Intestinal Epithelial Cell Homeostasis Research Articles

Body Mass Index and Other Anthropomorphic Variables in Relation to Risk of Colorectal Carcinoma Subtypes Classified by Tumor Differentiation Status

Metabolic energy dysregulation may affect intestinal epithelial cell homeostasis, and thus promote colorectal carcinogenesis. Yet, little is known about the association between obesity and enterocyte differentiation during tumor development.We hypothesized that an association between anthropomorphic measurements and colorectal carcinoma incidence might differ according to tumor histopathologic differentiation status. Anthropomorphic variables evaluated included Body Mass Index (BMI), Hip circumference (HC), Waist circumference (WC), and Waist‐Hip Ratio (WHR).Using data from the Nurses' Health Study and Health Professionals' Follow‐up Study, and performing duplication‐method Cox proportional hazard models, we prospectively examined an association between BMI, mean waist/hip circumference (WC/HC), and waist‐hip ratio (WHR) with the incidence of colorectal carcinoma subtypes classified by differentiation status. 120,813 participants were followed for 26 or 32 years and 1528 colorectal cancer cases with available tumor pathological data were documented.The association between BMI and colorectal cancer risk significantly differed depending on the presence or absence of poorly differentiated tumor foci (Pheterogeneity = 0.006). Higher BMI was associated with higher risk of colorectal carcinoma without poorly differentiated foci (≥30.0 vs. 18.5–22.4 kg/m2: multivariable‐adjusted hazard ratio, 1.87; 95% confidence interval, 1.49–2.34; Ptrend <0.001), but not with risk of carcinoma with poorly‐differentiated foci (Ptrend = 0.56). While in men, high WC, HC, and WHR were associated with overall incidence of colorectal carcinoma (Ptrend ≤ 0.01), there was no statistically significant association between these variables and tumor differentiation status (Pheterogeneity >0.27). Furthermore, the association between anthropomorphic variables and colorectal carcinoma did not differ significantly by overall tumor grade, mucinous differentiation, or the presence of a signet‐ring‐cell component (PHeterogeneity > 0.03, with the adjusted α of 0.01). The differential association of BMI with tumor differentiation appeared to be consistent in strata of tumor microsatellite instability and fatty acid synthase (FASN) expression, although statistical power was limited.Whilst BMI was associated with increased risk of the colorectal cancer subtype lacking poorly differentiated foci, there was no independent association between tumor differentiation and measures of central adiposity. Our findings suggest that the carcinogenic effect of generalized excess body adiposity may be stronger for tumors that retain greater degrees of intestinal differentiation.Support or Funding InformationThis work was supported by U.S. National Institutes of Health (NIH) grants (P01 CA87969 to M.J. Stampfer; UM1 CA186107 to M.J. Stampfer; P01 CA55075 to W.C. Willett; UM1 CA167552 to W.C. Willett; U01 CA167552 to W.C. Willett and L.A. Mucci; P50 CA127003 to C.S.F.; R01 CA118553 to C.S.F.; R01 CA169141 to C.S.F.; R01 CA137178 to A.T.C.; K24 DK098311 to A.T.C.; R35 CA197735 to S.O.; R01 CA151993 to S.O.; K07 CA190673 to R.N.; and K07 CA188126 to X.Z.); by Nodal Award from the Dana‐Farber Harvard Cancer Center (to S.O.); and by grants from the Project P Fund, The Friends of the Dana‐Farber Cancer Institute, Bennett Family Fund, and the Entertainment Industry Foundation through National Colorectal Cancer Research Alliance, and by Stand Up to Cancer Colorectal Cancer Dream Team Translational Research Grant (to C.S.F. and M.Gi.). J.B. was supported by the Australia Awards‐Endeavour Scholarships and Fellowships Program. K.K. was supported by grants from Overseas Research Fellowship (JP2017–775) from Japan Society for the Promotion of Science. T.H. was supported by a fellowship grant from the Mitsukoshi Health and Welfare Foundation.T.H. was supported by a fellowship grant from the Mitsukoshi Health and Welfare Foundation. L.L. was supported by a scholarship grant from Chinese Scholarship Council and a fellowship grant from Huazhong University of Science and Technology. The content is solely the responsibility of the authors and does not necessarily represent the official views of NIH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

P2Y6 Receptors Regulate CXCL10 Expression and Secretion in Mouse Intestinal Epithelial Cells.

