Altered Erythropoiesis in Mouse Models of Type 3...

Research ArticleAltered Erythropoiesis in Mouse Models ofType 3 Hemochromatosis

R M Pellegrino1 F Riondato2 L Ferbo1 M Boero1 A Palmieri1 L Osella2 P Pollicino2

B Miniscalco2 G Saglio1 and A Roetto1

1Department of Clinical and Biological Sciences AOU San Luigi Gonzaga University of Torino Orbassano Torino Italy2Department of Veterinary Sciences University of Torino Grugliasco Torino Italy

Correspondence should be addressed to A Roetto antonellaroettounitoit

Received 7 September 2016 Revised 1 March 2017 Accepted 4 April 2017 Published 2 May 2017

Academic Editor Francesco Onida

Copyright copy 2017 R M Pellegrino et alThis is an open access article distributed under theCreativeCommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Type 3 haemochromatosis (HFE3) is a rare genetic iron overload disease which ultimately lead to compromised organs functioningHFE3 is caused bymutations in transferrin receptor 2 (TFR2) gene that codes for twomain isoforms (Tfr2120572 and Tfr2120573) Tfr2120572 is oneof the hepatic regulators of iron inhibitor hepcidin Tfr2120573 is an intracellular isoform of the protein involved in the regulation of ironlevels in reticuloendothelial cells It has been recently demonstrated that Tfr2 is also involved in erythropoiesis This study aims tofurther investigate Tfr2 erythropoietic role by evaluating the erythropoiesis of two Tfr2 murine models wherein either one or bothof Tfr2 isoforms have been selectively silenced (Tfr2 KI and Tfr2 KO)The evaluations were performed in bone marrow and spleenin 14 daysrsquo and 10 weeksrsquo old mice to assess erythropoiesis in young versus adult animals The lack of Tfr2120572 leads to macrocytosiswith low reticulocyte number and increased hemoglobin values together with an anticipation of adult BM erythropoiesis andan increased splenic erythropoiesis On the other hand lack of Tfr2120573 (Tfr2 KI mice) causes an increased and immature splenicerythropoiesis Taken together these data confirm the role of Tfr2120572 in modulation of erythropoiesis and of Tfr2120573 in favoring ironavailability for erythropoiesis

1 Introduction

Type 3 haemochromatosis (HFE3) is an autosomal recessivegenetic disorder that leads to an accumulation of iron both inthe blood and in several tissues especially in the liver Becauseof this HFE3 patients have elevated transferrin saturation(TS) and serum ferritin level (sFt) [1]

This disease is caused bymutations in transferrin receptor2 (TFR2) gene [2] that codes for two main isoforms namelyTfr2 alpha (Tfr2120572) and Tfr2 beta (Tfr2120573) that showmoderatehomology to the type 1 transferrin receptor (Tfr1) [3] UnlikeTfr1 TFR2 gene expression itself is not directly regulated byiron [4] and TFR2 mRNA does not have iron responsiveelements (IREs) Thus there is no IRE-dependent posttran-scriptional regulation of the protein levels [5] Several in vitrostudies have demonstrated that Tfr2120572 can bind iron loadedtransferrin however with an affinity remarkably lower thanthat of Tfr1 [3] Additionally the levels of this protein inplasma membrane are regulated by TS with an increased

stabilization in the presence of highly saturated transferrin [67] The transcription of the entire TFR2 gene gives rise to theTfr2120572 isoform which is a transmembrane protein Tfr2120573 onthe other hand is a shorter isoform lacking the cytoplasmicand transmembrane domains and it is currently unknownif its activity is intracellular or extracellular The expressionpatterns of these two isoforms are also very different Tfr2120572is highly transcribed in the liver and in erythroleukemic cellline (K562) while Tfr2120573 is mainly transcribed in liver brainheart [3] and splenicmacrophages [8]Most TFR2mutationscompromise the production of both Tfr2 isoforms Howeversome mutations affect only the Tfr2120572 isoform while otherssuch as M172K abolish the Tfr2120573methionine starting codon[9 10]

Studies on murine models of HFE3 have demonstratedthat Tfr2120572 and Tfr2120573 isoforms have distinct functions in ironhomeostasis While Tfr2120572 is involved in the hepatic pathwayregulating iron hormone hepcidin (Hamp) [7] Tfr2120573 isinvolved in iron efflux from reticuloendothelial cells [8]

HindawiBioMed Research InternationalVolume 2017 Article ID 2408941 12 pageshttpsdoiorg10115520172408941

2 BioMed Research International

A recent study has demonstrated that the Tfr2120572 isoform isalso expressed in erythroid progenitors Here it interactswith and stabilizes the erythropoietin receptor (EpoR) henceestablishing a correlation between Tfr2120572 and erythropoiesisfor the first time [11] Furthermore it is thought to be directlyinvolved in the mechanisms of control of erythropoiesisespecially in conditions of iron deprivation [12 13]

It is well known that erythropoiesis as well as irondemand changes throughout life in humans as a consequenceof an increase in blood volume Also complete blood cellcount (CBC) values differ slightly depending upon the age[14] To our knowledge no data exist to date on variationsin erythropoiesis during ageing in mice Some regulatorsof erythropoiesis have been characterized in recent yearsOne of them named Erythroferrone (Erfe) is a hormoneproduced by erythroblasts that is able to regulate Hamp levelsas a consequence of erythropoietic demand due to blood loss[15] or anemia of inflammation [16]

To investigate how Tfr2120572 silencing influences erythrocyteproduction across lifespan inmice we studied erythropoiesisin two primary erythropoietic organs the bonemarrow (BM)and the spleen at different ages using twomousemodels withinactivated Tfr2120572 andor Tfr2120573 The findings of this studycould be an important step toward gaining a better insightinto Tfr2 involvement in erythropoiesis in humans

Our results indicate that the lack of Tfr2120572 influences BMand splenic erythropoiesis starting from an early stage of lifeMoreover Tfr2120573 also influences erythropoiesis by the mod-ulation of iron availability for erythrocyte maturation Moreimportantly we now show that Erfe expression is regulatedby erythropoiesis not only in adult animals as previouslydemonstrated [15] but also in young age Additionally Erfeappears to be negatively modulated by erythropoietic tissuesrsquoiron availability Lastly we also describe the physiologicalvariations of erythropoietic activity in WT mice duringageing

2 Materials and Methods

21 Animals Two Tfr2 mouse models on 129X1svJ strainwere studied (1) Tfr2 KI that has the Tfr2120573 isoform inacti-vated (120572+1205730) and (2) Tfr2 KO that has both Tfr2 isoformsinactivated (12057201205730) Selective targeting of Tfr2 isoforms wasobtained starting from the same target construct in whichmurine M163K mutation (homologous to M172K humanvariant) was inserted in murine Tfr2 gene exon 4 flanked by3 loxP sites activated through Crelox recombination system[8]

At adult age (10 w) Tfr2 KI mice have normal Hampand normal serum iron parameters but splenic iron overloadwhile Tfr2 KO animals have normal Hamp but high serumferritin and transferrin saturation as well as hepatic ironoverload [8]

All animals were housed at Department of VeterinarySciences University of Torino Animal housing and all theexperimental procedures were performed in accordance withEuropean (Official Journal of the European Union L276del 20102010 Vol 53 p 33ndash80) and National Legislation

(Gazzetta Ufficiale n∘ 61 del 14032014 p 2ndash68) for the pro-tection of animals used for scientific purposes

Tfr2-targeted mice and controls were maintained onstandard conditions and with ad libitum access to food andwater They were analyzed at 14 days and 10 weeks of ageOnlymalemice were used in this study tominimize potentialvariability related to sex

At least 6 animals were analyzed for each experimentalcondition

22 Hematological Analysis Peripheral blood from the ani-mals was subjected to complete blood cell count (CBC) anal-ysis Hemoglobin concentration (HB) hematocrit (HCT)erythrocytes number (RBC) and other indices (MCVMCH MCHC and reticulocytes) were measured using anADVIA120 Hematology System (Siemens Diagnostics)

