Heme b inhibits class III adenylyl cyclases.

Acidic lipid extracts from mouse liver, kidney, heart, brain, and lung inhibited human pseudoheterodimeric adenylyl cyclases (hACs) expressed in HEK293 cells. Using an acidic lipid extract from bovine lung, a combined MS- and bioassay-guided fractionation identified heme b as inhibitor of membrane-bound ACs. IC50 concentrations were 8-12 µM for the hAC isoforms. Hemopexin and bacterial hemophore attenuated heme b inhibition of hAC5. Structurally related compounds, such as hematin, protoporphyrin IX, and biliverdin, were significantly less effective. Monomeric bacterial class III ACs (mycobacterial ACs Rv1625c; Rv3645; Rv1264; cyanobacterial AC CyaG) were inhibited by heme b with similar efficiency. Surprisingly, structurally related chlorophyll a similarly inhibited hAC5. Heme b inhibited isoproterenol-stimulated cAMP accumulation in HEK293 cells. Using cortical membranes from mouse brain hemin efficiently and reversibly inhibited basal and Gsα-stimulated AC activity. The physiological relevance of heme b inhibition of the cAMP generating system in certain pathologies is discussed.

Trained innate immunity, long-lasting epigenetic modulation, and skewed myelopoiesis by heme.

Trained immunity defines long-lasting adaptations of innate immunity based on transcriptional and epigenetic modifications of myeloid cells and their bone marrow progenitors [M. Divangahi et al., Nat. Immunol. 22, 2-6 (2021)]. Innate immune cells, however, do not exclusively differentiate between foreign and self but also react to host-derived molecules referred to as alarmins. Extracellular "labile" heme, released during infections, is a bona fide alarmin promoting myeloid cell activation [M. P. Soares, M. T. Bozza, Curr. Opin. Immunol. 38, 94-100 (2016)]. Here, we report that labile heme is a previously unrecognized inducer of trained immunity that confers long-term regulation of lineage specification of hematopoietic stem cells and progenitor cells. In contrast to previous reports on trained immunity, essentially mediated by pathogen-associated molecular patterns, heme training depends on spleen tyrosine kinase signal transduction pathway acting upstream of c-Jun N-terminal kinases. Heme training promotes resistance to sepsis, is associated with the expansion of self-renewing hematopoetic stem cells primed toward myelopoiesis and to the occurrence of a specific myeloid cell population. This is potentially evoked by sustained activity of Nfix, Runx1, and Nfe2l2 and dissociation of the transcriptional repressor Bach2. Previously reported trained immunity inducers are, however, infrequently present in the host, whereas heme abundantly occurs during noninfectious and infectious disease. This difference might explain the vanishing protection exerted by heme training in sepsis over time with sustained long-term myeloid adaptations. Hence, we propose that trained immunity is an integral component of innate immunity with distinct functional differences on infectious disease outcome depending on its induction by pathogenic or endogenous molecules.

Mechanistic insights into heme-mediated transcriptional regulation via a bacterial manganese-binding iron regulator, iron response regulator (Irr).

The transcription factor iron response regulator (Irr) is a key regulator of iron homeostasis in the nitrogen-fixating bacterium Bradyrhizobium japonicum Irr acts by binding to target genes, including the iron control element (ICE), and is degraded in response to heme binding. Here, we examined this binding activity using fluorescence anisotropy with a 6-carboxyfluorescein-labeled ICE-like oligomer (FAM-ICE). In the presence of Mn2+, Irr addition increased the fluorescence anisotropy, corresponding to formation of the Irr-ICE complex. The addition of EDTA to the Irr-ICE complex reduced fluorescence anisotropy, but fluorescence was recovered after Mn2+ addition, indicating that Mn2+ binding is a prerequisite for complex formation. Binding activity toward ICE was lost upon introduction of substitutions in a His-cluster region of Irr, revealing that Mn2+ binds to this region. We observed that the His-cluster region is also the heme binding site; results from fluorescence anisotropy and electrophoretic mobility shift analyses disclosed that the addition of a half-equivalent of heme dissociates Irr from ICE, likely because of Mn2+ release due to heme binding. We hypothesized that heme binding to another heme binding site, Cys-29, would also inhibit the formation of the Irr-ICE complex because it is proximal to the ICE binding site, which was supported by the loss of ICE binding activity in a Cys-29-mutated Irr. These results indicate that Irr requires Mn2+ binding to form the Irr-ICE complex and that the addition of heme dissociates Irr from ICE by replacing Mn2+ with heme or by heme binding to Cys-29.

