Colonic Mucosa Of Ulcerative Colitis Patients Research Articles

Abstract LB360: Establishment of predictive biomarkers for carcinogenesis in ulcerative colitis using RNA editing analyses

Abstract Background: Ulcerative colitis (UC) is a chronic inflammatory disease continuous from the rectum. We previously reported that chronic inflammation may induce the RNA-editing enzyme ADAR1 and promote carcinogenesis (Journal of Crohn's and Colitis, 2023). However, the relationship between RNA editing and carcinogenesis in UC remains unresolved because we could not identify specific RNA editing sites. Therefore, in this study, we performed a new comprehensive RNA analysis using RNA sequencing to clarify the role of RNA editing in the carcinogenesis process in UC and to construct a prediction model of carcinogenic risk. Methods: We performed immunostaining of ADAR1, an RNA-editing enzyme, in 139 cases of ulcerative colitis. In addition, normal colorectal tissue, UC inflammatory phase, normal rectal mucosa of UC carcinoma cases, and UC carcinoma sites were submitted to RNA-Seq testing in 6 cases each (24 cases total). Bioinformatics analysis was performed to compare RNA editing sites in each group. Results: RNA-seq analysis showed a significant increase in global RNA editing sites in the inflammatory phase compared to normal colon tissue (p = 0.02). This phenomenon was due to increased ADAR1 expression associated with type 1 interferon. We hypothesized that global RNA editing was decreased in normal colonic mucosa during the remission phase but that some sites of prolonged RNA editing might promote carcinogenesis. We attempted to identify carcinogenesis-specific RNA editing sites. We identified 72 cancer-specific RNA editing sites, mainly in long noncoding RNAs such as NEAT1 and in the 3'UTR region of mRNAs such as CTSS. Assuming that the accumulation of these RNA editing leads to carcinogenesis, pathway analysis revealed that pathways promoting cancer invasion and stemness are activated. When ROC curves were generated using these editing sites as markers, the AUC was 0.935, suggesting that analysis of RNA editing in the colonic mucosa of UC patients may be able to predict the risk of carcinogenesis. Conclusions: We identified potential editing sites for carcinogenesis in UC, allowing the identification of carcinogenic mechanisms in UC and the establishment of predictive markers for carcinogenic risk. Citation Format: Toshiaki Takahashi, Kunitoshi Shigeyasu, Yoshitaka Kondo, Hibiki Umeda, Kazuya Moriwake, Masashi Kayano, Kaori Nitta, Fuminori Teraishi, Yuzo Umeda, Hiroshi Tazawa, Toshiyoshi Fujiwara. Establishment of predictive biomarkers for carcinogenesis in ulcerative colitis using RNA editing analyses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB360.

Comprehensive analysis of endoplasmic reticulum stress-associated genes signature of ulcerative colitis.

Endoplasmic reticulum stress (ERS) is a critical factor in the development of ulcerative colitis (UC); however, the underlying molecular mechanisms remain unclear. This study aims to identify pivotal molecular mechanisms related to ERS in UC pathogenesis and provide novel therapeutic targets for UC. Colon tissue gene expression profiles and clinical information of UC patients and healthy controls were obtained from the Gene Expression Omnibus (GEO) database, and the ERS-related gene set was downloaded from GeneCards for analysis. Weighted gene co-expression network analysis (WGCNA) and differential expression analysis were utilized to identify pivotal modules and genes associated with UC. A consensus clustering algorithm was used to classify UC patients. The CIBERSORT algorithm was employed to evaluate the immune cell infiltration. Gene Set Variation Analysis (GSVA), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to explore potential biological mechanisms. The external sets were used to validate and identify the relationship of ERS-related genes with biologics. Small molecule compounds were predicted using the Connectivity Map (CMap) database. Molecular docking was performed to simulate the binding conformation of small molecule compounds and key targets. The study identified 915 differentially expressed genes (DEGs) and 11 ERS-related genes (ERSRGs) from the colonic mucosa of UC patients and healthy controls, and these genes had good diagnostic value and were highly correlated. Five potential small-molecule drugs sharing tubulin inhibitors were identified, including albendazole, fenbendazole, flubendazole, griseofulvin, and noscapine, among which noscapine exhibited the highest correlation with a high binding affinity to the targets. Active UC and 10 ERSRGs were associated with a large number of immune cells, and ERS was also associated with colon mucosal invasion of active UC. Significant differences in gene expression patterns and immune cell infiltration abundance were observed among ERS-related subtypes. The results suggest that ERS plays a vital role in UC pathogenesis, and noscapine may be a promising therapeutic agent for UC by affecting ERS.

