Zinc deficiency induces production of the proinflammatory cytokines IL-1β and TNFα in promyeloid cells via epigenetic and redox-dependent mechanisms

Zinc deficiency induces production of the proinflammatory cytokines IL-1β and TNFα in promyeloid cells via epigenetic and redox-dependent mechanisms

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  Zinc deficiency induces production of the proinflammatory cytokines IL-1 β  and TNF α in promyeloid cells via epigenetic and redox-dependent mechanisms Inga Wessels, Hajo Haase, Gabriela Engelhardt, Lothar Rink, Peter Uciechowski ⁎ Institute of Immunology, Medical Faculty, RWTH Aachen University, Pauwelsstr. 30, D-52074 Aachen, Germany Received 13 December 2011; received in revised form 6 June 2012; accepted 6 June 2012  Abstract The deprivation of zinc, caused by malnutrition or as a consequence of aging or disease, strongly affects immune cell functions, causing higher frequency of infections. Among other effects, an increased production of reactive oxygen species (ROS) and proinflammatory cytokines has been observed in zinc-deficientpatients, but the underlying mechanisms were unknown. The aim of the current study was to define mechanisms explaining the increase in proinflammatorycytokine production during zinc deficiency, focusing on the role of epigenetic and redox-mediated mechanisms.Interleukin (IL)-1 β  and tumor necrosis factor (TNF) α  production was increased in HL-60 cells under zinc deficiency. Analyses of the chromatin structuredemonstrated that the elevated cytokine production was due to increased accessibilities of IL-1 β  and TNF α  promoters in zinc-deficient cells. Moreover, the levelof nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) oxidase-produced ROS was elevated under zinc deficiency, subsequently leading to p38mitogen-activated protein kinase (MAPK) phosphorylation. The increased activation of p38 MAPK appeared to be necessary for posttranscriptional processes inIL-1 β  and TNF α  synthesis.These data demonstrate that IL-1 β  and TNF α  expression under zinc deficiency is regulated via epigenetic and redox-mediated mechanisms. Assuming animportant role of zinc in proinflammatory cytokine regulation, this should encourage research in the use of zinc supplementation for treatment of inflammatory diseases.© 2013 Elsevier Inc. All rights reserved. Keywords:  Zinc deficiency; Epigenetics; Gene regulation; Cytokines; Reactive oxygen species 1. Introduction Zinc is an essential trace element important for a variety of cellularfunctions such as apoptosis, signal transduction, transcription, differ-entiation and replication in all organ systems and during embryonicdevelopment [1 – 5]. Therefore, zinc de 󿬁 ciency caused by malnutritionor as a consequence of aging, pregnancy or disease is detrimental forhuman health [3,5,6] and is currently one of the leading causes of morbidity and mortality in developing countries [7,8].It has been demonstrated that zinc is necessary for the structureand function of over 300 enzymes [9], including a number of DNAmethyltransferases, methyl-binding proteins and histone-modifyingenzymes such as acetylases, deacetylases or methylases [10 – 13]. Thisalong with the observation that zinc de 󿬁 ciency induces global DNAhypermethylation [11,13] points to a role of zinc in epigeneticprocesses such as chromatin remodeling, DNA methylation, histonemodi 󿬁 cation and noncoding RNA synthesis [10 – 13].In addition to its in 󿬂 uence on epigenetic