iron is an essential nutrient required for numerous cell functions and in order to avoid metal toxicity iron metabolism is tightly regulated . altered iron homeostasis and dysregulated cellular iron metabolism are associated with many chronic diseases including diabetes mellitus , cardiovascular diseases , arthritis , and neurodegenerative diseases . altered iron homeostasis in chronic inflammation is characterized by misdistribution of iron that is manifested in hypoferremia , commonly called anemia of chronic inflammation , and iron retention in macrophages and the reticuloendothelial system [ 2 , 3 ] . macrophages play a central role in iron homeostasis by uptake of damaged and senescent red blood cells and recycle large amounts of iron daily [ 4 , 5 ] . macrophages also play a very important role in host defense by detecting invading pathogens and responding in order to clear infection . since iron is essential for bacterial growth , macrophages retain iron to limit its extracellular bioavailability which thereby limits bacterial growth . hepcidin binds to ferroportin , the only known iron exporter , leading to its internalization and degradation and consequent iron retention . therefore , cellular iron homeostasis and iron egress via the hepcidin - ferroportin axis are very tightly regulated during infection and inflammation [ 811 ] . iron is a highly reactive metal ; thus , to reduce metal toxicity , iron is maintained bound to hemoproteins and in iron - sulfur clusters or safely stored in ferritin cages . free intracellular iron or poorly liganded iron is toxic since it catalyzes the production of reactive oxygen species ( ros ) . recently , the presence of a mammalian siderophore , an iron - chelating small molecule , was reported and the iron - binding moiety of this molecule was identified as 2,5-dihydroxybenzoic acid ( 2,5dhba ) . this mammalian siderophore captures free cytosolic iron and thus protects the cells from oxidant stress . the enzyme that catalyzes the synthesis of 2,5dhba is a member of the short - chain dehydrogenase / reductase family ( dhrs6 )   called 3-hydroxybutyrate dehydrogenase type 2 ( bdh2 ) . inhibition of bdh2 expression in murine pro - b lymphocytic cells led to the depletion of the mammalian siderophore and the accumulation of labile iron . the physiological role of bdh2 in sensing cellular iron was further confirmed by the discovery that the bdh2 enzyme contains an iron responsive element that interacts with iron regulatory proteins that control posttranscriptional gene expression . recently , zughaier et al . reported that bdh2 expression in macrophages is suppressed upon bacterial infection suggesting an important role for bdh2 in iron - limiting innate immunity . more recently , the devireddy group further confirmed the role of bdh2 in cellular iron homeostasis and showed that depletion of the endogenous siderophore 2,5-dhba in a mouse model resulted in microcytic anemia and iron overload in the spleen [ 16 , 17 ] . they also reported that bdh2 and ferritin - h synergistically regulate intracellular iron levels in an in vitro experimental cell culture model . however , the role of bdh2 in iron - limiting innate immune defenses in macrophages is not known . macrophages engulf senescent rbcs and recycle hemoglobin ; thus , they express heme oxygenase-1 ( ho-1 ) , the catalytic enzyme that degrades heme , consequently liberating iron and producing carbon monoxide . is highly induced in response to inflammation , oxidative stress , and hypoxia [ 19 , 20 ] due to its cytoprotective effects and anti - inflammatory activity [ 21 , 22 ] . therefore , ho-1 , as a stress - inducible protein , is an important player in intracellular iron homeostasis . further , neutrophil gelatinase - associated lipocalin ( ngal )   functions as an iron carrier protein that shuttles bound iron across tissues by internalization with a membrane - localized receptor [ 24 , 25 ] and also plays a critical physiological role in intracellular iron homeostasis by trafficking the iron - bound siderophore 2,5dhba   and iron - catecholamine complexes . ngal plays a crucial role in iron - limiting innate immune defenses since it limits iron availability by also scavenging bacterial siderophores and thereby exerts an antibacterial function [ 2729 ] . intracellular iron homeostasis is disrupted by endoplasmic reticulum ( er ) stress . er stress induced upon cellular perturbations is regulated by activation of the unfolded protein response to restore