Iron Reduces M1 Macrophage Polarization in RAW264.7 ...downloads.hindawi.com/journals/mi/2017/8570818.pdf · MediatorsofInflammation 5 IL-6 mRNA expression uns FAC M1 FAC + M1 0 5

Research ArticleIron Reduces M1 Macrophage Polarization in RAW2647Macrophages Associated with Inhibition of STAT1

Zhen-Shun Gan Qian-Qian Wang Jia-Hui Li Xu-Liang WangYi-Zhen Wang and Hua-Hua Du

Key Laboratory of Animal Nutrition and Feed Science (Eastern of China) Ministry of Agriculture College of Animal ScienceZhejiang University Hangzhou 310058 China

Correspondence should be addressed to Hua-Hua Du huahuaduzjueducn

Received 26 October 2016 Accepted 18 January 2017 Published 13 February 2017

Academic Editor Julio Galvez

Copyright copy 2017 Zhen-Shun Gan et alThis is an open access article distributed under theCreative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Ironmetabolism in inflammation has beenmostly characterized inmacrophages exposed to pathogens or inflammatory conditionsThe aim of this study is to investigate the cross-regulatory interactions between M1 macrophage polarization and iron metabolismFirstly we characterized the transcription of genes related to iron homeostasis inM1 RAW2647 macrophages stimulated by IFN-120574Themolecular signature ofM1macrophages showed high levels of iron storage (ferritin) a low level of iron export (ferroportin) andchanges of iron regulators (hepcidin and transferrin receptors) which favour iron sequestration in the reticuloendothelial systemand are benefit for inflammatory disordersThen we evaluated the effect of iron onM1 macrophage polarization Iron significantlyreduced mRNA levels of IL-6 IL-1120573 TNF-120572 and iNOS produced by IFN-120574-polarized M1 macrophages Immunofluorescenceanalysis showed that iron also reduced iNOS production However iron did not compromise but enhanced the ability of M1-polarizedmacrophages to phagocytose FITC-dextranMoreover we demonstrated that STAT1 inhibitionwas required for reductionof iNOS and M1-related cytokines production by the present of iron Together these findings indicated that iron decreasedpolarization of M1 macrophages and inhibited the production of the proinflammatory cytokines The results expanded ourknowledge about the role of iron in macrophage polarization

1 Introduction

Macrophages have long been considered to be importantimmune effector cells Depending on the microenviron-ment macrophages can acquire distinct morphological andfunctional properties Different inflammatory stimuli cantemporarily induce distinct subsets of macrophages withpolarized inflammatory phenotypes The Th1 cytokines suchas IFN-120574 stimulate the classic polarization and activationof macrophages into proinflammatory cells which are oftenreferred to as classically activated M1 macrophages [1]M1 phenotype is characterized by high capacity to presentantigen high levels of inflammatory cytokines (TNF-120572 IL-6)secretion and increased levels of NO production enhancedcapacity to kill intracellular pathogens and tumor cellsand promotion of polarized Th1 immune responses [2 3]Macrophages can also be alternatively activated by Th2cytokines such as IL-4 and IL-13 and are characterized by

minimal production of inflammatory molecules and woundhealing and repairThese alternatively activatedmacrophagesare referred to as M2 macrophages [4] Generally M1macrophages are considered proinflammatory cells whereasM2 macrophages are anti-inflammatory

Macrophages also play a critical role in body iron home-ostasis by recovering iron from old red blood cells andreturning it to the circulationThey are prodigious phagocyticcells that clear approximately 2 times 1011 erythrocytes each daywhich equate almost 3 kg of iron and hemoglobin per yearthat is ldquorecycledrdquo for the host to reuse [5 6] Iron is an essentialtrace element for multicellular organisms and nearly allmicroorganisms in which it functions as a catalytic compo-nent of enzymes that mediate many redox reactions that arecrucial for energy production and intermediary metabolism[7] Iron retention in the reticuloendothelial system is themain response of body iron homeostasis to inflammationand is regarded as a hostrsquos attempt to withhold iron from

HindawiMediators of InflammationVolume 2017 Article ID 8570818 9 pageshttpsdoiorg10115520178570818

2 Mediators of Inflammation

the invading pathogens [8] Increased iron retention withininflammatory macrophages is due to increased iron uptakeand decreased iron export and is favoured by the inductionof the iron storage protein ferritin (Ft) [9 10]

So far cytokines which drive macrophage polarizationhave been reported to ultimately control iron handling [1112] However there is limited information about the effect ofiron on the polarization and function of M1 macrophagesIn the present study we characterized the changes in irontrafficking in M1 macrophages and investigated the effects ofiron onM1polarization of RAW2647macrophage stimulatedby interferon-gamma (IFN-120574) We found that iron dramati-cally inhibited the transcription of proinflammatory cytokineand the production of enzyme inducible nitric oxide synthase(iNOS) in M1 macrophages We further demonstrated thatiron decreased M1 macrophage polarization involves inhi-bition of signal transducer and activator of transcription 1(STAT1) pathway

2 Materials and Methods

21 Reagents Ferric ammonium citrate (FAC)was purchasedfrom Sigma (USA) Recombinant murine interferon-gamma(IFN-120574) was purchased from PeproTech (USA)

22 Cell Culture and Stimulation The RAW2647 macro-phage cell line was generously offered by professorWeifenLirsquosLaboratory (College of Animal Science Zhejiang Univer-sity Hangzhou China) RAW2647 cells were cultured inDMEM with 10 FBS (Gibco USA) 100UmL penicillinand 100 120583gmL streptomycin at 37∘C in 5CO

2in humidified

incubator In a 6-well bottom plate 1 times 106 cells per wellwere seeded and incubated at 37∘C for 12 h The RAW2647cells were stimulated with FAC (0sim400120583gmL) for 24 hand the cytotoxicity assays were performed to ensure theappropriate FAC concentration The RAW2647 macrophagecells were treated with 25120583gmL FAC or IFN-120574 (20 ngmL)with or without FAC for 24 h which were used for geneexpression western blot analysis and immunofluorescenceor phagocytosis test

23 Cytotoxicity Assay Cell proliferation assay was evaluatedby a Cell Counting Kit-8 (CCK-8) Kit (Dojindo Japan)Briefly monolayers of RAW2647 cells in 96-well microplatewere cultured in DMEM supplemented with 10 FBS andincubatedwith FAC (3125sim400120583gmL) for 24 hThemediumwas replaced with fresh DMEM containing CCK-8 CCK-8 being nonradioactive allows sensitive colorimetric assaysfor the determination of the number of viable cells in cellproliferation and cytotoxicity assays After 2 h of incubationthe optical density was measured at OD

