Translation initiation factor eIF2Bε promotes Wnt-mediated clonogenicity and global translation in intestinal epithelial cells.

Modulation of global mRNA translation, which is essential for intestinal stem cell function, is controlled by Wnt signaling. Loss of tumor supressor APC in stem cells drives adenoma formation through hyperactivion of Wnt signaling and dysregulated translational control. It is unclear whether factors that coordinate global translation in the intestinal epithelium are needed for APC-driven malignant transformation. Here we identified nucleotide exchange factor eIF2Bε as a translation initiation factor involved in Wnt-mediated intestinal epithelial stemness. Using eIF2BεArg191His mice with a homozygous point mutation that leads to dysfunction in the enzymatic activity, we demonstrate that eIF2Bε is involved in small intestinal crypt formation, stemness marker expression, and secreted Paneth cell-derived granule formation. Wnt hyperactivation in ex vivo eIF2BεArg191His organoids, using a GSK3ß inhibitor to mimic Apc driven transformation, shows that eIF2Bε is essential for Wnt-mediated clonogenicity and associated increase of the global translational capacity. Finally, we observe high eIF2Bε expression in human colonic adenoma tissues, exposing eIF2Bε as a potential target of CRC stem cells with aberrant Wnt signaling.

Risk factors for proximal disease extension and colectomy in left-sided ulcerative colitis.

OBJECTIVE: The primary objective of this study was to assess proximal disease extension of ulcerative colitis (UC) over time, with disease behaviour pattern and risk factors for proximal disease extension and colectomy as secondary aims. METHODS: All cumulative incident cases diagnosed with UC at the Academic Medical Center between January 1990 and December 2009 were studied. The cumulative risk of colectomy was calculated by Kaplan-Meier analysis. The Cox proportional hazards regression was used to identify risk factors associated with proximal disease extension and colectomy. RESULTS: In total, 506 UC patients were included with a median age of 33 years (IQR 23-41) at diagnosis. Ninety-five (18.8%) patients underwent colectomy during follow-up. Median follow-up was 10 years (IQR 5-15). Initial disease extent was evaluable in 416 patients, of whom 142 (34.1%) had proctitis, 155 (37.3%) left-sided colitis and 119 (28.6%) pancolitis. Proximal disease extension was observed in 120 (28.8%) patients during follow-up (51 proctitis to left-sided colitis, 39 proctitis to extensive colitis and 30 left-sided to extensive colitis). Disease behaviour was evaluable in 378 patients, of whom 244 (64.6%) had less than one relapse per year. Younger age at diagnosis (HR 0.98, 95% CI 0.96-0.99) and continuous active disease (HR 2.18, 95% CI 1.27-3.73) were independent risk factors for proximal disease extension. The cumulative risk of colectomy did not change over time between patients diagnosed before and after the year 2000 (p = 0.341). Continuous active disease (HR 7.05, 95% CI 4.23-11.77), systemic steroids (HR 3.25, 95% CI 1.37-7.71) and cyclosporine treatment (HR 2.80, 95% CI 1.66-4.72) were independent risk factors for colectomy, whereas proctitis at diagnosis (HR 0.43, 95% CI 0.22-0.86) carried a lower risk. CONCLUSION: In one-third of UC patients, left-sided disease at diagnosis will extend proximally during 10 years of follow-up. Proximal disease extension was not a risk factor for colectomy, but the risk of colectomy is rather determined by continuous disease activity, and use of systemic steroids and cyclosporine.

Profoundly Expanded T-cell Clones in the Inflamed and Uninflamed Intestine of Patients With Crohn's Disease.

BACKGROUND AND AIM: T cells are key players in the chronic intestinal inflammation that characterises Crohn's disease. Here we aim to map the intestinal T-cell receptor [TCR] repertoire in patients with Crohn's disease, using next-generation sequencing technology to examine the clonality of the T-cell compartment in relation to mucosal inflammation and response to therapy. METHODS: Biopsies were taken from endoscopically inflamed and uninflamed ileum and colon of 19 patients with Crohn's disease. From this cohort, additional biopsies were taken after 8 weeks of remission induction therapy from eight responders and eight non-responders. Control biopsies from 11 patients without inflammatory bowel disease [IBD] were included. The TCRß repertoire was analysed by next-generation sequencing of biopsy RNA. RESULTS: Both in Crohn's disease patients and in non-IBD controls, a broad intestinal T-cell repertoire was found, with a considerable part consisting of expanded clones. Clones in Crohn's disease were more expanded [p = 0.008], with the largest clones representing up to as much as 58% of the total repertoire. There was a substantial overlap of the repertoire between inflamed and uninflamed tissue and between ileum and colon. Following therapy, responders showed larger changes in the T-cell repertoire than non-responders, although a considerable part of the repertoire remained unchanged in both groups. CONCLUSIONS: The intestinal T-cell repertoire distribution in Crohn's disease is different from that in the normal gut, containing profoundly expanded T-cell clones that take up a large part of the repertoire. The T-cell repertoire is fairly stable regardless of endoscopic mucosal inflammation or response to therapy.

