Disruption of hemochromatosis protein and transferrin receptor 2 … · Disruption of Hemochromatosis Protein and Transferrin Receptor 2 Causes Iron-Induced Liver Injury in Mice Roheeth

Disruption of Hemochromatosis Protein andTransferrin Receptor 2 Causes Iron-Induced

Liver Injury in MiceRoheeth D. Delima,1,2 Anita C.G. Chua,1,2 Janina E.E. Tirnitz-Parker,1,2,3 Eng K. Gan,1,4 Kevin D. Croft,5

Ross M. Graham,1,2,3 John K. Olynyk,1,2,4,6 and Debbie Trinder1,2

Mutations in hemochromatosis protein (HFE) or transferrin receptor 2 (TFR2) cause he-reditary hemochromatosis (HH) by impeding production of the liver iron-regulatory hor-mone, hepcidin (HAMP). This study examined the effects of disruption of Hfe or Tfr2,either alone or together, on liver iron loading and injury in mouse models of HH. Iron sta-tus was determined in Hfe knockout (Hfe2/2), Tfr2 Y245X mutant (Tfr2mut), and double-mutant (Hfe2/23Tfr2mut) mice by measuring plasma and liver iron levels. Plasma alaninetransaminase (ALT) activity, liver histology, and collagen deposition were evaluated toassess liver injury. Hepatic oxidative stress was assessed by measuring superoxide dismutase(SOD) activity and F2-isoprostane levels. Gene expression was measured by real-time poly-merase chain reaction. Hfe2/23Tfr2mut mice had elevated hepatic iron with a periportaldistribution and increased plasma iron, transferrin saturation, and non-transferrin-boundiron, compared with Hfe2/2, Tfr2mut, and wild-type (WT) mice. Hamp1 expression wasreduced to 40% (Hfe2/2 and Tfr2mut) and 1% (Hfe2/23Tfr2mut) of WT values. Hfe2/2

3Tfr2mut mice had elevated plasma ALT activity and mild hepatic inflammation with scat-tered aggregates of infiltrating inflammatory cluster of differentiation 45 (CD45)–positivecells. Increased hepatic hydoxyproline levels as well as Sirius red and Masson’s Trichromestaining demonstrated advanced portal collagen deposition. Hfe2/2 and Tfr2mut mice hadless hepatic inflammation and collagen deposition. Liver F2-isoprostane levels were ele-vated, and copper/zinc and manganese SOD activities decreased in Hfe2/23Tfr2mut,Tfr2mut, and Hfe2/2 mice, compared with WTmice. Conclusion: Disruption of both Hfeand Tfr2 caused more severe hepatic iron overload with more advanced lipid peroxidation,inflammation, and portal fibrosis than was observed with the disruption of either genealone. The Hfe2/23Tfr2mut mouse model of iron-induced liver injury reflects the liverinjury phenotype observed in human HH. (HEPATOLOGY 2012;56:585-593)

Primary and secondary iron overload disorders areimportant causes of liver disease and associatedmorbidity worldwide.1 The most common pri-

mary iron overload disorder is hereditary hemochro-

matosis (HH), which affects approximately 1 in 200individuals of Northern European descent.2 There arefour types of HH; the most common, HH type 1, iscaused primarily by homozygosity for the C282Y

Abbreviations: ALT, alanine transaminase; BMP6, bone morphogenic protein 6; CD45, cluster of differentiation 45; DAPI, 40,6-diamidino-2-phenylindole;HAMP, hepcidin; H&E, hematoxylin and eosin; HFE, hemochromatosis protein; HH, hereditary hemochromatosis; HIC, hepatic iron concentration; HJV,hemojuvelin; HSCs, hepatic stellate cells; Idl, inhibitor of differentiation 1; LPO, lipid peroxidation; mRNA, messenger RNA; NTBI, non-transferrin-bound iron;ROS, reactive oxygen species; SEM, standard error of the mean; SMAD, mothers against decapentaplegic; SOD, superoxide dismutase; TFR1, transferrin receptor1; TFR2, transferrin receptor 2; WT, wild type.From the 1School of Medicine and Pharmacology, Fremantle Hospital, University of Western Australia, Fremantle, Western Australia, Australia; 2Western Australian