processes, zinc alsoregulatesgeneexpressionviaitsinvolvementinintracellularsignaling[2,14 – 17]. Zinc is reported to stabilize but also inhibit transcriptionfactors, kinases and phosphatases or the assembly of multiproteincomplexes [2,15]. Moreover, the direct regulatory role of zinc in geneexpression as a second messenger and its indirect role via modifyingcalcium 󿬂 uxincellshavebeenreported[2].Finally,thereareanumberof studies describing the role of zinc as an antioxidant as well as theincrease of oxidative stress during zinc de 󿬁 ciency [16 – 19], providingalternative mechanisms for the regulation of gene expression by zinc.The majority of zinc-regulated genes are involved in signaltransduction, in responses to oxidative stress or in growth andenergy utilization [3], all known to be particularly important duringregulation of the immune response [15]. A variety of studies havealready shown a strong impact of zinc de 󿬁 ciency on cell-mediatedimmunity, including various T-cell defects [1,4,15,20,21]. In contrast,the number and reactivity of myeloid cells increase during zincde 󿬁 ciency [20]. It has been shown that zinc de 󿬁 ciency inducesproin 󿬂 ammatory cytokine synthesis and reactive oxygen productionin myeloid cells, but the number of studies is limited and theunderlying mechanisms are not completely understood [3,17,22 – 25].Therefore, we investigated the in 󿬂 uence of zinc de 󿬁 ciency on theproduction of the proin 󿬂 ammatory cytokines tumor necrosis factor(TNF) α  and interleukin (IL)-1 β  in promyeloid cells, focusing on therole of epigenetic and redox-mediated mechanisms as possibleexplanations for zinc-de 󿬁 ciency-induced changes.  Available online at www.sciencedirect.com  Journal of Nutritional Biochemistry 24 (2013) 289 – 297 ⁎  Corresponding author. Tel.: +49 241 8080203; fax:+49 241 8082613. E-mail address:  PUciechowski@ukaachen.de (P. Uciechowski). 0955-2863/$ - see front matter © 2013 Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.jnutbio.2012.06.007  2. Methods and materials  2.1. Cell culture Zinc-suf  󿬁 cient HL-60 cells (HL-60 suf  ) were grown in RPMI 1640 medium (Lonza,Verviers, Belgium) supplemented with 10% low-endotoxin fetal calf serum (PAA,Coelbe, Germany), 2 mM  L  -glutamine, 100 U/ml penicillin and 100  μ  g/ml streptomycin(all Lonza) in a 5% CO 2  humidi 󿬁 ed atmosphere at 37°C. For zinc-suf  󿬁 cient THP-1 cells,0.5%  β -mercaptoethanol (Merck, Darmstadt, Germany) was added to the medium. Toinducezincde 󿬁 ciency,cellswereculturedwiththemembranepermeablezincchelator N  , N  ´, N  ´-tetrakis-(2-pyridyl-methyl)ethylenediamine (TPEN, 1  μ  M, Sigma-Aldrich,Taufkirchen, Germany) for 7 days and are denoted as HL-60 def  and THP-1 def  . Tomeasure cellular viability, cells were treated as indicated in each experiment andsubsequently incubated with propidium iodide (10  μ  g/ml in phosphate-bufferedsaline) for 10 min at 4°C in the dark. The dye is membrane impermeable in intact cellsand stains dead cells as a result of the loss of plasma membrane integrity. Staining wasdetectedby 󿬂 owcytometryusingaFACSCalibur 󿬂 owcytometer.Forzincreconstitution(HL-60 rec ), HL-60 def  cells were washed and incubated in control medium for another 7days. Zinc-de 󿬁 cient medium was obtained by treatment with CHELEX 100 ionexchange resin (Sigma-Aldrich), known to bind divalent cations, for 1 h at roomtemperature, followed by reconstitution of 500  μ  M CaCl 2  and 400  μ  M MgCl 2  [25], beingessential for cell proliferation. Other cations need not be reconstituted as shown by Yuiet al. [26]. HL-60 cells cultured in chelexed medium are denoted HL-60 CHE . As a zinc-suf  󿬁 cientcontrol, chelexed mediumwas supplemented with8 μ  M ZnSO 4  (HL-60 CHE+ ).The differentiation of HL-60 cells into monocytic cells using 1 α ,25-dihydroxyvitaminD 3  (VD3; 100 nM) for 72 h was performed and monitored as described [25,27].  