|M.Sc Student||Orly Savion|
|Subject||Iron Homeostasis in Cell Models for Intestinal Inflammation|
|Department||Department of Biotechnology and Food Engineering||Supervisor||Dr. Meyron Holtz Esther|
|Full Thesis text|
Crohn’s disease (CD) is an inflammatory bowel disease that causes transmural inflammation of the gastrointestinal mucosa and is often accompanied by anemia. Treating this anemia with oral iron supplementation is usually not well received and accumulation of reactive iron and elevated oxidative stress markers were found in the inflamed tissue of CD patients. In addition, in animal models of inflammatory bowel disease, iron supplements have aggravated the inflammation.
To determine the mechanism of iron accumulation in inflammation, we hypothesized that activation of the cytosolic iron regulatory protein (IRP) 1 generates upregulation of the iron importers transferrin receptor 1 (TfR) and divalent metal transporter 1 (DMT1) and downregulation of the iron storage protein ferritin. We therefore induced an inflammation condition in two types of cell-models: An intestinal human epithelial colon carcinoma cell line (Caco-2 cells) as a cell-type which represents intestinal epithelial cells, and bone marrow derived macrophages (BMDM) from wild type mice and from mice with targeted deletions of IRP1 (IRP1-/-) as a cell-type which represents the first line of the intestinal immune response.
Increased immune stimulation in CD mediates inducible nitric oxide synthase iNOS activation resulting in nitric oxide (NO) synthesis during inflammation. Therefore, in this study we decided to induce inflammation on Caco-2 cells by exposing cells directly to the NO donor, S-nitroso-N-acetylpenicillamine (SNAP).
SNAP treatment led to the induction of inflammation, evaluated by the elevation of an inflammation marker cyclooxygenase-2. Both cell models showed elevated IRP1-RNA binding activity after SNAP treatment, which led to iron homeostasis mis-regulation: In Caco-2 cells treated with SNAP iron importers TfR and DMT1 expression was increased, while iron exporter protein FPT levels were possibly decreased. In wild type BMDMs levels of the iron storage protein ferritin were decreased. These findings led us to the conclusion that reactive oxygen or nitrogen species, present during inflammation, activate IRP1 and deactivate IRP2, leading to a total increase of IRP-RNA binding activity and that this activation perturbs iron homeostasis and may causes an accumulation of reactive iron, which can add to the severity of the inflammation.
In contrast to wild type cells, BMDMs from IRP1-/- mice showed increased levels of ferritin expression after SNAP treatment compared to control, indicating the major contribution of IRP1 activation to the depression of Ft expression in the WT BMDMs.