450 Lactate dehydro-

genase (LDH) release from damaged cells was determined48 h after treatment with FAC (3125sim400 120583gmL) LDHactivity in the culture supernatant was measured using aCytotoxicity LDH Assay Kit-WST (Dojindo Japan)

24 Total RNA Isolation and Real-Time PCR Total RNAisolated from RAW2647 cells was reverse transcribed usingMMLV Reverse Transcriptase (Thermo Fisher Scientific

USA) Real-time PCR was performed using FastStrat Univer-sal SYBR Green Master (ROX) (Roche USA) and the ABI7500 real-timePCR system (AppliedBiosystems)The follow-ing primers were used Hamp2 forward 51015840-ATCCCAATG-CAGAAGAGAAGG-31015840 and reverse 51015840-CAGATACCACAG-GAGGGTTTG-31015840 FPN forward 51015840-GGGTGGATAAGA-ATGCCAGACTT-31015840 and reverse 51015840-GTCAGGAGCTCA-TTCTTGTGTAGGA-31015840 FtH forward 51015840-TGGAACTGC-ACAAACTGGCTACT-31015840 and reverse 51015840-ATGGATTTC-ACCTGTTCACTCAGATAA-31015840 FtL forward 51015840-CGTGGA-TCTGTGTCTTGCTTCA-31015840 and reverse 51015840-GCGAAGAGA-CGGTGCAGACT-31015840 IRP1 forward 51015840-ACTTTGAAAGCT-GCCTTGG-31015840 and reverse 51015840-CTCCACTTCCAGGAG-ACAGG-31015840 IRP2 forward 51015840-TGAAGAAACGGACCT-GCTCT-31015840 and reverse 51015840-GCTCACATCCAACCACCT-CT-31015840 IL-6 forward 51015840-CTCCGACTTGTGAAGTGGTAT-AG-31015840 and reverse 51015840-CCACCTCAATGGACAGAATAT-CA-31015840 IL-1120573 forward 51015840-AGTTGACGGACCCCAAAA-G-31015840 and reverse 51015840-TTTGAAGCTGGATGCTCTCAT-31015840TNF-120572 forward 51015840-GCTCTTCTGTCTACTGAACTTCGG-31015840 and reverse 51015840-ATGATCTGAGTGTGAGGGTCTGG-31015840iNOS forward 51015840-CAGCTGGGCTGTACAAACCTT-31015840 andreverse 51015840-CATTGGAAGTGAAGCGTTTCG-31015840 120573-actinforward 51015840-CCACCATGTACCCAGGCATT-31015840 and reserse51015840-AGGGTGTAAAACGCAGCTCA-31015840 Fold changes werecalculated after normalizing the change in expression of thegene of interest to the housekeeping gene 120573-actin using thethreshold cycle values

25 Western Blot Analysis Total cell protein was preparedusing a Whole Protein Extraction Kit (KeyGEN China)Protein concentrations were determined using a BCA AssayKit (KeyGEN China) Equal amounts of proteins from eachsample were subjected to SDS-PAGE followed by transfer ofproteins to polyvinylidene difluoride (PVDF) membranesMembraneswere blocked in 5 skimmedmilk and incubatedwith a primary antibody overnight at 4∘C After washing withTBST membranes were incubated with secondary antibodylinked to HRP The blots were then developed with an ECLdetection system (Santa Cruz USA)

26 Immunofluorescence Analysis iNOS protein expressionlevels of the RAW2647 cells were evaluated by confocalimmunofluorescencemicroscopy Briefly theRAW2647 cellswere incubated with a rabbit monoclonal anti-iNOS anti-bodies (Abcam USA) for overnight at 4∘C and then withgoat anti-rabbit IgGCy3 secondary antibodies for 1 h Afterwashing with PBS the cells were incubated in a mediumcontaining 40mgml DAPI for 5min and examined with alaser-scanning microscope (ZEISS Germany)

27 Phagocytosis Assay To analyze the phagocytic activity ofmacrophages The RAW2647 macrophage cells were polar-ized by IFN-120574 (20 ngmL) in the presence of 25120583gmL FACfor 24 h and then incubated with fluorescein isothiocyanate-(FITC-) dextran (1mgmL) at 37∘C for 1 h After incubationthe cells were washed twice with PBS and the percentage ofintracellular FITC-dextran was determined by FluorescenceActivating Cell Sorter (FACS)

Mediators of Inflammation 3Ex

pres

sion

uns

uns M100

15

30

45

60lowastlowast

Hamp2 mRNA

(a)Ex

pres

sion

uns

uns M100

05

10

15lowast

FPN mRNA

(b)

Expr

essio

nun

s

uns M100

05

10

15

20

25lowastlowast

FtH mRNA

(c)

Expr

essio

nun

s

uns M100

15

30

45lowastlowast

FtL mRNA

(d)

Expr

essio

nun

s

uns M100

05

10

15

lowast

IRP1 mRNA

(e)Ex

pres

sion

uns

uns

IRP2 mRNA

M100

05

10

15

lowastlowast

(f)

Figure 1 Differential expression of ironmetabolism-related genes inM1macrophages (a) Hamp2 (b) FPN (c) FtH (d) FtL (e) IRP1 and (f)IRP2 mRNA levels in 24 h cells after polarization The expression was normalized to 120573-actin and then expressed in relation to unstimulatedmacrophages arbitrarily defined as 1 Data are mean plusmn SD for three independent experiments (uns unstimulated macrophages lowast119901 lt 005lowastlowast119901 lt 001)

28 Data Analysis Thedata were expressed as themeanplusmn SDof three independent experiments Statistical analyses wereperformed using two-tailed Studentrsquos 119905-test Values of 119901 lt005 were considered significant

