P0046From stress to healing: upadacitinib ameliorates epithelial endoplasmic reticulum stress and suppresses pro-fibrotic signalling in ulcerative colitis

Abstract Background Intestinal organoids provide a powerful tool to study epithelial responses in inflammatory bowel disease (IBD), preserving patient-specific and disease-relevant characteristics. As inflammation diminishes during culture, we previously developed inflammatory and endoplasmic reticulum (ER) stress-inducing cytokine mixes to mimic mucosal stress1-2. Here, we explored the direct epithelial effect of upadacitinib (UPA) – a JAK1 inhibitor – under inflammatory and ER stress conditions. Methods Ulcerative colitis (UC) -derived intestinal organoid (n = 3, non-inflamed biopsies) were cultured in human expansion media (HM, 2 days), followed by differentiation media (DM, 4 days). From day 6, organoids were exposed for 48 hours to either DM only (control, CTRL), inflammation (INF: 50 ng/ml TNFα, 20 ng/ml IL1β, 1 µg/ml Flagellin) or ER stress (ER: 10 ng/ml TNFα, 20 ng/ml IL1β, 1 µg/ml Flagellin, 25 ng/ml Tunicamycin) 1. UPA (10 µM) was administered for 48 hours (INFUPA48, ERUPA48), or the final 24 hours (INFUPA24, ERUPA24). Organoids were harvested on day 8 for RNA sequencing (Illumina NovaSeq 6000, DESeq2; Foldchange 1. 5, FDR 0. 05) (Fig. 1A). Results Principal component (PC) analysis revealed clustering primarily by inflammatory treatment (PC1 28%, PC2 19. 5%) (Fig. 1B). In inflammatory settings, continuous UPA exposure (INFUPA48 vs. INF) led to 103 differentially expressed genes (DEGS, 49 up, 54 down) including AKAP12, IFITM1, MFGE8, TJP1. Similarly, UPA supplementation in the final 24 hours (INFUPA24), resulted in 8 DEGs (4 up, 4 down) including AKAP12, IFITM1, MFGE8, MX1 (Fig. 1C). Under ER stress, 48 hours of combined UPA and ER exposure (ERUPA48 vs. ER) altered 68 genes (20 up, 48 down) including MX, MFGE8, IFITM1, EIF2AK2, while 24 hours exposure (ERUPA24) affected 36 genes (7 up, 29 down) such as MX1, IFITM1, IFI44, MFGE8 (Fig. 1D). Pathway analysis revealed strong downregulation of interferon (IFN) alpha/beta and gamma signalling, and modulation of host-responses in both ERUPA conditions (Fig. 2A). Overlap across all UPA conditions, identified 4 genes consistently regulated by UPA, with greater transcriptional impact under ER stress (Fig. 2B). Across UPA-treated groups, JAK1 inhibition markers were downregulated, while MFGE8, encoding for a protein with both anti-inflammatory and antifibrotic properties2, was upregulated. When INF was used as a trigger, UPA upregulated TJP1, a key marker of intestinal barrier restoration3, while in the ER exposed condition EIF2AK2, an ER marker gene4, was downregulated by UPA (Fig. 2C). Conclusion Upadacitinib exerts context-dependent epithelial effects that extend beyond immunosuppression, directly enhancing epithelial resilience and tissue restoration. References: 1. Giorio, Arnauts et al. , abstract Journal of Crohn’s and Colitis 2025; 2. Lin, Wang et al. Gut 2024; 3. Kuo et al. Gastroenterology 2021; 4. Yim et al. Frontiers in Immunology 2023 LG and KA are joint first authors Conflict of interest: Mr. Giorio, Lorenzo: Research support by Galapagos NV Arnauts, Kaline: Research support by Galapagos NV Van De Perre, Caitlin: No conflict of interest Guedelha Sabino, João: Speaker’s fees: Lilly, Pfizer, Abbvie, Ferring, Falk, Takeda, Janssen, Fresenius, and Galapagos. Consultancy fees: Takeda, Pfizer, Janssen, Ferring, Fresenius, Abbvie, Galapagos, Celltrion, Pharmacosmos, and Pharmanovia. Research support: Galapagos, Viatris, and Eurogenerics. JS is supported by a Senior Clinical researcher grant from the Research foundation – Flanders. Ferrante, Marc: Research grants from AbbVie, EG Pharma, Celltrion, Janssen, Pfizer, Takeda and Viatris Consultancy fees from AbbVie, AgomAb Therapeutics, Boehringer Ingelheim, Celgene, Celltrion, Eli Lilly, Janssen-Cilag, MRM Health, Merck Sharp and Dohme, Pfizer, Takeda and ThermoFisher Speakers’ fees from AbbVie, Biogen, Boehringer Ingelheim, Dr Falk Pharma, Ferring, Janssen-Cilag, Merck Sharp and Dohme, Pfizer, Takeda, Truvion Healthcare and Viatris Vermeire, Séverine: Grant: AbbVie, Pfizer, Takeda, J&J, Galapagos Personal Fees: AbbVie - AbolerIS Pharma - AgomAb - Alimentiv - Arena Pharmaceuticals - AstraZeneca - Avaxia- BMS - Boehringer Ingelheim - Celgene - CVasThera - Dr Falk Pharma - Ferring - Galapagos - Genentech-Roche - Gilead - GSK - Hospira - Imidomics - Janssen - J&J - Lilly - Materia Prima - MiroBio - Morphic - MrMHealth - Mundipharma - MSD - Pfizer - Prodigest - Progenity - Promakhos Therapeutics - Prometheus - Robarts Clinical Trials - Second Genome - Shire - Surrozen - Takeda - Theravance - Tillots Pharma AG - Zealand Pharma - Other: AbbVie, MSD, Takeda, Ferring, Genentech/Roche, Shire, Pfizer Inc, Galapagos, Mundipharma, Verstockt, Bram: - Research support from AbbVie, Biora Therapeutics, Celltrion, Landos, Pfizer, Sanofi, Sossei Heptares/Nxera and Takeda. - Speaker’s fees from Abbvie, Agomab, Alfasigma, Biogen, Bristol Myers Squibb, Celltrion, Eli Lily, Falk, Ferring, Galapagos, Materia Prima, Johnson and Johnson, Pfizer, Sandoz, Takeda, Tillots Pharma, Truvion and Viatris. - Consultancy fees from Abbvie, Alfasigma, Alimentiv, Anaptys Bio, Applied Strategic, Astrazeneca, Atheneum, BenevolentAI, Biora Therapeutics, Boxer Capital, Bristol Myers Squibb, Domain Therapeutics, Eli Lily, Galapagos, Guidepont, Landos, Merck, Mirador Therapeutics, Mylan, Nxera, Inotrem, Ipsos, Johnson and Johnson, Pfizer, Sandoz, Sanofi, Santa Ana Bio, Sapphire Therapeutics, Sosei Heptares, Takeda, Tillots Pharma and Viatris. - Stock options Vagustim and Thethis Pharma.