Produção Nacional
Revisado por pares
Heme Amplifies the Innate Immune Response to Microbial Molecules through Spleen Tyrosine Kinase (Syk)-dependent Reactive Oxygen Species Generation*
2010; Elsevier BV; Volume: 285; Issue: 43 Linguagem: Inglês
10.1074/jbc.m110.146076
ISSN1083-351X
AutoresPatricia L. Fernández, Fabiano Ferreira, Letícia S. Alves, Rodrigo T. Figueiredo, Diego Mourão‐Sá, Guilherme B. Fortes, Sophie Bergstrand, David Lönn, Ricardo R. Cevallos, Renata M. Pereira, Ulisses Gazos Lopes, Leonardo H. Travassos, Cláudia N. Paiva, Marcelo T. Bozza,
Tópico(s)Inflammasome and immune disorders
ResumoInfectious 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. 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. IntroductionA general consequence of infectious diseases that cause hemolysis, internal hemorrhage, or extensive cell damage is the release of hemeproteins. Upon oxidation, hemeproteins release heme, a potentially harmful molecule (1Ferreira A. Balla J. Jeney V. Balla G. Soares M.P. J. Mol. Med. 2008; 86: 1097-1111Crossref PubMed Scopus (155) Google Scholar). Heme-binding plasma proteins, such as hemopexin or albumin, remove the intravascular free heme, subsequently degraded by heme oxygenase-1 (HO-1), generating equimolar amounts of biliverdin, carbon monoxide, and free iron (2Muller-Eberhard U. Fraig M. Am. J. Hematol. 1993; 42: 59-62Crossref PubMed Scopus (67) Google Scholar, 3Morse D. Choi A.M. Am. J. Respir. Cell Mol. Biol. 2002; 27: 8-16Crossref PubMed Scopus (442) Google Scholar). HO-1-deficient mice (Hmox−/−) have high plasma concentrations of heme and show increased susceptibility to LPS-induced lethality, associated with inflammation and oxidative damage (4Poss K.D. Tonegawa S. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 10925-10930Crossref PubMed Scopus (1100) Google Scholar). Accumulation of large amounts of heme might overwhelm the capacity of heme scavengers and degrading system, thus causing oxidative stress and inflammation (5Muller-Eberhard U. Javid J. Liem H.H. Hanstein A. Hanna M. Blood. 1968; 32: 811-815Crossref PubMed Google Scholar, 6Jeney V. Balla J. Yachie A. Varga Z. Vercellotti G.M. Eaton J.W. Balla G. Blood. 2002; 100: 879-887Crossref PubMed Scopus (501) Google Scholar). In fact, recent studies suggest that heme, in combination with ROS 3The abbreviations used are: ROSreactive oxygen speciesDCdendritic cellDPIdiphenyleneiodoniumNACN-acetyl-l-cysteineTLRToll-like receptorsPRRpattern recognition receptorCM-H2DCFDA5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl esterITAMimmunoreceptor tyrosine-based activation motifNODnucleotide-binding oligomerization domain. and inflammatory mediators, increase blood brain barrier leakage and hepatocyte necrosis in models of malarial infection (7Pamplona A. Ferreira A. Balla J. Jeney V. Balla G. Epiphanio S. Chora A. Rodrigues C.D. Gregoire I.P. Cunha-Rodrigues M. Portugal S. Soares M.P. Mota M.M. Nat. Med. 2007; 13: 703-710Crossref PubMed Scopus (419) Google Scholar, 8Seixas E. Gozzelino R. Chora A. Ferreira A. Silva G. Larsen R. Rebelo S. Penido C. Smith N.R. Coutinho A. Soares M.P. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 15837-15842Crossref PubMed Scopus (213) Google Scholar).Hemolysis or hemoglobinemia are associated with increased mortality in septic patients (9Griffiths E. Cortes A. Gilbert N. Stevenson P. MacDonald S. Pepper D. Lancet. 1995; 345: 158-160Abstract PubMed Google Scholar, 10Su D. Roth R.I. Yoshida M. Levin J. Infect. Immun. 1997; 65: 1258-1266Crossref PubMed Google Scholar). Hemoglobin increases the secretion of TNF triggered by LPS, whereas globin has an inhibitory effect (11Yang H. Wang H. Bernik T.R. Ivanova S. Wang H. Ulloa L. Roth J. Eaton J.W. Tracey K.J. Shock. 2002; 17: 485-490Crossref PubMed Scopus (31) Google Scholar), 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 induction of cytokine expression (12Graça-Souza A.V. Arruda M.A. de Freitas M.S. Barja-Fidalgo C. Oliveira P.L. Blood. 