David Bernardo Ordiz

Tu1159 Unravelling the Mechanism of Action of Infliximab in Crohn's Disease and Healthy Controls Following In Vitro Culture With Blood-Enriched Dendritic Cells

Introduction Dendritic cells (DC) play a key role in discriminating between commensal microorganisms and harmful pathogens. DC phenotype and cytokine production determine the type of immune response elicited by T-cells following antigen presentation. DC also direct the T-cells to target tissues to perform their function via imprinting tissue-specific homing markers. In CD, dysregulation of the immune response to gut microbiota & aberrant immune cell trafficking play a role in disease pathogenesis. Infliximab (IFX) is an effective treatment for CD but its mechanism of action is unclear. In this study we investigated the in-vitro effect of IFX on phenotype & ongoing cytokine production of human blood-enriched DC from patients with active CD and healthy controls (HC) Methods Low density cells (LDC), enriched for DC, were obtained following Ficoll & Nycoprep separation of blood from patients with active CD (CDAI > 220) and HC. LDC were cultured (0.5x106cells/ml) with IFX (1.10&100μg/ml&basal) for 24hr. Activation marker (CD40, CD80, HLADR), TLR receptor (TLR2.4) and homing marker (CCR4.5.7.8.9.10, β7) expression was quantified by flow cytometry. Natural ongoing intracellular cytokine production (TNFα, TGFβ&IL-6.10.12.15) was assessed via intracellular staining and flow cytometry. Cytokine secretion was measured on cell-free culture supernatants via Multiplex. Unpaired t-test and one-way ANOVA statistical analyses were applied Results TNFα and IL-6 were increased in culture supernatants from CD although their intracellular ongoing cytokine production was decreased. LDC from CD had decreased β7 (gut-homing integrin) expression. Following IFX culture, LDC decreased β7 expression & CCR9 intensity ratio (dose-dependent). There was a trend towards reduction in TLR2 and 4 expression (not statistically significant). IL-12 production by LDC from HC was increased following IFX culture. There was a marked reduction in TNFα and IL-12 in cell supernatant following culture with IFX Conclusion Increased TNFα and IL-6 in culture supernatants from CD patients coupled with a decrease in ongoing production by DC suggests a negative cytokine feedback system. Reduced β7 expression on LDC from CD patients may suggest that DC have already been recruited to the site of mucosal inflammation. Further experiments are needed to confirm this. Reduced expression of CCR9 and elevated production of IL-12 in LDC cultured with IFX, shows reduced affinity for gut-homing and increased immunogenicity and may suggest a possible mechanism for IFX-induced paradoxical inflammation. The most dramatic effect of IFX was a reduction in TNFα in the supernatant, which is likely to represent neutralisation of the cytokine Disclosure of Interest None Declared.

Mo1763 Clinical Risk Factors for Crohn's Disease Postoperative Recurrence are Reflected in Alterations in Mucosally Adherent Microbiota at Surgical Resection

Introduction Clinical risk factors for Crohn9s disease (CD) recurrence after ileo-caecal resection (ICR) include smoking status, perforating disease and >1 surgical resection. The underlying mechanisms contributing to clinical risk are unknown. We aimed to study the relationship between risk factors and gut microbiota. Methods Samples of macroscopically inflamed and non-inflamed small bowel from patients undergoing surgical resection for CD were analysed. Samples were snap frozen in liquid nitrogen. Cryosections were cut and the frozen sections were hybridised with oligonucleotide probes targeting the microbial 16S rRNA of total bacteria, Escherichia coli , Bacteroides-Prevotella, Faecalibacterium prausnitzii , Clostrium coccoides - Eubacterium rectale and bifidobacteria. The hybridised mucosa associated microbiota (MAM) were identified and quantified. Patients with ≥1 risk factor were classified as high risk for disease recurrence. Results Fifteen patients underwent ICR (10 female); 9 were high risk (6 smokers, 4 fistulating disease and 2 recurrent resection- 3 patients had multiple risk factors). Faecalibacterium prausnitzii numbers in inflamed operative samples were lower in smokers compared with non-smokers (p=0.036). High-risk patients had lower numbers of bifidobacteria in both inflamed (p=0.006) and non-inflamed (p=0.01) operative samples compared with low risk patients. Conclusion The risk of post-operative CD recurrence may be predetermined at a pre-operative stage due to dysbiosis. The role of MAM as a tool to stratify risk requires further study. Drugs that modulate MAM may, in future, play a role in reducing post-operative recurrence. Competing interests None declared.

