Isabelle Cleynen1, Emilie Vazeille2, Marta Artieda3, Hein W Verspaget4, Magdalena Szczypiorska3, Marie-Agnès Bringer5, Peter L Lakatos6, Frank Seibold7, Kirstie Parnell8, Rinse K Weersma9, Jestinah M Mahachie John10, Rebecca Morgan-Walsh11, Dominiek Staelens1, Ingrid Arijs1, Gert De Hertogh12, Stefan Müller13, Atilla Tordai14, Daniel W Hommes15, Tariq Ahmad8, Cisca Wijmenga16, Sylvia Pender11, Paul Rutgeerts1, Kristel Van Steen10, Daniel Lottaz17, Severine Vermeire1, Arlette Darfeuille-Michaud2. 1. Department of Clinical and Experimental Medicine, TARGID, KU Leuven, Leuven, Belgium. 2. Clermont Université, Inserm U1071, Université d'Auvergne, INRA USC 2018, Clermont-Ferrand, France Centre Hospitalier Universitaire, Clermont-Ferrand, France. 3. Progenika Biopharma, S.A., Derio, Spain. 4. Department of Gastroenterology and Hepatology, Leiden University Medical Center, Leiden, The Netherlands Dutch Initiative on Crohn and Colitis (ICC). 5. Clermont Université, Inserm U1071, Université d'Auvergne, INRA USC 2018, Clermont-Ferrand, France. 6. 1st Department of Medicine, Semmelweis University, Budapest, Hungary. 7. Department of Gastroenterology, Spitalnetz Bern, Switzerland. 8. Peninsula Medical School, University of Exeter & Plymouth, Exeter, UK. 9. Dutch Initiative on Crohn and Colitis (ICC) Department of Gastroenterology and Hepatology, University Medical Center Groningen and the University of Groningen, Groningen, The Netherlands. 10. Systems and Modeling Unit, Montefiore Institute, University of Liège, Liège, Belgium Bioinformatics and Modeling, GIGA-R, University of Liège, Liège, Belgium. 11. Clinical and Experimental Sciences, Faculty of medicine, University of Southampton, Southampton, UK. 12. Department of Morphology and Molecular Pathology, University Hospital Gasthuisberg, Leuven, Belgium. 13. Department of Clinical Research, University of Bern, Bern, Switzerland. 14. Hungarian National Blood Transfusion Service, Molecular Diagnostics, Budapest, Hungary. 15. Department of Gastroenterology and Hepatology, Leiden University Medical Center, Leiden, The Netherlands Dutch Initiative on Crohn and Colitis (ICC) Division of Digestive Diseases, Inflammatory Bowel Diseases Center, UCLA, Los Angeles, USA. 16. Dutch Initiative on Crohn and Colitis (ICC) Department of Genetics, University Medical Center Groningen and the University of Groningen, Groningen, The Netherlands. 17. Department of Rheumatology, Clinical Immunology and Allergology, University Hospital of Bern, Inselspital, Switzerland.
Abstract
OBJECTIVE: Altered microbiota composition, changes in immune responses and impaired intestinal barrier functions are observed in IBD. Most of these features are controlled by proteases and their inhibitors to maintain gut homeostasis. Unrestrained or excessive proteolysis can lead to pathological gastrointestinal conditions. The aim was to validate the identified protease IBD candidates from a previously performed systematic review through a genetic association study and functional follow-up. DESIGN: We performed a genetic association study in a large multicentre cohort of patients with Crohn's disease (CD) and UC from five European IBD referral centres in a total of 2320 CD patients, 2112 UC patients and 1796 healthy controls. Subsequently, we did an extensive functional assessment of the candidate genes to explore their causality in IBD pathogenesis. RESULTS: Ten single nucleotide polymorphisms (SNPs) in four genes were significantly associated with CD: CYLD, USP40, APEH and USP3. CYLD was the most significant gene with the intronically located rs12324931 the strongest associated SNP (p(FDR)=1.74e-17, OR=2.24 (1.83 to 2.74)). Five SNPs in four genes were significantly associated with UC: USP40, APEH, DAG1 and USP3. CYLD, as well as some of the other associated genes, is part of the ubiquitin proteasome system (UPS). We therefore determined if the IBD-associated adherent-invasive Escherichia coli (AIEC) can modulate the UPS functioning. Infection of intestinal epithelial cells with the AIEC LF82 reference strain modulated the UPS turnover by reducing poly-ubiquitin conjugate accumulation, increasing 26S proteasome activities and decreasing protein levels of the NF-κB regulator CYLD. This resulted in IκB-α degradation and NF-κB activation. This activity was very important for the pathogenicity of AIEC since decreased CYLD resulted in increased ability of AIEC LF82 to replicate intracellularly. CONCLUSIONS: Our results reveal the UPS, and CYLD specifically, as an important contributor to IBD pathogenesis, which is favoured by both genetic and microbial factors. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.
OBJECTIVE: Altered microbiota composition, changes in immune responses and impaired intestinal barrier functions are observed in IBD. Most of these features are controlled by proteases and their inhibitors to maintain gut homeostasis. Unrestrained or excessive proteolysis can lead to pathological gastrointestinal conditions. The aim was to validate the identified protease IBD candidates from a previously performed systematic review through a genetic association study and functional follow-up. DESIGN: We performed a genetic association study in a large multicentre cohort of patients with Crohn's disease (CD) and UC from five European IBD referral centres in a total of 2320 CDpatients, 2112 UC patients and 1796 healthy controls. Subsequently, we did an extensive functional assessment of the candidate genes to explore their causality in IBD pathogenesis. RESULTS: Ten single nucleotide polymorphisms (SNPs) in four genes were significantly associated with CD: CYLD, USP40, APEH and USP3. CYLD was the most significant gene with the intronically located rs12324931 the strongest associated SNP (p(FDR)=1.74e-17, OR=2.24 (1.83 to 2.74)). Five SNPs in four genes were significantly associated with UC: USP40, APEH, DAG1 and USP3. CYLD, as well as some of the other associated genes, is part of the ubiquitin proteasome system (UPS). We therefore determined if the IBD-associated adherent-invasive Escherichia coli (AIEC) can modulate the UPS functioning. Infection of intestinal epithelial cells with the AIEC LF82 reference strain modulated the UPS turnover by reducing poly-ubiquitin conjugate accumulation, increasing 26S proteasome activities and decreasing protein levels of the NF-κB regulator CYLD. This resulted in IκB-α degradation and NF-κB activation. This activity was very important for the pathogenicity of AIEC since decreased CYLD resulted in increased ability of AIEC LF82 to replicate intracellularly. CONCLUSIONS: Our results reveal the UPS, and CYLD specifically, as an important contributor to IBD pathogenesis, which is favoured by both genetic and microbial factors. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.
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