Tu1922 Genome-Wide Epigenomic Analysis of Lamina Propria Mononuclear Cells of Inflammatory Bowel Disease

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using Student's T test. Results: RQ is derived from deltaCT and demonstrates the fold change of gene expression between the two genotypes. RQ and p values were as follows: ERAP2 (1.92, <0.0001), CARD9 (2.51, 0.0006), FASLG (1.11, 0.69), CDKAL1 (1.44, 0.079), RANKL (1.48, 0.37). Conclusions: We have identified two IBD-associated SNP's (rs2549794, rs4077515) where variation correlates with expression of adjacent candidate genes ERAP2 and CARD9 in ileal biopsies. These results link GWAS risk SNPs with a potential mechanism in immunologically relevant genes, and help to explain their impact on IBD pathogenesis.

colitis. Endogenous and exogenous cathelicidin ameliorate intestinal inflammation by inhibiting NF-kappaB-dependent TNFalpha synthesis, suggesting a novel therapy for colitis. Supported by a Pilot and Feasibility Study grant from UCLA-CURE Center, a Career Development Award grant from Crohn's and Colitis Foundation of America, and NIH K01DK084256 Award (HWK). Tu1920 a Farnesoid-X-Receptor (FXR)- Glucocorticoid Receptor (GR) Cascade Regulates Intestinal Innate Immunity in Response to FXR Activation Barbara Renga, Andrea Mencarelli, Claudio D'Amore, Angela Zampella, Eleonora Distrutti, Stefano Fiorucci Background. Nuclear receptors (NRs) are transcription factors that exert homeostatic functions at the interface between nutrient metabolism and innate immunity. The GR is prototypic of a subset of ligand-dependent NRs that integrate host immune responses with steroid metabolism. Glucocorticoids are widely used for the treatment of inflammatory, autoimmune and neoplastic diseases, and remain the first-line treatment for inducing moderate to severe active Crohn's disease and ulcerative colitis. Anti-inflammatory activities have been documented for other NRs, including the FXR, a bile acid sensor that regulates intestinal innate immunity and homeostasis. Aim. To investigate whether FXR regulation of intestinal innate immunity is mediated by GR gene activation. Methods. Colitis was induced in mice by intrarectal administration of TNBS. Mice were administered 6-ECDCA, a semi-synthetic FXR ligand (5 mg/kg) and the GR antagonist mifepristone (5 mg/kg) starting 3 days before TNBS. Relative mRNA expression of GR and pro-inflammatory genes was assayed by RT-PCR. The promoter analysis of human GR gene was performed with the Nubiscan program. Electrophoretic Mobility shift assay (EMSA) and Chromatin Immunoprecipitation (ChIP) were carried out in THP1 monocytic cell line. Results. In Vivo activation of FXR protects against the development of colitis by down-regulating the expression of pro-inflammatory cytokines (TNFα, IL1β, IFNγ) and by increasing the expression of GR mRNA. Mifepristone reversed the protection exerted by the FXR agonist indicating a role for GR in the effects of FXR. Exposure of THP-1 cells to natural or synthetic FXR ligands increased GR mRNA expression. Analysis of both human and mouse GR promoters revealed the presence of a conserved ER-8 sequence (an FXRE). Co-transfection of FXR/RXR significantly enhanced FXR-induced luciferase activity while the transfection with an heterologous promoter construct containing the mutated FXRE failed to be activated by FXR ligand. ChIP experiment demonstrated that FXR is recruited on the GR promoter following FXR activation. EMSA experiment showed that FXR was able to bind to the ER-8 motif and failed to bind to an oligonucleotide bearing the mutation in the ER-8 FXRE. Conclusions. Our results demonstrated that regulation of intestinal innate immunity by FXR involves the GR.

Tu1918 Intestinal Dendritic Cells Survey Circulatory Antigens Prior to Induction of Regulatory CD8α/β T Cells Joo Hye Song, Sun Young Chang, Bayasi Guleng, Carmen Alonso, Seiji Arihiro, Christpher Karp, Atul K. Bhan, Cox Terhorst, Hans-Christian Reinecker Background: Antigen processing by DCs in the intestinal immune system has been linked to the development of tolerance to self-antigens, food and commensal microbiota. It remains controversial which intestinal DC subsets control the generation of regulatory CD4+ or CD8+ T cells to govern mucosal and systemic immune responses. Moreover, antigens in the intestine transition from fenestrated capillaries through the lamina propria into the draining lymphatics, but whether or how this process controls mucosal immune responses is unclear. CX3CR1+ DCs are uniquely positioned for the recognition of environmental and circulatory antigens in the lamina propria as they interact with both the epithelium and with the capillary vessel system in the lamina propria.

