferation count in the intestinal tissues. Intraperitoneal injection of dexmedetomidine significantly protected intestine IR injury in rats by inhibiting the inflammatory response, intestinal epithelial apoptosis, and maintaining structural integrity of intestinal cells.

Protective Role of Rheum Tanguticum Polysaccharide 1 in Radiation- induced Intestinal Mucosal Injury.

The protective effects of Rheum tanguticum polysaccharide 1 (RTP1), which is extracted from the Chinese traditional medicine Rheum tanguticum, on radiation-induced intestinal mucosal injury was investigated.Rat intestinal crypt epithelial cells (IEC-6 cells) and Sprague-Dawley rats were each divided into control, irradiated and RTP1-pretreated irradiated groups. After irradiation, cell survival was determined by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide). assay, and the intracellular reactive oxygen species (ROS) was detected by fluorescent probe method. Apoptosis was observed by acridine orange staining, and cell cycle was analysed by flow cytometry. Histological analysis of the rat intestinal mucosa was conducted by haematoxylin and eosin staining. Irradiation at 8 Gy(Gray) decreased cell survival rate to only 54%, significantly increased intracellular ROS levels and induced apoptosis. RTP1 pretreatment significantly inhibited cell death, reduced the formation of intracellular ROS and partially inhibited apoptosis. Irradiation markedly reduced the height and quantity of rat intestinal villi, but it could be antagonised by RTP1 pretreatment. RTP1 can promote the recovery of intestinal mucosa damage, possibly by inhibiting radiation-induced intestinal epithelial apoptosis and intracellular ROS production.

Vitamin D/VDR signaling pathway ameliorates 2,4,6-trinitrobenzene sulfonic acid-induced colitis by inhibiting intestinal epithelial apoptosis.

Increasing epidemiological data have suggested a link between vitamin D deficiency and the incidence of inflammatory bowel disease (IBD). In the present study, we confirmed that vitamin D deficiency, as well as the decreased local expression of vitamin D receptor (VDR), was prevalent in an IBD cohort. The excessive apoptosis of intestinal epithelial cells (IECs) partly accounts for the development of colonic inflammation and eventually results in IBD. Based on the established inhibitory effects of the vitamin D/VDR pathway on IEC apoptosis, we treated mice with 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis with paricalcitol, a vitamin D analog, in order to investigate the mechanisms responsible for the inhibitory effects of the vitamin D/VDR pathway. We observed that following treatment with vitamin D, the mice presented with only minor bodyweight loss, and the mice also showed improved histological scores and decreased intestinal epithelial permeability compared with the vehicle-treated group. The colonic mRNA expression of inflammatory cytokines and chemokines was markedly suppressed, indicating less severe colitis in the vitamin D-treated mice. Subsequently, we investigated p53 upregulated modulator of apoptosis (PUMA) and p53, two major independent pathways of apoptosis, as well as caspase-3. We found that the vitamin D-treated mice had lower expression levels of caspase-3 than the vehicle-treated mice. PUMA expression showed the same tendency; however, the p53 protein level was not altered. The present study indicates that vitamin D attenuates the development of TNBS-induced colitis by inhibiting the apoptosis of IECs. The mechanisms involved include the downregulation of PUMA expression. Our data provide experimental support for the clinical trials of vitamin D intervention in patients with IBD.

M1 to M2 macrophage polarization in heparin-binding epidermal growth factor-like growth factor therapy for necrotizing enterocolitis

