International Journal for Parasitology 40 (2010) 1003–1012

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International Journal for Parasitology

journal homepage: www.elsevier .com/locate / i jpara

The IL-12p70/IL-10 interplay is differentially regulated by free hemeand hemozoin in murine bone-marrow-derived macrophages

Mathieu Cambos a,1, Stefany Bazinet a,1, Elie Abed a, Jaime Sanchez-Dardon a, Charlotte Bernard a,Robert Moreau a, Martin Olivier b, Tatiana Scorza a,*

a Department of Biological Sciences, Université du Québec à Montreal, Montreal, Que., Canadab Department of Microbiology and Immunology, McGill University, Montreal, Que., Canada

a r t i c l e i n f o

Article history:Received 22 December 2009Received in revised form 8 February 2010Accepted 9 February 2010

Keywords:HeminHemozoinMacrophagesIL-12IL-10P38 MAPK

0020-7519/$36.00 � 2010 Australian Society for Paradoi:10.1016/j.ijpara.2010.02.007

* Corresponding author. Tel.: +1 514 9873000x191E-mail address: scorza.tatiana@uqam.ca (T. Scorza

1 These authors contributed equally to this work.

a b s t r a c t

The outcome of malarial anemia is determined by a complex interplay between pro-inflammatory andanti-inflammatory cytokines, its severity associated with accumulation of hemozoin (Hz) in macro-phages, elevated IL-10 responses and impaired IL-12 production. Although free heme contributes tomalarial anemia by inducing oxidative damage of red blood cells (RBCs) and enhancing their clearanceby phagocytes, its impact on IL-12/IL-10 interactions has not been fully characterized. Herein, the effectof hemin (HE) on IL-12 and IL-10 responses was studied in murine bone marrow-derived macrophages(BMDM) and compared with synthetic Hz. Our data reveal that HE induces modest inhibition of IL-12p70 responses to lipopolysaccharide (LPS) whereas Hz significantly impairs IL-12p70 responses toIFNc/LPS through down-regulation of IL-12p35 and p40 gene expression. Although reactive oxygen spe-cies (ROS) are generated after short-term exposure to HE and Hz, prolonged exposure to these iron pro-toporphyrins has opposite effects on the cellular redox status, HE being the only compound able topromote persistent ROS production. Accordingly, the inhibitory effect of HE on IL-12p70 seems sustainedby redox-dependent induction of IL-10 and is partially controlled by the p38 mitogen-activated proteinkinase (MAPK) signalling pathway. Indeed, treatment with n-acetylcysteine (NAC) or with the p38 MAPKinhibitor SB203580 inhibits IL-10 responses and significantly restores IL-12p70 responses to IFNc/LPS inHE-conditioned BMDM. Our results suggest that oxidant stress induced by free heme may potentiallycontribute to sustained production of IL-10 and down-regulation of IL-12 responses in malaria.

� 2010 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction

Dysfunctional erythropoiesis is frequently observed in inflam-matory disorders such as rheumatoid arthritis, inflammatory bo-wel disease and cancer (Caro et al., 2001; Han et al., 2007;Nikolaisen et al., 2008; Bergamaschi et al., 2009). In this context,the systemic inflammation triggered by Plasmodium parasites hasbeen shown to inhibit erythropoiesis in response to anemia. Circu-lating levels of TNFa and IFNc are elevated in humans with severemalarial anemia, (Othoro et al., 1999; Ageely et al., 2008) and thesecytokines impair the responsiveness of erythroid precursors toerythropoietin or promote their apoptosis (Felli et al., 2005; Bucket al., 2008).

IL-12 is a cytokine required for the generation of T helper 1 re-sponses and for parasite clearance (Stevenson et al., 1995; Yoshim-oto et al., 1998). In addition, IL-12 promotes the differentiation andgrowth of erythroid precursors in vivo and in vitro (Dybedal et al.,

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1995; Jacobsen et al., 1996), which seems relevant for the controlof malarial anemia, as low levels of IL-12 correlate with anemiaseverity in humans (Luty et al., 2000; Perkins et al., 2000; Chaisa-vaneeyakorn et al., 2003; Chaiyaroj et al., 2004).

Hemozoin (Hz), a bio-crystal of hemoglobin-derived heme (Fe3+

protoporphyrin) and the most studied player in malarial inflamma-tion, is known to persist in tissues for several months followingparasite clearance (MacCallum, 1969; Levesque et al., 1999). Hzcontributes to the severity of anemia as it impairs IL-12 responsesby enhancing secretion of anti-inflammatory IL-10 (Keller et al.,2006), and stimulates TNFa production in macrophages and mono-cytes (Prada et al., 1995; Sherry et al., 1995; Nti et al., 2005; Pratoet al., 2005).

Recently, justified attention has been given to free hemoglobinand heme in malaria pathology (Ferreira et al., 2008; Pamplonaet al., 2009; Seixas et al., 2009). These two iron containingcompounds are released during infection and in conditions ofoxidative stress and high hemolysis as in malaria, free hemoglobinoxidizes to methemoglobin, liberating its prosthetic groups andfurther generating free heme (Jeney et al., 2002). Free heme is ahighly hydrophobic molecule that induces oxidative damage of

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1004 M. Cambos et al. / International Journal for Parasitology 40 (2010) 1003–1012

non-infected and infected red blood cell (RBC) membranes and en-hances their removal by phagocytes, thus contributing to anemia(Chiu et al., 1989; Hebbel and Eaton, 1989; Gatidis et al., 2009).