3 Results

31 Differentially Expressed Genes of Iron Metabolism in M1Macrophages We relied on established protocols to polarizeRAW2647 macrophages into M1 cells by exposure to IFN-120574[13] Polarization ofmacrophages skews the expression profileof genes involved in iron metabolism In comparison withunstimulated macrophages the transcript levels of hepcidin(Hamp2) which is the master regulator of iron homeostasiswere increased in M1 cells (Figure 1(a)) Accordingly thetranscripts of ferroportin (FPN) which is the main andpossibly exclusive iron exporter and is functionally involvedin modulating iron release were decreased in M1 cells(Figure 1(b)) The mRNA levels of ferritin heavy chain (FtH)and ferritin light chain (FtL) which are associated with ironstorage were increased in IFN-120574 stimulated macrophages(Figures 1(c)-1(d)) We also analyzed iron regulatory proteins(IRP1 and IRP2) which are proteins of iron metabolism

important in intracellular iron homeostasis and known tobe primarily regulated at the posttranscriptional level [14]Lower mRNA expressions of IRP1 and IRP2 were detected incells exposed to IFN-120574 (Figures 1(e)-1(f))

32 Determination of the Noncytotoxic Dose of Iron inMacrophages We evaluated the cytotoxicity of iron (FAC)ranging from 313 to 400 120583gmL on RAW2647 cells andfound that the optimal viability was 25120583gmL showing 100survival (Figure 2(a)) Moreover we confirmed cell damageby measuring the release of the cytosolic marker lactatedehydrogenase (LDH) Treatment with less than 100120583gmLFAC for 48 h showed no significant difference of LDHrelease when compared with the control group (Figure 2(b))Therefore 25 120583gmL FAC was used for the next experiments

33 Iron Reduced the Transcription of Proinflammatory Medi-ators by M1-Polarized Macrophage As transcriptions havebeen shown to be a major mechanism in monocytic cells forLPS or IFN-120574 stimulation we used qRT-PCR to determine ifiron was affecting the transcription of cytokines Proinflam-matory M1 RAW2647 macrophages increased high mRNAlevels of IL-6 IL-1120573 TNF-120572 and iNOS in response to the


NS

Cel

l via

bilit

y (

of c

ontro

l)

313 625 125 25 50 100 200 4000

20

40

60

80

100

120

140lowast

Concentration of iron (120583M)

(a)

Cell

cyto

toxi

city

( o

f con

trol)

NS

313 625 125 25 50 100 200 4000

10

20

30

40

lowastlowast


(b)

Figure 2 Determination of the noncytotoxic dose of iron (a) RAW2647 macrophage cells were incubated with FAC at range from 0 to400 120583gmL for 24 h Cell viability was determined by CCK-8 methodThe results are expressed as the percentage of viable cells and representmean plusmn SD of four samples (b) Cell death was confirmed by measuring the release of the cytosolic marker LDH LDH activity in thesupernatant was measured as described in methods lowast119901 lt 005 and lowastlowast119901 lt 001 (119905-test)

stimulation of IFN-120574 whereas unstimulated macrophagesproduced significantly lower levels (Figure 3) The additionof 25 120583gml FAC during the polarization of M1 macrophagesby IFN-120574 resulted in a severe blockage of the transcriptionof cytokines production (Figure 3) IL-1120573 mRNA was sig-nificantly inhibited by 77 (Figure 3(b)) FAC reduced IL-6 mRNA by 57 (Figure 3(a)) and TNF-120572 mRNA by 51(Figure 3(c)) It had amuch lesser effect on iNOS productionalthough it still significantly reduced the transcription by 43(Figure 3(d))

34 Iron Inhibited iNOS Production in M1 Macrophages M1macrophages produce inducible nitric oxide synthase (iNOS)that enables the cell to kill intracellular pathogens throughthe production of NO We further investigated whetheriron regulates iNOS production In line with the results oftranscription (Figure 3(d)) the M1-polarized macrophagesproduced higher iNOS in response to IFN-120574 compared withunstimulated cells However the presence of 25120583gml FACduring the polarization resulted in a blockage of iNOSproduction with 10 inhibition (Figure 4(a)) To confirm theoutcomes at a protein level and to determine if the changes inprotein expression have occurred uniformly across the entiremacrophage population cells were stimulated by IFN-120574 inthe presence of 25 120583gml FAC and examined by immunoflu-orescence microscopy with antibody specific for iNOSConsistent with mRNA expression outcomes (Figure 3(d))cells stimulated with IFN-120574 expressed higher level of iNOS(Figure 4(b) bottom left side red) The presence of FACsignificantly blocked the production of iNOS (Figure 4(b)bottom right side red) Of note staining levels were uniformin most cells within the same condition indicating that themRNA or protein expression patterns result from changesacross the entire populations

35 Iron Inhibited STAT1 Pathway in M1 MacrophagesMacrophage polarization is a complex process includingstimuli recognition and activation of the transcription factors[15] Recent studies have shown that STAT1 signaling path-ways are involved in M1 macrophage polarization [16] Toinvestigate whether iron affects these cascades we performedwestern blot to examine the phosphorylation of STAT1 M1-polarized macrophages increased the phosphorylation formsof STAT1 in response to IFN-120574 compared with unstimulatedcells However the presence of 25120583gml FAC during thepolarization resulted in a blockage the phosphorylation ofSTAT1 with 66 inhibition (Figure 5) indicating that FACdecreased M1 macrophage polarization was dependent onSTAT1 signaling

36 Iron Enhanced Phagocytosis Capacity of M1 PhagocytosisPhagocytosis plays a crucial role in macrophage-mediatedhost defense which leads to internalization and distractionof pathogens To determine if iron affected the phagocytosisof M1-polarized macrophages we examined the internal-ization of FITC-labeled dextran by FACS FAC-treated M1macrophages showed markedly increased uptake of FITC-dextran (Figure 6) Therefore iron inhibits the production ofthe proinflammatory cytokines in M1 macrophages withoutaffecting their phagocytosis functions

4 Discussion

Macrophages are important for immune responses and arewidely distributed in peripheral tissues where they play anindispensable role in the defense against pathogens This isat least partially achieved through the control of intracellulariron availability which limits pathogen growth [17] Asan essential trace element for microbes proliferation and

Mediators of Inflammation 5

IL-6

mRN

A ex

pres

sion

uns FAC M1 FAC + M10

5

10

15 lowastlowastlowastlowast

(a)uns FAC M1 FAC + M1

0

2

4

6

8 lowastlowast lowastlowast

IL-1120573

mRN

A ex

pres

sion

(b)


1

2

3

4


TNF-

120572m

RNA

expr

essio

n

(c)

iNO

S m

RNA

expr

essio

n


1

2

3


(d)