homeostatic cellular functions . control of er stress is required for cell survival and uncontrolled er stress leads to cell death [ 31 , 32 ] . er stress alters cellular iron homeostasis by inducing hepcidin expression [ 33 , 34 ] . altered iron homeostasis and er stress are associated with chronic neurodegenerative diseases such as huntington 's disease and alzheimer 's disease [ 35 , 36 ] . the goal of this study was to investigate whether er stress and inflammation modulate bdh2 expression in macrophages . here , we show that lps - induced inflammation combined with chemically induced er stress led to bdh2 downregulation , increased the expression of er stress markers including chop , upregulated hepcidin expression , downregulated ferroportin expression , caused iron retention in macrophages , and dysregulated cytokine release from macrophages . further , we show that er stress combined with inflammation dramatically upregulated ngal expression as well as the inducible heme oxygenase-1 enzyme in macrophages . this is the first report to show that er stress and inflammation downregulate the expression of bdh2 in human thp-1 macrophages . the results suggest that bdh2 plays an important role in modulating iron - limiting innate immune defenses . rpmi 1640 medium , dulbecco 's modified eagle medium ( d - mem ) , hbss , fetal bovine serum ( fbs ) , penicillin / streptomycin , sodium pyruvate , and nonessential amino acids were obtained from cellgro mediatech ( herdon , va ) . magnetic beads for a panel of human cytokines and chemokines were from invitrogen ( carlsbad , ca ) . tunicamycin , calcein - am , and thiazolyl blue tetrazolium bromide were purchased from sigma aldrich ( st . louis , mo ) . thp-1 macrophage - like cells were grown in rpmi 1640 medium supplemented with 10% fbs , 50  g / ml penicillin , and 50  iu / ml of streptomycin . hek293 cells were grown in d - mem supplemented with 10% fbs , 50  g / ml penicillin , and 50  iu / ml of streptomycin . freshly grown cells were harvested and adjusted to 1 million cells / ml and then transferred into 12-well tissue culture plates at 2  ml / well . to induce er stress cells were treated with tunicamycin ( 10  g / ml in dmso ) and incubated overnight at 37c . control cells were treated with dmso only and incubated similarly . following overnight exposure to tunicamycin , cells were exposed to lps ( 40  ng / ml ) to induce inflammation and incubated for 6  hr at 37c . harvested cells were washed with pbs and then placed in rlt buffer ( qiagen ; hilden , germany ) containing 1% -mercaptoethanol , passed over qiashredder columns , and the resulting lysates were saved at 80c for mrna extraction . rna was isolated using rneasy mini kits ( qiagen ) following the manufacturer 's instructions as previously described . briefly , cell lysates saved in rlt buffer were mixed in 70% ethanol and then passed over rneasy columns . columns were washed and treated with 10  l of rnase - free dnase ( qiagen ) for 15  min at room temperature prior to rna extraction , followed by additional washing and centrifugation . rna was eluted in 35  l of rnase - free water and then was reverse transcribed to cdna using quantitect reverse transcription kit ( qiagen ) following the manufacturer 's instructions . relative gene expression was determined by quantitative rt - pcr performed on resulting cdna using sybr green ( promega ; madison , wi ) following the manufacturer 's instructions . the mrna level was calculated in reference to -actin and fold change gene expression was calculated in reference to controls using the ct method . results were normalized to unstimulated dmso - treated cells which were used as controls for basal gene expression level . the following primers were used for qrt - pcr reactions : human hepcidin 5-gaccagtggctctgttttcc-3 and 5-cacatcccacactttgatcg-3 ; human bdh2 5-cagcgtcaaaggagttgtga-3 and 5-tggcgtatcaactgttcctg-3 ; human ngal 5-atgacatgaacctgctcgata-3 and 5-tcatagtcgttcattatcttc-3 ; human -actin 5-tcttccagccttccttcct-3 and 5-agcactgtgttggcgtacag-3. primers for er stress markers used in this study are as follows : human hsxbp-1 5-ttccggagctgggtatctca-3 and 5-gaacccccgtatccacagtc-3 ; human grp78 5-acggcagctgctattgctta-3 and 5-tcctgacatctttgcccgtc-3 ; human chop 5-gacctgcaagaggtcctgtc-3 and 5-gcagggtcaagagtggtgaa-3. ferroportin ( hs_slc40a1_1_sg ) , transferrin receptor ( hs_tfrc_1_sg ) , and heme oxygenase ( hs_hmox1_1_sg ) quantitect primers were purchased from qiagen . freshly grown thp-1 cells