Structure-activity relationship of heme and its analogues in membrane damage and inhibition of fusion.

Under pathological conditions, such as sickle cell disease and malaria, heme concentration increases considerably, and it induces membrane damage. As sickled and normal erythrocytes contain high cholesterol: phospholipid ratio, we investigated the role of lipid composition, chain length, and unsaturation on the partitioning and leakage of hemin in phospholipid vesicles. To establish structure-activity relationship in membrane damage, experiments with two other analogues, protoporphyrin-IX and hematoporphyrin (HP) were also carried out. Hemin and its analogues localize differently in membranes and exhibit distinct roles in partitioning, leakage and fusion. Hemin and HP trigger more leakage in the presence of aminophospholipids, whereas cholesterol buffers the destabilizing effect remarkably. Inhibition of fusion by hemin further suggests its unexplored and important role in membrane trafficking, particularly under diseased conditions.

Revisiting the putative role of heme as a trigger of inflammation.

Activation of the innate immune system by free heme has been proposed as one of the principal consequences of cell-free hemoglobin (Hb) exposure. Nonetheless, in the absence of infection, heme exposures within a hematoma, during hemolysis, or upon systemic administration of Hb (eg, as a Hb-based oxygen carrier) are typically not accompanied by uncontrolled inflammation, challenging the assumption that heme is a major proinflammatory mediator in vivo. Because of its hydrophobic nature, heme liberated from oxidized hemoglobin is rapidly transferred to alternative protein-binding sites (eg, albumin) or to hydrophobic lipid compartments minimizing protein-free heme under in vivo equilibrium conditions. We demonstrate that the capacity of heme to activate human neutrophil granulocytes strictly depends on the availability of non protein-associated heme. In human endothelial cells as well as in mouse macrophage cell cultures and in mouse models of local and systemic heme exposure, protein-associated heme or Hb do not induce inflammatory gene expression over a broad range of exposure conditions. Only experiments in protein-free culture medium demonstrated a weak capacity of heme-solutions to induce toll-like receptor-(TLR4) dependent TNF-alpha expression in macrophages. Our data suggests that the equilibrium-state of free and protein-associated heme critically determines the proinflammatory capacity of the metallo-porphyrin. Based on these data it appears unlikely that inflammation-promoting equilibrium conditions could ever occur in vivo.

The macrophage heme-heme oxygenase-1 system and its role in inflammation.

Heme oxygenase (HO)-1, the inducible isoform of the heme-degrading enzyme HO, plays a critical role in inflammation and iron homeostasis. Regulatory functions of HO-1 are mediated via the catalytic breakdown of heme, which is an iron-containing tetrapyrrole complex with potential pro-oxidant and pro-inflammatory effects. In addition, the HO reaction produces the antioxidant and anti-inflammatory compounds carbon monoxide (CO) and biliverdin, subsequently converted into bilirubin, along with iron, which is reutilized for erythropoiesis. HO-1 is up-regulated by a plethora of stimuli and injuries in most cell types and tissues and provides salutary effects by restoring physiological homeostasis. Notably, HO-1 exhibits critical immuno-modulatory functions in macrophages, which are a major cell population of the mononuclear phagocyte system. Macrophages play key roles as sentinels and regulators of the immune system and HO-1 in these cells appears to be of critical importance for driving resolution of inflammatory responses. In this review, the complex functions and regulatory mechanisms of HO-1 in macrophages will be high-lighted. A particular focus will be the intricate interactions of HO-1 with its substrate heme, which play a contradictory role in distinct physiological and pathophysiological settings. The therapeutic potential of targeted modulation of the macrophage heme-HO-1 system will be discussed in the context of inflammatory disorders.

Fur regulation of Staphylococcus aureus heme oxygenases is required for heme homeostasis.