Institute for Medical Research, Perth, Western Australia, Australia; 3School of Biomedical Sciences, Curtin University, Perth, Western Australia, Australia; 4Departmentof Gastroenterology, Fremantle Hospital, Fremantle, Western Australia, Australia; 5School of Medicine and Pharmacology, Royal Perth Hospital, University of WesternAustralia, Perth, Western Australia, Australia; and 6Curtin Health Innovation Research Institute, Curtin University, Perth, Western Australia, Australia.Received November 15, 2011; accepted February 19, 2012.This study was supported by grants from the National Health and Medical Research Council of Australia (NHMRC 572601; to D.T., J.K.O., A.C.G.C., and

R.M.G.) and the Fremantle Hospital Medical Research Foundation (FHMRF; to R.M.G., D.T., J.K.O., and R.D.D.). R.D.D. is the recipient of an FHMRFWarren Jones Ph.D. Scholarship. A.C.G.C. is the recipient of a Bushell Postdoctoral Fellowship from the Gastroenterological Society of Australia (GESA). J.T.P. isthe recipient of a Curtin University Research Fellowship. E.K.G. is the recipient of a GESA Postgraduate Clinical Research Scholarship. J.K.O. is the recipient of aNHMRC Practitioner Fellowship (513761). D.T. is the recipient of a NHMRC Senior Research Fellowship (1020437).

585

mutation in the hemochromatosis protein (HFE).3

Iron overload disease develops in up to 30% of theseindividuals and can result in significant hepatic, pan-creatic, cardiac, or musculoskeletal tissue damage.4 Ju-venile, or HH type 2, is rare and is caused by muta-tions in hemojuvelin (HJV) or hepcidin (HAMP).5

HH type 3 is also rare and is caused by mutations intransferrin receptor 2 (TFR2),6 whereas HH type 4 iscaused by mutations in ferroportin.7 Almost all casesof HH result in impaired HAMP synthesis. DecreasedHAMP levels in HH cause increased iron absorptionfrom the duodenum, with the excess iron being depos-ited mainly in the liver.1,8

Studies have shown that when hepatic iron concen-tration exceeds 60 lmol/g, hepatic stellate cells(HSCs) begin to exhibit early signs of activation, anintegral event in the initiation of hepatic fibrosis.9 Ashepatic iron levels increase further, the risk of signifi-cant liver fibrosis and, ultimately, cirrhosis increases.10

Although the exact mechanisms of liver injury inducedby iron overload have not yet been fully elucidated, itis thought that the accumulation of excess iron-cata-lyzed reactive oxygen species (ROS) plays a significantrole. Previous studies have demonstrated decreased he-patic levels of antioxidants, such as superoxide dismu-tase (SOD), ascorbate, b-carotene, and vitamins E andA in iron overload conditions.11,12 Furthermore, ironincreases the level of lipid peroxidation (LPO) prod-ucts, such as malondialdehyde and F2-isoprostanes,

13

which can cause mutagenesis in DNA.14 LPO-inducedDNA lesions are increased 2- to 3-fold in the livers ofHH patients and, together with the iron overloadobserved in HH, are associated with an approximately20-fold increased risk of hepatocellular carcinoma.15,16

Oxidative stress has been shown to activate apoptosisand necrosis, promoting the synthesis and release ofproinflammatory and fibrogenic factors that alterKupffer cell and hepatocyte functions, triggering theactivation of HSCs and fibrogenesis.8

There are a number of murine models that recapitu-late the disturbed iron metabolism of HH.17 The firstHH mouse developed was an Hfe knockout (Hfe�/�)mouse model of HH type 1.18 Hjv and Hamp knock-out mouse models effectively reflect HH type 2.17

There are several models of HH type 3, including the

Tfr2 Y245X mutant (Tfr2mut) mouse that is ortholo-gous to the Y250X mutation identified in somepatients with HH type 3.19 Knockout of ferroportin isembryonically lethal; however, the flatiron mouse,which has a missense mutation (H32R) in ferroportin,exhibits a phenotype similar to that observed in HHtype 4.18 To date, there is no report on the inductionof liver toxicity, injury, or fibrosis in any untreatedgenetic mouse models of HH. Tan et al., however,recently reported early signs of fibrosis in Hfe�/� micefed a modified fat diet.20 In the present study, wedescribe iron-induced liver injury in Hfe�/��Tfr2mut

mice, where disruption of both Hfe and Tfr2 causesmore severe iron loading than disruption of either Hfeor Tfr2 alone, leading to enhanced liver injury andfibrosis.

