Heme amplifies the innate immune response to microbial molecules through spleen tyrosine kinase (Syk)-dependent reactive oxygen species generation.

Abstract

Infectious diseases that cause hemolysis are among the most threatening human diseases, because of severity and/or global distribution. In these conditions, hemeproteins and heme are released, but whether heme affects the inflammatory response to microorganism molecules remains to be characterized. Here, we show that heme increased the lethality and cytokine secretion induced by LPS in vivo and enhanced the secretion of cytokines by macrophages stimulated with various agonists of innate immune receptors. Activation of nuclear factor κB (NF-κB) and MAPKs and the generation of reactive oxygen species were essential to the increase in cytokine production induced by heme plus LPS. This synergistic effect of heme and LPS was blocked by a selective inhibitor of spleen tyrosine kinase (Syk) and was abrogated in dendritic cells deficient in Syk. Moreover, inhibition of Syk and the downstream molecules PKC and PI3K reduced the reactive oxygen species generation by heme. Our results highlight a mechanism by which heme amplifies the secretion of cytokines triggered by microbial molecule activation and indicates possible pathways for therapeutic intervention during hemolytic infectious diseases.

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Heme Amplifies the Innate Immune Response to Microbial
Molecules through Spleen Tyrosine Kinase (Syk)-dependent
Reactive Oxygen Species Generation*□
Receivedforpublication,May21,2010,andinrevisedform,July16,2010 Published,JBCPapersinPress,August20,2010,DOI10.1074/jbc.M110.146076
Patricia L. Fernandez‡§1, Fabianno F. Dutra‡1, Letícia Alves‡, Rodrigo T. Figueiredo‡¶, Diego Moura ˜o-Sa?,
Guilherme B. Fortes‡, Sophie Bergstrand‡, David Lo ¨nn‡, Ricardo R. Cevallos§, Renata M. S. Pereira**,
Ulisses G. Lopes**, Leonardo H. Travassos‡**, Claudia N. Paiva‡, and Marcelo T. Bozza‡2
Fromthe‡Laborato ´riodeInflamac ¸a ˜oeImunidade,DepartamentodeImunologia,InstitutodeMicrobiologia,Universidade
FederaldoRiodeJaneiro,21941-902RiodeJaneiro,Brazil,the§InstitutodeInvestigacionesCientíficasyServiciosdeAlta
Tecnologia,0843-01103CiudaddePanama ´,Panama ´,the¶Po ´lodeXere ´m,InstitutodeCie ˆnciasBiome ´dicas,UniversidadeFederal
doRiodeJaneiro,21941-902RiodeJaneiro,Brazil,the?ImmunobiologyLaboratory,CancerResearchUK,LondonResearch
Institute,Lincoln’sInnFieldsLaboratories,LondonWC2A3PX,UnitedKingdom,andthe**InstitutodeBiofísicaCarlosChagas
Filho,UniversidadeFederaldoRiodeJaneiro,21941-902 Rio de Janeiro, Brazil
S
Infectious diseases that cause hemolysis are among the most
threatening human diseases, because of severity and/or global
distribution. In these conditions, hemeproteins and heme are
released, but whether heme affects the inflammatory response
to microorganism molecules remains to be characterized. Here,
we show that heme increased the lethality and cytokine secre-
tion induced by LPS in vivo and enhanced the secretion of cyto-
kinesbymacrophagesstimulatedwithvariousagonistsofinnate
immunereceptors.Activationofnuclearfactor?B(NF-?B)and
MAPKs and the generation of reactive oxygen species were
essential to the increase in cytokine production induced by
heme plus LPS. This synergistic effect of heme and LPS was
blocked by a selective inhibitor of spleen tyrosine kinase (Syk)
and was abrogated in dendritic cells deficient in Syk. Moreover,
inhibition of Syk and the downstream molecules PKC and PI3K
reduced the reactive oxygen species generation by heme. Our
results highlight a mechanism by which heme amplifies the
secretion of cytokines triggered by microbial molecule activa-
tion and indicates possible pathways for therapeutic interven-
tion during hemolytic infectious diseases.
A general consequence of infectious diseases that cause
hemolysis,internalhemorrhage,orextensivecelldamageisthe
releaseofhemeproteins.Uponoxidation,hemeproteinsrelease
heme, a potentially harmful molecule (1). Heme-binding
plasma proteins, such as hemopexin or albumin, remove the
intravascular free heme, subsequently degraded by heme oxy-
genase-1 (HO-1), generating equimolar amounts of biliverdin,
carbon monoxide, and free iron (2, 3). HO-1-deficient mice
(Hmox?/?)havehighplasmaconcentrationsofhemeandshow
increased susceptibility to LPS-induced lethality, associated
with inflammation and oxidative damage (4). Accumulation of
large amounts of heme might overwhelm the capacity of heme
scavengers and degrading system, thus causing oxidative stress
and inflammation (5, 6). In fact, recent studies suggest that
heme,incombinationwithROS3andinflammatorymediators,
increase blood brain barrier leakage and hepatocyte necrosis in
models of malarial infection (7, 8).
Hemolysis or hemoglobinemia are associated with increased
mortality in septic patients (9, 10). Hemoglobin increases the
secretionofTNFtriggeredbyLPS,whereasglobinhasaninhib-
itory effect (11), suggesting that heme is responsible for the
cytokine amplification. Heme has several pro-inflammatory
activities, including leukocyte activation and migration, up-
regulation of adhesion molecules, ROS production, and induc-
tion of cytokine expression (12–14). Recently, we have shown
that heme is able to activate Toll-like receptor 4 (TLR4) induc-
ing TNF on macrophages and dendritic cells (DC) (15).
