| 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-1β 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.Hemolysis, hemorrhage, and rhabdomyolysis cause the release of large amounts of hemoproteins to the extracellular space, which, once oxidized, release the heme moiety, a potentially harmful molecule due to its prooxidant, cytotoxic, and inflammatory effects (1, 2). Scavenging proteins such as haptoglobin and hemopexin bind hemoglobin and heme, respectively, promoting their clearance from the circulation and delivery to cells involved with heme catabolism. Heme oxygenase cleaves heme and generates equimolar amounts of biliverdin, carbon monoxide (CO) and iron (2). Studies using mice deficient for haptoglobin (Hp), hemopexin (Hx), and heme oxygenase 1 (HO-1) demonstrate the importance of these proteins in controlling the deleterious effects of heme. Both Hp−/− and Hx−/− mice have increased renal damage after acute hemolysis induced by phenyhydrazine (Phz) compared with wild-type mice (3, 4). Mice lacking both proteins present splenomegaly and liver inflammation composed of several foci with leukocyte infiltration after intravascular hemolysis (5). Hx protect mice against heme-induced endothelial damage improving liver and cardiovascular function (6–8). Lack of heme oxygenase 1 (Hmox1−/−) causes iron overload, increased cell death, and tissue inflammation under basal conditions and upon inflammatory stimuli (9–15). This salutary effect of HO-1 has been attributed to its effect of reducing heme amounts as well as generating the cytoprotective molecules, biliverdin and CO.Heme induces neutrophil migration in vivo and in vitro (16, 17), inhibits neutrophil apoptosis (18), triggers cytokine and lipid mediator production by macrophages (19, 20), and increases the expression of adhesion molecules and tissue factor on endothelial cells (21–23). Heme cooperates with TNF, causing hepatocyte apoptosis in a mechanism dependent on reactive oxygen species (ROS) generation (12). Whereas heme-induced TNF production depends on functional toll-like receptor 4 (TLR4), ROS generation in response to heme is TLR4 independent (19). We recently observed that heme triggers receptor-interacting protein (RIP)1/3-dependent macrophage-programmed necrosis through the induction of TNF and ROS (15). The highly unstable nature of iron is considered critical for the ability of heme to generate ROS and to cause inflammation. ROS generated by heme has been mainly attributed to the Fenton reaction. However, recent studies suggest that heme can generate ROS through multiple sources, including NADPH oxidase and mitochondria (22, 24–27).Heme causes inflammation in sterile and infectious conditions, contributing to the pathogenesis of hemolytic diseases, subarachnoid hemorrhage, malaria, and sepsis (11, 13, 24, 28), but the mechanisms by which heme operates in different conditions are not completely understood. Blocking the prooxidant effects of heme protects cells from death and prevents tissue damage and lethality in models of malaria and sepsis (12, 13, 15). Importantly, two recent studies demonstrated the pathogenic role of heme-induced TLR4 activation in a mouse model of sickle cell disease (29, 30). These results highlight the great potential of understanding the molecular mechanisms of heme-induced inflammation and cell death as a way to identify new therapeutic targets.Hemolysis and heme synergize with microbial molecules for the induction of inflammatory cytokine production and inflammation in a mechanism dependent on ROS and Syk (24). Processing of pro–IL-1β is dependent on caspase-1 activity, requiring assembly of the inflammasome, a cytosolic multiprotein complex composed of a NOD-like receptor, the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC), and caspase-1 (31–33). The most extensively studied inflammasome is the nucleotide-binding domain and leucine rich repeat containing family, pyrin domain containing 3 (NLRP3). NLRP3 and pro–IL-1β expression are increased in innate immune cells primed with NF-κB inducers such as TLR agonists and TNF (34, 35). NLRP3 inflammasome is activated by several structurally nonrelated stimuli, such as endogenous and microbial molecules, pore-forming toxins, and particulate matter (34, 35). The activation of NLRP3 involves K+ efflux, increase of ROS and Syk phosphorylation. Importantly, critical roles of NLRP3 have been demonstrated in a vast number of diseases (34, 36). We hypothesize that heme causes the activation of the inflammasome and secretion of IL-1β. Here we found that heme triggered the processing and secretion of IL-1β dependently on NLRP3 inflammasome in vitro and in vivo. The activation of NLRP3 by heme was dependent on Syk, ROS, and K+ efflux, but independent of lysosomal leakage, ATP release, or cell death. Finally, our results indicated the critical role of macrophages, the NLRP3 inflammasome, and IL-1R to the lethality caused by sterile hemolysis. |
