nearly all organisms require iron because of its ability to catalyze redox reactions , and humans have evolved mechanisms to recycle almost all the iron contained within hemoglobin and cellular enzymes with only minimal daily loss through the gastrointestinal tract . nevertheless , iron - deficiency anemia and anemia of chronic disease ( caused in part by iron - restricted erythropoiesis ) together are responsible for the majority of anemia cases worldwide , and while these conditions cause morbidity and mortality , it has become clear that limiting iron availability is in fact an innate immune strategy against microbes . indeed studies in humans and mice have shown that oral iron supplementation leads to increased mortality due to infection [ 3 , 4 ] . pathogens that enter and proliferate within a host must acquire iron from the host and have evolved a large and diverse number of mechanisms to accomplish this , and in response , mammals have developed complex mechanisms to keep iron from pathogens [ 6 , 7 ] . over the last two decades , there has been much effort aimed at understanding the pathophysiology of anemia associated with chronic inflammation / disease , characterized by hypoferremia . this led to the finding that a small peptide , hepcidin , initially identified as an antimicrobial peptide , is a master regulator of systemic iron stores . hepcidin is produced mainly by the liver and its production is regulated by inflammation / iron levels , hypoxia , and erythropoiesis ; hepcidin is elevated in anemia of chronic inflammation . hepcidin can bind to ferroportin 1 ( fpn1 ) , an iron export protein found on macrophages , enterocytes , and other cell types , and in doing so causes fpn1 internalization and degradation . the vast majority of bodily iron stores is complexed with heme in hemoglobin in red cells , and senescent red cells are broken down and phagocytosed by specialized macrophages in the spleen , which are highly efficient in recycling iron from hemoglobin . nevertheless , pathogens have evolved complex mechanisms to obtain heme from blood or tissues as an alternate source of iron . while the heme synthesis pathway has been well characterized in mammals , much less we identified the feline leukemia virus subgroup c receptor ( flvcr ) , a 12-transmembrane domain protein and member of the major facilitator superfamily , as a heme exporter in mammalian cells [ 13 , 14 ] . heme exported from cells through flvcr is rapidly bound by plasma proteins including hemopexin and albumin , which can then transport the heme to other sites for utilization . although macrophages express high levels of flvcr , consistent with a role for macrophages in recycling heme / heme iron from phagocytosed senescent red cells , the role of flvcr in regulating heme - iron after infection remains unexplored . macrophages upregulate heme oxygenase-1 ( hmox1 ) , a heme - degrading enzyme , in response to inflammation or infection ; therefore we hypothesized that macrophages dynamically regulate flvcr in response to inflammation or infection . we analyzed flvcr ( also referred to as mfsd7b ) mrna levels as well as that of the other key heme and iron regulatory genes , fpn1 ( also referred to as slc40a1 ) , hmox1 , and ferritin light chain ( ftl ) in macrophages treated with lps . we found that flvcr mrna decreases quickly upon lps stimulation , similar to fpn1 , before recovering to baseline 2448 hours later . the return to baseline flvcr expression coincides with the major increase in hmox1 and ftl1 expression , suggesting an initial need for increased heme in macrophages after infection accomplished by flvcr downregulation , then at later time points , heme is degraded by hmox1 and iron is sequestered in ferritin . while macrophage sequestration of heme and iron may be one aspect of antimicrobial defense , macrophages need heme and iron for the reactive oxygen ( ros ) production and bacterial killing [ 19 , 20 ] . these observations suggest that , upon infection , macrophages initially transiently increase intracellular heme and iron in order to kill bacteria before shifting to a strategy focused on iron sequestration . the j774a.1 cell line ( henceforth referred to as j774 ) was obtained from atcc and maintained in cdmem : dmem medium ( gibco ) supplemented with 10% inactivated fbs , penicillin / streptomycin / l - glutamine ( 1 unit / ml , 1  g / ml , and 2  mm ; gibco ) , hepes ( 10  mm ; gibco ) , -mercaptoethanol ( 0.05  m ; sigma ) , and mem - non - essential amino acids ( 0.1  mm ; gibco ) . bone - marrow - derived macrophages ( bmdm ) were prepared by euthanizing 68-week - old male c57bl/6 mice and sterilely dissecting both femurs . the bone marrow was flushed from the femur with hbss , homogenized into single cell suspension by pipetting , and centrifuged at 400  g  4 minutes ( 4c ) and supernatant aspirated . the cells were then resuspended at 5  10 cells / ml in bmdm media : rpmi1640 ( gibco ) supplemented with 20% inactivated fbs , 30% l929-conditioned media ( lcm ) , penicillin / streptomycin / l - glutamine ( 1 unit / ml , 1  g / ml , 2  mm ; gibco ) , -mercaptoethanol ( 0.05  m ; sigma ) , and 10  ml plated/10  cm sterile nontissue culture - treated petri dish ( corning ) . after 4 - 5 days bmdm were harvested by trypsin / versene treatment and replated at lower density as below for stimulation assays . all mice were bred and maintained in a specific pathogen - free barrier facility at the university of washington . experiments were performed in compliance with the university of washington institutional animal care and use committee regulations . j774 or bmdm were plated at 5  10 cells / well in 12-well tissue culture plates in cdmem ( j774 ) or bmdm media and allowed to adhere overnight . the following day , the media were exchanged for fresh cdmem with varying concentrations of hemin or lps ( e. coli o111:b4 ; sigma ) for varying durations . for later experiments , including those using tlr4 ; ly96 bmdm , o111:b4 ultrapure lps from invivogen was used . pam3csk4 was obtained from emc microcollections and polyinosinic : polycytidylic acid ( pic ) from amersham . at the appropriate time points , media were aspirated from stimulation wells and macrophages lysed with rlt buffer ( qiagen ) and stored at 80c . rna was then purified from lysate using rneasy plus mini kit ( qiagen ) and reverse - transcribed using iscript reverse transcriptase ( biorad ) . multiplex quantitative real - time pcr ( qpcr ) was performed on cdna using the kapa probefast biorad icycler reaction mix ( kapa biosystems ) with gene - specific primers obtained from integrated dna technology ( idt ) . primer sequences :  -actin ( f 5-accttctacaatgagctgcg-3 , r 5-ctggatggctacgtacatgg-3 , 5-/5cy5/tctgggtcatcttttcacggttggc/3iabrqsp/-3 ; flvcr ( f 5-atctggaacctgtgcagaaaca-3 , r 5-attgaataaaatgctccagtcatgat-3 , probe 5/hex / cccctttgttctcctgctggtcagttatg / iabkfq/-3 ) ; hmox1 ( f 5-ctgctagcctggtgcaagatact-3 , r 5-gtctgggatgagctagtgctgat-3 , probe 5-/fam / agacaccccgagggaaacccca / iabkfq/-3 ) ; fpn1 ( f 5-ccaaccggaaataaaaccacag-3 ) , ( r 5-aggagaaaacaggagcagattag-3 ) , ( probe 5-/fam / ccaaccggaaataaaaccacag / iabkfq/-3 ) ; and ftl1 ( f 5-cagccatgacctctcagattc-3 ) , ( r 5-ccacgtcatcccgatcaaaa-3 ) , ( probe 5-/hex / cgcctggtcaacttgcacctg / iabkfq/-3 ) . flvcr , hmox1 , and -actin primer sets were run together , and fpn1 , ftl1 , and -actin primer sets were run together . gene expression ( mrna rq ) was quantified as fold - change expression using the pffafl method ; -actin was the reference gene and untreated ( 0  ng / ml ) cells were the reference sample . a dilution series of untreated c57bl/6 macrophage cdna was run for every assay to determine the reaction efficiency for the pfaffl calculation , to ensure that amplification was linear , and to ensure that the samples being assayed were within the linear range of the assay . polarization of bmdm to the m1 and m2 states was performed using established protocols   as follows . the following day , either ifn/lps ( 100  u / ml ; 100  ng / ml ) , il4 ( 100  u / ml ) , or nothing was added and cells were lysed at various times afterward as above for rna isolation , cdna production , and qrt - pcr . the promoter binding tf profiling plate array ( signosis ) was used to assess tf binding to the murine flvcr promoter . in brief , this 96-well plate - based competition assay utilizes an array of biotinylated oligos specific for 48 transcription factors ( in duplicate ) . nuclear extracts from the cell of interest are incubated with purified , pcr - amplified flvcr promoter . the purified promoter competes with biotinylated tf - specific oligos for binding to tfs present in the nuclear extract . if there is no competition , each tf - bound oligo can hybridize to its specific complementary dna on the 96-well plate and be detected through luminescence . tf that are present in the extract and bind to the flvcr promoter will show decreased signal in the presence of the promoter compared to no promoter . nuclear extracts were prepared from 1  10 bmdm using the nuclear extraction kit ( signosis ) , and the protein content was determined by the bradford assay . 