BackgroundMacrophages can be polarized into proinflammatory (M1) and anti-inflammatory (M2) subtypes. However, whether macrophage polarization plays a role in necrotizing enterocolitis (NEC) remains unknown. Materials and methodsMacrophages were derived from the THP-1 human monocyte cell line. Apoptosis of human fetal small intestinal epithelial FHs-74 cells was determined by Annexin V/propidium iodide flow cytometry and by Western blotting to detect cleaved caspase-3. The effect of heparin-binding epidermal growth factor-like growth factor (HB-EGF) on macrophage polarization was determined by flow cytometry with M1/M2 markers and real time polymerase chain reaction. In vivo, experimental NEC was induced in mouse pups by repeated exposure to hypoxia, hypothermia, and hypertonic feedings. Intestinal histologic sections were subjected to immunohistochemical staining for the detection of M1 and M2 macrophages. ResultsIn vitro, FHs-74 cell apoptosis was increased after coculture with macrophages and lipopolysaccharide (LPS). This apoptosis was increased by exposure to M1-conditioned medium and suppressed by exposure to M2-conditioned medium. HB-EGF significantly decreased LPS-induced M1 polarization and promoted M2 polarization via signal transducers and activators of transcription 3 activation. Addition of HB-EGF to LPS-stimulated macrophages suppressed the proapoptotic effects of the macrophages on FHs-74 cells. In vivo, we found enhanced intestinal macrophage infiltration in pups subjected to NEC, most of which were M1 macrophages. HB-EGF treatment of pups subjected to experimental NEC significantly reduced M1 and increased M2 polarization and protected the intestines from NEC. ConclusionsM1 macrophages promote NEC by increasing intestinal epithelial apoptosis. HB-EGF protects the intestines from NEC by preventing M1 and promoting M2 polarization.

Sevoflurane protects against intestinal ischemia–reperfusion injury partly by phosphatidylinositol 3 kinases/Akt pathway in rats

Intestinal ischemia-reperfusion injury (IRI) is a clinical challenge with high morbidity and mortality, leading to intestine damage, systemic inflammation, and multiorgan failure. Previous research has shown that the inhaled anesthetic sevoflurane protects various organs from IRI. However, whether sevoflurane protects against intestinal IRI and which application condition is the most effective are not completely clear. Thus, we investigated the effects of sevoflurane on intestinal IRI with sevoflurane given before, during or after intestinal ischemia, and the role of phosphatidylinositol 3 kinases (PI3K)/Akt pathway in these effects. Rat model of intestinal ischemia-reperfusion (IR) was used in this study. The superior mesenteric artery was clamped for 60 minutes followed by 120 minutes of reperfusion. Sevoflurane at 0.25, 0.5, and 1.0 minimum alveolar concentration (MAC) was inhaled for 30 minutes before, during, or after ischemic insult. LY294002, a PI3K inhibitor, was injected intraperitoneally before sevoflurane inhalation. Intestinal IR caused a significant decrease of mean arterial blood pressure, severe intestinal mucosa injury and epithelial cell apoptosis, downregulation of the levels of phospho-Akt and phospho-Bad proteins. Exposure to 0.5 or 1.0 MAC sevoflurane before or after intestinal ischemia or 0.5 MAC during intestinal ischemia significantly ameliorated IR-induced histopathologic changes and decreased Chiu's scores. Pretreatment with 0.5 MAC sevoflurane also inhibited intestinal IR-induced increase of terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling-positive cells and activation of caspase-3 and restored expression of phospho-Akt and phospho-Bad. LY294002 partly blocked the protective effects induced by 0.5 MAC sevoflurane pretreatment. Our results suggest that sevoflurane inhalation at clinical related concentration before, during, or after ischemia protects against IR-induced intestinal injury. The pretreatment-induced protection was partly mediated by inhibiting intestinal mucosal epithelial apoptosis via activation of the PI3K/Akt pathway.

Apelin's effects on young rat gastrointestinal tract maturation

Apelin is considered an important gut regulatory peptide with potential physiological roles in gastrointestinal cytoprotection and regulation of food intake and drinking behavior. The aim of this study was to determine the effects of intraperitoneal or intragastric apelin administration on gastric and intestinal epithelial apoptosis, mitosis and DNA repair enzyme 8-oxoguanine (OGG 1/2) expression in young Wistar rats (50±5gb.wt.). Apelin-13 was intraperitoneally or intragastrically administered twice a day for 10 days (100nmol/kgb.wt./2×day), and control groups received physiological saline as a placebo. The rats were sacrificed after treatment, and the gastric fundus, duodenum, middle jejunum and colon tissue samples were harvested for immunofluorescence studies. Intragastric administration of apelin-13 increased the apoptotic index in the stomach and colon tissues (P≤0.001) but decreased apoptosis in the duodenum and jejunum (P<0.001); this approach reduced the number of mitotic cells in the jejunum and colon but increased mitoses (P<0.001) in the duodenum. Finally, intragastric apelin-13 increased (P<0.001) OGG 1/2 enzyme expression in the stomach and jejunum and decreased its expression in the colon (P<0.01). However, intraperitoneal apelin-13 injection caused the opposite effect in the same regions of the gastrointestinal tract. In conclusion, apelin inhibits gastrointestinal tissue maturation in young rats, regardless of the administration route. However, further studies are required to clarify the mechanism of apelin action on gastrointestinal tract maturation in young rats.