A15 GENETIC OR DIET-ASSOCIATED DEFECTS IN MUCUS FACILITATE ULCERATIVE COLITIS PATHOBIONT-DRIVEN COLITIS

Abstract Background The Inflammatory Bowel Diseases (IBD), Crohn’s Disease (CD) and ulcerative colitis (UC) affect > 270,000 Canadians and are increasing in incidence. Ileal CD has been linked to the overgrowth of mucosal adherent E. coli. Recent studies have also implicated the adherence of Escherichia coli pathobionts to the colonic mucosa of UC patients. Using the representative UC E. coli pathobiont p19A, we recently demonstrated it aggravated chemical-induced colitis in susceptible mice, through the actions of the toxin alpha-hemolysin, and by adhering to the inflamed colonic mucosa via the adhesin FimH. It is less clear what host factors control susceptibility to the UC pathobionts. One of the potential candidates is the glycosylated mucin (Muc2) which forms the mucus layer that covers the colonic epithelium and is often impaired in UC patients. Purpose To define the role of mucus structure and function in determining susceptibility to the p19A pathobiont, and its ability to cause colitis in mice. Method In vitro growth was assessed to test p19A’s ability to utilize mucin-derived sugars as nutrients. Susceptibility to p19A was tested in two mouse models of colonic mucus impairment. The first is a mouse strain deficient in core 1 derived O-glycans in their intestinal epithelial cells (IEC C1galt1-/-), resulting in reduced Muc2 glycosylation, and thus a thin and impaired mucus barrier. The second model involves feeding wildtype (WT) C57BL/6CR mice a fiber-free (FF) diet, resulting in a significantly thinner colonic mucus layer. The mice were subsequently orally gavaged with p19A and their susceptibility determined by p19A burdens, intestinal histopathology, inflammatory cytokine and short chain fatty acid (SCFA) production. Result(s) When tested in vitro, the p19A pathobiont was found to use an array of mucin-derived sugars as sole carbon source to proliferate. Following oral gavage of WT mice fed a normal diet, immunostaining identified p19A within the colonic mucus but it did not reach the colonic mucosa or cause disease. In contrast, p19A was found at the colonic mucosal surface of mucus-defective IEC C1galt1-/- mice (as compared to IEC C1galt1flox/flox mice) and in WT mice fed a fiber-free diet. This mucosal adherence was associated with increased body weight loss during the course of infection, as well as increased p19A burdens, colonic pathology and pro-inflammatory cytokine expression. Especially fiber-free diet-fed mice showed reduced SCFA levels in their feces at baseline. When the mice were given exogenous SCFA (tributyrin) orally, p19A infection was reduced. Conclusion(s) Our results indicate that UC E. coli pathobionts are able to dwell within colonic mucus and utilize mucin sugars as nutrients. Moreover, they can reach the mucosal surface and induce colitis in hosts suffering genetic or diet-based mucus dysfunction. In part, this susceptibility reflects the important role played by mucus in the production of SCFA, suggesting potential therapeutic approaches for patients suffering UC. Please acknowledge all funding agencies by checking the applicable boxes below CCC, CIHR Disclosure of Interest None Declared

Endoplasmic Reticulum Stress in Colonic Mucosa of Ulcerative Colitis Patients Is Mediated by PERK and IRE1 Pathway Activation.

Ulcerative colitis (UC) is characterized by a chronic overproduction of proinflammatory cytokines. During an acute phase, the endoplasmic reticulum (ER) is overloaded and the protein folding process is impaired, a condition named ER stress. This state induces a response (unfolded protein response (UPR)), initiated by the activation of IRE1/Xbp-1, PERK/eIF2α, and ATF6 pathways, which has previously been linked to intestinal inflammation in experimental models. ER stress and UPR activation trigger the activation of proinflammatory, autophagy, and apoptosis genes, in addition to promoting protein degradation. Therefore, the goal of this study was to evaluate the activation of ER stress and UPR in colonic mucosa of UC patients. Patient and Methods. Transcriptional analysis of ER stress- and UPR-related genes was performed by qPCR from intestinal mucosa of patients with UC. We also performed in situ hybridization (ISH) and immunohistochemistry (IHQ) of PERK/eIF2α and IRE1/Xbp-1 pathways and UPR-related chaperones. Results. We first evaluated inflammatory genes via qPCR, and we observed that all analyzed proinflammatory transcripts were upregulated in UC patients. ISH and IHQ images showed that ER stress is activated via PERK/eIF2α and IRE1/Xbp-1 pathways not only in intestinal epithelial cells but also in cells of the lamina propria of UC colonic mucosa. Transcriptional analysis confirmed that EIF2AK3 was upregulated in UC patients. UPR-related genes, such as ATF3, STC2, and DDIT3, along with the chaperones and cochaperones DNAJC3, CALR, HSP90B1, and HSPA5, were also upregulated in UC patients. In addition, we observed that proapoptotic and autophagy genes (Bax and ATG6L1, respectively) were also upregulated. Conclusion. Our results suggest that ER stress and UPR are indeed activated in UC patients and this may contribute to the chronic inflammatory process seen in UC. The increased apoptosis and autophagy markers further support the activation of these findings once they are activated to counterbalance tissue damage. These findings provide new insights into the molecular mechanisms that maintain UC activity and open new possibilities to attenuate intestinal inflammation.

