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1.
mSphere ; 9(8): e0046724, 2024 Aug 28.
Article in English | MEDLINE | ID: mdl-39037263

ABSTRACT

Systemic candidiasis remains a significant public health concern worldwide, with high mortality rates despite available antifungal drugs. Drug-resistant strains add to the urgency for alternative therapies. In this context, vaccination has reemerged as a prominent immune-based strategy. Extracellular vesicles (EVs), nanosized lipid bilayer particles, carry a diverse array of native fungal antigens, including proteins, nucleic acids, lipids, and glycans. Previous studies from our laboratory demonstrated that Candida albicans EVs triggered the innate immune response, activating bone marrow-derived dendritic cells (BMDCs) and potentially acting as a bridge between innate and adaptive immunity. Vaccination with C. albicans EVs induced the production of specific antibodies, modulated cytokine production, and provided protection in immunosuppressed mice infected with lethal C. albicans inoculum. To elucidate the mechanisms underlying EV-induced immune activation, our study investigated pathogen-associated molecular patterns (PAMPs) and pattern recognition receptors (PRRs) involved in EVs-phagocyte engagement. EVs from wild-type and mutant C. albicans strains with truncated mannoproteins were compared for their ability to stimulate BMDCs. Our findings revealed that EV decoration with O- and N-linked mannans and the presence of ß-1,3-glucans and chitin oligomers may modulate the activation of specific PRRs, in particular Toll-like receptor 4 (TLR4) and dectin-1. The protective effect of vaccination with wild-type EVs was found to be dependent on TLR4. These results suggest that fungal EVs can be harnessed in vaccine formulations to selectively activate PRRs in phagocytes, offering potential avenues for combating or preventing candidiasis.IMPORTANCESystemic candidiasis is a serious global health concern with high mortality rates and growing drug resistance. Vaccination offers a promising solution. A unique approach involves using tiny lipid-coated particles called extracellular vesicles (EVs), which carry various fungal components. Previous studies found that Candida albicans EVs activate the immune response and may bridge the gap between innate and adaptive immunity. To understand this better, we investigated how these EVs activate immune cells. We demonstrated that specific components on EV surfaces, such as mannans and glucans, interact with receptors on immune cells, including Toll-like receptor 4 (TLR4) and dectin-1. Moreover, vaccinating with these EVs led to strong immune responses and full protection in mice infected with Candida. This work shows how harnessing fungal EVs might lead to effective vaccines against candidiasis.


Subject(s)
Candida albicans , Candidiasis , Dendritic Cells , Extracellular Vesicles , Fungal Vaccines , Receptors, Pattern Recognition , Toll-Like Receptor 4 , Animals , Candida albicans/immunology , Extracellular Vesicles/immunology , Toll-Like Receptor 4/immunology , Toll-Like Receptor 4/metabolism , Mice , Candidiasis/immunology , Candidiasis/prevention & control , Candidiasis/microbiology , Fungal Vaccines/immunology , Fungal Vaccines/administration & dosage , Dendritic Cells/immunology , Receptors, Pattern Recognition/immunology , Mice, Inbred C57BL , Female , Immunity, Innate , Disease Models, Animal
2.
J Immunol ; 205(10): 2795-2805, 2020 11 15.
Article in English | MEDLINE | ID: mdl-33037139

ABSTRACT

Hemolysis causes an increase of intravascular heme, oxidative damage, and inflammation in which macrophages play a critical role. In these cells, heme can act as a prototypical damage-associated molecular pattern, inducing TLR4-dependent cytokine production through the MyD88 pathway, independently of TRIF. Heme promotes reactive oxygen species (ROS) generation independently of TLR4. ROS and TNF production contribute to heme-induced necroptosis and inflammasome activation; however, the role of ROS in proinflammatory signaling and cytokine production remains unknown. In this study, we demonstrate that heme activates at least three signaling pathways that contribute to a robust MAPK phosphorylation and cytokine expression in mouse macrophages. Although heme did not induce a detectable Myddosome formation, the TLR4/MyD88 axis was important for phosphorylation of p38 and secretion of cytokines. ROS generation and spleen tyrosine kinase (Syk) activation induced by heme were critical for most proinflammatory signaling pathways, as the antioxidant N-acetyl-l-cysteine and a Syk inhibitor differentially blocked heme-induced ROS, MAPK phosphorylation, and cytokine production in macrophages. Early generated mitochondrial ROS induced by heme was Syk dependent, selectively promoted the phosphorylation of ERK1/2 without affecting JNK or p38, and contributed to CXCL1 and TNF production. Finally, lethality caused by sterile hemolysis in mice required TLR4, TNFR1, and mitochondrial ROS, supporting the rationale to target these pathways to mitigate tissue damage of hemolytic disorders.


