P0041Intestinal epithelial glutathione peroxidase 4 expression as a druggable target in Crohn’s disease after ileocolonic resection

Abstract Background Postoperative disease recurrence is frequent in Crohn’s disease (CD) following ileocolonic resection (ICR), underlining the need for new treatment options. Recent evidence suggests that reduced Glutathione peroxidase 4 (GPX4), a selenoprotein and antioxidative enzyme that protects against enteritis1, 2, is associated with disease recurrence after ICR3. Here, we aimed to identify factors that impair mucosal GPX4 expression and to develop therapeutic strategies to boost its function in the gut to treat ileitis. Methods RNA sequencing data of mucosal specimens of the neo-terminal ileum from 36 patients after ICR were used to define transcriptional ­signatures associated with reduced GPX4 expression. To model human findings, we used intestinal epithelial cell (IEC) -specific transgenic mice lacking Gpx4 (Gpx4-/-IEC ΔERT), mice prone to endoplasmic reticulum (ER) stress (lacking X-box-binding protein 1; Xbp1-/-IEC) and double mutant (i. e. Xbp1-/-IEC/Gpx4+/-IEC) mice. GPX4 expression and enteritis severity were assessed by histology, confocal microscopy, immunoblotting, and flow cytometry. Therapeutic rescue of enteritis was tested using oral selenium supplementation and reactive oxygen species scavengers. Results An ER stress-related signature was inversely correlated with GPX4 expression in human post-ICR samples (Fig. 1A). To investigate the consequences of ER stress in intestinal epithelium exhibiting reduced GPX4 activity (as observed in Crohn’s ileitis), we generated mice that delete Xbp1 and one allele of Gpx4 specifically in IECs (i. e. , Xbp1-/-IEC/Gpx4+/-IEC mice). Xbp1-/-IEC/Gpx4+/-IEC mice developed severe spontaneous ileitis with a patchy discontinuous ileitis and crypt hyperplasia (Fig. 1B, C) that was associated with increased ER stress and related c-Jun N terminal kinase (JNK) activity (Fig. 1D, E). Moreover, treatment with the antioxidants N-acetylcysteine or α-tocopherol ameliorated enteritis in Xbp1-/-IEC mice which exhibit reduced epithelial GPX4 levels (Fig. 2A, B), suggesting that impaired GPX4 activity promotes enteritis in these mice. More importantly, oral selenium supplementation boosted epithelial GPX4 expression and ameliorated enteritis in Xbp1-/-IEC mice (Fig. 2C-F), whereas oral selenium did not improve enteritis in Gpx4-/-IEC ΔERT mice (Fig. 2G), confirming that its anti-inflammatory effect depends on intestinal epithelial GPX4 function. Conclusion Our study identifies a functional link between intestinal epithelial ER stress and GPX4-mediated antioxidative actions in controlling ileitis in mice and likely contributing to small intestinal CD. This pathway is druggable by oral selenium supplementation in mice, establishing a basis for non-immunosuppressive therapy after ICR. References: 1. Mayr, L. et al. Dietary lipids fuel GPX4-restricted enteritis resembling Crohn’s disease. Nature communications 11, 1775, doi: 10. 1038/s41467-020-15646-6 (2020). 2. Schwärzler, J. et al. PUFA-Induced Metabolic Enteritis as a Fuel for Crohn’s Disease. Gastroenterology 162, 1690-1704, doi: 10. 1053/j. gastro. 2022. 01. 004 (2022). 3. Verstockt, S. et al. Op01 sequencing-based gene network analysis reveals a profound role for ferroptosis key gene gpx4 in post-operative endoscopic recurrence in crohn’s disease (ecco conference). Journal of Crohn’s and Colitis 17, Issue Supplement₁: i1-i3 (2023). Conflict of interest: Dr. Schwärzler, Julian Peter: No conflicts of interest to declare. Verstockt, Sare: Grant: Postdoctoral fellowship of the Research foundation – Flanders (FWO), Belgium Willeit, Peter: consultancy fees from Novartis Pharmaceuticals Bislenghi, Gabriele: no conflict of interest Meyer, Moritz: No conflict of interest Mayr, Lisa: No conflict of interest Machiels, Kathleen: postdoctoral fellow of the Fund for Scientific Research-Flanders, Belgium (FWO-Vlaanderen) until 5/01/2021. Pfizer Medical advisor employee in inflammation and immunology since 5/01/2021. De Hertogh, Gert: Other: Fees to my institution KULeuven for my activities as central pathology reviewer for: Centocor and Eli Lilly Lenfant, Matthias: None Scheffauer, Laura: No conflict of interest Grabherr, Felix: No conflict of interest Jans, Deborah: No conflict of interest Abdurahiman, Saeed: None to declare Cleynen, Isabelle: Grants: research support from FWO/SBO and FWO large infrastructure. Personal fees: none. Non-financial support: none. Other: none. Jukic, Almina: No conflict of interest Zundel, Luis: No conflict of interest Moser, Patrizia: No conflict of interest Forer, Lukas: consultancy fees from Novartis Pharmaceuticals Witsch-Baumgartner, Martina: No conflict of interest 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 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. D’Hoore, André Jan Louis: Personal Fees: Takeda, Janssens Hess, Michael: No conflict of interest Kaser, Arthur: Personal Fees: MiroBio, Merck, Janssen, GSK, Pfizer, ONO Pharmaceuticals, Ferring, Nimbus, Astrazeneca, Imhotex, Boehringer Ingelheim, Novartis, Applied Molecular Transport, Eli Lilly, Galapagos Blumberg, Richard S.: No conflict of interest Koch, Robert: No conflict of interest Le Bourhis, Lionel: No conflict of interest Hammoudi, Nassim: Other: Honoraria from Janssen Cazal-Hatem, Dominique: No conflict of interest 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, Tilg, Herbert: No conflict of interest Allez, Matthieu: Grant: Janssen, Genentech/Roche, Takeda Personal Fees: Abbvie, Amgen, Astra-Zeneca, Biogen, Boehringer-Ingelheim, Bristol Myers Squibb, Celgene, Celltrion, Ferring, Galapagos, Genentech, Gilead, IQVIA, Janssen, Novartis, Pfizer, Spyre therapeutics, Roche, Takeda, Tillots 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.