Materials and Methods

Animals. Hfe�/� mice were generated by the dis-ruption of the Hfe gene using homologous recombina-tion, as described by Zhou et al.18 Tfr2mut mice weregenerated with a Y245X mutation in Tfr2, as reportedpreviously.19 Hfe�/� and Tfr2mut mice were back-crossed for 10 generations onto an AKR genetic back-ground (Animal Resource Center, Murdoch, WesternAustralia, Australia). Hfe�/� and Tfr2mut mice werethen crossed to generate Hfe�/��Tfr2mut double-mu-tant mice. Hfe�/�, Tfr2mut, Hfe�/��Tfr2mut, andwild-type (WT) mice (AKR background) were fedstandard mouse chow (200 mg iron/kg diet; SpecialtyFeeds, Glen Forrest, Western Australia, Australia) adlibitum from 4 weeks of age. An additional group ofWTmice was fed an iron-supplemented diet (20 g car-bonyl iron/kg diet; Specialty Feeds) for 3 weeks from8 weeks of age. At 11 weeks of age, after overnightfasting, blood was collected by cardiac puncture andorgans were perfused in situ with isotonic saline. Liverswere collected and snap-frozen in liquid nitrogen orfixed in formalin. This study was approved by TheUniversity of Western Australia (Perth, Western Aus-tralia, Australia) Animal Ethics Committee.RNA Expression. Total RNA was isolated from liver

tissue using TRI Reagent (Ambion Biosystems, Score-sby, Victoria, Australia) and reverse-transcribed using

Address reprint requests to: Debbie Trinder, Ph.D., School of Medicine and Pharmacology, Fremantle Hospital, University of Western Australia, P.O. Box 480,Fremantle, Western Australia 6959, Australia. E-mail: [email protected]; fax: 618-94312977.; .CopyrightVC 2012 by the American Association for the Study of Liver Diseases.View this article online at wileyonlinelibrary.com.DOI 10.1002/hep.25689Potential conflict of interest: Nothing to report.Additional Supporting Information may be found in the online version of this article.

586 DELIMA ET AL. HEPATOLOGY, August 2012

Superscript III (Invitrogen, Mulgrave, Victoria, Aus-tralia), as described previously.21 transferrin receptor 1(Tfr1), Tfr2, Hamp1, b-actin,21 bone morphogenicprotein (Bmp6),22 and inhibitor of differentiation 1(Id1)23 transcripts were measured by real-timepolymerase chain reaction in a Rotor-Gene 3000(Qiagen, Doncaster, Victoria, Australia) using primers,as previously described. Hfe expression was measuredusing a forward primer, 5’-CAGCTGAAACGGCTCCTG-3’, and a reverse primer, 5’-CGAGTCACTTTCACCAAAGTAGG-3’. Gene expression was quantifiedusing standard curves generated using plasmids con-taining complementary DNA of the gene of interestand normalized against b-actin messenger RNA(mRNA) expression.Iron Parameters. Plasma iron, transferrin satura-

tion, and non-transferrin-bound iron (NTBI) concen-tration were measured as previously described.24 He-patic nonheme iron concentration (HIC) wasmeasured using bathophenanthroline disulfonic acidby the method of Kaldor.25

Histology. Liver tissue was fixed in 10% neutralbuffered formalin for 12 hours before being subjectedto routine histological processing and stained with he-matoxylin and eosin (H&E). Liver sections werestained with Perls’ Prussian blue for the detection oftissue iron as well as with Sirius red and Masson’s tri-chrome for the detection of collagen.26 Stained sec-tions were digitized using an Aperio Scanscope XT(Aperio Technologies, Vista, CA) and analyzed using apositive pixel-count algorithm supplied by the manu-facturer. Pixel positivity was determined by the num-ber of pixels representing stained tissue divided by thetotal number of pixels in the whole liver section.Immunofluorescence. Cluster of differentiation 45–

positive (CD45þ) staining was performed on methanol/acetone (1:1) fixed liver cryosections using a rat anti-CD45 antibody (Ly-5, 1:150; BD Pharmingen, SanDiego, CA) and detected with goat antirat Alexa Fluor594 or goat antirat Alexa Fluor 488 (1:200; Invitrogen,Mulgrave, Victoria, Australia) and mounted with LongGold antifade reagent, containing 40,6-diamidino-2-phe-nylindole (DAPI; Invitrogen), for nuclear quantitation.Quantification was performed by the acquisition of sixrandom, nonoverlapping fields of view per tissue sam-ple, followed by colocalization analysis of CD45 andDAPI (nuclear quantification) using the AnalySIS LifeScience Professional program (Olympus, Melbourne,Victoria, Australia). Ferritin staining was performedusing a rabbit antiferritin antibody (1:800; Dako,Glostrup, Denmark) and detected using a goat antirab-bit Alexa Fluor 594 (1:200; Invitrogen).