were harvested and adjusted to 1 million cells / ml and 8  ml of cells was transferred into 6-well tissue culture plates . to induce er stress , cells were treated with tunicamycin ( 10  g / ml ) and incubated overnight at 37c . some cells were exposed to lps ( 40  ng / ml ) to induce inflammation and incubated for 6  hr at 37c . harvested thp-1 cells were lysed in ripa buffer ( boston bioproducts ; boston , ma ) and centrifuged to remove nuclei and debris , and the protein concentration of the supernatant was measured using a bca protein assay ( thermo ; waltham , ma ) . equal amounts of protein were removed from each macrophage treatment group , mixed with 6x laemlli buffer , and boiled for 15 minutes . for untransfected hek lysate and the bdh2 transfected hek positive control groups ( origene technologies ; rockville , md ) , 50  ng of protein was loaded to adjust for overexpression . samples were loaded onto a 12% miniprotean tgx gel ( biorad ; hercules , ca ) , electrophoresed , and transferred onto nitrocellulose membranes ( biorad ; hercules , ca ) . to detect bdh2 , membranes were incubated with anti - bdh2 antibody ( truemab antibody clone 2g1 from origene ) . to detect actin , membranes were incubated with a mouse anti- actin antibody ( sigma ) . in both cases , membranes were incubated with an hrp - conjugated anti - mouse secondary antibody ( cell signaling ; danvers , ma ) , incubated in amersham western blot detection reagent ( ge healthcare ; cleveland , oh ) , and visualized with hyblot cl autoradiography film ( denville scientific ; metuchen , nj ) . cytokines and chemokines released from thp-1 macrophages treated with tunicamycin alone , lps alone , or both tunicamycin and lps as described above were measured using luminex magnetic beads ( invitrogen ) following the manufacturer 's instructions . intracellular accumulation of labile iron in macrophages was measured using the well - established calcein - am method . freshly grown thp-1 cells were treated with tunicamycin ( 10  g / ml ) and incubated overnight prior to stimulation with lps ( 40  ng / ml ) for 6  hr . control cells were treated with dmso only . harvested cells were resuspended in rpmi 1640 medium supplemented with 10% fbs , and 0.5  m calcein - am was added and incubated for 15  min at 37c . calcein - loaded cells were washed twice with pbs to remove extracellular calcein - am and resuspended in hbss . 100  l aliquots , containing 0.25 million cells , were transferred into quadruplicate wells in black 96-well plates . after a 20  min incubation at 37c , fluorescence was determined at 485  nm ( excitation ) and 535  nm ( emission ) wavelengths using a bio - tek synergy 2 instrument ( winooski ; vt ) . the tetrazolium salt dye is cleaved by live and metabolically active cells but not by dead cells ; therefore , the mtt reduction assay was used to measure the viability of thp-1 cells treated with tunicamycin with or without lps [ 41 , 42 ] . briefly , freshly grown thp-1 cells adjusted to 1 million cells / ml were treated with tunicamycin doses ranging from 0.5 to 10  g / ml and incubated overnight followed in some cases by 6  hr of exposure to lps . five mg of thiazolyl blue tetrazolium bromide was dissolved in 1  ml of pbs and 15  l was added to 150  l of treated thp-1 cells which were further incubated at 37c for one hour . cells were centrifuged to remove extracellular dye and then lysed in 150  l of dmso to dissolve the intracellular dye . the absorbance of reduced dye was measured at 591  nm wavelength using a bio - tek spectrophotometer . mean values  sd of three independent experiments , or two in few cases , were calculated with microsoft excel . statistical analysis was performed with prism v5.0 software using one - way anova followed by bonferroni multiple comparisons post hoc analysis . recently , bdh2 was reported to play a crucial role in intracellular iron trafficking by catalyzing the synthesis of 2,5-dhba . however , bdh2 expression and regulation in macrophages during er stress and inflammation have not yet been described . to investigate bdh2 expression in macrophages and its regulation by er stress and inflammation , we employed human macrophage - like monocytic cells ( thp-1 ) treated with tunicamycin overnight prior to lps exposure for 6  hr to induce er stress and inflammation , respectively . here we show that bdh2 protein and gene expression in macrophages was downregulated by er stress and inflammation ( figure 1 ) . reduced bdh2 protein levels