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GRK 1482 Jahrbuch 2011-2014

Publications [1] Werner T, Wagner SJ, Martínez I, Walter J, Chang JS, Clavel T, Kiesling S, Schümann K, Haller D. Depletion of luminal iron alters the gut microbiota and prevents Crohn’s disease-like ileitis. Gut. 2011, 60: 325-333. [2] Wagner SJ, Schmidt A, Effenberger JP, Gruber L, Danier J, Haller D. Semisynthetic diet ameliorates Crohn’s di- sease-like ileitis in TNF∆ARE/WT mice through antigen- independent mechanisms of gluten. Inflamm Bowel Dis. 2013 Apr 4. (epub). [3] Coplin M, Schuette S, Leichtmann G; Lashner B. Tolerability of iron: a comparison of bis-glycino iron II andironsulfate.ClinicalTherapeutics.1991,13:606-612. [4] Darshan D, Vanoaica L, Richman L, Beermann F, Kühn CL. Conditional deletion of ferritin H in mice induces loss of iron storage and liver damage. Hepatoloy. 2009, 50 (3): 852-86. PhD FELLOWS GRK Progress Report 2011-2014 | Page 45 Aim In this project we evaluate the role of luminal iron on intestinal inflammatory processes. The focus is a better understanding of the molecular mechanisms of dietary iron on epithelial cell level. We will further dissect the influence of different iron formula- tions (e.g. iron sulfate, heme iron). To follow up the influence of luminal iron on the intestinal microbiota we are planning to work with Desulfovibrio bacteria in germ free mice. Above, we parti- cipate in a clinical trial with IBD patients to compare our pre- clinical findings from animal studies with human patient’s data. Methods and Results For the characterization of the molecular mechanisms of the effect of luminal iron on inflammatory processes we focus on the iron storage protein ferritin. Mice carrying a conditional H-ferritinknockoutintheintestinalepithelialcells(Fth∆/∆ mice;4) will be challenged with dextran sodium sulfate (DSS) to look at the susceptibility of Fth∆/∆ mice for intestinal inflammation. Tissue samples will be analyzed on histological and molecular levels. The intestine-specific deletion of H-ferritin triggers the loss of mucosal iron storage along with increasing serum iron and body iron load. We will further titrate the dietary iron and thereby trying to avoid the development hemochromatosis in this mouse model. To dissect the role of different nutritional iron sources on the development of experimental ileitis we performed feeding stu- dies using TNF∆ARE/WT and Rag-/- mice (in cooperation with PD Dr. A. Krug, Klinikum Rechts der Isar). For the histological examination H&E staining of distal ileal tissue sections was prepared. Inflammatory markers (TNF, IFNγ) were analyzed on RNA and protein expression levels via q-RT-PCR and western blotting. There were no significant differences between the feeding groups in the TNF∆ARE/WT receiving diets fortified with either ferrous sulfate or hemin (180 mg/kg diet each) in terms of weight development, histological scoring and expression of proinflammatory cytokines. We observed an impaired iron sta- tus in the TNF∆ARE/WT mice compared to the wildtype control mice measured by the hematocrit level and the hemoglobin level in the plasma. BiopsysamplesofanemicpatientssufferingfromeitherCDorUC on two different iron replacement therapies (oral ferrous sulfate vs. systemic iron sucrose) were analyzed for proinflammatory cytokines (TNF, IL-8), ER stress markers (Grp78, XBP-1s) and oxidative stress markers (catalase, superoxidase dismutase) with neither significant differences between the treatment groups and nor significant differences between IBD patients and control patients. Outlook In the next experiments the gut microbiota of anemic IBD pa- tients receiving either an oral iron sulfate or a systemic iron sucrose therapy will be characterized via a sequencing tech- nique to further investigate the effect of luminal iron on the bacterial composition. Working with Fth∆/∆ mice we will not only evaluate the suscep- tibility for intestinal inflammation after a DSS challenge but also try to avoid the development of hemochromatosis by titrating the iron content in the diet. By colonizing germ-free IL10-/- mice with bacteria of the genus Desulfovibrio we will examine the role of single iron-dependent bacteria in the development of experi- mental IBD. The main readouts will be the phenotypically deve- lopment as well as the histological examination of the intestine. Supervisors Prof. Dr. Dirk Haller I TUM I Nutrition and Immunology PD Dr. Anne Krug I TUM I Klinikum rechts der Isar I Internal Medicine II Start of project: November 2011 Academic background: Studies of Nutritional Science at Technische Universität München Figure 1: Principle component analysis (PCA) of the cecal microbiota by pyrosequencing of 16S rRNA tags in WT and TNF∆ARE/WT mice re- vealed differences in the microbial composition between the different diet groups. Red: iron adequate diet, green: iron deficient diet.