The Relationship between Mucins and Ulcerative Colitis: A Systematic Review.

Mucins are a family of glycosylated proteins which are the primary constituents of mucus and play a dynamic role in the regulation of the protective mucosal barriers throughout the human body. Ulcerative colitis (UC) is an Inflammatory Bowel Disease (IBD) characterised by continuous inflammation of the inner layer of the large intestine, and in this systematic review we analyse currently available data to determine whether alterations exist in mucin activity in the colonic mucosa of UC patients. Database searches were conducted to identify studies published between 1990 and 2020 that assess the role of mucins in cohorts of UC patients, where biopsy specimens were resected for analysis and control groups were included for comparison. 5497 articles were initially identified and of these 14 studies were systematically selected for analysis, a further 2 articles were identified through citation chaining. Therefore, 16 studies were critically reviewed. 13 of these studies assessed the role of MUC2 in UC and the majority of articles indicated that alterations in MUC2 structure or synthesis had an impact on the colonic mucosa, although conflicting results were presented regarding MUC2 expression. This review highlights the importance of further research to enhance our understanding of mucin regulation in UC and summarises data that may inform future studies.

Relationship between matrix Gla protein and clinical features of ulcerative colitis

Objective: To analyze the relationship between matrix Gla protein(MGP) and clinical characteristics of patients with ulcerative colitis (UC). Methods: Fifty-one UC patients who were admitted to the gastroenterology department of Peking union medical college hospital from July 1, 2015 to May 31, 2017 were included. Twenty-seven healthy subjects in the same period were included as normal controls. The expression of MGP mRNA in the colonic mucosa was detected by real-time fluorescence quantitative PCR. Clinical data of the patients were collected during a 2-year follow-up. The public data set containing MGP gene expression profile of UC patients was downloaded from the GEO database, and was divided into four groups according to the microscopic Mayo score. The differential expression of MGP in each group was analyzed. Results: All the fifty-one UC patients were followed up. The expression of MGP mRNA in the colonic mucosa of UC patients treated with hormone and immunosuppressive agents or biological agents or surgery was higher than that of patients treated with mesalazine. MGP mRNA expression was positively correlated with C-reactive protein level. It was also higher in the colonic mucosa of UC patients with clostridium difficilis or cytomegalovirus infection than that of patients without opportunistic infection. The difference of MGP mRNA expression between groups in GEO public data set was statistically significant(P<0.01), showing an up-regulation trend with the aggravation of inflammation. The expression level of MGP was moderately correlated with the microscopic Mayo score. The relationship between MGP mRNA expression and lifestyle, lesion range, erythrocyte sedimentation rate, parenteral manifestations and recurrence frequency of UC patients was not obvious. Conclusions: MGP is associated with colonic inflammation and its abnormal expression can help to predict the disease activity of patients with UC.

MiR-125b and miR-223 Contribute to Inflammation by Targeting the Key Molecules of NFκB Pathway.

The contribution of miRNA in the pathogenesis of ulcerative colitis (UC) has emerged in the past few decades. Differential miRNA expression has been demonstrated in UC patients, and their ability to target the genes involved in inflammatory pathway has also been explored in recent years. miR-125b and miR-223 have been demonstrated to get upregulated within the colonic mucosa of UC patients. Here, we explored the biological relevance of miR-125b and miR-223 altered expression during UC by identifying the potential gene targets for miR-125b and miR-223. TRAF6 and A20, the signaling molecules involved in the NFκB pathway, were identified as target genes for miR-125b while IKKα was identified as a gene target for miR-223. The colonic mucosal samples from UC patients exhibited a significant rise in miR-125b and miR-223 expression while a subsequent downregulation was observed in the expression of TRAF6, A20, and IKKα. This negative correlation between miRNAs and their respective target genes was validated by co-transfecting miR-125b and miR-223 in HT29 cells. Co-transfection with miR-125b resulted in a marked decline in the expression of TRAF6 and A20, while the miR-223 co-transfected cells exhibited lower IKKα expression levels. Additionally, co-transfection with miR-125b or miR-223 in HT29 cells caused higher p65 and pro-inflammatory cytokines (IL-8 and IL-1β) expression upon LPS stimulation. From our findings, we highlight the possible contribution of miR-125b and miR-223 in regulating the inflammatory response during UC by negatively regulating the expression of TRAF6, A20, and IKKα. Therefore, we conclude that these two miRNAs could be considered as potential candidates for developing promising biomarkers for screening and diagnosis of UC.

