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An RNAi screen identifies TLR2/6 as mediators of a novel inflammatory pathway for rapid hepcidin-independent hypoferremia

Guida, Claudia

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Abstract

Systemic iron homeostasis is essential for human health. Its maintenance critically depends on the interaction between the hepatic hormone hepcidin and the sole known iron exporter ferroportin (FPN) predominantly expressed in hepatocytes, duodenal enterocytes and macrophages. Hepcidin binding leads to FPN internalization and degradation resulting in cellular iron retention. Iron is an essential nutrient also for pathogens and plays a central role in host-pathogen interactions. The innate immune system fights infections by sequestration of iron in macrophages of the reticuloendothelial system. The resulting hypoferremia represents a major host defence strategy. A current model posits that hepcidin is the crucial effector of this response, as its release from macrophages and hepatocytes provokes FPN protein decrease and, consequently, tissue iron retention. The aim of my PhD project was to identify novel cellular regulators of hepcidin-mediated ferroportin (FPN) degradation, a fundamental process that controls systemic iron homeostasis. To reach this aim I generated a HeLa cell line expressing a hFPN-renilla fusion protein, which was used for a focused high-throughput RNAi screen targeting kinases and related proteins. Out of 779 genes tested, the screen identified 71 putative regulators of FPN protein stability. Validation experiments confirmed the phenotype of 24 genes. Interestingly, most validated regulators of FPN expression conferred hepcidin-independent FPN regulation. From these I selected 14 genes associated with immune processes for further characterization in murine bone marrow-derived macrophages (BMDMs). Finally, my studies focused on Toll-like receptor 6 (TLR6) as an effective regulator of FPN expression in BMDMs and I investigated how the TLR6 activation pathway modulates iron regulation in the inflammatory context. TLR2/6 ligation by the synthetic lipoprotein derived from Mycoplasma: FSL1 triggered a profound decrease in FPN mRNA and protein expression in BMDMs as well as in the liver and the spleen of mice. Unexpectedly hepcidin expression remained unchanged. Hepcidin-independent FPN down regulation was a conserved response to different microbial lipopeptides and elicited a fast, hepcidin-independent hypoferremia pathway. These findings were further confirmed in C326S FPN knock-in mice with a disrupted hepcidin/FPN regulatory circuitry. This work challenges the prevailing role of hepcidin in inflammatory hypoferremia and suggests that rapid hepcidin-independent FPN down regulation may represent the first line response to restrict iron access to pathogens.

Item Type: Dissertation
Supervisor: Muckenthaler, Prof. Dr. Martina
Date of thesis defense: 24 October 2014
Date Deposited: 09 Dec 2014 14:21
Date: 2014
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Subjects: 500 Natural sciences and mathematics
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