Liver Injury. Plasma alanine aminotransferase(ALT) was measured as an indicator of liver injuryusing a kit according to the manufacturer’s instructions(Sigma-Aldrich, St. Louis, MO). Liver F2-isoprostanes,a marker of LPO, was measured by gas chromatogra-phy/mass spectrometry using a deuterium-labeled in-ternal standard, as previously described.27 The antioxi-dant, butylated hydroxyl toluene, was added to livertissue to scavenge any ROS generated during tissuestorage and processing. Activities of antioxidantenzymes copper/zinc and manganese SOD were meas-ured in the liver as an index of oxidative stress using akit according to the manufacturer’s instructions (Cay-man Chemical, Sydney, New South Wales, Australia).Liver hydroxyproline content was measured as a bio-chemical marker of liver collagen using a kit accordingto the manufacturer’s instructions (QuickZyme Bio-sciences, Leiden, Netherlands).Statistical Analysis. Results are expressed as mean

6 standard error of the mean (SEM), where n ¼ 5-15mice per group. Differences between groups were ana-lyzed using analysis of variance with Tukey’s multiplecomparison post-test or an unpaired Student’s t test(GraphPad Prism; GraphPad Software, Inc., La Jolla,CA). Differences between groups were defined as stat-istically significant for P < 0.05.

Results

Liver Gene Expression. Expression of Hfe, Tfr1,Tfr2, Bmp6, Id1, and Hamp1 genes is shown in Table 1.Hfe expression in Tfr2mut and WTmice was similar andundetectable in Hfe�/� and Hfe�/��Tfr2mut mice (P <0.001). Tfr2 mRNA expression in Tfr2mut and Hfe�/�

�Tfr2mut mice was decreased by approximately 65%,compared with non-iron-loaded WTmice (P < 0.001).Tfr2 mRNA expression in Hfe�/� and iron-loaded WTmice was also lower than non-iron-loaded WTmice (P< 0.05). Tfr1 mRNA expression was reduced in alltypes of HH mice and iron-loaded WTmice, comparedwith non-loaded WT mice (P < 0.05), consistent withliver iron loading. Expression of the gene encoding theiron-regulatory hormone, Hamp1, was decreased toapproximately 40% in Hfe�/� and Tfr2mut mice, com-pared with non-iron-loaded WT mice. In Hfe�/�

�Tfr2mut mice, Hamp1 expression was almost abolished,being further reduced to approximately 1% or 3% ofthat observed in non-iron-loaded WTmice (P < 0.01)or Hfe�/� and Tfr2mut mice (P < 0.05), respectively.Hamp1 expression, as expected, was increased in iron-loaded WT mice, compared with non-iron-loaded WTmice (P < 0.05) and HH mice (P < 0.001). Bmp6

HEPATOLOGY, Vol. 56, No. 2, 2012 DELIMA ET AL. 587

expression was increased in all HH and iron-loaded WTmice (p < 0.05), compared with non-iron-loaded WTmice, consistent with iron-dependent regulation ofBmp6. However, phosphorylated mothers against decap-entaplegic (Smad)1/5/8 protein levels were decreasedsignificantly in Hfe�/��Tfr2mut mice, compared withall other types of mice (P < 0.05), and inhibited inHfe�/� and Tfr2mut mice, compared with iron-loadedWT mice (P < 0.05; Supporting Fig. 1). Id1 (Bmp6/pSmad1/5/8 target), as with Hamp1 expression, wasdecreased in all HH mice, compared with non-iron-loaded WT mice (P < 0.05). This is consistent withimpaired pSmad1/5/8 signaling in HH mice.Plasma Iron Parameters. Plasma iron concentration

and transferrin saturation were higher in Hfe�/�

�Tfr2mut, Tfr2mut, Hfe�/�, and iron-loaded WT mice,compared with non-iron-loaded WT mice (P < 0.05;Fig. 1A,B). Iron concentration and transferrin saturationwere greatest in Hfe�/��Tfr2mut mice (P < 0.05; Fig.1A,B). Plasma iron concentration in Tfr2mut mice wasincreased, compared to Hfe�/� mice (P < 0.05). PlasmaNTBI concentration was also elevated in all iron-loadedmice (P < 0.05). In Hfe�/��Tfr2mut mice, NTBI levelswere 7-fold higher than non-iron-loaded WT mice andmore than 2-fold higher than Hfe�/�, Tfr2mut, and iron-loaded WTmice (P < 0.001; Fig. 1C).