P276 Reactivation of Epstein–Barr virus and cytomegalovirus behaves differently in pathophysiology of ulcerative colitis

Abstract Background Cytomegalovirus (CMV) and Epstein–Barr virus (EBV) are members of the herpesvirus family. CMV reactivation is often complicated with ulcerative colitis (UC) and is known as one of exacerbation factors. However, the association between EBV reactivation and pathophysiology of UC is still unclear. Methods This study enrolled 116 active UC patients who received colonoscopy between January 2005 and January 2019 in Kyoto University Hospital. 244 biopsy specimens were obtained from inflamed colonic mucosa to assess EVB and CMV reactivation. Viral loads of EBV and CMV in inflamed mucosa were measured by real-time PCR assay. The reactivation of those viruses was defined as DNA quantity more than 10 copies/μg DNA. Clinical severity was assessed by Lichtiger index and defined as follow: 4–8 as mild, 9–12 as moderate, and more than 12 as severe. Endoscopic severity was assessed by Mayo endoscopic score. We examined the correlation between the positivity of each viral reactivation and patients’ characteristics or prognosis of UC. Results (1) Median age, Lichtiger score and Mayo endoscopic score at the time to assess the viral reactivations were 36 years-old, 8, and 3, respectively. (2) EBV and CMV reactivation were observed in 127 samples (52.0%) and 73 samples (29.9%), respectively. There was no correlation between EBV and CMV viral load (correlation coefficient 0.19), although a significant correlation between those viral reactivations was observed in active colonic mucosa of UC patients (p = 0.002). (3) The proportion of EBV reactivation was higher in both clinically and endoscopically severe UC patients compared with those with mild activity. On the other hands, there was no association between CMV reactivation and clinical or endoscopic severity. (4) Multivariate analysis indicated risk factors for EBV reactivation as receiving anti-TNF-α antibodies (odds ratio [OR] 4.2) or calcineurin inhibitors (OR 3.5), and CMV reactivation (OR 2.1), respectively. (5) Multivariate analysis also indicated risks for CMV reactivation as steroid-refractory (OR 4.7) and EBV reactivation (OR 2.0). (6) EBV and CMV reactivation did not affect clinical outcomes including the requirement of colectomy or intensification of immunosuppressive treatments and the incidence of colitis-associated cancer, dysplasia and lymphoproliferative disease. Conclusion Reactivation of EBV or CMV may behave differently in pathophysiology of UC. Further studies are required to clarify the role of EBV reactivation on colonic inflammation in UC patients.

STMN1 accumulation is associated with dysplastic and neoplastic lesions in patients with ulcerative colitis.

Ulcerative colitis (UC) is thought to be associated with precancerous lesions that can ultimately lead to colon cancer. Therefore, diagnostic markers for colorectal dysplasia and cancer are urgently needed for patients with UC. Stathmin 1 (STMN1) is a novel cancer biomarker that is also a novel target for treatment in several cancers, including colon cancer. However, few studies have investigated the relationship between STMN1 expression and clinical features in colorectal dysplasia and cancer in patients with UC. The present study examined the clinical significance of STMN1 expression in colorectal dysplasia and cancer with UC. The present study performed an immunohistochemical analysis of 31 clinical colorectal samples from eight patients with colorectal dysplasia and/or cancer to assess the relationships between STMN1 expression and clinicopathological features including mismatch repair protein expression, rate of Ki-67 positivity, differentiation level, TNM stage, and UC duration. STNM1 expression was detected in 95.7% of dysplastic and cancerous lesions, whereas p53, the current diagnostic marker, was not expressed in 39.1% of dysplastic and cancerous lesions. Furthermore, STMN1 expression was associated with a high rate of positivity for Ki-67, a proliferation marker. Our data suggest that STMN1 in the colonic mucosa of UC patients may be useful as an early diagnostic marker of dysplasia and colitic cancer.

Weak Cytotoxic T Cells Activation Predicts Low-Grade Dysplasia Persistence in Ulcerative Colitis.

