A263 IRONING OUT THE DETAILS: CROHN’S DISEASE PATIENT DERIVED MACROPHAGES ARE MORE SUSCEPTIBLE TO FERROPTOSIS

TL;DR

Macrophages from Crohn's disease patients are more likely to undergo a type of cell death called ferroptosis, possibly due to lower levels of a protective protein.

Contribution

The study identifies ferroptosis as a novel mechanism of macrophage death in Crohn’s disease linked to reduced GPx1 expression.

Findings

CD macrophages are twice as susceptible to H2O2-induced cell death compared to healthy controls.

Ferroptosis markers like SLC7A11 and PTGS2 are induced in CD macrophages, and lipid peroxidation can be blocked with liproxstatin-1.

GPX1 mRNA and protein levels are reduced in CD macrophages despite higher serum GPx activity in patients.

Abstract

Death of intestinal epithelial cells is ubiquitous in Crohn’s disease (CD) but the death of immune cells such as macrophages is less explored. We have found that monocyte derived macrophages from patients with active CD are more susceptible to H2O2-induced cytotoxicity but the form of regulated cell death these cells undergo remains to be elucidated. Selenium (Se)—a common micronutrient deficiency in patients with CD—is used in the synthesis of selenoproteins that have antioxidant properties (e.g., glutathione peroxidases (GPx)) and are highly expressed in macrophages. Whether Se deficiency plays a role in the increased cytotoxicity also remains to be elucidated.

To determine the form of cell death of macrophages in response to H2O2-induced cytotoxicity and whether selenium deficiency plays a role in the increased susceptibility to H2O2-induced cytotoxicity.

Blood collected from healthy volunteers and patients with active CD was analyzed for serum GPx activity. Monocytes isolated by plastic adherence were treated with M-CSF (10ng/ml, 7d) to derive macrophages. Macrophages were treated with H2O2 (500μm, 2h) and lactate dehydrogenase release was measured. GPX1, SLC7A11 and PTGS2 mRNA expression were determined by qPCR. The presence of cleaved caspase 3 was determined by immunoblotting and IL-1β by ELISA. Lipid peroxidation a marker of ferroptosis was assessed by staining macrophages with Bodipy C-11 (± liproxstatin-1, an inhibitor of ferroptosis).

CD macrophages were two times more susceptible to H2O2-evoked cell death. In response to H2O2 macrophages did not express cleaved caspase 3 or IL-1β, but there was an induction of ferroptosis markers (SLC7A11 and PTGS2). Moreover, lipid peroxidation was induced and could be blocked with liproxstatin-1. Dietary Se intake did not differ between groups but serum GPx activity was greater in patients with CD compared to control. In contrast, GPX1 mRNA expression and GPx1 protein expression were decreased in CD macrophages compared to healthy controls.

Macrophages derived from patients with CD are inherently more sensitive to ferroptosis potentially through reduced GPx1 expression. Future studies warrant testing if GPx1 prevents ferroptotic macrophage cell death and if it holds therapeutic relevance in CD pathophysiology.

Helmsley Charitable Trust, Crohn's and Colitis Foundation of America