Heme is a cofactor that is essential for cellular respiration and for the function of many enzymes. If heme levels become too low within the cell, S. aureus switches from producing energy via respiration to producing energy by fermentation. S. aureus encodes two heme oxygenases, IsdI and IsdG, which cleave the porphyrin heme ring releasing iron for use as a nutrient source. Both isdI and isdG are only expressed under low iron conditions and are regulated by the canonical Ferric Uptake Regulator (Fur). Here we demonstrate that unregulated expression of isdI and isdG within S. aureus leads to reduced growth under low iron conditions. Additionally, the constitutive expression of these enzymes leads to decreased heme abundance in S. aureus, an increase in the fermentation product lactate, and increased resistance to gentamicin. This work demonstrates that S. aureus has developed tuning mechanisms, such as Fur regulation, to ensure that the cell has sufficient quantities of heme for efficient ATP production through aerobic respiration.

Role of Heme Iron in the Association Between Red Meat Consumption and Colorectal Cancer.

The growing incidence of colorectal cancer (CRC) in the world seems to be related to the spread of Westernized lifestyles, particularly dietary habits. Several studies have found that high consumption of red meat-especially processed red meat, a mainstay of Western diets-is associated with an increased risk of developing CRC. One possible reason for the association are the adverse effects exerted by the heme iron contained in red meat, which has the potential to affect homeostasis and colonic epithelial cell renewal and to promote the formation of mutagenic and carcinogenic agents. A correlation has also emerged between CRC and alterations of the gut microbiota, since the micro-organisms found in the intestinal lumen seem to influence the activation of enterocyte genes involved in the initiation and progression of carcinogenesis. Dietary habits can therefore modify the gut microbiota, affecting gene activation and favoring the development of cancer cells. We review and discuss the most recent literature on the hypothesis that heme iron can exert adverse effects by altering the gut microbiota and colorectal epithelial cell homeostasis.

Insights into functions of the H channel of cytochrome c oxidase from atomistic molecular dynamics simulations.

Proton pumping A-type cytochrome c oxidase (CcO) terminates the respiratory chains of mitochondria and many bacteria. Three possible proton transfer pathways (D, K, and H channels) have been identified based on structural, functional, and mutational data. Whereas the D channel provides the route for all pumped protons in bacterial A-type CcOs, studies of bovine mitochondrial CcO have led to suggestions that its H channel instead provides this route. Here, we have studied H-channel function by performing atomistic molecular dynamics simulations on the entire, as well as core, structure of bovine CcO in a lipid-solvent environment. The majority of residues in the H channel do not undergo large conformational fluctuations. Its upper and middle regions have adequate hydration and H-bonding residues to form potential proton-conducting channels, and Asp51 exhibits conformational fluctuations that have been observed crystallographically. In contrast, throughout the simulations, we do not observe transient water networks that could support proton transfer from the N phase toward heme a via neutral His413, regardless of a labile H bond between Ser382 and the hydroxyethylfarnesyl group of heme a In fact, the region around His413 only became sufficiently hydrated when His413 was fixed in its protonated imidazolium state, but its calculated pKa is too low for this to provide the means to create a proton transfer pathway. Our simulations show that the electric dipole moment of residues around heme a changes with the redox state, hence suggesting that the H channel could play a more general role as a dielectric well.

Haem-Based Sensors of O2: Lessons and Perspectives.

Haem-based sensors have emerged during the last 15 years as being a large family of proteins that occur in all kingdoms of life. These sensors are responsible mainly for detecting binding of O2, CO and NO and reporting the ligation status to an output domain with an enzymatic or macromolecule-binding property. A myriad of biological functions have been associated with these sensors, which are involved in vasodilation, bacterial symbiosis, chemotaxis and biofilm formation, among others. Here, we critically review several bacterial systems for O2 sensing that are extensively studied in many respects, focusing on the lessons that are important to advance the field.

A protective role of Heme-regulated eIF2α kinase in cadmium-induced liver and kidney injuries.