Methods: To investigate the uptake of circulatory antigen by CX3CR1+ DCs, we utilized In Vivo imaging system and electron microscopy after intravenous injection of Alexa 647 labeled OVA. OT-I CD8 T cells were cultured with CX3CR1+ DCs isolated from OVA injected CX3CR1-GFP mice and tested for their capacity to inhibit T cell activation In Vitro and to prevent IBD induced by injection of CD4+ CD45RBhigh cells into syngeneic immunodeficient mice.

Results: We demonstrate that small intestinal CX3CR1+ dendritic cells (DCs) acquire and process both circulatory and luminal antigens in the lamina propria. Through cross-presentation of antigen, CX3CR1+ DCs induce differentiation of IL-10 secreting CD8α/β T regulatory cells, that is dependent upon Programmed Death ligand 1/2 and its counter-structure PD1 of CD8 T cells. Differentiated CD8+ T regulatory cells represent a subset of intraepithelial lymphocytes (IELs) which express the specific adhesion molecule CD103. The CD8+ Tregs inhibit antigen specific CD4+ T cell activation through IL-10 secretion and prevent CD45RBhigh CD4 T cell-mediated small intestinal inflammation. Conclusion: Our finding shows that the lamina propria CX3CR1+ DCs facilitate the surveillance of circulatory antigens and act as a conduit for the processing of selfand intestinally- absorbed antigens leading to the induction of CD8+ T regulatory cells, which govern tolerance in the mucosal immune system.

Tu1921 Selective Inhibitors of RIP2 Kinase Activity Reduce PRO-Inflammatory Cytokine Production in IBD Mucosal Biopsy Cultures and Inflammation in Murine TNBS-Induced Colitis Peter Gough, Bart Votta, Linda Casillas, Pamela Haile, David Lipshutz, Biva Desai, Barbara Swift, Carol Ann Capriotti, Michael Reilly, Kevin P. Foley, Paolo Biancheri, Anna Vossenkämper, Thomas T. MacDonald, Robert W. Marquis, John Bertin A breakdown in intestinal immune homeostasis, associated with epithelial barrier disruption, is a common feature among various forms of human inflammatory bowel disease. The resulting interactions between commensal bacteria and mucosal immune cells trigger inflammation via activation of pattern recognition receptors (PRRs). The PRRs NOD1 and NOD2, and their associated effector kinase RIP2, have the potential to be key drivers of intestinal inflammation through their ability to detect intracellular bacterial products such as peptidoglycan derivatives. In order to elucidate the role of NOD1 and NOD2 in mediating this PRRdriven inflammatory response, we have generated and characterized potent and highly selective small molecule inhibitors of RIP2 kinase. One such inhibitor, GSK'214, inhibited RIP2 kinase autophosphorylation (IC50 = 3.2 nM), and showed a high degree of selectivity (>2000-fold) when profiled against a panel of 300 kinases. GSK'214 blocked NOD1 and NOD2-dependent inflammatory cytokine production In Vitro and In Vivo, but did not inhibit TLR-2, -4, -7, -9 or TNFR-mediated signaling. To explore the role of RIP2 kinase in human inflammatory bowel disease we applied our specific inhibitors to cultured intestinal biopsies obtained from Crohn's Disease and Ulcerative Colitis patients. Strikingly, inhibition of RIP2 kinase activity in these cultures attenuated the spontaneous release of pro-inflammatory cytokines in a manner comparable to the steroid prednisilone. Furthermore, oral dosing of a RIP2 kinase inhibitor was able to reduce intestinal inflammation in a murine TNBS-induced colitis model. These results demonstrate the importance of RIP2 kinase activity in modulating intestinal inflammation and suggest that RIP2 kinase inhibition may be a novel therapeutic approach for the treatment of IBD.