Mammalian pattern recognition receptors (PRRs) recognize
conserved microbial molecules from all classes of microorgan-
isms (16, 17). The activation of these receptors elicits selective
intracellular signaling cascades that result in the production of
cytokines, chemokines, lipid mediators, and reactive oxygen/
nitrogen species. The synergistic effect of certain associations
of PRR agonists on cytokine secretion by macrophages and DC
promotes the inflammatory response and the activation of the
adaptive immune system (18). The increased secretion of cyto-
kines is also achieved by combinations of microbial molecules
with molecules from host origin such as cytokines, ATP, and
ROS (19, 20). This activation of the immune system is consid-
ered essential for pathogen killing but is also critically involved
intissuedamageandsepsis(19).Inthisstudy,weinvestigatedif
heme would be able to affect the inflammatory response trig-
* This work was supported by Conselho de Desenvolvimento Científico e
Tecnolo ´gico,Fundac ¸a ˜odeAmparoa ` PesquisadoEstadodoRiodeJaneiro,
Fundac ¸a ˜o Jose ´ Bonifa ´cio, and Programa de Nu ´cleos de Excele ˆncia, Secre-
taria Nacional de Ciencia Tecnologia e Innovacion de Panama.
□
STheon-lineversionofthisarticle(availableathttp://www.jbc.org)contains
supplemental Figs. 1–4.
1Both authors contributed equally to this work.
2To whom correspondence should be addressed. Tel.: 55-21-22700990; Fax:
55-21-2560834; E-mail: mbozza@micro.ufrj.br.
3Theabbreviationsusedare:ROS,reactiveoxygenspecies;DC,dendriticcell;
DPI, diphenyleneiodonium; NAC, N-acetyl-L-cysteine; TLR, Toll-like recep-
tors; PRR, pattern recognition receptor; CM-H2DCFDA, 5-(and-6)-chloro-
methyl-2?,7?-dichlorodihydrofluorescein diacetate, acetyl ester; ITAM,
immunoreceptor tyrosine-based activation motif; NOD, nucleotide-bind-
ing oligomerization domain.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 43, pp. 32844–32851, October 22, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc.Printed in the U.S.A.
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gered by microbial molecules. Heme caused a remarkable
increase in the production of cytokines induced by microbial
molecules and the lethality induced by LPS. We demonstrate
that the heme-LPS synergistic effect on cytokine secretion and
the generation of ROS by heme were both dependent on Syk
activation.
EXPERIMENTAL PROCEDURES
Mice—C57BL/6 mice were supplied by the UFRJ breeding
facilities(RiodeJaneiro,Brazil)orbredatCancerResearchUK
in specific pathogen-free conditions. Fetal liver cells from WT
embryos or Syk?/?embryos were used to generate radiation
chimeras in C57BL/6 hosts, as described previously (22).
Tlr4?/?, Myd88?/?, Asc?/?, Rip2?/?, and Syk?/?mice were
kindly provided by S. Akira, A. Coyle, V. Tybulewicz, D. Zam-
boni,andN.Camera.Theanimalswerekeptataconstanttem-
perature (25 °C) with free access to chow and water in a room
with a 12-h light/dark cycle. The animal protocols were
approved by the Centro de Cie ˆncias da Sau ´de, Universidade
Federal do Rio de Janeiro Animal Ethics Committee and by
the London Research Institute Animal Ethics Committee and
were performed under the authority of the Animal Scientific
Procedures Act of 1986 (United Kingdom).
Reagents—LPS0111:B4fromEscherichiacoli,CpG,synthetic
bacterial lipopeptide (Pan3Cys-Ser-(Lys)4), poly(I-C), were
obtained from InvivoGen; FK565 was from Astellas Pharma-
ceutical,andMDPwasfromCalbiochem.Hemewaspurchased
from Frontier Scientific, Logan, UT. Heme was dissolved in
NaOH (0.1 M), diluted in RPMI 1640 medium, and filtered.
Stock solutions of heme were prepared immediately before use
inthedarktoavoidthegenerationoffreeradicals.Diphenylene
iodonium (DPI), N-acetyl-L-cysteine (NAC), rotenone, allo-
purinol, L-NAME, piceatannol, paraquat, and SP600125 were
obtained from Sigma. CM-H2DCFDA was obtained from
Invitrogen. PD98059, SB203580, calphostin C, wortmannin,
and wedelolactone were from Calbiochem. RPMI 1640 me-
diumformacrophageculturewasobtainedfromSigmaandwas
supplemented with penicillin/streptomycin and fetal calf
serum (FCS) from Invitrogen.
Hemolysis and Heme in Endotoxemic Mice—Mice (n ? 10)
C57Bl/6 received an intraperitoneal injection of phenylhydr-
azine (0.15 mg/g in a volume of 200 ?l) and were injected with
LPS (10 mg/kg) 4 h later. Control groups received an intraper-
itoneal injection of LPS or phenylhydrazine alone. Mice were
monitored for 72 h. Mice (n ? 10) C57Bl/6 were injected intra-
peritoneallywithheme(50mg/kg)andLPS(10mg/kg).Control
groups received an intraperitoneal injection of LPS or heme
alone. Mice were monitored for 72 h. C67Bl/6 mice were
injected intraperitoneally with saline solution, heme (50
mg/kg), LPS (1 mg/kg), or heme plus LPS. Three and 6 h after
injection,animalsweresacrificed,andtheirblood(toisolatethe
plasma) was collected and stored at ?20 °C for cytokine
determinations.