INTRODUCTION:In patients with ulcerative colitis (UC), dysplasia develops in 10%–20% of cases. The persistence of low-grade dysplasia (LGD) in UC in 2 consecutive observations is still an indication for restorative proctocolectomy. Our hypothesis is that in the case of weak cytotoxic activation, dysplasia persists. We aimed to identify possible immunological markers of LGD presence and persistence.METHODS:We prospectively enrolled 112 UC patients who underwent screening colonoscopy (T0) who had biopsies taken from their sigmoid colon. Ninety of them had at least a second colonoscopy (T1) with biopsies taken in the sigmoid colon and 8 patients had dysplasia in both examinations suggesting a persistence of LGD in their colon. Immunohistochemistry and real time polymerase chain reaction for CD4, CD69, CD107, and CD8β messenger RNA (mRNA) expression and flow cytometry for epithelial cells expressing CD80 or HLA avidin-biotin complex were performed. Non-parametric statistics, receiver operating characteristic curves analysis, and logistic multiple regression analysis were used.RESULTS:Thirteen patients had LGD diagnosed at T0. The mucosal mRNA expression of CD4, CD69, and CD8β was significantly lower than in patients without dysplasia (P = 0.033, P = 0.046 and P = 0.007, respectively). A second colonoscopy was performed in 90 patients after a median follow-up of 17 (12–25) months and 14 of the patients were diagnosed with LGD. In these patients, CD8β mRNA expression at T0 was significantly lower in patients without dysplasia (P = 0.004). A multivariate survival analysis in a model including CD8β mRNA levels and age >50 demonstrated that both items were independent predictors of dysplasia at follow-up (hazard ratio [HR] = 0.47 [95% confidence interval [CI]: 0.26–0.86], P = 0.014, and HR = 13.32 [95% CI: 1.72–102.92], P = 0.013).DISCUSSION:These data suggest a low cytotoxic T cell activation in the colonic mucosa of UC patients who do not manage to clear dysplasia. Thus, low level of CD8β mRNA expression in non-dysplastic colonic mucosa might be considered in future studies about the decision making of management of LGD in UC.

Anti-commensal IgG Drives Intestinal Inflammation and Type 17 Immunity in Ulcerative Colitis

SummaryInflammatory bowel disease is a chronic, relapsing condition with two subtypes, Crohn’s disease (CD) and ulcerative colitis (UC). Genome-wide association studies (GWASs) in UC implicate a FCGR2A variant that alters the binding affinity of the antibody receptor it encodes, FcγRIIA, for immunoglobulin G (IgG). Here, we aimed to understand the mechanisms whereby changes in FcγRIIA affinity would affect inflammation in an IgA-dominated organ. We found a profound induction of anti-commensal IgG and a concomitant increase in activating FcγR signaling in the colonic mucosa of UC patients. Commensal-IgG immune complexes engaged gut-resident FcγR-expressing macrophages, inducing NLRP3- and reactive-oxygen-species-dependent production of interleukin-1β (IL-1β) and neutrophil-recruiting chemokines. These responses were modulated by the FCGR2A genotype. In vivo manipulation of macrophage FcγR signal strength in a mouse model of UC determined the magnitude of intestinal inflammation and IL-1β-dependent type 17 immunity. The identification of an important contribution of IgG-FcγR-dependent inflammation to UC has therapeutic implications.

Colonic hypereosinophilia in ulcerative colitis may help to predict the failure of steroid therapy.

Although glucocorticosteroids (GS) and mesalazine are effective and widely employed to treat moderate-to-severe ulcerative colitis (UC), information regarding the factors responsible for response to such therapy is still scarce. One of these factors is thought to be an increased number of mucosal eosinophils. The aim of our study was to determine whether the presence of hypereosinophilia in colonic mucosa of UC patients might influence the short-term response to l treatment with GS and mesasalazine. Clinical, endoscopic, and pathologic data from patients with a recent diagnosis of moderate UC, who had not undergone treatment, were obtained, and the short-term outcome after 1 month of conventional first-line treatment (mesalazine plus GS) was evaluated. There were 53 patients with a median age of 37 years (95% CI 30-47).Overall, at the end of treatment period 16 (30%) patients responded, whereas a response was not observed in the other 37 (70%) patients. Interestingly, all patients of this latter group had colonic mucosal hypereosinophilia. No significant differences were found between the two groups concerning sex and age at diagnosis, but hypereosinophilia was inversely correlated with the duration of the disease (p = 0.054), and significantly correlated to the localization of UC (p = 0.0023). In addition, The Mayo score was significantly higher in patients with hypereosinophilia (median 8; 95% CI 8-9;) when compared to patients without hypereosinophilia (median 7; 95% CI 7-7, p < 0.0001) including the Mayo endoscopic subscore (median 3; 95% CI 2-3 vs median 2; 95% CI 2-2, respectively; p = 0.007). The presence of colonic mucosal hypereosinophilia may be useful to predict the short-term outcome to conventional first-line therapy in treatment-naïve UC patients. It remains to be seen whether this might be important in modifying the first-line therapy in this subgroup of patients.