A number of studies have reported that cadmium (Cd) can incur liver and kidney injuries. The recruitment and activation of leukocytes have been demonstrated to be involved in Cd-induced biological effects. Ironically, activated leukocytes and secreted cytokines are also reported to be required for the later recovery of the damaged tissues. Yet, the mechanisms driving the production of leukocytes have not been fully elucidated. Heme-regulated eIF2α kinase (HRI) is essential for translational regulation and stressed erythropoiesis in iron deficiency. Meanwhile, HRI is important in the maturation and function of macrophages, indicating that HRI might be indispensable for the development and function of other myeloid lineages. Apart from macrophages, whether HRI regulates the production of leukocytes and further affects Cd-induced tissue injuries is still elusive. In this study, we aimed to elucidate the role of HRI in liver and kidney injuries and the associated mechanisms upon Cd exposure. We found that Cd-exposed mice showed impaired production of leukocytes and developed morphological disorders in liver and kidney. Furthermore, Hri null mice exhibited a reduced number of monocytes and neutrophils and compromised cytokine production, relative to wild-type mice. Absence of Hri also exacerbated the impairments of liver and kidney upon Cd treatment. Together, these results highlighted a crucial role of HRI in protecting liver and kidney against Cd-induced injuries through inducing the development of monocytes and neutrophils. Our results further extended the understanding of HRI on the regulation of non-erythroid lineages and might provide new aspects for preventing and treating Cd-induced detrimental effects.

The impact of metagenomic interplay on the mosquito redox homeostasis.

Mosquitoes are exposed to oxidative challenges throughout their life cycle. The primary challenge comes from a blood meal. The blood digestion turns the midgut into an oxidative environment, which imposes pressure not only on mosquito fecundity and other physiological traits but also on the microbiota in the midgut. During evolution, mosquitoes have developed numerous oxidative defense mechanisms to maintain redox homeostasis in the midgut. In addition to antioxidants, SOD, catalase, and glutathione system, sufficient supply of the reducing agent, NADPH, is vital for a successful defense against oxidative stress. Increasing evidence indicates that in response to oxidative stress, cells reconfigure metabolic pathways to increase the generation of NADPH through NADP-reducing networks including the pentose phosphate pathway and others. The microbial homeostasis is critical for the functional contributions to various host phenotypes. The symbiotic microbiota is regulated largely by the Duox-ROS pathway in Drosophila. In mosquitoes, Duox-ROS pathway, heme-mediated signaling, antimicrobial peptide production and C-type lectins work in concert to maintain the dynamic microbial community in the midgut. Microbial mechanisms against oxidative stress in this context are not well understood. Emerging evidence that microbial metabolites trigger host oxidative response warrants further study on the metagenomic interplay in an oxidative environment like mosquito gut ecosystem. Besides the classical Drosophila model, hematophagous insects like mosquitoes provide an alternative model system to study redox homeostasis in a symbiotic metagenomic context.

Correlation of BACH1 and Hemoglobin E/Beta-Thalassemia Globin Expression.

OBJECTIVE: The diverse clinical phenotype of hemoglobin E (HbE)/ß-thalassemia has not only confounded clinicians in matters of patient management but has also led scientists to investigate the complex mechanisms involved in maintaining the delicate red cell environment where, even with apparent similarities of α- and ß-globin genotypes, the phenotype tells a different story. The BTB and CNC homology 1 (BACH1) protein is known to regulate α- and ß-globin gene transcriptions during the terminal differentiation of erythroid cells. With the mutations involved in HbE/ß-thalassemia disorder, we studied the role of BACH1 in compensating for the globin chain imbalance, albeit for fine-tuning purposes. MATERIALS AND METHODS: A total of 47 HbE/ß-thalassemia samples were analyzed using real-time quantitative polymerase chain reaction and correlated with age, sex, red blood cell parameters, globin gene expressions, and some clinical data. RESULTS: The BACH1 expression among the ß-thalassemia intermedia patients varied by up to 2-log differences and was positively correlated to age; α-, ß-, and γ-globin gene expression level; and heme oxygenase 1 protein. BACH1 was also negatively correlated to reticulocyte level and had a significant correlation with splenectomy. CONCLUSION: This study indicates that the expression of BACH1 could be elevated as a compensatory mechanism to decrease the globin chain imbalance as well as to reduce the oxidative stress found in HbE/ß-thalassemia.

Biology of Heme in Mammalian Erythroid Cells and Related Disorders.