23 Flow Cytometry Spleen and bone marrow (BM) wereextracted from sacrificed animals and used for flow cyto-metric analysis using APC-Ter119 and PE-CD71 Fc-receptorwas previously blocked using anti-mouse CD16CD32 amix of FITC-conjugated lymphoid and myeloid markers(CD3e CD45R CD41 CD11b and Gr-1) was used to excludeleukocytes and 7-AAD was used to exclude dead cells Allreagents were purchased from eBiosciences Ter119-positiveevents were allocated into five subsets representing sequentialmaturation stages (ProE EryA EryB and EryC) accordingto CD71 intensity and FSC properties [17ndash19] An EryDregion was added according to the results by Chen et alwho localized a gate where orthochromic erythroblasts withnuclear-cytoplasmic dyssynchrony fall [19]

24 Histology and Perlrsquos Staining Animals livers and spleenswere explanted fixed in 4 PFA cryoprotected by a sucrosegradient (75 15 and 30) and embedded in OCT priorto cryosectioning at 30 120583m Tissue sections were stained withPerlrsquos Prussian blue method (Bio-Optica) Images were takenat 20x magnification using a LEICA DFC208 microscope

25 MonocytesMacrophages Isolation For each group (Tfr2KI Tfr2 KO and control litter-mates) monocytesmacro-phages were separated from a pool of 4 spleens using MACSCD11b MicroBeads (Milteni Biotec)

26 Molecular Analysis Hamp gene expression was evalu-ated in Tfr2 targeted and in WT mouse liver For reverse-transcription 1120583g of total RNA 25 120583M random hexamersand 100Uof reverse transcriptase (Applied Biosystems USA)were used

Hamp expression levels were measured by quantita-tive real-time reverse-transcription (RT-PCR) with CFX96Real-Time System (BIO-RAD) using a quantitative RT-PCRassay (Assays-on-Demand Applied Biosystems USA) Erfeexpression was evaluated using SYBR Green PCR technol-ogy (EVAGreen BIO-RAD) using the following primersmErfe F1 51015840ATGGGGCTGGAGAACAGC31015840 and mErfe R151015840TGGCATTGTCCAAGAAGACA31015840 All analyses were car-ried out in triplicate and results showing a discrepancy greater

BioMed Research International 3

than 1 threshold cycle in 1 of the wells were excluded Gus(120573-glucuronidase) gene was used as housekeeping controlThe results were analyzed using the ΔΔ threshold cycle (119862

119905)

method [20]Western blots of BM spleen and monocytesmacro-

phages lysates (50 120583g) for Cleaved Caspase-3 (5A1E CellSignaling) Bcl-xL (H-5) divalentmetal transporter 1 (DMT1)(H-108) Fpn1 (G-16) Tfr2 (S-20) 120573-actin (C-4) (Santa CruzBiotechnology) and Ft-L (kindly provided by S Levi Milan)proteins were performed through standard protocols

Data from Western blot quantification (Image Lab Soft-ware BIO-RAD) were obtained after normalizing on 120573-actinlevels and expressed as fold increase relative to the meanvalue obtained from the WT mice

27 Statistical Analysis For hematological analysis and flowcytometry experiments statistical comparisons among geno-types and age groups were performed using nonparametrictests (Kruskal-Wallis or MannndashWhitney resp) using SPSSversion 21 software Differences of mRNA expression andprotein production between controls and targeted mice wereevaluated with a nonparametric Studentrsquos 119905-test (unpairedtwo-tailed) using GraphPad Prism software 119875 lt 005 wasconsidered to be statistically significant

3 Results

31 Peripheral Blood Cell Count The pattern of erythro-poiesis was observed to be different in WT and Tfr2 miceacross the lifespanAdditionally Tfr2mice showed significantvariations in erythropoiesis compared to age-matched WTcontrols in the CBC assay

311 Young versus Adult Animals 14 d old mice had lowerRBC HB and HCT and higher MCV and reticulocytescompared to adults in all genotypes analyzed (Figure 1)Young WT mice showed similar MCH but lower MCHCas compared to adult WT mice demonstrating that normalmouse erythropoiesis during animal growth follows the sametrend as that of normal human erythropoiesis [14]

Young Tfr2 KI and KO mice demonstrated higher MCHbut similar MCHC compared to adult animals with the samegenotype (Figure 1)Thus youngTfr2mice have increased thelevels of hemoglobin in RBC compared to age-matched WTanimals However the differences are statistically significantonly in Tfr2 KO mice

312 Young Tfr2 Mice The comparison of 14 d old Tfr2 KIto WT mice did not reveal any significant differences in allCBC parameters analyzed On the contrary 14 d old Tfr2 KOmice had higher RBC HB HCT MCH and MCHC valuesthan age-matched WT mice (significant differences for HBMCH and MCHC only) (Figures 1(b) 1(e) and 1(g)) and asignificantly lower number of reticulocytes (Figure 1(f))

To investigate if this difference in the number of retic-ulocytes in young Tfr2 KO animals could be due to analteration of the apoptosis pathway in the BM we analyzedCleaved Caspase-3 (CC-3) [21] and Bcl-xL [22] The level of

the proapoptotic marker CC-3 was comparable to that of age-matched WT mice while the antiapoptotic marker Bcl-xLincreased about 15-fold in Tfr2 KO young animals versusWT (Figure S1 A in Supplementary Material available onlineat httpsdoiorg10115520172408941) In conclusion anincrease in apoptotic death is not the underlying cause for thedecreased reticulocyte production in these animals

313 Adult Tfr2 Mice Adult Tfr2 KI animals had signifi-cantly higher number of RBC but lower MCH and MCHCcompared to age-matched WT (Figures 1(a) 1(e) and 1(g))however these differences were not statistically significant

Adult Tfr2 KO mice had statistically significantlyincreased MCV and MCH compared to WT litter-matesof the same age (Figures 1(d) and 1(e)) All the other CBCparameters were comparable to those of age-matched WTanimals

32 BM and Spleen Erythropoiesis Flow cytometry analysisof the BM and spleen erythropoiesis revealed several unex-pected results in both young and adult animals

The BM erythropoiesis was increased in adult micecompared to the young ones (Figure 2(a)) in WT and KImice alike In contrast Tfr2 KO mice had high levels oferythropoiesis already at 14 d of age with values similar toadults (Figure 2(a))

Both Tfr2 KI and Tfr2 KO 14 d old mice had increasedsplenic erythropoiesis compared to age-matchedWT thoughthe difference was statistically significant only for the Tfr2 KOgroup Interestingly splenic erythropoiesis similarly reducedin adulthood for the two genotypes reaching comparablevalues to the WT (Figure 2(b))

From the qualitative point of view no evident differenceswere found in the BM of young Tfr2 mice except for a highernumber of EryD cells in Tfr2 KO (Figure 2(c)) Unexpectedlyan enhanced splenic erythropoiesis was observed in youngTfr2 KI mice characterized by a left shift in the erythroidlineage resulting in a higher number of precursor erythroidcells and a lower number of mature cells (Figure 2(d)) Sim-ilarly young Tfr2 KO mice also presented increased splenicerythropoiesis as mentioned above (Figure 2(b)) Howeverno statistically different values were found compared to WT(Figure 2(d))

Surprisingly the erythropoiesis in adult Tfr2 KO micewasmarkedly altered Although an increase in the percentageof early precursors (Figures 2(c) and 2(d)) was found noreticulocytosis could be seen in these mice This could bebecause of an increase in apoptosis as is evidenced by thedoubling of the levels of the apoptotic marker Caspase-3 inadult Tfr2 KO mice compared to age-matched WT On thecontrary the antiapoptotic marker Bcl-xL was significantlydecreased in Tfr2 KO mice compared to WT (Figure S1 B)

Finally flow cytometry analysis revealed that in bothyoung and adult Tfr2 KO mice CD71 MFI was significantlylower compared to WT in BM as well as in the spleen andin all erythroid maturation stages except for EryC in adultanimals CD71 MFI in Tfr2 KO mice was lower compared to


KOKIWT

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(g)