were observed in thp-1 cells after treatment with lps and tunicamycin as compared to cells treated with dmso alone ( figure 1(a ) ) . consistent with this observation , bdh2 gene expression was significantly downregulated when thp-1 macrophages were treated with tunicamycin and lps either alone or in combination ( figure 1(b ) ) . treatment with tunicamycin alone reduced bdh2 mrna expression in a dose - dependent manner ( figure 1(c ) ) . further , we examined the activation of er stress markers and as expected overnight tunicamycin treatment significantly induced er stress markers chop , hsxbp-1 , grp78 ( figure 2 ) , and atf6 ( data not shown ) . in contrast , 6  hr lps treatment alone did not upregulate er stress markers in thp-1 cells ( figure 2 ) , as documented previously . cellular viability was assessed using the mtt reduction assay   which showed that the doses of tunicamycin and lps used here did not reduce cellular viability ( see supplementary figure 1a in supplementary material available online at http://dx.doi.org/10.1155/2014/140728 ) . er stress controls iron homeostasis via the induction of hepcidin , the master iron regulating hormone . hepcidin binds to the only known cellular iron exporter , ferroportin , and leads to its internalization and degradation , consequently inhibiting iron egress from macrophages . the dysregulation of the hepcidin - ferroportin axis leads to iron retention in macrophages and increases the cytosolic labile iron pool which is highly reactive and causes oxidative stress . therefore , detoxifying labile iron is essential to maintaining cellular iron homeostasis . to investigate intracellular iron status in tunicamycin and lps treated thp-1 macrophages we determined the expression of hepcidin and ferroportin . the data show that tunicamycin - induced er stress as well as lps - induced inflammation led to significant upregulation of hepcidin gene expression in thp-1 macrophages ( figure 3(a ) ) . in contrast , tunicamycin and lps treatment led to a dramatic reduction in ferroportin expression in macrophages ( figure 3(b ) ) . ferroportin allows iron egress from macrophages ; therefore , its degradation leads to iron retention . we measured labile iron accumulation in macrophages using calcein - am   and found that tunicamycin and lps treatment led to significant iron retention ( figure 4 and supplementary figure 1b ) . a synergistic effect on iron retention the results suggest that chemically induced er stress and inflammation alter intracellular iron homeostasis in macrophages . er stress is also reported to highly induce ngal expression . however , the impact of er stress combined with inflammation on ngal expression is not yet known . to this end , we determined ngal gene expression in tunicamycin and lps treated macrophages . as expected , lps treatment led to significant upregulation of ngal gene expression ( ~12-fold ) but tunicamycin treatment led to a dramatic ~1500-fold increase in ngal gene expression ( figure 5(a ) ) . tunicamycin induced ngal gene expression in a dose - dependent manner ( figure 5(b ) ) . a synergistic effect was observed when thp1 cells were cotreated with tunicamycin and lps which resulted in an approximately ~3000-fold increase in ngal gene expression ( figure 5(a ) ) . tunicamycin - induced er stress led to ~100-fold greater increase in ngal gene expression compared to lps . the synergistic cross - talk between tunicamycin - induced er stress and lps - induced inflammation led to ~200-fold increase in ngal gene expression compared to lps alone . these data suggest that chemically induced er stress dramatically increases ngal expression more than inflammation but when combined a synergistic upregulation of ngal is observed . ho-1 is a stress - induced anti - inflammatory [ 19 , 21 , 22 ] cytoprotective enzyme that catalyzes the degradation of heme and consequently liberates iron and carbon monoxide . the released carbon monoxide suppresses hepcidin expression ; therefore , ho-1 plays a role in regulating iron homeostasis . therefore , we investigated the impact of er stress and inflammation on ho-1 expression in macrophages as an anti - inflammatory immune response . here we show that ho-1 gene expression is significantly upregulated in thp-1 cells treated with tunicamycin or lps alone ( figure 6 ) . however , the combination of tunicamycin and lps treatment synergistically upregulated ho-1 expression in thp-1 cells ( figure 6 ) suggesting a potent response to combat cellular