Pathogenesis Of Crohn's Disease Research Articles

Nuclear factor-κB - importance, induction of inflammation, and effects of pharmacological modulators in Crohn's disease.

Crohn's disease (CD) is a chronic inflammatory disease of unknown etiology that covers the entire digestive tract and occurs with periods of remission and clinical exacerbation. CD is most common in North America and Europe, but its incidence is rising rapidly in Asian countries. The pathogenesis of CD is unclear, while genetic predisposition, immune imbalance, and host-intestinal microbiota interactions are taken into account. Incorrect activation of κB nuclear factor (NF-κB) signaling pathways is associated with CD initiation and progression. NF-κB leads to excessive production of pro-inflammatory cytokines that cause a chronic inflammatory process of the intestines. It is currently believed that the NF-κB pathway plays a key role in the pathogenesis of CD, hence current treatments aim to block this pathway. Studies have shown that activation of NF-κB is reduced by treatment with, among others, mesalazine and glucocorticoids. This review presents epidemiology and pathogenesis of CD, the participation of NF-κB in this disease, as well as modern methods of treatment aimed at inhibiting NF-κB activation.

Under-expression of microRNA-146a and 21 and their association with Crohn's disease.

MicroRNAs (miRNAs) can post-transcriptionally regulate gene expression and are involved in the immune response. Excessive immune response to the gut microbiota plays a major role in the pathogenesis of Crohn's disease (CD). Regarding the role of miRNAs in immune response, this study aimed to investigate the contribution of miRNAs in the pathogenesis of CD. A total of 53 participants, including 23 CD patients and 30 healthy controls (HCs) were enrolled in this study. miRNAs, including miR-21, miR-29a, miR-29b, miR-31, miR-146a, miR-155, miR-181a, and miR-181c were evaluated via TaqMan MicroRNA Assays. Among the eight miRNAs, the amounts of miR-146a and miR-21 were significantly decreased in the CD patients relative to HC subjects. Moreover, we showed that there was a negative correlation between miR-146a and Harvey-Bradshaw index (HBI), as well as a positive correlation of miR-21 and miR-29b with HBI. Under-expression of miR-146a and miR-21, which are critical for the regulatory function of regulatory T cells (Tregs), is remarkably associated with CD.

Monocytes from infliximab-resistant patients with Crohn\u2019s disease exhibit a disordered cytokine profile

Crohn's disease (CD) is a chronic inflammatory disorder characterized by immune response dysregulation. Tumor necrosis factor-α (TNFα) is a key cytokine in the pathogenesis of CD, as indicated by the efficacy of anti-TNF-α therapy with infliximab (IFX). However, approximately 30–40% of CD patients fail to respond to IFX with still unclear underlying mechanisms. This study compares the inflammatory phenotype of monocytes from CD patients, who respond or non-respond to IFX. Under basal conditions, the mRNA for the cytokines TNFα, IL-23, IL-1β and the chemokines CXCL8/IL-8, CCL5/RANTES and CCL2/MCP-1 was up-regulated in monocytes from non-responders than responders. The expression of the same cytokines and CCL2/MCP-1 was higher in non-responders also upon LPS treatment. Moreover, higher secretion of TNFα, IL-1β, IFNγ and IL-2 proteins occurred in the supernatants of LPS-treated non-responders cells. Resistance to IFX in CD may result from a transcriptional dysregulation of circulating monocytes, leading to hyperactivation of pro-inflammatory pathways. Monocytes’ cytokine profile may thus represent a predictive marker of response to IFX. Monocytes were isolated from blood samples of 19 CD patients (11 responders, 8 non-responders) and incubated with or without LPS. Cytokine profiles were assessed by RT-qPCR and, in the supernatants, by ELISA assay.