In order to evaluate a possible contribution for free heme incytokine-induced malarial dyserythropoiesis, we investigated theeffect of exposure to hemin (HE) on IL-12 and IL-10 responses aswell as on the redox status of murine bone-marrow-derived mac-rophages (BMDM). We hypothesized that HE, a potent oxidant, im-pairs IL-12p40 responses and enhances IL-10 responses inmacrophages through oxidative stress. The effect of Hz was as-sessed in parallel since initial interaction with Hz is known to trig-ger the release of chemokines and pro-inflammatory cytokines inmacrophages partially through redox-dependent signalling (Jara-millo et al., 2005). Our data reveal that free heme down-regulatesIL-12 responses through redox/p38 MAPK-dependent induction ofIL-10, which contrasts with the inhibitory effects that Hz has bothon IL-12 and IL-10 secretion.

2. Materials and methods

2.1. Chemicals

All chemicals including Hemin chloride (Fluka) and LPS (Esche-richia coli, serotype 0111:B4) were purchased from Sigma Aldrich(Canada). SB203580 (4-(4-Fluorophenyl)-2-(4-methylsulfinyl phe-nyl)-5-(4-pyridyl)-1H-imidazole) was purchased from Cell Signal-ling (USA).

2.2. Preparation of synthetic Hz

Synthetic Hz was prepared as previously described (Jaramilloet al., 2009; Shio et al., 2009) using high purity chemical reagents(>99% purity). Briefly, 0.8 mmol HE (Fluka) was dissolved in de-gassed NaOH (0.1 M) for 30 min with mild stirring, and the pHwas adjusted to four by drop-wise addition of propionic acid. Themixture was allowed to anneal at 70 �C for 18 h and washed threetimes with NaHCO3 (0.1 M) for 3 h, after which it was washed withMetOH. All washes were alternated with distilled H2O. The samplewas finally dried in a vacuum oven overnight over phosphorouspentoxide. All synthetic Hz samples were analyzed by X-ray pow-der diffraction, field emission gun scanning electron microscopy,and infra-red spectroscopy to characterize the crystalline state ofHz, and their purity was assessed by elemental analysis as previ-ously reported (Jaramillo et al., 2009; Shio et al., 2009).

2.3. Preparation of BMDM

Femoral marrows from 4 to 6 weeks old female BALB/c mice(Charles River Laboratories, Canada) were flushed and cells wereplated at 1 � 106 cells/ml in culture Petri dishes (Saarstedt, Can-ada) using DMEM supplemented with 10% FBS, 1% HEPES, penicil-lin and streptomycin as culture medium (Invitrogen, Canada). Thecells were differentiated with 50% mouse L929 fibroblast cell lineculture supernatant as a source for macrophage-colony stimulatingfactor. Following 6 days culture, non-adherent cells were removedand adherent cells (BMDM) were recovered by detachment with arubber policeman, after which the cells were washed twice withPBS (Invitrogen, Canada). Analysis of the cell preparation by flowcytometry revealed P95% CD11b+ F4-80+ cells (data not shown).All mice used in this study were housed and handled in accordancewith procedures approved by the Animal Care Committee of theUniversity of Quebec in Montreal, protocol 562-R3-0510.

2.4. Reactive oxygen species (ROS) measurement

BMDM suspensions were treated with HE or Hz (12.5, 25 and50 lg/ml) for 4 or 18 h. The cells were recovered and labelled with20,70-dichlorodihydrofluorescein diacetate (H2-DCFDA, MolecularProbe, Invitrogen, Canada) (100 lM) for 30 min at 37 �C, and themean cellular fluorescence was measured in a FACscancytofluorometer.

2.5. Determination of intracellular glutathione/glutathione disulphide(GSH/GSSG) ratios

BMDM were plated at 2 � 106 cells/ml in complete RPMI 1640media in 24 well plates (Saarstedt, Canada) and were treated withHE or Hz (25 lg/ml) for 18 h. Supernatants were removed and thecells were rinsed with PBS prior to direct sonication in cold MESbuffer (0.2 M 2-(N-morpholino) ethanesulphonic acid, 50 mMphosphate and 1 mM EDTA, pH 6.0). The cell homogenate wasrecovered and centrifuged and the particle clear supernatant wasdeproteinated by addition of equal volumes of 10% metaphospho-ric acid. Fifty microliters of TEAM (4 M solution of triethanolamine)was added per milliliter of sample and the mixture was vortexedimmediately. For GSSG determination, derivatization of GSH waseffectuated with 2-vinylpyridine. Total GSH and GSSG in sampleswere then assessed with the Glutathione determination kit (Cay-man Chemicals, USA).

2.6. SDS–PAGE and Western blot analysis of HO-1, p38 mitogen-activated protein kinase (MAPK) and MAPKAPK-2

For detection of heme oxygenase-1 (HO-1) 1 � 106 BMDM/well(six well plates, Saarstedt, Canada) were treated with HE or Hz for6 h (12.5–50 lg/ml) or 18 h (25 lg/ml) after which the cells weredirectly lyzed in 200 ll of Laemli buffer (Bio-Rad). Equal volumesof lysate were loaded onto 12% polyacrylamide gels for SDS–PAGE.Proteins were transferred onto nitrocellulose membranes (Hy-bond-C; Amersham Biosciences), after which the membranes wereblocked overnight in SuperBlotto (Pierce Chemical Co., USA). Anti-HO-1 rabbit antibody (Assay Designs/Stressgen Bioreagents, USA)was used at 1 lg/ml in Tris-buffered saline/Tween 20 for 2 h afterwhich membranes were washed and further incubated with bio-tinylated anti-rabbit polyclonal antibody (Sigma). Membraneswere then washed prior to the addition of substrate.