Figure 1 (a) Red blood cells (RBC times10E06 cells120583L) (b) hemoglobin concentration (HB gdL) (c) hematocrit (HCT ) (d) meancorpuscular volume (MCV fL) (e) mean corpuscular hemoglobin (MCH pg) (f) reticulocytes (retic times10E09 cellsL) and (g) meancorpuscular hemoglobin concentration (MCHC gdL) values obtained from animals CBC WT wild type (blue) KI Tfr2 KI (green) KOTfr2 KO (yellow) 14 d 14 days 10 w 10 weeks lowast ∘ and indicate statistically significant differences (119875 lt 005) compared to age-matched WTKO and KI respectively

KI mice for EryC in young animals and for EryA and EryB inadults (Figure S2)

33 Iron Levels in Tfr2 Animals Since iron availability couldinfluence erythropoietic stimulus we analyzed the iron levelsand hepatic Hamp production in erythropoietic tissues ofTfr2 animals as compared to controls

Perlrsquos histological staining (Figures 3(a) and 3(b)) revealedsurprisingly high iron levels in livers from Tfr2 KO micealready at 14 d of age (Figure 3(a)) and as expected in adultanimals as well (Figure 3(b)) In contrast no significant liveriron deposit was visible in Tfr2 KI young and adult animals(Figures 3(a) and 3(b)) A significant splenic iron overloadwas evidenced only in Tfr2 KI adult animals (Figure 3(b))


BM

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en

14 d 10 w

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(d)

Figure 2 Flow cytometry analysis of quantitative (a b) and qualitative (c d) erythropoiesis in bonemarrow (BM) and spleen of 14 days (14 d)and 10 weeks (10w) oldmiceWT wild type KI Tfr2 KI KO Tfr2 KO ProE EryA EryB EryC and EryD represent sequential erythropoieticmaturation stages lowast and indicate statistically significant differences (119875 lt 005) compared to age-matched WT and KI respectively

BM iron staining with Perlrsquos did not reveal any obviouslarge iron deposit (data not shown) In addition L ferritin(Ft-L) levels were found to be significantly elevated in theBM of both Tfr2 young mice (Figure 3(c)) However duringadulthood it remained high only in the BM of Tfr2 KO mice(Figure 3(d))

34 Hamp and Erfe Analysis The hepatic Hamp expressionwas significantly decreased in Tfr2 KI and KO animalscompared to age-matched WT litter-mates The same wastrue for adult animals although to a lesser degree (Figures4(a) and 4(b))

Additionally Erfe transcript levels were significantly dif-ferent in the three genotypes they were significantly higherin the BM and the spleen of young Tfr2 KI animals andsignificantly lower in young Tfr2 KO mice as compared toWT (Figures 4(c) and 4(d)) In adult Tfr2 KI and KO miceErfe transcription was similar to adult WT in both tissuesanalyzed (Figures 4(c) and 4(d))

Longitudinal comparison between the two ages revealedthat Erfe transcription was significantly increased in theyoung compared to genotype matched adult mice with theexception of Tfr2 KO BM whose Erfe amount remained

constant during the growth period of the animal (Figures 4(c)and 4(d))

35 Erythropoietic Tissues Monocytes To unravel the roleof Tfr2120573 isoform in the iron flux in macrophages duringerythropoiesis this cell type was isolated from the spleen ofWT and Tfr2 targeted mice at the two experimental timepoints Tfr2120573 levels were evaluated together with the mainproteins involved in cellular iron traffic namely the irondeposit protein ferritin (Ft-L) the iron importer DMT1 andthe iron exporter Ferroportin 1 (Fpn1)

In WT mice Tfr2120573 isoform was observed to decrease insplenic macrophages of adult animals as compared to theyoung ones (Figure 5(a)) In the same cells DMT1 and Fpn1decreased as well while Ft-L levels increased (Figure 5(b))

In young Tfr2 KI mice splenic macrophages presenteda lower DMT1 and higher Fpn1 and Ft-L compared to age-matched WT sib pairs (Figure 5(b)) During the growthperiod DMT1 and Ft-L consistently increased while Fpn1significantly decreased

On the other hand in young Tfr2 KO mice splenicmacrophages presented a lower DMT1 and Fpn1 and com-parable Ft-L in comparison to age-matched WT sib pairs


(A) (B) (C)

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en14

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)Bo

ne m

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Figure 3 Perlrsquos Prussian blue staining of liver and spleen sections from wild type (WT) Tfr2 KI (KI) and Tfr2 KO (KO) animals at (a) 14days (14 d) and (b) 10 weeks (10w) of ageWestern blot analysis shows ferritin L (Ft-L) protein production in bonemarrow ofWT KI and KOmice at (c) 14 days (14 d) and (d) 10 weeks (10w) of age au arbitrary unitThe following symbols indicate statistically significant differenceslowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 compared to age-matched WT mice


Hamp

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Figure 4 Hepcidin (Hamp) gene expression in Tfr2 targeted and in WT mouse liver at (a) 14 days (14 d) and (b) 10 weeks (10w) of ageErythroferrone (Erfe) gene expression in bone marrow (c) and spleen (d) of WT KI and KO mice at 14 days (14 d) and 10 weeks (10w) ofage WT wild type KI Tfr2 KI KO Tfr2 KO The following symbols indicate statistically significant differences lowast119875 lt 005 and lowastlowast119875 lt 001compared to age-matchedWTmice and119875 lt 005 andand119875 lt 001 and andandand119875 lt 0001 compared to animals with the same genotype at 14 days of age

(Figure 5(b)) During the growth period DMT1 and Fpn1decreased while Ft-L was obviously increased

4 Discussion

It is well known that iron is essential for adequate ery-thropoiesis In the condition of iron deficiency the mostimportant pathway that is impaired is RBC productionfirstly in the bone marrow (BM) followed by the spleenErythropoiesis itself undergoes physiological changes thatreflect the requirements of an organism throughout itslifespan It increases during youth when there is massivebody growth but remains roughly constant during adult lifeand tends to decrease during ageing [23ndash25] The adequateiron availability for this dynamic erythropoiesis is achievedthrough the modulation of hepcidin one of the chief ironregulators [7]

Among the different hepcidin regulators transferrinreceptor 2 alpha (Tfr2120572) has been shown to play a role as aniron sensor in the liver [7] and as erythropoiesis regulatorin erythropoietic tissues [26] Notably the gene encodingTFR2 is transcribed in two main isoforms the alpha form

expressed in the liver and few other tissues and the shorterbeta form with a low ubiquitous expression However it isfound to be in significantly higher levels in the spleen [3] Inthe liver Tfr2120572 exerts its action on the plasma membrane Itis not directly responsive to iron levels [4] but is stabilized onplasmamembrane by iron loaded transferrin [27] Accordingto the most recent functional models hepatic Tfr2120572 interactswith the other iron proteins as Tfr1 and Hfe to sense bodyiron levels and to transduce the signal of iron excess throughthe activation of the Smad 158 andor the Erk12 pathwayscausing an increase in the hepatic hepcidin [7]

Recent data has demonstrated that Tfr2120572 also has anextrahepatic function It is well expressed in BM where itinteracts with erythropoietin receptor (EpoR) thereby beinginvolved in regulation of erythropoiesis [11] Further severalstudies have demonstrated the role of Tfr2120572 in regulatingRBC production in mouse models particularly in conditionof iron deficiency [12 13 28]

In contrast very little is known about the function ofthe second TFR2 isoform Tfr2120573 It is significantly producedin splenic macrophages and its silencing in the Tfr2 KImice does not cause any variation in serum iron parameters


Tfr2 beta

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Figure 5 (a) Comparison between 14-day (14 d) and 10-week (10w) Tfr2120573 expression (on the left) and production (on the right) in splenicmacrophages of wild type (WT) animals (b) Quantification of divalent metal transporter 1 (DMT1) Ferroportin 1 (Fpn1) and ferritin L (Ft-L)protein production resulting fromWestern blot analysis of splenic macrophages isolated fromWT KI and KOmice at 14 d and 10w of agesau arbitrary unit SPL total spleen lowast indicates statistically significant difference (119875 lt 005)

and liver iron content Nevertheless these animals presentiron retention in the macrophages probably through thedownregulation of iron exporter Fpn1 [8]