Suppression of anti-drug antibodies to infliximab or adalimumab with the addition of an immunomodulator in patients with inflammatory bowel disease.

BACKGROUND: Loss of response to anti-tumour necrosis factor (TNF) therapy in patients with inflammatory bowel disease (IBD) is often caused by anti-drug antibody formation with neutralisation of drug effect. Addition of an immunomodulator has been suggested to reduce immunogenicity, leading to regained response. AIM: To investigate whether addition of an immunomodulator to anti-TNF monotherapy could lead to anti-drug antibody suppression and regained clinical response in IBD patients. METHODS: We retrospectively collected measurements of infliximab or adalimumab serum concentrations and anti-drug antibodies to identify anti-drug positive patients with loss response who were given an immunomodulator. RESULTS: Anti-drug antibodies against infliximab and adalimumab were detected in 98/376 (26%) and in 61/226 (27%) patients, respectively. Immunomodulators were given to 17/159 patients. Clinical response was recaptured in 6/10 patients receiving a thiopurine and in all (7/7) patients receiving methotrexate. In 7/8 patients on infliximab, serum concentrations increased (median 2.84 µg/mL; IQR: 1.19-4.98) and in 6/9 patients on adalimumab (median 3.10 µg/mL; IQR: 1.45-4.45). This was accompanied by a decrease in anti-drug antibodies to undetectable levels (median 11 months for both anti-TNF agents). In 23 patients, no immunomodulator was added but anti-TNF interval was shortened (17/23) or dosage was increased (6/23), which resulted in a clinical response in 10/17 and 2/6 patients, respectively. CONCLUSION: In 77% of IBD patients with loss of response due to immunogenicity, addition of immunomodulator resulted in undetectable anti-drug antibody levels, increased serum drug concentrations and regained clinical response. This strategy should be considered in this patient population before switching to other agents.

The ATG16L1 risk allele associated with Crohn's disease results in a Rac1-dependent defect in dendritic cell migration that is corrected by thiopurines.

Thiopurines are commonly used drugs in the therapy of Crohn's disease, but unfortunately only show a 30% response rate. The biological basis for the thiopurine response is unclear, thus hampering patient selection prior to treatment. A genetic risk factor associated specifically with Crohn's disease is a variant in ATG16L1 that reduces autophagy. We have previously shown that autophagy is involved in dendritic cell (DC)-T-cell interactions and cytoskeletal regulation. Here we further investigated the role of autophagy in DC cytoskeletal modulation and cellular trafficking. Autophagy-deficient DC displayed loss of filopodia, altered podosome distribution, and increased membrane ruffling, all consistent with increased cellular adhesion. Consequently, autophagy-deficient DC showed reduced migration. The cytoskeletal aberrations were mediated through hyperactivation of Rac1, a known thiopurine target. Indeed thiopurines restored the migratory defects in autophagy-deficient DC. Clinically, the ATG16L1 risk variant associated with increased response to thiopurine treatment in patients with Crohn's disease but not ulcerative colitis. These results suggest that the association between ATG16L1 and Crohn's disease is mediated at least in part through Rac1 hyperactivation and subsequent defective DC migration. As this phenotype can be corrected using thiopurines, ATG16L1 genotyping may be useful in the identification of patients that will benefit most from thiopurine treatment.

Induction of endoplasmic reticulum stress by deletion of Grp78 depletes Apc mutant intestinal epithelial stem cells.