CellCultureandStimulation—Peritonealmacrophageswere
obtained 4 days after intraperitoneal inoculation of 2 ml of 3%
thioglycollate by peritoneal washes with chilled RPMI 1640
medium. Cells were seeded at 2 ? 105/well in 96-well plates in
RPMI 1640 medium with 10% FCS. Nonadherent cells were
removed by washing, and adherent cells were stimulated as
indicated in the figure legends. Supernatants were collected at
the intervals indicated in the figure legends and frozen until
cytokine determination. Bone marrow was harvested from the
tibias and femurs from WT mice and differentiated into bone
marrow-derived macrophages as described previously (21).
Briefly, the cells were resuspended at 4 ? 106/10 ml in RPMI
1640mediumsupplementedwith20%ofFCSand30%ofsuper-
natantfromL929cellscultures.After4days,freshmediumwas
addedtothecellcultures.Macrophageswerecollectedatday7,
and 2 ? 105/well were plated in 96-well plates in RPMI 1640
medium with 10% of FCS. Nonadherent cells were removed,
and adherent cells were stimulated as indicated in the figure
legends. Supernatants were collected and frozen for cytokine
determinations. Bone marrow-derived dendritic cells (DC)
were prepared as described previously (22). Briefly, bone mar-
row was harvested from the tibias and femora from WT or
chimericmiceanddifferentiatedintoDC.Thecellswereresus-
pendedina24-mlfinalvolumeofRPMI1640mediumcontain-
ing 10% FCS, 50 ?M ?-mercaptoethanol, 100 units/ml penicil-
lin/streptomycin, 1 mM sodium pyruvate, 10 mM HEPES,
nonessentialaminoacids,and200units/mlGM-CSF.Fourmil-
lilitersofcellsuspensionwereplatedperwellintoa6-wellplate
(1?plate/mouse).Cellswereincubatedat37 °Cand5%CO2.A
partial medium change was performed after 2 days of culture.
On day 3 of culture, all nonadherent cells were removed, and 4
ml of a new complete medium and 200 units/ml rmGM-CSF
wereaddedtoeachwell.Onday5,DCwereharvestedinPBS,2
mM EDTA and enriched using CD11c-microbeads (Miltenyi
Biotec).DCwereplatedat2?105/wellina96-wellplatefor2h
in RPMI 1640 medium with 10% of FCS. Cells were stimulated
as indicated in the figure legends. Supernatants were collected
and frozen for cytokine determination. Cell viability was ana-
lyzed by LDH measurement in supernatants collected after the
4-h stimulation period for all treatments with selective inhibi-
torsincombinationwithhemeandLPS.Cellviabilityoftreated
groups was similar to the nonstimulated controls.
Cytokine Measurements—The concentrations of TNF, IL-6,
andIP-10weredeterminedusingELISA(PeproTechandR&D
Systems). All the measurements were performed in duplicate
according to the manufacturer’s instructions.
Western Blot Analysis for Syk, MAPKs Phosphorylation, and
I?B? Degradation—Elicited peritoneal macrophages were
plated in 6-well plates at a density of 2.0 ? 106cells/well. Non-
adherent cells were removed by washing with medium, and
adherent cells were stimulated as indicated in the figure leg-
ends. After 30 min, cells were lysed in a buffer consisting of
Tris-HCl (50 mM), NaCl (150 mM), 1% Nonidet P-40, 0.25%
sodium deoxycholate, EDTA (1 mM), aprotinin (5 mg/ml), leu-
peptin (5 mg/ml), pepstatin (5 mg/ml), PMSF (1 mM), sodium
orthovanadate (1 mM), and NaF (1 mM), pH 7.5. Cell lysates
were centrifuged, and 20 ?g of protein contained in the super-
natants were boiled and subjected to electrophoresis in SDS-
polyacrylamidegelinreducingconditions.Proteinsweretrans-
ferredtoanitrocellulosemembraneat4 °Cfor2h.Membranes
were blocked with Tris-buffered saline solution with 0.05% of
Tween20(TBS-T)and5%offat-freemilk,incubatedovernight
with anti-phospho (p)-ERK1/2 (1:1000), anti-p-JNK (1:1000)
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(Santa Cruz Biotechnology), anti-p-p38 (1:1000), or anti-p-Syk
(1:1000) (Cell Signaling Technology) diluted in blocking solu-
tion. Membranes were washed in TBS-T and incubated for 2 h
with horseradish peroxidase-conjugated goat anti-rabbit
(1:10,000) or goat anti-mouse (1:10,000) IgG polyclonal anti-
bodies(SantaCruzBiotechnology).Thebandswererevealedby
chemiluminescence, using ECL substrate (Santa Cruz Biotech-
nology). The normalization of Syk and MAPKs phosphoryla-
tion was performed by stripping membranes during 30 min at
50 °C in stripping buffer (100 mM ?-mercaptoethanol, 2% SDS,
62.5 mM Tris-HCl, pH 6.7). After stripping, membranes were
washed with TBS-T, blocked with 5% fat-free milk TBS-T,
incubated overnight with rabbit anti-?-actin (1:1000) (Sigma)
forp-Sykmembranesorrabbitanti-ERK2(1:1000)(SantaCruz
Biotechnology) for MAPK membranes. Detection was per-
formedasdescribedabove.ThedegradationofI?Bproteinwas
analyzed by Western blot with polyclonal rabbit anti-I?B
(Sigma) diluted (1:1000) in block solution. Detection of non-
phosphorylated ERK2 was used as loading control.