Therefore in the presentmanuscript we aimed to analyzethe role of both Tfr2 isoforms in erythropoiesis and thecontribution of available iron in the modulation of ery-thropoiesis We used the Tfr2 KI animals (120572+1205730) in whichcirculating iron levels are normal and the Tfr2 KO mice(12057201205730) that have severe iron overload in addition to increasedserum ferritin and transferrin saturation We compared theerythropoiesis in these animals to that of WT litter-matesFurthermore we evaluated these two Tfr2 mouse models atyoung age (14 d) when iron demand is high to fulfill growthrequirements and at adult age (10 w) when iron is neededprimarily for themaintenance of erythropoiesis Our findingsdemonstrate that adult Tfr2 KOmice show normal erythroidparameters at CBC except for an increasedMCV and a higherhemoglobin content (MCH) This indicates that in Tfr2 KOmice the maximum amount of HB is produced in the RBCin the early stages of erythropoiesis when cells are larger Onthe contrary in WT animals the same hemoglobin amountreaches the final concentration through the reduction of

RBC dimension This phenomenon could be associated withan attempt of the body to eliminate the excess iron Inthe same animals BM and splenic erythroid productionis quantitatively normal but it is characterized by a shifttoward immature precursors The left shift in the maturationsequence could be an evidence of a delayed erythropoiesisin accordance with the results of previous studies [12 13]On the other hand lack of reticulocytosis in these animalscan be explained by an increase in the total BM apoptosisconfirmed by an increase of apoptotic marker Caspase-3 anda simultaneous decrease of the antiapoptotic protein Bcl-xLThis could represent a late stage control mechanism that mayaccount for the depletion of late precursors that is ultimatelyresponsible for an ineffective erythropoiesis

Importantly these findings are in contrast with the studyby Nai et al in which BM specific Tfr2 KO mice (Tfr2BMKO)present an increased number of RBCs decreased volume andhemoglobin content and increased splenic stress erythro-poiesis in the presence of normal serum iron parameters[28]

The primary genetic differences between these two mod-els are that Tfr2BMKO mice maintain Tfr2120572 and 120573 isoforms


function in the other body organs particularly in the liver andin the spleen whereas Tfr2 KO mice have a total silencing ofboth that causes an increased iron availability

Therefore the comparison between these two animalmodels is useful to unravel the role of iron in inducing RBCproduction in both the BM and the spleen It is clear from thedata that an increased iron availability in Tfr2 KOmice causeserythropoiesis since 14 d oldTfr2KOanimals already have anerythropoietic activity similar to adult WTmice In additionit also causes erythropoietic changes an increased numberof immature cells and increased apoptosis as is evident inTfr2 KO adult animals compared to age-matched WT Theincreased iron levels in the BM of Tfr2 KO animals couldtrigger EryA erythroblasts production but the increasedapoptosis finally normalizes RBC output Also the presenceof macrocytosis together with a low reticulocytes numberand increased BM apoptosis in Tfr2 KO mice resembles theerythropoiesis of myelodysplastic syndromes (MDS) [29] Inthese conditions iron overload has been demonstrated tohave a causative role In fact iron chelation of these MDSpatients ameliorates their BM dysfunction [30]

It would be interesting to induce iron normalization inthe Tfr2 KO animals to evaluate if a phenotype comparableto that of Nai et al [28] can be achieved

Another important difference between the two models isrelated to erythropoietic regulator Erythroferrone (Erfe) [15]Erfe levels are increased in Tfr2BMKO mice while decreasedin our Tfr2 KO animals Erfe increases due to an increasediron demand for erythropoiesis and causes a downregulationof hepcidin [28] However to our knowledge this is the firsttime it has been demonstrated that Erfe reduction can alsobe a consequence of an adequate erythropoiesis as we couldobserve in Tfr2 KO young animals

Furthermore the trend of Erfe in animals of different agesin both WT and Tfr2 targeted animals is very interesting Itclearly appears that Erfe has an important role in erythro-poiesis regulation not only at adult age as has been alreadydemonstrated [16] but also at young age Also its expressioncorrelates very well with erythropoietic boost inWT animalsbeing high in 14 d old animals and decreasing significantly inadult animals Notably BM and splenic Erfe transcription issignificantly reduced in young Tfr2 KO animals and remainsconstant during their growth period in agreement with theearly achievement of adult erythropoiesis pattern in thesemice

The relationship between Erfe and Tfr2 has been demon-strated through several experimental approaches [13 28]Ourdata shows that this relationship is far more evident in younganimalsMoreover we demonstrate that the presence of Tfr2120572in the liver is essential to have the hepcidin response to ErfeIn fact Hamp is decreased in response to an increase of Erfein Tfr2 KI mice that produces the Tfr2 alpha isoform asexpected Similarly in Tfr2BMKO animal model a decreasedErfe amount corresponds to and increases Hamp level [28]On the contrary in Tfr2 KO animals in which Tfr2120572 is absentin the liver Hamp does not increase in response to low Erfelevels

Lastly the lack of Tfr2120573 leads to an evident splenic ironaccumulation only in adult Tfr2 KI animals as previouslydemonstrated [8] Furthermore it is surprising to see thatlack of Tfr2120572 causes an iron accumulation as early as 14 d ofage in the liver of Tfr2 KO animals

We have focused at least a part of our analysis on younganimals because very little is known about erythropoiesis atthis stage of life even in WT animals It is important to notethat in the latter the erythropoietic stimulus is mainly iron-dependent since an adult erythropoiesis becomes evident iniron enriched Tfr2 KO young animals

Surprisingly the data from the analysis of erythropoiesisin Tfr2 KI mice at young age is the most interesting whenthese animals present normal serum iron parameters normalBM and splenic iron amount and normal CBC In spiteof this their splenic erythropoiesis appears to be increasedand immature compared to age-matched WTThese data aresupported by a significant increase of BM and splenic Erfeas well Tfr2 KO mice also present an early increased splenicerythropoiesis but it is qualitatively normal This could be aconsequence of the increased circulating iron availability thatcan ensure a qualitatively normal but relatively high splenicerythropoietic maturation

Next we hypothesized that enhanced splenic immatureerythropoiesis in the absence of Tfr2120573 in young Tfr2 KImice could be caused by low iron availability because ofiron retention in splenic macrophages This situation shouldhave been evident in the spleen where reticuloendotelial cellsare particularly abundant So to confirm this hypothesis weevaluated iron levels in splenic monocytes of Tfr2 mice Wedetected an increase in ferritin and a decrease in Fpn1 in thesplenicmonocytes of youngTfr2KI andKO compared toWTage-matched mice which confirms our hypothesis

Interesting speculations can bemade from this analysis ofthe evolution of splenic erythropoiesis during the lifespan ofanimals

From the analysis of WT animals it is evident that thespleen eventually loses its role as erythropoietic organ andbecomes a deposit site for iron that derives from erythrocytecatabolism Ft-L in fact increases in splenicmonocytes ofWTmice during ageing due to Fpn1 decrease

The same is true in Tfr2 micersquos splenic monocytes buthere the increase of iron is far more evident in splenicmonocytes of adult Tfr2 KI mice compared to age-matchedWT and Tfr2 KO Also iron importer DMT1 is increasedin the splenic monocytes of Tfr2 KI mice Thus it couldbe interesting to further investigate the relationship betweenTfr2120573 and this divalent metal transporter

On the basis of the data obtained in the presentmanuscript a model for the role of iron in the stimulation oferythropoiesis at different ages and the involvement of Tfr2isoforms in erythropoietic organs is illustrated in Figure 6

5 Conclusions

An analysis of erythropoiesis in mice with inactivationof one or both of Tfr2 isoforms confirms that there is aspecific function of Tfr2120572 in erythropoiesis which has been


WTYoung Adult

FeFe Fe

Normal Normal erythropoiesis

Fe FeFe

erythropoiesisNormal Normal

erythropoiesis erythropoiesis

(a)

FeFe Fe Fe Fe

Fe

ErfeErfe

Young Adult

Normal erythropoiesis

Normal Normal erythropoiesis erythropoiesis

Tfr2 KI (훼+훽0)

Immatureerythropoiesis

(b)

FeFe Fe Fe

FeFe

FeFe Fe

Fe FeFe

ApoptosisApoptosis

Fe Fe

ErfeErfe

Young AdultFeFe




Tfr2 KO (훼 훽00 )