Tu1919 Anti-Inflammatory Effects of Anti-Microbial Peptide Cathelicidin in Mice With Acute Colitis Hon Wai Koon, Jeremy Chen, Ryan Ichikawa, David Q. Shih, Samantha Ho, Michelle Cheng, Tressia Hing, Michelle Vu, Ivy Law, Dezheng Zhao, Richard Gallo, Stephan R. Targan, Charalabos Pothoulakis Background and Aims: Cathelicidins (LL-37 in human and mCRAMP in mice) represent a family of endogenous peptides with well-established anti-microbial activity. We previously reported an anti-inflammatory role for endogenous cathelicidin in the dextran sulfate (DSS) mouse model, resembling ulcerative colitis (UC) (Gastroenterology 2011 141:1852-63). Here we examined the role of endogenous and exogenous cathelicidin in the trinitrobenzene sulphonic acid (TNBS) mouse model, resembling Crohn's disease (CD), and determined the anti-inflammatory mechanism of this response. Methods: Wild-type (WT) and mCRAMP deficient (Camp-/-) mice were injected with TNBS intracolonically (100mg/kg, 5 mice/ group). Some mice were treated intracolonically with mCRAMP (5 mg/kg). After 7 days mouse colons were collected for analysis. Results: In colonic biopsies from UC and CD patients (n=10/group), high TNFalpha was statistically associated with elevated LL-37 protein level, suggesting colitis-mediated cathelicidin expression. Following TNBS administration, colonic mCRAMP mRNA levels in WT mice were significantly increased (~15 fold, p= 0.0001), compared to ethanol-treated groups. TNBS exposed Camp-/- mice had significantly higher colitis histology score (by 35%, p=0.04) and TNFalpha levels (by ~25%, p=0.03) compared to WT mice. On the other hand, intracolonic mCRAMP ameliorated acute colitis evidenced by significantly reduced colonic histology score (~40%, p=0.0006) and tissue TNFalpha protein level (~40%, p=0.03). Exposure of human primary colonic epithelial cells to a proinflammatory cytokine cocktail (TNFalpha, IL-1beta and IFNgamma, 10 ng/ml each) increased TNFalpha mRNA and protein expression (by ~10 fold, p=0.0001) which was reduced (by ~20%, p=0.02) in the presence of LL-37 (1 microM). Similarly, incubation (46 hrs) of NCM460 human colonocytes with LL-37 (5-10 microM) completely abolished proinflammatory cytokine cocktail induced TNFalpha with inhibition of NF-kappaB phosphorylation (by ~40%, p=0.04) and transcriptional activity (by ~50%, p=0.01). Inflamed colons from IBD patients (by ~10 fold, p=0.03) and TNBS exposed mice (~2 fold, p=0.0008) had increased expression of phosphorylated NF-kappaB, compared to controls. At day 7 of TNBS colitis, Camp-/- mice developed stronger NF-kappaB phosphorylation (by 50%, p= 0.04) than WT mice but all were significantly diminished by (>50%, p=0.004) intracolonic mCRAMP. Conclusions: Endogenous cathelicidin is induced in colons in response to acute

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Tu1922 Genome-Wide Epigenomic Analysis of Lamina Propria Mononuclear Cells of Inflammatory Bowel Disease Takeshi Otsubo, Yuki I. Kawamura, Kenshiro Oshima, Takaho A. Endo, Tetsuro Toyoda, Teruki Hagiwara, Yutaka J. Kawamura, Fumio Konishi, Hideaki Yano, Yukio Saito, Masahira Hattori, Taeko Dohi Background & Aim: Inflammatory bowel diseases (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), are caused by an inappropriate inflammatory response in a genetically susceptible host triggered by various environmental factors. We assume that epigenetic changes in the immune system may reflect the influence of these environmental factors. Accumulating evidences have demonstrated that chronic inflammation is associated with epigenetic changes, and it may contribute to IBD etiology; however, to our knowledge, epigenetic alterations of inflammatory immune cells in IBD had not been reported. To find out aberrant DNA methylation and chromatin modification in lamina propria mononuclear