Subject(s)
Heme/metabolism , Hemolysis/immunology , Reactive Oxygen Species/metabolism , Signal Transduction/immunology , Animals , Chemokine CXCL1/metabolism , Disease Models, Animal , Humans , Macrophages/cytology , Macrophages/immunology , Mice , Mice, Knockout , Mitochondria/immunology , Mitochondria/metabolism , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Phosphorylation/immunology , Receptors, Tumor Necrosis Factor, Type I/genetics , Receptors, Tumor Necrosis Factor, Type I/metabolism , Syk Kinase/metabolism , Toll-Like Receptor 4/genetics , Toll-Like Receptor 4/metabolism , Tumor Necrosis Factor-alpha/metabolism
3.
Proc Natl Acad Sci U S A ; 113(47): E7474-E7482, 2016 11 22.
Article in English | MEDLINE | ID: mdl-27821769

ABSTRACT

Hemolytic diseases include a variety of conditions with diverse etiologies in which red blood cells are destroyed and large amounts of hemeproteins are released. Heme has been described as a potent proinflammatory molecule that is able to induce multiple innate immune responses, such as those triggered by TLR4 and the NLRP3 inflammasome, as well as necroptosis in macrophages. The mechanisms by which eukaryotic cells respond to the toxic effects induced by heme to maintain homeostasis are not fully understood, however. Here we describe a previously uncharacterized cellular response induced by heme: the formation of p62/SQTM1 aggregates containing ubiquitinated proteins in structures known as aggresome-like induced structures (ALIS). This action is part of a response driven by the transcription factor NRF2 to the excessive generation of reactive oxygen species induced by heme that results in the expression of genes involved in antioxidant responses, including p62/SQTM1. Furthermore, we show that heme degradation by HO-1 is required for ALIS formation, and that the free iron released on heme degradation is necessary and sufficient to induce ALIS. Moreover, ferritin, a key protein in iron metabolism, prevents excessive ALIS formation. Finally, in vivo, hemolysis promotes an increase in ALIS formation in target tissues. Our data unravel a poorly understood aspect of the cellular responses induced by heme that can be explored to better understand the effects of free heme and free iron during hemolytic diseases such as sickle cell disease, dengue fever, malaria, and sepsis.


Subject(s)
Heme Oxygenase-1/metabolism , Heme/metabolism , Iron/metabolism , NF-E2-Related Factor 2/metabolism , Reactive Oxygen Species/metabolism , Sequestosome-1 Protein/metabolism , Animals , Ferritins/metabolism , HEK293 Cells , Heme/chemistry , Humans , Mice , Oxidative Stress , Protein Aggregates , Proteolysis , RAW 264.7 Cells , Sequestosome-1 Protein/chemistry , Ubiquitination , Up-Regulation
4.
Proc Natl Acad Sci U S A ; 111(39): E4110-8, 2014 Sep 30.
Article in English | MEDLINE | ID: mdl-25225402

ABSTRACT

The increase of extracellular heme is a hallmark of hemolysis or extensive cell damage. Heme has prooxidant, cytotoxic, and inflammatory effects, playing a central role in the pathogenesis of malaria, sepsis, and sickle cell disease. However, the mechanisms by which heme is sensed by innate immune cells contributing to these diseases are not fully characterized. We found that heme, but not porphyrins without iron, activated LPS-primed macrophages promoting the processing of IL-1ß dependent on nucleotide-binding domain and leucine rich repeat containing family, pyrin domain containing 3 (NLRP3). The activation of NLRP3 by heme required spleen tyrosine kinase, NADPH oxidase-2, mitochondrial reactive oxygen species, and K(+) efflux, whereas it was independent of heme internalization, lysosomal damage, ATP release, the purinergic receptor P2X7, and cell death. Importantly, our results indicated the participation of macrophages, NLRP3 inflammasome components, and IL-1R in the lethality caused by sterile hemolysis. Thus, understanding the molecular pathways affected by heme in innate immune cells might prove useful to identify new therapeutic targets for diseases that have heme release.


