Goo Lee

Phosphoinositide 3-Kinase Signaling Mediates β-Catenin Activation in Intestinal Epithelial Stem and Progenitor Cells in Colitis

BACKGROUND & AIMS Mechanisms responsible for crypt architectural distortion in chronic ulcerative colitis (CUC) are not well understood. Data indicate that serine/threonine protein kinase Akt (Akt) signaling cooperates with Wingless (Wnt) to activate beta-catenin in intestinal stem and progenitor cells through phosphorylation at Ser552 (P-beta-catenin(552)). We investigated whether phosphoinositide 3-kinase (PI3K) is required for Akt-mediated activation of beta-catenin during intestinal inflammation. METHODS The class IA subunit of PI3K was conditionally deleted from intestinal epithelial cells in mice named I-pik3r1KO. Acute inflammation was induced in mice and intestines were analyzed by biochemical and histologic methods. The effects of chemically blocking PI3K in colitic interleukin-10(-/-) mice were examined. Biopsy samples from patients were examined. RESULTS Compared with wild-type, I-pik3r1KO mice had reduced T-cell-mediated Akt and beta-catenin signaling in intestinal stem and progenitor cells and limited crypt epithelial proliferation. Biochemical analyses indicated that PI3K-Akt signaling increased nuclear total beta-catenin and P-beta-catenin(552) levels and reduced N-terminal beta-catenin phosphorylation, which is associated with degradation. PI3K inhibition in interleukin-10(-/-) mice impaired colitis-induced epithelial Akt and beta-catenin activation, reduced progenitor cell expansion, and prevented dysplasia. Human samples had increased numbers of progenitor cells with P-beta-catenin(552) throughout expanded crypts and increased messenger RNA expression of beta-catenin target genes in CUC, colitis-associated cancer, tubular adenomas, and sporadic colorectal cancer, compared with control samples. CONCLUSIONS PI3K-Akt signaling cooperates with Wnt to increase beta-catenin signaling during inflammation. PI3K-induced and Akt-mediated beta-catenin signaling are required for progenitor cell activation during the progression from CUC to CAC; these factors might be used as biomarkers of dysplastic transformation in the colon.

Anti-interferon-inducible chemokine, CXCL10, reduces colitis by impairing T helper-1 induction and recruitment in mice.

Background: Colitis in interleukin (IL)‐10−/− mice is a CD4+ T helper 1 (TH1)‐mediated disease characterized by intermittent, transmural inflammation reminiscent of human Crohns disease. In this study, we investigated the hypothesis that production of the CXC chemokine CXCL10 (interferon [IFN]&ggr;‐inducible protein 10) enhances induction of inflammatory responses in draining lymph nodes (LNs) and promotes colonic TH1 cell recruitment. Methods: Colitis was induced in B6 IL‐10−/− mice. Mice were given anti‐CXCL10 mAb in 2‐week intervals before and after peak colitis. Colitis severity was graded and cytokine/chemokine levels were analyzed by real‐time polymerase chain reaction. Cell yields were quantitated and effector cell recruitment was assessed by recovery of transferred D011.10 TH1 cells shortly (72 h) after transfer. Results: Treatment with anti‐CXCL10 during colitis development decreased clinical and histologic disease severity as well as cytokine/chemokine mRNA and accumulation of mononuclear cells in LNs and colon. Treatment of mice with severe colitis reduced colitis scores and cell yields to lesser degrees. Anti‐CXCL10 specifically decreased recruitment of transferred TH1 cells into mesenteric LNs (MLNs) and colon of IL‐10−/− mice by 75% (P < 0.05). Conclusion: These results suggest that CXCL10 plays a dual role in colitis development by enhancing TH1 cell generation in inductive sites and promoting effector cell recruitment to inflamed tissue. Blockade of CXCL10 may be a useful adjunct to remission‐inducing therapies in inflammatory bowel disease (IBD) by impairing disease recurrence through selective inhibition of effector cell generation and trafficking in vivo.