For analyses of p38 MAPK and MAPKAPK-2, BMDM were trea-ted for 18 h with HE or Hz (25 lg/ml), after which recombinantIFNc (100 U/ml; Cedarlane, Canada) was added 15 min prior tostimulation with LPS (50 ng/ml). In parallel, BMDM were treated1 h with the p38 MAPK inhibitor SB203580 (1 lM) prior to stimu-lation. At 0, 30 and 60 min post-stimulation, the cells were washedtwice with cold PBS (Invitrogen, Canada) and lysed in 40 mM Tris–HCl (pH 6.8), 275 mM NaCl, 20% glycerol (v/v), 2% IGEPAL (SigmaAldrich, Canada), 1 mM phenylmethylsulphonyl fluoride, 20 lg/ml aprotinine, 20 lg/ml leupeptine, 2 mM Na3VO4 and 50 mMNaF for 30 min on ice. The cell lysates were centrifuged (10,000g,15 min) and supernatant protein concentrations were quantifiedwith the DC protein assay (Bio-Rad, Canada). About 20–60 lg pro-teins/lane were separated by SDS–PAGE and transferred to nitro-cellulose membranes, and membranes were blocked as describedabove. Anti-p38, anti-phospho p38, anti-MAPKAPK-2 and anti-phospho MAPKAPK-2 antibodies (Cell Signaling Technology) wereused following the manufacturer’s instructions. Membranes werewashed and incubated with anti-rabbit IgG horseradish peroxi-dase-conjugated antibody (Sigma). Reactions were carried out withSuperSignal West pico chemiluminescent Substrate (ThermoScientific). As controls for loading, anti-b-actin (Biolegend) wasused in combination with an anti-mouse Ig biotinylated polyclonal

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antibody (Amersham Biosciences) and a Streptavidin horseradishperoxidase conjugate (GE Healthcare). Relative expression levelsof HO-1 were normalized with respect to b-actin by densitometry.

2.7. Induction of IL-12p40, IL-12p70 and IL-10

BMDM were seeded at a density of 5 � 105 cells/well in a 96-well plate in complete medium (Invitrogen), and were treated withHE or Hz (25 lg/ml) for 18 h. Secretion of IL-12p40, IL-12p70 andIL-10 was induced by stimulation with LPS (50 ng/ml; Sigma Al-drich) or recombinant murine IFNc (100 U/ml; Cederlane) 15 minprior to addition of LPS. IL-12p70, IL-12p40 and IL-10 levels in cul-ture supernatants were determined 24 h later using the ELISA kitsBD OpTEIA set mouse IL-12 (p70), and mouse IL-10 (BD Biosci-ences) and ELISA MAX™ Set Deluxe Mouse IL-12p40 (BioLegend).In some experiments BMDM were treated with 1 lg/ml anti-IL-10 monoclonal antibody (mAb) or control isotype antibody (Bio-Legend), NAC (0.25 mM, Sigma Aldrich), or 1 lM p38 MAPK inhib-itor (SB203580, Cell Signalling) 1 h prior to stimulation.

2.8. Reverse-transcription PCR (RT-PCR)

Total RNA was extracted from cells using TriZol (Invitrogen)according to the manufacturer’s instructions. Reverse transcription(RT) reactions were carried out with an Omniscript RT kit (Qiagen,Canada). The PCR amplifications were conducted with a Taq PCRcore kit (Qiagen) using specific primers. IL-12p35 F: 50-AAGCTCTGCATCCTGCTTCAC-30, R: 50-GATAGCCCATCACCCTGTTGA-30.IL-12p40, F: 50-CTCGCAGCAAAGCAAGGTAAG-30, R: 50-CTGAATCTGGACAGCC TCTAGATG-30. GAPDH F: 50-GGAGATTGTTGCCATCAACGA-30; R: 50-TGGGAGTTGCTGTTGAAGTCG-30. Each primer was de-signed in distinct exons to ensure specific transcript amplifications.Amplifications were carried out for 40 cycles with incubation for1 min at 94 �C, 30 s at 58 �C and 1 min at 72 �C. Amplification prod-ucts were resolved in a 2% agarose gel with ethidium bromide rev-elation. Relative expression of IL-12p35 and IL-12p40 wasdetermined by densitometry.

2.9. Statistical analyses

Data was analyzed with GraphPad Prism software, using ANO-VA for group comparison. Values represent the mean ± SEM of fourindependent experiments. A P value 60.05 was consideredsignificant.

Fig. 1. Hemin (HE) has a major negative impact on IL-12p70 responses to IFNc/lipopolysmarrow-derived macrophages (BMDM) (1 � 105/well) were cultured in 96-well culture pLPS (50 ng/ml) (A and C) or with IFN-c (100 U/ml) 15 min prior to LPS treatment for 24 hELISA. Error bars represent the mean ± SEM of six independent experiments. Cytokine corespect to controls (Ctrl) are indicated by underlined numbers. ***P < 0.001, **P < 0.01; *

3. Results

3.1. HE impairs IL-12p70 responses in BMDM through down-regulation of IL-12p35 and IL-12p40 gene expression

To characterize the possible contributions of free heme on thedown-regulation of IL-12, we evaluated the capacity of HE (Fe3+

protoporphyrin monomer) to modulate the secretion of IL-12p70.For this, BMDM were conditioned with 25 lg/ml HE, a concentra-tion considered comparable with the level of hemoglobin-derivedheme released during mild Plasmodium infection (Sherry et al.,1995; Sullivan et al., 1996). Eighteen hours later, the cells werestimulated with LPS or IFNc/LPS for 24 h after which IL-12p70 con-tent was assessed in culture supernatants by ELISA.