2.2. Enzyme-linked immunosorbent assay (ELISA) Supernatants were harvested and stored at  − 20°C, and IL-1 β  and TNF α  werequanti 󿬁 ed by ELISA (BD Pharmingen).  2.3. Reverse transcription and real-time polymerase chain reaction (PCR) RNA was isolated using RNA II-Kit (Macherey-Nagel, Düren, Germany) andreverse-transcribed using qScript cDNA Synthesis Kit in reactions containing 50 ng/ μ  lRNA (Quanta Bioscience, Gaithersburg, MD, USA). Primers for IL-1 β  [28], TNF α (forward primer: 5´-ATGAGCACTGAAAGCATGATCC-3´; reverse primer: 5´-GAGGGCT-GATTAGAGAGAGGTC-3´) and the housekeeping gene  Porphobilinogen-deaminase (PBGD) [29] were added at  󿬁 nal concentrations of 0.1  μ  M. To exclude ampli 󿬁 cationof genomic DNA or hnRNA, primer pairs which exclusively bind to exon – exon bordersorwithindifferentexonswerechosen. IL-1 β andTNF α real-time PCRswereperformedwith 2  μ  l cDNA in 25- μ  l reaction volumes in duplicates using Brilliant Sybr Green qPCR MasterMix(AppliedBiosystems,Darmstadt,Germany)withthefollowingparameters:95°Cfor15minfollowedby40cyclesof95°Cfor30sand56°Cfor30s.Standardcurveswere generated using 10-fold serial dilutions of cDNA from peripheral bloodmononuclear cells. The mRNA levels of the cytokines were normalized to PBGD levels.  2.4. Chromatin accessibility by real-time (CHART)-PCR assay MNase accessibility assays were performed, and results were plotted as describedpreviously [27]. Real-time PCR was performed in 25- μ  l reaction volumes in duplicatesusing Brilliant Sybr Green qPCR Master Mix (Applied Biosystems) containing 100 ng of DNA. Primers for IL-1 β  promoter regions IL-1 β  I, II, IV and VIII [30]; TNF α  promoterregions TNFI – IV [31];or theGAPDH promoter [32]wereadded ata 󿬁 nal concentrationof 0.1  μ  M. For quanti 󿬁 cation, a standard curve was generated using serial dilutions of genomic DNA. MNase accessibility was calculated by the following formula: a  =  j 100 −  quantity MNase+ ð Þ quantity MNase − ð Þ  ⋅ 100   j %  2.5. Measurement of free intracellular zinc with FluoZin-3AM  Free zinc was measured as described previously [33] using FluoZin-3 AM ester (1 μ  M, Invitrogen, Karlsruhe, Germany). The zinc-dependent  󿬂 uorescence was analyzedwith FACScan (BD Bioscience) using Cellquest software 3.0. The concentration of intracellular labile zinc was calculated from the mean  󿬂 uorescence with the formula[Zn]= K  D ×[( F  − F  min )/( F  max − F  )] using a dissociation constant for the Zn/FluoZin-3AMcomplex of 8.9 nM [34] and determining the maximal and minimal  󿬂 uorescence byaddition of zinc (100  μ  M) and pyrithione (50  μ  M) or TPEN (50  μ  M), respectively.  2.6. Measurement of reactive oxygen species (ROS) production using dihydrorhodamine123 (DHR) A total of1×10 6 cells/ml were loaded with DHR (1  μ  g/ml,Invitrogen) in incubationbuffer (5 mM glucose, 1 mM MgCl 2 , 1 mM NaH 2 PO 4 , 1.3 mM CaCl 2 , 120 mM NaCl, 25mM Hepes, 5.4 mM KCl, 0.3% bovine serum albumin; pH 7.35) for 30 min at 37°C.Subsequently, cells were washed with measurement buffer (incubation buffer withoutalbumin) and transferred into a 96-well plate at a density of 1×10 6 cells/ml. Theresulting  󿬂 uorescence was recorded on a  󿬂 uorescence well plate reader (excitationwavelength: 485 nm, emission: 535 nm, Ultra 384, Tecan, Crailsheim, Germany).  