Abstract
The inflammatory bowel diseases (IBD) Crohn’s disease (CD) and ulcerative colitis (UC) are disorders that cause chronic inflammation of the gastrointestinal tract. Both genetic and environmental factors contribute to the pathogenesis of IBD. There are currently 238 known genetic susceptibility loci for the development of IBD. The physiological impact of most of these loci remains a gap in our knowledge.
The single nucleotide polymorphism rs1077773 is one such locus and is located ~56kbp downstream from the aryl hydrocarbon receptor (AHR) gene. AHR is a ligand-activated transcription factor that is crucial to maintaining intestinal homeostasis. We hypothesized that rs1077773 enhances AHR activity to regulate mucosal immune response and maintain intestinal homeostasis.
All study procedures and reagents were approved by the Washington University Institutional Review Board (#202011003). Patient biopsies were collected and genotyped using the IBD Genetics Consortium custom GSA SNP chip (Broad Institute). SNP rs1077773 was imputed through TopMed Imputation Server at University of Michigan. Patient derived organoids (PDOs) were derived and maintained in 3D culture and supplemented with 50% L-WRN conditioned medium with passage every 3-4 days as previously described. PDOs were treated with AHR agonist 6-Formylindolo[3,2-b]carbazole (FICZ) or vehicle for 48h. Expression of canonical AHR transcriptional target Cytochrome P450 1A1 (CYP1A1) was assessed by RT-qPCR. Peripheral blood mononuclear cells (PBMCs) were isolated from pediatric surgical patients homozygous for reference allele (N=2) and patients heterozygous for rs1077773 (N=2) and treated with lipopolysaccharide in the presence or absence of FICZ for 24h. Cytokine levels in culture supernatant were measured via LEGENDplex human essential immune response panel.
Variant rs1077773 is associated with enhanced CYP1A1 expression following AHR activation in PDOs (average fold change relative to untreated 2.87 ± 3.2 v 8.99 ± 2.27 for G/G and G/A vs A/A, respectively; p=0.0293). In PBMCs, LPS treatment caused increased IL6 and IL8 secretion, which was abrogated in response to AHR activation. Patients heterozygous for rs1077773 exhibit enhanced reduction in IL6 and IL8 levels and stabilization of IL10 levels.
This work demonstrates that rs1077773 is a quantitative trait locus for AHR activity and modulates mucosal immune response. Further mechanistic understanding of this locus and its correlates could improve our understanding of IBD susceptibility and may lead to novel therapeutic approaches specific to the intestinal epithelium.
Expression of AHR and CYP1A1 by rs1077773 genotype. RFC, relative fold change.
Percent change in cytokine levels following AHR activation.