In this study, we investigated the role of extracellular nucleotides in chemokine (KC, MIP-2, MCP-1, and CXCL10) expression and secretion by murine primary intestinal epithelial cells (IECs) with a focus on P2Y6 receptors. qRT-PCR experiments showed that P2Y6 was the dominant nucleotide receptor expressed in mouse IEC. In addition, the P2Y6 ligand UDP induced expression and secretion of CXCL10. For the other studies, we took advantage of mice deficient in P2Y6 (P2ry6-/-). Similar expression levels of P2Y1, P2Y2, P2X2, P2X4, and A2A were detected in P2ry6-/- and WT IEC. Agonists of TLR3 (poly(I:C)), TLR4 (LPS), P2Y1, and P2Y2 increased the expression and secretion of CXCL10 more prominently in P2ry6-/- IEC than in WT IEC. CXCL10 expression and secretion induced by poly(I:C) in both P2ry6-/- and WT IEC were inhibited by general P2 antagonists (suramin and Reactive-Blue-2), by apyrase, and by specific antagonists of P2Y1, P2Y2, P2Y6 (only in WT), and P2X4. Neither adenosine nor an A2A antagonist had an effect on CXCL10 expression and secretion. Macrophage chemotaxis was induced by the supernatant of poly(I:C)-treated IEC which was consistent with the level of CXCL10 secreted. Finally, the non-nucleotide agonist FGF2 induced MMP9 mRNA expression also at a higher level in P2ry6-/- IEC than in WT IEC. In conclusion, extracellular nucleotides regulate CXCL10 expression and secretion by IEC. In the absence of P2Y6, these effects are modulated by other P2 receptors also present on IEC. These data suggest that the presence of P2Y6 regulates chemokine secretion and may also regulate IEC homeostasis.

Regulation of Intestinal Epithelial Cells Properties and Functions by Amino Acids.

Intestinal epithelial cells (IECs) line the surface of intestinal epithelium, where they play important roles in the digestion of food, absorption of nutrients, and protection of the human body from microbial infections, and others. Dysfunction of IECs can cause diseases. The development, maintenance, and functions of IECs are strongly influenced by external nutrition, such as amino acids. Amino acids play important roles in regulating the properties and functions of IECs. In this article, we briefly reviewed the current understanding of the roles of amino acids in the regulation of IECs' properties and functions in physiological state, including in IECs homeostasis (differentiation, proliferation, and renewal), in intestinal epithelial barrier structure and functions, and in immune responses. We also summarized some important findings on the effects of amino acids supplementation (e.g., glutamine and arginine) in restoring IECs' and intestine functions in some diseased states. These findings will further our understanding of the important roles of amino acids in the homeostasis of IECs and could potentially help identify novel targets and reagents for the therapeutic interventions of diseases associated with dysfunctional IECs.

Chromofungin Ameliorates the Progression of Colitis by Regulating Alternatively Activated Macrophages.

Ulcerative colitis (UC) is characterized by a functional dysregulation of alternatively activated macrophage (AAM) and intestinal epithelial cells (IECs) homeostasis. Chromogranin-A (CHGA) secreted by neuroendocrine cells is implicated in intestinal inflammation and immune dysregulation. CHGA undergoes proteolytic processing to generate CHGA-derived peptides. Chromofungin (CHR: CHGA47–66) is a short CHGA-derived peptide encoded by CHGA Exon-IV and is involved in innate immune regulation, but the basis is poorly investigated. We investigated the expression of CHR in colonic tissue of patients with active UC and assessed the effects of the CHR in dextran sulfate sodium (DSS) colitis in mice and on macrophages and human colonic epithelial cells. We found that mRNA expression of CHR correlated positively with mRNA levels of AAM markers and gene expression of tight junction (TJ) proteins and negatively with mRNA levels of interleukin (IL)-8, IL-18, and collagen in patients with active UC. Moreover, AAM markers correlated positively with gene expression of TJ proteins and negatively with IL-8, IL-18, and collagen gene expression. Experimentally, intracolonic administration of CHR protected against DSS-induced colitis by priming macrophages into AAM, reducing colonic collagen deposition, and maintaining IECs homeostasis. This effect was associated with a significant increase of AAM markers, reduction of colonic IL-18 release and conservation of gene expression of TJ proteins. In vitro, CHR enhanced AAM polarization and increased the production of anti-inflammatory mediators. CHR-treated AAM conditioned medium increased Caco-2 cell migration, viability, proliferation, and mRNA levels of TJ proteins, and decreased oxidative stress-induced apoptosis and proinflammatory cytokines release. Direct CHR treatments had the same effect. In conclusion, CHR treatment reduces the severity of colitis and the inflammatory process via enhancing AAM functions and maintaining IECs homeostasis. CHR is involved in the pathogenesis of inflammation in experimental colitis. These findings provide insight into the mechanisms of colonic inflammation and could lead to new therapeutic strategies for UC.