Proteinase-activated Receptor 2 (PAR2) Decreases Apoptosis in Colonic Epithelial Cells

Mucosal biopsies from inflamed colon of inflammatory bowel disease patients exhibit elevated epithelial apoptosis compared with those from healthy individuals, disrupting mucosal homeostasis and perpetuating disease. Therapies that decrease intestinal epithelial apoptosis may, therefore, ameliorate inflammatory bowel disease, but treatments that specifically target apoptotic pathways are lacking. Proteinase-activated receptor-2 (PAR2), a G protein-coupled receptor activated by trypsin-like serine proteinases, is expressed on intestinal epithelial cells and stimulates mitogenic pathways upon activation. We sought to determine whether PAR2 activation and signaling could rescue colonic epithelial (HT-29) cells from apoptosis induced by proapoptotic cytokines that are increased during inflammatory bowel disease. The PAR2 agonists 2-furoyl-LIGRLO (2f-LI), SLIGKV and trypsin all significantly reduced cleavage of caspase-3, -8, and -9, poly(ADP-ribose) polymerase, and the externalization of phosphatidylserine after treatment of cells with IFN-γ and TNF-α. Knockdown of PAR2 with siRNA eliminated the anti-apoptotic effect of 2f-LI and increased the sensitivity of HT-29 cells to cytokine-induced apoptosis. Concurrent inhibition of both MEK1/2 and PI3K was necessary to inhibit PAR2-induced survival. 2f-LI was found to increase phosphorylation and inactivation of pro-apoptotic BAD at Ser(112) and Ser(136) by MEK1/2 and PI3K-dependent signaling, respectively. PAR2 activation also increased the expression of anti-apoptotic MCL-1. Simultaneous knockdown of both BAD and MCL-1 had minimal effects on PAR2-induced survival, whereas single knockdown had no effect. We conclude that PAR2 activation reduces cytokine-induced epithelial apoptosis via concurrent stimulation of MEK1/2 and PI3K but little involvement of MCL-1 and BAD. Our findings represent a novel mechanism whereby serine proteinases facilitate epithelial cell survival and may be important in the context of colonic healing.

TU-100 (Daikenchuto) and ginger ameliorate anti-CD3 antibody induced T cell-mediated murine enteritis: microbe-independent effects involving Akt and NF-κB suppression.

The Japanese traditional medicine daikenchuto (TU-100) has anti-inflammatory activities, but the mechanisms remain incompletely understood. TU-100 includes ginger, ginseng, and Japanese pepper, each component possessing bioactive properties. The effects of TU-100 and individual components were investigated in a model of intestinal T lymphocyte activation using anti-CD3 antibody. To determine contribution of intestinal bacteria, specific pathogen free (SPF) and germ free (GF) mice were used. TU-100 or its components were delivered by diet or by gavage. Anti-CD3 antibody increased jejunal accumulation of fluid, increased TNFα, and induced intestinal epithelial apoptosis in both SPF and GF mice, which was blocked by either TU-100 or ginger, but not by ginseng or Japanese pepper. TU-100 and ginger also blocked anti-CD3-stimulated Akt and NF-κB activation. A co-culture system of colonic Caco2BBE and Jurkat-1 cells was used to examine T-lymphocyte/epithelial cells interactions. Jurkat-1 cells were stimulated with anti-CD3 to produce TNFα that activates epithelial cell NF-κB. TU-100 and ginger blocked anti-CD3 antibody activation of Akt in Jurkat cells, decreasing their TNFα production. Additionally, TU-100 and ginger alone blocked direct TNFα stimulation of Caco2BBE cells and decreased activation of caspase-3 and polyADP ribose. The present studies demonstrate a new anti-inflammatory action of TU-100 that is microbe-independent and due to its ginger component.