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expressed on cells vital to mucosal immune responses. Its expression was up-regulated on activated human T cells, and was further influenced by pharmaceutical TNFα inhibitors. Conclusion We demonstrate, for the first time, a role for GP2 in immune regulation which could be a platform for therapeutic medication for the treatment of IBD. Involvement of the GP2 in bacterial handling could yield great benefit for medical intervention. Last, antiGP2 antibodies might serve as a diagnostic tool for recognition of CD sub-types. Tu1925 MicroRNA Expression in Treatment Naive Active and Inactive Ulcerative Colitis Andrew Claridge, Markus Gwiggner, Rebecca Morgan-Walsh, Sylvia L. Pender, Tilman Sanchez-Elsner, JR Fraser Cummings Introduction MicroRNAs are a group of small non coding RNAs that are becoming recognised as key players in inflammatory pathways through their ability to regulate gene expression. Numerous microRNAs have been shown to be dysregulated in Ulcerative Colitis (UC), although published results are inconsistent possibly due to the combining of different sub-phenotypes and concomitant medication. Many other factors can influence microRNA expression including the origin of the tissue and the degree of inflammatory activity present at the site of biopsy. Defining the microRNA profile in untreated UC patients may give a more accurate understanding of the disease mechanisms underpining the pathophysiology of UC. Aim To identify the microRNA signature in UC patients who are treatment naïve. Methods Sigmoid biopsies were taken from 20 patients with active (Baron grade 2 or 3) UC, 10 patients with previous active UC who had no current activity (macroscopically and microscopically inactive disease) and 20 normal controls (patients investigated for anaemia or rectal bleeding with normal endoscopy). All patients were demographically and ethnically matched and taking no medication. The differential expression of 377 microRNAs were calculated and ranked in active UC (n=6) and controls (n=6) using Taqman® Array Human MicroRNA card A. The 20 microRNAs with the greatest fold increase and fold decrease in expression were then compared across the array cards and consistently dysregulated microRNAs were quantified using RT-qPCR in active UC (n=20), inactive UC (n=10) and normal control samples (n=20). Statistical analysis was performed using the Mann-Whitney test. Results 7 microRNAs that were consistently dysregulated on the array cards were quantified using RT-qPCR. This confirmed an increased fold change in 3 microRNAs; miR-31 (11.67, p<0.001), miR-223 (3.10, p<0.001) and miR-146b-5p (2.15, p=0.002) in active UC compared with normal controls. Only miR-31 and -223 were significantly dysregulated in inactive sigmoid UC compared with normal controls (fold increase of 3.52, p=0.009 and 2.35, p= 0.020 respectively). Four microRNAs were shown to have significantly decreased expression by RT-qPCR. Conclusions and discussion (1) A distinct subset of microRNAs are dysregulated in the mucosa of actively inflamed sigmoid UC in patients who are on no treatment compared to inactive UC patients and controls. (2) miR-31 and -223 have an increased expression in sigmoid UC in the absence of active inflammation. This may give an insight into the relapsing remitting disease course of UC. (3) Manipulating microRNA expression offers promise as a potential new therapeutic pathway in active disease. (4) It is essential when investigating the pathophysiology of UC to define a tight and homogeneous group of patients not only for disease activity but also current treatments.