Reelin protects from colon pathology by maintaining the intestinal barrier integrity and repressing tumorigenic genes

We previously reported that reelin, an extracellular matrix protein first known for its key role in neuronal migration, reduces the susceptibility to dextran sulphate sodium (DSS)-colitis. The aim of the current study was to determine whether reelin protects from colorectal cancer and how reelin defends from colon pathology. In the colon of wild-type and of mice lacking reelin (reeler mice) we have analysed the: i) epithelium cell renewal processes, ii) morphology, iii) Sox9, Cdx2, Smad5, Cyclin D1, IL-6 and IFNγ mRNA abundance in DSS-treated and untreated mice, and iv) development of azoxymethane/DSS-induced colorectal cancer, using histological and real time-PCR methodologies. The reeler mutation increases colitis-associated tumorigenesis, with increased tumours number and size. It also impairs the intestinal barrier because it reduces cell proliferation, migration, differentiation and apoptosis; decreases the number and maturation of goblet cells, and expands the intercellular space of the desmosomes. The intestinal barrier impairment might explain the increased susceptibility to colon pathology exhibited by the reeler mice and is at least mediated by the down-regulation of Sox9 and Cdx2. In response to DSS-colitis, the reeler colon increases the mRNA abundance of IL-6, Smad5 and Cyclin D1 and decreases that of IFNγ, conditions that might result in the increased colitis-associated tumorigenesis found in the reeler mice. In conclusion, the results highlight a role for reelin in maintaining intestinal epithelial cell homeostasis and providing resistance against colon pathology.

HVEM expression by intestinal epithelial cells modulates the microbiome and epithelial immunity

Abstract The herpes virus entry mediator (HVEM), a member of the tumor necrosis factor receptor superfamily (TNFRSF), is highly expressed by intestinal epithelial cells (IEC). Previously, we reported that HVEM expression by epithelial cells was required for innate immune defense from acute infections in the lung and the intestine (Nature 488:222,2012). Here, we demonstrate a novel, constitutive role of HVEM expression by IEC in microbial homeostasis and epithelial immunity. Mice with an epithelial-specific deletion of the gene encoding HVEM (HvemΔIEC) had significantly increased segmented filamentous bacteria (SFB). This caused an increase in Th17 cells and IL-22+ILC3s in the intestine lamina propria. HvemΔIEC mice also exhibited increased goblet cell hypertrophy and hyperplasia, crypt hyperplasia, villous atrophy as they aged, and they showed a severe defect in fucosylation of cell surface proteins, even at younger ages. Treatment with the antibiotic vancomycin eliminated SFB and decreased Th17 cells and ILC3, but did not reverse the defect in fucosylation. Our findings suggest that epithelial cell expression of HVEM is important at steady state both for shaping the intestinal microbiota and for the homeostasis of IEC in the intestine.

Mend Your Fences: The Epithelial Barrier and its Relationship With Mucosal Immunity in Inflammatory Bowel Disease.