10  g of bmdm nuclear extract was incubated with or without 15  pmol of purified pcr - amplified murine flvcr promoter , processed , and hybridized to the tf array plate following the manufacturer 's protocol . previously , we observed that macrophages expressed high levels of flvcr , consistent with a role for macrophages in recycling heme / heme iron from phagocytosed senescent red cells . to determine whether flvcr expression in macrophages is regulated by heme , j774 macrophages were exposed to increasing doses of hemin for different times and then flvcr , hmox1 , fpn1 , and ftl1 mrna levels were determined by multiplex quantitative rt - pcr ( qpcr ) . while the hmox1 , fpn1 , and ftl1 mrna showed a dose - responsive increase to hemin exposure for 10 hours , flvcr mrna levels did not change ( figure 1 ) . this led us to ask whether flvcr in macrophages might have another role aside from heme regulation after erythrophagocytosis . macrophages are key regulators of systemic iron balance that maintain organismal iron supply while sequestering iron from pathogens . given that much of the body iron store is found in heme , we next tested whether macrophages modulate flvcr expression in response to infection . fpn1 transcription is downregulated in macrophages stimulated by lps [ 24 , 25 ] ; therefore we stimulated j774 with varying concentrations of lps for different durations and quantified mrna levels of flvcr and key heme / iron regulatory genes . we found that flvcr expression decreased rapidly upon lps stimulation before recovering to baseline at 2448 hours ( figure 2(a ) ) . fpn1 increased rapidly and transiently before then decreasing over the first 24 hours ; fpn1 recovery was slower and not complete by 48 hours ( figure 2(a ) ) . ftl1 kinetics were similar to those of flvcr though the initial decrease in expression was not as marked as flvcr and at later time points ftl1 increased above baseline ( figure 2(a ) ) . the decrease in flvcr expression was dose - responsive between 0 and 100  ng with no further decrease at higher lps doses , as seen in figure 2(b ) , which shows heme and iron regulatory gene expression at 10 hours . both flvcr and hmox1 show a dose - responsive decrease and increase in mrna expression , respectively , whereas fpn1 and ftl1 had the maximal drop in expression at the lowest dose of lps , 10  ng / ml ( figure 2(b ) ) . we next assessed the effect of lps stimulation on primary murine bone - marrow - derived macrophages ( bmdm ) and again observed a dose - responsive decrease in flvcr similar to the decrease in fpn1 expression ( figure 3 ) . the kinetics of flvcr and fpn1 downregulation in response to lps were similar to that seen in j774 ( data not shown ) . thus , both primary bmdm and macrophage cell lines respond to lps signaling by downregulating heme and iron export . activation of macrophages in vitro or in vivo by various stimuli leads to distinct gene expression patterns , a process referred to as macrophage polarization . lps and ifn treatment leads to m1 macrophage polarization , suited for combating infection and acute inflammation , whereas il4 treatment leads to m2 polarization . most studies on macrophage regulation of iron balance have been done on m1 macrophages , but one study found that in contrast to m1 macrophages , m2-polarized human macrophages do not sequester iron but rather release iron to the surrounding tissues , promoting proliferation . to determine whether flvcr was differentially regulated in m1- versus m2-polarized macrophages , we generated primary murine undifferentiated macrophages ( m0 ) and then treated with either lps and ifn or il4 to generate m1- and m2-polarized macrophages . similar to previous reports , we found that hmox1 and ftl1 expression was not upregulated in m2 macrophages ( figure 4 ) . however , both m1 and m2 macrophages downregulated fpn1 mrna , and in contrast to lps treatment alone , fpn1 mrna remained suppressed at 48 hours . interestingly , flvcr expression decreased later and to a much lower extent in m2 versus m1 macrophages . thus , m2 macrophages maintain flvcr expression , possibly to export heme to cells in regenerating tissues . lps binds to the tlr4 receptor complex on macrophages to activate multiple downstream signaling pathways . to confirm that lps - induced flvcr downregulation was mediated by the tlr4 pathway , we stimulated bmdm from tlr4 ; ly96 mice or controls with lps or lps / ifn. ly96 encodes md2 , a coreceptor with tlr4 required for lps signaling . loss of tlr4 and md2 completely reversed lps - induced downregulation of flvcr and fpn1 ( figure 5 ) . to demonstrate specificity , we stimulated wild - type and tlr4 ; ly96 bmdm with the tlr1/2 agonist pam3csk4 ( pam3 ) and tlr3 agonist polyinosinic : polycytidylic acid ( pic ) . we found that both pam3 and pic treatment led to flvcr and fpn1 downregulation in both wild - type and tlr4 ; ly96 bmdm ( figure 5 ) . another group recently showed that tlr2 and tlr6 agonists cause fpn1 mrna downregulation in macrophages . thus , decreased transcription of the genes encoding both heme and iron export proteins is a common pathway following tlr activation in macrophages . interestingly , hmox1 was upregulated by lps and pam3 , but not by pic ( figure 5 ) , suggesting distinct regulation of flvcr as compared to other heme regulatory genes . to explore the connection between lps stimulation and flvcr mrna transcription , we used the epdnew eukaryotic promoter database   to identify the human flvcr promoter sequence and then queried the promoter sequence for transcription factor ( tf ) binding sites using promo [ 30 , 31 ] . this analysis revealed many potential binding sites motifs for transcription factors known to be expressed in macrophages such as nf-b , irf-1 , and c / ebp   ( see supplemental figure 1 of the supplementary material available online at http://dx.doi.org/10.1155/2016/4039038 ) . we next used a multiplex tf binding assay to identify tf present in murine bmdm that can bind to the murine flvcr promoter . several of the tf with highest in vitro flvcr promoter binding activity ( figure 6 ) such as stat4 , ap2 , sp-1 , and irf-1 had predicted binding sites with the human flvcr promoter ( supplemental figure 1 ) . moreover , stat4   and irf-1   are known downstream mediators of lps / tlr4 signaling in macrophages . in this study , we found that primary and immortalized murine macrophages downregulate flvcr mrna levels upon lps stimulation , similar to the downregulation of fpn1 expression previously described [ 24 , 25 ] . macrophages also downregulated flvcr and fpn1 in response to tlr1/2 and tlr3 agonists , suggesting that heme and iron sequestration in macrophages is a general response to inflammatory / infectious stimuli . it was previously reported that m1 polarization causes human macrophages to sequester iron through fpn1 downregulation while m2 polarization leads to increased fpn1 protein expression . a more recent study   found that fpn1 expression was decreased in murine m0 macrophages polarized to both the m1 and m2 states . interestingly , this study also found that heme ( in the form of rbc or free heme ) polarizes m0 macrophages to the m1 state . we found that , in murine macrophages , both m1 and m2 polarization caused decreased expression of fpn1 and flvcr , though notably the decrease in flvcr expression occurred later and was less marked under m2 polarizing conditions . the hypothesis is that macrophages sequester iron in response to infection ( m1 ) and export iron and heme under m2 conditions in which tissue regeneration and proliferating cells have higher demand for heme and iron . future studies could explore flvcr and fpn1 expression in vivo under physiologic conditions of m1 ( acute infection ) and m2 polarization ( late - stage wound healing ) or investigate how tumor - associated macrophages regulate heme and iron . surprisingly , flvcr mrna levels in j774 macrophages did not change significantly in response to heme treatment . while free heme may not alter flvcr mrna expression in macrophages , it is possible that flvcr expression may change in response to macrophage erythrophagocytosis , especially in splenic macrophages specialized to take up senescent rbc . heme has been shown to regulate fpn1 transcription through binding the transcriptional factors btb and cnc homology 1 ( bach1 ) and nuclear factor erythroid 2-like ( nrf2 ) , which associate with a conserved maf recognition element ( mare)/antioxidant response element ( are ) in the fpn1 promoter . thus , while hmox1 and fpn1 in macrophages may respond to changes in intracellular heme and iron levels in addition to inflammatory / infectious signals , the primary role of flvcr in macrophages may be the regulation of heme in response to infection / inflammation . it is notable that the decrease in flvcr expression occurs rapidly within 10 hours after lps stimulation , while fpn1 expression had an initially small increase followed by a slower decrease in expression ( figures 2(a ) and 4 ) . whether this transient fpn1 increase prior to the subsequent downregulation is functionally