Heme induces programmed necrosis on macrophages through autocrine TNF and ROS production

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 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...

Red alert: labile heme is an alarmin

“…Mø also undergo programmed cell death when exposed to labile heme [58]. However, labile heme induces Mø to undergo necroptosis, rather than apoptosis.…”

Hemolysis and free hemoglobin revisited: exploring hemoglobin and hemin scavengers as a novel class of therapeutic proteins

“…In addition, activation of TLRs and downstream inflammatory signaling, particularly of the TLR-4 pathway, can be triggered by free hemin in some models. [22][23][24] In summary, the definitive pathophysiology of extracellular Hb is dependent on timing, quantity, and tissue localization of Hb/ hemin exposure in a specific clinical condition and may result from cumulative effects largely described by the 4 mechanisms discussed in the sections above. For example, systemic and, to some extent, pulmonary hypertension is the most apparent and readily measurable effect of free Hb after intravascular hemolysis.…”

Hemolysis-induced lethality involves inflammasome activation by heme

“…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.…”

“…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).…”

“…Hx protect mice against hemeinduced endothelial damage improving liver and cardiovascular function (6)(7)(8). Lack of heme oxygenase 1 (Hmox1 −/− ) causes iron overload, increased cell death, and tissue inflammation under basal conditions and upon inflammatory stimuli (9)(10)(11)(12)(13)(14)(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.…”