| Human hepcidin,a 25-amino acid,cysteine-rich cationic peptide mainly secreted by the liver.The synthesis of hepcidin is greatly stimulated by inflammation or by iron overload,while suppressed by hypoxia and anemia.By internalization and degradation of feroportin,which is the only known iron exporter in vertebrates,presents on macrophages and enterocytes,hepcidin suppressed iron uptake and release respectively. Previous studies have identified a vital link in the acute phase activation of hepcidin and established IL-6 and STAT3 as the key effector of baseline hepcidin expression and during inflammatory conditions in hepatocytes.Sepsis is a common and major cause of mortality in critical illness,initiation of sepsis occurs as microbial components are recognized pattern recognition molecules or receptors,such as Toll-like receptors (TLRs),activation of which induces the transcription of inflammatory and immune response genes,often via NF-κB mediated mechanisms,resulting in the release of a number of endogenous mediators including a series of cationic antimicrobial peptides. Although hepcidin expression in hepatocytes upregulated significantly in septic animal models,the recent evidence showed that the hepatogenic hepcidin synthesis by bacterial stimulation is not mediated by TLR4,which is one of the crucial PAMP recognized receptors in the pathogenesis of sepsis.Conversely,bacterial stimulation triggered a TLR4 independent hepcdin transcription production in myeloid cells in the TLR4 gene knockout models.So we hypothesized that the mechanisms of hepcidin production in myeloid cells may differ from that in hepatocytes and the regulable expression of hepcidin in activatory peripheral blood mononuclear cells(PBMCs) and neutrophils may significantly contribute to the process as well as the outcome of severe sepsis.The present study was designed to investigate the mRNA levels of hepcidin in peripheral blood cells in vivo from patients with severe sepsis compared with those in health controls by semi-quantitative RT-PCR assay.In addition,LPS treatment was carried out in peripheral blood at 1-1000 ng/mL with time course from 0 to 24 hours. To explore the differences of hepcidin expression in peripheral blood cells induced by individual inflammatory cytokines,whole blood was co-cultured with 100ng/mL LPS, 20ng/mL TNF-α,20ng/mL IL-1β,20ng/mL IL-6,or 50ng/mL IFN-γfor 6 hours.We further explore possible transcriptional factors involved in the transcriptional regulation of hepcidin in myeloid cells,two inhibitors Ro106-9920 and rapamycin which specifically inhibit the biological activity of transcription factors NF-κB and STAT3 respectively were treated with PBMCs and neutriphils which were separated from whole blood by Ficoll-Hypaque density centrifugation.Meanwhile,10μg/mL TNF-αAb combined with 100ng/ml LPS was also used to treat the cells.The present study showed that the mRNA levels of hepcidin in peripheral blood cells from patients with severe sepsis significantly increased compared to those from controls(p＜0.01).In untreated peripheral blood cells,only a low level of hepcidin mRNA was detected with a Hepcidin-to-hHPRT1 ratio from 0 to 0.12.However,an LPS stimulated increase of hepcidin mRNA was observed under from low to high LPS concentrations and as much as 15- to 20-fold increase was detected after the peripheral blood cells were exposed to LPS for 6 hours.More interestingly,IFN-γand IL-1βas well as IL-6,which are important in hepcidin augmentation in hepatocytes,also did not affect hepcidin expression in peripheral blood cells(p＞0.05).Conversely,TNF-αstimulation increased the expression of hepcidin strongly(p＜0.01).We showed that inhibition of STAT3 did not significantly affect the mRNA level of hepcidin both in neutrophils and PBMCs,while inhibition of NF-κB did completely abrogate the inducibility of hepcidin by LPS.We also found that neutraliztion of TNF-αeffect partly suppress the hepcidin expression upgrate in peripheral blood cells co-incubation with LPS by treatment with TNF-αAb.In summary,hepcidin mRNA was dramatically induced by proposed microbial components and cytokins in peripheral blood cells from severe sepsis patients and LPS/ TNF-α/NF-κB axis potentially play an important role for the inducible expression of hepcidin in neutrophils and PBMCs in sepsis.
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