In six independent experiments, HE had modest inhibitory ef-fects on IL-12p70 responses to LPS (Fig. 1A; P < 0.05) but signifi-cantly inhibited its secretion in response to IFNc/LPS (Fig. 1B;P < 0.001). In contrast, exposure to Hz resulted in impaired IL-12p70 responses to both LPS (Fig. 1A; P < 0.01) and IFNc/LPS(Fig. 1B; P < 0.001).

Since biologically active IL-12p70 is formed from a 35 kDa (p35)and a 40 kDa (p40) subunit, we assessed the effect of HE on IL-12p40 secretion (as homodimer and heterodimer). In response toLPS stimulation, inhibition of IL-12p40 secretion was induced byHE (Fig. 1C; P < 0.001), an effect that was also caused by Hz(Fig. 1C; P < 0.001). Comparable modest inhibition was inducedby HE and Hz on IL-12p40 responses to IFNc/LPS (Fig. 1D;P < 0.01 and P < 0.05, respectively). Comparable effects were mea-sured in response to stimulation with the Toll-like receptor 2(TLR2) agonist Pam3Cys (data not shown). The effects of HE orHz on IL-12p70 responses were sustained for more than 72 h andwere induced after 2 h treatment with the protoporphyrins (datanot shown).

As IL-12p70 and not IL-12p40 secretion was significantly af-fected by HE, IL-12p40 and IL-12p35 gene expression were studiedby RT-PCR (Fig. 2). No apparent effect was revealed on IL-12p40gene expression by RT-PCR in two independent experiments ofwhich a representative result is shown in Fig. 2A. Although IL-12p35 mRNA remained undetectable in resting BMDM, compara-ble levels of induction were measured for control, HE and Hz-con-ditioned cells in response to LPS stimulation (Fig. 2A). Differencesin IL-12p40 and IL-12p35 gene expression with respect to controlBMDM became apparent in response to IFNc/LPS, for which com-parable inhibition was induced by HE and Hz on both IL-12p40(Fig. 2B) and IL-12p35 mRNA expression (Fig. 2C). Altogether, theseresults indicate that, similar to Hz, HE significantly down-regulatesIL-12p40 and IL-12p35 gene expression and consequently IL-12p70 secretion.

accharide (LPS), which is unrelated to down-regulation of IL-12p40 secretion. Bonelates in the presence of HE or hemozoin (Hz) (25 lg/ml) for 18 h and stimulated with(B and D). Culture supernatants were assessed for IL-12p70 and IL-12p40 content byncentrations were compared using one-way ANOVA; percentages of inhibition withP < 0.05.

Fig. 2. Hemin (HE) and hemozoin (Hz) down-regulate IL-12p35 and IL-12p40 geneexpression in response to IFNc/lipopolysaccharide (LPS) stimulation. (A) Control(Ctrl), HE and Hz-conditioned bone marrow-derived macrophages (BMDM)(3 � 106/well) were stimulated for 6 h with LPS (50 ng/ml) or pre-stimulated15 min with IFNc (100 U/ml) prior to addition of LPS. Total RNA was isolated fromthe cells and the levels of IL-12p35, IL-12p40 and GADPH transcripts weredetermined by semi-quantitative reverse-transcription PCR (RT-PCR) as describedin Section 2. Relative expression levels of IL-12p40 (B) and IL-12p35 (C) in HE andHz-conditioned BMDM (25 lg/ml, 18 h) stimulated with IFNc/LPS are expressed asthe mean ± SEM of three independent experiments. The percentages of inhibition ingene expression relative to controls are indicated by underlined numbers.

Fig. 3. IL-10 secretion in hemin (HE)-conditioned bone marrow-derived macro-phages (BMDM) is refractory to the inhibitory effects of IFNc. BMDM (1 � 105/well)remained untreated (Ctrl) or were cultured in the presence of HE or hemozoin (Hz)(25 lg/ml) for 18 h after which the cells were stimulated with lipopolysaccharide(LPS) (50 ng/ml) or primed with IFNc (100 U/ml) 15 min prior to LPS treatment.Twenty-four hour culture supernatants were harvested and assessed for IL-10 byELISA. Error bars represent the mean ± SEM of four independent experiments. IL-10concentrations in culture supernatants were compared using one-way ANOVA.Percentages of IL-10 inhibition induced by IFNc and percentages of inhibition withrespect to control BMDM are indicated by underlined numbers. ***P < 0.001;**P < 0.01; *P < 0.05.

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3.2. HE inhibits IL-12p70 responses through an IL-10-dependentmechanism

IL-10 is a primary physiological inhibitor of IL-12. Importantly, theintracellular redox status of antigen presenting cells seems to influ-ence in an IL-10-dependent manner their capacity to produce IL-12as low levels of reduced GSH down-regulate IL-12 production and en-hance IL-10 responses (Murata et al., 2002). Based on this informationwe determined the effects of HE and Hz on IL-10 secretion.

In BMDM, exposure to HE increased IL-10 secretion both in re-sponse to LPS and IFNc/LPS (Fig. 3; P < 0.01), whereas production ofthis anti-inflammatory cytokine was significantly impaired in Hz-conditioned BMDM (Fig 3; P < 0.01 and P < 0.05, for LPS andIFNc/LPS stimulation, respectively). Overall, IFNc efficiently re-duced IL-10 secretion in control BMDM and Hz-conditioned BMDM(Fig. 3; P < 0.001), an effect that was absent in HE-conditionedBMDM (Fig. 3). In the absence of TLR stimulation, IFNc did not in-duce IL-12p40 or IL-12p70 secretion.