Genetic variation in the farnesoid X-receptor predicts Crohn\u2019s disease severity in female patients

The farnesoid X receptor (FXR) is implicated in Crohn's disease (CD) pathogenesis. It is unclear how genetic variation in FXR impacts CD severity versus genetic variation in nuclear receptors such as pregnane X receptor (PXR) and the multi-drug resistance protein 1 (MDR1, ABCB1). To evaluate FXR-1G > T as a genomic biomarker of severity in CD and propose a plausible molecular mechanism. A retrospective study (n = 542) was conducted in a Canadian cohort of CD patients. Genotypic analysis (FXR-1G > T, MDR1 3435C > T and PXR -25385C > T) as well as determination of the FXR downstream product, fibroblast growth factor (FGF) 19 was performed. Primary outcomes included risk and time to first CD-related surgery. The effect of estrogen on wild type and variant FXR activity was assessed in HepG2 cells. The FXR-1GT genotype was associated with the risk of (odds ratio, OR = 3.34, 95% CI = 1.58–7.05, p = 0.002) and earlier progression to surgery (hazard ratio, HR = 3.00, 95% CI = 1.86–4.83, p < 0.0001) in CD. Female carriers of the FXR-1GT genotype had the greatest risk of surgery (OR = 14.87 95% CI = 4.22–52.38, p < 0.0001) and early progression to surgery (HR = 6.28, 95% CI = 3.62–10.90, p < 0.0001). Women carriers of FXR-1GT polymorphism had a three-fold lower FGF19 plasma concentration versus women with FXR-1GG genotype (p < 0.0001). In HepG2 cells cotransfected with estrogen receptor (ER) and FXR, presence of estradiol further attenuated variant FXR activity. MDR1 and PXR genotypes were not associated with surgical risk. Unlike MDR1 and PXR, FXR-1GT genetic variation is associated with earlier and more frequent surgery in women with CD. This may be through ER-mediated attenuation of FXR activation.

Propionate Induces Virulent Properties of Crohn's Disease-Associated Escherichia coli.

Crohn’s disease (CD) is a severe chronic immune-mediated granulomatous inflammatory disease of the gastrointestinal tract. The mechanisms of CD pathogenesis remain obscure. Metagenomic analysis of samples from CD patients revealed that several of them have the elevated level of Escherichia coli with adhesive–invasive phenotype (AIEC). Previously, we isolated an E. coli strain CD isolate ZvL2 from a patient with CD, which features AIEC phenotype. Here, we demonstrate that prolonged growth on propionate containing medium stimulates virulent properties of CD isolate ZvL2, while prolonged growth on glucose reduces these properties to levels indistinguishable from laboratory strain K-12 MG1655. Propionate presence also boosts the ability of CD isolate ZvL2 to penetrate and colonize macrophages. The effect of propionate is reversible, re-passaging of CD isolate on M9 medium supplemented with glucose leads to the loss of its virulent properties. Proteome analysis of CD isolate ZvL2 growth in medium supplemented with propionate or glucose revealed that propionate induces expression porins OmpA and OmpW, transcription factors PhoP and OmpR, and universal stress protein UspE, which were previously found to be important for macrophage colonization by enteropathogenic bacteria.

A sequence-dependent classification algorithm for Crohn’s Disease – causing NOD2 protein mutations

Certain NOD2 protein mutations have been associated with the onset of the inflammatory bowel disease, Crohn’s Disease (CD). NOD2 is involved in the inflammatory response of the gut to the microbial community, wherein its functional impairment through mutations may lead to CD progression. Considering the significant role that NOD2 plays in CD pathogenesis, predicting whether a specific type of NOD2 mutation is the cause of CD can greatly aid the accuracy of the disease diagnosis. Hence, a novel sequence-based classification algorithm built on artificial neural network (ANN) is herein presented that can predict whether a specific NOD2 mutation can cause CD or not. The NOD2 mutant types and their association with CD were taken from literature, and the calculated sequence-order coupling numbers were used as the classification predictors. The formulated ANN classifier exhibited satisfactory predictive ability, with 82.4 % accuracy, 62.5 % sensitivity, 100 % specificity, 100 % positive predictive value, and 75 % negative predictive value. The presented ANN classifier provides a proof-of-concept that predicting the onset of CD from NOD2 protein variant is possible.