Subject(s)
Heme/metabolism , Hemolysis/physiology , Inflammasomes/metabolism , Animals , Carrier Proteins/genetics , Carrier Proteins/metabolism , Caspase 1/deficiency , Caspase 1/genetics , Caspase 1/metabolism , Heme/chemistry , Heme/immunology , Hemolysis/immunology , Humans , Inflammasomes/immunology , Interleukin-1beta/metabolism , Macrophage Activation , Macrophages/immunology , Macrophages/metabolism , Male , Membrane Glycoproteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Models, Biological , NADPH Oxidase 2 , NADPH Oxidases/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein , Potassium/metabolism , Protoporphyrins/chemistry , Protoporphyrins/metabolism , Reactive Oxygen Species/metabolism
5.
Front Pharmacol ; 5: 115, 2014.
Article in English | MEDLINE | ID: mdl-24904418

ABSTRACT

Heme is an essential molecule expressed ubiquitously all through our tissues. Heme plays major functions in cellular physiology and metabolism as the prosthetic group of diverse proteins. Once released from cells and from hemeproteins free heme causes oxidative damage and inflammation, thus acting as a prototypic damage-associated molecular pattern. In this context, free heme is a critical component of the pathological process of sterile and infectious hemolytic conditions including malaria, hemolytic anemias, ischemia-reperfusion, and hemorrhage. The plasma scavenger proteins hemopexin and albumin reduce heme toxicity and are responsible for transporting free heme to intracellular compartments where it is catabolized by heme-oxygenase enzymes. Upon hemolysis or severe cellular damage the serum capacity to scavenge heme may saturate and increase free heme to sufficient amounts to cause tissue damage in various organs. The mechanism by which heme causes reactive oxygen generation, activation of cells of the innate immune system and cell death are not fully understood. Although heme can directly promote lipid peroxidation by its iron atom, heme can also induce reactive oxygen species generation and production of inflammatory mediators through the activation of selective signaling pathways. Heme activates innate immune cells such as macrophages and neutrophils through activation of innate immune receptors. The importance of these events has been demonstrated in infectious and non-infectious diseases models. In this review, we will discuss the mechanisms behind heme-induced cytotoxicity and inflammation and the consequences of these events on different tissues and diseases.

6.
Future Microbiol ; 9(2): 147-61, 2014.
Article in English | MEDLINE | ID: mdl-24571070

ABSTRACT

AIMS: Glucuronoxylomannan (GXM) is the major polysaccharide component of Cryptococcus neoformans. We evaluated in this study whether GXM fractions of different molecular masses were functionally distinct. MATERIALS & METHODS: GXM samples isolated from C. neoformans cultures were fractionated to generate polysaccharide preparations differing in molecular mass. These fractions were used in experiments focused on the association of GXM with cell wall components of C. neoformans, as well as on the interaction of the polysaccharide with host cells. RESULTS & CONCLUSION: GXM fractions of variable molecular masses bound to the surface of a C. neoformans acapsular mutant in a punctate pattern that is in contrast to the usual annular pattern of surface coating observed when GXM samples containing the full molecular mass range were used. The polysaccharide samples were also significantly different in their ability to stimulate cytokine production by host cells. Our findings indicate that GXM fractions are functionally distinct depending on their mass.


Subject(s)
Cryptococcus neoformans/pathogenicity , Fungal Capsules/immunology , Polysaccharides/immunology , Animals , Cryptococcosis/pathology , Cryptococcus neoformans/metabolism , Cytokines/biosynthesis , Fungal Capsules/chemistry , Fungal Capsules/pathology , Macrophages/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Molecular Weight , Polysaccharides/chemistry , Protein Binding , Subcellular Fractions/chemistry
7.
Fungal Genet Biol ; 60: 64-73, 2013 Nov.
Article in English | MEDLINE | ID: mdl-23608320

ABSTRACT

The principal capsular component of Cryptococcus neoformans, glucuronoxylomannan (GXM), interacts with surface glycans, including chitin-like oligomers. Although the role of GXM in cryptococcal infection has been well explored, there is no information on how chitooligomers affect fungal pathogenesis. In this study, surface chitooligomers of C. neoformans were blocked through the use of the wheat germ lectin (WGA) and the effects on animal pathogenesis, interaction with host cells, fungal growth and capsule formation were analyzed. Treatment of C. neoformans cells with WGA followed by infection of mice delayed mortality relative to animals infected with untreated fungal cells. This observation was associated with reduced brain colonization by lectin-treated cryptococci. Blocking chitooligomers also rendered yeast cells less efficient in their ability to associate with phagocytes. WGA did not affect fungal viability, but inhibited GXM release to the extracellular space and capsule formation. In WGA-treated yeast cells, genes that are involved in capsule formation and GXM traffic had their transcription levels decreased in comparison with untreated cells. Our results suggest that cellular pathways required for capsule formation and pathogenic mechanisms are affected by blocking chitin-derived structures at the cell surface of C. neoformans. Targeting chitooligomers with specific ligands may reveal new therapeutic alternatives to control cryptococcosis.