The intestinal epithelium can be easily disrupted during gut inflammation as seen in inflammatory bowel disease (IBD), such as ulcerative colitis or Crohn’s disease. For a long time, research into the pathophysiology of IBD has been focused on immune cell–mediated mechanisms. Recent evidence, however, suggests that the intestinal epithelium might play a major role in the development and perpetuation of IBD. It is now clear that IBD can be triggered by disturbances in epithelial barrier integrity via dysfunctions in intestinal epithelial cell–intrinsic molecular circuits that control the homeostasis, renewal, and repair of intestinal epithelial cells. The intestinal epithelium in the healthy individual represents a semi-permeable physical barrier shielding the interior of the body from invasions of pathogens on the one hand and allowing selective passage of nutrients on the other hand. However, the intestinal epithelium must be considered much more than a simple physical barrier. Instead, the epithelium is a highly dynamic tissue that responds to a plenitude of signals including the intestinal microbiota and signals from the immune system. This epithelial response to these signals regulates barrier function, the composition of the microbiota, and mucosal immune homeostasis within the lamina propria. The epithelium can thus be regarded as a translator between the microbiota and the immune system and aberrant signal transduction between the epithelium and adjacent immune cells might promote immune dysregulation in IBD. This review summarizes the important cellular and molecular barrier components of the intestinal epithelium and emphasizes the mechanisms leading to barrier dysfunction during intestinal inflammation.

Huangqin-tang ameliorates dextran sodium sulphate-induced colitis by regulating intestinal epithelial cell homeostasis, inflammation and immune response.

Huangqin-tang (HQT) is a traditional Chinese medicine (TCM) formula widely used for the treatment of inflammatory bowel disease in China. However, the molecular mechanisms by which HQT protects the colon are unclear. We studied the protective effects of HQT and the underlying mechanisms in an experimental mouse model and in vitro. In vivo, dextran sodium sulphate (DSS)-induced acute and chronic colitis were significantly ameliorated by HQT as gauged by phenotypic, histopathologic and inflammatory manifestations of the disease. Mechanistically, DSS-induced nuclear factor-κB (NF-κB) signalling was inhibited by HQT. Moreover, HQT-treated mice demonstrated significant changes in cell apoptosis, expression of apoptosis-associated genes such as caspase-3, bax, bcl-2, and intestinal permeability. HQT also increased occluding and zonula occludens-1 (ZO-1), inhibited cell proliferation (Ki67), and increased regulatory T cells numbers, protein expression of Foxp3 and IL-10 in the colonic tissue. In vitro, HQT down-regulated production of pro-inflammatory cytokines and supressed the NF-κB signalling pathway in lipopolysaccharides-induced RAW 264.7 macrophages. Our study suggests that HQT plays a critical role in regulating intestinal epithelial cell homeostasis, inflammation and immune response in colitis and offers novel therapeutic options in the management of inflammatory bowel disease.

The miR-196b miRNA inhibits the GATA6 intestinal transcription factor and is upregulated in colon cancer patients.

ObjectiveTo explore the possible misexpression of the microRNA miR-196b in colorectal cancer (CRC) and its role in controlling the expression of GATA6, a putative target gene crucial to intestinal cell homeostasis and tumorigenesis.DesignThe expression of miR-196b was analysed by qRT-PCR in surgical resection samples from a cohort of sporadic colon cancer patients. Manipulations of miR-196b expression were performed to demonstrate its inhibition of GATA6 protein levels.ResultsWe found that miR-196b is significantly upregulated in pre-treatment surgical resection samples from a cohort of sporadic colon cancer patients. The upregulation of miR-196b correlates with less severe clinicopathological characteristics, such as early tumor stage and absence of lymph node metastases. We show that in CRC cells, miR-196b targets the mRNA of GATA6, a transcription factor involved in the homeostasis and differentiation of intestinal epithelial cells, and a positive regulator of the Wnt/β-catenin pathway. We moreover found that the increase of miR-196b correlates with a reduced GATA6 protein expression in colon cancer patients.ConclusionOur results establish miR-196b as a post-transcriptional inhibitor of GATA6 in CRC cells, implicating miR-196b function in gene regulatory pathways crucial to intestinal cell homeostasis and tumorigenesis. Our results furthermore suggest a role of miR-196b expression in CRC, as an antagonist of GATA6 function in tumor cells, thus providing the basis for a potential targeting strategy for the treatment of CRC.

