Patients With Microscopic Colitis Have Altered Levels of Inhibitory and Stimulatory Biomarkers in Colon Biopsies and Sera Compared to Non-inflamed Controls

Introduction: Microscopic colitis (MC) is an inflammatory bowel condition with two subtypes, lymphocytic colitis (LC) and collagenous colitis (CC). Unlike patients with ulcerative colitis (UC) and non-inflamed individuals, MC patients have reduced risk of developing colorectal cancer, possibly due to increased immune surveillance in MC patients.Aim: To examine differences in levels of immunomodulatory molecules, including those involved in immune checkpoint mechanisms, in sera from patients with MC and in colonic biopsies from patients with MC and UC compared with controls.Methods: Using Luminex, 23 analytes (4-1BB, 4-1BBL, APRIL, BAFF, BTLA, CD27, CD28, CD80, CTLA-4, E-cadherin, Galectin-3, GITR, HVEM, IDO, IL-2Rα, LAG-3, MICA, MICB, PD-1, PD-L1, PD-L2, sCD40L and TIM-3) were studied in serum from patients with active MC (n = 35) and controls (n = 23), and in colonic biopsies from patients with active LC (n = 9), active CC (n = 16) and MC in histological remission (LC n = 6, CC n = 6), active UC (n = 15) and UC in remission (n = 12) and controls (n = 58).Results: In serum, IDO, PD-1, TIM-3, 4-1BB, CD27, and CD80 were decreased whereas 4-1BBL and IL-2Rα were increased in MC patients compared with controls. In contrast, in biopsies, levels of PD-L2 and 4-1BB were increased in MC and UC patients with active disease. Furthermore, in biopsies from CC and UC but not LC patients with active disease, CTLA-4, PD-1, APRIL, BAFF, and IL-2Rα were increased compared with controls. PD-L1 was increased in CC but not UC or LC patients. CD27 and TIM-3 were decreased in biopsies from MC patients in comparison to controls whereas levels of MICB were decreased in patients with active UC compared with controls.Conclusions: Compared with non-inflamed controls, levels of soluble and membrane-bound immunomodulatory molecules were systemically and locally altered in MC and UC patients, with most analytes being decreased in serum but enhanced in colonic biopsies. These findings contribute to knowledge about checkpoint molecules and their role as biomarkers in MC and may also contribute to knowledge about possible mechanisms behind the seemingly protective effects of MC against colorectal cancer.

Increased NOX1 and DOUX2 Expression in the Colonic Mucosa of Patients with Chronic Functional Constipation

Aim To determine whether oxidative stress and inflammation are associated with constipation by examining the expression of the main producers of reactive oxygen species, NADPH oxidases, and pro-inflammatory cytokines in the colon of patients with chronic functional constipation. Methods The colonic biopsies were collected from 32 patients with chronic functional constipation and 30 healthy subjects who underwent colonoscopy. Colonic mucosal histology was observed. IL-1β, IL-6, IL-8 mRNA, and four members of NADPH oxidase (NOX1, NOX2, DOUX2 and NOX4) protein and mRNA were assessed by immunohistochemistry, western blotting and RT-PCR. Results The tissues from both patients and healthy subjects showed normal histological structure without increase of inflammatory cells. NOX1 protein and mRNA levels were significantly increased compared to controls (P<0.05). DOUX2 protein, but not mRNA, was increased by twofold compared to controls (P<0.05). The levels of NOX2 and NOX4 protein and mRNA demonstrated no significant difference between patients and control subjects. The levels of IL-1β and IL-6 mRNA were significantly higher in constipation patients (P<0.05), while IL-8 mRNA level was no different between the two groups. Conclusion NADPH oxidase and pro-inflammatory cytokine might be involved in the pathogeneses of chronic functional constipation.