2002; 99: 4160-4165Crossref PubMed Scopus (222) Google Scholar, 13Wagener F.A. Volk H.D. Willis D. Abraham N.G. Soares M.P. Adema G.J. Figdor C.G. Pharmacol. Rev. 2003; 55: 551-571Crossref PubMed Scopus (465) Google Scholar, 14Porto B.N. Alves L.S. Fernández P.L. Dutra T.P. Figueiredo R.T. Graça-Souza A.V. Bozza M.T. J. Biol. Chem. 2007; 282: 24430-24436Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Recently, we have shown that heme is able to activate Toll-like receptor 4 (TLR4) inducing TNF on macrophages and dendritic cells (DC) (15Figueiredo R.T. Fernandez P.L. Mourao-Sa D.S. Porto B.N. Dutra F.F. Alves L.S. Oliveira M.F. Oliveira P.L. Graça-Souza A.V. Bozza M.T. J. Biol. Chem. 2007; 282: 20221-20229Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar).Mammalian pattern recognition receptors (PRRs) recognize conserved microbial molecules from all classes of microorganisms (16Medzhitov R. Nature. 2007; 449: 819-826Crossref PubMed Scopus (1966) Google Scholar, 17Akira S. Uematsu S. Takeuchi O. Cell. 2006; 124: 783-801Abstract Full Text Full Text PDF PubMed Scopus (8561) Google Scholar). 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 (18Trinchieri G. Sher A. Nat. Rev. Immunol. 2007; 7: 179-190Crossref PubMed Scopus (1065) Google Scholar). The increased secretion of cytokines is also achieved by combinations of microbial molecules with molecules from host origin such as cytokines, ATP, and ROS (19Nathan C. Nature. 2002; 420: 846-852Crossref PubMed Scopus (1980) Google Scholar, 20Powers K.A. Szászi K. Khadaroo R.G. Tawadros P.S. Marshall J.C. Kapus A. Rotstein O.D. J. Exp. Med. 2006; 8: 1951-1961Crossref Scopus (152) Google Scholar). This activation of the immune system is considered essential for pathogen killing but is also critically involved in tissue damage and sepsis (19Nathan C. Nature. 2002; 420: 846-852Crossref PubMed Scopus (1980) Google Scholar). In this study, we investigated if heme would be able to affect the inflammatory response triggered 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.DISCUSSIONIn 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 microbial products increased lethality and cytokine production in vivo and in vitro. Hemolysis induced by phenylhydrazine or treatment with heme caused an increased lethality when combined with a sublethal dose of LPS. The effect of heme on cytokine 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 earlier and heightened activation of NF-κB and with phosphorylation of MAPKs on macrophages treated with heme and LPS. 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 mechanism of ROS generation by heme and heme-LPS synergism to increase cytokine production involved the activation of Syk, PI3K, and PKC. Together, these results revealed a critical and previously unrecognized role of heme in enhancing innate immune response by affecting the signaling of PRRs through Syk activation and subsequent change of the redox state of macrophages.A low dose of LPS alone resulted in insignificant cytokine secretion, whereas the same dose of LPS together with heme resulted in substantial production of cytokines, thus suggesting that this synergism may be particularly important under conditions of low agonist concentrations. Heme affected MyD88 and TIR domain-containing adaptor-inducing interferon β (TRIF) pathways induced by LPS upon activation of TLR4, increasing the secretion of TNF, IL-6, and IP-10. Heme also enhanced cytokine secretion induced by TLR2, TLR9, and TLR3 agonists. 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 β only (TLR3), or with that able to induce both pathways simultaneously (TLR4). Moreover, heme increased cytokine secretion induced by NOD agonists. These receptors, in contrast to TLRs, are localized to the cytosol, instead of being associated to cell membranes, and use RICK/RIP2 as adaptor protein (30Carneiro L.A. Magalhaes J.G. Tattoli I. Philpott D.J. Travassos L.H. J. Pathol. 