Subject(s)
Cryptococcus neoformans/pathogenicity , Fungal Capsules/metabolism , Phagocytosis/drug effects , Polysaccharides/metabolism , Wheat Germ Agglutinins/pharmacology , Animals , Brain/microbiology , Chitin/metabolism , Cryptococcosis/drug therapy , Cryptococcosis/pathology , Cryptococcus neoformans/growth & development , Cryptococcus neoformans/metabolism , Fungal Capsules/drug effects , Macrophages/drug effects , Macrophages/immunology , Mice , Mice, Inbred C57BL , Wheat Germ Agglutinins/metabolism
8.
J Clin Invest ; 122(7): 2531-42, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22728935

ABSTRACT

Oxidative damage contributes to microbe elimination during macrophage respiratory burst. Nuclear factor, erythroid-derived 2, like 2 (NRF2) orchestrates antioxidant defenses, including the expression of heme-oxygenase-1 (HO-1). Unexpectedly, the activation of NRF2 and HO-1 reduces infection by a number of pathogens, although the mechanism responsible for this effect is largely unknown. We studied Trypanosoma cruzi infection in mice in which NRF2/HO-1 was induced with cobalt protoporphyrin (CoPP). CoPP reduced parasitemia and tissue parasitism, while an inhibitor of HO-1 activity increased T. cruzi parasitemia in blood. CoPP-induced effects did not depend on the adaptive immunity, nor were parasites directly targeted. We also found that CoPP reduced macrophage parasitism, which depended on NRF2 expression but not on classical mechanisms such as apoptosis of infected cells, induction of type I IFN, or NO. We found that exogenous expression of NRF2 or HO-1 also reduced macrophage parasitism. Several antioxidants, including NRF2 activators, reduced macrophage parasite burden, while pro-oxidants promoted it. Reducing the intracellular labile iron pool decreased parasitism, and antioxidants increased the expression of ferritin and ferroportin in infected macrophages. Ferrous sulfate reversed the CoPP-induced decrease in macrophage parasite burden and, given in vivo, reversed their protective effects. Our results indicate that oxidative stress contributes to parasite persistence in host tissues and open a new avenue for the development of anti-T. cruzi drugs.


Subject(s)
Chagas Disease/parasitology , Oxidative Stress , Parasitemia/parasitology , Trypanosoma cruzi/physiology , Animals , Antioxidants/metabolism , Antiparasitic Agents/pharmacology , Antiparasitic Agents/therapeutic use , Cation Transport Proteins/genetics , Cation Transport Proteins/metabolism , Chagas Disease/drug therapy , Ferritins/genetics , Ferritins/metabolism , Ferrous Compounds/pharmacology , Heart/parasitology , Heme Oxygenase-1/antagonists & inhibitors , Heme Oxygenase-1/genetics , Heme Oxygenase-1/metabolism , Host-Parasite Interactions , Iron/metabolism , Macrophages, Peritoneal/drug effects , Macrophages, Peritoneal/enzymology , Macrophages, Peritoneal/parasitology , Mice , Mice, Inbred C57BL , Muscle, Skeletal/parasitology , NF-E2-Related Factor 2/genetics , NF-E2-Related Factor 2/metabolism , Parasitemia/drug therapy , Protoporphyrins/pharmacology , Protoporphyrins/therapeutic use , Reactive Oxygen Species/metabolism , Receptors, Immunologic/metabolism , Respiratory Burst , Trypanosoma cruzi/drug effects
9.
Blood ; 119(10): 2368-75, 2012 Mar 08.
Article in English | MEDLINE | ID: mdl-22262768