stress . in this report we show that tunicamycin - induced er stress and lps - induced inflammation downregulate bdh2 expression in macrophages and alter intracellular iron homeostasis . vitamin d possesses immune modulatory activity and exerts an anti - inflammatory effect [ 4749 ] . found that vitamin d plays an important role in regulating the iron - hepcidin - ferroportin axis in monocytes by restoring hepcidin and ferroportin gene expression and reducing il-6 and il-1 cytokine release . vitamin d was also reported to suppress er stress in macrophages and therefore is a natural er stress reliever [ 51 , 52 ] . we hypothesized that vitamin d may restore bdh2 expression in lps stimulated macrophages . to test our hypothesis , thp-1 macrophages were treated with 20  nm of 1,25 dihydroxy vitamin d3 ( 1,25(oh)2d3 ) , the hormonally active form of vitamin d3 , for 24  hr prior to lps exposure . here we report for the first time that vitamin d3 pretreatment protects macrophages from the lps - induced reduction in bdh2 gene expression ( figure 7 ) . treatment with vitamin d3 alone did not significantly induce bdh2 expression but lps treatment led to significant downregulation of bdh2 expression which was absent in cells pretreated with vitamin d3 prior to lps . taken together , the data suggest that downregulation of bdh2 gene expression by lps , which is a common model of inflammation , can be restored by vitamin d3 . upon exposure to lps , macrophages respond by releasing large quantities of inflammatory mediators , an innate immune response known as the cytokine storm . macrophages downregulate de novo protein synthesis as an adaptation process to restore er homeostasis , which results in reduced secretion of some inflammatory mediators [ 53 , 54 ] . in this report therefore , we examined the impact of er stress combined with inflammation on release of inflammatory mediators from macrophages . using luminex technology , we evaluated cytokine and chemokine release from cells treated with tunicamycin ( 10  g / ml ) to induce er stress prior to lps ( 40  ng / ml ) exposure for 6  hr to induce the cytokine storm . as expected , lps exposure without tunicamycin treatment induced massive release of cytokines ( il-1 , tnf , il-6 , il-12p40 , ifn , il-1r antagonist , and il-2r ) and chemokines ( rantes , mcp-1 , mip1 , mip1 , ip-10/cxcl10 , and il-8 ) compared to unstimulated thp-1 macrophages ( table 1 ) . tunicamycin treatment alone ( without lps exposure ) also induced cytokine / chemokine release which was typically ~2- to 5-fold less than lps treatment ( figures 8(a ) , 8(b ) , 8(c ) , and 8(d ) and table 1 ) . since er stress downregulates protein synthesis machinery , tunicamycin treatment followed by lps exposure led to significant reduction in cytokines ( il-12p40 , il-1r antagonist ) and chemokines ( mip1 , mip1 ) compared to lps exposure alone . a slight reduction was observed in other cytokines and chemokines released from macrophages exposed to both tunicamycin and lps ( table 1 ) . in contrast , a significant and synergistic increase in il-6 release was observed when thp-1 macrophages were exposed to both tunicamycin and lps . il-6 is a proinflammatory cytokine that plays a significant role in systemic iron metabolism by directly inducing the expression of hepcidin , the master iron - regulating hormone . therefore , tunicamycin - induced er stress combined with inflammation upregulates il-6 , consequently contributing to altered iron homeostasis . limiting the bioavailability of iron during infection is a host defense mechanism driven by the hepcidin - ferroportin axis [ 2 , 7 , 15 ] that leads to iron retention in macrophages . in this report we describe the downregulation of bdh2 in macrophages in response to er stress and inflammation . we show that chemically induced er stress combined with lps - induced inflammation downregulates bdh2 expression in human thp-1 macrophages . bdh2 catalyzes the synthesis of the mammalian siderophore 2,5dhba that protects from oxidant stress by capturing free toxic iron . cells lacking a functional siderophore retain high levels of free iron leading to increased ros production and iron - deficient mitochondria . more recently , liu and coworkers also reported that bdh2 contains an iron - responsive element and thereby is regulated by intracellular iron levels . reported the downregulation of bdh2 gene expression in macrophages in response to bacterial infection . here we report a synergistic effect of er