Figure 3 Effects of iron on mRNA expression in M1 macrophage (a) IL-6 (b) IL-1120573 (c) TNF-120572 and (d) iNOS mRNA expression wereassessed by real-time PCR Gene expression is represented as fold-change compared to unstimulated macrophages Data are mean plusmn SD forthree independent experiments (uns unstimulated macrophages lowastlowast119901 lt 001)

pathogenicity iron affects cell-mediated immune functionand thus host response toward pathogens On the one handthe polarization of macrophages can have important effectson ironmetabolism but on the other hand iron can influencedirectly macrophage polarization [18] The aim of this studyis to investigate the cross-regulatory interactions betweenM1macrophage polarization and iron metabolism

Cellular iron homeostasis in macrophages is regulated atmultiple steps and by numerous genes [19] Macrophages canacquire iron via the divalent metal transporter 1 and phago-cytosis of senescent erythrocytes with subsequent recyclingof iron The diversion of cellular iron is then orchestratedby the IRPIRE interaction resulting in reutilization ironstorage within ferritin or iron export FPN is only onewell-characterized pathway for iron export from cells [20]Hepcidin induced by iron and cytokines and master regu-lator of body iron homeostasis exerts its regulatory effectsvia binding to its receptor FPN [21] The transcriptionaland posttranscriptional control of many of the genes areresponsible for these functions [13] so we detected themRNA levels of iron related genes to characterize the changesof iron metabolism in M1 macrophages In this study M1macrophages derived by recombinant IFN-120574 alone expresshigh mRNA levels of hepcidin (Hamp2) FtH and FtL and

low levels of FPN IRP1 and IRP2 M1 macrophages whichdirectly deal with microbes at sites of infection upregulatehepcidin and downregulate FPN thus limiting release ofiron which could favour invading pathogens Meanwhile byupregulating FtH and FtL expression and limiting IRP1 andIRP2 M1 cells possibly protect themselves against oxidativedamage and further limit the availability of the moleculeto internalized microbes These observations are consistentwith experiments on human peripheral bloodmonocytes andmice macrophages [11 12]

It is well known that M1 macrophages have enhancedmicrobicidal capacity secrete high levels of proinflammatorycytokines and produce great amount of oxygen and nitrogenradicals to increase their killing activity [22] In this study wefound that iron dramatically inhibited the transcriptions ofproinflammatory cytokine IL-6 IL-1120573 and TNF-120572 in IFN-120574-stimulated M1 macrophages Proinflammatory cytokinesas markers of activated polarized M1 macrophages areregarded as the effector molecules to mediate resistanceagainst pathogens [16] Prevention of the transcription ofthese proinflammatory cytokines indicated that amajor effectof iron is to prevent IFN-120574-induced activation of these genesMeanwhile M1 macrophages produce high levels of iNOSwhich is a major component of the antimicrobial effector


iNOS

M1

FACM2

00

05

10

15

20

25

30

uns FAC M1 FAC + M1

lowast

iNO

S120573

-act

in (f

old

of u

ns)

120573-Actin

+

++

+minusminusminus minus minusminus minus

minus

(a)

uns FAC

M1 (IFN-120574) M1 (IFN-120574) + FAC

(b)

Figure 4 Effects of iron on iNOS expression in M1 macrophage RAW2647 macrophage cells were incubated with the stimulus either FACIFN-120574 or no stimulus for 24 h Cells were harvested and analyzed for iNOS (a) by western blot or fixed and stained with antibodies for iNOS(b) Protein expression is represented as fold-change compared to unstimulated macrophages In each sample group cell was stained with thered fluorescent-labeled antibody for the targeted protein and the blue fluorescent-labeled DAPI for nucleus Data are representative imagesof three (a) or two (b) independent experiments (uns unstimulated macrophages lowast119901 lt 005)

p-ST

AT1

GA

PDH

(fol

d of

uns

)


05

10

15

20

25

30lowastlowast

Control M1 FAC FAC + M1

p-STAT1

STAT1

GAPDH

Figure 5 Effect of iron on STAT1 activation in M1 macrophages RAW2647 macrophage cells were incubated with the stimulus eitherFAC IFN-120574 or no stimulus for 24 h Cell lysates were prepared and phosphorylation of STAT1 (p-STAT1) was analyzed by western blotOne representative blot and the densitometric quantification are shown Data are mean plusmn SD for three independent experiments (unsunstimulated macrophages lowastlowast119901 lt 001)

machinery and the formation of NO has been shown tomediate protection from infection [23] We observed thatiron also had a mild suppressive effect on iNOS productionwhich led to a decreased production ofNO Ironmight inhibitthe expression of iNOS by a transcriptional mechanisminvolving the deactivation of the transcription factor nuclearfactor (NF)-IL-6 [24]However the potent inhibitory effect ofironwas restricted to specific functions of classically activated

macrophages namely proinflammatory cytokines and iNOSproduction as it did not affect the phagocytosis capacity ofM1 macrophages

STAT1 is an important signaling molecule that playsa major role in mediating proinflammatory responses fol-lowing ligation of the Th1-type cytokine IFN-120574 which isimportant for modulating protective immune responses tomultiple pathogens [25] Here we showed that iron also


Relat

ive p

hago

cyto

sis ra

te

uns M1 FAC + M100

05

10

15

20

lowastlowast

lowast

lowast

ControlC

ount

s200

160

120

80

40

0

FITC100 101 102 103 104

Cou

nts

200

160

120

80

40

0

Cou

nts

200

160

120

80

40

0

FITC100 101 102 103 104

FITC100 101 102 103 104

M1

M1

M1

IFN-120574 (20ngmL)

FAC (25120583M) + IFN-120574 (20ngmL)

Figure 6 Effect of iron onM1macrophages phagocytosis activity RAW2647macrophages were polarized to anM1 phenotype in the absence(uns) or presence of 25 120583gmL of FAC washed and incubated with FITC-dextran at 37∘C for 1 h the intracellular FITC-dextran wasmeasuredby FACS Data are mean plusmn SD for three independent experiments (uns unstimulated macrophages lowast119901 lt 005 lowastlowast119901 lt 001)

decreased the phosphorylation of STAT1 in IFN-120574-inducedmacrophages which suggested that STAT1 inhibition mightbe required for reduction of iNOS and M1-related cytokinesproduction This result was in line with previous reportthat STAT1 signaling in macrophages during C neoformansinfection is critical for the induction of M1 macrophageactivation and the production of NO [26]