ABSTRACT

Diseases that cause hemolysis or myonecrosis lead to the leakage of large amounts of heme proteins. Free heme has proinflammatory and cytotoxic effects. Heme induces TLR4-dependent production of tumor necrosis factor (TNF), whereas heme cytotoxicity has been attributed to its ability to intercalate into cell membranes and cause oxidative stress. We show that heme caused early macrophage death characterized by the loss of plasma membrane integrity and morphologic features resembling necrosis. Heme-induced cell death required TNFR1 and TLR4/MyD88-dependent TNF production. Addition of TNF to Tlr4(-/-) or to Myd88(-/-) macrophages restored heme-induced cell death. The use of necrostatin-1, a selective inhibitor of receptor-interacting protein 1 (RIP1, also known as RIPK1), or cells deficient in Rip1 or Rip3 revealed a critical role for RIP proteins in heme-induced cell death. Serum, antioxidants, iron chelation, or inhibition of c-Jun N-terminal kinase (JNK) ameliorated heme-induced oxidative burst and blocked macrophage cell death. Macrophages from heme oxygenase-1 deficient mice (Hmox1(-/-)) had increased oxidative stress and were more sensitive to heme. Taken together, these results revealed that heme induces macrophage necrosis through 2 synergistic mechanisms: TLR4/Myd88-dependent expression of TNF and TLR4-independent generation of ROS.


Subject(s)
Heme/pharmacology , Macrophages/drug effects , Reactive Oxygen Species/metabolism , Tumor Necrosis Factors/metabolism , Animals , Blotting, Western , Cell Survival/drug effects , Cells, Cultured , Dose-Response Relationship, Drug , Heme Oxygenase-1/genetics , Heme Oxygenase-1/metabolism , Imidazoles/pharmacology , Indoles/pharmacology , JNK Mitogen-Activated Protein Kinases/metabolism , Macrophages/cytology , Macrophages/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Myeloid Differentiation Factor 88/genetics , Myeloid Differentiation Factor 88/metabolism , NIH 3T3 Cells , Necrosis , Receptor-Interacting Protein Serine-Threonine Kinases/antagonists & inhibitors , Receptor-Interacting Protein Serine-Threonine Kinases/genetics , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , Receptors, Tumor Necrosis Factor, Type I/genetics , Receptors, Tumor Necrosis Factor, Type I/metabolism , Toll-Like Receptor 4/genetics , Toll-Like Receptor 4/metabolism , Tumor Necrosis Factors/pharmacology
10.
J Biol Chem ; 285(52): 40714-23, 2010 Dec 24.
Article in English | MEDLINE | ID: mdl-20959459

ABSTRACT

Pseudallescheria boydii (Scedosporium apiospermum) is a saprophytic fungus widespread in the environment, and has recently emerged as an agent of localized as well as disseminated infections, particularly mycetoma, in immunocompromised and immunocompetent hosts. We have previously shown that highly purified α-glucan from P. boydii activates macrophages through Toll-like receptor TLR2, however, the mechanism of P. boydii recognition by macrophage is largely unknown. In this work, we investigated the role of innate immune receptors in the recognition of P. boydii. Macrophages responded to P. boydii conidia and hyphae with secretion of proinflammatory cytokines. The activation of macrophages by P. boydii conidia required functional MyD88, TLR4, and CD14, whereas stimulation by hyphae was independent of TLR4 and TLR2 signaling. Removal of peptidorhamnomannans from P. boydii conidia abolished induction of cytokines by macrophages. A fraction highly enriched in rhamnomannans was obtained and characterized by NMR, high performance TLC, and GC-MS. Preparation of rhamnomannans derived from P. boydii triggered cytokine release by macrophages, as well as MAPKs phosphorylation and IκBα degradation. Cytokine release induced by P. boydii-derived rhamnomannans was dependent on TLR4 recognition and required the presence of non-reducing end units of rhamnose of the rhamnomannan, but not O-linked oligosaccharides from the peptidorhamnomannan. These results imply that TLR4 recognizes P. boydii conidia and this recognition is at least in part due to rhamnomannans expressed on the surface of P. boydii.


Subject(s)
Macrophage Activation/immunology , Macrophages, Peritoneal/immunology , Mannans/immunology , Pseudallescheria/immunology , Spores, Fungal/immunology , Toll-Like Receptor 4/immunology , Animals , Cytokines/immunology , Cytokines/metabolism , Hyphae/immunology , Hyphae/metabolism , Immunity, Innate/immunology , Lipopolysaccharide Receptors/immunology , Lipopolysaccharide Receptors/metabolism , Macrophages, Peritoneal/metabolism , Mannans/metabolism , Mice , Mice, Knockout , Mitogen-Activated Protein Kinase Kinases/immunology , Mitogen-Activated Protein Kinase Kinases/metabolism , Myeloid Differentiation Factor 88/immunology , Myeloid Differentiation Factor 88/metabolism , Pseudallescheria/metabolism , Spores, Fungal/metabolism , Toll-Like Receptor 2/immunology , Toll-Like Receptor 2/metabolism , Toll-Like Receptor 4/metabolism
11.
J Biol Chem ; 285(43): 32844-32851, 2010 Oct 22.
Article in English | MEDLINE | ID: mdl-20729208

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.