In order to confirm a possible link between enhanced IL-10 re-sponses and the down-regulation of IL-12p70 responses in HE-con-ditioned cells, BMDM were treated with anti-IL-10 neutralizingmAb or control isotype mAb 1 h prior to stimulation. AlthoughIL-10 neutralization significantly enhanced IL-12p70 responses toLPS in control BMDM (Fig. 4A, P < 0.05) it did not modify the re-sponse in HE-treated BMDM nor in Hz-treated cells (Fig. 4A). Incontrast, neutralization of IL-10 had a major positive impact onIL-12p70 responses to IFNc/LPS in HE-conditioned BMDM(Fig. 4B; P < 0.001) but did not improve these responses in controlor Hz-conditioned BMDM (Fig. 4B).

3.3. Prolonged exposure to HE induces ROS and decreases GSH/GSSGratios on BMDM

HE is a potent inducer of HO-1 (Lang et al., 2005; Desbuardset al., 2007; Lakkisto et al., 2009), an enzyme that attenuates sev-

eral pathologies of an inflammatory nature (Desbuards et al., 2007;Zhen-Wei et al., 2007; Kubulus et al., 2008), and restores cellularredox balance (Wu et al., 2008; Brunt et al., 2009). In order to eval-uate the capacity of BMDM to control HE-induced oxidant stress,intracellular levels of ROS were determined in BMDM treated for4 and 18 h with HE. As Hz has been shown to modulate chemokineand cytokine responses in macrophages partially through redox-dependent stress (Jaramillo et al., 2005), BMDM were also treatedwith Hz for comparative purposes.

Levels of ROS were first analyzed in H2-DCFDA-labelled BMDMafter a 4 h treatment with 12.5–50 lg/ml of HE or Hz, and themean channel fluorescence was measured by flow cytometry. At12.5–50 lg/ml, HE induced important levels of ROS compared withcontrol BMDM (Fig. 5A, P < 0.001). Exposure to Hz also inducedROS (Fig. 5A, P < 0.001), but the effects were significantly lowerthan those conferred by HE (Fig. 5A; P < 0.001).

ROS levels were also measured in BMDM conditioned with HEor Hz for 18 h. Relative to the 4 h treatment, ROS sharply decreasedin HE-conditioned cells, but remained significantly higher than incontrol BMDM (Fig. 5B; P < 0.01 for 12.5 lg/ml and P < 0.001 for25 and 50 lg/ml). In contrast, after 18 h treatment, exposure toHz decreased intracellular ROS to levels below those measured incontrol cells (Fig. 5B; P < 0.001). The inhibitory effect of Hz onROS levels became apparent after 5 h of treatment and was sus-tained for more than 48 h (data not shown).

Total GSH and intracellular GSH/GSSG ratios were also deter-mined in BMDM treated with HE or Hz for 18 h (Fig. 6). AlthoughHE failed to modify total GSH or reduced GSH concentrations, theirlevels were significantly elevated in Hz-conditioned cells (Fig. 6A,B; P < 0.001 and P < 0.05, respectively). In contrast, the concentra-tions of GSSG increased in HE-conditioned BMDM (Fig. 5C;P < 0.001) but were not affected by Hz. In accordance with theROS response measured at 18 h post-treatment with HE, GSH/GSSGratios were significantly decreased in these cells (Fig. 6D;P < 0.001), whereas elevated GSH/GSSG ratios were measured inHz-conditioned BMDM (Fig. 6D; P < 0.001).

As mentioned previously, HE is a known inducer of HO-1, theenzyme that catalyzes the cleavage of the protoporphyrin ring,generating carbon monoxide (CO), biliverdin and free iron (Fe2+)

Fig. 4. Neutralization of IL-10 restores IL-12p70 responses to IFNc/lipopolysaccharide (LPS) in hemin (HE)-conditioned bone marrow-derived macrophages (BMDM). BMDM(1 � 105/well) remained untreated (Control) or were cultured in the presence of HE or hemozoin (Hz) (25 lg/ml) for 18 h and stimulated with LPS (50 ng/ml) (A) or with IFNc(100 U/ml) for 15 min prior to LPS stimulation (B). Anti-IL-10 neutralizing monoclonal antibody or control isotype antibody (1 lg/ml) was added to the culture medium 1 hbefore stimulation and 24 h culture supernatants were assessed for IL-12p70 content by ELISA. Error bars represent the mean ± SEM of four independent experiments.Percentages of inhibition with respect to controls are indicated in underlined numbers, and the effect of anti-IL-10 treatment on IL-12p70 concentrations was compared usinga one-way ANOVA. ***P < 0.001; *P < 0.05.

Fig. 5. Reactive oxygen species (ROS) are generated after short-term and long-term exposure to hemin (HE). Bone marrow-derived macrophages (BMDM) (1 � 106/well) wereincubated for 4 h (A) or 18 h (B) with HE or hemozoin (Hz), (12.5–50 lg/ml) and then treated with 20 ,70-dichlorodihydrofluorescein diacetate (H2-DCFDA) for estimation ofratios of mean channel fluorescence intensity with respect to control BMDM. Values were compared using one-way ANOVA. ***P < 0.001; **P < 0.01. Values from BMDMtreated with equal HE/Hz concentrations were compared with a Student’s t test (underlined asterisks).

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(Otterbein and Choi, 2000). Biliverdin is further converted to biliru-bin by biliverdin reductase whereas Fe2+ induces up-regulation ofiron-binding ferritin and iron exclusion (Bach, 2005). Importantly,these products contribute to the control of inflammation throughvarious mechanisms including IL-10 up-regulation (Otterbeinet al., 2000; Otterbein and Choi, 2000). In BMDM, 6 h exposure toHE promoted dose-dependent induction of HO-1 protein expres-sion (Fig. 7A). At 25 lg/ml, the effect was significant in three inde-pendent experiments (Fig. 7C, P < 0.01). Six hours exposure to Hzalso induced HO-1 expression in a dose-dependent manner, butas with the ROS response, the effect was milder compared withHE. Following 18 h exposure, HO-1 protein levels were relativelyhigher only in BMDM conditioned with 25 lg/ml of HE althoughthe differences with respect to BMDM were not statistically signif-icant (Fig. 7C).