S100B Inhibition Attenuates Intestinal Damage and Diarrhea Severity During Clostridioides difficile Infection by Modulating Inflammatory Response

The involvement of the enteric nervous system, which is a source of S100B, in Clostridioides difficile (C. difficile) infection (CDI) is poorly understood although intestinal motility dysfunctions are known to occur following infection. Here, we investigated the role of S100B in CDI and examined the S100B signaling pathways activated in C. difficile toxin A (TcdA)- and B (TcdB)-induced enteric glial cell (EGC) inflammatory response. The expression of S100B was measured in colon tissues and fecal samples of patients with and without CDI, as well as in colon tissues from C. difficile-infected mice. To investigate the role of S100B signaling in IL-6 expression induced by TcdA and TcdB, rat EGCs were used. Increased S100B was found in colonic biopsies from patients with CDI and colon tissues from C. difficile-infected mice. Patients with CDI-promoted diarrhea exhibited higher levels of fecal S100B compared to non-CDI cases. Inhibition of S100B by pentamidine reduced the synthesis of IL-1β, IL-18, IL-6, GMCSF, TNF-α, IL-17, IL-23, and IL-2 and downregulated a variety of NFκB-related genes, increased the transcription (SOCS2 and Bcl-2) of protective mediators, reduced neutrophil recruitment, and ameliorated intestinal damage and diarrhea severity in mice. In EGCs, TcdA and TcdB upregulated S100B-mediated IL-6 expression via activation of RAGE/PI3K/NFκB. Thus, CDI appears to upregulate colonic S100B signaling in EGCs, which in turn augment inflammatory response. Inhibition of S100B activity attenuates the intestinal injury and diarrhea caused by C. difficile toxins. Our findings provide new insight into the role of S100B in CDI pathogenesis and opens novel avenues for therapeutic interventions.

Landscapes and bacterial signatures of mucosa-associated intestinal microbiota in Chilean and Spanish patients with inflammatory bowel disease

Inflammatory bowel diseases (IBDs), which include ulcerative colitis (UC) and Crohn’s disease (CD), cause chronic inflammation of the gut, affecting millions of people worldwide. IBDs have been frequently associated with an alteration of the gut microbiota, termed dysbiosis, which is generally characterized by an increase in abundance of Proteobacteria such as Escherichia coli, and a decrease in abundance of Firmicutes such as Faecalibacterium prausnitzii (an indicator of a healthy colonic microbiota). The mechanisms behind the development of IBDs and dysbiosis are incompletely understood. Using samples from colonic biopsies, we studied the mucosa-associated intestinal microbiota in Chilean and Spanish patients with IBD. In agreement with previous studies, microbiome comparison between IBD patients and non-IBD controls indicated that dysbiosis in these patients is characterized by an increase of pro-inflammatory bacteria (mostly Proteobacteria) and a decrease of commensal beneficial bacteria (mostly Firmicutes). Notably, bacteria typically residing on the mucosa of healthy individuals were mostly obligate anaerobes, whereas in the inflamed mucosa an increase of facultative anaerobe and aerobic bacteria was observed. We also identify potential co-occurring and mutually exclusive interactions between bacteria associated with the healthy and inflamed mucosa, which appear to be determined by the oxygen availability and the type of respiration. Finally, we identified a panel of bacterial biomarkers that allow the discrimination between eubiosis from dysbiosis with a high diagnostic performance (96% accurately), which could be used for the development of non-invasive diagnostic methods. Thus, this study is a step forward towards understanding the landscapes and alterations of mucosa-associated intestinal microbiota in patients with IBDs.

1123 An Audit of Pick-Up Rate of Random Colonic Biopsies

Aim We aimed to evaluate optimal random biopsy criteria are being followed in our institution to increase the diagnostic yield of a subsequent histopathological examination and to reduce the number of unnecessary biopsies in which histopathology is unlikely to deliver clinically useful information and causing a burden on health resources in terms of cost and manpower. Method Our study was a retrospective on 419 random colonoscopy biopsies performed over 6 months. Data collection included variables such as age, gender, indications, request of urgency, and histology findings. Data analysis was done descriptively. Results Out of 419 random biopsies, only 10.02% had positive findings. The total number of histology results with microscopic colitis was 10. The main indication of the random colonic biopsy was a change in bowel habits (328 cases) followed by significant diarrhea greater than 50 years in 20 cases. In patients with a change in bowel habits, 2.44% of histopathology specimens revealed microscopic colitis. The percentage of random colonic biopsy histology in patients greater than 50 years with significant diarrhea showed microscopic colitis was 10%. Conclusions Our study revealed random biopsy during colonoscopy should only be done in selected patients otherwise it has low diagnostic yields biopsy and should only be reserved for patients with risk factors for optimum utilization of health resources and to reduce the cost burden. A scoring system may be helpful to risk-stratify patients in low and high risk for MC to determine which patients qualify for RCB.