Subject(s)
Heme/immunology , Immunity, Innate/physiology , Intracellular Signaling Peptides and Proteins/immunology , Lipopolysaccharides/immunology , Macrophages, Peritoneal/immunology , Protein-Tyrosine Kinases/immunology , Reactive Oxygen Species/immunology , Animals , Cytokines/immunology , Cytokines/metabolism , Dendritic Cells/immunology , Dendritic Cells/metabolism , Heme/agonists , Heme/metabolism , Heme/pharmacology , Humans , Immunity, Innate/drug effects , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Lipopolysaccharides/agonists , Lipopolysaccharides/pharmacology , Macrophages, Peritoneal/metabolism , Mice , Mice, Knockout , NF-kappa B/genetics , NF-kappa B/immunology , NF-kappa B/metabolism , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/immunology , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Protein Kinase C/genetics , Protein Kinase C/immunology , Protein Kinase C/metabolism , Protein Kinase Inhibitors/pharmacology , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Reactive Oxygen Species/metabolism , Syk Kinase
12.
J Biol Chem ; 282(28): 20221-9, 2007 Jul 13.
Article in English | MEDLINE | ID: mdl-17502383

ABSTRACT

Heme is an ancient and ubiquitous molecule present in organisms of all kingdoms, composed of an atom of iron linked to four ligand groups of porphyrin. A high amount of free heme, a potential amplifier of the inflammatory response, is a characteristic feature of diseases with increased hemolysis or extensive cell damage. Here we demonstrate that heme, but not its analogs/precursors, induced tumor necrosis factor-alpha (TNF-alpha) secretion by macrophages dependently on MyD88, TLR4, and CD14. The activation of TLR4 by heme is exquisitely strict, requiring its coordinated iron and the vinyl groups of the porphyrin ring. Signaling of heme through TLR4 depended on an interaction distinct from the one established between TLR4 and lipopolysaccharide (LPS) since anti-TLR4/MD2 antibody or a lipid A antagonist inhibited LPS-induced TNF-alpha secretion but not heme activity. Conversely, protoporphyrin IX antagonized heme without affecting LPS-induced activation. Moreover, heme induced TNF-alpha and keratinocyte chemokine but was ineffective to induce interleukin-6, interleukin-12, and interferon-inducible protein-10 secretion or co-stimulatory molecule expression. These findings support the concept that the broad ligand specificity of TLR4 and the different activation profiles might in part reside in its ability to recognize different ligands in different binding sites. Finally, heme induced oxidative burst, neutrophil recruitment, and heme oxygenase-1 expression independently of TLR4. Thus, our results presented here reveal a previous unrecognized role of heme as an extracellular signaling molecule that affects the innate immune response through a receptor-mediated mechanism.


Subject(s)
Heme/immunology , Immunity, Innate/immunology , Macrophage Activation/immunology , Macrophages/immunology , Signal Transduction/immunology , Toll-Like Receptor 4/immunology , Animals , Cells, Cultured , Cytokines/immunology , Drug Antagonism , Gene Expression Regulation, Enzymologic , Heme/antagonists & inhibitors , Heme/pharmacology , Heme Oxygenase-1/immunology , Hemolysis/genetics , Hemolysis/immunology , Immunity, Innate/drug effects , Immunity, Innate/genetics , Inflammation/genetics , Inflammation/immunology , Lipopolysaccharide Receptors/immunology , Lipopolysaccharides/pharmacology , Macrophage Activation/drug effects , Macrophage Activation/genetics , Mice , Mice, Knockout , Myeloid Differentiation Factor 88/immunology , Neutrophils/immunology , Photosensitizing Agents/antagonists & inhibitors , Photosensitizing Agents/pharmacology , Protoporphyrins/antagonists & inhibitors , Protoporphyrins/pharmacology , Respiratory Burst/drug effects , Respiratory Burst/genetics , Respiratory Burst/immunology , Signal Transduction/drug effects , Signal Transduction/genetics , Toll-Like Receptor 4/deficiency
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