3.4. NAC reverts the inhibitory effect of HE on IL-12p70 responses

We hypothesized that the down-regulation of IL-12p70 re-sponses to IFNc/LPS in HE-conditioned BMDM was concurrent toredox-dependent induction of IL-10. We therefore evaluatedwhether treatment with the GSH precursor NAC reverted the IL-12p70 inhibition in HE-treated BMDM (Fig. 8). Interestingly, aswith IL-10 neutralization, treatment with NAC enhanced IL-

12p70 responses in control BMDM (Fig. 8A; P < 0.05), but did notrevert the inhibitory effects of HE nor Hz on IL-12p70 responseto LPS (Fig. 8A). However, NAC restored IL-12p70 response toIFNc/LPS in HE-conditioned BMDM (Fig. 8B; P < 0.001) whereas itdid not modify the responses in Hz-conditioned BMDM. Treatmentwith NAC did not affect IL-10 responses to LPS in any of the groups(Fig. 8C), but it significantly decreased IL-10 responses to IFNc/LPSin HE-conditioned BMDM (Fig. 8D; P < 0.001). Altogether these re-sults suggest that, albeit inducing HO-1 expression, exposure to HEincreases ROS and decreases intracellular GSH/GSSG ratios, andthat these effects are associated with up-regulation of IL-10 secre-tion and concomitant inhibition of IL-12p70 responses.

3.5. Inhibition of p38 MAPK restores IL-12p70 responses to IFNc/LPS inHE-conditioned BMDM

Mitogen-activated Protein Kinase 3 (MKK3)-deficient macro-phages are deficient in IL-12p40/IL-12p35 responses, suggestinga pivotal role for the MKK3/p38 MAPK pathway in IL-12p70 pro-duction (Lu et al., 1999). As IL-12p40 responses in human periph-eral blood mononuclear cells (PBMC) are enhanced by the p38MAPK selective inhibitor SB203580 (<10 lM range) (Marriottet al., 2001), it has been suggested that LPS activation of p38 MAPKmay have a dual impact on IL-12 (Salmon et al., 2001). Indeed by

Fig. 6. Hemin (HE) decreases intracellular glutathione/glutathione disulphide(GSH/GSSG) ratios in bone marrow-derived macrophages (BMDM). BMDM(2 � 106/well) were treated with HE or hemozoin (Hz) (25 lg/ml) for 18 h afterwhich cell lysates were deproteinized and assessed for intracellular levels of totalglutathione (A), GSH (B) and GSSG (C). GSH/GSSG ratios were estimated accordingly(D). Error bars represent the mean ± SEM of four independent experiments. A one-way ANOVA was used to compare values with respect to control (Ctrl) BMDM.***P < 0.001; *P < 0.05.

Fig. 7. HO-1 protein levels significantly increase after 6 h exposure to hemin (HE).Bone marrow-derived macrophages (BMDM) (2 � 106/well) were treated with HEor hemozoin (Hz) (12.5–50 lg/ml) for 6 h (A) or for 18 h (25 lg/ml) (B) after whichthe culture supernatant was removed and the cells were lyzed in equal volumes ofLaemli buffer. Proteins were resolved by SDS–PAGE, transferred onto nitrocellulosemembranes and processed for detection using specific polyclonal antibodies tomurine heme oxygenase-1 (HO-1) or b-actin. Relative expression levels of HO-1were normalized with respect to b-actin by densitometry and data from fourindependent experiments were analyzed using a one-way ANOVA (C). **P < 0.01.Ctrl, control.

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interfering with IL-10 production, inhibition of p38 MAPK maystimulate IL-12 secretion in conditions associated with robustIL-10 responses (Salmon et al., 2001). The stimulatory effect ofSB203580 on IL-12 is obvious at concentrations lower than 5 lM,and correlates in a dose-dependent manner with drops in IL-10levels (Salmon et al., 2001). Based on this information, we evalu-ated whether SB203580 could rescue IL-12p70 responses in HE-conditioned BMDM. As high concentrations of SB203580 may af-fect kinases other than p38 MAPK (Lee et al., 1999; Lali et al.,2000), a 1 lM concentration of inhibitor was used. When assessedat the 1 lM concentration, SB203580 decreased in a reproduciblemanner IL-10 secretion in control BMDM without compromisingthe IL-12p70 response (data not shown). When used 1 h prior toIFNc/LPS stimulation, 1 lM of SB203580 reduced more than 50%phosphorylation of MAP kinase-activated protein kinase 2 (MAP-KAPK-2), which is a specific downstream substrate of p38 MAPK(Fig. 9).

Pre-treatment with SB203580 significantly increased IL-12p70responses to LPS in both control BMDM and HE-conditionedBMDM (Fig. 10A; P < 0.001). However, SB203580 rather accentu-ated the deficiency induced by HE with respect to control BMDMtreated with the inhibitor (Fig. 10A; P < 0.001), whereas it did notaffect IL-12p70 responses to LPS in Hz-conditioned BMDM. In con-trast, inhibition of the p38 MAPK enhanced IL-12p70 responses toIFNc/LPS in HE-conditioned BMDM (Fig. 10B; P < 0.05) abrogatingthe relative inhibitory effects of HE, whereas it further impairedthose responses in Hz-treated BMDM (Fig. 10B). In parallel,SB203580 significantly reduced IL-10 responses to LPS in all groups(Fig. 10C; P < 0.001), and had only a significant negative effect onIL-10 responses to IFNc/LPS in HE-conditioned BMDM (Fig. 10D;P < 0.001).