Iron exertsmultiple effects on immune effector functionsThis is on the one hand based on the role of iron for thedifferentiation and proliferation of immune cells includingantigen presenting cells and lymphocytes [27] Moreoveriron affects antimicrobial immune function of macrophagesvia inhibition of IFN-120574 inducible effector pathways [28 29]The relevance of these observations was substantiated byexperiments which demonstrated that macrophages loadedwith iron lose their ability to kill intracellular pathogenssuch as Salmonella Mycobacteria Chlamydia or Legionellaby IFN-120574-mediated pathways [17 30 31] To the best ofour knowledge there is not previous report on the effectof iron addition on the macrophage polarization Our data

provide evidence that iron inhibits the polarization of M1macrophages stimulated by IFN-120574 and then impairs theproinflammatory responses of macrophages

Obviously the retention of iron in the M1 macrophagesreduces circulating iron levels and thus the availability ofthis essential nutrient for extracellular microbes This ironwithholding strategy appears to be of benefit to combat infec-tions with circulating pathogensThus iron supplementationduring infections is inadvisable and even hazardous Clinicaltrials demonstrated that iron supplementation resulted inhigher incidence of or higher mortality from infectionssuch as malaria diarrhea or bacterial meningitis [32 33]The pathways underlying these devastating outcomes remainelusive However they may be linked to iron mediated mod-ulation of antimicrobial immune defense of macrophagesor traced back to increased availability of the metal forpathogens

In conclusion based on discovering iron sequestration inM1macrophages we have demonstrated that iron suppressedIFN-120574 induced M1 polarization of RAW2647 macrophages


with decreased proinflammatory responses and iNOS pro-duction while preserving their phagocytosis activity It sug-gested that iron loading duringM1 polarization would impairthe antimicrobial immune defense of macrophages Thusa certain balance of iron not too less and not too muchis needed to strengthen immune response to successfullycombat infections

Competing Interests

The authors declare that there is no conflict of interests

Acknowledgments

This work was supported by Natural Science Foundation ofZhejiang province of China (no LR16C170001) and NationalNatural Science Foundation of China (nos 31272455 and31572411)

References

[1] AMantovani A Sica S Sozzani P Allavena A Vecchi andMLocati ldquoThe chemokine system in diverse forms of macrophageactivation and polarizationrdquo Trends in Immunology vol 25 no12 pp 677ndash686 2004

[2] M Naito ldquoMacrophage differentiation and function in healthand diseaserdquo Pathology International vol 58 no 3 pp 143ndash1552008

[3] H Qin A T Holdbrooks Y Liu S L Reynolds L LYanagisawa and E N Benveniste ldquoSOCS3 deficiency promotesM1 macrophage polarization and inflammationrdquo Journal ofImmunology vol 189 no 7 pp 3439ndash3448 2012

[4] S Gordon ldquoAlternative activation of macrophagesrdquo NatureReviews Immunology vol 3 no 1 pp 23ndash35 2003

[5] N C Andrews ldquoForging a field the golden age of iron biologyrdquoBlood vol 112 no 2 pp 219ndash230 2008

[6] I De Domenico D McVey Ward and J Kaplan ldquoRegulationof iron acquisition and storage consequences for iron-linkeddisordersrdquo Nature Reviews Molecular Cell Biology vol 9 no 1pp 72ndash81 2008

[7] T Ganz and E Nemeth ldquoIron homeostasis in host defence andinflammationrdquo Nature Reviews Immunology vol 15 no 8 pp500ndash510 2015

[8] G Cairo F Bernuzzi and S Recalcati ldquoA precious metal ironan essential nutrient for all cellsrdquo Genes amp Nutrition vol 1 no1 pp 25ndash39 2006

[9] F M Torti and S V Torti ldquoRegulation of ferritin genes andproteinrdquo Blood vol 99 no 10 pp 3505ndash3516 2002

[10] M Knutson and M Wessling-Resnick ldquoIron metabolism inthe reticuloendothelial systemrdquoCritical Reviews in Biochemistryand Molecular Biology vol 38 no 1 pp 61ndash88 2003

[11] G Corna L Campana E Pignatti et al ldquoPolarization dictatesiron handling by inflammatory and alternatively activatedmacrophagesrdquo Haematologica vol 95 no 11 pp 1814ndash18222010

[12] S Recalcati M Locati A Marini et al ldquoDifferential regulationof iron homeostasis during human macrophage polarizedactivationrdquo European Journal of Immunology vol 40 no 3 pp824ndash835 2010

[13] F O Martinez S Gordon M Locati and A Mantovani ldquoTran-scriptional profiling of the human monocyte-to-macrophagedifferentiation and polarization new molecules and patterns ofgene expressionrdquo Journal of Immunology vol 177 no 10 pp7303ndash7311 2006

[14] M L Wallander E A Leibold and R S Eisenstein ldquoMolecularcontrol of vertebrate iron homeostasis by iron regulatory pro-teinsrdquo Biochimica et Biophysica ActamdashMolecular Cell Researchvol 1763 no 7 pp 668ndash689 2006

[15] S K Biswas and A Mantovani ldquoMacrophage plasticity andinteraction with lymphocyte subsets cancer as a paradigmrdquoNature Immunology vol 11 no 10 pp 889ndash896 2010

[16] D M Mosser and J P Edwards ldquoExploring the full spectrumof macrophage activationrdquo Nature Reviews Immunology vol 8no 12 pp 958ndash969 2008

[17] P N Paradkar I De Domenico N Durchfort I Zohn JKaplan and D M Ward ldquoIron depletion limits intracellularbacterial growth inmacrophagesrdquoBlood vol 112 no 3 pp 866ndash874 2008

[18] S RecalcatiM Locati E Gammella P Invernizzi andG CairoldquoIron levels in polarized macrophages regulation of immunityand autoimmunityrdquo Autoimmunity Reviews vol 11 no 12 pp883ndash889 2012

[19] G Weiss ldquoIron metabolism in the anemia of chronic diseaserdquoBiochimica et BiophysicaActa vol 1790 no 7 pp 682ndash693 2009

[20] MWHentzeMUMuckenthaler BGaly andCCamaschellaldquoTwo to tango regulation of Mammalian iron metabolismrdquoCell vol 142 no 1 pp 24ndash38 2010