Heme is a prosthetic group comprising ferrous iron (Fe(2+)) and protoporphyrin IX and is an essential cofactor in various biological processes such as oxygen transport (hemoglobin) and storage (myoglobin) and electron transfer (respiratory cytochromes) in addition to its role as a structural component of hemoproteins. Heme biosynthesis is induced during erythroid differentiation and is coordinated with the expression of genes involved in globin formation and iron acquisition/transport. However, erythroid and nonerythroid cells exhibit distinct differences in the heme biosynthetic pathway regulation. Defects of heme biosynthesis in developing erythroblasts can have profound medical implications, as represented by sideroblastic anemia. This review will focus on the biology of heme in mammalian erythroid cells, including the heme biosynthetic pathway as well as the regulatory role of heme and human disorders that arise from defective heme synthesis.

Heme-Mediated Induction of CXCL10 and Depletion of CD34+ Progenitor Cells Is Toll-Like Receptor 4 Dependent.

Plasmodium falciparum infection can cause microvascular dysfunction, cerebral encephalopathy and death if untreated. We have previously shown that high concentrations of free heme, and C-X-C motif chemokine 10 (CXCL10) in sera of malaria patients induce apoptosis in microvascular endothelial and neuronal cells contributing to vascular dysfunction, blood-brain barrier (BBB) damage and mortality. Endothelial progenitor cells (EPC) are microvascular endothelial cell precursors partly responsible for repair and regeneration of damaged BBB endothelium. Studies have shown that EPC's are depleted in severe malaria patients, but the mechanisms mediating this phenomenon are unknown. Toll-like receptors recognize a wide variety of pathogen-associated molecular patterns generated by pathogens such as bacteria and parasites. We tested the hypothesis that EPC depletion during malaria pathogenesis is a function of heme-induced apoptosis mediated by CXCL10 induction and toll-like receptor (TLR) activation. Heme and CXCL10 concentrations in plasma obtained from malaria patients were elevated compared with non-malaria subjects. EPC numbers were significantly decreased in malaria patients (P < 0.02) and TLR4 expression was significantly elevated in vivo. These findings were confirmed in EPC precursors in vitro; where it was determined that heme-induced apoptosis and CXCL10 expression was TLR4-mediated. We conclude that increased serum heme mediates depletion of EPC during malaria pathogenesis.

Ativação da heme oxigenase-1 e via da necroptose como mecanismos imunopatogênicos na infecção de macrófagos por leishmania infantum / heme oxygenase-1 activation and via the necroptose as immunopathogenics mechanisms for the infection of macrophages by Leishmania infantum

A Leishmaniose visceral (LV) apresenta ampla distribuição geográfica e é fatal caso não seja tratada. As manifestações hematológicas são constantes na LV e em casos não tratados os pacientes evoluem à óbito por sangramento maciço ou anemia grave. Neste cenário, mecanismos ligados à morte celular, hemólise, metabolismo do heme e atividade da enzima heme oxigenase podem estar envolvidos na imunopatogênese da LV. A heme oxigenase (HO) tem importantes propriedades regulatórias e está envolvida em processos fisiológicos e patofisiológicos como citoproteção e inflamação. Nesse projeto testamos a hipótese de que a ativação da enzima heme oxigenase-1 (HO-1) favorece a infecção por Leishmania infantum chagasi, principal agente etiológico da LV humana no Brasil e de que mecanismos de morte celular inflamatória induzida por heme estão associados com a resistência ao parasita. Nossas observações nesse trabalho indicam que a enzima HO-1 é induzida em macrófagos durante a infecção por L. chagasi e que a indução farmacológica da HO-1, pela CoPP aumenta a carga parasitária de macrófagos infectados por L. chagasi e reduz a produção de mediadores próinflamatórios. Além disso, a HO-1 favorece um ambiente anti-inflamatório onde prevalece a presença de IL-10...

Visceral leishmaniasis (VL) is a widespread disease and is fatal if left untreated. Hematological manifestations are common in VL and untreated patients evolve to death from massive bleeding and severe anemia. In this scenario, mechanisms related to cell death pathways, hemolysis, heme metabolism and enzymatic activity of heme oxygenase may be involved in the immunopathogenesis of the disease. Heme oxygenase (HO) has important regulatory properties and is involved in patho-physiological processes such as cytoprotection and inflammation. This project tested the hypothesis that heme oxygenase- 1 (HO-1) activation favors Leishmania infantum chagasi infection, the main etiologic agent of human VL in Brazil, we also tested whether heme induced inflammatory cell death pathways are involved in resistance to Leishmania infection. Our observations indicate that HO-1 is induced in macrophages infected with L. infantum chagasi and pharmacological induction for HO-1 by CoPP increases parasite load of infected macrophages and reduces production on inflammatory mediators. In addition, HO-1 contributes to the anti inflammatory pathway that favors L. chagasi replication through a higher IL-10/TNF-α...

