Western-diet-derived arachidonic acid induces epithelial ferroptosis which is a feature of Crohn's disease

Introduction:

Glutathione peroxidase 4 (GPX4) is an anti-oxidative enzyme which is a key regulator of ferroptosis. Ferroptosis is a recently described cell death mechanism, defined by iron dependency and increased lipid peroxidation (LPO). A genome wide association study described GPX4 as a risk gene for Crohn's disease (CD). Up until now, there has been no link between LPO and ferroptosis in the intestine of CD patients.

Methods:

Biopsies collected from CD patients and healthy controls undergoing routine colonoscopy were used for IHC analysis, protein and RNA expression measurements. MODE-K cells, a murine small intestinal epithelial cell line was used for in vitro experiments. Cells were stimulated 24 hours after GPX4 silencing was done in cells to achieve decreased GPX4 levels. For further work up, an ACSL4 knockout MODE-K cell was developed, using CrisprCas9. LPO in IHC was measured with 4HNE staining and in live cells with a BodipyC11 probe via flow cytometry.

Results:

Mucosa obtained from lesional small intestine of CD patients, showed reduced GPX4 activity and expression compared to healthy controls. In line with this, CD patients displayed increased LPO. Silencing of GPX4 in MODE-K cells (siGPX4) induced ferroptosis, indicated by increased LPO and cell death. Stimulating siGPX4 cells with polyunsaturated fatty acids such as arachidonic acid (AA) led to a marked upregulation of IL-6 and the IL-8 homologue CXCL1. ACSL4 and lipoxygenases controlled this inflammatory response.

Conclusion:

Crohn's disease patients have a reduction of GPX4 in lesional areas of their gut. Reduction of GPX4 activity in intestinal epithelial cells evokes ferroptosis. Moreover, AA induced inflammation was restricted by GPX4 and driven by ACSL4 and lipoxygenase-mediated LPO in these cells. We conclude, that dietary derived compounds, such as fatty acids, could trigger ferroptosis in Crohn's disease.