TH1/TH17 profiles in crohn's disease: a cross sectional single centre study in postoperative crohn's disease

Introduction Th1 and Th17 pathways are implicated in Crohn9s disease (CD). In operative resection samples healthy ileum shows high TGFβ levels in patients who develop recurrence, with TGFβ being a known activator of the Th17 response. Other studies in CD show a dominant Th1 cytokine profile, with high levels of IFNγ, which reduce Th17 response and augment Th1 response. The relationship of Th1/Th17 cytokine profiles in postoperative CD has not been examined. The authors aimed to study tissue Th1/Th17 cytokine secretion after in vitro biopsy culture in postoperative CD. Methods Colonoscopy was undertaken in postoperative CD patients. Recurrence graded as no/minimal inflammation (Rutgeert Score (RS) ≥1) or progressive inflammation (RS≥2). Ileal biopsies were cultured overnight and cell free supernatants obtained. Supernatant cytokines (IL-2, IL-4, IL-10, IL-17 TNFα, INFg and IL-6) were assessed by flow cytometry using cytometric bead array (Becton Dickinson). Statistical analysis was via unpaired t tests. Results Consecutive patients attending endoscopy (n=24, 9M/15F) were identified. Mean age 45.0 years and time from I to C resection was 5.8 years; 5 patients were smokers. Drugs were thiopurines 13, Infliximab 1 and nil 10. Endoscopic severity was i0 n=5, i1 n=6, i2 n=5, i3 n=3, i4 n=5. Mean cytokine concentrations from supernatants are shown in the table 1. Comparison between RS≥1 and ≥2 showed that pro-inflammatory cytokines IL-17a (p Conclusion Cytokine profiles in those with RS≥2, show higher levels of IL-17a and IFNγ and reduced IL-10 compared to RS≥1. This profile supports a Th17 and Th1 mediated response as one of the early instigators of endoscopic progression in postoperative CD. The authors9 observation is consistent with recent findings of a T cell subset able to produce cytokines involved in both Th1 and Th17 responses. Previous therapies directed at Th1 pathway, for example, anti-IL-12p40 antibody ustekinumab and anti-IFNγ Fontolizumab failed to show significant clinical benefit in CD. Given our findings targeting the Th17 response, for example, with anti-IL-23 antibodies and anti-IL-17 may deliver improved therapeutic outcome.

Increase in Dendritic Cell Migration Markers CCR7 and CCR9 in the Neo-Terminal Ileum of Postoperative Crohn's Disease: An Adaptive Response to Bacterial Exposure?