MiR-30 Family Controls Proliferation and Differentiation of Intestinal Epithelial Cell Models by Directing a Broad Gene Expression Program That Includes SOX9 and the Ubiquitin Ligase Pathway

Proliferation and differentiation of intestinal epithelial cells (IECs) occur in part through precise regulation of key transcription factors, such as SOX9. MicroRNAs (miRNAs) have emerged as prominent fine-tuners of transcription factor expression and activity. We hypothesized that miRNAs, in part through the regulation of SOX9, may mediate IEC homeostasis. Bioinformatic analyses of the SOX9 3′-UTR revealed highly conserved target sites for nine different miRNAs. Of these, only the miR-30 family members were both robustly and variably expressed across functionally distinct cell types of the murine jejunal epithelium. Inhibition of miR-30 using complementary locked nucleic acids (LNA30bcd) in both human IECs and human colorectal adenocarcinoma-derived Caco-2 cells resulted in significant up-regulation of SOX9 mRNA but, interestingly, significant down-regulation of SOX9 protein. To gain mechanistic insight into this non-intuitive finding, we performed RNA sequencing on LNA30bcd-treated human IECs and found 2440 significantly increased genes and 2651 significantly decreased genes across three time points. The up-regulated genes are highly enriched for both predicted miR-30 targets, as well as genes in the ubiquitin-proteasome pathway. Chemical suppression of the proteasome rescued the effect of LNA30bcd on SOX9 protein levels, indicating that the regulation of SOX9 protein by miR-30 is largely indirect through the proteasome pathway. Inhibition of the miR-30 family led to significantly reduced IEC proliferation and a dramatic increase in markers of enterocyte differentiation. This in-depth analysis of a complex miRNA regulatory program in intestinal epithelial cell models provides novel evidence that the miR-30 family likely plays an important role in IEC homeostasis.

426 Epithelial Cell Adhesion Molecule (EpCAM) regulates epithelial homeostasis via Rho-associated protein kinase (ROCK)

EpCAM (Epithelial Cell Adhesion Molecule) is a cell surface protein that is expressed by some normal epithelial cells and many carcinomas. EpCAM is not a classical adhesion molecule and it has been reported to modulate epithelial cell physiology via several mechanisms. Some patients with a congenital diarrheal syndrome termed congenital tufting enteropathy (CTE) have EpCAM mutations, and EpCAM knockout mice have a CTE-like phenotype, documenting the importance of EpCAM in intestinal epithelial cell (IEC) homeostasis. For this reason, we are studying primary IEC to understand EpCAM function in greater detail. IEC organoids can be readily propagated from mouse intestine and they recapitulate many facets of normal IEC development and physiology. Treatment of crypts from ROSA CreER EpCAMfl/fl mice with tamoxifen for the first 3 days of culture leads to loss of EpCAM expression and organoids that exhibit smaller sizes, lower forming efficiencies, and markedly reduced passaging efficiencies as compared with control organoids. Although cellular polarity is maintained and intercellular junctions are not strikingly abnormal in EpCAM-deficient organoids, organoid architecture is disorganized and epithelial integrity is lost. Effects of EpCAM loss are selectively attenuated in EpCAM-deficient organoids treated with Rho-associated protein kinase (ROCK) inhibitors and a myosin II inhibitor. Consistent with this, phosphorylation of targets that are downstream of ROCK, including myosin light chain II (Ser19 and Thr18/Ser19), is enhanced in EpCAM-deficient organoids. Fluorescence in situ hybridization experiments suggest that stem cell numbers are decreased in EpCAM-deficient organoids but Paneth cells, an important source of Wnt in vitro, are also abnormal. Similar results have been obtained after acute EpCAM deletion in intestines of ROSA CreER EpCAMfl/fl mice treated with tamoxifen for 3 days. We are in the process of determining if our results reflect regulation of ROCK by EpCAM in stem cells, Paneth cells or perhaps both cells, and elucidating the mechanism by which this occurs.

Flavaglines Ameliorate Experimental Colitis and Protect Against Intestinal Epithelial Cell Apoptosis and Mitochondrial Dysfunction.