A Novel Topical PPARγ Agonist Induces PPARγ Activity in Ulcerative Colitis Mucosa and Prevents and Reverses Inflammation in Induced Colitis Models

Peroxisome proliferator-activated receptor-gamma (PPARγ) exerts anti-inflammatory effects and is therefore a potential target in ulcerative colitis (UC). A novel PPARγ agonist (AS002) developed for local action was evaluated ex vivo in biopsies from UC patients and in vivo in mice with low-grade dextran sodium sulfate (DSS)- and trinitrobenzene sulfonic acid (TNBS)-induced colitis. Colonic biopsies from UC patients (n = 18) and healthy controls (n = 6) were incubated with AS002 or rosiglitazone (positive control) to measure mRNA expression of the PPARγ-responsive gene ADIPOPHILIN and protein levels of UC-related cytokines (enzyme-linked immunosorbent assay). AS002 absorption was determined in the colonic mucosa of UC patients. DSS-colitis mice received PPARγ agonists or vehicle daily by intrarectal administration starting 2 days before induction of colitis (preventive) or from days 3 to 8 (curative). Myeloperoxidase (MPO) and cytokine levels in colonic mucosa were determined. In addition, AS002 effects were studied in TNBS colitis. AS002 displayed an absorption pattern of a lipophilic drug totally metabolized in the mucosa. AS002 and rosiglitazone increased ADIPOPHILIN mRNA expression (3-fold) and decreased TNF-α, IL-1β, and IL-13 levels in human UC biopsies. In DSS, in both preventive and curative treatment and in TNBS colitis, AS002 protected against macroscopic and histological damage and lowered MPO and TNF-α, IL-1β, and IL-13 levels. AS002 triggers anti-inflammatory PPARγ activity in the human colonic mucosa of UC patients and prevents and reverses colitis in mice. Our data suggest that AS002 has potential for topical maintenance treatment of UC, which warrants further studies in vivo in patients.

The Expression of the Short Isoform of Thymic Stromal Lymphopoietin in the Colon Is Regulated by the Nuclear Receptor Peroxisome Proliferator Activated Receptor-Gamma and Is Impaired during Ulcerative Colitis.

The etiology of inflammatory bowel diseases remains largely unknown. We previously demonstrated that the expression of the peroxisome proliferator activated receptor-gamma (PPARγ) is downregulated in colonic epithelial cells of patients with ulcerative colitis (UC). PPARγ is a nuclear receptor that modulates inflammation. We hypothesized that its deficiency may play a role in the loss of intestinal homeostasis through the control of immunomodulatory factors. We found that thymic stromal lymphopoietin (TSLP), an epithelial cytokine with pleiotropic functions, is regulated by PPARγ. While this cytokine possesses two isoforms, only the short form (sfTSLP) was regulated by PPARγ. sfTSLP mRNA expression was decreased both in PPARγ knock-down Caco2 cells and cells treated with PPARγ antagonist, whereas PPARγ agonists induced the expression of sfTSLP in Caco2 and T-84 cells. The response element activated by PPARγ was identified in the promoter of the sfTSLP gene by chromatin immunoprecipitation and gene reporter assays. The expression of sfTSLP was significantly decreased in the colonic mucosa of UC patients compared to controls and was correlated with PPARγ expression. Our results identified sfTSLP as a new PPARγ-target gene and support the hypothesis that, in UC, PPARγ deficiency in colonic mucosa could play a role in the loss of intestinal tolerance through an impaired sfTSLP expression.

IL-1β-Induced Downregulation of the Multifunctional PDZ Adaptor PDZK1 Is Attenuated by ERK Inhibition, RXRα, or PPARα Stimulation in Enterocytes.

Background: The PDZ adaptor protein PDZK1 modulates the membrane expression and function of a variety of intestinal receptors and ion/nutrient transporters. Its expression is strongly decreased in inflamed intestinal mucosa of mice and IBD patients.Aim and Methods: We investigated whether the inflammation-associated PDZK1 downregulation is a direct consequence of proinflammatory cytokine release by treating intestinal Caco-2BBE cells with TNF-α, IFN-γ, and IL-1β, and analysing PDZK1 promotor activity, mRNA and protein expression.Results: IL-1β was found to significantly decrease PDZK1 promoter activity, mRNA and protein expression in Caco-2BBE cells. A distal region of the hPDZK1 promoter was identified to be important for basal expression and IL-1β-responsiveness. This region harbors the retinoid acid response element RARE as well as binding sites for transcription factors involved in IL-β downstream signaling. ERK1/2 inhibition by the specific MEK1/2 inhibitors PD98059/U0126 significantly attenuated the IL-1β mediated downregulation of PDZK1, while NF-κB, p38 MAPK, and JNK inhibition did not. Expression of the nuclear receptors RXRα and PPARα was decreased in inflamed colonic-mucosa of ulcerative colitis patients and in IL-1β-treated Caco2-BBE cells. Moreover, the RAR/RXR ligand 9-cis retinoic acid and the PPARα-agonist GW7647 stimulated PDZK1 mRNA and protein expression and attenuated IL-1β-mediated inhibition.Conclusions: The strong decrease in PDZK1 expression during intestinal inflammation may be in part a consequence of IL-1β-mediated RXRα and PPARα repression and can be attenuated by agonists for either nuclear receptor, or by ERK1/2 inhibition. The negative consequences of inflammation-induced PDZK1 downregulation on epithelial transport-function may thus be amenable to pharmacological therapy.