2.7. Cell extracts and Western blotting  Atotal of2×10 6 cellswerelysedandsonicatedin100 μ  llysisbuffer[0.5MTris – HCl(pH 6.8), 26.6% glycerin, 10% sodium dodecyl sulfate (SDS), 1 mM Na 3 VO 4  and 1%  β -mercaptoethanol] [27]. SDS – polyacrylamide gel electrophoresis using an equivalent of 4×10 5 cells and Western Blot analysis were performed as described previously [27].Membranes were incubated withhorseradish-peroxidase (HRP)-linked anti-rabbit IgGsecondary antibody and HRP-coupled anti-biotin antibody for detection of biotin-labeledmolecularweight standardfor1h,followedbydetectionwithLumiGloreagent(Cell Signaling Technology) on aLAS-3000 (Fuji 󿬁 lm Lifescience, Düsseldorf, Germany).The membrane was stripped, blocked and then reprobed for  β -actin as described [27].  2.8. Statistical analysis Statistical signi 󿬁 cance of experimental results was analyzed by Student's  t   test or,in caseof multiple comparisons, by one-wayanalysis ofvariance (ANOVA)followed byTukey's or Dunnett´s honestly signi 󿬁 cant difference post hoc test using GraphPadPrism software version 5 (GraphPad software, La Jolla, CA, USA). For singlecomparisons,* P  b .05and* P  b .01areusedfordatasigni 󿬁 cantlydifferentfromtherespectiveHL-60 suf  as determined by ANOVA/Dunnett's honestly signi 󿬁 cant difference test orStudent's  t   test. For multiple comparisons, signi 󿬁 cant differences at P  b .05, determined byANOVA/Tukey's honestly signi 󿬁 cant difference test, are indicated by different letters. 3. Results  3.1. Impact of zinc deficiency on intracellular free zinc as well as IL-1  β  and TNF  α   expression Zinc de 󿬁 ciency induces proin 󿬂 ammatory cytokine expression inmyeloid cells [22,27], but the underlying mechanisms are unknown.To examine the effect of zinc de 󿬁 ciency on IL-1 β  and TNF α expression, HL-60 cells, producing negligible proin 󿬂 ammatory IL-1 β and TNF α  amounts [27], were incubated with TPEN.First, we veri 󿬁 ed that long-term depletion of zinc by TPENsigni 󿬁 cantly decreased free intracellular zinc levels in HL-60 cellscompared to HL-60 suf  (Fig. 1A), while not affecting viability of thecells (Supplemental  󿬁 gure 1). Moreover, unstimulated HL-60 def  produced small amounts of IL-1 β  and TNF α  mRNA which weresigni 󿬁 cantly increased after stimulation with phorbol-12-myristate-13-acetate (PMA)only (Fig. 1B, C). Low expressionof IL-1 β  and TNF α mRNA was also detected in unstimulated HL-60 suf  , but no signi 󿬁 cantincrease was observed after stimulation with PMA (Fig. 1B, C).Lipopolysaccharide (LPS) generally had no effect, indicating that HL-60 def  cells were not differentiated and CD14 negative [25]. Thissuggests a positive regulatory role of long-term zinc de 󿬁 ciency inPMA-induced IL-1 β  and TNF α  transcription.Tocheckwhetherzincde 󿬁 ciencyalsoinducedthesecretionofIL-1 β and TNF α  by HL-60 cells, we analyzed their quantities in thesupernatants of the cells by ELISA. The basal amount of IL-1 β  releasedby HL-60 def  was higher than by HL-60 suf  (Fig. 2A), but did not reachsigni 󿬁 cance. IL-1 β  protein levels further increased after PMA stimula-tioninHL-60 def  only(Fig.2A).PMAtreatmentincreasedTNF α secretionby HL-60 def  and HL-60 suf  (Fig. 2B). However, the amounts of TNF α detectedinthesupernatantsofHL-60 def  weresigni 󿬁 cantlyhigherthanthose of PMA-stimulated HL-60 suf  , re 󿬂 ectingthe mRNAdata. Reconsti-tutionofHL-60 def  cellswithcontrolmediumforanother7days(Fig.2A,B) showed the reversibility of the changes induced by zinc de 󿬁 ciency.The lowlevelsofIL-1 β and TNF α  proteininthesupernatants after zincreconstitution were comparable to those detected for HL-60 suf  .  