Flavaglines are a family of natural compounds shown to have anti-inflammatory and cytoprotective effects in neurons and cardiomyocytes. Flavaglines target prohibitins as ligands, which are scaffold proteins that regulate mitochondrial function, cell survival, and transcription. This study tested the therapeutic potential of flavaglines to promote intestinal epithelial cell homeostasis and to protect against a model of experimental colitis in which inflammation is driven by epithelial ulceration. Survival and homeostasis of Caco2-BBE and IEC-6 intestinal epithelial cell lines were measured during treatment with the flavaglines FL3 or FL37 alone and in combination with the proinflammatory cytokines tumor necrosis factor (TNF) α and interferon γ. Wild-type mice were intraperitoneally injected with 0.1 mg/kg FL3 or vehicle once daily for 4 days during dextran sodium sulfate-induced colitis to test the in vivo anti-inflammatory effect of FL3. FL3 and FL37 increased basal Caco2-BBE and IEC-6 cell viability, decreased apoptosis, and decreased epithelial monolayer permeability. FL3 and FL37 inhibited TNFα- and interferon γ-induced nuclear factor kappa B and Cox2 expression, apoptosis, and increased permeability in Caco2-BBE cells. FL3 and FL37 protected against TNFα-induced mitochondrial superoxide generation by preserving respiratory chain complex I activity and prohibitin expression. p38-MAPK activation was essential for the protective effect of FL3 and FL37 on barrier permeability and mitochondrial-derived reactive oxygen species production during TNFα treatment. Mice administered FL3 during dextran sodium sulfate colitis exhibited increased colonic prohibitin expression and p38-MAPK activation, preserved barrier function, and less inflammation. These results suggest that flavaglines exhibit therapeutic potential against colitis and preserve intestinal epithelial cell survival, mitochondrial function, and barrier integrity.

Apoptotic pathways as a therapeutic target for colorectal cancer treatment.

Colorectal cancer is the second leading cause of death from cancer among adults. The disease begins as a benign adenomatous polyp, which develops into an advanced adenoma with high-grade dysplasia and then progresses to an invasive cancer. Appropriate apoptotic signaling is fundamentally important to preserve a healthy balance between cell death and cell survival and in maintaining genome integrity. Evasion of apoptotic pathway has been established as a prominent hallmark of several cancers. During colorectal cancer development, the balance between the rates of cell growth and apoptosis that maintains intestinal epithelial cell homeostasis gets progressively disturbed. Evidences are increasingly available to support the hypothesis that failure of apoptosis may be an important factor in the evolution of colorectal cancer and its poor response to chemotherapy and radiation. The other reason for targeting apoptotic pathway in the treatment of cancer is based on the observation that this process is deregulated in cancer cells but not in normal cells. As a result, colorectal cancer therapies designed to stimulate apoptosis in target cells would play a critical role in controlling its development and progression. A better understanding of the apoptotic signaling pathways, and the mechanisms by which cancer cells evade apoptotic death might lead to effective therapeutic strategies to inhibit cancer cell proliferation with minimal toxicity and high responses to chemotherapy. In this review, we analyzed the current understanding and future promises of apoptotic pathways as a therapeutic target in colorectal cancer treatment.

Intestinal Epithelial Cell Tyrosine Kinase 2 Transduces IL-22 Signals To Protect from Acute Colitis.

In the intestinal tract, IL-22 activates STAT3 to promote intestinal epithelial cell (IEC) homeostasis and tissue healing. The mechanism has remained obscure, but we demonstrate that IL-22 acts via tyrosine kinase 2 (Tyk2), a member of the Jak family. Using a mouse model for colitis, we show that Tyk2 deficiency is associated with an altered composition of the gut microbiota and exacerbates inflammatory bowel disease. Colitic Tyk2(-/-) mice have less p-STAT3 in colon tissue and their IECs proliferate less efficiently. Tyk2-deficient primary IECs show reduced p-STAT3 in response to IL-22 stimulation, and expression of IL-22-STAT3 target genes is reduced in IECs from healthy and colitic Tyk2(-/-) mice. Experiments with conditional Tyk2(-/-) mice reveal that IEC-specific depletion of Tyk2 aggravates colitis. Disease symptoms can be alleviated by administering high doses of rIL-22-Fc, indicating that Tyk2 deficiency can be rescued via the IL-22 receptor complex. The pivotal function of Tyk2 in IL-22-dependent colitis was confirmed in Citrobacter rodentium-induced disease. Thus, Tyk2 protects against acute colitis in part by amplifying inflammation-induced epithelial IL-22 signaling to STAT3.