Epithelial NF-κB Enhances Transmucosal Fluid Movement by Altering Tight Junction Protein Composition after T Cell Activation

In inflammatory bowel disease (IBD), aberrant activation of innate and adaptive immune responses enhances mucosal permeability through mechanisms not completely understood. To examine the role of epithelial nuclear factor (NF-kappaB) in IBD-induced enhanced permeability, epithelial-specific IkappaBalpha mutant (NF-kappaB super repressor) transgenic (TG) mice were generated. NF-kB activation was inhibited in TG mice, relative to wild-type mice, following T cell-mediated immune cell activation using an anti-CD3 monoclonal antibody. Furthermore, epithelial NF-kappaB super repressor protein inhibited diarrhea and blocked changes in transepithelial resistance and transmucosal flux of alexa350 (0.35 kDa) and dextran3000 (3 kDa). In vivo perfusion loop studies in TG mice revealed reversed net water secretion and reduced lumenal flux of different molecular probes (bovine serum albumin, alexa350, and dextran3000). Cell-imaging and immunoblotting of low-density, detergent-insoluble membrane fractions confirmed that tight junction proteins (occludin, claudin-1 and zona occludens-1) are internalized through an NF-kappaB-dependent pathway. Taken together, these data suggest that IBD-associated diarrhea results from NF-kappaB-mediated tight junction protein internalization and increased paracellular permeability. Thus, reduction of epithelial NF-kappaB activation in IBD may repair defects in epithelial barrier function, reduce diarrhea, and limit protein (eg, serum albumin) losses. Epithelial NF-kappaB activation induced by mucosal T cells, therefore, actively plays a role in opening paracellular spaces to promote transmucosal fluid effux into the intestinal lumen.

PI3K/AKT Signaling Is Essential for Communication between Tissue-Infiltrating Mast Cells, Macrophages, and Epithelial Cells in Colitis-Induced Cancer

Purpose: To understand signaling pathways that shape inflamed tissue and predispose to cancer is critical for effective prevention and therapy for chronic inflammatory diseases. We have explored phosphoinositide 3-kinase (PI3K) activity in human inflammatory bowel diseases and mouse colitis models. Experimental Design: We conducted immunostaining of phosphorylated AKT (pAKT) and unbiased high-throughput image acquisition and quantitative analysis of samples of noninflamed normal colon, colitis, dysplasia, and colorectal cancer. Mechanistic insights were gained from ex vivo studies of cell interactions, the piroxicam/IL-10−/− mouse model of progressive colitis, and use of the PI3K inhibitor LY294002. Results: Progressive increase in densities of pAKT-positive tumor-associated macrophages (TAM) and increase in densities of mast cells in the colonic submucosa were noted with colitis and progression to dysplasia and cancer. Mast cells recruited macrophages in ex vivo migration assays, and both mast cells and TAMs promoted invasion of cancer cells. Pretreatment of mast cells with LY294002 blocked recruitment of TAMs. LY294002 inhibited mast cell and TAM-mediated tumor invasion, and in mice, blocked stromal PI3K, colitis, and cancer. Conclusion: The PI3K/AKT pathway is active in cells infiltrating inflamed human colon tissue. This pathway sustains the recruitment of inflammatory cells through a positive feedback loop. The PI3K/AKT pathway is essential for tumor invasion and the malignant features of the piroxicam/IL-10−/− mouse model. LY294002 targets the PI3K pathway and hinders progressive colitis. These findings indicate that colitis and progression to cancer are dependent on stromal PI3K and sensitive to treatment with LY294002. Clin Cancer Res; 19(9); 2342–54. ©2013 AACR.

Therapeutic benefit of pentostatin in severe IL-10−/− Colitis

Background: Pentostatin, an adenosine deaminase (ADA) inhibitor, is a purine antimetabolite used for the treatment of leukemias. ADA inhibition blunts expansion of proliferating lymphocytes and increases adenosine release, a potent anti‐inflammatory molecule. Human inflammatory bowel disease (IBD) is driven by expansion of effector T cells (Teff) that overwhelm reulatory T cells (Treg) and propagate innate immune reponses. Here we study the therapeutic benefits of ADA inhibition to impair Teff cell expansion and reduce inflammatory cytokine release in IL‐10‐deficient (IL‐10‐/‐) mice. Methods: Colitis was induced in IL‐10‐/‐ mice by administering piroxicam for two weeks. Mice were treated with daily pentostatin or phosphate‐buffered saline for 1 week and effects on tissue inflammation, lymphocyte numbers and cytokine production examined. Results: Pentostatin reduced inflammation by >50% and nearly normalized serum amyloid A levels. Lymphocyte expansions in the colon and mesenteric lymph node (MLN) (3.5‐fold and >5‐fold respectively) dropped by >50‐90%. Pro‐inflammatory factors in the colon and MLN (IL‐1&bgr;, IFN‐&ggr;, IL‐6, CXCL10, TNF) dropped whereas FoxP3 and TGF‐&bgr; were unchanged. Reductions in cytokine production from equivalent numbers of T cells from pentostatin‐treated mice after in vitro (36h) or in vivo (3h) activation suggested anti‐inflammatory effects of pentostatin independent of lymphodepletion contributed to its therapeutic benefit. Analysis of mucosal lymphocyte subsets suggested pentostatin reduced numbers of effector CD4+ CD69+ T cells, while sparing CD4+ CD62L+ T cells. Conclusions: Pentostatin dosages that avoid severe lymphocyte depletion effectively treat colitis by impairing Teff cell expansion and reducing pro‐inflammatory cytokine production while preserving regulatory Treg populations and function.