3.2. Chromatin remodeling within IL-1  β   and TNF  α   promoters Recent results showed that chromatin remodeling within IL-1 β and TNF α  promoters into an open structure is important for theactivation of IL-1 β  and TNF α  expression [27,31]. Because zinc isinvolved in epigenetic processes such as chromatin remodeling [11 – 13,35], we compared the chromatin structures of IL-1 β  and TNF α promoters in HL-60 def  and HL-60 suf  . Accessibilities of promoterregions IL-1 β  I ( − 107 to  − 17), IL-1 β  II ( − 199 to  − 109) (Fig. 3A), 290  I. Wessels et al. / Journal of Nutritional Biochemistry 24 (2013) 289 –  297   TNF α  I (+99/ – 42) and TNF α  II (+32/ – 119) (Fig. 3B) weresigni 󿬁 cantly increased under zinc de 󿬁 ciency, demonstrating that IL-1 β  and TNF α  promoters become highly accessible under zincde 󿬁 ciency near the transcriptional start sites. Zinc reconstitutiondecreased the accessibilities of promoter regions IL-1 β  I, IL-1 β  II,TNF α  I and TNF α  II compared to the structures observed in HL-60 def  ,resembling the inaccessible structure detected in HL-60 suf  .The accessibility of promoter region TNF α  III ( − 100/ − 250) rosefrom 43% to 59% under zinc de 󿬁 ciency (Fig. 3B), without reachingsigni 󿬁 cance. Analyses of regions IL-1 β  IV ( − 347 to  − 257), IL-1 β  VIII( − 673 to  − 583) andTNF IV ( − 195/ – 345),which arelocatedfurtherupstream,revealedtheircompleteinaccessibilityunderallconditions(datanotshown).Additionally,zincde 󿬁 ciencyhadnoeffectonhumanGAPDH promoter accessibility (data not shown), demonstrating thespeci 󿬁 c in 󿬂 uence of zinc on these proin 󿬂 ammatory gene promoters.  3.3. Specificity of the zinc-deficiency-induced changes To exclude TPEN speci 󿬁 c side effects other than intracellular zincchelation, we analyzed HL-60 cells cultured in zinc-de 󿬁 cient,chelexed medium (HL-60 CHE ) in comparison to HL-60 CHE+ supple-mented with 8  μ  M ZnSO 4  (Supplemental Figure 2). Moreover, to ruleout a cell-speci 󿬁 c phenomenon, the monocytic cell line THP-1 wasused. Signi 󿬁 cantly decreased intracellular zinc content in TPEN-cultured THP-1 def  (Supplemental Figure 3A) and in HL-60 CHE (Supplemental Figure 2A) as well as an increase in IL-1 β  and TNF α secretion(SupplementalFigures2B – Cand3B – C)inthesecellscouldbe Fig. 1. Impact of intracellular free zinc levels on IL-1 β  and TNF α  mRNA expression. (A)HL-60 suf  (black) and HL-60 def  (light grey) cells were loaded with the zinc-speci 󿬁 c 󿬂 uorescent probe FluoZin-3, and zinc-dependent  󿬂 uorescence was recorded by  󿬂 owcytometry. Results represent means±S.E. (S.E.M.) of   n =7 independent experiments.Signi 󿬁 cant differences at ** P  b .01 were determined by Student's  t   test. (B-C) HL-60 suf  (black)andHL-60 def  (lightgrey)cellswerestimulatedwithLPS(250ng/ml)orPMA(10ng/ml) for 3 h. IL-1 β  (B) and TNF α  (C) mRNA was analyzed by quantitative real-timePCR.ValueswerenormalizedtohousekeepinggenePBGDandarepresentedasmeans±S.E. of   n =8 independent experiments. Signi 󿬁 cant differences at  P  b .001, determined byANOVA/Tukey's honestly signi 󿬁 cant difference test, do not share the same letters.Fig. 2. Zinc de 󿬁 ciency and proin 󿬂 ammatory IL-1 β  and TNF α  release. HL-60 suf  (black),HL-60 def  (light grey) and HL-60 rec (dark grey) cells were cultured as described inMaterials and methods. After stimulation with PMA (10 ng/ml) for 3 h, the amounts of (A) IL-1 β  and (B) TNF α  in the culture supernatants were measured by ELISA. Resultsshown are means±S.E.M. of   n =6 independent experiments. 