Abstracts of the 15th Congress of ECCO – European Crohn’s and Colitis Organisation S087 increased from 19% to 26% for L1 (p < 0.0001) and from 50% to
58% for L3 (p < 0.0001), while L2 locations decreased from 31% to 16% (p < 0.0001). In the UC group, the proportion of patients with E1 location decreased from 42% to 34%, whereas the propor-tion with E3 locapropor-tion increased from 19% to 28% (p < 0.0001).
Conclusion: In a large, population-based study over a period of
27 years, we found that the incidences of CD and UC in the young women in northern France are still rising quickly. Our present results suggest that one or more environmental factors may significantly predispose women to IBD in our region. Among patients with CD, we observed an increase in ileal involvement and a concomitant de-crease in colonic involvement.
DOP48
Gene expression landscape of epithelial
monolayer in inflammatory bowel disease.
S. Sæterstad1, A.E. Østvik2,3,A.K. Sandvik1,2,3, A. Granlund Van Beelen*1
1Centre of Molecular Inflammation Research, Norwegian University of Science and Technology NTNU, Trondheim, Norway, 2 Department of Clinical and Molecular Medicine, Norwegian University of Science and Technology NTNU,Trondheim, Norway, 3Department of Gastroenterology, St. Olav’s University Hospital, Trondheim, Norway
Background: Gene expression analyses on IBD tissue are dominated
by analyses of biopsy homogenates that are highly heterogeneous due to a difference in cell populations when contrasting inflamed vs. healthy mucosa. We wished to characterise the inflammatory activity of the isolated epithelial monolayer (EM), avoiding the infiltrating inflammatory cells, thereby form a comprehensive understanding of the epithelium’s role in IBD pathobiology.
Methods: Total RNA was isolated from laser capture microdissected
(LCM) colon EM, and sequencing libraries were prepared using TruSeq RNA access kit (Illumina, CA, USA). Gene cluster analysis (WGCNA) was used to stratify the expression data in genetic mod-ules. Following this, we characterised the modules correlated to IBD status, using Metacore pathway analysis, in order to suggest what underlying biological processes contribute to the observed correl-ation of expression data. Finally, we used immunohistochemistry to verify the protein expression pattern of key players in the suggested pathways, thereby confirming EM involvement in these processes during inflammation.
Results: Biopsies from active (a) IBD (7 UCa, 5 CDa) and from
un-inflamed (u) IBD and healthy control (7 UCu, 5 CDu and 6 HC) were included in the analysis. 6386 genes were differen-tially expressed (adjusted p-value (pval.) <0.05) in EM from active inflamed colon vs. un-inflamed (Figure 1). Six gene mod-ules showed a significant correlation between inflammatory status and gene expression (Figure 2). Of note was a broad, sig-nificant up-regulation of MHC class II presentation machinery, e.g. STAT1 (log2 fold change (lfc) 0.93, p < 0.001), CIITA (lfc 2.57, p < 0.001) and HLA-DRB1 (lfc 3.69, p < 0.001), and a down-regulation unique to epithelial cells of the retinoic acid receptors RARA (lfc −0.79, p < 0.001) and RXRA (log fc −0.56, p < 0.001). The most significantly up-regulated pathway was ‘Immune Response’, induction of antigen presentation machinery by IFN-γ (FDR 2.5 × 10–18), while ‘RXR-dependent regulation
of lipid metabolism’ (FDR 2.6 × 10–8) was the most significantly
down-regulated pathway.
Figure 1. Principal component analysis of expression data
Figure 2. Correlation (p-value) for Module-inflammatory status relationship.
Related processes given in title of significant modules.
Conclusion: Our analyses suggest that the involvement of the EM
in IBD inflammation goes far beyond acting as a barrier. The largest genetic modules are dominated by genes important for inflamma-tory regulation, suggesting that the EM is much more involved in these processes than what is commonly understood. This study lends support to further analyses of interplay between epithelial cells and immune cells, particularly in a pro-inflammatory environment.
DOP49
Quantitative proteomics analysis of
macrophages from Crohn’s disease patients and
infected with adherent-invasive Escherichia coli
C. Douadi*1, E. Vazeille1, C. Chambon2, M. Hébraud3,M. Dodel4, B. Pereira4, D. Coban4, A. Buisson4, N. Barnich1
1Auvergne University, M2iSH Laboratory, Clermont-Ferrand, France, 2INRA, Plateforme d’Exploration du Métabolisme- Composante Protéomique PFEMcp, Saint-Genès-Champanelle, France, 3INRA, UMR Microbiologie Environnement Digestif
S088 Digital oral presentations Santé MEDiS, Saint-Genès-Champanelle, France, 4CHU,
Gastro-entérologie, Clermont-Ferrand, France
Background: Crohn’s disease (CD) is a disabling inflammatory
bowel disease. Currently, the available treatments only alleviate the symptoms. The anti-TNFα represent the most efficient therapeutic class, but its mechanisms are poorly known. Experimental data have highlighted the role of intestinal macrophages in the patho-genesis of CD. These macrophages present a defect in the control of CD-associated adherent-invasive E. coli (AIEC) replication, which is linked to altered autophagy. The main aim of this study was to com-pare the proteomic profile of macrophages from CD patients with and without anti-TNFα treatment, to those from ulcerative colitis (UC) patients or healthy subjects, both at the basal state and after AIEC infection.