Epithelioid Cell Granulomas in Crohn's Disease Are Differentially Associated With Blood Vessels and Lymphatic Vessels: A Sequential Double Immunostaining Study.

Crohn's disease (CD) is a gastrointestinal disorder of unknown etiology. CD-specific longitudinal ulcers show an association between disease pathogenesis and vasculature dysfunction. Granulomatous lymphangitis may also contribute to CD pathogenesis; meanwhile, vasculitis is the primary CD lesion. We investigated the association between granulomas and lymphatic and blood vessels to assess the role of vasculature in CD pathogenesis. Two small and large intestine specimens were obtained from four CD patients. From each specimen, 160 sequential sections were obtained and double immunohistochemical stained to label lymphatic and blood vessels in association with granulomas. We found that 289 of 342 granulomas (85%) were associated with a lymphatic vessel and 313 of 364 granulomas (86%) were associated with a blood vessel. Although intrablood vessel granulomas were not detected, intralymphatic vessel granulomas were. In the internal region of the granuloma, we found more blood vessels than lymphatic vessels. Hence, these results cumulatively demonstrate that CD epithelioid cell granulomas are differentially associated with lymphatic and blood vessels, suggesting both as essential for the formation and maintenance of these granulomas. Moreover, both lymphatic and blood vessels may participate in granulomatous inflammation in the primary CD lesions; however, additional studies with larger numbers of participants are required to validate our findings.

Hsa_circRNA_102610 upregulation in Crohn's disease promotes transforming growth factor-β1-induced epithelial-mesenchymal transition via sponging of hsa-miR-130a-3p.

BACKGROUNDThe incidence of inflammatory bowel disease, a chronic intestinal inflammatory disorder that includes Crohn’s disease (CD) and ulcerative colitis, is rising. Circular RNAs are considered valuable diagnostic biomarkers for CD. Current evidence supports the views that epithelial-mesenchymal transition (EMT) plays an important role in CD pathogenesis, and that hsa-miR-130a-3p can inhibit transforming growth factor-β1 (TGF-β1)-induced EMT. Our previous study revealed that hsa_circRNA_102610 was upregulated in CD patients. Moreover, we predicted an interaction between hsa_circRNA_102610 and hsa-miR-130a-3p. Thus, we hypothesized that hsa_circRNA_102610 may play roles in the proliferation and EMT of intestinal epithelial cells by sponging hsa-miR-130a-3p to participate in the pathogenesis of CD.AIMTo explore the mechanism of hsa_circRNA_102610 in the pathogenesis of CD.METHODSThe relative expression levels of hsa_circRNA_102610 and hsa-miR-130a-3p in patients were detected by quantitative reverse transcription-polymerase chain reaction. The proliferation of human intestinal epithelial cells (HIECs) and normal-derived colon mucosa cell line 460 (NCM460) cells was detected by cell counting kit-8, 5-ethynyl-2’-deoxyuridine staining and cell cycle assays following overexpression or downregulation of hsa_circRNA_102610. Cell proliferation assays were performed as described above in a rescue experiment with hsa-miR-130a-3p mimics. The interaction of hsa_circRNA_102610 and hsa-miR-130a-3p was verified by fluorescence in situ hybridization and dual luciferase reporter assays. The relative expression levels of CyclinD1, mothers against decapentaplegic homolog 4 (SMAD4), E-cadherin, N-cadherin and Vimentin were detected by western blotting following hsa_circRNA_102610 overexpression, TGF-β1-induced EMT or hsa-miR-130a-3p mimic transfection (in rescue experiments).RESULTSUpregulation of hsa_circRNA_102610 was determined to be positively correlated with elevated fecal calprotectin levels in CD (r = 0.359, P = 0.007) by Pearson correlation analysis. Hsa_circRNA_102610 promoted the proliferation of HIECs and NCM460 cells, while hsa-miR-130a-3p reversed the cell proliferation-promoting effects of hsa_circRNA_102610. Fluorescence in situ hybridization and dual luciferase reporter assays showed that hsa_circRNA_102610 directly bound hsa-miR-130a-3p in NCM460 and 293T cells. An inverse correlation between downregulation of hsa-miR-130a-3p and upregulation of hsa_circRNA_102610 in CD patients was observed (r = -0.290, P = 0.024) by Pearson correlation analysis. Moreover, overexpression of hsa_circRNA_102610 promoted SMAD4 and CyclinD1 protein expression validated by western-blotting. Furthermore, over-expression of hsa_circRNA_102610 promoted TGF-β1 induced EMT in HIECs and NCM460 cells via targeting of hsa-miR-130a-3p, with increased expression of Vimentin and N-cadherin and decreased expression of E-cadherin.CONCLUSIONHsa_circRNA_102610 upregulation in CD patients could promote the proliferation and EMT of intestinal epithelial cells via sponging of hsa-miR-130a-3p.