Calmodulin Antagonist W-7 Enhances Intermediate Conductance Ca2+- Sensitive Basolateral Potassium Channel (IKCa) Activity in Human Colonic Crypts

Intermediate conductance potassium (IKCa) channels are exquisitively Ca2+ sensitive, intracellular Ca2+ regulating channel activity by complexing with calmodulin (CaM), which is bound to the cytosolic carboxyl tail. Although CaM antagonists might be expected to decrease IKCa channel activity, the effect of W-7 in human T lymphocytes are conflicting. We therefore evaluated the effect of W-7 on basolateral IKCa channels in human colonic crypt cells. Intact crypts obtained from normal human colonic biopsies by Ca2+ chelation were used for patch clamp studies of basolateral IKCa channels in the cell-attached configuration. IKCa channel activity was studied when the bath Ca2+ concentration was changed from 1.2 mmol/L to 100 μmol/L and back to 1.2 mmol/L, as well as from 100 μmol/L to 1.2 mmol/L and back to 100 μmol/L, both in the absence and presence of 25 μmol/L W-7. Decreasing bath Ca2+ from 1.2 mmol/L to 100 μmol/L decreased IKCa channel activity reversibly in the absence of W-7, whereas there was a uniformly high level of channel activity at both bath Ca2+ concentrations in the presence of W-7. In separate experiments, increasing bath Ca2+ from 100 μmol/L to 1.2 mmol/L increased IKCa channel activity reversibly in the absence of W-7, whereas there was again a uniformly high level of channel activity at both bath Ca2+ concentrations in the presence of W-7. We, therefore, propose that W-7 has a specific stimulatory effect on basolateral IKCa channel activity, despite its ability to inhibit Ca2+/CaM-mediated, IKCa channel-dependent Cl− secretion in human colonic epithelial cells. Graphic Abstract

Reverse translation approach generates a signature of penetrating fibrosis in Crohn’s disease that is associated with anti-TNF response

ObjectiveFibrosis is a common feature of Crohn’s disease (CD) which can involve the mesenteric fat. However, the molecular signature of this process remains unclear. Our goal was to define the transcriptional signature of mesenteric fibrosis in CD subjects and to model mesenteric fibrosis in mice to improve our understanding of CD pathogenesis.DesignWe performed histological and transcriptional analysis of fibrosis in CD samples. We modelled a CD-like fibrosis phenotype by performing repeated colonic biopsies in mice and analysed the model by histology, type I collagen-targeted positron emission tomography (PET) and global gene expression. We generated a gene set list of essential features of mesenteric fibrosis and compared it to mucosal biopsy datasets from inflammatory bowel disease patients to identify a refined gene set that correlated with clinical outcomes.ResultsMesenteric fibrosis in CD was interconnected to areas of fibrosis in all layers of the intestine, defined as penetrating fibrosis. We found a transcriptional signature of differentially expressed genes enriched in areas of the mesenteric fat of CD subjects with high levels of fibrosis. Mice subjected to repeated colonic biopsies showed penetrating fibrosis as shown by histology, PET imaging and transcriptional analysis. Finally, we composed a composite 24-gene set list that was linked to inflammatory fibroblasts and correlated with treatment response.ConclusionWe linked histopathological and molecular features of CD penetrating fibrosis to a mouse model of repeated biopsy injuries. This experimental system provides an innovative approach for functional investigations of underlying profibrotic mechanisms and therapeutic concepts in CD.