[21] E Nemeth M S Tuttle J Powelson et al ldquoHepcidin regulatescellular iron efflux by binding to ferroportin and inducing itsinternalizationrdquo Science vol 306 no 5704 pp 2090ndash20932004

[22] S Gordon and F O Martinez ldquoAlternative activation ofmacrophagesmechanism and functionsrdquo Immunity vol 32 no5 pp 593ndash604 2010

[23] K A Shirey L E Cole A D Keegan and S N VogelldquoFrancisella tularensis live vaccine strain induces macrophagealternative activation as a survival mechanismrdquo Journal ofImmunology vol 181 no 6 pp 4159ndash4167 2008

[24] M Dlaska and G Weiss ldquoCentral role of transcription factorNF-IL6 for cytokine and iron- mediated regulation of murineinducible nitric oxide synthase expressionrdquo Journal of Immunol-ogy vol 162 no 10 pp 6171ndash6177 1999

[25] B Saha S J Prasanna B Chandrasekar and D NandildquoGenemodulation and immunoregulatory roles of interferon120574rdquoCytokine vol 50 no 1 pp 1ndash14 2010

[26] C M L Wager C R Hole K L Wozniak M A OlszewskiM Mueller and F L Wormley ldquoSTAT1 signaling withinmacrophages is required for antifungal activity against Cryp-tococcus neoformansrdquo Infection and Immunity vol 83 no 12pp 4513ndash4527 2015

[27] G Cairo S Recalcati A Mantovani and M Locati ldquoIrontrafficking andmetabolism inmacrophages contribution to thepolarized phenotyperdquo Trends in Immunology vol 32 no 6 pp241ndash247 2011

[28] FOOmara andB R Blakley ldquoThe effects of iron deficiency andiron overload on cell-mediated immunity in the mouserdquo BritishJournal of Nutrition vol 72 no 6 pp 899ndash909 1994

[29] H Oexle A Kaser J Most et al ldquoPathways for the regulation ofinterferon-120574-inducible genes by iron in humanmonocytic cellsrdquoJournal of Leukocyte Biology vol 74 no 2 pp 287ndash294 2003


[30] S Chlosta D S Fishman L Harrington et al ldquoThe ironefflux protein ferroportin regulates the intracellular growth ofSalmonella entericardquo Infection amp Immunity vol 74 no 5 pp3065ndash3067 2006

[31] M Nairz D Haschka E Demetz and G Weiss ldquoIron at theinterface of immunity and infectionrdquo Frontiers in Pharmacol-ogy vol 5 article no 152 2014

[32] S Sazawal R E Black M Ramsan et al ldquoEffects of routineprophylactic supplementation with iron and folic acid onadmission to hospital and mortality in preschool children ina high malaria transmission setting community-based ran-domised placebo-controlled trialrdquoLancet vol 367 no 9505 pp133ndash143 2006

[33] S Soofi S Cousens S P Iqbal et al ldquoEffect of provisionof daily zinc and iron with several micronutrients on growthand morbidity among young children in Pakistan a cluster-randomised trialrdquo The Lancet vol 382 no 9886 pp 29ndash402013

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


the invading pathogens [8] Increased iron retention withininflammatory macrophages is due to increased iron uptakeand decreased iron export and is favoured by the inductionof the iron storage protein ferritin (Ft) [9 10]

So far cytokines which drive macrophage polarizationhave been reported to ultimately control iron handling [1112] However there is limited information about the effect ofiron on the polarization and function of M1 macrophagesIn the present study we characterized the changes in irontrafficking in M1 macrophages and investigated the effects ofiron onM1polarization of RAW2647macrophage stimulatedby interferon-gamma (IFN-120574) We found that iron dramati-cally inhibited the transcription of proinflammatory cytokineand the production of enzyme inducible nitric oxide synthase(iNOS) in M1 macrophages We further demonstrated thatiron decreased M1 macrophage polarization involves inhi-bition of signal transducer and activator of transcription 1(STAT1) pathway

2 Materials and Methods

21 Reagents Ferric ammonium citrate (FAC)was purchasedfrom Sigma (USA) Recombinant murine interferon-gamma(IFN-120574) was purchased from PeproTech (USA)

22 Cell Culture and Stimulation The RAW2647 macro-phage cell line was generously offered by professorWeifenLirsquosLaboratory (College of Animal Science Zhejiang Univer-sity Hangzhou China) RAW2647 cells were cultured inDMEM with 10 FBS (Gibco USA) 100UmL penicillinand 100 120583gmL streptomycin at 37∘C in 5CO

2in humidified

incubator In a 6-well bottom plate 1 times 106 cells per wellwere seeded and incubated at 37∘C for 12 h The RAW2647cells were stimulated with FAC (0sim400120583gmL) for 24 hand the cytotoxicity assays were performed to ensure theappropriate FAC concentration The RAW2647 macrophagecells were treated with 25120583gmL FAC or IFN-120574 (20 ngmL)with or without FAC for 24 h which were used for geneexpression western blot analysis and immunofluorescenceor phagocytosis test

23 Cytotoxicity Assay Cell proliferation assay was evaluatedby a Cell Counting Kit-8 (CCK-8) Kit (Dojindo Japan)Briefly monolayers of RAW2647 cells in 96-well microplatewere cultured in DMEM supplemented with 10 FBS andincubatedwith FAC (3125sim400120583gmL) for 24 hThemediumwas replaced with fresh DMEM containing CCK-8 CCK-8 being nonradioactive allows sensitive colorimetric assaysfor the determination of the number of viable cells in cellproliferation and cytotoxicity assays After 2 h of incubationthe optical density was measured at OD

450 Lactate dehydro-

genase (LDH) release from damaged cells was determined48 h after treatment with FAC (3125sim400 120583gmL) LDHactivity in the culture supernatant was measured using aCytotoxicity LDH Assay Kit-WST (Dojindo Japan)

24 Total RNA Isolation and Real-Time PCR Total RNAisolated from RAW2647 cells was reverse transcribed usingMMLV Reverse Transcriptase (Thermo Fisher Scientific