Mucin gene expression in the intestine of ulcerative colitis patients: a systematic review and meta-analysis.

The mucus layer of the colon is the main barrier between luminal microbes and the mucosa, and plays a significant role in the body defense mechanisms. Several studies have examined mucin gene (MUC) expression in ulcerative colitis (UC) without conclusive results. The aim of the study was to establish the knowledge of mucin expression in UC as a basis for further investigation. English medical literature searches were performed for mucin expression in the colonic mucosa of UC patients in comparison with controls. Case-control studies were included. A meta-analysis was carried out using 'Comprehensive meta-analysis' software. Pooled odds ratios (ORs) and 95% confidence intervals were calculated. Altogether, we found 311 eligible studies. Only 10 case-control studies from five countries fulfilled the inclusion criteria. A moderate heterogeneity was found in the studies included: Q=52.703, d.f. (Q)=15.000, I=71.539%. OR for mucin expression in UC patients versus healthy controls was 1.868 with a 95% confidence interval (CI) 1.263-2.764, P=0.002. Thus, we could find a significant increase of 87% of mucin expression in UC patients. OR for MUC2 was 2.520, 95% CI 1.320-4.809, P<0.001. MUC3 was also increased with OR 2.599, 95% CI 1.389-4.861, P=0.003. Funnel plot did not indicate a significant publication bias. We found a global increase in mucin expression in UC patients, specifically in MUC2 and MUC3. Further studies are needed, especially in patients treated with biologics for mucosal healing, to understand the role of mucin expression in the natural history of UC.

Downregulation of serotonin reuptake transporter gene expression in healing colonic mucosa in presence of remaining low-grade inflammation in ulcerative colitis.

The serotonin reuptake transporter (SERT) terminates serotonin activity by removing it from interstitial space. Downregulated colonic SERT expression has been reported in irritable bowel disease (IBS), and symptoms resembling IBS occur in cases of inflammatory bowel disease (IBD) in remission; thus, a common pathogenesis for IBS and IBD is possible. However, little is known regarding SERT expression in colonic mucosa of IBD patients during healing. Twenty-two ulcerative colitis (UC) patients underwent colonoscopy examinations, during which inflamed mucosa was distinguished from that undergoing healing. Healing mucosa was classified into regular and irregular vessel patterns by narrowband imaging magnifying colonoscopy. Expressions of SERT and various inflammation-related genes in biopsy samples were assessed using a polymerase chain reaction array system and real-time polymerase chain reaction. Colitis model mice were established by administration of dextran sodium sulfate or transfer of CD4(+) T cells isolated from SAMP1 mice, then time-course changes of SERT and inflammatory gene expressions were observed in colonic mucosa. In UC patients, SERT expression in inflamed mucosa was significantly lower than in healing mucosa. SERT expression was decreased in healing mucosa with an irregular vessel pattern with mildly increased levels of inflammatory gene expression. In mice, SERT expression was suppressed in inflamed mucosa and continuously observed with low-grade mucosal inflammation during colitis healing. Sserotonin reuptake transporter expression is downregulated in healing colonic mucosa of UC patients and that suppression may be dependent on the presence of remaining low-grade colonic inflammation.

Correlation of Serum Vitamin A Levels with Disease Activity Indices and Colonic IL-23R and FOXP3 mRNA Expression in Ulcerative Colitis Patients.

Genome-wide association studies have identified IL-23 receptor (IL-23R) as a susceptibility locus for the pathogenesis of ulcerative colitis (UC), which is characterized by exaggerated Th2/Th17 response. Studies have shown that vitamin A (VA) reduces disease progression by promoting FOXP3⁺ T cells and curbing Th17 cells. In this study, we explored the association of colonic IL-23R and FOXP3 expression in fifty-one UC patients (23 in remission and 28 with active disease) with serum VA levels and disease activity. We observed that decreased serum VA levels were associated with increased disease activity. However, there was no significant difference in mucosal IL-23R and FOXP3 expression in UC patients with moderate-to-severe disease activity compared to those in remission. Also, no significant correlation was drawn between serum VA levels and mucosal IL-23R and FOXP3 expression. Our study suggests that even after an established role of VA in inhibiting Th17 responses in mice models and humans, serum VA levels and disease activity do not correlate with FOXP3 and IL-23R expression in colonic mucosa of UC patients.