Utilizing Deep Learning to Analyze Whole Slide Images of Colonic Biopsies for Associations Between Eosinophil Density and Clinicopathologic Features in Active Ulcerative Colitis

Background Eosinophils have been implicated in the pathogenesis of ulcerative colitis and have been associated with disease course and therapeutic response. However, associations between eosinophil density, histologic activity, and clinical features have not been rigorously studied. Methods A deep learning algorithm was trained to identify eosinophils in colonic biopsies and validated against pathologists’ interpretations. The algorithm was applied to sigmoid colon biopsies from a cross-sectional cohort of 88 ulcerative colitis patients with histologically active disease as measured by the Geboes score and Robarts histopathology index (RHI). Associations between eosinophil density, histologic activity, and clinical features were determined. Results The eosinophil deep learning algorithm demonstrated almost perfect agreement with manual eosinophil counts determined by 4 pathologists (interclass correlation coefficients: 0.805–0.917). Eosinophil density varied widely across patients (median 113.5 cells per mm2, interquartile range 108.9). There was no association between eosinophil density and RHI (P = 0.5). Significant differences in eosinophil density were seen between patients with Montreal E3 vs E2 disease (146.2 cells per mm2 vs 88.2 cells per mm2, P = 0.005). Patients on corticosteroids had significantly lower eosinophil density (62.9 cells per mm2 vs 124.1 cells per mm2, P = 0.006). No association between eosinophil density and biologic use was observed (P = 0.5). Conclusions We developed a deep learning algorithm to quantify eosinophils in colonic biopsies. Eosinophil density did not correlate with histologic activity but did correlate with disease extent and corticosteroid use. Future studies applying this algorithm in larger cohorts with longitudinal follow-up are needed to further elucidate the role of eosinophils in ulcerative colitis.

OP0035 ASSESSMENT OF THE INTESTINAL PERMEABILITY IN PATIENTS WITH RHEUMATOID ARTHRITIS USING COLONIC TISSUES AND SERA

Background:Patients with rheumatoid arthritis (RA) have an altered gut microbiota (dysbiosis) (1-3). This microbiota interacts with intestinal epithelium which can lead to an increased intestinal permeability, responsible for the passage of antigens and inflammatory molecules, and can therefore promote systemic inflammation. Gut microbiota tends to normalize with disease control (2), suggesting that systemic inflammation may directly influence the composition of microbiota and the gut barrier. It was shown in many inflammatory diseases that intestinal permeability is impaired, but to date there is very little data in RA.Objectives:In the present study, we evaluate the intestinal permeability in RA patients by analyzing tight junctions in colonic biopsies and serum markers.Methods:Colonic biopsies from 20 RA patients who underwent coloscopy for screening with normal histology were compared with those from 20 age and sex matched controls. ZO-1, occludin and claudin 2 junction proteins were evaluated by immunohistochemistry. The staining intensity was assessed by two blinded independent readers. The serum concentrations of LPS-binding protein (LBP), CD14s and zonulin were evaluated by ELISA in 25 patients naive of DMARDs, 41 patients before and after introduction of a DMARDs and 21 controls. Elevated zonulin in serum indicates an increase in intestinal permeability while LBP and CD14s indicate bacterial translocation.Results:ZO-1 expression was significantly lower in biopsies from patients with RA than controls (mean score ± SD of 1.6 ± 0.56 vs 2.0 ± 0.43; p = 0.01). Age, sex, disease duration and immunological status did not significantly influence the expression of colonic junction proteins. LBP and CD14s were higher in serum from RA patients naive of DMARDs than controls (p = 0.002 and p = 0.003). LBP, CD14s and zonulin levels significantly correlated with DAS28 (r = 0.61, p = 0.005; r = 0.51, p = 0.030 and r = 0.46, p = 0.049, respectively). After treatment, unlike non-responders, LBP and CD14s were significantly reduced in DMARD responders and variations in LBP and CD14s significantly correlated with changes in DAS28 (r = 0.46, p = 0.002 and r = 0, 33 and p = 0.030, respectively).Conclusion:This work is one of the first to explore intestinal permeability in RA and to show altered tight junction in colonic tissue from RA. This increased intestinal permeability appears to be related to the systemic inflammation. Improving the gut microbiota through food or probiotics could enhance the effect of treatments by limiting this amplification loop of inflammation.References:[1]Horta-Baas G, Romero-Figueroa MDS, Montiel-Jarquin AJ, Pizano-Zarate ML, Garcia-Mena J, Ramirez-Duran N. Intestinal Dysbiosis and Rheumatoid Arthritis: A Link between Gut Microbiota and the Pathogenesis of Rheumatoid Arthritis. J Immunol Res. 2017;2017:4835189.[2]Zhang X, Zhang D, Jia H, Feng Q, Wang D, Liang D, et al. The oral and gut microbiomes are perturbed in rheumatoid arthritis and partly normalized after treatment. Nat Med. 2015;21(8):895-905.[3]Maeda Y, Kurakawa T, Umemoto E, Motooka D, Ito Y, Gotoh K, et al. Dysbiosis Contributes to Arthritis Development via Activation of Autoreactive T Cells in the Intestine. Arthritis Rheumatol. 2016;68(11):2646-61.Disclosure of Interests:Rachel Audo: None declared, Pauline Sanchez: None declared, Julie Mielle: None declared, Laurence Macia: None declared, Benjamin Rivière: None declared, Cédric Lukas: None declared, Bernard Combe: None declared, Jacques Morel: None declared, Claire Daien Speakers bureau: Pfizer roche chugai fresenius BMS msd Novartis galapagos, Consultant of: Abivax abbbvie BMS roche chugai, Grant/research support from: Pfizer, roche-chugai, fresenius, msd