Nicotine-induced cellular stresses and autophagy in human cancer colon cells: A supportive effect on cell homeostasis via up-regulation of Cox-2 and PGE2 production

Nicotine, one of the active components in cigarette smoke, has been described to contribute to the protective effect of smoking in ulcerative colitis (UC) patients. Furthermore, the nicotinic acetylcholine receptor α7 subunit (α7nAChR) expressed on immune cells, is an essential regulator of inflammation. As intestinal epithelial cells also express α7nAChR, we investigated how nicotine could participate in the homeostasis of intestinal epithelial cells. First, using the human adenocarcinoma cell line HT-29, we revealed that nicotine, which triggers an influx of extracellular Ca2+ following α7nAChR stimulation, induces mitochondrial reactive oxygen species (ROS) production associated with a disruption of the mitochondrial membrane potential and endoplasmic reticulum stress. This results in caspase-3 activation, which in turn induces apoptosis. Additionally, we have shown that nicotine induces a PI3-K dependent up-regulation of cyclooxygenase-2 (Cox-2) expression and prostaglandin E2 (PGE2) production. In this context, we suggest that this key mediator participates in the cytoprotective effects of nicotine against apoptosis by stimulating autophagy in colon cancer cells. Our results provide new insight into one potential mechanism by which nicotine could protect from UC and suggest an anti-inflammatory role for the cholinergic pathway at the epithelial cell level.

Bioactive molecules in the <10kDa fraction of the Lactobacillus helveticus R0389 and Lactobacillus rhamnosus R0011 secretomes down-regulate IL-8 production by HT-29 human intestinal epithelial cells induced by pro-inflammatory stimuli (MUC9P.752)

Abstract The human gut microbiome has recently been associated with maintaining intestinal epithelial cell (IEC) homeostasis at the gastrointestinal interface by modulating host mucosal immune responses through varied mechanisms. The aim of this study was to compare the immunomodulatory activities of the L. helveticus R0389 and L. rhamosus R0011 secretomes on IEC. The human IEC line HT-29 was activated with a range of innate immune stimulants, to allow delineation of differential effects of the secretomes on Interleukin-8 (IL-8) production induced by varied stimuli. HT-29 IEC were co-incubated with either L. helveticus R0389 or L. rhamnosus R0011 secretomes and one of the following pro-inflammatory stimuli: interleukin 1-β (IL-1β), tumor necrosis factor-α (TNF-α); the toll-like receptor (TLR) agonists poly (I:C) (TLR3) or lipopolysaccharide (LPS) (TLR4), and alterations in IL-8 production were measured via enzyme-linked immunosorbant assay (ELISA). The L. helveticus R0389 and L. rhamnosus R0011 secretomes were also subjected to size fractionation to determine the size of the bioactive constituent of the secretome. Down-regulation of IL-8 was observed in HT-29 IEC co-incubated with the <10kDa fraction of the L. helveticus R0389 secretome or the L. rhamnosus R0011 secretome, for all of the innate immune stimulants tested. These results suggest that both Lactobacilli may secrete a low molecular weight bioactive molecule that modulates host immune activity by IEC.

Demonstrating a Role for Nuclear APC in Intestinal Cellular Differentiation

Colon cancer is the second leading cause of cancer‐related mortality in the United States, resulting in over 50,000 deaths annually. Approximately 80% of colon cancers begin with a mutation in the gene coding for the tumor suppressor, Adenomatous Polyposis Coli, APC. Facilitated by two nuclear localization signals (NLS) and multiple nuclear export signals, APC protein can shuttle between the cytoplasm and the nucleus of cells. To understand the functions of nuclear APC, our lab generated a knock‐in mouse model with mutations introduced that inactivate both Apc NLS, thus dramatically decreasing their amount of nuclear Apc. We have previously shown increased proliferation and Wnt target gene expression in intestinal epithelial cells from ApcmNLS/mNLS mice, suggesting a role for nuclear APC in inhibition of proliferation and Wnt signaling. Here we examine the role of nuclear Apc in intestinal epithelial differentiation and stem cell homeostasis. Paraffin‐embedded tissue from the intestines of Apc+/+ and ApcmNLS/mNLS mice was sectioned and stained for markers of enterocytes (alkaline phosphatase), goblet cells (alcian blue), enteroendocrine cells (chromogranin A) and Paneth cells (lysozyme C). Quantification of positive cells indicated that ApcmNLS/mNLS mice had fewer differentiated enterocytes, goblet cells, and Paneth cells, yet more enteroendocrine cells than their wild‐type littermates. Together, these data support a role for nuclear Apc in promoting enterocyte, goblet cell, and Paneth cell differentiation while inhibiting enteroendocrine cell differentiation. No significant change was observed in quiescent stem cell (DCAMKL‐1) populations. Information gathered from this analysis will contribute to our understanding of the mechanism of intestinal differentiation and tumorigenesis.