Quantitative Real Time RT-PCR—Total RNA was extracted
using TRIzol (Invitrogen), and 1 ?g was reverse-transcribed
using M-MLV reverse transcriptase and random hexamer
primers (Invitrogen). Subsequent quantitative real time PCR
analysis was performed on an ABI 7500 (Applied Biosystems)
using SYBR Green master mix (Applied Biosystems). Amplifi-
cation conditions were as follows: 95 °C (10 min), 40 cycles of
95 °C (15 s), and 60 °C (60 s). All data were normalized to the
corresponding HPRT expression, and the fold difference rela-
tivetothecontrollevelwasshown.Theanalysesofrelativegene
expression data were performed by the 2???CTmethod. TNF
primers are forward 5?-GGTCCCCAAAGGGATGAGAAG-
TTC-3? and reverse 5?-CCACTTGGTGGTTTACTACGACG-
3?; HPRT primers are forward 5?-GCTGGTGAAAAGGACC-
TCT-3? and reverse 5?-CACAGGACTAGAACACCTGC-3?.
Electrophoretic Mobility Shift Assay—Nuclear extracts were
obtained and analyzed for NF-?B activation by EMSA, as
described previously (23). Binding reactions were performed
using 1 ?g of nuclear protein in the presence of 40,000 cpm of
32P-end-labeled double-stranded consensus NF-?B oligonu-
cleotide (sequence 5?-AGT TGA GGG GAC TTT CCC AGG
C-3?, Santa Cruz Biotechnology) and 1 ?g of poly(dI-dC)?(dI-
dC) (Amersham Biosciences) in binding buffer (50 mM HEPES,
pH 7.9, 20% glycerol, 5 mM DTT, 5 mM EDTA, and 0.5 ?g of
BSA) for 30 min at room temperature. The DNA-protein com-
plex formed was separated from free oligonucleotide on 4%
native polyacrylamide gel.
Reactive Oxygen Species Generation—Macrophages (106
cells/well) were stimulated in the presence of 10% FCS with
hemeduring1hafterthetreatmentwithdeferoxamine,piceat-
annol, wortmannin, or calphostin C. After the stimulus, cells
were incubated with 2 ?M 5-(and-6)-chloromethyl-2?,7?-
dichlorodihydrofluoresceindiacetate,
H2DCFDA) in RPMI 1640 medium for 30 min at 37 °C under
5% CO2atmosphere. ROS production was analyzed by flow
cytometry using FACScan flow cytometer (BD Biosciences).
Statistical Analysis—Data are presented as means ? S.E.
Results were analyzed using a statistical software package
(GraphPad Prism 4). Statistical analyses were performed by
acetyl ester (CM-
one-way analysis of variance and Newman-Keuls post test. A
significant difference between groups was considered if p ?
0.05.Comparisonsofsurvivalcurveswereanalyzedbylog-rank
(Mantel-Cox test).
RESULTS
HemolysisandHemeIncreasedLethalityInducedbyaSuble-
thal Dose of LPS—Previous studies have shown that hemolysis
or hemoglobinemia is associated with increased mortality in
patientswithsepsis(9,10).Toinvestigatewhetherendogenous
heme affects the host response to microbial products without
interferingwithbacterialgrowth,weusedanestablishedmodel
of hemolysis caused by phenylhydrazine injection and chal-
lengedmicewithasublethaldoseofLPS.Thisprotocolinduced
a striking increase in lethality compared with LPS or phenylhy-
drazine alone (Fig. 1A). To define if exogenous heme could
synergize with LPS in vivo, to cause mortality, we treated mice
with heme and challenged them with a sublethal dose of LPS.
This combination also caused a significant increase in lethality
compared with the groups receiving LPS or heme alone (Fig.
1B). The combined treatment with heme and LPS caused a
significant increase in plasma TNF and IL-6 compared with
LPSorhemealoneat3and6hafterchallenge,respectively(Fig.
1,CandD).Theseresultsindicatethathemeincreasesboththe
lethality and cytokine concentration induced by LPS in vivo.
Heme Increased the Secretion of Cytokines Induced by LPS in
Macrophages—Macrophages are important sources of inflam-
matorycytokinesduringinfectionandinflammation.Todefine
the mechanism by which heme enhances cytokine secretion
induced by LPS, macrophages were stimulated with increasing
concentrations of heme and LPS. In the presence of serum,
stimulation with heme or low concentrations of LPS caused a
negligiblesecretionofTNForIL-6,whereasthecombinationof
treatmentsinducedarobustsecretionofthesecytokines(Fig.2,
A and B). This potentiating effect of heme upon LPS stimula-
tion was dose-dependent and was observed at concentrations
of 30 and 100 ?M. Heme also potentiated the secretion of IP-10
induced by LPS (data not shown). These results indicate that
heme increases the secretion of MyD88- and TIR domain-con-
taining adaptor-inducing interferon ?-dependent cytokines
induced by LPS on macrophages.