4. Discussion

The interplay between IL-12 and IL-10 seems relevant for thecontrol of malarial anemia, as its severity is associated with lowlevels of the hematopoietic cytokine IL-12 (Chaisavaneeyakornet al., 2003; Chaiyaroj et al., 2004; Casals-Pascual et al., 2006),and high levels of IL-10 (Dodoo et al., 2002; Lyke et al., 2004; On-g’echa et al., 2008), suggesting a direct detrimental inhibitory ef-fect of IL-10 on IL-12 responses. However, several studies havereported elevated IL-10 levels in patients with uncomplicatedmalarial anemia and a positive contribution by this cytokine inerythropoiesis homeostasis cannot be ruled out (Othoro et al.,1999; Nussenblatt et al., 2001; Lyke et al., 2004; Ageely et al.,2008; Ouma et al., 2008).

Plasmodium infections are characterized by high hemolysis andelevated plasma levels of free heme. Heme is a harmful hydropho-bic molecule that induces ROS once Fe is released in response toHO-1 activity or oxidative degradation (Tenhunen et al., 1968). Inaddition, free heme promotes the recruitment and activation ofneutrophils which further contribute to superoxide productionand oxidative damage (Porto et al., 2007). Herein, the possibleimplications for free heme in malaria dyserythropoiesis wereinvestigated by assessing the regulatory effects of HE on IL-12and IL-10 responses in BMDM.

We hypothesized that HE would modulate IL-12 and IL-10 re-sponses by altering the redox status in macrophages as describedfor diesel exhaust particles (Yin et al., 2004). Indeed, low levelsof reduced GSH may down-regulate IL-12 production and favorIL-10 responses (Murata et al., 2002). Moreover, the priming effectof IFN-c on IL-12p70 production requires elevated GSH/GSSG ra-tios in human monocytes (Utsugi et al., 2002). Our data associatesoxidant properties with HE, characterized by robust ROS responses

Fig. 8. n-Acetylcysteine (NAC) restores IL-12p70 responses and decreases IL-10 responses to IFNc/lipopolysaccharide (LPS) in hemin (HE)-conditioned bone marrow-derivedmacrophages (BMDM). BMDM (1 � 105/well) remained untreated (Ctrl) or were cultured with HE or hemozoin (Hz) (25 lg/ml) for 18 h prior to LPS (A and C) or IFNc/LPS (Band D) stimulation. NAC (0.25 mM) was added into the culture medium 1 h before stimulation. Twenty-four hour culture supernatants were harvested and assessed for IL-12p70 (A and B) and IL-10 content (C and D) by ELISA. Error bars represent the mean ± SEM of four independent experiments. A one-way ANOVA was used to compare IL-12p70 and IL-10 values with respect to respective BMDM stimulated in the absence of NAC. The percentage of inhibition on IL-12p70 responses with respect to controls areindicated by underlined numbers. ***P < 0.001; *P < 0.05.

Fig. 9. 4-[5-(4-Fluorophenyl)-2-[4-(methylsulfonyl)phenyl]-1H-imidazol-4-yl]pyridine (SB203580) efficiently inhibits p38 mitogen-activated protein kinase (MAPK) activityin bone marrow-derived macrophages (BMDM). BMDM (4 � 106/well) were treated with hemin (HE) or hemozoin (Hz) (25 lg/mL) for 18 h and stimulated with IFNc (100 U/ml) and lipopolysaccharide (LPS) (50 ng/ml) for 30 and 60 min. Proteins in total cell lysates were separated by SDS–PAGE and the levels of phospho-p38 MAPK, phospho-MAPkinase-activated protein kinase 2 (MAPKAPK-2) (in the absence of, or with 1 lM SB203580 pre-exposure for 1 h), and total p38 MAPK were revealed by Western blots usingspecific monoclonal antibodies. Results are representative of two independent experiments.

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and enhanced intracellular levels of GSSG, which persist despitethe induction of HO-1.

HE has important inhibitory effects on the priming of IL-12p70responses to LPS by IFNc and this inhibition associates both withdecreases in IL-12p40 and IL-12p35 mRNA levels and enhancedIL-10 responses. These results suggest that this inhibition is conse-quent to the fact that HE-conditioned BMDM are rendered insensi-tive to the inhibitory effects of IFNc on IL-10 responses. In thiscontext, HE may possibly interfere with activation of the glycogensynthase kinase-3 (GSK-3), an enzyme known to repress the activ-ity of the transcriptional factors AP-1 and CRP, required for IL-10gene expression (Hu et al., 2006). In addition, since secretion ofIL-12p40 is minimally affected by HE, we also assume that itsinhibitory effect on IL-12p35 gene transcription is the limiting fac-

tor for production of bioactive IL-12p70. This assumption is furthercorroborated by the fact that IL-12p40 gene expression and secre-tion is known to largely exceed IL-12p70 levels and that post-tran-scriptional control of gene expression may be induced by IL-10 inmurine macrophages (Bogdan et al., 1992; Aste-Amezaga et al.,1998).