USA) Real-time PCR was performed using FastStrat Univer-sal SYBR Green Master (ROX) (Roche USA) and the ABI7500 real-timePCR system (AppliedBiosystems)The follow-ing primers were used Hamp2 forward 51015840-ATCCCAATG-CAGAAGAGAAGG-31015840 and reverse 51015840-CAGATACCACAG-GAGGGTTTG-31015840 FPN forward 51015840-GGGTGGATAAGA-ATGCCAGACTT-31015840 and reverse 51015840-GTCAGGAGCTCA-TTCTTGTGTAGGA-31015840 FtH forward 51015840-TGGAACTGC-ACAAACTGGCTACT-31015840 and reverse 51015840-ATGGATTTC-ACCTGTTCACTCAGATAA-31015840 FtL forward 51015840-CGTGGA-TCTGTGTCTTGCTTCA-31015840 and reverse 51015840-GCGAAGAGA-CGGTGCAGACT-31015840 IRP1 forward 51015840-ACTTTGAAAGCT-GCCTTGG-31015840 and reverse 51015840-CTCCACTTCCAGGAG-ACAGG-31015840 IRP2 forward 51015840-TGAAGAAACGGACCT-GCTCT-31015840 and reverse 51015840-GCTCACATCCAACCACCT-CT-31015840 IL-6 forward 51015840-CTCCGACTTGTGAAGTGGTAT-AG-31015840 and reverse 51015840-CCACCTCAATGGACAGAATAT-CA-31015840 IL-1120573 forward 51015840-AGTTGACGGACCCCAAAA-G-31015840 and reverse 51015840-TTTGAAGCTGGATGCTCTCAT-31015840TNF-120572 forward 51015840-GCTCTTCTGTCTACTGAACTTCGG-31015840 and reverse 51015840-ATGATCTGAGTGTGAGGGTCTGG-31015840iNOS forward 51015840-CAGCTGGGCTGTACAAACCTT-31015840 andreverse 51015840-CATTGGAAGTGAAGCGTTTCG-31015840 120573-actinforward 51015840-CCACCATGTACCCAGGCATT-31015840 and reserse51015840-AGGGTGTAAAACGCAGCTCA-31015840 Fold changes werecalculated after normalizing the change in expression of thegene of interest to the housekeeping gene 120573-actin using thethreshold cycle values

25 Western Blot Analysis Total cell protein was preparedusing a Whole Protein Extraction Kit (KeyGEN China)Protein concentrations were determined using a BCA AssayKit (KeyGEN China) Equal amounts of proteins from eachsample were subjected to SDS-PAGE followed by transfer ofproteins to polyvinylidene difluoride (PVDF) membranesMembraneswere blocked in 5 skimmedmilk and incubatedwith a primary antibody overnight at 4∘C After washing withTBST membranes were incubated with secondary antibodylinked to HRP The blots were then developed with an ECLdetection system (Santa Cruz USA)

26 Immunofluorescence Analysis iNOS protein expressionlevels of the RAW2647 cells were evaluated by confocalimmunofluorescencemicroscopy Briefly theRAW2647 cellswere incubated with a rabbit monoclonal anti-iNOS anti-bodies (Abcam USA) for overnight at 4∘C and then withgoat anti-rabbit IgGCy3 secondary antibodies for 1 h Afterwashing with PBS the cells were incubated in a mediumcontaining 40mgml DAPI for 5min and examined with alaser-scanning microscope (ZEISS Germany)

27 Phagocytosis Assay To analyze the phagocytic activity ofmacrophages The RAW2647 macrophage cells were polar-ized by IFN-120574 (20 ngmL) in the presence of 25120583gmL FACfor 24 h and then incubated with fluorescein isothiocyanate-(FITC-) dextran (1mgmL) at 37∘C for 1 h After incubationthe cells were washed twice with PBS and the percentage ofintracellular FITC-dextran was determined by FluorescenceActivating Cell Sorter (FACS)


pres

sion

uns

uns M100

15

30

45

60lowastlowast

Hamp2 mRNA

(a)Ex

pres

sion

uns

uns M100

05

10

15lowast

FPN mRNA

(b)

Expr

essio

nun

s

uns M100

05

10

15

20

25lowastlowast

FtH mRNA

(c)

Expr

essio

nun

s

uns M100

15

30

45lowastlowast

FtL mRNA

(d)

Expr

essio

nun

s

uns M100

05

10

15

lowast

IRP1 mRNA

(e)Ex

pres

sion

uns

uns

IRP2 mRNA

M100

05

10

15

lowastlowast

(f)



3 Results






NS

Cel

l via

bilit

y (

of c

ontro

l)

313 625 125 25 50 100 200 4000

20

40

60

80

100

120

140lowast


(a)

Cell

cyto

toxi

city

( o

f con

trol)

NS

313 625 125 25 50 100 200 4000

10

20

30

40

lowastlowast


(b)






4 Discussion



IL-6

mRN

A ex

pres

sion


5

10



0

2

4

6


IL-1120573

mRN

A ex

pres

sion

(b)


1

2

3

4


TNF-

120572m

RNA

expr

essio

n

(c)

iNO

S m

RNA

expr

essio

n


1

2

3


(d)







iNOS

M1

FACM2

00

05

10

15

20

25

30

uns FAC M1 FAC + M1

lowast

iNO

S120573

-act

in (f

old

of u

ns)

120573-Actin

+

++


minus

(a)

uns FAC

M1 (IFN-120574) M1 (IFN-120574) + FAC

(b)


p-ST

AT1

GA

PDH

(fol

d of

uns

)


05

10

15

20

25

30lowastlowast


p-STAT1

STAT1

GAPDH






Relat

ive p

hago

cyto

sis ra

te

uns M1 FAC + M100

05

10

15

20

lowastlowast

lowast

lowast

ControlC

ount

s200

160

120

80

40

0

FITC100 101 102 103 104

Cou

nts

200

160

120

80

40

0

Cou

nts

200

160

120

80

40

0

FITC100 101 102 103 104

FITC100 101 102 103 104

M1

M1

M1

IFN-120574 (20ngmL)

FAC (25120583M) + IFN-120574 (20ngmL)









Competing Interests


Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















pres

sion

uns

uns M100

15

30

45

60lowastlowast

Hamp2 mRNA

(a)Ex

pres

sion

uns

uns M100

05

10

15lowast

FPN mRNA

(b)

Expr

essio

nun

s

uns M100

05

10

15

20

25lowastlowast

FtH mRNA

(c)

Expr

essio

nun

s

uns M100

15

30

45lowastlowast

FtL mRNA

(d)

Expr

essio

nun

s

uns M100

05

10

15

lowast

IRP1 mRNA

(e)Ex

pres

sion

uns

uns

IRP2 mRNA

M100

05

10

15

lowastlowast

(f)