(c)

Figure 6 Model depicting the involvement of iron and Tfr2 isoforms in erythropoiesis (a) In young (14 d) WT mice erythropoiesis takesplace in both bonemarrow (BM) and spleenwhile in adult animals BMerythropoiesis is predominant as compared to splenic RBCproduction(blue arrows) (b) in the presence of normal circulating iron levels lack of Tfr2120573 provokes iron retention inmacrophages that causes immaturesplenic erythropoiesis and Erythroferrone (Erfe) increase in young mice that generally have a high iron requirement This is normalized inadult animals although an increased amount of iron is retained in the spleen (c) the same should be observed in the spleen of Tfr2 KOanimals but they have sufficient circulating iron amount to normalize splenic erythropoiesis (Erfe is decreased) High iron availability alsocauses an increase of the early stage of BM erythropoiesis in adult mice that does not result in reticulocytosis because of increased apoptosis


previously demonstrated aswell Germinal lack of Tfr2120572 (Tfr2KO) causes an anticipation of adult erythropoiesis in youngmice in both BM and the spleen On the other hand lack ofTrf2120573 is responsible for an increased but immature splenicerythropoiesis that is normalized during animal growthThiseffect due to Trf2120573 absence in Tfr2 KO mice is compensatedby the increased amount of circulating iron available forerythrocyte production

Conflicts of Interest

The authors declare that they have no conflicts of interest

Authorsrsquo Contributions

Authors RM Pellegrino and F Riondato contributed equallyto this work as the first authors

Acknowledgments

This work was supported by grants from University of TurinProgetti di AteneoCSP 2012 (12-CSP-C03-065) and AIRC(IG2011 cod 12141) to G Saglio University of Turin RILO2015 (RIcerca LOcale 2015) project acronymMeCCaSARiC_3to A Roetto The authors wish to thank Professor Marco DeGobbi for the helpful discussion and Dr Ishira Nanavaty forthe final manuscript editing

References

[1] C Camaschella and A Roetto ldquoTFR2-related hereditaryhemochromatosisrdquo in GeneReviews R A Pagon M P AdamH H Ardinger et al Eds University of Washington SeattleWA USA 2014

[2] C Camaschella A Roetto A Calı et al ldquoThe gene TFR2 ismutated in a new type of haemochromatosis mapping to 7q22rdquoNature Genetics vol 25 no 1 pp 14ndash15 2000

[3] H Kawabata R Yang T Hirama et al ldquoMolecular cloningof transferrin receptor 2 A new member of the transferrinreceptor-like familyrdquo Journal of Biological Chemistry vol 274no 30 pp 20826ndash20832 1999

[4] R E Fleming M C Migas C C Holden et al ldquoTransferrinreceptor 2 continued expression in mouse liver in the face ofiron overload and in hereditary hemochromatosisrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 97 no 5 pp 2214ndash2219 2000

[5] M U Muckenthaler B Galy and MW Hentze ldquoSystemic ironhomeostasis and the iron-responsive elementiron-regulatoryprotein (IREIRP) regulatory networkrdquoAnnual Review of Nutri-tion vol 28 pp 197ndash213 2008

[6] J Chen J Wang K R Meyers and C A Enns ldquoTransferrin-directed internalization and cycling of transferrin receptor 2rdquoTraffic vol 10 no 10 pp 1488ndash1501 2009

[7] MWHentzeMUMuckenthaler BGaly andCCamaschellaldquoTwo to tango regulation ofmammalian ironmetabolismrdquoCellvol 142 no 1 pp 24ndash38 2010

[8] A Roetto F Di Cunto R M Pellegrino et al ldquoComparison of3 Tfr2-deficient murine models suggests distinct functions forTfr2-120572 and Tfr2-120573 isoforms in different tissuesrdquo Blood vol 115no 16 pp 3382ndash3389 2010

[9] A Roetto A Totaro A Piperno et al ldquoNew mutations inacti-vating transferrin receptor 2 in hemochromatosis type 3rdquoBloodvol 97 no 9 pp 2555ndash2560 2001

[10] S Majore F Milano F Binni et al ldquoHomozygous pM172Kmutation of the TFR2 gene in an Italian family with type 3hereditary hemochromatosis and early onset iron overloadrdquoHaematologica vol 91 8 Article ID ECR33 2006

[11] H Forejtnikova M Vieillevoye Y Zermati et al ldquoTransferrinreceptor 2 is a component of the erythropoietin receptorcomplex and is required for efficient erythropoiesisrdquo Blood vol116 no 24 pp 5357ndash5367 2010

[12] ANai RM PellegrinoMRausa et al ldquoThe erythroid functionof transferrin receptor 2 revealed by Tmprss6 inactivation indifferent models of transferrin receptor 2 knockout micerdquoHaematologica vol 99 no 6 pp 1016ndash1021 2014

[13] D F Wallace E S Secondes G Rishi et al ldquoA critical rolefor murine transferrin receptor 2 in erythropoiesis during ironrestrictionrdquo British Journal of Haematology vol 168 no 6 pp891ndash901 2015

[14] X Troussard S Vol E Cornet et al ldquoFull blood count nor-mal reference values for adults in Francerdquo Journal of ClinicalPathology vol 67 no 4 pp 341ndash344 2014

[15] L Kautz G Jung E V Valore S Rivella E Nemeth and TGanz ldquoIdentification of erythroferrone as an erythroid regu-lator of iron metabolismrdquo Nature Genetics vol 46 no 7 pp678ndash684 2014

[16] L Kautz G Jung E Nemeth and T Ganz ldquoErythroferronecontributes to recovery from anemia of inflammationrdquo Bloodvol 124 no 16 pp 2569ndash2574 2014

[17] Y Liu R Pop C Sadegh C Brugnara V H Haase and MSocolovsky ldquoSuppression of Fas-FasL coexpression by erythro-poietinmediates erythroblast expansion during the erythropoi-etic stress response in vivordquo Blood vol 108 no 1 pp 123ndash1332006

[18] H-C Chu H-Y Lee Y-S Huang et al ldquoErythroid differ-entiation is augmented in Reelin-deficient K562 cells andhomozygous reeler micerdquo FEBS Letters vol 588 no 1 pp 58ndash64 2014

[19] M L Chen T D Logan M L Hochberg et al ldquoErythroiddysplasia megaloblastic anemia and impaired lymphopoiesisarising frommitochondrial dysfunctionrdquo Blood vol 114 no 19pp 4045ndash4053 2009

[20] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCRand the 2minusΔΔ119862TmethodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[21] U Testa ldquoApoptotic mechanisms in the control of erythro-poiesisrdquo Leukemia vol 18 no 7 pp 1176ndash1199 2004

[22] M Silva A Benito C Sanz et al ldquoErythropoietin can inducethe expression of Bcl-x(L) through Stat5 in erythropoietin-dependent progenitor cell linesrdquo Journal of Biological Chemistryvol 274 no 32 pp 22165ndash22169 1999

[23] J L Beard ldquoIron requirements in adolescent femalesrdquo TheJournal of Nutrition vol 130 pp 440Sndash442S 2000

[24] J M Guralnik R S Eisenstaedt L Ferrucci H G Klein andR C Woodman ldquoPrevalence of anemia in persons 65 yearsand older in the United States evidence for a high rate ofunexplained anemiardquo Blood vol 104 no 8 pp 2263ndash22682004

[25] H Nilsson-Ehle R Jagenburg S Landahl and A SvanborgldquoBlood haemoglobin declines in the elderly implications forreference intervals from age 70 to 88rdquo European Journal ofHaematology vol 65 no 5 pp 297ndash305 2000


[26] L Silvestri A Nai A Pagani and C Camaschella ldquoTheextrahepatic role of TFR2 in iron homeostasisrdquo Frontiers inPharmacology vol 5 p 93 2014

[27] M B Johnson J Chen N Murchison F A Green and CA Enns ldquoTransferrin receptor 2 evidence for ligand-inducedstabilization and redirection to a recycling pathwayrdquoMolecularBiology of the Cell vol 18 no 3 pp 743ndash754 2007

