Siri Sæterstad [1], Ann Elisabeth Østvik [1,2], Marianne Doré Hansen [1,3], Torunn Bruland [1,2], Atle van Beelen Granlund [1,2,4,5]

Affiliates

[1] Department of Clinical and Molecular Medicine, Norwegian University of Science and Technology (NTNU), Trondheim, Norway. [2] Department of Gastroenterology and Hepatology, Clinic of Medicine, St. Olav’s University Hospital, Trondheim, Norway. [3] Clinic of Laboratory Medicine, St. Olav's University Hospital, Trondheim, Norway. [4] Department of Pathology, St. Olav’s University Hospital, Trondheim, Norway. [5] Centre of Molecular Inflammation Research, Norwegian University of Science and Technology (NTNU), Trondheim, Norway.

Abstract

Introduction
Genetics is a known, however complex, etiological factor in inflammatory bowel disease (IBD). Variants in Endoplasmic Reticulum Aminopeptidase 2 (ERAP2) have been associated with multiple inflammatory conditions, including Crohn’s disease and ulcerative colitis. Several of these variants are associated with ERAP2 expression levels, where increased expression of ERAP2 is associated with inflammation. How ERAP2 contributes to pathogenesis is however unclear. While primarily known for its role in major histocompatibility class I antigen-presentation, accumulating evidence suggest that ERAP2 harbors pleiotropic functions beyond peptide trimming. Additional functions of ERAP2 have mainly been demonstrated in cancer cell lines or immune cells, while the role of ERAP2 in epithelial cells remain unexplored. As the involvement of epithelial cells in development of IBD is increasingly acknowledged, we set out to investigate the effects of ERAP2-proficiency and -deficiency in human-derived colon organoids (colonoids) after proinflammatory stimulation.

Methods
Two ERAP2-linked expression quantitative trait loci (eQTLs) were analyzed in a cohort of IBD patients, namely rs2910686 and rs2248374. Genotypes for both variants were correlated with colonic ERAP2 expression levels in uninflamed and inflamed biopsies. Based on rs2910686 genotype, ERAP2-proficient (n=4) and ERAP2-deficient (n=4) colonoids were established to study differential expression between proficient and deficient donors post IFNɣ-stimulation.

Results
An in-house analysis of colonic biopsies derived from inflamed and uninflamed IBD patients identified rs2910686 as a strong eQTL of ERAP2. Stratifying patients based on rs2910686 genotype revealed ERAP2 as upregulated in the inflamed mucosa of IBD patients, a finding that is masked when not taking genotype into account. We also confirm that mechanisms independent of the well-established eQTL rs2248374 can lead to ERAP2-deficiency. We found a total of 586 genes to be differentially expressed between ERAP2-proficient and -deficient colonoids post IFNɣ-stimulation, a known inducer of ERAP2.

Conclusions
Our results demonstrate that ERAP2 is upregulated in the inflamed colon mucosa when taking rs2910686 genotype into account. We confirm that mechanisms independent of the eQTL rs2248374 can lead to ERAP2-deficiency, underscoring the importance of evaluating multiple variants when determining ERAP2 status. We further demonstrate that ERAP2-proficient and ERAP2-deficient human-derived colonoids display differential expression post IFNɣ-stimulation, suggesting that ERAP2 presence affects the inflammatory response in epithelial cells. Lastly, we demonstrate that stratification of organoids based on genotype serve as a useful and physiologically relevant model system for evaluating the effects of disease-associated genetic variants.

Poster_P7_Siri Sæterstad