Heme Increased the Cytokine Secretion Induced by Different
TLR and NOD Agonists—To define whether the effect of heme
was exclusive to LPS stimulation, we used agonists of TLR2
(Pan3-Cys), TLR3 (poly(I-C)), and TLR9 (CpG). Heme signifi-
cantly enhanced the secretion of TNF and IL-6 induced by all
tested TLR agonists (supplemental Fig. 1, A–C, and data not
shown).NOD-likereceptorsarecytosolicreceptorsinvolvedin
the recognition of microorganisms and viral molecules present
in the cytosol (17). Treatment of macrophages with heme also
increasedtheproductionofTNFinducedbyNOD1andNOD2
agonists (supplemental Fig. 1, D and E). Together, these results
indicate that heme increases the cytokine secretion by macro-
phages activated with TLR and NOD agonists.
Heme Advanced and Increased the Activation of NF-?B and
MAPKs Induced by LPS—Activation of NF-?B is essential for
cytokine production induced by several PRR agonists (16, 17).
To determine the involvement of this pathway on macrophage
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activationinducedbyhemeplusLPS,weinitiallycharacterized
the content of I?B? by Western blot. Treatment of macro-
phageswithhemeplusLPScausedanearlydestructionofI?B?
comparedwiththeeffectofhemeorLPSalone(Fig.3,AandB).
Next,wedeterminedthebindingofNF-?BbyEMSAinnuclear
extracts of macrophages stimulated with heme, LPS, or heme
plus LPS. As early as 30 min of stimulation, heme and LPS
induced a strong activation of NF-?B compared with single
stimulations (Fig. 3C). Treatment with an inhibitor of IKK
inhibited the secretion of TNF in a
concentration-dependent
(Fig. 3D).
Activation of macrophages with
LPS causes the phosphorylation of
MAPKs, considered essential for
the production of several cytokines,
including TNF (16, 17). Thus, we
investigated the effect of heme on
the phosphorylation of MAPKs
stimulatedbyLPS.At15and30min
after stimulation with heme plus
LPS, we observed an increased
phosphorylation of ERK and p38
compared with controls receiving
heme or LPS alone (Fig. 4A). An
increase in JNK was observed at 30
and 60 min after treatment with
heme and LPS (Fig. 4A). Treatment
with ERK, p38, or JNK inhibitors
blocked the potentiating effect of
heme on LPS-induced cytokine
secretion (Fig. 4, B and C). As
expected, we also observed an
increase in TNF mRNA on macro-
phages treated with heme plus LPS
compared with single treatments
(supplemental Fig. 2). These results
indicate that NF-?B and MAPKs
activation are essential to the coop-
erative effect of heme and LPS on
cytokine production.
Induction of ROS Was Involved in
thePotentiatingEffectofHemeupon
LPS Stimulation—ROS participate
as second messengers in several sig-
naling pathways, affecting the acti-
vation of downstream molecules
such as NF-?B and MAPKs and
priming macrophages to subse-
quent activation by LPS (20, 24, 25).
Heme induced a significant genera-
tion of ROS in macrophages (Fig.
5A).Thus,wetestedtheroleofROS
on heme-induced cytokine amplifi-
cation triggered by LPS. Pretreat-
ment with the antioxidant NAC
blocked thecytokine
induced by heme and LPS, whereas
fashion
secretion
DPI, a flavoprotein inhibitor, or rotenone, a mitochondrial
inhibitor, partially inhibited the enhancing effect of heme on
LPS-induced TNF and IL-6 (Fig. 5B and data not shown). Sim-
ilarly, apocynin, a NADPH inhibitor, also blocked the synergis-
tic effect of heme and LPS (data not shown). Treatment with
allopurinol, a xanthine oxidase inhibitor, or with L-NAME, a
NO synthase inhibitor, did not affect the ability of heme to
increase cytokine production induced by LPS (Fig. 5B). The
treatmentofmacrophageswithparaquat,adrugthatcausesthe
FIGURE 1. Hemolysis and heme potentiated the lethal effect of LPS. A, mice (n ? 10) C57Bl/6 received an
intraperitoneal injection of phenylhydrazine (Phz) (0.15 mg/g) in a volume of 200 ?l. Four hours later the
animals were injected with LPS (10 mg/kg). Control (C) groups received an intraperitoneal injection of LPS or
phenylhydrazinealone.Miceweremonitoredfor72h.*,p?0.0001comparedwithLPSinjection.B,mice(n?
10)C57Bl/6wereinjectedintraperitoneallywithheme(50mg/kg)andLPS(10mg/kg).Controlgroupsreceived
an intraperitoneal injection of LPS or heme alone. Mice were monitored for 72 h. *, p ? 0.001 compared with
LPSinjection.CandD,micereceivedheme(50mg/kg)and/orLPS(1mg/kg),andplasmaconcentrationsofTNF
and IL-6 were evaluated 3 and 6 h after the challenge, respectively. n ? 4 *, p ? 0.05 compared with LPS
injection. H, heme; L, LPS.
FIGURE2.HemeincreasedthesecretionofcytokinesinducedbyLPSinmacrophages.A,peritonealmacro-
phagesfromC57Bl/6micewerestimulatedwithheme(10,30,or100?M)1hbeforethestimuluswithLPS(0.1
or 1 ng/ml). After 4 h of stimulus with LPS, the supernatants were collected for TNF (A) and IL-6 (B) determina-
tions by ELISA. Results represents mean ? S.E. for cytokines determinations and are representative of three
different experiments. C, control.