Notch receptor regulation of intestinal stem cell homeostasis and crypt regeneration

The Notch signaling pathway regulates intestinal epithelial cell homeostasis, including stem cell maintenance, progenitor cell proliferation and differentiation. Notch1 and Notch2 receptors are expressed in the epithelium, but individual contributions to these functions are unclear. We used genetic deletion to define receptor roles on stem cell function, cell proliferation/differentiation, and repair after injury. Loss of Notch1 induced a transient secretory cell hyperplasia that spontaneously resolved over time. In contrast, deletion of Notch2 had no secretory cell effect. Compound deletions of Notch1 and Notch2 resulted in a more severe secretory cell hyperplasia than deletion of Notch1 alone. Furthermore, only double deletion of Notch1 and Notch2 decreased cell proliferation, suggesting a low threshold for maintenance of proliferation compared to differentiation. Stem cells were affected by deletion of Notch1, with reduced expression of Olfm4 and fewer LGR5+ stem cells. Deletion of Notch2 had no apparent affect on stem cell homeostasis. However, we observed impaired crypt regeneration after radiation in both Notch1- and Notch2-deleted intestine, suggesting that higher Notch activity is required post-injury. These findings suggest that Notch1 is the primary receptor regulating intestinal stem cell function and that Notch1 and Notch2 together regulate epithelial cell proliferation, cell fate determination, and post-injury regeneration.

Intestinal epithelial suppressor of cytokine signaling 3 enhances microbial-induced inflammatory tumor necrosis factor-α, contributing to epithelial barrier dysfunction.

A single layer of intestinal epithelial cells (IEC) lines the entire gastrointestinal tract and provides the first line of defense and barrier against an abundance of microbial stimuli. IEC homeostasis and repair are mediated through microbe-sensing Toll-like receptor (TLR)-induced inflammatory pathways. Increasing evidence supports a role of suppressor of cytokine signaling 3 (SOCS3) as a modulator of IEC turnover, balancing controlled repair and replenishment with excessive IEC proliferation predisposing to dysplasia and cancer. Our data indicate that SOCS3 can limit microbial-induced IEC repair, potentially through promoting tumor necrosis factor-α (TNF-α) and limiting TNFR2 expression. Activation of TLR5 signaling pathways, compared with other TLR, increases TNF-α mRNA in a dose-dependent manner and SOCS3 enhances TLR5-induced TNF-α. We also show that flagellin promotes transcription of TNFR2 and that SOCS3 limits this expression, presenting a mechanism of SOCS3 action. Our data also support the role of microbial ligands in epithelial wound healing and suggest that a functional consequence of increased TNF-α is reduced wound healing. These results provide further evidence to support the regulatory role of epithelial SOCS3 in intestinal health and suggest that the increased expression of SOCS3 observed in IBD may serve to perpetuate “inflammation” by promoting TNF-α production and limiting epithelial repair in response to commensal microflora.

Lymphotoxin beta receptor signaling limits mucosal damage through driving IL-23 production by epithelial cells.

The immune mechanisms regulating epithelial cell repair after injury remain poorly defined. We demonstrate here that lymphotoxin beta receptor (LTβR) signaling in intestinal epithelial cells promotes self-repair after mucosal damage. Using a conditional gene-targeted approach, we demonstrate that LTβR signaling in intestinal epithelial cells is essential for epithelial IL-23 production and protection against epithelial injury. We further show that epithelial-derived IL-23 promotes mucosal wound healing by inducing the IL-22-mediated proliferation and survival of epithelial cells and mucus production. Additionally, we identified CD4−CCR6+T-bet−RORγt+ lymphoid tissue inducer cells as the main producers of protective IL-22 after epithelial damage. Thus, our results reveal a novel role for LTβR signaling in epithelial cells in the regulation of intestinal epithelial cell homeostasis to limit mucosal damage.