HIC. HIC was elevated in all iron-loaded mice,compared with non-iron-loaded mice. HIC in Hfe�/�,Tfr2mut, and iron-loaded WT mice was similar andapproximately 3-fold higher than non-loaded WTmice (P < 0.001; Fig. 2A). HIC was greater in Hfe�/�

�Tfr2mut mice, compared with either Hfe�/� or Tfr2mut

mice (P < 0.01; Fig. 2A) and approximately 5-fold thatof the non-iron-loaded WT mice. Perls’ Prussian bluestaining of liver sections from Hfe�/��Tfr2mut micedemonstrated a periportal distribution of iron, similarto that observed in Hfe�/�, Tfr2mut, and iron-loadedWT mice. However, the intensity of iron staining wasgreater in Hfe�/��Tfr2mut than in the other types ofmice (Fig. 2B-D). These results indicate an increasediron burden in Hfe�/��Tfr2mut mice.Liver Injury. H&E-stained liver sections from

Hfe�/��Tfr2mut mice demonstrated mild inflamma-tion with evidence of scattered foci of infiltratinginflammatory cells throughout the liver parenchyma(Fig. 3). Immunofluorescent detection of the pan leu-kocyte marker, CD45, revealed that the cell aggregatesconsisted mainly of CD45þ inflammatory cells (Fig.3A,E) that colocalized predominately, but not exclu-sively, with the iron storage protein, ferritin, in peri-portal regions of the liver (Supporting Fig. 2). Thenumber of CD45þ inflammatory cells was significantly

Table 1. mRNA Expression of Iron Genes

Gene WT WTþFe Hfe�/� Tfr2mut Hfe�/��Tfr2mut

Hfe 33 6 2 29 6 1 —*,† 34 6 1‡ —*,†,§

Tfr2 85 6 5 65 6 5* 62 6 10* 30 6 4*,†,‡ 28 6 6*,†,‡

Tfr1 98 6 17 59 6 5* 35 6 9*,† 43 6 6*,† 43 6 8*,†

Hamp1 73 6 14 120 6 12* 34 6 13*,† 29 6 8*,† 0.9 6 0.4*,†,‡,§

Bmp6 1.8 6 0.2 5.2 6 0.3* 3.3 6 0.0*,† 3.1 6 0.3*,† 3.7 6 0.4*,†

Id1 91 6 3 487 6 34* 42 6 3*,† 62 6 12*,† 52 6 6*,†

Results are expressed as mean 6 SEM (n ¼ 5-15) mRNA copy number of the gene of interest relative to b-actin.*P < 0.05 versus WT.

†P < 0.05 versus WTþFe.

‡P < 0.05 versus Hfe�/�.§P < 0.05 versus Tfr2mut denote significance between groups.

Fig. 1. Plasma iron parameters. Plasma iron concentration (A), transferrin saturation (B), and NTBI concentration (C) were measured in WT,iron-loaded WT (WTþFe), Hfe�/�, Tfr2mut, and Hfe�/��Tfr2mut mice. Results are expressed as mean 6 SEM (n ¼ 5-15). a, P < 0.05 versusWT; b, P < 0.05 versus WTþFe; c, P < 0.05 versus Hfe�/�; d, P < 0.05 versus Tfr2mut.


increased in the livers from Hfe�/��Tfr2mut mice,compared with the other groups of mice (P < 0.05),whereas the number of CD45þ cells in Hfe�/�,Tfr2mut, and iron-loaded WT mice was not signifi-cantly different from those in non-iron-loaded WTmice (Fig. 3F). Another unique feature of Hfe�/�

�Tfr2mut mice was the evidence of inflammatory side-ronecrosis of hepatocytes, which was not observed inany other group of mice (Fig. 3E).Liver injury was assessed by examining plasma ALT

as well as hepatic SOD and F2-isoprostane levels.Plasma ALT activity was increased in Hfe�/��Tfr2mut

mice by at least 1.8-fold, compared with all othertypes of mice (P < 0.001; Fig. 4A). Both hepatic cop-per/zinc (cytosolic) and manganese (mitochondrial)SOD activities were significantly decreased in all HHmice. In Hfe�/��Tfr2mut mice copper/zinc SOD levelswere similar, whereas manganese SOD levels were sig-nificantly lower than Hfe�/� and Tfr2mut mice (P <0.01; Fig. 4B). Liver F2-isoprostanes were elevated inall groups of HH mice, compared with non-iron-loaded WT mice (P < 0.01), with Hfe�/� �Tfr2mut