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generation of superoxide, also induced an increase in TNF
secretion triggered by LPS (Fig. 5C). Next, we analyzed the role
of the coordinated iron of heme on cytokine production and
ROS generation. The use of an iron chelator, deferoxamine,
suppressed the effect of heme on cytokine secretion and ROS
generation (Fig. 5, D and E). These results suggest that induc-
tionofROSbyhemeisinvolvedintheincreasedcytokinesecre-
tion triggered by LPS.
Synergistic Effect of Heme Combined with Microbial Mole-
cules on TNF Secretion Required Syk—Macrophages from
Tlr4?/?mice stimulated with heme plus CpG or from
Myd88?/?micestimulatedwithhemepluspoly(I-C)showeda
marked increase in the TNF secretion compared with macro-
phages stimulated with CpG or poly(I-C) alone (supplemental
Fig. 3, A and B). Heme also potentiated the TNF production by
Asc?/?and Rick?/?macrophages activated with LPS (supple-
mentalFig.3,CandD).Together,thesedataindicatethatTLR4
and the adaptor proteins MyD88, ASC, or RICK/Rip2 are
not essential to the potentiating effect of heme upon LPS
stimulation.
Recent studies indicate that receptors that signal through
immunoreceptor tyrosine-based activation motifs (ITAMs)
regulate the signaling by heterologous receptors such as TLRs,
modulating cytokine production by macrophages and DC (18,
26–28).ThiseffectofITAM-associatedreceptorsrequiresSyk,
an essential molecule in downstream activation of NF-?B via
theCARD9adaptor,andofPKCandcalciumsignalingviaacti-
vation of phospholipase C?. These pathways participate on
activationofMAPKs,andSykalsoactivatesPI3KandROSpro-
duction (29). The stimulation of macrophages with heme
causedthephosphorylationofSyk,suggestingaputativeroleof
this pathway on the heme potentiating effect upon LPS stimu-
lation (Fig. 6A). To directly evaluate the role of Syk, we used
piceatannol,aselectiveSykinhibitor(29).Treatmentofmacro-
phages with piceatannol abolished the amplifying effect of
heme on cytokine secretion induced by LPS, CpG, and
poly(I-C) (Fig. 6B and supplemental Fig. 4). The synergistic
inductionofTNFbyhemeandLPSwasalsoobservedusingDC
and was abolished after treatment with piceatannol (Fig. 6C).
More important, the synergism of heme and LPS was absent in
Syk-deficientDC(Fig.6D).ConsideringthecriticalroleofROS
and Syk on heme-induced cytokine amplification, we analyzed
the involvement of Syk in ROS generation induced by heme.
Treatment of macrophages with piceatannol abrogated the
FIGURE 3. Heme increased the activation of NF-?B induced by LPS.
A, macrophages from C57Bl/6 mice were stimulated with heme (H) (100 ?M)
and/or LPS (L) (0.1 ng/ml) in the time intervals indicated. Cell extracts were
submitted to SDS-PAGE. I?B degradation was detected by immunoblotting.
Detection of nonphosphorylated ERK2 was used as loading control (C).
B, densitometric analysis of I?B? degradation relative to negative control in
the 15-min interval. Values are mean ? S.E. of three different experiments. *,
p ? 0.05. C, NF-?B activation was analyzed by EMSA from nuclear extracts in
the time intervals indicated. The figures are representatives of two different
experiments with similar results. D, production of TNF was evaluated in peri-
toneal macrophages treated with wedelolactone (40 or 80 ?M) for 30 min
beforethestimuliwithheme(100?M)andLPS(0.1ng/ml).Thesupernatants
were collected 4 h after the stimuli. Results represent mean ? S.E. for TNF
determination and are representatives of two different experiments. *, p ?
0.05. H, heme; L, LPS; WED, wedelolactone.
FIGURE 4. Heme anticipates and increases the activation of MAPKs
induced by LPS. A, macrophages from C57Bl/6 mice were stimulated with
heme (100 ?M) and/or LPS (0.1 ng/ml) in the time intervals indicated. Cell
extracts were submitted to SDS-PAGE. ERK1/2, JNK, and p38 phosphoryla-
tions were detected by immunoblotting. Detection of nonphosphorylated
ERK2 was used as loading control. The figures are representatives of three
different experiments with similar results. B and C, production of TNF was
evaluated in peritoneal macrophages treated with SB203580 (10 ?M),
PD98059 (30 ?M) (B), or SP600125 (30 ?M) (C) for 30 min before the stimuli
withheme(100?M)andLPS(0.1ng/ml).Thesupernatantswerecollected4h
after the stimuli. Results represent means ? S.E. for TNF determination and
are representative of two different experiments. *, p ? 0.05. H, heme; L, LPS;
SB, SB203580; PD, PD98059; SP, SP600125.
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ROSgenerationbyheme(Fig.6E).Together,theseresultsindi-
cated that activation of Syk is essential to ROS generation by
heme affecting the cytokine production induced by microbial
ligands.