Modern principles of Crohn's disease diagnosis in children

The aim of our work is to review and analyze scientific literature to highlight the world experience in diagnosing Crohn’s disease (CD) in children, given the need to maintain a unified strategy in diagnostic tactics for this pathology.CD is an autoimmune disease with a chronic progressive transmural inflammatory process of unknown etiology, which can affect the entire gastrointestinal tract from the oral cavity to the anal canal in patients of any age. Approximately 20–25 % of CD cases manifest in patients under the age of 18 years. The pathogenesis of CD is a multifocal process in which genetic mutations of more than 50 genes take place. Morphological changes are diverse and not fully understood. CD is characterized by systemic, local manifestations and the presence of surgical complications. The main diagnostic methods are endoscopic, radiological, morphological and MRI. Differential diagnosis of CD should include ulcerative colitis, inflammatory and autoimmune intestinal diseases in children, congenital malformations of the gastrointestinal tract. Today, the diagnosis of CD in children remains debatable.Conclusions. In the lack of a clear idea of the etiological factors, pathogenesis of CD in children and its consequences, finding diagnostic tactics for this pathology determines the urgency of the issue in pediatrics and pediatric surgery. Diagnostic search in children with CD is a lengthy and materially expensive, requiring powerful facilities and equipment as well as fundamental competencies of a physician. The cascade approach to the diagnosis of CD proposed by the World Gastroenterology Organization (WGO) is the best option for determining the optimal targeted diagnostic algorithm for each patient, taking into account the existing logistic support at a hospital and the financial capacity of a patient.

Adherent-Invasive E. coli: Update on the Lifestyle of a Troublemaker in Crohn's Disease.

Besides genetic polymorphisms and environmental factors, the intestinal microbiota is an important factor in the etiology of Crohn’s disease (CD). Among microbiota alterations, a particular pathotype of Escherichia coli involved in the pathogenesis of CD abnormally colonizes the intestinal mucosa of patients: the adherent-invasive Escherichia coli (AIEC) pathobiont bacteria, which have the abilities to adhere to and to invade intestinal epithelial cells (IECs), as well as to survive and replicate within macrophages. AIEC have been the subject of many studies in recent years to unveil some genes linked to AIEC virulence and to understand the impact of AIEC infection on the gut and consequently their involvement in CD. In this review, we describe the lifestyle of AIEC bacteria within the intestine, from the interaction with intestinal epithelial and immune cells with an emphasis on environmental and genetic factors favoring their implantation, to their lifestyle in the intestinal lumen. Finally, we discuss AIEC-targeting strategies such as the use of FimH antagonists, bacteriophages, or antibiotics, which could constitute therapeutic options to prevent and limit AIEC colonization in CD patients.

KLRG1 and CD103 Expressions Define Distinct Intestinal Tissue-Resident Memory CD8 T Cell Subsets Modulated in Crohn's Disease.

