Monocyte expression of iron related genes in the Dysmetabolic Hepatic Iron Overload syndrome Monocyte mRNA expression of iron related genes in the very common Dysmetabolic Hepatic Iron Overload syndrome: an alternative source of hepcidin production explaining the pathogenesis of D-HIO and its iron phenotype?
Background
Hepcidin, the master regulator of iron homeostasis, is mainly produced by hepatocytes. Interestingly, tissues other than the liver can also synthesize hepcidin, including the kidney, the right hearth atrium and the spinal cord. Moreover, significant hepdicin expression has been found in spleen, alveolar and bone marrow-derived murine macrophages. Recently, it was shown that monocyte derived hepcidin exerts autocrine or paracrine regulation towards cellular iron metabolism by modulating ferroportin expression in inflammatory monocytes, thus contributing to iron sequestration within monocytes as found in anemia of chronic diseases [Theurl I et al, Blood 2008;111:2392-9].
The most common form of iron overload is associated with metabolic abnormalities, insulin resistance and hepatic steatosis, and variably named Insulin Resistance- or Dysmetabolic-Hepatic Iron Overload [IR-HIO or D-HIO] syndrome. It is characterised by hyperferritinemia with normal or slightly increased transferrin saturation and a mixed pattern of hepatic iron overload involving either hepatocytes and mesenchimal cells. D-HIO phenotype suggests the existence of defects in cellular iron excretion as observed in ferroportin disease, but the pathogenetic mechanisms leading to iron overload and the relationship between insulin resistance and iron overload is still unknown. We recently observed in patients with D-HIO, that urinary hepcidin concentrations were significantly higher than in controls and they did not correlate with the mRNA level in the liver suggesting the existence of altered hepcidin synthesis and possibly the existence of an hepcidin source other that the liver.
Aim
Based on these observation we will investigate whether monocyte derived hepcidin may effectively exerts regulation towards cellular iron metabolism in D-HIO patients. Hepcidin formation by activated monocytes/macrophages might result in a biologically significant accumulation of this peptide in the inflammatory environment associated with insulin resistance and steatohepatitis which then affects iron homeostasis of monocytes/tissue macrophages in an autocrine and paracrine fashion. Actually, expression data are not available on this field, yet. In particular we will focus on iron-related genes expression in monocytes of D-HIO patients at baseline and after two different phases of treatment: diet to obtain regulation of the metabolic abnormalities and iron depletion. Data will be compared with those observed in HFE hemochromatosis and control subjects. A sample of patients’ monocytes mRNAs will be collected separately to perform wide expression arrays in order to explore the existence of still unknown pathways of interaction between iron and monocyte activation in D-HIO. A sample of hepatic tissue, obtained by diagnostic liver biopsy from the same patients will be collected to compare data at the hepatic level.
Patients and Methods
Five patients from each group will be studied: D-HIO, HFE-Hemochromatosis (C282Y homozygotes), and healthy controls matched for age and sex at diagnosis. Monocytes will be also collected in D-HIO patients after diet and iron depletion and in HFE-hemochromatosis after iron depletion. Monocyte samples will be collected at least one month after the last phlebotomy. Monocytes will be separated from buffycoat polymorphonucleate cells by Ficoll gradient. Then, microbeads coated with CD14 antibodies will be used to positively select target cells. Purity and integrity will be assessed by cytofluorometry analysis. A piece of needle liver biopsy (0.5-1 cm), performed for diagnostic purposes, will be immediately snap-frozen in liquid nitrogen for mRNA analysis. RNA from hepatic tissue and CD14-positive monocytes will be extracted using TRIzol (Invitrogen, Grand Island, NY, USA), quantified by spectrophotometry and its integrity assessed by non-denaturing agarose gel. mRNA expression levels will be evaluated by quantitative real time PCR (qRT-PCR) will be performed to validate results. Relative quantities present in each sample will be assessed using the Ct method. According to antibodies availability, Western Blots will be performed to assess protein expression.
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