Dietary lipids fuel GPX4-restricted enteritis resembling Crohn’s disease

The increased incidence of inflammatory bowel disease (IBD) has become a global phenomenon that could be related to adoption of a Western life-style. Westernization of dietary habits is partly characterized by enrichment with the ω-6 polyunsaturated fatty acid (PUFA) arachidonic acid (AA), which entails risk for developing IBD. Glutathione peroxidase 4 (GPX4) protects against lipid peroxidation (LPO) and cell death termed ferroptosis. We report that small intestinal epithelial cells (IECs) in Crohn’s disease (CD) exhibit impaired GPX4 activity and signs of LPO. PUFAs and specifically AA trigger a cytokine response of IECs which is restricted by GPX4. While GPX4 does not control AA metabolism, cytokine production is governed by similar mechanisms as ferroptosis. A PUFA-enriched Western diet triggers focal granuloma-like neutrophilic enteritis in mice that lack one allele of Gpx4 in IECs. Our study identifies dietary PUFAs as a trigger of GPX4-restricted mucosal inflammation phenocopying aspects of human CD.

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Male and female mice were used, all included mice had a C57BL6/J background. GPX4flox/flox mice were crossed with VillinCre positive mice to obtain GPX4flox/wt VillinCre+ and GPX4flox/wt VillinCre-mice. Mice were 7 to 10 week old at the start of the experiments. Housing was done under SPF conditions with a 12/12 hour dark/light cycle.
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Human studies were approved by the Ethics Committee of the Medical University of Innsbruck LPO and cell death labelling. Cells derived from cell culture or from IEC isolation procedures were incubated with BODIPY 581/591 C11 or the surface labelling antibodies (see below) at 37°C in the dark for ten to 30 minutes in flow cytometry buffer (2% FCS, 2mM EDTA in PBS). Cells were subsequently washed with PBS, resuspended in FACS buffer and transferred through a 40µm cell strainer for flow cytometry. Annexin V, PI or 7AAD were used for cell death analysis. Mouse LPMCs were isolated according to previously published protocols 60,61. In short, the proximal small intestine was flushed with ice cold PBS, opened longitudinally, cut into small pieces and transferred to HBSS (Gibco, 14175-053) containing 10% FCS, DTT (1mM) and EDTA (2mM) followed by shaking for 20 minutes at room temperature. Samples were vortexed to remove IELs and the tissue was washed and collected in IMDM (Gibco, 21056-023) 20% FCS. Tissue was washed and 10U/ml DNAse (10U/ml, Sigma, D8764) and 128 U/ml collagenase (128U/ml, Sigma, C1889) digested on a shaker for 60 minutes at 37°C. Cells were passed through cell strainers (100µm) and washed twice before transferring to cytometry buffer for staining (see below). Human IEC isolation. The biopsies were moved from RPMI (Biochrome, FG1385) to HBSS-CMF buffer (Gibco, 14175-053, 0.5% BSA, 2mM EDTA and DTT). Samples were incubated on a shaker for 20 minutes at room temperature. The samples were then vortexed vigorously and supernatant (containing IECs) was collected through a 100µm cell strainer, which was repeated for a total of three times. Supernatant was then spun down at 300g and the pellet was used for GPX4 activity assay and western blot analysis as detailed in the respective sections.