Intestinal tissue-resident memory CD8 T cells (Trm) are non-recirculating effector cells ideally positioned to detect and react to microbial infections in the gut mucosa. There is an emerging understanding of Trm cell differentiation and functions, but their implication in inflammatory bowel diseases, such as Crohn's disease (CD), is still unknown. Here, we describe CD8 cells in the human intestine expressing KLRG1 or CD103, two receptors of E-cadherin. While CD103 CD8 T cells are present in high numbers in the mucosa of CD patients and controls, KLRG1 CD8 T cells are increased in inflammatory conditions. Mucosal CD103 CD8 T cells are more responsive to TCR restimulation, but KLRG1 CD8 T cells show increased cytotoxic and proliferative potential. CD103 CD8 T cells originate mostly from KLRG1 negative cells after TCR triggering and TGFβ stimulation. Interestingly, mucosal CD103 CD8 T cells from CD patients display major changes in their transcriptomic landscape compared to controls. They express Th17 related genes including CCL20, IL22, and IL26, which could contribute to the pathogenesis of CD. Overall, these findings suggest that CD103 CD8 T cells in CD induce a tissue-wide alert increasing innate immune responses and recruitment of effector cells such as KLRG1 CD8 T cells.

Aspects of free radical oxidation in the large bowel in ulcerative colitis and Crohn’s disease

Research into the accumulation patterns of protein oxidative modification (POM) products and lipids in Crohn’s disease (CD) and ulcerative colitis (UC) could have important implications for understanding the pathogenesis and improving the diagnosis and therapy for these diseases. The aim of this study was to investigate the aspects of free radical oxidation (FRO) in the large bowel and their possible correlations with clinical symptoms of UC and CD. In the Wistar rat model used in the experiment, CD was induced with 2,4,6-trinitrobenzenesulfonic acid, and UC was induced with oxazolone. Clinical status was assessed using the Disease activity index (DAI). Lipid peroxidation (LPO) products were measured in the heptane and isopropanol phases of the intestinal mucosa extract. POM products were measured following spontaneous and stimulated oxidation. The DAI ( Me (Q25–Q75)) was increased in both CD and UC on days 3 and 7 of the experiment: for CD, it was equally increased on days 3 and 7 (7 (3-7)) and was 11 (11–11) and 11 (9–11) for UC on days 3 and 7, respectively. The amount of primary, secondary and end LPO products in the heptane and isopropanol phases, as well as the total amount of POM products, was increased in the homogenized mucosa of the large bowel. In the CD group, the relative content of secondary basic POM products was increased on day 7 of the experiment. The following patterns of FRO were revealed: accumulation of LPO products in the UC group and accumulation of POM products in the CD group; UC is characterized by the accumulation of mostly LPO products in the heptane phase and secondary LPO products in the isopropanol phase; CD is characterized by the accumulation of secondary basic POM products. DAI scores were correlated with the amount of LPO products in the isopropanol phase and the amount of POM products in the spontaneous oxidation mode. The highest number of strong correlations was observed in the UC group. Our findings suggest a very serious contribution of FRO changes to the pathogenesis of UC and CD, meaning that LPO and POM products could be regarded as diagnostic markers and indicators of treatment efficacy.

NOD2 modulates immune tolerance via the GM-CSF–dependent generation of CD103+ dendritic cells

Four decades ago, it was identified that muramyl dipeptide (MDP), a peptidoglycan-derived bacterial cell wall component, could display immunosuppressive functions in animals through mechanisms that remain unexplored. We sought to revisit these pioneering observations because mutations in NOD2, the gene encoding the host sensor of MDP, are associated with increased risk of developing the inflammatory bowel disease Crohn's disease, thus suggesting that the loss of the immunomodulatory functions of NOD2 could contribute to the development of inflammatory disease. Here, we demonstrate that intraperitoneal (i.p.) administration of MDP triggered regulatory T cells and the accumulation of a population of tolerogenic CD103+ dendritic cells (DCs) in the spleen. This was found to occur not through direct sensing of MDP by DCs themselves, but rather via the production of the cytokine GM-CSF, another factor with an established regulatory role in Crohn's disease pathogenesis. Moreover, we demonstrate that populations of CD103-expressing DCs in the gut lamina propria are enhanced by the activation of NOD2, indicating that MDP sensing plays a critical role in shaping the immune response to intestinal antigens by promoting a tolerogenic environment via manipulation of DC populations.