[28] A NaiM R Lidonnici M Rausa et al ldquoThe second transferrinreceptor regulates red blood cell production inmicerdquoBlood vol125 no 7 pp 1170ndash1179 2015

[29] S DNimer ldquoMyelodysplastic syndromesrdquoBlood vol 111 no 10pp 4841ndash4851 2008

[30] E Messa D Cilloni F Messa F Arruga A Roetto and GSaglio ldquoDeferasirox treatment improved the hemoglobin leveland decreased transfusion requirements in four patients withthemyelodysplastic syndrome and primarymyelofibrosisrdquoActaHaematologica vol 120 no 2 pp 70ndash74 2008

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


A recent study has demonstrated that the Tfr2120572 isoform isalso expressed in erythroid progenitors Here it interactswith and stabilizes the erythropoietin receptor (EpoR) henceestablishing a correlation between Tfr2120572 and erythropoiesisfor the first time [11] Furthermore it is thought to be directlyinvolved in the mechanisms of control of erythropoiesisespecially in conditions of iron deprivation [12 13]

It is well known that erythropoiesis as well as irondemand changes throughout life in humans as a consequenceof an increase in blood volume Also complete blood cellcount (CBC) values differ slightly depending upon the age[14] To our knowledge no data exist to date on variationsin erythropoiesis during ageing in mice Some regulatorsof erythropoiesis have been characterized in recent yearsOne of them named Erythroferrone (Erfe) is a hormoneproduced by erythroblasts that is able to regulate Hamp levelsas a consequence of erythropoietic demand due to blood loss[15] or anemia of inflammation [16]

To investigate how Tfr2120572 silencing influences erythrocyteproduction across lifespan inmice we studied erythropoiesisin two primary erythropoietic organs the bonemarrow (BM)and the spleen at different ages using twomousemodels withinactivated Tfr2120572 andor Tfr2120573 The findings of this studycould be an important step toward gaining a better insightinto Tfr2 involvement in erythropoiesis in humans

Our results indicate that the lack of Tfr2120572 influences BMand splenic erythropoiesis starting from an early stage of lifeMoreover Tfr2120573 also influences erythropoiesis by the mod-ulation of iron availability for erythrocyte maturation Moreimportantly we now show that Erfe expression is regulatedby erythropoiesis not only in adult animals as previouslydemonstrated [15] but also in young age Additionally Erfeappears to be negatively modulated by erythropoietic tissuesrsquoiron availability Lastly we also describe the physiologicalvariations of erythropoietic activity in WT mice duringageing

2 Materials and Methods

21 Animals Two Tfr2 mouse models on 129X1svJ strainwere studied (1) Tfr2 KI that has the Tfr2120573 isoform inacti-vated (120572+1205730) and (2) Tfr2 KO that has both Tfr2 isoformsinactivated (12057201205730) Selective targeting of Tfr2 isoforms wasobtained starting from the same target construct in whichmurine M163K mutation (homologous to M172K humanvariant) was inserted in murine Tfr2 gene exon 4 flanked by3 loxP sites activated through Crelox recombination system[8]

At adult age (10 w) Tfr2 KI mice have normal Hampand normal serum iron parameters but splenic iron overloadwhile Tfr2 KO animals have normal Hamp but high serumferritin and transferrin saturation as well as hepatic ironoverload [8]

All animals were housed at Department of VeterinarySciences University of Torino Animal housing and all theexperimental procedures were performed in accordance withEuropean (Official Journal of the European Union L276del 20102010 Vol 53 p 33ndash80) and National Legislation

(Gazzetta Ufficiale n∘ 61 del 14032014 p 2ndash68) for the pro-tection of animals used for scientific purposes

Tfr2-targeted mice and controls were maintained onstandard conditions and with ad libitum access to food andwater They were analyzed at 14 days and 10 weeks of ageOnlymalemice were used in this study tominimize potentialvariability related to sex

At least 6 animals were analyzed for each experimentalcondition

22 Hematological Analysis Peripheral blood from the ani-mals was subjected to complete blood cell count (CBC) anal-ysis Hemoglobin concentration (HB) hematocrit (HCT)erythrocytes number (RBC) and other indices (MCVMCH MCHC and reticulocytes) were measured using anADVIA120 Hematology System (Siemens Diagnostics)

23 Flow Cytometry Spleen and bone marrow (BM) wereextracted from sacrificed animals and used for flow cyto-metric analysis using APC-Ter119 and PE-CD71 Fc-receptorwas previously blocked using anti-mouse CD16CD32 amix of FITC-conjugated lymphoid and myeloid markers(CD3e CD45R CD41 CD11b and Gr-1) was used to excludeleukocytes and 7-AAD was used to exclude dead cells Allreagents were purchased from eBiosciences Ter119-positiveevents were allocated into five subsets representing sequentialmaturation stages (ProE EryA EryB and EryC) accordingto CD71 intensity and FSC properties [17ndash19] An EryDregion was added according to the results by Chen et alwho localized a gate where orthochromic erythroblasts withnuclear-cytoplasmic dyssynchrony fall [19]

24 Histology and Perlrsquos Staining Animals livers and spleenswere explanted fixed in 4 PFA cryoprotected by a sucrosegradient (75 15 and 30) and embedded in OCT priorto cryosectioning at 30 120583m Tissue sections were stained withPerlrsquos Prussian blue method (Bio-Optica) Images were takenat 20x magnification using a LEICA DFC208 microscope

25 MonocytesMacrophages Isolation For each group (Tfr2KI Tfr2 KO and control litter-mates) monocytesmacro-phages were separated from a pool of 4 spleens using MACSCD11b MicroBeads (Milteni Biotec)

26 Molecular Analysis Hamp gene expression was evalu-ated in Tfr2 targeted and in WT mouse liver For reverse-transcription 1120583g of total RNA 25 120583M random hexamersand 100Uof reverse transcriptase (Applied Biosystems USA)were used

Hamp expression levels were measured by quantita-tive real-time reverse-transcription (RT-PCR) with CFX96Real-Time System (BIO-RAD) using a quantitative RT-PCRassay (Assays-on-Demand Applied Biosystems USA) Erfeexpression was evaluated using SYBR Green PCR technol-ogy (EVAGreen BIO-RAD) using the following primersmErfe F1 51015840ATGGGGCTGGAGAACAGC31015840 and mErfe R151015840TGGCATTGTCCAAGAAGACA31015840 All analyses were car-ried out in triplicate and results showing a discrepancy greater



119905)





3 Results
















KOKIWT

14 d 10 w

lowast∘

400

600

800

1000

1200

RBC

(a)KOKIWT

14 d 10 w

lowast

80

100

120

140

160

180

HB

(b)KOKIWT

14 d 10 w300

350

400

450

500

550

HCT

(c)

KOKIWT

14 d 10 w

lowast

420

470

520

570

620

MCV

^

(d)

14 d 10 w

KOKIWT

lowast

1300

1400

1500

1600

1700

1800

1900

MCH

^

(e)

14 d 10 w

KOKIWT

0

2500

5000

7500

10000

12500

15000

Retic

lowast^

(f)

KOKIWT

14 d 10 w

lowast^

250

270

290

310

330

350

MCH

C

(g)






BM

KOKIWT

14 d 10 w00

200

400

600

800

TER1

19po

s7A

AD

neg

mea

n

lowast

lowast^

(a)

Sple

en

KOKIWT

14 d 10 w00

200

400

600

800

TER1

19po

s7A

AD

neg

mea

n

lowast

(b)

BM

14 d 10 w

KOKIWT KOKIWT00

200

400

600

800

1000

CD71

mea

n

lowast

lowastlowast

^

ProEEryA

EryBEryCEryD

Indice1

(c)

KOKIWT KOKIWT

ProEEryA

EryBEryCEryD

Sple

en

14 d 10 w

00

200

400

600

800

1000

CD71

mea

n

lowast

lowast

lowastlowast

Indice1

(d)












(A) (B) (C)

(D) (E) (F)

KOKIWT

Sple

en14

dLi

ver1

4d

(a)

(A) (B) (C)

(D) (E) (F)

KOKIWT

Live

r10w

Sple

en10

w

(b)

Ft-L

Ft-L

WT KI KO

lowast

lowast

lowastlowast

Actin

0

2

4

6

Bone

mar

row

14d

(au

)