stress combined with inflammation on the downregulation of bdh2 gene and protein expression in macrophages which led to dysregulated cytokine release and iron retention . our finding is novel and sheds light on the role of bdh2 in iron - limiting innate immunity . recent evidence showed that iron retention dictates macrophage polarization [ 5 , 57 ] and induces unrestrained proinflammatory macrophages that impair wound healing . hepcidin is directly induced by lps via tlr4 activation   and during inflammation hepcidin is induced by il-6 . our data show that chemically induced er stress and lps - induced inflammation dysregulated the hepcidin - ferroportin axis and led to iron retention in thp-1 macrophages and consequently downregulated bdh2 expression . devireddy and coworkers showed that downregulating bdh2 expression using sirna resulted in the depletion of the mammalian siderophore which caused iron retention in a murine pro - b lymphocytic cell line , reduced mitochondrial iron , and consequently reduced heme synthesis . bdh2 is a homolog of the bacterial enzyme enta that catalyzes the synthesis of the bacterial siderophore , enterobactin . ngal sequesters enterobactin thereby limiting bacterial growth and traffics iron - bound complexes across tissues . thus , in addition to their physiological roles in iron homeostasis , ngal and bdh2 constitute the iron - limiting arm of innate immune defense . further , ngal is highly induced as an antioxidant response during oxidative stress   and in response to er stress . our data demonstrate a synergistic effect of er stress and inflammation on the dramatic upregulation of ngal . elevated levels of ngal are associated with many chronic diseases like kidney disease , heart disease , arthritis , chronic obstructive pulmonary disease , and cancer [ 6264 ] . high levels of ngal were shown to deactivate macrophages and worsen outcomes of pneumococcal pneumonia . we recently reported that circulating ngal levels are elevated in subjects with cystic fibrosis compared to healthy donors and demonstrated that peripheral monocytes secrete ngal in response to lps and to pseudomonas aeruginosa infection . moreover , ngal expression is upregulated whereas bdh2 expression is downregulated in macrophages upon bacterial infection with neisseria gonorrhoeae . taken together , er stress combined with inflammation downregulates bdh2 expression consequent to the dysregulation of hepcidin - ferroportin axis leading to iron retention in macrophages and dramatic ngal upregulation which contribute to altered intracellular iron homeostasis ( figure 9 ) . er stress has been shown to induce chronic inflammation in metabolic and chronic heart diseases [ 66 , 67 ] . tunicamycin , which inhibits protein glycosylation and triggers the unfolded protein response , combined with lps synergistically increased il-6 release from macrophages . previous study showed that treatment with anti - il-6 antibody lowered hepcidin levels which alleviated hypoferremia and restored iron homeostasis [ 2 , 69 ] . among many inflammatory mediators il-6 is a predictor of mortality in chronic kidney disease , in hiv , and in other chronic diseases associated with immune activation due to microbial translocation [ 72 , 73 ] . our results suggest that er stress combined with inflammation increases il-6 that induces hepcidin expression leading to increased intracellular iron which downregulates bdh2 expression . further , ngal possesses an immunomodulatory activity and exerts a suppressive effect on macrophages leading to reduced cytokine release [ 65 , 74 ] . we observed a synergistic effect of er stress combined with inflammation on the dramatic upregulation of ngal expression which may contribute to dysregulated cytokine and chemokine response in macrophages . in this report we describe a mechanism by which er stress combined with inflammation contributes to dysfunctional iron - limiting innate immune responses in macrophages . vitamin d possesses immunomodulatory activities and lowers circulating cytokines in vivo [ 47 , 48 , 75 ] . we reported that the hormonally active form of vitamin d , 1,25 dihydroxyvitamin d3 , reduced mcp-1 release from macrophages treated with lps or infected with pseudomonas aeruginosa bacteria . more recently , zughaier et al . showed that 1,25 dihydroxyvitamin d3 treatment reduced il-6 release from lps activated macrophages and reduced hepcidin gene expression while upregulating ferroportin gene expression in a dose - dependent