2008; 214: 136-148Crossref PubMed Scopus (151) Google Scholar, 31Shaw M.H. Reimer T. Kim Y.G. Nuñez G. Curr. Opin. Immunol. 2008; 20: 377-382Crossref PubMed Scopus (238) Google Scholar). Together, these results establish that heme amplifies cytokine secretion by PRRs situated on distinct locations and using distinct signal transduction pathways.The combination of heme and LPS caused an enhanced IκBα degradation, NF-κB 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. ROS affect the activation of NF-κB, a key component in TLR and NLR signaling pathways, and are required for cytokine production upon receptor activation (16Medzhitov R. Nature. 2007; 449: 819-826Crossref PubMed Scopus (1966) Google Scholar, 17Akira S. Uematsu S. Takeuchi O. Cell. 2006; 124: 783-801Abstract Full Text Full Text PDF PubMed Scopus (8561) Google Scholar, 18Trinchieri G. Sher A. Nat. Rev. Immunol. 2007; 7: 179-190Crossref PubMed Scopus (1065) Google Scholar). Exogenously added H2O2 causes the activation of NF-κB and increases the secretion of cytokines by macrophages stimulated with LPS, whereas antioxidants have an inhibitory effect (20Powers K.A. Szászi K. Khadaroo R.G. Tawadros P.S. Marshall J.C. Kapus A. Rotstein O.D. J. Exp. Med. 2006; 8: 1951-1961Crossref Scopus (152) Google Scholar). The generation of ROS by heme was involved in the potentiating effect on LPS-induced cytokine secretion. NAC, DPI, and rotenone impaired this enhanced response, whereas inhibition of xanthine oxidase or NOS had no effect. These results suggest that ROS generated by NADPH and mitochondria are involved on heme-LPS synergism to produce cytokines.Recent studies demonstrated an intense cooperation of several PRRs and ITAM-associated receptors, demonstrating both positive and negative regulation (22Rogers N.C. Slack E.C. Edwards A.D. Nolte M.A. Schulz O. Schweighoffer E. Williams D.L. Gordon S. Tybulewicz V.L. Brown G.D. Reis e Sousa C. Immunity. 2005; 22: 507-517Abstract Full Text Full Text PDF PubMed Scopus (725) Google Scholar, 26Gantner B.N. Simmons R.M. Canavera S.J. Akira S. Underhill D.M. J. Exp. Med. 2003; 197: 1107-1117Crossref PubMed Scopus (1309) Google Scholar, 27Brown G.D. Herre J. Williams D.L. Willment J.A. Marshall A.S. Gordon S. J. Exp. Med. 2003; 197: 1119-1124Crossref PubMed Scopus (983) Google Scholar, 28Turnbull I.R. McDunn J.E. Takai T. Townsend R.R. Cobb J.P. Colonna M. J. Exp. Med. 2005; 202: 363-369Crossref PubMed Scopus (79) Google Scholar, 29Ivashkiv L.B. Nat. Immunol. 2009; 10: 340-347Crossref PubMed Scopus (174) Google Scholar, 32Gross O. Poeck H. Bscheider M. Dostert C. Hannesschläger N. Endres S. Hartmann G. Tardivel A. Schweighoffer E. Tybulewicz V. Mocsai A. Tschopp J. Ruland J. Nature. 2009; 459: 433-436Crossref PubMed Scopus (684) Google Scholar, 33Hise A.G. Tomalka J. Ganesan S. Patel K. Hall B.A. Brown G.D. Fitzgerald K.A. Cell Host Microbe. 2009; 5: 487-497Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar, 34Wang L. Gordon R.A. Huynh L. Su X. Park Min K.H. Han J. Arthur J.S. Kalliolias G.D. Ivashkiv L.B. Immunity. 2010; 32: 518-530Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). It has been suggested that activation of ITAM-associated receptors with high affinity ligands causes cross-inhibition of TLR receptors, whereas low affinity ligands potentiate the response (34Wang L. Gordon R.A. Huynh L. Su X. Park Min K.H. Han J. Arthur J.S. Kalliolias G.D. Ivashkiv L.B. Immunity. 2010; 32: 518-530Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). The mechanism of the synergistic induction of cytokine production by TLRs and ITAM-associated receptors seems to be an enhanced activation of NF-κB and MAPKs (29Ivashkiv L.B. Nat. Immunol. 