0 indicates that nocytokine secretion could be detected. Signi 󿬁 cant differences at  P  b .05, determined byANOVA/Tukey's honestly signi 󿬁 cant difference test, do not share the same letters.291 I. Wessels et al. / Journal of Nutritional Biochemistry 24 (2013) 289 –  297   demonstrated. Additionally, promoter accessibilities of IL-1 β  I, IL-1 β  II,TNF α IandTNF α IIwerehigherinTHP-1 def  andHL-60 CHE thaninTHP-1 suf  orHL-60 CHE+ ,respectively(SupplementalFigures2D – Eand3D – E).  3.4. Role of zinc deficiency in HL-60 differentiation into monocytic cells Since VD3-induced differentiation of HL-60 cells leads to IL-1 β promoter remodeling and IL-1 β  and TNF α  production, and combinedzinc de 󿬁 ciency enhances CD11b/CD14 surface expression [27,38],CD14expressionwasinvestigatedunderzinc-de 󿬁 cientconditions.Noexpression of the monocyte marker CD14 on stimulated andunstimulated HL-60 def  or HL-60 CHE (Supplemental Figure 4A – D) orafter TPEN incubation of HL-60 cells for 14 days could be detected. Incontrast,VD3incubatedcellsshowed58%CD14 + cells(SupplementalFigure 4E – F). This suggests that the remodeling of IL-1 β  and TNF α promoters induced by zinc de 󿬁 ciency was a separate effect indepen-dent from complete differentiation.  3.5. ROS production of zinc-deficient HL-60 cells Zinc de 󿬁 ciency is described to elevate oxidative stress in differentcell types [18,19]. We observed that the basal level of ROS wassigni 󿬁 cantly higherin HL-60 def  than in HL-60 suf  (Fig. 4A), indicating ashift to a more intracellular oxidative milieu under zinc de 󿬁 ciency.WhereasnochangesinDHRoxidationafterLPSorPMAstimulationofHL-60 suf  (Fig.4B)couldbedetected,ROSconcentrationsinHL-60 def  steadily increased only after PMA stimulation until the end of theexperiment (Fig. 4C). In HL-60 rec , only a small increase of DHR oxidation could be detected shortly after PMA stimulation (Fig. 4D),indicating the reversibility of the changes in PMA-induced ROSsynthesis that we observed in HL-60 def  . LPS had no in 󿬂 uence on ROSproduction in HL-60 rec as observed in HL-60 suf  and HL-60 def  (Fig. 4D).In myeloid cells, ROS are primarily produced by nicotinamideadenine dinucleotide phosphate-oxidase (NADPH) oxidase (NOX)during oxidative burst [36]. Preincubation of HL-60 suf  , HL-60 def  andHL-60 rec with the NOX inhibitor diphenyleneiodonium (DPI) beforePMA stimulation did not alter the basal levels of ROS production inthese cells (Fig. 4E – G). In contrast, DPI preincubation almostcompletely abrogated the PMA-induced increase in DHR oxidationin HL-60 def  (Fig. 4F). This indicates that ROS are produced by NOXin HL-60 def  .  