Intestinal epithelial stem cells are highly sensitive to differentiation induced by endoplasmic reticulum (ER) stress. Colorectal cancer develops from mutated intestinal epithelial stem cells. The most frequent initiating mutation occurs in Apc, which results in hyperactivated Wnt signalling. This causes hyperproliferation and reduced sensitivity to chemotherapy, but whether these mutated stem cells are sensitive to ER stress induced differentiation remains unknown. Here we examined this by generating mice in which both Apc and ER stress repressor chaperone Grp78 can be conditionally deleted from the intestinal epithelium. For molecular studies, we used intestinal organoids derived from these mice. Homozygous loss of Apc alone resulted in crypt elongation, activation of the Wnt signature and accumulation of intestinal epithelial stem cells, as expected. This phenotype was however completely rescued on activation of ER stress by additional deletion of Grp78. In these Apc-Grp78 double mutant animals, stem cells were rapidly lost and repopulation occurred by non-mutant cells that had escaped recombination, suggesting that Apc-Grp78 double mutant stem cells had lost self-renewal capacity. Although in Apc-Grp78 double mutant mice the Wnt signature was lost, these intestines exhibited ubiquitous epithelial presence of nuclear ß-catenin. This suggests that ER stress interferes with Wnt signalling downstream of nuclear ß-catenin. In conclusion, our findings indicate that ER stress signalling results in loss of Apc mutated intestinal epithelial stem cells by interference with the Wnt signature. In contrast to many known inhibitors of Wnt signalling, ER stress acts downstream of ß-catenin. Therefore, ER stress poses a promising target in colorectal cancers, which develop as a result of Wnt activating mutations.

The Link between the Appendix and Ulcerative Colitis: Clinical Relevance and Potential Immunological Mechanisms.

The human appendix has long been considered as a vestigial organ, an organ that has lost its function during evolution. In recent years, however, reports have emerged that link the appendix to numerous immunological functions in humans. Evidence has been presented for an important role of the appendix in maintaining intestinal health. This theory suggests that the appendix may be a reservoir or 'safe house' from which the commensal gut flora can rapidly be reestablished if it is eradicated from the colon. However, the appendix may also have a role in the development of inflammatory bowel disease (IBD). Several large epidemiological cohort studies have demonstrated the preventive effect of appendectomy on the development of ulcerative colitis, a finding that has been confirmed in murine colitis models. In addition, current studies are examining the possible therapeutic effect of an appendectomy to modulate disease course in patients with ulcerative colitis. This literature review assesses the current knowledge about the clinical and immunological aspects of the vermiform appendix in IBD and suggests that the idea of the appendix as a vestigial remnant should be discarded.

Oral tolerance is inefficient in neonatal mice due to a physiological vitamin A deficiency.

Increased risk of allergy during early life indicates deficient immune regulation in this period of life. To date, the cause for inefficient neonatal immune regulation has never been elucidated. We aimed to define the ontogeny of oral tolerance and to identify necessary conditions specific for this stage of life. Ovalbumin (OVA) was administered orally to mice through breast milk and efficiency of systemic tolerance to OVA was assessed in adulthood using a model of allergic airway inflammation. Oral tolerance induction was fully efficient starting third week of life. Inefficiency in neonates was a consequence of abnormal antigen transfer across the gut barrier and retinaldehyde dehydrogenase expression by mesenteric lymph node CD103(+) neonatal dendritic cells, resulting in inefficient T-cell activation. Neonates' serum retinol levels were three times lower than in adult mice, and vitamin A supplementation was sufficient to rescue neonatal defects and allow tolerance induction from birth. The establishment of oral tolerance required the differentiation of Th1 lymphocytes in both vitamin A-supplemented neonates and 3-week-old unsupplemented mice. This knowledge should guide the design of interventions for allergy prevention that are adapted to the neonatal stage of life such as vitamin A supplementation.

The BMP pathway either enhances or inhibits the Wnt pathway depending on the SMAD4 and p53 status in CRC.

BACKGROUND: Constitutive Wnt activation is essential for colorectal cancer (CRC) initiation but also underlies the cancer stem cell phenotype, metastasis and chemosensitivity. Importantly Wnt activity is still modulated as evidenced by higher Wnt activity at the invasive front of clonal tumours termed the ß-catenin paradox. SMAD4 and p53 mutation status and the bone morphogenetic protein (BMP) pathway are known to affect Wnt activity. The combination of SMAD4 loss, p53 mutations and BMP signalling may integrate to influence Wnt signalling and explain the ß-catenin paradox. METHODS: We analysed the expression patterns of SMAD4, p53 and ß-catenin at the invasive front of CRCs using immunohistochemistry. We activated BMP signalling in CRC cells in vitro and measured BMP/Wnt activity using luciferase reporters. MTT assays were performed to study the effect of BMP signalling on CRC chemosensitivity. RESULTS: Eighty-four percent of CRCs with high nuclear ß-catenin staining are SMAD4 negative and/or p53 aberrant. BMP signalling inhibits Wnt signalling in CRC only when p53 and SMAD4 are unaffected. In the absence of SMAD4, BMP signalling activates Wnt signalling. When p53 is lost or mutated, BMP signalling no longer influences Wnt signalling. The cytotoxic effects of 5-FU are influenced in a similar manner. CONCLUSIONS: The BMP signalling pathway differentially modulates Wnt signalling dependent on the SMAD4 and p53 status. The use of BMPs in cancer therapy, as has been proposed by previous studies, should be targeted to individual cancers based on the mutational status of p53 and SMAD4.