3 Results






NS

Cel

l via

bilit

y (

of c

ontro

l)

313 625 125 25 50 100 200 4000

20

40

60

80

100

120

140lowast


(a)

Cell

cyto

toxi

city

( o

f con

trol)

NS

313 625 125 25 50 100 200 4000

10

20

30

40

lowastlowast


(b)






4 Discussion



IL-6

mRN

A ex

pres

sion


5

10



0

2

4

6


IL-1120573

mRN

A ex

pres

sion

(b)


1

2

3

4


TNF-

120572m

RNA

expr

essio

n

(c)

iNO

S m

RNA

expr

essio

n


1

2

3


(d)







iNOS

M1

FACM2

00

05

10

15

20

25

30

uns FAC M1 FAC + M1

lowast

iNO

S120573

-act

in (f

old

of u

ns)

120573-Actin

+

++


minus

(a)

uns FAC

M1 (IFN-120574) M1 (IFN-120574) + FAC

(b)


p-ST

AT1

GA

PDH

(fol

d of

uns

)


05

10

15

20

25

30lowastlowast


p-STAT1

STAT1

GAPDH






Relat

ive p

hago

cyto

sis ra

te

uns M1 FAC + M100

05

10

15

20

lowastlowast

lowast

lowast

ControlC

ount

s200

160

120

80

40

0

FITC100 101 102 103 104

Cou

nts

200

160

120

80

40

0

Cou

nts

200

160

120

80

40

0

FITC100 101 102 103 104

FITC100 101 102 103 104

M1

M1

M1

IFN-120574 (20ngmL)

FAC (25120583M) + IFN-120574 (20ngmL)









Competing Interests


Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















NS

Cel

l via

bilit

y (

of c

ontro

l)

313 625 125 25 50 100 200 4000

20

40

60

80

100

120

140lowast


(a)

Cell

cyto

toxi

city

( o

f con

trol)

NS

313 625 125 25 50 100 200 4000

10

20

30

40

lowastlowast


(b)






4 Discussion



IL-6

mRN

A ex

pres

sion


5

10



0

2

4

6


IL-1120573

mRN

A ex

pres

sion

(b)


1

2

3

4


TNF-

120572m

RNA

expr

essio

n

(c)

iNO

S m

RNA

expr

essio

n


1

2

3


(d)







iNOS

M1

FACM2

00

05

10

15

20

25

30

uns FAC M1 FAC + M1

lowast

iNO

S120573

-act

in (f

old

of u

ns)

120573-Actin

+

++


minus

(a)

uns FAC

M1 (IFN-120574) M1 (IFN-120574) + FAC

(b)


p-ST

AT1

GA

PDH

(fol

d of

uns

)


05

10

15

20

25

30lowastlowast


p-STAT1

STAT1

GAPDH






Relat

ive p

hago

cyto

sis ra

te

uns M1 FAC + M100

05

10

15

20

lowastlowast

lowast

lowast

ControlC

ount

s200

160

120

80

40

0

FITC100 101 102 103 104

Cou

nts

200

160

120

80

40

0

Cou

nts

200

160

120

80

40

0

FITC100 101 102 103 104

FITC100 101 102 103 104

M1

M1

M1

IFN-120574 (20ngmL)

FAC (25120583M) + IFN-120574 (20ngmL)









Competing Interests


Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















IL-6

mRN

A ex

pres

sion


5

10



0

2

4

6


IL-1120573

mRN

A ex

pres

sion

(b)


1

2

3

4


TNF-

120572m

RNA

expr

essio

n

(c)

iNO

S m

RNA

expr

essio

n


1

2

3


(d)







iNOS

M1

FACM2

00

05

10

15

20

25

30

uns FAC M1 FAC + M1

lowast

iNO

S120573

-act

in (f

old

of u

ns)

120573-Actin

+

++


minus

(a)

uns FAC

M1 (IFN-120574) M1 (IFN-120574) + FAC

(b)


p-ST

AT1

GA

PDH

(fol

d of

uns

)


05

10

15

20

25

30lowastlowast


p-STAT1

STAT1

GAPDH






Relat

ive p

hago

cyto

sis ra

te

uns M1 FAC + M100

05

10

15

20

lowastlowast

lowast

lowast

ControlC

ount

s200

160

120

80

40

0

FITC100 101 102 103 104

Cou

nts

200

160

120

80

40

0

Cou

nts

200

160

120

80

40

0

FITC100 101 102 103 104

FITC100 101 102 103 104

M1

M1

M1

IFN-120574 (20ngmL)

FAC (25120583M) + IFN-120574 (20ngmL)









Competing Interests


Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















iNOS

M1

FACM2

00

05

10

15

20

25

30

uns FAC M1 FAC + M1

lowast

iNO

S120573

-act

in (f

old

of u

ns)

120573-Actin

+

++


minus

(a)

uns FAC

M1 (IFN-120574) M1 (IFN-120574) + FAC

(b)


p-ST

AT1

GA

PDH

(fol

d of

uns

)


05

10

15

20

25

30lowastlowast


p-STAT1

STAT1

GAPDH






Relat

ive p

hago

cyto

sis ra

te

uns M1 FAC + M100

05

10

15

20

lowastlowast

lowast

lowast

ControlC

ount

s200

160

120

80

40

0

FITC100 101 102 103 104

Cou

nts

200

160

120

80

40

0

Cou

nts

200

160

120

80

40

0

FITC100 101 102 103 104

FITC100 101 102 103 104

M1

M1

M1

IFN-120574 (20ngmL)

FAC (25120583M) + IFN-120574 (20ngmL)









Competing Interests


Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Relat

ive p

hago

cyto

sis ra

te

uns M1 FAC + M100

05

10

15

20

lowastlowast

lowast

lowast

ControlC

ount

s200

160

120

80

40

0

FITC100 101 102 103 104

Cou

nts

200

160

120

80

40

0

Cou

nts

200

160

120

80

40

0

FITC100 101 102 103 104

FITC100 101 102 103 104

M1

M1

M1

IFN-120574 (20ngmL)

FAC (25120583M) + IFN-120574 (20ngmL)









Competing Interests


Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Competing Interests


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
















Iron Reduces M1 Macrophage Polarization in RAW264.7 ...downloads.hindawi.com/journals/mi/2017/8570818.pdf · MediatorsofInflammation 5 IL-6 mRNA expression uns FAC M1 FAC + M1 0 5

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