mice having similar liver F2-isoprostane levels to iron-

loaded WTmice and significantly higher levels than ei-ther Hfe�/� or Tfr2mut mice (P < 0.01; Fig. 4C).Collagen Deposition. Hepatic collagen deposition, a

marker of fibrosis, was examined by histology using Sir-ius red and Masson’s trichrome staining and by bio-chemical measurement of hydroxyproline levels.Hydroxyproline levels were increased in all iron-loadedmice. In Hfe�/��Tfr2mut mice, hydroxyproline levelswere significantly increased, compared with Tfr2mut

mice, and both were elevated, compared with Hfe�/�

and iron-loaded WT mice (Fig. 4D; P < 0.05). Like-wise, Hfe�/��Tfr2mut mice had significantly increasedSirius red staining, compared with Hfe�/�, Tfr2mut, andiron-loaded WT mice (P < 0.05), which, in turn,exhibited greater collagen deposition than non-iron-loaded WT mice (P < 0.01; Fig. 5A-F). Sirius redstaining revealed portal tract thickening and periportalfibrosis in Hfe�/��Tfr2mut mice. In addition, there wasevidence of portal tract bridging in Hfe�/� �Tfr2mut

mice, which was not evident in other groups. Quantifi-cation of Sirius red staining correlated with HIC (r2 ¼0.98; P ¼ 0.001), plasma NTBI (r2 ¼ 0.82; P ¼0.033), as well as hydroxyproline (r2 ¼ 0.89; P ¼0.015) and F2-isoprotane levels (r

2 ¼ 0.77; P ¼ 0.048)in HH mice. This suggests that the collagen levelsmeasured by biochemical assay were consistent with his-tological observations using Sirius red staining and weredependent on both plasma NTBI and HIC in HHmice. Furthermore, the intensity of trichrome staining,a commonly used, but less sensitive, marker of fibrosis,was also significantly enhanced in Hfe�/��Tfr2mut andTfr2mut mice (Fig. 6F), with evidence of collagen thick-ening in the periportal region of the liver (Fig. 6A-E).

Discussion

In this study, we have used Hfe�/� and Tfr2mut

mouse models of HH types 1 and 3, respectively, anda Hfe�/��Tfr2mut mouse model to examine the effectsof disruption of Hfe and Tfr2, either alone or in com-bination, on liver iron loading and iron-induced liverinjury. We describe, to our knowledge, the first reportof a genetic HH mouse model of iron-induced liverinjury, the Hfe�/��Tfr2mut mouse, which reflects boththe iron-loaded phenotype and increased liver injuryobserved in HH patients.Hfe�/��Tfr2mut mice had elevated plasma and he-

patic iron levels, determined by both biochemical andhistological methods, compared with Hfe�/� andTfr2mut mice. Hamp1 levels were reduced in Hfe�/�

and Tfr2mut mice and almost abolished in Hfe�/�

�Tfr2mut mice. Hepcidin, the peptide encoded byHamp1, is a negative regulator of iron absorption and

Fig. 2. Hepatic iron concentration. Hepatic iron concentration wasdetermined biochemically and by Perls’ Prussian blue staining. (A)Results are expressed as mean 6 SEM (n ¼ 5-15). a, P < 0.001versus WT; b, P < 0.001 versus WTþFe; c, P < 0.01 versus Hfe�/�;d, P < 0.001 versus Tfr2mut. Staining was conducted on WT (B), iron-loaded WT (WTþFe; C), Hfe�/� (D), Tfr2mut (E), and Hfe�/��Tfr2mut

(F) mice. Each panel is representative of staining from 6-8 animals.


reduced hepcidin levels in Hfe�/�, Tfr2mut, and Hfe�/�

�Tfr2mut mice would lead to increased iron absorptionand hepatic iron deposition.8 In association withincreased liver iron loading, there was a pronouncedelevation of plasma ALT activity, a marker of liverinjury, in Hfe�/��Tfr2mut mice. There was also mildhepatic inflammatory cell infiltration with scattered fociof CD45þ leukocytes and some evidence of hepatocytesideronecrosis in Hfe�/��Tfr2mut mice. Elevatedhydroxyproline levels as well as Sirius red and tri-chrome staining showing marked portal tract collagendeposition and portal bridging in Hfe�/��Tfr2mut

mice clearly demonstrates the presence of liver fibrosisin areas of greatest iron accumulation. In comparison,Hfe�/� and Tfr2mut mice had less collagen depositionand inflammation. Histological evidence of a more pro-nounced liver damage in Hfe�/��Tfr2mut mice wascorroborated by decreased SOD activity and enhanced