Endogenous c-Myc is essential for p53-induced apoptosis in response to DNA damage in vivo.

Recent studies have suggested that C-MYC may be an excellent therapeutic cancer target and a number of new agents targeting C-MYC are in preclinical development. Given most therapeutic regimes would combine C-MYC inhibition with genotoxic damage, it is important to assess the importance of C-MYC function for DNA damage signalling in vivo. In this study, we have conditionally deleted the c-Myc gene in the adult murine intestine and investigated the apoptotic response of intestinal enterocytes to DNA damage. Remarkably, c-Myc deletion completely abrogated the immediate wave of apoptosis following both ionizing irradiation and cisplatin treatment, recapitulating the phenotype of p53 deficiency in the intestine. Consistent with this, c-Myc-deficient intestinal enterocytes did not upregulate p53. Mechanistically, this was linked to an upregulation of the E3 Ubiquitin ligase Mdm2, which targets p53 for degradation in c-Myc-deficient intestinal enterocytes. Further, low level overexpression of c-Myc, which does not impact on basal levels of apoptosis, elicited sustained apoptosis in response to DNA damage, suggesting c-Myc activity acts as a crucial cell survival rheostat following DNA damage. We also identify the importance of MYC during DNA damage-induced apoptosis in several other tissues, including the thymus and spleen, using systemic deletion of c-Myc throughout the adult mouse. Together, we have elucidated for the first time in vivo an essential role for endogenous c-Myc in signalling DNA damage-induced apoptosis through the control of the p53 tumour suppressor protein.

Rage signalling promotes intestinal tumourigenesis.

Development of colon cancer is a multistep process that is regulated by intrinsic and extrinsic cellular signals. Extrinsic factors include molecular patterns that are derived from either pathogens (PAMPs) or cellular damage (DAMPs). These molecules can promote tumourigenesis by activation of the innate immune system, but the individual contribution of ligands and their receptors remains elusive. The receptor for advanced glycation end products (Rage) is a pattern recognition receptor that binds multiple ligands derived from a damaged cell environment such as Hmgb1 and S100 protein. Here we show that Rage signalling has a critical role in sporadic development of intestinal adenomas, as Apc(Min/+) Rage(-/-) mice are protected against tumourigenesis.

Role of mast cells in colorectal cancer development, the jury is still out.

The link between inflammation and colorectal cancer development is becoming increasingly clear. It had long been recognized that patients with inflammatory bowel disease are at an increased risk of colon cancer. Evidence from experimental animals now also implicates the innate immune system in the development of sporadically occurring intestinal adenomas, the precursors to colorectal cancer. Here we discuss the interaction between the immune system and the adenoma to carcinoma sequence with a special emphasis on the role of mast cells which may play a key role in adenoma development. This article is part of a Special Issue entitled: Mast cells in inflammation.

Key elements of the BMP/SMAD pathway co-localize with CDX2 in intestinal metaplasia and regulate CDX2 expression in human gastric cell lines.

Helicobacter pylori infection induces intestinal metaplasia of the stomach, a preneoplastic lesion associated with an increased risk for gastric cancer development. Intestinal metaplasia is induced by the intestine-specific transcription factor CDX2 but the mechanisms responsible for this ectopic expression have never been described. We hypothesized that the BMP/SMAD pathway has a role in CDX2 regulation, in this context, for the following reasons: (1) the BMP pathway is crucial for normal intestinal differentiation and (2) there is an influx of BMP2 and BMP4-producing cells to the stomach upon Helicobacter pylori infection. We evaluated the expression of key elements of the BMP pathway in human stomach specimens with IM. Growth factor treatments, with BMP2 and BMP4, were performed in cultured cells and a knock-down experiment of SMAD4 was done using RNAi. We showed overexpression in IM of BMP2/4, BMPR1A, and SMAD4 in 56% of IM foci, and pSMAD1/5/8 in 100% of IM foci as compared to adjacent mucosa. In vitro, treatment of AGS cells with BMP2 and BMP4 increased endogenous CDX2 expression as well as the intestinal differentiation markers MUC2 and LI-cadherin. On the other hand, SMAD4 knock-down led to decreased endogenous CDX2, MUC2, and LI-cadherin in AGS. Treatment of the SMAD4 knock-down cells had no influence on CDX2 expression as opposed to wild-type cells. A 9.3 kb CDX2 promoter could be transactivated by SMAD4 and SMAD1 in a cell-dependent manner. In conclusion, we identified for the first time that the BMP pathway is active in intestinal metaplasia and that BMP2 and BMP4 regulate CDX2 expression and promote intestinal differentiation through the canonical signal transducers.