Taken together, our results suggest that free heme may contrib-ute to malarial anemia by inhibiting IL-12p70 responses and limit-ing its beneficial effects on erythropoiesis. As administration ofIL-12 has shown major stimulatory effects on erythropoiesis inIFNc deficient mice, a beneficial effect for free heme on erythropoi-esis cannot be excluded. It is possible that IFNc may exertdetrimental effects on erythropoiesis, i.e., by down-regulatingthe production of anti-inflammatory IL-10. Thus, by rendering

Fig. 10. Inhibition of p38 mitogen-activated protein kinase (MAPK) restores IL-12p70 responses to IFNc/lipopolysaccharide (LPS only in hemin (HE)-conditioned bonemarrow-derived macrophages (BMDM). BMDM (1 � 105/well) remained untreated (Ctrl) or were cultured in the presence of HE or hemozoin (Hz) (25 lg/ml) for 18 h andstimulated with LPS (50 ng/ml) (A and C) or pre-treated with IFNc (100 U/ml) for 15 min prior to stimulation with LPS (B and D). 4-[5-(4-Fluorophenyl)-2-[4-(methylsulfonyl)phenyl]-1H-imidazol-4-yl]pyridine (SB203580) (1 lM) was added to the culture medium 1 h before stimulation. Twenty-four hour culture supernatantswere harvested and assessed for IL-12p70 (A and B) and IL-10 content (C and D) by ELISA. Error bars represent the mean ± SEM of four independent experiments; thepercentages of inhibition with respect to control BMDM are indicated with underlined numbers. A one-way ANOVA was used to compare values with respect to BMDM in theabsence of SB203580 treatment. ***P < 0.001; *P < 0.05.

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macrophages refractory to the inhibitory effects of IFNc on IL-10responses, free heme may also contribute to decreased TNFa pro-duction (Kontoyiannis et al., 2001; Denys et al., 2002) which is amajor player in malarial dyserythropoiesis (Felli et al., 2005; Bucket al., 2008). Indeed, HE significantly inhibits TNFa responses toIFNc/LPS in BMDM (data not shown), an effect that has been cor-roborated in the human monocytic cell line THP-1 (Silver et al.,1997).

A collateral observation in our study concerns the detrimentaleffect that Hz has on IL-10 responses in BMDM, which contrastswith the stimulatory effects measured in human PBMC, whichhave been linked to the inhibition of IL-12p40 gene expressionand to impaired IL-12p70 responses (Keller et al., 2006). In thiscontext, we also measured over-production of IL-10 and impairedIL-12p70 responses to LPS and IFNc/LPS in murine peritoneal exu-dates cells pre-conditioned with Hz (data not shown), which maysuggest functional differences between BMDM and peritonealmacrophages/PBMC. However, when using total cell exudates,the contribution of other cells (T cells and B cells) in the IL-10 re-sponse cannot be excluded. As accumulation of Hz in intervilliousBMC correlates with impaired IL-10 and TNFa responses duringnatural infection (Perkins et al., 2003), we suggest that the regula-tory effects of Hz on cytokines may indeed differ among cell types,and the anti-oxidant defences of target cells may contribute to thereported variability. In agreement with our data, the deficient IL-12response measured in peritoneal macrophages from Plasmodiumberghei-infected mice, which may involve both the activities ofHE and Hz, is not efficiently reverted by in vitro neutralization ofIL-10 compared with the enhanced response measured in controlmacrophages treated similarly (Xu et al., 2001). As Hz impairs bothIL-12p70 and IL-10 responses to LPS, it may possibly affect an earlyevent in TLR signalling. In this context, by increasing GSH/GSSG

ratios and hampering ROS responses in BMDM, Hz may affect theactivity of the NADPH oxidase complex, as has been observed inhuman monocyte-derived macrophages (Schwarzer and Arese,1996). Direct interaction of NADPH oxidase with TLR-4 is requiredfor LPS-dependent ROS generation and concomitant activation ofnuclear factor-jB (NF-jB) (Park et al., 2004), highlighting the pos-sibility for long-term treatment with Hz to interfere with NF-jBROS-dependent signalling.

Although inhibition of the p38 MAPK enhanced IL-12p40 re-sponses to LPS in HE and Hz-conditioned BMDM (data not shown),it had a major stimulatory effect on IL-12p70 responses to LPS incontrol BMDM. A comparable effect was measured in response toNAC, for which this reducing agent also had minor stimulatory ef-fects on IL-12p70 responses to LPS in HE-treated cells comparedwith control BMDM, but significantly restored these responses inHE-conditioned BMDM stimulated with IFNc/LPS. The significanceof these differences remains to be investigated, but it is temptingto speculate that HE may up-regulate IL-10 responses in LPS-stim-ulated cells, partially through a p38 MAPK-independentmechanism.

In conclusion, our data strongly suggest that HE inhibits IL-12p70 responses, partially through a redox-dependent mechanismthat sustains IL-10 responses, despite the presence of IFNc, andwhich in contrast to oxidants such as diesel exhaust particles, doesnot rely on modulation of IL-12p40 gene expression (Ohtani et al.,2005). The impact of these effects on erythropoiesis in vivo re-mains to be evaluated, as HE stimulates the development of earlystage erythroid precursors in vitro (Monet and Holden, 1982).In addition, the relative contributions of HE and Hz in dyserythro-poiesis may also depend on their mechanism of entry into macro-phages and on their interaction with hematopoietic precursors. Hzis readily phagocytosed and accumulates rapidly in macrophages,

M. Cambos et al. / International Journal for Parasitology 40 (2010) 1003–1012 1011

also interacting with cell membranes (Schwarzer et al., 1999). Onthe other hand, HE is an amphipathic molecule with high affinitywith cell membranes, binding to lipoproteins in plasma, includinglow density lipoproteins (LDL) which may represent a relevantadditional entrance to macrophages in vivo (Camejo et al., 1998).Thus, Hz and HE might not interact in comparable amounts withmacrophages in vivo. Finally, due to its affinity with membranes,it is possible that HE may alter the function of cells other thanphagocytes, such as T and B cells, which contribute to the systemiccytokine response in malaria.

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