LPO in the liver, indicating elevated hepatic oxidativestress.The iron-dependent regulation of HAMP is con-

trolled by HFE and TFR2, as well as BMP6/SMADcell-signaling pathways.22,23,28 It has been demon-strated that HFE can interact with TFR1 and TFR2to form a complex that is hypothesized to sense plasmatransferrin saturation and modulate hepcidin synthesisaccordingly.1,8 However, the nature of this mechanismis yet to be fully elucidated. Our findings support pre-vious studies that suggest there is cross-talk betweenHFE/TFR2- and BMP6/SMAD-signaling pathways,because the absence of functional HFE and/or TFR2attenuated iron-induced phosphorylation of SMAD1/5/8 and hepcidin expression.23,28

Mice with deletions in both Hfe and Tfr2 have beengenerated on other genetic backgrounds.23,28 Thesemice, as with our HH murine model, exhibited

Fig. 3. Liver histology. H&Estaining of liver sections from WT(left panel, A), iron-loaded WT(WTþFe; B), Hfe�/� (C), Tfr2mut

(D), and Hfe�/��Tfr2mut (leftpanel, E) mice. Arrows indicateinflammatory sideronecrosis of he-patocytes (left panel, E). CD45-stained (red) liver sections from WT(right panel, A) and Hfe�/�

�Tfr2mut (right panel, E) mice.Each panel shows a representativephotomicrograph of staining from 6animals. The number of CD45þ

cells (F) is expressed as mean 6SEM (n ¼ 6). a, P < 0.05 versusWT; b, P < 0.05 versus WTþFe; c,P < 0.05 versus Hfe�/�; d, P <0.05 versus Tfr2mut.


elevated plasma and liver iron levels, compared withmice with the appropriate deletion of Hfe or Tfr2, aswell as a marked reduction in Hamp1 expression, con-sistent with increased liver iron accumulation. How-ever, the degree of regulation of Hamp1 observed inHfe�/� and Tfr2mut mice, and its near abrogationinduced by the disruption of both Hfe and Tfr2, sug-gests that the formation of a Hfe and Tfr2 complex isnot a prerequisite for the initiation of iron-dependentHAMP synthesis. The degree of iron overload, how-ever, varies between strains, which is consistent withprevious observations that iron metabolism is modifiedby genetic background.29 Our HH mice were gener-ated on an AKR background and have relatively highplasma and liver iron levels, compared with otherstrains of mice. Colocalization of a more markedfibrotic process in areas of greatest iron deposition inthe hepatic periportal regions in our Hfe�/��Tfr2mut

mice provides further evidence of the importance ofgenetic background and phenotypic expression of ironoverload in the pathogenesis of liver injury in HH.Rodents are generally relatively resistant to iron-

induced liver injury. Dietary carbonyl iron loading ofrats for 3 months produced iron loading in hepato-cytes, similar to the levels observed in the Hfe�/�

�Tfr2mut mice in the present study, but demonstratedonly early signs of liver injury, including increasedLPO and collagen gene expression. Long-term ironloading was required for up to 12 months before mor-phological evidence of fibrosis was observed.30,31 Die-tary iron supplementation in combination with hepa-totoxins, such as ethanol and carbon tetrachloride, was

Fig. 4. Biochemical markers of liver injury.Plasma ALT (A), liver copper/zinc (open bars)and manganese (hatched bars) SOD (B), liverF2-isoprotane (C), and hydroxyproline (D) lev-els were measured in WT, iron-loaded WT(WTþFe), Hfe�/�, Tfr2mut, and Hfe�/

��Tfr2mut mice. Results are expressed asmean 6 SEM (n ¼ 5-15). a, P < 0.05 ver-sus WT; b, P < 0.05 versus WTþFe; c, P <0.05 versus Hfe�/�; d, P < 0.05 versusTfr2mut. For manganese SOD: 1, P < 0.05versus WT; 2, P < 0.01 versus WTþFe; 3, P< 0.01 versus Hfe�/�; 4, P < 0.01 versusTfr2mut.