2009; 10: 340-347Crossref PubMed Scopus (174) Google Scholar). We observed that heme caused the phosphorylation of Syk and that treatment 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 production by macrophages. These results resemble the ROS production induced by Dectin-1 and TGF-β-activated kinase (TAK1)-mediated signal in cooperation with TLR2, which enhances activation of NF-κB (26Gantner B.N. Simmons R.M. Canavera S.J. Akira S. Underhill D.M. J. Exp. Med. 2003; 197: 1107-1117Crossref PubMed Scopus (1309) Google Scholar).The mechanism of ROS generation by heme is considered to be at least partially dependent on the Fenton reaction (13Wagener F.A. Volk H.D. Willis D. Abraham N.G. Soares M.P. Adema G.J. Figdor C.G. Pharmacol. Rev. 2003; 55: 551-571Crossref PubMed Scopus (465) Google Scholar). However, we recently observed that PKC and PI3K are also involved on ROS generation induced by heme on neutrophils, thus also suggesting a receptor-mediated process (14Porto B.N. Alves L.S. Fernández P.L. Dutra T.P. Figueiredo R.T. Graça-Souza A.V. Bozza M.T. J. Biol. Chem. 2007; 282: 24430-24436Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). A recent report indicates that monosodium urate crystals activate Syk in DC by a mechanism independent of protein receptors in the plasma membrane and dependent on cholesterol in lipid rafts (35Ng G. Sharma K. Ward S.M. Desrosiers M.D. Stephens L.A. Schoel W.M. Li T. Lowell C.A. Ling C.C. Amrein M.W. Shi Y. Immunity. 2008; 29: 807-818Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). H2O2 also activates NF-κB, a process dependent on Syk (36Takada Y. Mukhopadhyay A. Kundu G.C. Mahabeleshwar G.H. Singh S. Aggarwal B.B. J. Biol. Chem. 2003; 278: 24233-24241Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar), thus suggesting a possible involvement of this pathway on H2O2-induced synergism on cytokine production induced by LPS. At present there is no evidence indicating that H2O2 uses a receptor to activate this pathway. The involvement of a putative specific ITAM-associated receptor activated by heme or, alternatively, Syk activation by heme in a membrane receptor-independent effect requires further investigation.In conclusion, our results show that heme is capable of potentiating cytokine production induced by different innate receptors irrespective of their subcellular localization. This phenomenon ascribes to heme a general role on modulation of innate responses to pathogens and/or NF-κB activation. The molecular mechanism behind this effect of heme is the activation of Syk and subsequent ROS generation. IntroductionA general consequence of infectious diseases that cause hemolysis, internal hemorrhage, or extensive cell damage is the release of hemeproteins. Upon oxidation, hemeproteins release heme, a potentially harmful molecule (1Ferreira A. Balla J. Jeney V. Balla G. Soares M.P. J. Mol. Med. 2008; 86: 1097-1111Crossref PubMed Scopus (155) Google Scholar). Heme-binding plasma proteins, such as hemopexin or albumin, remove the intravascular free heme, subsequently degraded by heme oxygenase-1 (HO-1), generating equimolar amounts of biliverdin, carbon monoxide, and free iron (2Muller-Eberhard U. Fraig M. Am. J. Hematol. 1993; 42: 59-62Crossref PubMed Scopus (67) Google Scholar, 3Morse D. Choi A.M. Am. J. Respir. Cell Mol. Biol. 2002; 27: 8-16Crossref PubMed Scopus (442) Google Scholar). HO-1-deficient mice (Hmox−/−) have high plasma concentrations of heme and show increased susceptibility to LPS-induced lethality, associated with inflammation and oxidative damage (4Poss K.D. Tonegawa S. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 10925-10930Crossref PubMed Scopus (1100) Google Scholar). Accumulation of large amounts of heme might overwhelm the capacity of heme scavengers and degrading system, thus causing oxidative stress and inflammation (5Muller-Eberhard U. Javid J. Liem H.H. Hanstein A. Hanna M. Blood. 1968; 32: 811-815Crossref PubMed Google Scholar, 6Jeney V. Balla J. Yachie A. Varga Z. Vercellotti G.M. Eaton J.W. Balla G. Blood. 