3.6. Connection between zinc, ROS and cytokine expression To elucidate whetherchangesin theROSproductionarerelatedtocytokine expression under zinc de 󿬁 ciency, cells were again preincu-bated with DPI before stimulation, and IL-1 β  and TNF α  release wasmeasured by ELISA. DPI abrogated the PMA-induced increase of IL-1 β secretion in HL-60 def  (Fig. 5A), whereas the basal IL-1 β  expressionremained unchanged. In addition, we found a signi 󿬁 cant decrease inPMA-induced TNF α  synthesis in HL-60 suf  and HL-60 def  preincubatedwith DPI (Fig. 5B), suggesting that ROS-dependent and ROS-independent pathways are involved in PMA-induced TNF α  synthesis.To more precisely de 󿬁 ne how ROS in 󿬂 uence IL-1 β  and TNF α expression under zinc de 󿬁 ciency, we assessed IL-1 β  and TNF α promoter accessibilities after treatment with  N  -acetylcysteine(NAC) for 7 days. The strong antioxidant NAC was able to abrogatePMA-induced DHR 123 oxidation in HL-60 def  (Supplemental Figure5). As depicted in Fig. 6A – B, coincubation of HL-60 def  with NAC didnotinhibittheremodelingofpromoterregionsIL-1 β I,IL-1 β II,TNF α Iand TNF α  II observed in HL-60 def  , but further increased the promoteraccessibilitiesof these regions. Control regionsIL-1 β IV, IL-1 β  VIII andTNF α IVremainedinaccessible(datanotshown).Hence,theseresultsexcluded that ROS are involved in chromatinremodeling of IL-1 β  andTNF α  promoters.Next, we investigated whether ROS induced transcription of IL-1 β and TNF α . Fig. 7 shows that NAC had no in 󿬂 uence on IL-1 β  and TNF α mRNA levels in HL-60 def  . Additionally, preincubation of HL-60 def  withDPIhadalsonosigni 󿬁 canteffectonPMA-inducedIL-1 β orTNF α mRNAexpression (Supplemental Figure 6). Hence, it was excluded that theROS-dependentincreaseofIL-1 β andTNF α secretionwasregulatedviachromatin remodeling (Fig. 6) or changes in transcription (Fig. 7). Fig.3.Impactofzincde 󿬁 ciencyonIL-1 β andTNF α promoterconformation.CHART-PCR analyses of IL-1 β  and TNF α  promoters in HL-60 cells are shown. HL-60 suf  (black), HL-60 def  (light grey) and HL-60 rec (dark grey bars) cells were cultured as described inMaterials and methods. Real-time PCR was performed using primer sets for (A) IL-1 β promoter regions I and II and for (B) TNF α  promoter regions I, II and III. Meancalculated accessibilities and S.E.M. for  n =4 independent experiments are presented.* P  b .05 for data signi 󿬁 cantly different from respective HL-60 suf  were determined byANOVA/Dunnett's honestly signi 󿬁 cant difference test.Fig.4.Changesintheredoxstatusduringzincde 󿬁 ciency.HL-60 suf  ,HL-60 def  andHL-60 rec cellswereculturedasdescribedinMaterialsandmethodsandloadedwithDHR123for30min.The  󿬂 uorescence resulting from DHR123 oxidation was measured in a well plate reader. (A) Basal levels of oxidated DHR123  󿬂 uorescence are shown as mean ±S.E.M. of   n =7independentexperiments.Signi 󿬁 cantdifferencesfromHL-60 suf  at** P  b .01weredeterminedbyStudent's t  test.(B – D)After10minofrecordingofthebaseline 󿬂 uorescenceforHL-60 suf  (B), HL-60 def  (C) and HL-60 rec (D), the cells were stimulated with buffer (circles), LPS (250 ng/ml, triangles) or PMA (10 ng/ml, squares) for another 45 min, and  󿬂 uorescence wasmonitored and normalized to untreated controls. One representative example of   n =3 for each approach is shown. (E – G) DHR123 loaded HL-60 suf  (E), HL-60 def  (F) and HL-60 rec (G)cellswerepreincubated withbuffer(circles)ortheNOXinhibitorDPI(10 μ  M,triangles)for30min.After10minofrecordingofthebaseline, buffer( 󿬁 lledsymbols)orPMA(10ng/ml,open symbols) was added, and  󿬂 uorescence was monitored for another 45 min and normalized to buffer-treated controls. Shown are mean ±S.E.M. of at least  n =3 independentexperiments for each type of cell culture.292  I. Wessels et al. / Journal of Nutritional Biochemistry 24 (2013) 289 –  297   293 I. Wessels et al. / Journal of Nutritional Biochemistry 24 (2013) 289 –  297 
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