InvolvementofDownstreamEffectorsofSykonHeme-induced
ROS—PKC and PI3K are downstream effectors in the Syk sig-
naling pathway. On neutrophils, we previously observed a crit-
ical role of these molecules on ROS generation (14). Similarly,
PKCandPI3KinhibitorsreducedROSgenerationbyhemeand
blocked the synergistic effect of heme and LPS on cytokine
production (Fig. 7, A–C). Together, these results indicate that
Syk, PKC, and PI3K activation and the subsequent ROS gener-
ation are involved in the potentiating effect of heme upon LPS
stimulation.
DISCUSSION
In malaria, hemorrhagic fevers, leptospirosis, and septic
shock, high quantities of hemoproteins are released, probably
affecting the inflammatory response triggered by microbial
molecules. Here, we showed that heme synergized with micro-
bial products increased lethality and cytokine production in
vivo and in vitro. Hemolysis induced by phenylhydrazine or
treatment with heme caused an increased lethality when com-
bined with a sublethal dose of LPS. The effect of heme on cyto-
kine secretion was not restricted to LPS, because heme also
synergizes with agonists of TLR2, TLR3, TLR9, NOD1, and
NOD2. The increased cytokine production correlated with an
earlierandheightenedactivationofNF-?Bandwithphosphor-
ylationofMAPKsonmacrophagestreatedwithhemeandLPS.
Moreover, inhibition of these pathways abrogated the
increased secretion of TNF. Furthermore, we demonstrated
that generation of oxidative stress by heme was essential for its
potentiating effects upon LPS stimulation. The upstream mecha-
nism of ROS generation by heme and heme-LPS synergism to
increasecytokineproductioninvolvedtheactivationofSyk,PI3K,
andPKC.Together,theseresultsrevealedacriticalandpreviously
unrecognizedroleofhemeinenhancinginnateimmuneresponse
byaffectingthesignalingofPRRsthroughSykactivationandsub-
sequentchangeoftheredoxstateofmacrophages.
A low dose of LPS alone resulted in insignificant cytokine
secretion, whereas the same dose of LPS together with heme
resultedinsubstantialproductionofcytokines,thussuggesting
thatthissynergismmaybeparticularlyimportantundercondi-
tionsoflowagonistconcentrations.HemeaffectedMyD88and
TIR domain-containing adaptor-inducing interferon ? (TRIF)
pathways induced by LPS upon activation of TLR4, increasing
the secretion of TNF, IL-6, and IP-10. Heme also enhanced
cytokinesecretioninducedbyTLR2,TLR9,andTLR3agonists.
These results indicate that heme synergizes with those TLRs
that use exclusively MyD88 (TLR2 and -9), with the TLR that
activates TIR domain-containing adaptor-inducing interferon
?only(TLR3),orwiththatabletoinducebothpathwayssimul-
taneously (TLR4). Moreover, heme increased cytokine secre-
tion induced by NOD agonists. These receptors, in contrast to
TLRs,arelocalizedtothecytosol,insteadofbeingassociatedto
cellmembranes,anduseRICK/RIP2asadaptorprotein(30,31).
Together, these results establish that heme amplifies cytokine
secretion by PRRs situated on distinct locations and using dis-
tinct signal transduction pathways.
ThecombinationofhemeandLPScausedanenhancedI?B?
degradation, NF-?B activation, and MAPKs phosphorylation.
The activation of these pathways was essential to the effect of
heme, because treatment with selective inhibitors abrogated
the increase in cytokine secretion. The redox state severely
affects the response of leukocytes to microbial molecules and
cytokines.ROSaffecttheactivationofNF-?B,akeycomponent
in TLR and NLR signaling pathways, and are required for cyto-
kine production upon receptor activation (16–18). Exog-
enously added H2O2causes the activation of NF-?B and
increasesthesecretionofcytokinesbymacrophagesstimulated
with LPS, whereas antioxidants have an inhibitory effect (20).
The generation of ROS by heme was involved in the potentiat-
ing effect on LPS-induced cytokine secretion. NAC, DPI, and
rotenone impaired this enhanced response, whereas inhibition
ofxanthineoxidaseorNOShadnoeffect.Theseresultssuggest
thatROSgeneratedbyNADPHandmitochondriaareinvolved
on heme-LPS synergism to produce cytokines.
Recent studies demonstrated an intense cooperation of sev-
eralPRRsandITAM-associatedreceptors,demonstratingboth
positiveandnegativeregulation(22,26–29,32–34).Ithasbeen
suggested that activation of ITAM-associated receptors with
high affinity ligands causes cross-inhibition of TLR receptors,
whereas low affinity ligands potentiate the response (34). The
mechanismofthesynergisticinductionofcytokineproduction
FIGURE 5. Heme amplifies the production of cytokines dependently of
ROS. A, macrophages were stimulated for 1 h with heme (100 ?M), and ROS
generation was evaluated by flow cytometry using the probe CM-H2DCFDA (2
?M). B, macrophages were preincubated for 30 min with NAC (10 mM), DPI (10
?M),rotenone(1?M),allopurinol(10?M),orL-NAME(100?M)andstimulatedwith
heme(100?M)andLPS(0.1ng/ml),andafter4hthesupernatantswerecollected
forposteriordeterminationbyELISA.C,macrophageswerestimulatedwithpara-
quat(100?M)andLPS(0.1ng/ml),andafter4hthesupernatantswerecollected
forTNFdeterminationbyELISA.D,macrophageswerepreincubatedfor30min
with deferoxamine (2 ?M) and stimulated with heme (100 ?M) and LPS (0.1
ng/ml),andafter4hthesupernatantswerecollectedforposteriordetermination
byELISA.E,macrophageswerepretreatedwithdeferoxamine(2?M)andstimu-
lated for 1 h with heme (100 ?M). The ROS generation was evaluated by flow
cytometry using the probe CM-H2DCFDA (2 ?M). All stimuli were performed in
the presence of 10% FCS. Results represent mean ? S.E. for cytokines and are
representativeoftwoorthreedifferentexperiments.*,p?0.05.H,heme;L,LPS;
Pq,paraquat;DFO,deferoxamine.