P079 Valproic acid increases MiR449a expression in intestinal mucosa from patients with inflammatory bowel disease

Background Histone acetylation/deacetylation and microRNAs (MiRNA) are epigenetic mechanisms involved in the pathogenesis of inflammatory bowel diseases (IBD). Histone deacetylase (HDAC) inhibitors, such as valproic acid (VPA), demonstrated anti-inflammatory and anti-tumour effects in animal models of colitis.1,2 Modulation of MiRNAs is hypothesized to be a possible mechanism of action of HDAC inhibition.3 Recently, VPA was shown to modulate histone-3 (H3) acetylation and cytokine production in the human gut.4 In this study, we analysed whether VPA influences MiRNA expression in ex-vivo biopsies from IBD patients. Methods Colonic biopsies from IBD patients collected during routine endoscopic procedures were cultured with VPA (5 mM) or control media for 24 hours. Total RNA was extracted and a MiRNA array was performed (miScript™ miRNA PCR Array), profiling the expression of 84 MiRNAs predicted to regulate pro- or anti-inflammatory genes. Significantly altered MiRNAs were then validated in an independent cohort of samples using quantitative (q) RT-PCR. Caco-2 (ATCC® HTB-37™) cell cultures treated with VPA (5 mM) or control media were used to confirm changes of H3 acetylation and MiRNA expression. After 24 hours, H3 acetylation was evaluated by immunofluorescence and apoptotic markers were analysed by qPCR. Results Colonic biopsies from inflamed mucosa of 6 IBD patients (2 with Crohn’s disease (CD) and 4 with ulcerative colitis (UC)) were cultured with VPA or control media. MiRNA analysis showed a significant increase of miR449a, miR373-3p, miR300 and miR520d-3p in treated biopsies compared to control (p=0.004, p=0.024, p=0.04 and p=0.014, respectively). Validation was performed in an independent cohort of 14 IBD patients (7 CD; 7 UC), confirming a significant increase of MiR449a (p=0.026) in VPA treated biopsies. VPA treated Caco-2 cell cultures showed a significant increase of H3 acetylation density level (p=0.02), confirming the HDAC inhibitory activity. Gene expression analysis showed increased Caspase-3 (p=0.015), a pro-apoptotic marker, and decreased BCL-3 (p&lt;0.01), an anti-apoptotic marker. The level of MiR449a expression in VPA treated cells was significantly elevated (p&lt;0.01). Conclusion VPA increases the expression of MiR449a in cultured ex-vivo biopsies and in Caco-2 cells. This MiRNA has recently demonstrated to be involved in IBD pathogenesis and in colitis-associated colon cancer. This may represent a new insight into IBD pathogenesis and colitis-associated colon cancer development. Further work is required to determine the functional significance of miR-499a in the human intestine. References