Sa2004 Effects of AVX-470m, an Oral Polyclonal Anti-TNF Antibody, on Survival and Biomarkers in a Murine Model of Gastrointestinal Acute Radiation Syndrome

Intestinal mucositis is a common side-effect in patients undergoing radiotherapy and poses a severe dose-limiting factor in treatment1. Identification of compounds that prevent radiationinduced mucositis and improve intestinal restitution may offer new therapeutic strategies for maintaining intestinalmucosal integrity in patients undergoing radiotherapy. Pathogenesis of mucositis involves production of pro-inflammatory cytokines and reactive oxygen metabolites2,3 and increased apoptosis in intestinal epithelium, leading to loss of intestinal structure and function4. STW 5 is an herbal multi-component preparation having potent anti-oxidant and anti-inflammatory properties in intestinal inflammatory disorders,5,6, making it an attractive candidate to study its potential usefulness in mucositis. Intestinal mucositis was induced experimentally in rats by exposing them to whole body irradiation from a Caesium 137 source at a radiation dose of 6 Gray. Three days later, rats developed intestinal mucositis, judged by histological examination of the small intestine and measurement of associated parameters in intestinal homogenates and serum. STW5 was given orally for 5 days to rats in graded doses of 2 10 ml/kg before exposing them to radiation and continued for 3 days after exposure. Animals were sacrificed 24h later. Pretreatment with STW 5 led to dosedependent reduction in histological changes. A dose of 5 ml/kg was chosen to study in detail the protective effects of the preparation on relevant associated parameters. Histologically, irradiation caused shortening and fusion of villi, activation of mucus secreting glands, inflammatory cell infiltration of lamina propria and mucosal atrophy. Inflammation markers: tumor necrosis factor and myeloperoxidase activity in homogenates were also raised. The changes were associated with a rise in thiobarbituric acid reactive substances, reduction in glutathione and in protein content of intestinal homogenates. Biomarkers of intestinal tissue injury: plasma diamineoxidase was raised while citrulline7 was significantly reduced. Apoptosis was evidenced by a rise in cytosolic calcium, depletion of mitochondrial cytochrome c and B-cell lymphoma 2. Most histological changes and associated derangement in the parameters tested were largely prevented by STW 5. The findings pave the way to a new therapeutic approach in management of radiation induced mucositis. 1Costa G and Donaldson SS (1979) N Engl J Med 300, 1471-1474, 2MacNaughton WK (2000) Aliment Pharmacol Ther. 14, 523-528, 3Linard C et al (2003) Am J Physiol Gastrointest Liver Physiol. 285, 556-565, 4Wong TC et al (2006) J Pain Symptom Manage. 32, 27-37, 5Khayyal MT et al (2006) Phytomedicine. 13 Suppl 5, 56-66, 6Schempp H et al (2006) Phytomedicine. 2006;13 Suppl 5, 36-44 7Lutgens L and Lambin P (2007) World J. Gastroenterol., 13, 3033 3042

Regulation of intestinal epithelial apoptosis by protease‐activated receptor 2 involves EGFR‐transactivation and is associated with Mcl‐1 upregualtion (151.1)