important is not known . it is not known whether flvcr is also posttranslationally regulated , though one study using transfected cell lines found that flvcr had a long half - life ( > 16 hours ) . it has been difficult to study the localization and regulation of flvcr protein in vivo or from ex vivo murine cells because there is no antibody available . once alternative strategies ( such as genetic knock - in of epitope tag sequences ) make in vivo and ex vivo of murine flvcr detection and localization feasible , it will be important to study the kinetics and trafficking of flvcr and fpn1 protein after lps treatment . the decrease in flvcr expression precedes the major increase in hmox1 expression , at which point flvcr expression has returned to baseline . this suggests that the decrease in flvcr is functionally important in the first hours after infection , and as other mechanisms for systemic iron regulation ( hepcidin - mediated fpn1 degradation , increased ferritin expression ) are initiated , flvcr returns to baseline . one explanation is that early flvcr downregulation might be important for macrophage killing of intracellular pathogens . heme and iron - containing enzymes produce the reactive oxygen species and other compounds needed for intracellular killing of bacteria . macrophages that have just encountered and/or endocytosed bacteria may downregulate flvcr in order to increase heme available for cytolytic enzymes . a recent review highlighted the  macrophage paradox ,  that is the finding that many pathogens preferentially replicate inside macrophages in spite of their specialized killing function , and different bacteria have different intracellular niches within macrophages . given that heme and iron are also trafficked and regulated differently in intracellular compartments [ 12 , 3840 ] , there are likely several layers of regulation of heme and iron regulatory proteins at transcriptional , posttranscriptional , translational , and posttranslational ( including trafficking ) levels required to meet the challenges presented by specific pathogens . this is supported by the recent finding that the survival of listeria monocytogenes localized to different intracellular compartments in macrophages was differentially altered by fpn1 expression . moreover , macrophages are not a uniform population , but rather there are several subtypes and macrophage differentiation states which serve specific functions in different tissues and depending on the conditions . macrophages that are specialized for erythrophagocytosis may regulate heme and iron regulatory proteins in response to heme and iron levels   rather than inflammatory signals as we observed here . our finding that macrophages dynamically regulate flvcr expression in response to tlr signaling points to a new potential role for flvcr and heme export in macrophages during infection and inflammation . future studies aimed at elucidating the transcriptional and posttranscriptional regulation of flvcr in response to tlr and inflammatory signaling will improve our understanding of the complex interplay cell and tissue - localized demands for heme and iron and systemic heme / iron homeostasis .
pathogenic bacteria have evolved multiple mechanisms to capture iron or iron - containing heme from host tissues or blood . in response , 
 organisms have developed defense mechanisms to keep iron from pathogens . 
 very little of the body 's iron store is available as free heme ; rather nearly all body iron is complexed with heme or other proteins . 
 the feline leukemia virus , subgroup c ( felv - c ) receptor , flvcr , exports heme from cells . 
 it was unknown whether flvcr regulates heme - iron availability after infection , but given that other heme regulatory proteins are upregulated in macrophages in response to bacterial infection , we hypothesized that macrophages dynamically regulate flvcr . 
 we stimulated murine primary macrophages or macrophage cell lines with lps and found that flvcr is rapidly downregulated in a tlr4/md2-dependent manner ; tlr1/2 and tlr3 stimulation also decreased flvcr expression . 
 we identified several candidate tlr - activated transcription factors that can bind to the flvcr promoter . 
 macrophages must balance the need to sequester iron from systemic circulating or intracellular pathogens with the macrophage requirement for heme and iron to produce reactive oxygen species . 
 our findings underscore the complexity of this regulation and point to a new role for flvcr and heme export in macrophages responses to infection and inflammation .