[Role of HRI in apoptosis resistance]. / Rôle de l'heme regulated inhibitor (HRI) dans la résistance à l'apoptose.

When exposed to environmental stresses, cells activate defence mechanisms to adapt stress and inhibit apoptotic pathways leading to their survival. Stressed cells also reduce their general metabolism in part by inhibiting mRNA translation, thereby saving energy needed to repair stress-induced damages. Under stress conditions, the inhibition of mRNA translation occurs mainly at its initiation step through the phosphorylation of the translation initiation factor eIF2α. One of the four kinases known to phosphorylate eIF2α is heme-regulated inhibitor (HRI). The activation of HRI occurs under conditions of heme deficiency, oxidative stress and treatment with anti-cancer drugs such as proteasome inhibitors. In this article, we discuss the role of HRI in promoting cell resistance to stress-mediated apoptosis.

Phagocytic uptake of oxidized heme polymer is highly cytotoxic to macrophages.

Apoptosis in macrophages is responsible for immune-depression and pathological effects during malaria. Phagocytosis of PRBC causes induction of apoptosis in macrophages through release of cytosolic factors from infected cells. Heme polymer or ß-hematin causes dose-dependent death of macrophages with LC50 of 132 µg/ml and 182 µg/ml respectively. The toxicity of hemin or heme polymer was amplified several folds in the presence of non-toxic concentration of methemoglobin. ß-hematin uptake in macrophage through phagocytosis is crucial for enhanced toxicological effects in the presence of methemoglobin. Higher accumulation of ß-hematin is observed in macrophages treated with ß-hematin along with methemoglobin. Light and scanning electron microscopic observations further confirm accumulation of ß-hematin with cellular toxicity. Toxicological potentiation of pro-oxidant molecules toward macrophages depends on generation of H2O2 and independent to release of free iron from pro-oxidant molecules. Methemoglobin oxidizes ß-hematin to form oxidized ß-hematin (ßH*) through single electron transfer mechanism. Pre-treatment of reaction mixture with spin-trap Phenyl-N-t-butyl-nitrone dose-dependently reverses the ß-hematin toxicity, indicates crucial role of ßH* generation with the toxicological potentiation. Acridine orange/ethidium bromide staining and DNA fragmentation analysis indicate that macrophage follows an oxidative stress dependent apoptotic pathway to cause death. In summary, current work highlights mutual co-operation between methemoglobin and different pro-oxidant molecules to enhance toxicity towards macrophages. Hence, methemoglobin peroxidase activity can be probed for subduing cellular toxicity of pro-oxidant molecules and it may in-turn make up for host immune response against the malaria parasite.

Heme-induced neutrophil extracellular traps contribute to the pathogenesis of sickle cell disease.

Sickle cell disease (SCD) is characterized by recurring episodes of vascular occlusion in which neutrophil activation plays a major role. The disease is associated with chronic hemolysis with elevated cell-free hemoglobin and heme. The ensuing depletion of heme scavenger proteins leads to nonspecific heme uptake and heme-catalyzed generation of reactive oxygen species. Here, we have identified a novel role for heme in the induction of neutrophil extracellular trap (NET) formation in SCD. NETs are decondensed chromatin decorated by granular enzymes and are released by activated neutrophils. In humanized SCD mice, we have detected NETs in the lungs and soluble NET components in plasma. The presence of NETs was associated with hypothermia and death of these mice, which could be prevented and delayed, respectively, by dismantling NETs with DNase I treatment. We have identified heme as the plasma factor that stimulates neutrophils to release NETs in vitro and in vivo. Increasing or decreasing plasma heme concentrations can induce or prevent, respectively, in vivo NET formation, indicating that heme plays a crucial role in stimulating NET release in SCD. Our results thus suggest that NETs significantly contribute to SCD pathogenesis and can serve as a therapeutic target for treating SCD.