(c)

Ft-L

WT KI KO

Ft-L


Actin

00

05

10

15

20

(au

)Bo

ne m

arro

w10

w

(d)



Hamp

WT KI KO


00

05

10

15ΔΔ

CtLi

ver1

4d

(a)

Hamp

WT KI KO00

05

10

15

lowastlowast

lowast

Live

r10w

ΔΔ

Ct

(b)

Erfe

WT KI KO WT KI KO0

1

2

3

20

40

60

^ ^^

lowast

lowast

ΔΔ

Ct

10 w14 d

Bone

mar

row

(c)

Erfe


20

40

60

^^^^^^

^^

lowastlowast

lowastlowastΔΔ

Ct

10 w14 d

Sple

en

(d)



4 Discussion







Tfr2 beta

Tfr2 beta

Tfr2 beta

0

2

4

6

8

10

ΔΔ

Ct


lowastlowast

00

05

10

15

20

Actin

(au

)

(a)

Fpn1


DMT1

WT KI KO WT KI KO

Ft-L


00

05

10

15

(au

)

(au

)

0

1

2

3

4

5

0

1

2

3

4

5

(au

)(b)






















5 Conclusions



WTYoung Adult

FeFe Fe


Fe FeFe



(a)

FeFe Fe Fe Fe

Fe

ErfeErfe

Young Adult



Tfr2 KI (훼+훽0)


(b)

FeFe Fe Fe

FeFe

FeFe Fe

Fe FeFe

ApoptosisApoptosis

Fe Fe

ErfeErfe

Young AdultFeFe





(c)








Acknowledgments


References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















119905)





3 Results
















KOKIWT

14 d 10 w

lowast∘

400

600

800

1000

1200

RBC

(a)KOKIWT

14 d 10 w

lowast

80

100

120

140

160

180

HB

(b)KOKIWT

14 d 10 w300

350

400

450

500

550

HCT

(c)

KOKIWT

14 d 10 w

lowast

420

470

520

570

620

MCV

^

(d)

14 d 10 w

KOKIWT

lowast

1300

1400

1500

1600

1700

1800

1900

MCH

^

(e)

14 d 10 w

KOKIWT

0

2500

5000

7500

10000

12500

15000

Retic

lowast^

(f)

KOKIWT

14 d 10 w

lowast^

250

270

290

310

330

350

MCH

C

(g)






BM

KOKIWT

14 d 10 w00

200

400

600

800

TER1

19po

s7A

AD

neg

mea

n

lowast

lowast^

(a)

Sple

en

KOKIWT

14 d 10 w00

200

400

600

800

TER1

19po

s7A

AD

neg

mea

n

lowast

(b)

BM

14 d 10 w

KOKIWT KOKIWT00

200

400

600

800

1000

CD71

mea

n

lowast

lowastlowast

^

ProEEryA

EryBEryCEryD

Indice1

(c)

KOKIWT KOKIWT

ProEEryA

EryBEryCEryD

Sple

en

14 d 10 w

00

200

400

600

800

1000

CD71

mea

n

lowast

lowast

lowastlowast

Indice1

(d)












(A) (B) (C)

(D) (E) (F)

KOKIWT

Sple

en14

dLi

ver1

4d

(a)

(A) (B) (C)

(D) (E) (F)

KOKIWT

Live

r10w

Sple

en10

w

(b)

Ft-L

Ft-L

WT KI KO

lowast

lowast

lowastlowast

Actin

0

2

4

6

Bone

mar

row

14d

(au

)

(c)

Ft-L

WT KI KO

Ft-L


Actin

00

05

10

15

20

(au

)Bo

ne m

arro

w10

w

(d)



Hamp

WT KI KO


00

05

10

15ΔΔ

CtLi

ver1

4d

(a)

Hamp

WT KI KO00

05

10

15

lowastlowast

lowast

Live

r10w

ΔΔ

Ct

(b)

Erfe

WT KI KO WT KI KO0

1

2

3

20

40

60

^ ^^

lowast

lowast

ΔΔ

Ct

10 w14 d

Bone

mar

row

(c)

Erfe


20

40

60

^^^^^^

^^

lowastlowast

lowastlowastΔΔ

Ct

10 w14 d

Sple

en

(d)



4 Discussion







Tfr2 beta

Tfr2 beta

Tfr2 beta

0

2

4

6

8

10

ΔΔ

Ct


lowastlowast

00

05

10

15

20

Actin

(au

)

(a)

Fpn1


DMT1

WT KI KO WT KI KO

Ft-L


00

05

10

15

(au

)

(au

)

0

1

2

3

4

5

0

1

2

3

4

5

(au

)(b)






















5 Conclusions



WTYoung Adult

FeFe Fe


Fe FeFe



(a)

FeFe Fe Fe Fe

Fe

ErfeErfe

Young Adult



Tfr2 KI (훼+훽0)


(b)

FeFe Fe Fe

FeFe

FeFe Fe

Fe FeFe

ApoptosisApoptosis

Fe Fe

ErfeErfe

Young AdultFeFe





(c)








Acknowledgments


References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















KOKIWT

14 d 10 w

lowast∘

400

600

800

1000

1200

RBC

(a)KOKIWT

14 d 10 w

lowast

80

100

120

140

160

180

HB

(b)KOKIWT

14 d 10 w300

350

400

450

500

550

HCT

(c)

KOKIWT

14 d 10 w

lowast

420

470

520

570

620

MCV

^

(d)

14 d 10 w

KOKIWT

lowast

1300

1400

1500

1600

1700

1800

1900

MCH

^

(e)

14 d 10 w

KOKIWT

0

2500

5000

7500

10000

12500

15000

Retic

lowast^

(f)

KOKIWT

14 d 10 w

lowast^

250

270

290

310

330

350

MCH

C

(g)






BM

KOKIWT

14 d 10 w00

200

400

600

800

TER1

19po

s7A

AD

neg

mea

n

lowast

lowast^

(a)

Sple

en

KOKIWT

14 d 10 w00

200

400

600

800

TER1

19po

s7A

AD

neg

mea

n

lowast

(b)

BM

14 d 10 w

KOKIWT KOKIWT00

200

400

600

800

1000

CD71

mea

n

lowast

lowastlowast

^

ProEEryA

EryBEryCEryD

Indice1

(c)

KOKIWT KOKIWT

ProEEryA

EryBEryCEryD

Sple

en

14 d 10 w

00

200

400

600

800

1000

CD71

mea

n

lowast

lowast

lowastlowast

Indice1

(d)












(A) (B) (C)

(D) (E) (F)

KOKIWT

Sple

en14

dLi

ver1

4d

(a)

(A) (B) (C)

(D) (E) (F)

KOKIWT

Live

r10w

Sple

en10

w

(b)

Ft-L

Ft-L

WT KI KO

lowast

lowast

lowastlowast

Actin

0

2

4

6

Bone

mar

row

14d

(au

)

(c)

Ft-L

WT KI KO

Ft-L


Actin

00

05

10

15

20

(au

)Bo

ne m

arro

w10

w

(d)



Hamp

WT KI KO


00

05

10

15ΔΔ

CtLi

ver1

4d

(a)

Hamp

WT KI KO00

05

10

15

lowastlowast

lowast

Live

r10w

ΔΔ

Ct

(b)

Erfe

WT KI KO WT KI KO0

1

2

3

20

40

60

^ ^^

lowast

lowast

ΔΔ

Ct

10 w14 d

Bone

mar

row

(c)

Erfe


20

40

60

^^^^^^

^^

lowastlowast

lowastlowastΔΔ

Ct

10 w14 d

Sple

en

(d)



4 Discussion







Tfr2 beta

Tfr2 beta

Tfr2 beta

0

2

4

6

8

10

ΔΔ

Ct


lowastlowast

00

05

10

15

20

Actin

(au

)

(a)

Fpn1


DMT1

WT KI KO WT KI KO

Ft-L


00

05

10

15

(au

)

(au

)

0

1

2

3

4

5

0

1

2

3

4

5

(au

)(b)






















5 Conclusions



WTYoung Adult

FeFe Fe


Fe FeFe



(a)

