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...

For patients with leprosy, nerve damage is a major cause of morbidity. Although antibiotic therapy can eliminate the pathogen, Mycobacterium leprae, therapy is often initiated after nerve damage has occurred. Furthermore, nerve damage can occur during the administration of therapy, in particular, during the reactive states of erythema nodosum leprosum and the reversal reaction.In all forms of leprosy, M. leprae can be detected in nerves in active lesions (24). The M. leprae-Schwann cell interaction is a complex process, involving multiple bacterial ligands and cellular receptors (21). One initial target for the M. leprae interaction with peripheral nerves is laminin 2, located in the basal lamina of the Schwann cell axon unit (22). A specific glycolipid of M. leprae has been shown to mediate this interaction and hence determine the predilection of M. leprae for nerves (17). Other mycobacteria, including M. tuberculosis, M. chelonae, and M. smegmatis, exhibit laminin-binding capacity for adherence to Schwann cells (14). The colonization of Schwann cells by M. leprae also stimulates granuloma formation and cellmediated nerve injury (28). However, damage to cutaneous nerves can also occur in the absence of immune cells (23). Therefore, study of the M. leprae-Schwann cell interaction is essential for understanding the mechanisms of nerve injury in leprosy.

Excessive release of proinflammatory cytokines by innate immune cells is an important component of the pathogenic basis of malaria. Proinflammatory cytokines are a direct output of Toll-like receptor (TLR) activation during microbial infection. Thus, interference with TLR function is likely to render a better clinical outcome by preventing their aberrant activation and the excessive release of inflammatory mediators. Herein, we describe the protective effect and mechanism of action of E6446, a synthetic antagonist of nucleic acid-sensing TLRs, on experimental cerebral malaria (ECM) induced by Plasmodium berghei ANKA. We show that in vitro, low doses of E6446 specifically inhibited the activation of human and mouse TLR9. Tenfold higher concentrations of this compound also inhibited the human TLR8 response to single-stranded RNA. In vivo, therapy with E6446 diminished the activation of TLR9 and prevented the exacerbated cytokine response observed during acute Plasmodium infection. Furthermore, severe signs of ECM, such as limb paralysis, brain vascular leak, and death, were all prevented by oral treatment with E6446. Hence, we provide evidence that supports the involvement of nucleic acid-sensing TLRs in malaria pathogenesis and that interference with the activation of these receptors is a promising strategy to prevent deleterious inflammatory responses that mediate pathogenesis and severity of malaria.immunotherapy | innate immunity | nucleic acid recognition | inflammation

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High levels of circulating immunocomplexes (ICs) are found in patients with either infectious or sterile inflammation. We report that patients with either Plasmodium falciparum or Plasmodium vivax malaria have increased levels of circulating anti-DNA antibodies and ICs containing parasite DNA. Upon stimulation with malaria-induced ICs, monocytes express an NF-κB transcriptional signature. The main source of IC-induced proinflammatory cytokines (i.e., tumor necrosis factor alpha [TNF-α] and interleukin-1β [IL-1β])in peripheral blood mononuclear cells from acute malaria patients was found to be a CD14+ CD16 (FcγRIIIA)+ CD64 (FcγRI)high CD32 (FcγRIIB)low monocyte subset. Monocytes from convalescent patients were predominantly of the classical phenotype (CD14+ CD16−) that produces high levels of IL-10 and lower levels of TNF-α and IL-1β in response to ICs. Finally, we report a novel role for the proinflammatory activity of ICs by demonstrating their ability to induce inflammasome assembly and caspase-1 activation in human monocytes. These findings illuminate our understanding of the pathogenic role of ICs and monocyte subsets and may be relevant for future development of immunity-based interventions with broad applications to systemic inflammatory diseases.

The development of deformities during the course of leprosy disease is a major public health concern worldwide. It is possible that cytokine production and apoptosis of Schwann cells (SCs) directly affect nerve degeneration and regeneration leading to injury of the myelin sheath and axon. In the present study, the expression of TNFalpha, TGFbeta, and their receptors, in addition to cell death triggered by cytokines or whole Mycobacterium leprae were investigated in a human SC line. The results showed the presence of TNF-Rs and TGF-RII on the SC membrane and the shedding of TNF-Rs during the culture period. Evaluation of cell death was performed through TUNEL and flow cytometry techniques. TNFalpha/TGFbeta combination as well as M. leprae infection triggered an increase in the apoptosis rate in the cultured SC. Moreover, reverse transcriptase-polymerase chain reaction assay revealed that M. leprae upregulated the expression of such cytokines and their receptors on the SC line. Despite the detection of TNFalpha mRNA, no protein was found in the culture supernatants. The data indicate that induction of SC death after cell interaction with M. leprae may, in fact, be implicated in the pathogenesis of nerve damage, which can most likely be modulated by in vivo cytokine production.