2002; 100: 879-887Crossref PubMed Scopus (501) Google Scholar). In fact, recent studies suggest that heme, in combination with ROS 3The abbreviations used are: ROSreactive oxygen speciesDCdendritic cellDPIdiphenyleneiodoniumNACN-acetyl-l-cysteineTLRToll-like receptorsPRRpattern recognition receptorCM-H2DCFDA5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl esterITAMimmunoreceptor tyrosine-based activation motifNODnucleotide-binding oligomerization domain. and inflammatory mediators, increase blood brain barrier leakage and hepatocyte necrosis in models of malarial infection (7Pamplona A. Ferreira A. Balla J. Jeney V. Balla G. Epiphanio S. Chora A. Rodrigues C.D. Gregoire I.P. Cunha-Rodrigues M. Portugal S. Soares M.P. Mota M.M. Nat. Med. 2007; 13: 703-710Crossref PubMed Scopus (419) Google Scholar, 8Seixas E. Gozzelino R. Chora A. Ferreira A. Silva G. Larsen R. Rebelo S. Penido C. Smith N.R. Coutinho A. Soares M.P. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 15837-15842Crossref PubMed Scopus (213) Google Scholar).Hemolysis or hemoglobinemia are associated with increased mortality in septic patients (9Griffiths E. Cortes A. Gilbert N. Stevenson P. MacDonald S. Pepper D. Lancet. 1995; 345: 158-160Abstract PubMed Google Scholar, 10Su D. Roth R.I. Yoshida M. Levin J. Infect. Immun. 1997; 65: 1258-1266Crossref PubMed Google Scholar). Hemoglobin increases the secretion of TNF triggered by LPS, whereas globin has an inhibitory effect (11Yang H. Wang H. Bernik T.R. Ivanova S. Wang H. Ulloa L. Roth J. Eaton J.W. Tracey K.J. Shock. 2002; 17: 485-490Crossref PubMed Scopus (31) Google Scholar), 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 induction of cytokine expression (12Graça-Souza A.V. Arruda M.A. de Freitas M.S. Barja-Fidalgo C. Oliveira P.L. Blood. 2002; 99: 4160-4165Crossref PubMed Scopus (222) Google Scholar, 13Wagener F.A. Volk H.D. Willis D. Abraham N.G. Soares M.P. Adema G.J. Figdor C.G. Pharmacol. Rev. 2003; 55: 551-571Crossref PubMed Scopus (465) Google Scholar, 14Porto B.N. Alves L.S. Fernández P.L. Dutra T.P. Figueiredo R.T. Graça-Souza A.V. Bozza M.T. J. Biol. Chem. 2007; 282: 24430-24436Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Recently, we have shown that heme is able to activate Toll-like receptor 4 (TLR4) inducing TNF on macrophages and dendritic cells (DC) (15Figueiredo R.T. Fernandez P.L. Mourao-Sa D.S. Porto B.N. Dutra F.F. Alves L.S. Oliveira M.F. Oliveira P.L. Graça-Souza A.V. Bozza M.T. J. Biol. Chem. 2007; 282: 20221-20229Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar).Mammalian pattern recognition receptors (PRRs) recognize conserved microbial molecules from all classes of microorganisms (16Medzhitov R. Nature. 2007; 449: 819-826Crossref PubMed Scopus (1966) Google Scholar, 17Akira S. Uematsu S. Takeuchi O. Cell. 2006; 124: 783-801Abstract Full Text Full Text PDF PubMed Scopus (8561) Google Scholar). 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 (18Trinchieri G. Sher A. Nat. Rev. Immunol. 2007; 7: 179-190Crossref PubMed Scopus (1065) Google Scholar). The increased secretion of cytokines is also achieved by combinations of microbial molecules with molecules from host origin such as cytokines, ATP, and ROS (19Nathan C. Nature. 2002; 420: 846-852Crossref PubMed Scopus (1980) Google Scholar, 20Powers K.A. Szászi K. Khadaroo R.G. Tawadros P.S. Marshall J.C. Kapus A. Rotstein O.D. J. Exp. Med. 2006; 8: 1951-1961Crossref Scopus (152) Google Scholar). This activation of the immune system is considered essential for pathogen killing but is also critically involved in tissue damage and sepsis (19Nathan C. Nature. 2002; 420: 846-852Crossref PubMed Scopus (1980) Google Scholar). In this study, we investigated if heme would be able to affect the inflammatory response triggered 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.
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