FeFe Fe Fe Fe

Fe

ErfeErfe

Young Adult



Tfr2 KI (훼+훽0)


(b)

FeFe Fe Fe

FeFe

FeFe Fe

Fe FeFe

ApoptosisApoptosis

Fe Fe

ErfeErfe

Young AdultFeFe





(c)








Acknowledgments


References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















BM

KOKIWT

14 d 10 w00

200

400

600

800

TER1

19po

s7A

AD

neg

mea

n

lowast

lowast^

(a)

Sple

en

KOKIWT

14 d 10 w00

200

400

600

800

TER1

19po

s7A

AD

neg

mea

n

lowast

(b)

BM

14 d 10 w

KOKIWT KOKIWT00

200

400

600

800

1000

CD71

mea

n

lowast

lowastlowast

^

ProEEryA

EryBEryCEryD

Indice1

(c)

KOKIWT KOKIWT

ProEEryA

EryBEryCEryD

Sple

en

14 d 10 w

00

200

400

600

800

1000

CD71

mea

n

lowast

lowast

lowastlowast

Indice1

(d)












(A) (B) (C)

(D) (E) (F)

KOKIWT

Sple

en14

dLi

ver1

4d

(a)

(A) (B) (C)

(D) (E) (F)

KOKIWT

Live

r10w

Sple

en10

w

(b)

Ft-L

Ft-L

WT KI KO

lowast

lowast

lowastlowast

Actin

0

2

4

6

Bone

mar

row

14d

(au

)

(c)

Ft-L

WT KI KO

Ft-L


Actin

00

05

10

15

20

(au

)Bo

ne m

arro

w10

w

(d)



Hamp

WT KI KO


00

05

10

15ΔΔ

CtLi

ver1

4d

(a)

Hamp

WT KI KO00

05

10

15

lowastlowast

lowast

Live

r10w

ΔΔ

Ct

(b)

Erfe

WT KI KO WT KI KO0

1

2

3

20

40

60

^ ^^

lowast

lowast

ΔΔ

Ct

10 w14 d

Bone

mar

row

(c)

Erfe


20

40

60

^^^^^^

^^

lowastlowast

lowastlowastΔΔ

Ct

10 w14 d

Sple

en

(d)



4 Discussion







Tfr2 beta

Tfr2 beta

Tfr2 beta

0

2

4

6

8

10

ΔΔ

Ct


lowastlowast

00

05

10

15

20

Actin

(au

)

(a)

Fpn1


DMT1

WT KI KO WT KI KO

Ft-L


00

05

10

15

(au

)

(au

)

0

1

2

3

4

5

0

1

2

3

4

5

(au

)(b)






















5 Conclusions



WTYoung Adult

FeFe Fe


Fe FeFe



(a)

FeFe Fe Fe Fe

Fe

ErfeErfe

Young Adult



Tfr2 KI (훼+훽0)


(b)

FeFe Fe Fe

FeFe

FeFe Fe

Fe FeFe

ApoptosisApoptosis

Fe Fe

ErfeErfe

Young AdultFeFe





(c)








Acknowledgments


References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(A) (B) (C)

(D) (E) (F)

KOKIWT

Sple

en14

dLi

ver1

4d

(a)

(A) (B) (C)

(D) (E) (F)

KOKIWT

Live

r10w

Sple

en10

w

(b)

Ft-L

Ft-L

WT KI KO

lowast

lowast

lowastlowast

Actin

0

2

4

6

Bone

mar

row

14d

(au

)

(c)

Ft-L

WT KI KO

Ft-L


Actin

00

05

10

15

20

(au

)Bo

ne m

arro

w10

w

(d)



Hamp

WT KI KO


00

05

10

15ΔΔ

CtLi

ver1

4d

(a)

Hamp

WT KI KO00

05

10

15

lowastlowast

lowast

Live

r10w

ΔΔ

Ct

(b)

Erfe

WT KI KO WT KI KO0

1

2

3

20

40

60

^ ^^

lowast

lowast

ΔΔ

Ct

10 w14 d

Bone

mar

row

(c)

Erfe


20

40

60

^^^^^^

^^

lowastlowast

lowastlowastΔΔ

Ct

10 w14 d

Sple

en

(d)



4 Discussion







Tfr2 beta

Tfr2 beta

Tfr2 beta

0

2

4

6

8

10

ΔΔ

Ct


lowastlowast

00

05

10

15

20

Actin

(au

)

(a)

Fpn1


DMT1

WT KI KO WT KI KO

Ft-L


00

05

10

15

(au

)

(au

)

0

1

2

3

4

5

0

1

2

3

4

5

(au

)(b)






















5 Conclusions



WTYoung Adult

FeFe Fe


Fe FeFe



(a)

FeFe Fe Fe Fe

Fe

ErfeErfe

Young Adult



Tfr2 KI (훼+훽0)


(b)

FeFe Fe Fe

FeFe

FeFe Fe

Fe FeFe

ApoptosisApoptosis

Fe Fe

ErfeErfe

Young AdultFeFe





(c)








Acknowledgments


References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Hamp

WT KI KO


00

05

10

15ΔΔ

CtLi

ver1

4d

(a)

Hamp

WT KI KO00

05

10

15

lowastlowast

lowast

Live

r10w

ΔΔ

Ct

(b)

Erfe

WT KI KO WT KI KO0

1

2

3

20

40

60

^ ^^

lowast

lowast

ΔΔ

Ct

10 w14 d

Bone

mar

row

(c)

Erfe


20

40

60

^^^^^^

^^

lowastlowast

lowastlowastΔΔ

Ct

10 w14 d

Sple

en

(d)



4 Discussion







Tfr2 beta

Tfr2 beta

Tfr2 beta

0

2

4

6

8

10

ΔΔ

Ct


lowastlowast

00

05

10

15

20

Actin

(au

)

(a)

Fpn1


DMT1

WT KI KO WT KI KO

Ft-L


00

05

10

15

(au

)

(au

)

0

1

2

3

4

5

0

1

2

3

4

5

(au

)(b)






















5 Conclusions



WTYoung Adult

FeFe Fe


Fe FeFe



(a)

FeFe Fe Fe Fe

Fe

ErfeErfe

Young Adult



Tfr2 KI (훼+훽0)


(b)

FeFe Fe Fe

FeFe

FeFe Fe

Fe FeFe

ApoptosisApoptosis

Fe Fe

ErfeErfe

Young AdultFeFe





(c)








Acknowledgments


References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Tfr2 beta

Tfr2 beta

Tfr2 beta

0

2

4

6

8

10

ΔΔ

Ct


lowastlowast

00

05

10

15

20

Actin

(au

)

(a)

Fpn1


DMT1

WT KI KO WT KI KO

Ft-L


00

05

10

15

(au

)

(au

)

0

1

2

3

4

5

0

1

2

3

4

5

(au

)(b)






















5 Conclusions



WTYoung Adult

FeFe Fe


Fe FeFe



(a)

FeFe Fe Fe Fe

Fe

ErfeErfe

Young Adult



Tfr2 KI (훼+훽0)


(b)

FeFe Fe Fe

FeFe

FeFe Fe

Fe FeFe

ApoptosisApoptosis

Fe Fe

ErfeErfe

Young AdultFeFe





(c)








Acknowledgments


References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of































5 Conclusions



WTYoung Adult

FeFe Fe


Fe FeFe



(a)

FeFe Fe Fe Fe

Fe

ErfeErfe

Young Adult



Tfr2 KI (훼+훽0)


(b)

FeFe Fe Fe

FeFe

FeFe Fe

Fe FeFe

ApoptosisApoptosis

Fe Fe

ErfeErfe

Young AdultFeFe





(c)








Acknowledgments


References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















WTYoung Adult

FeFe Fe


Fe FeFe



(a)

FeFe Fe Fe Fe

Fe

ErfeErfe

Young Adult



Tfr2 KI (훼+훽0)


(b)

FeFe Fe Fe

FeFe

FeFe Fe

Fe FeFe

ApoptosisApoptosis

Fe Fe

ErfeErfe

Young AdultFeFe





(c)








Acknowledgments


References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















Acknowledgments


References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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Altered Erythropoiesis in Mouse Models of Type 3...

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