Stem cells as potential novel therapeutic strategy for inflammatory bowel disease.

Hematopoietic stem cell transplantation and mesenchymal stromal cell therapy are currently under investigation as novel therapies for inflammatory bowel diseases. Hematopoietic stem cells (HSC) are thought to repopulate the immune system and reset the immunological response to luminal antigens. Mesenchymal stromal cells (MSC) are cells that have the capacity to differentiate into wide variety of distinct cell lineages and suppress immune responses in vitro and in vivo. Recent results from animal models and early human experience in graft-versus-host disease but also Crohn's Disease suggest that ex vivo expanded MSCs may have clinically useful immunomodulatory effects.

Effect of aspirin on the Wnt/beta-catenin pathway is mediated via protein phosphatase 2A.

Nonsteroidal anti-inflammatory drugs show chemopreventive efficacy in colon cancer, but the mechanism behind this remains unclear. Elucidating this mechanism is seen as vital to the development of new chemopreventive agents. We studied the effects of aspirin on the oncogenic Wnt/beta-catenin pathway activity in colorectal cancer cell lines and observed that aspirin dose-dependently decreased the activity of this pathway, as judged by TCF-driven luciferase activity, reduced Wnt target gene expression and increased phosphorylation of beta-catenin by immunoblotting. Furthermore, the ubiquitination and cytoplasmic levels of beta-catenin were assessed by immunoblotting, and also the localization of beta-catenin was shown by green fluorescent protein-tagged beta-catenin and time-lapse fluorescent imaging. Importantly, aspirin treatment caused increased phosphorylation of protein phosphatase 2A (PP2A), an event associated with inhibition of PP2A enzymatic activity, which was confirmed by a reduction in enzymatic PP2A activity. Moreover, this inhibition of PP2A enzymatic activity was essential for the effects of aspirin on the Wnt/beta-catenin pathway as shown by transient transfection with PP2A constructs. The findings in this article provide a molecular explanation for the efficacy of aspirin in chemoprevention of colorectal cancer and shows biochemical evidence that PP2A is an important regulator of Wnt/beta-catenin pathway activity in these cells.

Altered bone morphogenetic protein signalling in the Helicobacter pylori-infected stomach.

Morphogens regulate epithelial cell fate decisions in the adult gastrointestinal tract. The authors hypothesized that influx of inflammatory cells into the lamina propria may disturb the normal expression gradients of morphogens (morphogenetic landscape) in gastrointestinal epithelia. Changes in the activity of the bone morphogenetic protein (BMP) pathway in normal and Helicobacter pylori-infected gastric mucosa were therefore examined. It is shown that BMP receptors, the activated (phosphorylated) form of the intracellular BMP signal transduction protein SMAD1, and BMP target ID2 all localize to gastric epithelial cells that are at the end of the axis of epithelial renewal in normal mucosa. Colonization of human gastric mucosa with H. pylori was associated with an increase in BMP2 expression due to influx of inflammatory cells that produce BMP2. Furthermore, whereas no BMP4 was detected in the normal antrum, focal infiltrates of BMP4-expressing cells were found in the H. pylori-infected stomach. This influx of BMP-expressing cells was associated with an increase in epithelial BMP signalling. Interestingly, a shift in activity of the BMP pathway was observed towards the precursor cell compartment (isthmus) of the gastric units. Thus, H. pylori infection results in an influx of inflammatory cells that disturb the normal activity gradient of a morphogenetic pathway with an established role in epithelial cell fate regulation. The data suggest that morphological changes in epithelial histology may result from alterations in the morphogenetic landscape secondary to changes in the cellular composition of the lamina propria.

Development and differentiation of the intestinal epithelium.

The gastrointestinal tract develops from a simple tube to a complex organ with patterns of differentiation along four axes of asymmetry. The organ is composed of all three germ layers signaling to each other during development to form the adult structure. The gut epithelium is a constitutively developing tissue, constantly differentiating from a stem cell in a progenitor pool throughout the life of the organism. Signals from the adjacent mesoderm and between epithelial cells are required for normal orderly development/differentiation, homeostasis, and apoptosis. Embryonically important patterning factors are used during adult stages for these processes. Such critical pathways as the hedgehog, bone morphogenetic protein, Notch, Sox, and Wnt systems are used both in embryologic and adult times of gut development. We focus on and review the roles of these factors in gut epithelial cell development and differentiation.