Colonic biopsies from inflamed colon have higher levels of epithelial apoptosis than biopsies from healthy individuals. Therapies that inhibit intestinal epithelial apoptosis may decrease the inflammation observed in inflammatory bowel disease. We previously showed that signaling by PAR2, a GPCR activated by serine proteases, delayed IFN‐γ &amp;amp; TNF‐α‐induced apoptosis in colonic epithelial (HT‐29) cells. The observed decrease in the cleavage of caspase‐8, 9, 3 and PARP following the activation of PAR2 by 2‐furoyl‐LIGRLO (2fLI) was shown to be dependent on the activities of PI3K and ERK1/2. In this study we found that selective inhibition of EGFR with AG1478 blocked the phosphorylation of PI3K and ERK1/2. Pre‐treatment with AG1478 prior to the addition of IFN‐γ &amp;amp; TNF‐α also blocked 2fLI‐induced intestinal epithelial survival, as determined by PARP cleavage. Modulation of BCL‐2 family members was then investigated due to their known function in regulating mitochondrial permeability and apoptosis. Protein expression of pro‐apoptotic BAK, BAX and BIM were not altered by either cytokine or 2fLI treatment. Likewise, no change in expression of anti‐apoptotic BCL‐XL was observed. Treatment with 2fLI stimulated phosphorylation and inactivation of the apoptotic BH3‐only protein, BAD. Knockdown of BAD with siRNA, however, did not alter 2fLI‐stimulated intestinal survival. Finally, 2fLI stimulated a rapid increase in anti‐apoptotic MCL‐1 expression. PAR2 signaling reduces epithelial apoptosis in response to inflammatory cytokines via EGFR transactivation and possibly by regulation of MCL‐1 expression. Our findings may represent a mechanism whereby proteases facilitate epithelial cell survival and colonic healing after inflammation.Grant Funding Source: Supported by the Crohn’s and Colitis Foundation of Canada

Stabilization of XIAP mRNA through the RNA binding protein HuR regulated by cellular polyamines

The X chromosome-linked inhibitor of apoptosis protein (XIAP) is the most potent intrinsic caspase inhibitor and plays an important role in the maintenance of intestinal epithelial integrity. The RNA binding protein, HuR, regulates the stability and translation of many target transcripts. Here, we report that HuR associated with both the 3'-untranslated region and coding sequence of the mRNA encoding XIAP, stabilized the XIAP transcript and elevated its expression in intestinal epithelial cells. Ectopic HuR overexpression or elevated cytoplasmic levels of endogenous HuR by decreasing cellular polyamines increased [HuR/XIAP mRNA] complexes, in turn promoting XIAP mRNA stability and increasing XIAP protein abundance. Conversely, HuR silencing in normal and polyamine-deficient cells rendered the XIAP mRNA unstable, thus reducing the steady state levels of XIAP. Inhibition of XIAP expression by XIAP silencing or by HuR silencing reversed the resistance of polyamine-deficient cells to apoptosis. Our findings demonstrate that HuR regulates XIAP expression by stabilizing its mRNA and implicates HuR-mediated XIAP in the control of intestinal epithelial apoptosis.

Intestinal epithelial apoptosis initiates gut mucosal injury during extracorporeal membrane oxygenation in the newborn piglet

Neonates and young infants exposed to extracorporeal circulation during extracorporeal membrane oxygenation (ECMO) and cardiopulmonary bypass are at risk of developing a systemic inflammatory response syndrome with multi-organ dysfunction. We used a piglet model of ECMO to investigate the hypothesis that epithelial apoptosis is an early event that precedes villous damage during ECMO-related bowel injury. Healthy 3-week-old piglets were subjected to ECMO for up to 8 h. Epithelial apoptosis was measured in histopathological analysis, nuclear imaging, and terminal deoxynucleotidyl transferase dUTP nick end labeling. Plasma intestinal fatty acid-binding protein (I-FABP) levels were measured by enzyme immunoassay. Intestinal mast cells were isolated by fluorescence-assisted cell sorting. Cleaved caspase-8, caspase-9, phospho-p38 MAPK, and fas ligand expression were investigated by immunohistochemistry, western blots, and reverse transcriptase-quantitative PCR. Piglet ECMO was associated with increased gut epithelial apoptosis. Extensive apoptotic changes were noted on villus tips and in scattered crypt cells after 2 h of ECMO. After 8 h, the villi were denuded and apoptotic changes were evident in a majority of crypt cells. Increased circulating I-FABP levels, a marker of gut epithelial injury, showed that epithelial injury occurred during ECMO. We detected increased cleaved caspase-8, but not cleaved caspase-9, in epithelial cells indicating that the extrinsic apoptotic pathway was active. ECMO was associated with increased fas ligand expression in intestinal mast cells, which was induced through activation of the p38 mitogen-activated protein kinase. We conclude that epithelial apoptosis is an early event that initiates gut mucosal injury in a piglet model of ECMO.

