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-␣ (TNF-␣) 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-␣ secretion but not heme activity. Conversely, protoporphyrin IX antagonized heme without affecting LPS-induced activation. Moreover, heme induced TNF-␣ and keratinocyte chemokine but was ineffective to induce interleukin-6, interleukin-12, and interferoninducible 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.

Significance Heme causes inflammation in sterile and infectious conditions, contributing to the pathogenesis of sickle cell disease, malaria, and sepsis, but the mechanisms by which heme operates are not completely understood. Here we show that heme induces IL-1β processing through the activation of the nucleotide-binding domain and leucine rich repeat containing family, pyrin domain containing 3 (NLRP3) inflammasome in macrophages. Our results suggest that among NLRP3 activators, heme has common as well as unique requirements to trigger inflammasome activation. In vivo, hemolysis and heme cause inflammasome activation. Importantly, macrophages, inflammasome components, and IL-1R contribute to hemolysis-induced lethality. These results highlight the potential of understanding the molecular mechanisms by which heme is sensed by innate immune receptors as a way to identify new therapeutic strategies to treat the pathological consequences of hemolytic diseases.

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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 hemeinduced 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. (Blood. 2012;119(10): 2368-2375) IntroductionThe term programmed cell death was used for many years as a synonym of apoptosis, whereas necrosis in the opposite extreme was considered an abrupt and uncontrolled type of cell death. However, recent evidence clearly shows that several nonapoptotic cell death modes including autophagy, pyroptosis, and necrosis also involve elaborate molecular circuitry. 1,2 This scenario was originally revealed in a study showing that depending on the cell type, tumor necrosis factor (TNF) could trigger different cellular fates including survival, apoptosis, and necrosis. 3 On blockage of protein synthesis or NF-B, activation of death cytokine receptors of the TNF superfamily triggers caspase-dependent apoptosis, whereas simultaneous inhibition of caspase reorients the cell death to necrosis. [4][5][6][7] Receptor-interacting protein 1 (RIP1, also known as RIPK1) regulates survival and cell death fates. Mice deficient in Rip1 present extensive apoptosis, dying early after birth. The increased sensitivity to TNF-mediated cell death in Rip1 Ϫ/Ϫ cells correlates with a failure to activate NF-B. 8 Recent work shows that necrotic cell death is highly regulated by the RIP1 and RIP3 kinases (also known as RIPK3). 6,7,9-11 Programmed necrosis can be initiated by several stimuli including DNA damage, oxidative stress, infection, and activation of pattern recognition receptors. 1,2,[12][13][14][15][16][17] Intra or extra vascular hemolysis, rhabdomyolysis, and extensive cell damage cause the release of large quantities of hemeproteins. The oxidation of some hemeproteins including hemoglobin and myoglobin can release the heme moiety promoting further oxidation an...

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 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 activation and indicates possible pathways for therapeutic intervention during hemolytic infectious diseases.A 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 (1). 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 (2, 3). HO-1-deficient mice (Hmox Ϫ/Ϫ ) have high plasma concentrations of heme and show 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, in combination with ROS 3 and inflammatory mediators, 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 secretion of TNF triggered by LPS, whereas globin has an inhibitory effect (11), suggesting that heme is responsible for the cytokine amplification. Heme has several pro-inflammatory activities, including leukocyte activation and migration, upregulation of adhesion molecules, ROS production, and induction of cytokine expression (12-14). Recently, we have shown that heme is able to activate Toll-like receptor 4 (TLR4) inducing TNF on macrophages and dendritic cells (DC) (15).Mammalian pattern recognition receptors (PRRs) recognize conserved microbial molecules from all classes of microorganisms (16,17). The activation of these receptors elicits selective intracellular signaling cascades that result in the production of cytokines, chemokin...