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 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...

Hemolysis-induced lethality involves inflammasome activation by heme

“…S1 A and B). We have previously shown that the synergistic effect of heme on cytokine production triggered by microbial molecules only occurs in the presence of serum (24). Thus, we tested the requirement of serum on heme-induced IL-1β processing.…”

“…Heme is a potent inducer of ROS production, activating different sources of ROS generation in macrophages (19,24). Recent studies indicate an essential role of mitochondrial ROS (mtROS) on NLRP3 activation induced by several stimuli (48,49).…”

“…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 apoptosisassociated speck-like protein containing a CARD (ASC), and caspase-1 (31)(32)(33).…”

“…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)(25)(26)(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).…”