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by TLRs and ITAM-associated receptors seems to be an
enhanced activation of NF-?B and MAPKs (29). We observed
that heme caused the phosphorylation of Syk and that treat-
ment with a selective inhibitor of Syk caused the abrogation
of heme-LPS synergistic effects on macrophages and DC.
Using Syk-deficient DC, we confirmed the critical role of Syk
on heme-LPS synergistic effects. The inhibitors of Syk and
downstream effectors, including
PKC and PI3K, all affected the
generation of ROS and the heme-
LPS synergism on cytokine pro-
duction by macrophages. These
results resemble the ROS produc-
tion induced by Dectin-1 and
TGF-?-activated kinase (TAK1)-
mediated signal in cooperation
with TLR2, which enhances acti-
vation of NF-?B (26).
The mechanism of ROS genera-
tion by heme is considered to be at
least partially dependent on the
Fenton reaction (13). However, we
recently observed that PKC and
PI3K are also involved on ROS gen-
erationinducedbyhemeonneutro-
phils, thus also suggesting a recep-
tor-mediated process (14). A recent
report indicates that monosodium
uratecrystalsactivateSykinDCbya
mechanism independent of protein
receptors in the plasma membrane
and dependent on cholesterol in
lipid rafts (35). H2O2also activates
NF-?B, a process dependent on Syk
(36), thus suggesting a possible
involvement of this pathway on
H2O2-induced synergism on cyto-
kine production induced by LPS. At
presentthereisnoevidenceindicat-
ing that H2O2uses a receptor to
activate this pathway. The involve-
ment of a putative specific ITAM-
associated receptor activated by
heme or, alternatively, Syk activa-
tion by heme in a membrane recep-
tor-independent effect requires fur-
ther investigation.
In conclusion, our results show
that heme is capable of potentiat-
ing cytokine production induced
by different innate receptors irre-
spective of their subcellular local-
ization. This phenomenon ascribes
to heme a general role on modula-
tion of innate responses to patho-
gens and/or NF-?B activation. The
molecular mechanism behind this
effect of heme is the activation of
Syk and subsequent ROS generation.
Acknowledgments—We thank Dr. Shizuo Akira, Dr. Anthony Coyle,
Dr. Victor Tybulewicz, Dr. Dario Zamboni, Dr. Patricia Bozza, and
Dr. Niels Camera for kindly providing mice strains used in this study
and Miguel Rodriguez for the critical review of the manuscript.
FIGURE 6. Syk is involved in the synergistic effect of heme on cytokine secretion. A, macrophages from
C57Bl/6micewerestimulatedwithheme(100?M)inthetimeintervalsindicated.Cellextractsweresubmitted
to SDS-PAGE and Syk phosphorylation was detected by immunoblotting. Detection of ?-actin was used as
loading control. The figure is representative of three different experiments with similar results. B, peritoneal
macrophages; C, dendritic cells from C57Bl/6 were stimulated with heme (100 ?M) and/or LPS (0.1 ng/ml) 30
min after the treatment with 10 ?M piceatannol. *, p ? 0.05. D, DC from Syk?/?and WT mice were stimulated
withheme(100?M)andLPS(1ng/ml),andsupernatantswerecollected4haftertreatment.TNFwasquantified
by ELISA. The stimuli were performed in the presence of 10% FCS. Results represent means ? S.E. and are
representativeoftwodifferentexperiments.*,p?0.05.E,peritonealmacrophagesfromC57Bl/6weretreated
with 10 ?M of the Syk inhibitor piceatannol for 30 min and stimulated with heme (100 ?M) for 1 h. The ROS
generation was evaluated by flow cytometry using the probe CM-H2DCFDA (2 ?M). Results are representative
of three different experiments. H, heme; L, LPS; Pice, piceatannol.
FIGURE7.PKCandPI3KareimportanttoROSgenerationinducedbyheme.Peritonealmacrophagesfrom
C57Bl/6weretreatedwith10?MofthePKCinhibitorcalphostin(A)or50?MofthePI3Kinhibitorwortmannin
(B)for30minandstimulatedwithheme(100?M)for1h.TheROSgenerationwasevaluatedbyflowcytometry
usingtheprobeCM-H2DCFDA(2?M).Inhibitorsplusheme(thickline),heme(filledareacurve),probe(fineline).
Theresultsarerepresentativeofthreedifferentexperiments.C,macrophageswerepretreatedfor30minwith
calphostin (10 ?M) or wortmannin (50 ?M) and were stimulated with heme, LPS, or heme plus LPS. TNF was
quantified by ELISA in supernatants collected 4 h after treatment with heme and LPS. All experiments were
performedwithmediumsupplementedwith10%FCS.Resultsrepresentmean?S.E.andarerepresentativeof
three different experiments. *, p ? 0.05.
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