Identification of Immune Cell Landscape and Construction of a Novel Diagnostic Nomogram for Crohn's Disease.

Crohn’s disease (CD) has an increasing incidence and prevalence worldwide. The etiology of CD remains unclear and there is no gold standard for diagnosis. The dysregulated immune response and different infiltration status of immune cells are critical for CD pathogenesis; therefore, it is important to provide an overview of immune-cell alterations in CD and explore a novel method for auxiliary diagnosis. Here we analyzed microarray datasets from Gene Expression Omnibus (GEO), and an extended version of Cell-type Identification By Estimating Relative Subsets Of RNA Transcripts (CIBERSORTx) was utilized to estimate the fraction of 22 types of immune cells. Differentially expressed genes (DEGs) and a protein-protein interaction (PPI) network were identified, and we performed gene set enrichment analysis (GSEA) and gene set variation analysis (GSVA) to identify differentially regulated pathways in CD. Least absolute shrinkage and selection operator (LASSO) regression was conducted to filter features, and a diagnostic nomogram based on logistic regression was built and validated in an independent validation cohort. In the derivation cohort, we found a proportion of 17 immune-cell types to be significantly altered between CD and healthy controls and a total of 150 DEGs were identified, which were mostly related to the immune response. Among the 15 hub genes based on the PPI network, C-X-C chemokine ligand 8 (CXCL8) and interleukin-1B (IL-1B) showed the highest degree of interaction. Additionally, GSEA and GSVA identified five significantly enriched pathways, among which the nucleotide-binding oligomerization domain (NOD)-like receptor signaling pathway was critical in the CD development. Furthermore, six variables comprising of CXCL8, IL-1B, M1 macrophages, regulatory T cells, CD8+ T cells, and plasma cells were identified by LASSO regression and incorporated into a logistic regression model. The nomogram displayed a good prediction, with a 0.915 area under the receiver operating curve (AUC) and a C-index of 0.915 [95% confidence interval (CI): 0.875–0.955]. Similar results were found in the validation cohort, with an AUC of 0.884 and a 0.884 C-index (95% CI: 0.843–0.924). These results provide novel in silico insight into cellular and molecular characteristics of CD and potential biomarkers for diagnosis and targeted therapy.

Paneth cell α-defensin misfolding correlates with dysbiosis and ileitis in Crohn’s disease model mice

Crohn's disease (CD) is an intractable inflammatory bowel disease, and dysbiosis, disruption of the intestinal microbiota, is associated with CD pathophysiology. ER stress, disruption of ER homeostasis in Paneth cells of the small intestine, and α-defensin misfolding have been reported in CD patients. Because α-defensins regulate the composition of the intestinal microbiota, their misfolding may cause dysbiosis. However, whether ER stress, α-defensin misfolding, and dysbiosis contribute to the pathophysiology of CD remains unknown. Here, we show that abnormal Paneth cells with markers of ER stress appear in SAMP1/YitFc, a mouse model of CD, along with disease progression. Those mice secrete reduced-form α-defensins that lack disulfide bonds into the intestinal lumen, a condition not found in normal mice, and reduced-form α-defensins correlate with dysbiosis during disease progression. Moreover, administration of reduced-form α-defensins to wild-type mice induces the dysbiosis. These data provide novel insights into CD pathogenesis induced by dysbiosis resulting from Paneth cell α-defensin misfolding and they suggest further that Paneth cells may be potential therapeutic targets.