Introduction Gut dendritic cells (DCs) are crucial in bacterial recognition, T cell signalling and inflammatory regulation. DC TLR expression is altered in CD: increased on myeloid DC (MDC) in colonic CD and reduced on plasmacytoid DCs (PDC) in postoperative CD (POCD) ileum. In active CD, peripheral CD4 and CD8 T cells showed increased intestinal homing with high CCR9 levels. In Crohn9s colonic tissues, CCR7, a homing marker crucial for DC trafficking to mesenteric lymph nodes, was elevated compared with controls. Additionally CCR7 expression on MDC is higher in ileal compared with colonic tissue in CD. CCR7 and CCR9 expression on DC in POCD is unknown. After ileo-caecal resection the neo-terminal ileum is exposed to the bacteria rich contents of the colon. The authors hypothesise that alteration in gut microflora after surgery may modulate expression of homing markers on DC from POCD patients. The authors aimed to examine homing marker expression on MDC and PDC from the ileum and the colon in healthy controls (HC) and POCD patients. Methods HC and POCD patients were identified at colonoscopy. Intestinal lamina propria mononuclear cells were collected using collagenase digestion and labelled with directly conjugated monoclonal antibodies to CCR7 and 9. PDC and MDC were characterised as CD11c+ve and –ve respectively and expression of CCR7 and 9 by multicolour flow cytometry measured. Statistical analysis was via unpaired t tests. In experiments with paired colonic and ileal samples paired t tests were performed. Results In paired samples, HC ileal CCR9+ve PDC concentrations were lower than colonic PDC (26.46±10.43/ml SEM vs 53.76±20.16/ml SEM, p There were significantly higher concentrations of CCR9+ve and CCR7+ve PDCs within ileal POCD compared with ileum normal controls (103.8±22.64/ml vs 37.68±7.434/ml, p=0.039 and 117.4±26.97/ml SEM vs 40.47±3.97/ml SEM, p=0.03). No differences in MDC concentrations of both homing markers in all types of tissue existed. Conclusion POCD neo-terminal ileum showed higher CCR7+ve and CCR9+ve PDC than normal ileum. This novel finding indicates a potential role for PDC in CD pathogenesis. The loss of the ileocaecal valve in POCD may alter microbiota flora exposure in the ileum with an adaptive response of CCR9+ve DC to a level seen in normal colonic tissue. Additionally, this may induce migration of PDC by upregulation of CCR7 expression. Further studies to examine the changes with disease progression may unravel the function of PDC in ileal POCD tissues.

W1816 Loss of TLR 2 and 4 on Ileal Plasmacytoid Dendritic Cells in Post-Operative Crohn's Disease Patients

Introduction Aberrant responses to bacterial flora are implicated in post-operative recurrence of Crohn9s disease (CD) after ileo-ceacal resection. Response to gut flora is conducted by dendritic cells (DC) which can induce a tolerogenic or active immune response. DC express Toll like receptors (TLR) which recognise bacterial products and are important in immune tolerance in health and highly expressed in IBD colonic tissue. Two subsets of DC are plasmacytoid (PDC) and myeloid cells (MDC). These DC show functional differences in their response to bacteria and are able to cross-talk. Expression of TLR on DC in the small intestine of post-operative CD patients is still largely unknown. We aimed to assess expression of TLR on DC from lamina propria mononuclear cells (LPMC) in post-operative CD patients as compared with normal controls. Methods Small intestine biopsies were obtained from 18 post-operative CD patients and 5 controls by colonoscopy. LPMC were collected using collagenase digestion, labelled with monoclonal antibodies to TLR2 and 4 and assessed by multicolour flow cytometry. Result A high percentage of DC from normal ileum expressed TLR2 and 4 with no significant differences in expression between PDC and MDC. However, expression of TLR 2 and 4 was significantly lower in PDC from post op CD patients compared with PDC in normal controls (mean 22.30% vs 60%, p=0.0003 and 38.55% vs 65.16%, p=0.02), respectively. Furthermore, PDC and MDC differed in expression of TLR2 and 4 in tissues from post-op CD patients; PDC expressed significantly less TLR2 and 4 compared with MDC (22.30% vs 38.11%, p=0.002 and 38.55% vs 50.89%, p=0.01), respectively. Conclusion Reduction of TLR2 and 4 expression in PDC compared with their counterparts in normal controls is likely to be the reason for the reduced expression of both TLRs on PDC compared with MDC in CD tissues. The lack of TLR2 and 4 expression on PDC maybe related to the disease activity as the normal controls did not show any statistical differences in expression of either TLR between the DC subsets. Other groups have suggested that functional differences exist between PDC and MDC in their activation by bacterial products and ability to produce innate and acquired inflammatory responses. In particular, blood PDC require the presence of MDC to respond to bacterial products to activate T cells rather than driving innate immunity. Our results indicate that PDC response to intestinal microflora may differ from that in MDC and provide initial background to the DC function and diversity in post-operative CD recurrence.