Fluorescence endoscopy of cathepsin activity discriminates dysplasia from colitis

Background:Surveillance colonoscopy using random biopsies to detect colitis-associated cancer (CAC) suffers from poor sensitivity. Although chromoendoscopy improves detection, acceptance in the community has been slow. Here, we examine the usefulness of near infrared fluorescence (NIRF) endoscopy to image molecular probes for cathepsin activity in colitis-induced dysplasia. Methods:In patient samples, cathepsin activity was correlated with colitis and dysplasia. In mice, cathepsin activity was detected as fluorescent hydrolysis product of substrate-based probes after injection into Il10−/− colitic mice. Fluorescence colonoscopy and colonic whole-mount imaging were performed before complete sectioning and pathology review of resected colons. Results:Cathepsin activity was 5-fold and 8-fold higher in dysplasia and CAC, respectively, compared with areas of mild colitis in patient tissue sections. The signal-to-noise ratios for dysplastic lesions seen by endoscopy in Il10−/− mice were 5.2 ± 1.3 (P = 0.0001). Lesions with increased NIRF emissions were classified as raised or flat dysplasia, lymphoid tissue, and ulcers. Using images collected by endoscopy, a receiver operating characteristic curve for correctly diagnosing dysplasia was calculated. The area under the curve was 0.927. At a cutoff of 1000 mean fluorescence intensity, the sensitivity and specificity for detecting dysplasia were 100% and 83%, respectively. Analysis revealed that focally enhanced NIRF emissions derived from increased numbers of infiltrating myeloid-derived suppressor cells and macrophages with equivalent cathepsin activity. Conclusions:These studies indicate that cathepsin substrate-based probe imaging correctly identifies dysplastic foci within chronically inflamed colons. Combined white light and NIRF endoscopy presents unique advantages that may increase sensitivity and specificity of surveillance colonoscopy in patients with CAC.

DNA-driven nutritional therapy of inflammatory bowel disease.

Inflammatory bowel disease (IBD) consists of two main disorders, ulcerative colitis and Crohns disease, that cause chronic, recurrent inflammation of the intestine. An inappropriate immune response to the enteric ecosystem has been postulated to cause IBD. Genomewide association studies provide the information of diverse genetic variations and susceptibilities to patients with IBD. Through the application of these studies, the pathogenesis of IBD may result in part from genetic abnormalities that regulate epithelial barrier function and innate and adaptive immune responses. Crohns disease shows strong association with CARD15, ATG15L1, and IRGM, which are involved in the innate immunity. In the adaptive immune response, IL23R, MST1, IL12B, and STAT3 polymorphisms are associated with Crohns disease and ulcerative colitis. Current pharmacologic treatment of IBD, including 5-aminosalycylate, steroids, and immunomodulator therapy, are mainly aimed at suppressing inflammation non-specifically, except biologic therapies such as anti-tumor necrosis factor molecule, which block specific proinflammatory molecules. For nutritional issues in IBD, the mainstay of therapy has been supportive, with particular attention paid to the prevention, recognition, and therapy of nutritional deficiencies, and individual outcomes to specific dietary factors have been controversial. Parenteral nutritional support and exclusionary diets have been investigated and are not the subject of this review. The emerging concepts of nutrition-gene interaction gave birth to unique scientific fields, nutrigenetics and nutrigenomics. These studies provide information about 1) the genetic variability that induces an individuals response to nutrition according to particular states of health and disease and 2) changes in gene expression that develop as a result of nutrition-gene interaction. For IBD, the role of diet in the regulation of the immune response against gut flora is the subject of current intensive evaluation. These approaches may lead clinicians to derive a personalized nutritional prescription based on individual genetic variations and may result in a significant impact on IBD treatment.