Role of hypoxia-inducible factor-1α in pathogenesis and disease evaluation of ulcerative colitis.

The main aim of the present study was to measure hypoxia-inducible factor-1α (HIF-1α) in serum and colonic mucosa of ulcerative colitis (UC) patients and to analyze its role in the pathogenesis, disease activity and severity of UC. A total of 47 UC patients and 13 UC in remission patients were recruited for the present study. Ten healthy subjects were also included to serve as controls. HIF-1α in the serum was measured using ELISA. The citrate-microwave-SP immunohistochemical method was used to measure the expression of HIF-1α in colonic mucosa. The results showed that, HIF-1α in serum was notably higher in UC patients (73.21±28.65) than UC in remission patients (44.54±14.75) and controls (42.83±15.49). The difference between UC patients and UC in remission patients was significant (P<0.05). A correlation analysis revealed that, the HIF-1α level in serum was positively associated with disease activity, disease severity and endoscopic grade. The expression of HIF-1α in colonic mucosa of UC patients was (58.05±13.83) higher than that in UC in remission patients (3.00±2.72) and controls (3.04±2.69) and this difference was statistically significant (P<0.05). A positive correlation was identified between the expression of HIF-1α in colonic mucosa and the disease activity, severity and endoscopic grade. Thus, the present findings indicated that, HIF-1α is likely to play an important role in the pathogenesis of UC and may serve as a biomarker to evaluate disease activity and severity in UC patients.

Abstract 1053: Potential utility of DNA methytlation as a biomarker for prediction of ulcerative colitis associated colitic cancer (UCAC)

Abstract Background: Patients with Ulcerative colitis (UC) have more increased risk for the development of colorectal cancers compared with the average-risk population. Methods or strategies other than intensive colonoscopic surveillance to diagnose ulcerative colitis-associated colorectal carcinoma (UCAC) at early stage are limited. DNA methylation is deeply involved in carcinogenesis in many tumor types and its potential utility as a biomarker has been suggested. Aim: To evaluate potential utility of DNA methylation as the risk marker for UCAC, we quantified DNA methylation of inflamed rectal mucosa, neoplastic tissue and adjacent colonic mucosa of UC patients. Method: Inflamed rectal mucosa (n = 84) and normal appealing proximal mucosa (n = 10) of 84 cancer-free UC patients were examined. Neoplastic tissue (n = 33) and adjacent colonic mucosa (n = 26) from 16 UC patients with neoplasia (UCAC or DALM) were also studied. Bisulfite pyro sequencing was performed for the quantification of 45 cancer or age-related candidate panels and LINE1 repetitive element. Illumina Infinium Human Methylation 450 platform was also used to determine 470,000 CpG sites distributed across the genome. Results: Inflamed mucosa in UC was characterized as hyper methylation in candidate panels using the helical unsupervised clustering. In several panels, hyper methylation was associated with severe clinical phenotypes including steroid dependent, refractory, longstanding severe inflammation etc. (DPYS and miR-1247: p&amp;lt;0.05. GSTP1: p&amp;lt;0.01. mean of three: p&amp;lt;0.0001). Notably, in many panels, hyper methylation was significantly associated with longer duration of disease in cancer-free patients. Among them, some panels showed hyper methyhlation in both neoplastic and adjacent tissue compared to rectal mucosa of cancer-free cases (DPYS and N33, both p values &amp;lt;0.0001). Genome scale analysis revealed that UC with severe inflammation or neoplasia was characterized as hyper methylation at CpG sites rather than outside CpG sites. Gene ontology analysis demonstrated frequently methylated promoter CpG sites in these cases frequently encoded protein related to biosynthetic process (Fold enrichment: 2.07. p value using Benjamini correction: 5.69E-05). Conclusion: Hyper methylation at CpG sites of non-neoplastic colonic mucosa in UC with severe inflammation and neoplasia suggested the potential utility of DNA methytlation as a biomarker for prediction of UCAC. Moreover, enrichment of specific pathway in methylated genes suggests the possibility of targeting therapy for treating UCAC. Citation Format: Tomomitsu Tahara, Naoko Nakano, Mitsuo Nagasaka, Yoshihito Nakagawa, Tomoyuki Shibata, Naoki Ohmiya, Ichiro Hirata. Potential utility of DNA methytlation as a biomarker for prediction of ulcerative colitis associated colitic cancer (UCAC). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1053. doi:10.1158/1538-7445.AM2015-1053