Mitochondrial impairment drives intestinal stem cell transition into dysfunctional Paneth cells predicting Crohn’s disease recurrence

ObjectiveReduced Paneth cell (PC) numbers are observed in inflammatory bowel diseases and impaired PC function contributes to the ileal pathogenesis of Crohn’s disease (CD). PCs reside in proximity to Lgr5+...

LncRNACNN3-206 activates intestinal epithelial cell apoptosis and invasion by sponging miR-212, an implication for Crohn's disease

BACKGROUNDStatistics indicate that the incidence of Crohn’s disease (CD) is rising in many countries. The poor understanding on the pathological mechanism has limited the development of effective therapy against this disease. Previous studies showed that long noncoding RNAs (lncRNAs) could be involved in autoimmune diseases including CD, but the detailed molecular mechanisms remain unclear.AIMTo identify the differentially expressed lncRNAs in the intestinal mucosa associated with CD, and to characterize their pathogenic role(s) and related mechanisms.METHODSThe differential expression of lncRNAs was screened by high-throughput RNA sequencing, and the top candidate genes were validated in an expanded cohort by real-time PCR. The regulatory network was predicted by bioinformatic software and competitive endogenous RNA analysis, and was characterized in Caco-2 and HT-29 cell culture using methods of cell transfection, real-time PCR, Western blotting analysis, flow cytometry, and cell migration and invasion assays. Finally, these findings were confirmed in vivo using a CD animal model.RESULTSThe 3' end of lncRNACNN3-206 and the 3’ UTR of Caspase10 contain high-affinity miR212 binding sites. lncRNACNN3-206 expression was found to be significantly increased in intestinal lesions of CD patients. Activation of the lncRNACNN3-206-miR-212-Caspase10 regulatory network led to increased apoptosis, migration and invasion in intestinal epithelial cells. Knockdown of lncRNACNN3-206 expression alleviated intestinal mucosal inflammation and tissue damage in the CD mouse model.CONCLUSIONlncRNACNN3-206 may play a key role in CD pathogenesis. lncRNACNN3-206 could be a therapeutic target for CD treatment.

Protein kinase 2 (CK2)controls CD4+ T cell effector function in the pathogenesis of colitis.

Crohn’s disease (CD), one of the major forms of inflammatory bowel disease (IBD), is characterized by chronic inflammation of the gastrointestinal tract and associated with aberrant CD4+ Th1 and Th17 responses. Protein kinase 2 (CK2) is a conserved serine-threonine kinase involved in signal transduction pathways which regulate immune responses. CK2 promotes Th17 cell differentiation and suppresses the generation of Foxp3+ regulatory T cells. The function of CK2 in CD4+ T-cells during the pathogenesis of CD is unknown. We utilized the T-cell induced colitis model, transferring CD45RBhi naïve CD4+ T-cells from CK2αfl/fl controls and CK2αfl/fldLck-Cre mice into Rag 1−/− mice. CD4+ T-cells from CK2αfl/fldLck-Cre mice failed to induce wasting disease and significant intestinal inflammation, which was associated with decreased IL-17A+, IFN-γ+ and double positive IL-17A+ IFN-γ+ CD4+ T-cells in the spleen and colon. We determined that CK2α regulates CD4+ T-cell proliferation through a cell-intrinsic manner. CK2α is also important in controlling CD4+ T-cell responses by regulating NFAT2, which is vital for T-cell activation and proliferation. Our findings indicate that CK2α contributes to the pathogenesis of colitis by promoting CD4+ T-cell proliferation and Th1 and Th17 responses, and that targeting CK2 may be a novel therapeutic treatment for patients with CD.