Methods: Peripheral blood monocyte-derived macrophages (MDM)
were obtained from 44 CD patients including 22 with and 22 without anti-TNFα treatment, 22 UC patients and 22 healthy sub-jects. MDM was infected or not with AIEC LF82 reference strain. The numbers of intracellular bacteria were determined at 1h and 6h post-infection, using gentamicin protection assay. The ‘bottom-up’ proteomic analysis of macrophages after 6h of AIEC LF82 infec-tion or not, was assessed by a mass spectrometer, using the label-free quantification.
Results: AIEC survival was reduced within MDM from CD patients
with anti-TNFα treatment compared with those from CD without TNFα treatment (p = 0.0235). The proteomic analysis identified 1359 proteins, of which, 71 were differentially expressed between MDM at the basal state and infected by AIEC LF82 whatever the origin, 30 proteins were specific in CD patients with anti-TNFα treatment and 16 in CD patients without anti-TNFα treatment (fold change >1.3). The bioinformatics analysis shows an impact of sev-eral pathways involved in cellular activation, immune process and lysosomal activity. Interestingly, CD macrophages expression levels of β3 integrin (ITGB3), glutathione peroxidase-1 (GPx-1) and per-oxiredoxin 3 (PRDX3) are significantly correlated with AIEC sur-vival at 6h post-infection and faecal calprotectin. The stratification of MDM from CD patients according to the level of faecal calpro-tectin highlighted that levels of calprocalpro-tectin and α-L-Fucosidase 1 (FUCA-1) increased in a similar way, both at the basal state and after AIEC LF82 infection.
Conclusion: Our study highlighted that the anti-TNFα treatment
limits bacteria replication and acts on macrophages function. For the first time, global proteomic analysis suggests that CD macrophages-AIEC interaction could be orchestrated by ITGB3, GPx-1, PRDX3 and FUCA-1. These proteins could represent potential therapeutic targets in CD patients that need to be further investigated.
DOP50
The landscape of somatic mutations in
non-neoplastic IBD-affected colon
S. Olafsson*1, R.E. McIntyre1, T. Coorens2, T. Butler2, P. Robinson2,
H. Lee-Six2, M. Sanders2, K. Arestang3, C. Dawson3, M. Tripathi4,
K. Strongili3, Y. Hooks2, M.R. Stratton2, M. Parkes3, I. Martincorena2,
T. Raine3, P.J. Campbell2, C.A. Anderson1
1Department of Human Genetics, Wellcome Trust Sanger Institute, Hinxton, UK, 2Cancer, Aging and Somatic Mutations, Wellcome Trust Sanger Institute, Hinxton, UK, 3Department of Gastroenterology,
Addenbrooke’s Hospital, Cambridge, UK, 4Department of Histopathology, Cambridge University Hospitals NHS Foundation Trust, Cambridge, UK
Background: Under normal physiological conditions, colonic crypts
accrue ~40 somatic mutations for every year of life. That somatic mutations contribute to the development of cancer is well estab-lished, but their patterns, burden and functional consequences in diseases other than cancer have not been extensively studied and our understanding of the effects of chronic inflammation on the mu-tational profile and clonal structure of the colon is limited. Here, we investigated how the recurrent cycles of inflammation, ulcer-ation and regenerulcer-ation seen in IBD impact the mutulcer-ational and clonal structure of intestinal epithelia.
Methods: We isolated and whole-genome sequenced ~400
indi-vidual colonic crypts from 46 IBD patients and compared these to ~400 crypts from 41 non-IBD controls. We compared the mutation burden, mutational signature exposure, clonal structure and cancer driver mutation landscape in crypts from actively and previously inflamed regions with crypts dissected from controls.
Results: We estimated the base substitution rate of affected colonic
epithelial cells to be doubled after IBD onset. This change was pri-marily driven by acceleration of mutational processes ubiquitously observed in normal colon (Figure 1), and we did not detect an IBD-specific mutational process. In contrast to the normal colon, where clonal expansions outside the confines of the crypt are rare, we observed widespread millimetre-scale clonal expansions, even in the absence of mutations in KRAS, TP53 and APC (Figure 2). We discovered that mutations in ARID1A, PIGR and ZC3H12A, and genes in the interleukin 17 and Toll-like receptor pathways, were under positive selection in colonic crypts from IBD patients (Figure 3). With the exception of ARID1A, these genes and pathways have not been previously associated with cancer risk. A previously pub-lished mouse model of ZC3H12A suggests that LoF mutations in this gene may facilitate healing of affected mucosa without promot-ing tumorigenesis. This could make the encoded protein an attractive drug target. The observed enrichment of mutations in PIGR and IL17 and TLR pathways suggests that somatic mutations may ini-tiate, maintain or perpetuate IBD pathogenesis through disruption of microbe-epithelial homeostasis.