Tu1923 The Effect of Antigen Oxidation and TGFβ on NaïVE T Cell Phenotype and Proliferation Sahil Khanna, William I. Tremaine, Maneesh Dave, Phyllis A. Svingen, Yuning Xiong, David R. Linden, William A. Faubion Background: A key component of the innate immune response is the oxidation of microbial and host tissue via myeloperoxidase derived hypochlorous acid (HOCl). Chronic inflammatory states involving adaptive immune responses are associated with protein oxidation; yet little data exist regarding the T cell response to oxidized antigens, particularly within the TGFβ-rich milieu of the intestine. Aim: To characterize phenotype & proliferation of naïve murine CD4+ T cells in response to oxidized ovalbumin and TGFβ. Methods: Naïve murine T-cells (CD4+ CD62+) from ovalbumin transgenic II (OTII) mice were exposed to ovalbumin (Ova) or ovalbumin exposed to HOCl (Cl-ova) fed irradiated antigen presenting cells (APCs), and T-cell proliferation & development of regulatory T-cells was measured. OTII mice were also fed with Ova or Cl-Ova and T-cell responses were measured in a delayed type hypersensitivity (DTH) model. Results: When compared to Ova-loaded, Cl-Ova loaded APCs led to enhanced proliferation of both naïve OTII and DO11.10 T cells measured by thymidine incorporation. OVA-loaded and Cl-OVA-loaded APCs demonstrated similar surface expression of MHCII, CD80, and CD86; thus Cl-OVA induced hyperproliferative response were independent of co-stimulatory signals. Furthermore, on treatment with TGFβ, Cl-OVA loaded APCs induced proliferation of both DO11.10 and OTII naïve T cells as opposed to suppression in response to OVA-loaded APCs. The enhanced proliferation effect was related to T-cell receptor strength of signaling enhancement measured at 18 hours. This effect on T-cell proliferation was not IL-2 dependent as there were no differences in IL-2 as measured by mRNA and cytokine production. Cl-OVA fed OTII mice had more CD4+ T cells within the mesenteric lymphatics compared to OVA-fed mice. The expanding T cell population in response to Cl-OVA-loaded APCs and TGFβ contained a significant proportion of FOXP3expressing cells. These cells demonstrated suppressive regulatory T-cell function in in-vitro suppression assays. Both Ova and Cl-ova fed mice had dampened responses to antigen challenge in a DTH model. In chronic inflammatory conditions outside the intestine, neutrophil activation/infiltration leads to HOCl-mediated protein oxidation demonstrated by elevated chloro-tyrosine levels. Despite heavy neutrophil infiltration, colonic tissue isolated from humans with chronic ulcerative colitis had no increase in chloro-tyrosine when compared to normal. Conclusion: These data demonstrate that HOCl-mediated oxidation of antigens enhances T cell proliferation particularly in response to TGFβ. Furthermore, TGFβ treatment leads to expansion of FOXP3+ Treg cells. The lack of evidence for HOCl-mediated oxidation in the diseased human colon suggests that the absence of this phenomenon may contribute to chronic intestinal inflammation.

Tu1926 Mucosal Gene Expression Profiles in Pouchitis and in Active Ileal Crohn's Disease are Very Similar Ingrid Arijs, Jan Van der Goten, Marc Ferrante, Kathleen Machiels, Vicky De Preter, Leentje Van Lommel, Gert Van Assche, Severine Vermeire, Frans C. Schuit, Paul J. Rutgeerts Introduction and aim: Pouchitis is the most important long-term complication of ileal pouchanal anastomosis (IPAA) for ulcerative colitis (UC). The etiology of pouchitis is not understood and pouchitis is a useful model to study early inflammatory bowel disease. We investigated the gene expression profiles of mucosal pouch biopsies in comparison with the profiles of ileal mucosa of Crohn's ileitis (CDi), colonic mucosa of UC and ileal mucosa of healthy controls, focusing specifically on gene expression of cell adhesion molecules (CAMs) and antimicrobial peptides (AMPs). Methods: Mucosal biopsies were obtained from the pouch of 15 IPAA patients (6 with endoscopic pouchitis and 9 with endoscopically normal pouch), from the (neo-)terminal ileum of 45 CDi patients [18 with mild disease (only aphthous lesions) and 27 with moderate to severe disease (presence of ulcers)] and 12 controls with normal ileum, and from the colon of 21 UC patients [10 with active disease (endoscopic Mayo score 2-3) and 11 with disease in remission (endoscopic Mayo score 0)]. Total RNA isolated from biopsies was used to analyze the gene expression via Affymetrix GeneChip® Human Gene 1.0 ST arrays. Data was analyzed with Bioconductor software. Pair-wise comparisons of the pouch gene expression profiles with the profiles of CDi, UC and control ileums were performed (significant genes: false discovery rate<5% and >2-fold change). Results: No CAM and AMP genes were significantly differentially expressed between normal pouch and mild active CDi, and between pouchitis and moderate to severe CDi. In contrast, many significant differences in gene expression of CAMs and AMPs were found between normal pouch/pouchitis and remission/active UC, e.g. gene expression of TECK, NTS, REG3A, LEAP2, DEFA5 and DEFA6 was >2-fold significantly increased, and SLPI gene expression was >2-fold significantly decreased in both pouch groups vs. UC groups. As compared to control ileums, gene expression of 13 CAMs (ICAM1, PECAM1, SELE, SELP, CCL2, CCL28, CXCL1, CXCL2, CXCL5, CXCL6, IL8, CXCR1 and CXCR2) and 7 AMPs (S100A8, S100A9, S100A12, C1R, NOS2, PI3 and LCN2) was >2-fold significantly increased, and only 2 AMPs (NTS and LEAP2) were >2-fold significantly decreased in both pouchitis and moderate to severe CDi. Moreover, gene expression of CCL28, NOS2 and LCN2 was already >2-fold significantly increased in normal pouch vs. control ileums. Conclusion: Our data demonstrate that patients with normal pouch have a similar gene expression profile as mild active CDi, and that the profiles of pouchitis are similar to the profiles of moderate to severe CDi. Many CAMs and AMPs are dysregulated in patients with pouchitis, and there is already upregulation of CCL28, NOS2 and LCN2 in patients with a normal pouch. This work can help to further refine therapeutic strategies in pouchitis and ileal CD.