P828 DNA methylation profile defines epigenetic characteristics and molecular targets of Crohn’s disease

Abstract Background Inflammatory bowel disease(IBD) is known to be caused by a genetic predisposition involving multiple genes; however, there is growing evidence that abnormal interaction with environmental, particularly epigenetic, factors can have a significant contribution during the development of IBD. Although many studies, particularly genome-wide association studies (GWAS), have been performed to identify the genetic changes underlying the pathogenesis of Crohn’s disease (CD), the role of epigenetic changes in the development of complications arising from CD is poorly understood. Methods Here, we employed an unbiased approach to define DNA methylation alteration in CD patients using the Human Methylation 450K Bead Chip platform. Compared to normal controls, the majority of differential DNA methylation in CD patient samples was in the promoter, intergenic, and gene body regions. Results The DNA methylation profile in CD revealed 134 probes (23 hypermethylated and 111 hypomethylated probes) that were differentially methylated. We validated the methylation levels of 19 genes that showed hypermethylation in CD patients compared with normal control. Technical validation was performed using quantitative MSP analysis and we finally identified that the Fragile Histidine Triad (FHIT) genes were hypermethylated in a disease-specific manner. Using a large cohort for CD patients samples (n = 207), we found that FHIT is frequently methylated in CD patients (71%) by MSP and significantly increasing methylation level in CD patient samples. In addition, we confirmed the methylation level of FHIT gene between normal colon and CD patients. Due to hypermethylation of FHIT gene promoter in CD patients, we observed that the level of FHIT protein is downregulated in CD patient samples compared with normal by IHC analysis. Gene network analysis by GO and metascape for hypermethylated genes in CD patients suggested putative cellular and molecular interactions relevant to IBD pathology. Conclusion Overall, our DNA methylation profile identifies newly hypermethylated genes in CD, as well as paves the way to a better understanding of the role of epigenetics in the pathogenesis of CD, and provides direction for future research in the diagnosis/prognosis or therapeutic treatments for CD.

DOP03 The impact of elemental diet on the human gut microbial structure and intestinal metabolites

Abstract Background The incidence and prevalence of Crohn’s disease (CD) are increasing globally. The efficacy of elemental nutrition (EN) for CD has been reported for decades. Recent studies demonstrated that elemental diet (ED), one of the types of EN, which contains individual amino acids, sugars (mono- or polysaccharides), and low fat, improves the anti-TNFa antibody therapy outcome by reducing the loss of response and has the preventive effect against the postoperative recurrence. Given its excellent safety, ED can be a promising concomitant therapeutic option for CD. However, the underlying mechanisms of ED remain unclear. Imbalance of the gut microbiota, dysbiosis, is thought to be involved in CD pathogenesis. Meanwhile, diet influences gut microbiota. We hypothesised that ED exerts clinical efficacy by shifting the gut microbiota to the anti-inflammatory environment. We also believe that investigating human samples is crucial to obtain mechanistic insights into ED therapy, considering species-specific differences in interactions between the host and microbiota. In this study, we examined the impact of ED on the intestinal bacterial compositions as well as metabolites in human subjects. Methods Four healthy individuals participated in the study. The subjects continued their dietary habits for 2 weeks after the registration of the study and then replaced 900 kcal/day of their regular diet with 900 kcal/day of ED (time point 1, T1). ED was continued for 2 weeks (T2). After the withdrawal of ED, subjects resumed their original dietary habits for 2 weeks until the endpoint of this study (T3). Faecal samples were collected from all subjects at all time points, T1-3. Faecal DNA and metabolites were extracted and analysed with 16S rRNA gene amplicon sequencing and metabolomic analysis, respectively. This study was approved by the Institutional Review Board of Kyorin University School of Medicine (IRB No. 720) and performed in accordance with the principles of the Declaration of Helsinki. Results Interindividual differences were observed in the gut bacterial compositions (Figure 1) as well as metabolites (Figure 2) at all time points. Changes over time varied among subjects. On the other hand, some bacteria (e.g. [Ruminococcus] gnavus group and Parabacteroides) and metabolites including short-chain fatty acids and bile acids changed significantly between time points among the 4 subjects. The bacterial and metabolite profiles at T3 were different from those T1 in all subjects. Conclusion The alterations of the gut microbiota induced by ED vary with each individual. Some microbes and metabolites change commonly across the subjects. ED may have a lasting effect on the gut environment even after the withdrawal.