Is RXRα Crucially Involved in Intestinal Inflammation?

Retinoid X receptors (RXRs) are nuclear receptors that control several physiological processes [1]. Three closely related RXR isotypes are encoded on chromosome 9: RXRα (activated by 9-cis-retinoic acid), RXRβ, and RXRγ. RXRs form heterodimers, either spontaneously or in a ligand-dependent manner, with other nuclear receptors including retinoic acid receptors (RAR), peroxisome proliferator-activated receptors (PPAR), estrogen receptors (ER), vitamin D receptors (VDR), thyroid hormone receptors (TR), farnesoid X receptors (FXR), liver X receptors (LXR), constitutive androstane receptors (CAR), and pregnane X receptors (PXR) [2, 3]. Dimerization of RXR with other nuclear receptors transcriptionally controls a variety of cellular processes by binding to specific DNA response elements within regions of target gene promoters [4]. Nuclear receptor binding to RXR can be functionally divided into “permissive” and “non-permissive” groups. Heterodimers that form when binding to a “permissive” receptor, for example PPAR, can be activated by agonists to either heterodimeric partner. In contrast, “non-permissive” RXR nuclear receptor heterodimers, for example VDR and THR, can be activated only by agonists binding to the partner of RXR [5]. 1,25(OH)2D3 binds to its receptor, VDR, which requires RXR to bind to vitamin D response elements for transcriptional regulation. Several pieces of evidence are indicative of loss of vitamin D-VDR signals in the pathogenesis of inflammatory bowel disease. First, VDR polymorphisms are associated with IBD risk in some populations [6]. In a murine model, VDR/IL-10 double knockout mice develop more severe colitis and mortality is higher [7]. In IL-10(−/−) mice, vitamin D deficiency exacerbates the symptoms of colitis, whereas vitamin D supplementation ameliorates symptoms and reduces inflammation [8]. Another mechanism whereby vitamin D protects against IBD development is by maintaining or restoring the integrity of the intestinal mucosal barrier by preventing intestinal epithelial apoptosis [9]. In this regard, 1,25(OH)2D3 has several effects on the immune system, including suppression of T cell activation and modulation of antigen presentation [10]. Furthermore, VDR signaling is also involved in regulation of the innate immune response in macrophages [11]. In this issue of Digestive Diseases and Sciences, Knack-stedt et al. report the potential involvement of RXRα in mucosal inflammation [12]. Although the study did not demonstrate differences in colitis between RXRα heterozygotes and wild-type mice in a murine model of early colitis, there were significant increases in Snail1 and Snail2 in the RXR+/− mice. Snail 1 and Snail 2 are transcription factors linked to the epithelial–mesenchymal transition that mediate cell migration during inflammation [13]. These transcription factors are up-regulated in ulcerative colitis and can silence VDR expression, which is often down-regulated in active mucosal inflammation and colon cancer [9, 14]. Through this mechanism, RXRα is likely to be important in modulating inflammation and carcinogenesis, because, for example, RXR and VDR polymorphisms alter the risk of sporadic colon cancer [15, 16]. It is difficult to understand, however, why mucosal inflammation was no different despite up-regulation of Snail 1 and Snail 2 in this study. Although the RXRα heterozygous model used in this work did not result in significant down-regulation of RXRα protein expression, a model with conditional RXRα homozygous deletion might reveal a greater effect on mucosal inflammation. Furthermore, extending the time of investigation from a model of early colitis to one of chronic colitis might reveal different effects of RXRα hemizygous deletion in colonic inflammation. Nevertheless, further mechanistic evaluation of the effect of RXRα in mucosal inflammation is needed, because it is unknown whether the RXR–VDR heterodimer, as suggested by the authors, or alternative nuclear receptors or other pathways regulate the inflammatory cascade or mediate carcinogenesis, as supported by the observation that RXR and PPARγ agonists reduced inflammation in a TNBS-induced colitis murine model [17]. There is increasing evidence that low serum vitamin D concentrations are a risk factor for the development of inflammatory bowel disease [18]. Although evidence for humans is lacking, in murine models, treatment with vitamin D does improve symptoms of colitis [8]. Potentially, therapy targeting RXRα, in conjunction with vitamin D, could also be used to treat bowel inflammation. Because VDR forms a non-permissive heterodimer with RXR, RXR agonists are unlikely to be beneficial in regulating transcription at VDREs. Furthermore, because RXRα seems to be excess in most cells, therapeutic intervention to increase RXR expression is unlikely to enhance RXR–VDR signaling [19]. For this reason, the greatest potential for therapeutic intervention involving this pathway is likely to involve increasing the interaction of RXR with its heterodimeric partners.

