Mechanism On Regulation Of Hepcidin By Commensal Bacteria

The liver hormone hepcidin is the central regulator of systemic iron metabolism.Its increased expression in inflammatory states leads to hypoferremia and anemia.Elucidation of the mechanisms that up-regulate hepcidin during inflammation is essential for developing rational therapies for this anemia.Using mouse models of inflammatory bowel disease,it has been shown previously that colitis-associated hepcidin induction is influenced by intestinal microbiota composition.Here we investigate how two commensal bacteria,Bifidobacterium infantis and Bacteroides fragilis,representative members of the gut microbiota,affect hepcidin expression.Based on the results of the study,new therapies for hepcidin-induced anemia could be developed.1 Mechanism on how B.inf antis and B.fragilis affect expression of hepatocyte hepcidinA human hepatocyte cell line(HuH7)was stimulated with TLR(Toll-like receptor)ligands or infected with B.infantis and B.fragilis,which were anaerobically cultured in MRS(dMan Rogosa Sharpe)and RCM(Reinforced Clostridial medium)broth respectively.By qRT-PCR,the expression level of hepcidin in HuH7 cells was determined.The results showed that TLR ligands or either of the bacteria had no effects on hepcidin mRNA expression in HuH7 cells.The supernatants of a human macrophage cell line(THP-1)infected with either B.infantis or B.fragilis were collected and added to HuH7 cells,then the expression level of hepcidin in HuH7 cell was evaluated by qRT-PCR.It indicated that supernatants of THP-1 cells infected with either of the bacteria significantly up-regulated hepcidin of HuH7 cells.The contents of cytokines in supernatants from B.infantis-or B.fragilis-infected THP-1 cells were detected by ELISA.We found that either of the bacteria induced production of IL-1β in the supernatants.When the IL-1β in the supernatant was neutralized by Anti-IL-1β antibody,the activity of supernatnat up-regulating hepcidin in HuH7 cell was abrogated.Through qPT-PCR,moreover,purified IL-1β increased hepcidin expression when added to the hepatocyte line or primary human hepatocytes and when injected into mice,and the iron level in mouse serum was significantly decreased.To uncover the pathway to up-regulate hepcidin in hepatocytes and in mouse liver by IL-1β,Western blot was performed.It showed that IL-1β activated the BMP(Bone Morphogenetic Protein)signaling pathway in hepatocytes and in mouse liver,as indicated by increased phosphorylation of SMAD1/5/8(Small Mothers Against Decapentaplegic 1/5/8),while this activity was declined by anti-IL-1β antibody.Activation of BMP signaling correlated with IL-1β-induced expression of BMP2 in human hepatocytes and Activin B in mouse liver.Treatment of hepatocytes with two different chemical inhibitors of BMP signaling,Dorsomorphin and LDN193189,or with a neutralizing antibody to BMP2 prevented IL-1β-induced up-regulation of hepcidin in HuH7 cells and primary human hepatocytes.2 Mechanism of how IL-1β is produced by B.infantis-and B.fragilis-infected macrophagesThe mouse BMDMs(Bone Marrow-Derived Macrophage)and IBMDMs(Immortalized Bone Marrow-Derived Macrophage)were infected by B.infantis or B.fragilis for 1h respectively,washed and incubated for 4h or overnight,the contents of IL-1β in supernatants and the expression levels of IL-1β mRNA in cells were determined by ELISA and qRT-PCR respectively.The results showed that the levels of IL-1β and IL-1β mRNA were significantly increased after overnight incubation,while increased IL-1β mRNA expression levels were only observed after 4h incubation.By Western Blot,the contents of intracellular and extracelluar IL-1β were detected.It indicated that both elevated intracellular and extracellular IL-1β concentrations were only observed after overnight incubation,whereas the caspase-1 inhibitor ZYVAD added 30min before infection prevented production of extracellular IL-1β.For killing bacteria,B.infantis and B.fragilis suspensions were incubated for 30min at 95 ℃,and IL-1β secretion also occurred in response to heat-killed bacteria.When the phagocytosis was inhibited by Cyt D(Cytochalasin D),IL-1β production was only partly reduced in IBMDMs infected with B.fragilis.Similar results were obtained with a wild-type IBMDMs but neither B.infantis nor B.fragilis induced IL-1β secretion in NLRP3 KO IBMDMs.IL-1β secretion in response to B.infantis and B.fragilis was significantly reduced when the wild-type macrophage line was treated with inhibitors of potassium efflux,either increased extracellular potassium concentrations or the channel blocker ruthenium red.The THP-1 cells and huamn MDMs(Monocyte-Derived Macrophage)were infected with live bacteria for 1h,washed and incubated for 4h,and the levels of IL-1β in supernatants or intracellular IL-1β mRNA expression were evaluated by ELISA and qRT-PCR respectively.Live B.infantis and B.fragilis induced IL-1β secretion by THP-1 cells and human MDMs after 4 h infection,and the secretion was inhibited by ZYVAD.The results on Western Blot indicated that either of the bacteria elevated the contents of caspase-1p10.And the IL-1β production by THP-1 cells and human MDMs were also inhibited by raised extracellular potassium and ruthenium red but not by Cyt D.Moreover,HK(Heat-Killed)B.infantis and B.fragilis also induced IL-1β secretion from THP-1,human MDMs and mouse BMDMs,similar with live bacteria.And the production of IL-1βinduced by HK B.infantis and B.fragilis was dependent on potassium efflux,not Cyt D.Also,separating live bacteria with macrophage by Transwell,after overnight incubation,the activity of live bacteria to induce IL-1β secretion was abolished.ConclusionThe potassium efflux was induced by contaction between commensal bacteria and macrophages,which resulted in the activation of NLRP3 inflammasome and caspase-1.Consequently,the production and secretion of mature IL-1β occurred.Following the contents of BMP2 in human hepatocytes and Activin B in mouse liver was increased by stimulation of purified IL-1(3,BMP signaling pathway was activated,by which the hepcidin expression level was up-regulated.It was the first time to show that the commensal bacteria,B.infantis and B.fragilis,induced IL-1β production of macrophages by activation of NLRP3 inflammasome,and IL-1β-induced up-regulation of hepcidin resulted from activating BMP signaling pathway.The data in this research indicated that IL-1β secreted by macrophages infected with commensal bacteria may play an important role in anemia of inflammation.Moreover,the results provided more information to develop more efficient therapies for anemia of inflammation.