Tu1924 Identification of Glycoprotein 2 as an Immunomodulator of Innate and Adaptive Immune Responses Lael Werner, Dirk Reinhold, Dirk Roggenbuck, Andreas Sturm Background: Pancreatic autoantibodies (PAB) are presumably Crohn's disease (CD)-specific serological markers. Their antigen, glycoprotein 2 (GP2), has only recently been identified. GP2 is secreted from pancreatic granules and is suggested to support bacterial handling. However, the functional roles of GP2 in mucosal immunity are unknown. We hypothesized that not only GP2 antibodies differentiate CD patients, but also that GP2 itself modulates innate and adaptive immune responses. Methods& Results: We initially confirmed anti-GP2 levels are significantly elevated in CD, using ELISA. For functional studies, we cultured stimulated peripheral blood, lamina propria, or intraepithelial T lymphocytes; as well as human intestinal epithelial cells or epithelial line T84 with or without GP2 for 48h. GP2 has an immunosupressive effect on T cells, as observed by decreased cell cycling (flow cytometric PI staining), activation (CD25 expression), pro-inflammatory cytokine secretion (ELISA) and apoptosis (annexinV/PI staining). GP2 also modifies epithelial function as observed by decreased cell cycling and IL8 secretion (ELISA), as well as modulation of TLR4, E-Cadherin and CD49d expression. No migratory influence was exerted by GP2 on either PBMCs (Transwell assay) or epithelial cells (wound healing assay). However, T84 stimulated with GP2 potently chemoattracts PBMCs (Transwell co-cultures). Moreover, GP2 increased antigen presentation by T84 as observed by increased expression of HLA-DR, CD40, and MICA. In phagocytosis, GP2 augments uptake of E.Coli by epithelial cells and even more efficiently by monocytes (Phagocytosis Assay Kit). Last, GP2 was ubiquitously

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cells (LPMCs) of IBD patients in detail, we performed genome-wide analyses by using the Next-Generation Sequencer (NGS). Method: LPMCs were isolated from both inflamed UC mucosa and macroscopically normal mucosa of non-IBD subjects. We obtained EpCAMCD3+ T cells and EpCAM-CD33+ macrophages (Mφ) and dendritic cells (DC) with a cell sorter. Purified CD3+T and CD33+ cells were immediately fixed with formalin for the chromatin-crosslinking followed by the chromatin shearing. The sheared chromatins were applied to a chromatin immunoprecipitation (ChIP) with anti-H3K4me3, anti-H3K9me3 and anti-H3K27me3 antibodies. For the methylated DNA, MeDIP analysis was performed. Immunoprecipitated-DNA was subjected to the SOLiD library construction system and sequenced by the NGS SOLiD4. Result We obtained 60-70% and 53-65% of reliable sequence tags for ChIP-sequencing (ChIP-seq) and MeDIP-sequencing (MeDIP-seq), respectively, on the reference Human genome, ensuring the fidelity of the sequence data. In the genomewide comparative analysis, we found that DNA methylation levels were globally lower in UC samples than in control non-IBD samples, especially in CD33+ cells. In addition, increased H3K4me3 marks were observed in the upstream of inflammatory cytokine genes, such as Il1b, in CD33+ cells but not in CD3+T cells. Conclusion: In this study, we established ChIPseq and MeDIP methods for genome-wide epigenetic analysis of immune cells in IBD mucosa. We found for the first time the genome-wide hypomethylation in the LPMC of the UC patient. Furthermore, the patterns of histone modifications and DNA methylation in specific loci were quite different between CD3+T and CD33+Mφ/DC cell fractions. Our results indicate that simultaneous integrated analysis of multiple epigenetic modifications in specific cell fractions is useful to find marks associated with disease pathogenesis and/or severity.