manner ; thereby , vitamin d restored the hepcidin - ferroportin axis in macrophages . taken together , our data show that er stress and inflammation downregulate bdh2 expression and vitamin d treatment restored bdh2 expression in lps activated macrophages . it is not known whether vitamin d exerts direct or indirect regulatory effects on bdh2 gene expression but it is worthy of further investigation . the important role of bdh2 in intracellular homeostasis relies on the production of the mammalian siderophore 2,5-dhba that binds free iron and thereby restores cellular iron homeostasis . we propose that the synthetic iron chelating drug deferiprone ( dfp : 3-hydroxy-1,2-dimethylpyridin-4(1h)-one ) [ 76 , 77 ] , commonly used to treat iron overload disorders , may emulate the mammalian siderophore and redistribute iron retained in macrophages . dfp is a small molecule that diffuses across the cellular membrane and scavenges free intracellular iron . this dfp - iron complex shares structural resemblance to enterobactin , the major bacterial siderophore that binds iron with very high affinity ( figure 10 ) . in response to infection and as a defense mechanism , the host upregulates ngal expression to scavenge enterobactin which in turn limits iron availability and inhibits bacterial growth . our data show that ngal is dramatically upregulated in response to er stress and inflammation . we hypothesize that , upon downregulation of bdh2 expression in macrophages , dfp may compensate for the reduction in mammalian siderophore production . dfp gains access to cells and scavenges free intracellular iron and then exits as a dfp - iron complex . based on the structural resemblance to enterobactin - iron complexes , the dfp - iron complex is expected to bind ngal by fitting into the hydrophobic pocket   ( figure 10 ) . therefore , dfp could be a potential therapeutic for altered cellular iron homeostasis that lowers both intracellular iron and abnormal ngal levels , consequently restoring intracellular iron homeostasis in macrophages . inflammation combined with er stress led to massive bdh2 downregulation and increased the expression of er stress markers , upregulated hepcidin expression , downregulated ferroportin expression , caused iron retention in macrophages , and dysregulated cytokine release from macrophages . er stress combined with inflammation synergistically upregulated the expression of the iron carrier protein ngal and the stress - inducible heme degrading enzyme heme oxygenase-1 ( ho-1 ) leading to iron liberation . bdh2 is expressed in many types of cells ; therefore , downregulation of bdh2 may have detrimental effects on cellular functions . downregulation of bdh2 during infection is due to activation of hepcidin and intracellular iron retention , indicating an important role for bdh2 in iron - limiting innate immunity and host defense . our data suggest that continued er stress combined with inflammation impacts on bdh2 expression and contributes to altered intracellular iron homeostasis .
macrophages play a very important role in host defense and in iron homeostasis by engulfing senescent red blood cells and recycling iron . 
 hepcidin is the master iron regulating hormone that limits dietary iron absorption from the gut and limits iron egress from macrophages . upon infection macrophages 
 retain iron to limit its bioavailability which limits bacterial growth . 
 recently , a short chain butyrate dehydrogenase type 2 ( bdh2 ) protein was reported to contain an iron responsive element and to mediate cellular iron trafficking by catalyzing the synthesis of the mammalian siderophore that binds labile iron ; therefore , bdh2 plays a crucial role in intracellular iron homeostasis . 
 however , bdh2 expression and regulation in macrophages have not yet been described . here 
 we show that lps - induced inflammation combined with er stress led to massive bdh2 downregulation , increased the expression of er stress markers , upregulated hepcidin expression , downregulated ferroportin expression , caused iron retention in macrophages , and dysregulated cytokine release from macrophages . 
 we also show that er stress combined with inflammation synergistically upregulated the expression of the iron carrier protein ngal and the stress - inducible heme degrading enzyme heme oxygenase-1 ( ho-1 ) leading to iron liberation . 
 this is the first report to show that inflammation and er stress downregulate the expression of bdh2 in human thp-1 macrophages .