P0003Linking cholesterol homeostasis to intestinal inflammation in inflammatory bowel diseases

Abstract Background Inflammatory bowel disease (IBD), comprising ulcerative colitis (UC) and Crohn’s disease (CD), is characterized by chronic gastrointestinal inflammation (1) driven by dysregulated interactions between the immune system and the gut microbiota. Cholesterol has emerged as a potential regulator of intestinal inflammation through its ability to modulate the NLRP3 inflammasome (2), a key pathway controlling IL-1β and IL-18 secretion (3). However, the relationship between cholesterol metabolism, inflammation, and microbiota-driven regulation remains poorly understood. We hypothesize that cholesterol accumulation in the colonic mucosa influences NLRP3 inflammasome activation and downstream cytokine release. Here, we investigate the role of cholesterol in inflammasome activation using IBD-relevant models including ex vivo cultured biopsies and human macrophages. Methods Transcriptomic data (bulk RNA-seq) from 1,000 IBD patients (4), publicly available scRNA-seq datasets (5, 6), and colonic biopsy samples were analyzed to assess cholesterol-related gene expression and IL-1β/IL-18 levels. Cholesterol depletion (methyl-β-cyclodextrin, MβCD) and NLRP3 inhibition (MCC950) were evaluated in colonic explants ex vivo and human macrophages in vivo. Statistical analyses were performed using Kruskal–Wallis tests. Results UC and CD samples showed increased expression of cholesterol transport and LDL-R–related genes, accompanied by reduced ABCA1 and ABCG1 expression. NLRP3, IL-1β, and IL-18 levels were elevated across IBD tissues. Cholesterol depletion with MβCD reduced cholesterol content and IL-18 secretion in UC explants (p 0.05) without affecting IL-1β, while no effect was observed in CD. Moreover, Cholesterol depletion with MβCD reduced cholesterol and inflammasome derived cytokines in in vitro cultures of macrophages. Additionally, MCC950 effectively inhibited cholesterol-induced inflammasome activation in vitro (p 0.0001). Conclusion Intracellular cholesterol activates the NLRP3 inflammasome and promotes IL-18–driven inflammation, particularly in UC. Targeting cholesterol-related pathways, while considering microbiome-driven influences, may offer new avenues for modulating inflammation in IBD. References: 1.West, N., Hegazy, A., Owens, B. et al. Oncostatin M drives intestinal inflammation and predicts response to tumor necrosis factor–neutralizing therapy in patients with inflammatory bowel disease. Nat Med 23, 579–589 (2017). https://doi.org/10.1038/nm.4307. 2. Astorga J, Gasaly N, Dubois-Camacho K, De la Fuente M, Landskron G, Faber KN, Urra KA, Hermoso, MA. The role of cholesterol and mitochondrial bioenergetics in activation of the inflammasome in IBD. Frontiers in immunology, 2022, DOI10.3389/fimmu.2022.1028953. 3. Aggeletopoulou, MK, Tsounis EP, Triantos C. Exploring the role of IL-1β in inflammatory bowel disease pathogenesis. Frontiers in Medicine 2024 DOI10.3389/fmed.2024.1307394. 4.Hu S, Bourgonje AR, Gacesa R, Jansen BH, Bjork JR, Bangma A, Hidding IJ, van Dullemen HM, Visschedijk MC, Faber KN et al. (2024) Mucosal host-microbe interactions associate with clinical phenotypes in inflammatory bowel disease. Nat Commun 15, 1470. 5.Smillie CS, Biton M, Ordovas-Montanes J, Sullivan KM, Burgin G, Graham DB, Herbst RH, Rogel N, Slyper M, Waldman J, Sud M, Andrews E, Velonias G, Haber AL, Jagadeesh K, Vickovic S, Yao J, Stevens C, Dionne D, Nguyen LT, Villani AC, Hofree M, Creasey EA, Huang H, Rozenblatt-Rosen O, Garber JJ, Khalili H, Desch AN, Daly MJ, Ananthakrishnan AN, Shalek AK, Xavier RJ, Regev A. Intra- and Inter-cellular Rewiring of the Human Colon during Ulcerative Colitis. Cell. 2019 Jul 25;178(3):714-730.e22. doi: 10.1016/j.cell.2019.06.029. PMID: 31348891; PMCID: PMC6662628. 6.Kong L, Pokatayev V, Lefkovith A, Carter GT, Creasey EA, Krishna C, Subramanian S, Kochar B, Ashenberg O, Lau H, Ananthakrishnan AN, Graham DB, Deguine J, Xavier RJ. The landscape of immune dysregulation in Crohn’s disease revealed through single-cell transcriptomic profiling in the ileum and colon. Immunity. 2023 Feb 14;56(2):444-458.e5. doi: 10.1016/j.immuni.2023.01.002. Epub 2023 Jan 30. Erratum in: Immunity. 2023 Dec 12;56(12):2855. doi: 10.1016/j.immuni.2023.10.017. PMID: 36720220; PMCID: PMC9957882. Conflict of interest: Molina, Hector: No conflict of interest Astorga, Jessica: No conflict of interest Lopez, Valentina Arlette: No conflict of interest Dubois-Camacho, Karen: No conflict of interest Landskron, Glauben: No conflict of interest De la Fuente, Marjorie: No conflict of interest Fernandez, Romina: No conflict of interest Flores, Lilian: No conflict of interest Festen, Eleonora: No conflict of interest Faber, Klaas-Nico: No conflict of interest Dijkstra, Gerard: No conflict of interest Weersma, Rinse K: No conflict of interest Lui, Moting: No conflict of interest Nuñez, Paulina: No conflict of interest Quera, Rodrigo Enrique: No conflict of interest Hermoso, Marcela: No conflict of interest