Fig. 5. Collagen deposition in the liver. Sirius red staining of liversections from WT (A), iron-loaded WT (WTþFe; B), Hfe�/� (C), Tfr2mut

(D), and Hfe�/��Tfr2mut (E) mice. Staining intensity is quantified in(F). There was increased collagen deposition in the portal tracts ofWTþFe, Hfe�/�, and Tfr2mut mice with advanced thickening of theportal tract in Hfe�/��Tfr2mut mice. Results are expressed as mean6 SEM (n ¼ 6). a, P < 0.01 versus WT; b, P < 0.05 versus WTþFe;c, P < 0.01 versus Hfe�/�; d, P < 0.05 versus Tfr2mut. Each panelis a representative photomicrograph of staining from 6 animals.


required to accelerate liver injury.32,33 In the presentstudy, the degree of liver fibrosis observed in Hfe�/�

�Tfr2mut mice at 3 months of age was similar to thatobserved after dietary loading of rodents for 12months.30,31 In our Hfe�/��Tfr2mut mice, hepaticinflammation, fibrosis, and LPO occurred in the pres-ence of marked elevation of both plasma NTBI andhepatic iron levels, similar to those observed in humanHFE-related HH.34,35 Furthermore, the degree offibrosis observed in the HH mice was dependent onboth HIC and NTBI levels.The observation that Hfe�/��Tfr2mut mice have

increased plasma ALT levels is consistent with previ-ous observations in HH patients, where the majorityof patients had mildly elevated ALT levels.36 Levels ofthe antioxidant enzymes, cytosolic copper/zinc andmitochondrial manganese SOD, were both decreasedin Hfe�/��Tfr2mut mice consistent with increasedoxidative stress. Earlier studies have also reporteddecreased copper/zinc SOD in dietary iron-over-loaded animals, whereas manganese SOD wasdecreased in Hfe knockout and increased in iron-loaded rodents.11,20,37 Furthermore, LPO was

increased in HH mice. Unexpectedly, the level of F2-isoprostanes in dietary iron-loaded mice was greaterthan in HH mice with similar HIC. This may be theresult of differences between dietary iron (i.e., highHAMP) and genetic HH (i.e., low HAMP) modelsof liver iron overload where variation in cellular irondistribution between parenchymal and Kupffer cellsoccurs, despite similar total HIC.Mild liver inflammation was observed only in Hfe�/�

�Tfr2mut mice, suggesting that there was an iron-con-centration threshold effect. Mild inflammation has beendocumented in human HH studies during the develop-ment of fibrosis and cirrhosis.38 Deugnier et al. reportedinflammatory infiltrates in approximately 50% of liverbiopsies from HH patients.39 Inflammation was pre-dominantly present in portal and periportal regions andcorrelated with histological iron scores, sideronecroticchanges in hepatocytes, and hepatic fibrosis. Anotherstudy showed that approximately 25% of liver biopsiesfrom untreated HH patients displayed moderate inflam-matory infiltration.40 Bridle et al. also reported that60% of liver biopsies from HH patients showed mildinflammation consisting of scattered inflammatory foci.Furthermore, patients with hepatic inflammation had ahigher incidence of hepatic fibrosis.41 Iron-loaded andapoptotic/necrotic hepatocytes are purported to inducethe activation of HSCs by various signaling mecha-nisms, resulting in enhanced production of proinflam-matory and -fibrogenic cytokines as well as the recruit-ment of inflammatory cells.8 Our study provides furthersupport for the direct hepatotoxic effects of iron over-load, which results from the disruption of Hfe andTfr2, manifesting as inflammation and increased colla-gen deposition, suggesting the activation of HSCs.Iron plays an important part in the progression of

hepatic injury, and it does this through its ability tocatalyze the formation of highly reactive, damagingROS. ROS induce tissue injury by promoting LPO aswell as protein and DNA modification, leading, ulti-mately, to apoptosis and necrosis. Further investigationinto the molecular mechanisms of iron toxicity andhow it causes liver injury will provide a better under-standing of the role iron plays in the progression ofliver disease. The Hfe�/��Tfr2mut mouse represents amodel of the genetic iron overload disorder, HH, thatmimics both iron overload and consequent liver injuryobserved in humans with HH.

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Fig. 6. Masson’s trichrome staining. Trichrome staining of liver sec-tions from WT (A), iron-loaded WT (WTþFe; B), Hfe�/� (C), Tfr2mut

(D), and Hfe�/��Tfr2mut (E) mice. Staining intensity is quantified in(F). Results are expressed as mean 6 SEM (n ¼ 6). a, P < 0.05 ver-sus WT; b, P < 0.01 versus WTþFe; c, P < 0.01 versus Hfe�/�.There is increased thickening of portal tract collagen in Tfr2mut andHfe�/��Tfr2mut mice. Each panel is a representative photomicrographof staining from 6 animals.


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Disruption of hemochromatosis protein and transferrin receptor 2 … · Disruption of Hemochromatosis Protein and Transferrin Receptor 2 Causes Iron-Induced Liver Injury in Mice Roheeth