Gut Microbiota Protects against Gastrointestinal Tumorigenesis Caused by Epithelial Injury

Inflammation is a critical player in the development of both colitis-associated and sporadic colon cancers. Several studies suggest that the microbiota contribute to inflammation and tumorigenesis; however, studies to understand the role of the microbiota in colon tumor development in germ-free (GF) mice are limited. We therefore studied the effects of the microbiota on the development of inflammation and tumors in GF and conventionally raised specific pathogen-free (SPF) mice treated with azoxymethane (AOM) and dextran sulfate sodium (DSS). We discovered that GF mice developed significantly more and larger tumors compared with that in SPF mice after AOM and DSS treatment despite the lack of early acute inflammation in response to chemically induced injury by DSS. Although the extent of intestinal epithelial damage and apoptosis was not significantly different in GF and SPF mice, there was a delay in intestinal epithelial repair to DSS-induced injury in GF mice resulting in a late onset of proinflammatory and protumorigenic responses and increased epithelial proliferation and microadenoma formation. Recolonization of GF mice with commensal bacteria or administration of lipopolysaccharide reduced tumorigenesis. Thus, although commensal bacteria are capable of driving chronic inflammation and tumorigenesis, the gut microbiota also have important roles in limiting chemically induced injury and proliferative responses that lead to tumor development.

Inhibition of Intestinal Epithelial Apoptosis Improves Survival in a Murine Model of Radiation Combined Injury

World conditions place large populations at risk from ionizing radiation (IR) from detonation of dirty bombs or nuclear devices. In a subgroup of patients, ionizing radiation exposure would be followed by a secondary infection. The effects of radiation combined injury are potentially more lethal than either insult in isolation. The purpose of this study was to determine mechanisms of mortality and possible therapeutic targets in radiation combined injury. Mice were exposed to IR with 2.5 Gray (Gy) followed four days later by intratracheal methicillin-resistant Staphylococcus aureus (MRSA). While either IR or MRSA alone yielded 100% survival, animals with radiation combined injury had 53% survival (p = 0.01). Compared to IR or MRSA alone, mice with radiation combined injury had increased gut apoptosis, local and systemic bacterial burden, decreased splenic CD4 T cells, CD8 T cells, B cells, NK cells, and dendritic cells, and increased BAL and systemic IL-6 and G-CSF. In contrast, radiation combined injury did not alter lymphocyte apoptosis, pulmonary injury, or intestinal proliferation compared to IR or MRSA alone. In light of the synergistic increase in gut apoptosis following radiation combined injury, transgenic mice that overexpress Bcl-2 in their intestine and wild type mice were subjected to IR followed by MRSA. Bcl-2 mice had decreased gut apoptosis and improved survival compared to WT mice (92% vs. 42%; p<0.01). These data demonstrate that radiation combined injury results in significantly higher mortality than could be predicted based upon either IR or MRSA infection alone, and that preventing gut apoptosis may be a potential therapeutic target.