Epithelial TNF Receptor Signaling Promotes Mucosal Repair in Inflammatory Bowel Disease

TNF plays an integral role in inflammatory bowel disease (IBD), as evidenced by the dramatic therapeutic responses in Crohn’s disease (CD) patients induced by chimeric anti-TNF mAbs. However, treatment of CD patients with etanercept, a decoy receptor that binds soluble TNF, fails to improve disease. To explore this discrepancy, we investigated the role of TNF signaling in Wnt/β-catenin–mediated intestinal stem cell and progenitor cell expansion in CD patients, human cells, and preclinical mouse models. We hypothesized that TNF exerts beneficial effects on intestinal epithelial cell (IEC) responses to injury. In CD patients, intestinal stem cell and progenitor cell Wnt/β-catenin signaling correlates with inflammation status. TNF-deficient (Tnf−/−) mice exhibited increased apoptosis, less IEC proliferation, and less Wnt signaling when stimulated with anti-CD3 mAb. Bone marrow (BM) chimera mice revealed that mucosal repair depended on TNF production by BM–derived cells and TNFR expression by radioresistant IECs. Wild-type→Tnfr1/2−/− BM chimera mice with chronic dextran sodium sulfate colitis exhibited delayed ulcer healing, more mucosal inflammation, and impaired Wnt/β-catenin signaling, consistent with the hypothesis that epithelial TNFR signaling participates in mucosal healing. The direct effect of TNF on stem cells was demonstrated by studies of TNF-induced Wnt/β-catenin target gene expression in murine enteroids and colonoid cultures and TNF-induced β-catenin activation in nontransformed human NCM460 cells (TOPFlash) and mice (TOP-GAL). Together, these data support the hypothesis that TNF plays a beneficial role in enhancing Wnt/β-catenin signaling during ulcer healing in IBD. These novel findings will inform clinicians and therapeutic chemists alike as they strive to develop novel therapies for IBD patients.

Su1992 TNF Mediates β-Catenin Activation in Intestinal Stem Cells During Colitis

G A A b st ra ct s in colonoid culture and we posit that activation of the TRIF signaling pathway is the main mediator of these changes. Understanding how innate immune recognition pathways crosstalk with epithelial stem cell self-renewal and differentiation pathways will be important for future studies examining the role of stem cells in homeostasis and tumor promotion. We would like to acknowledge the technical help and provision of reagents by Richard J. von Furstenberg and Susan J. Henning.

177 The Roles of TNFR Signaling and NOX1 in Epithelial ß-Catenin Activation During Colitis

TNF blockade in Crohns disease (CD) and ulcerative colitis (UC) patients reveals that TNF plays a central role in disease pathogenesis. Here we examine the potential that TNF activates epithelial stem and progenitor cell (ISC/PC) β-catenin through intestinal epithelial cell (IEC) TNFR1 and TNFR2 signaling. Tissue sections and purified IEC protein isolates were analyzed using Akt-phosphorylated β-catenin (P-β-catenin552 or P-β-cat) Ab. Results: IHC staining of biopsy tissue from control (uninflamed) and inflamed CD/UC patients revealed that Pβ-cat levels increased 2.8-fold in untreated colitis. However, in anti-TNF treated patients selected for ongoing active inflammation, P-β-cat levels were reduced by over 50% (p host) revealed that T cell activation increased numbers of P-βcat-stained IECs by 110% in WT->WT BMC with no increases seen in anti-CD3-treated WT-> TNFR1/2 KO mice. These findings suggest epithelial TNFR signaling regulates IEC β-catenin activation. In studies comparing colitic IL-10 KO (colitis score >3/4) mice to WT healthy controls (n=5 in each group) mRNA from CD44+-sorted IECs was used for genomewide expression array analysis revealing that levels of NADPH oxidase 1 (Nox1) were significantly induced (3.22-fold, p-value: 3 x106). We next tested whether Nox1 may participate in TNF-mediated activation of β-catenin in ISC. We confirmed Nox1 mRNA induction in IECs from IL-10 KO colitis mice (17-fold). Furthermore Nox1 mRNA was induced in purified IECs by anti-CD3 mAb treatment of WT mice (6-fold). Importantly, Nox1 mRNA induction was reduced by over 50% in T cell stimulated TNF-R1/R2 KO mice. Lastly, mRNA from purified CD44+-sorted IECs from Nox1 KO mice revealed that baseline levels of cMyc, cyclinD1, Ascl2 and Lrig1 expression were reduced compared to WT mice. Conclusion: Together the data suggest that epithelial TNF receptor signaling is required for optimal levels of IEC β-catenin activation in patients with active colitis. Data in IL-10 ko and WT mice suggest that epithelial TNFR signaling induces Nox1 which has recently been shown to play a role in Wnt/β-catenin signaling in the intestine (Coant et al. MCB 2010). Together these data are consistent with the hypothesis that Nox1 is a key mediator of TNFinduced epithelial β-catenin activation in UC and CD.