Inhibition of IKKβ in Enterocytes Exacerbates Sepsis-Induced Intestinal Injury and Worsens Mortality

Nuclear factor-κB is a critical regulator of cell-survival genes and the host inflammatory response. The purpose of this study was to investigate the role of enterocyte-specific NF-kB in sepsis through selective ablation of IkB kinase. Prospective, randomized controlled study. Animal laboratories in university medical centers. Mice lacking functional NF-kB in their intestinal epithelium (Vil-Cre/Ikkβ) and wild-type mice were subjected to sham laparotomy or cecal ligation and puncture. Animals were killed at 24 hours or followed 7 days for survival. Septic wild-type mice had decreased villus length compared with sham mice, whereas villus atrophy was further exacerbated in septic Vil-Cre/Ikkβ mice. Sepsis induced an increase in intestinal epithelial apoptosis compared with sham mice, which was further exacerbated in Vil-Cre/Ikkβ mice. Sepsis induced intestinal hyperpermeability in wild-type mice compared with sham mice, which was further exacerbated in septic Vil-Cre/Ikkβ mice. This was associated with increased intestinal expression of claudin-2 in septic wild-type mice, which was further increased in septic Vil-Cre/Ikkβ mice. Both, pro-inflammatory and anti-inflammatory cytokines were increased in serum following cecal ligation and puncture, and interleukin 10 and monocyte chemoattractant protein-1 levels were higher in septic Vil-Cre/Ikkβ mice than in septic wild-type mice. All septic mice were bacteremic, but no differences in bacterial load were identified between wild-type and Vil-Cre/Ikkβ mice. To determine the functional significance of these results, animals were followed for survival. Septic wild-type mice had lower mortality than septic Vil-Cre/Ikkβ mice (47% vs 80%, p<0.05). Antitumor necrosis factor administration decreased intestinal apoptosis, permeability, and mortality in wild-type septic mice, and a similar improvement in intestinal integrity and survival were seen when antitumor necrosis factor was given to Vil-Cre/Ikkβ mice. Enterocyte-specific NF-kB has a beneficial role in sepsis by partially preventing sepsis-induced increases in apoptosis and permeability, which are associated with worsening mortality.

Chronic Alcohol Ingestion Increases Mortality and Organ Injury in a Murine Model of Septic Peritonitis

BackgroundPatients admitted to the intensive care unit with alcohol use disorders have increased morbidity and mortality. The purpose of this study was to determine how chronic alcohol ingestion alters the host response to sepsis in mice.MethodsMice were randomized to receive either alcohol or water for 12 weeks and then subjected to cecal ligation and puncture. Mice were sacrificed 24 hours post-operatively or followed seven days for survival.ResultsSeptic alcohol-fed mice had a significantly higher mortality than septic water-fed mice (74% vs. 41%, p = 0.01). This was associated with worsened gut integrity in alcohol-fed mice with elevated intestinal epithelial apoptosis, decreased crypt proliferation and shortened villus length. Further, alcohol-fed mice had higher intestinal permeability with decreased ZO-1 and occludin protein expression in the intestinal tight junction. The frequency of splenic and bone marrow CD4+ T cells was similar between groups; however, splenic CD4+ T cells in septic alcohol-fed mice had a marked increase in both TNF and IFN-γ production following ex vivo stimulation. Neither the frequency nor function of CD8+ T cells differed between alcohol-fed and water-fed septic mice. NK cells were decreased in both the spleen and bone marrow of alcohol-fed septic mice. Pulmonary myeloperoxidase levels and BAL levels of G-CSF and TFG-β were higher in alcohol-fed mice. Pancreatic metabolomics demonstrated increased acetate, adenosine, xanthine, acetoacetate, 3-hydroxybutyrate and betaine in alcohol-fed mice and decreased cytidine, uracil, fumarate, creatine phosphate, creatine, and choline. Serum and peritoneal cytokines were generally similar between alcohol-fed and water-fed mice, and there were no differences in bacteremia, lung wet to dry weight, or pulmonary, liver or splenic histology.ConclusionsWhen subjected to the same septic insult, mice with chronic alcohol ingestion have increased mortality. Alterations in intestinal integrity, the host immune response, and pancreatic metabolomics may help explain this differential response.

Tu1745 Intestinal Epithelial Apoptosis and Immune Activation During HIV Infection

Tu1745 Intestinal Epithelial Apoptosis and Immune Activation During HIV Infection