Heme Release Research Articles

Serum Haptoglobin and Hemopexin Levels in Pediatric SS and SC Disease Patients: Biomarker of Hemolysis and Inflammation

▪INTRODUCTION: Hemolysis is a hallmark of sickle cell disease (SCD) pathogenesis, with the release of hemoglobin (Hb) and heme into the plasma that can promote vasculopathy, inflammation, thrombosis, and renal injury. Plasma haptoglobin (Hp) and hemopexin (Hpx) tightly bind free Hb and heme, respectively, thwarting these clinical sequelae. Hp-Hb and Hpx-heme complexes bind to CD163 and CD91 receptors found primarily on macrophages and hepatocytes, respectively, and are taken up by receptor-mediated endocytosis. In SCD patients it is generally believed that chronic hemolysis depletes plasma haptoglobin and hemopexin levels, however we found only one paper in the literature that has reported plasma Hp and Hpx levels in SCD patients, albeit in a limited number of SS (n=5) and SC (n=3) patients (Muller-Eberhard U et al. Blood 1968). Here we report serum Hp and Hpx levels in 272 Brazilian SS, SC, and AA children along with systemic biomarkers of hemolysis, iron metabolism, inflammation leukocyte and platelet counts, and lipid metabolism. We hypothesize that serum Hp and Hpx levels will be significantly lower and biomarkers of hemolysis, iron metabolism, and inflammation will be significantly higher in SS and SC children compared to AA children. METHODS: The present study used blood samples collected prospectively from 272 unrelated steady-state SCD children with SS (n=179) and SC (n=93) disease. Patients were followed at Bahia Pediatrics Centers, during January 2010 to November 2015. The study included 28 healthy AA volunteers randomly selected from the Clinical Laboratory of the Faculdade de Farmácia da Universidade Federal da Bahia. The study was approved by the Fundação de Pesquisa Oswaldo Cruz research board, and all procedures were in accordance with the Declaration of Helsinki of 1975, as revised in 2000. Biochemical and inflammatory analyses were performed by immunochemistry assay and immunoassay. Hematological analyses were carried out using an electronic cell counter and the Hb profile was investigated by high performance liquid chromatography. Serum Hp and Hpx levels were measured by enzyme-linked immunoassay following the manufacturer’s instructions. RESULTS: Evaluating the Hp and Hpx levels, we found that SS and SC patients have lower Hp and Hpx levels (SS<SC<AA) than AA controls (p<.001, Table 1). Similarly, SS and SC patients have lower hemoglobin and hematocrits (SS<SC<AA) and increased reticulocyte counts and serum LDH (SS>SC>AA) than AA controls (p<.001, Table 1). Total, direct, and indirect bilirubin, markers of heme degradation, followed a similar pattern (SS>SC>AA, p<.001, Table 1). These data may reflect more intravascular hemolysis in the following pattern: SS>SC>AA. Likewise, serum ferritin levels, a marker of body iron stores, were higher in SS and SC patients than AA controls (SS>SC>AA, p<.001, Table 1). CRP levels and white blood cells count, markers of inflammation, also reflected the same pattern (SS>SC>AA, p<.001, Table 1). In a different pattern, platelet counts were highest in SS patients and lowest in SC patients (SS>AA>SC). Finally, Total, HDL, and LDL cholesterol levels were lowest in patients with more hemolysis (SS<SC<AA, p<.001, Table 1). CONCLUSIONS: We report lower levels of Hp and Hpx in children with SCD (SS<SC<AA). These data support Muller-Eberhard’s 1968 brief report on Hp and Hpx levels in hemolytic conditions and demonstrate significant correlations of serum Hp and Hpx with biomarkers of hemolysis, inflammation, and cholesterol. We speculate that serum Hp and Hpx levels in SCD may serve as clinical indicators of hemolysis, inflammation, and disease severity. [Display omitted] DisclosuresVercellotti:Imara: Research Funding. Belcher:Cydan/Imara: Research Funding; CSL-Behring: Research Funding.

Modulation of Hemolytic and Hemoglobin/Heme Scavenging Profiles in Sickle Cell Anemia, Hereditary Spherocytosis and Paroxysmal Nocturnal Hemoglobinuria

Sickle Cell Anemia (SCA), Hereditary Spherocytosis (HS) and Paroxysmal Nocturnal Hemoglobinuria (PNH) can share some clinical complications, such as leg ulcers and priapism, due to hemolytic processes. Intravascular hemolysis (IVH) results in the release of cell-free hemoglobin (Hb) and heme into the circulation, potentially causing depletion of nitric oxide, oxidative stress and inflammation. We compared the hemolytic profile and heme/Hb scavenging profiles of patients with SCA on hydroxyurea therapy (SCAHU; 15-30 mg/kg/day for ≥ 2 months) or not (SCA), hereditary spherocytosis (HS), splenectomized HS (SHS) and paroxysmal nocturnal hemoglobinuria not on eculizumab therapy (PNH). Blood samples were collected from patients and from healthy volunteers (CON). Plasma levels of heme, haptoglobin (Hp) and hemopexin (Hpx) were quantified by colorimetric assay or ELISA. Fetal hemoglobin (HbF) was determined in the SCA groups by HPLC. Reticulocytes (RET) and cell counts were determined using an automated flow cytometry hematology system and serum levels of lactate dehydrogenase (LDH) and unconjugated bilirubin (UCB) were quantified by a modular colorimetric automation system. As expected, plasma heme was significantly elevated in the SCA group, compared to the CON group and with intermediate values in the SCAHU group (57.4±9.3, 24.5±2.9, 43.2±4.5µM, respectively, p<0.05, N≥10), correlating significantly and negatively with HbF levels in the SCA patients (r=-0.94, p<0.01). Accordingly, RET and UCB were significantly increased in SCA, compared to CON, with SCAHU presenting intermediate values (RET: 16.2±1.4; 1.9±0.1; 11.5±1.2%; UCB: 2.1±0.7; 0.2±0.0; 1.44±0.2 mg/dL; respectively, p<0.01, N≥10), indicating increased red cell turnover and heme catabolism in SCA. No significant elevations in plasma heme were observed in the HS and SHS groups, compared to CON (35.3±3.8 and 34.5±10.0µM; P>0.05, N≥10), although RET and UCB levels were significantly elevated in HS (8.27±1.07 % and 1.72±0.57 mg/dL, P<0.01, N≥13), but not SHS (2.80±0.17 % and 0.99±0.66 mg/dL, N≥11), perhaps indicating a redistribution of extravascular hemolysis (EHV) to low-level IVH in SHS. Surprisingly, plasma heme was not significantly elevated in PNH individuals studied (36.5±4.0 µM, N≥10), even though concentrations of plasma LDH were the highest in this patient group (1196±305 U/L; compared to 182±8, 352±73, 407±38. 211±19 and 182±28 U/L for CON, SCA, SCAHU, HS, SHS, respectively, P<0.01; N≥10) and LDH correlated significantly with plasma heme in this group (r=0.905, P<0.01 for PNH). RET and UCB presented and intermediate values in PNH (5.35±1.21% and 0.84±0.18 mg/dL, P<0.05, N≥10, compared to CON). To investigate whether hemolytic profiles may be modulated by the Hb and heme scavengers, Hp and Hpx, we measured these proteins in the circulations of patients. Hp was significantly depleted in SCA, SCAHU, HS and PNH (1.0±0.4, 1.0±0.6, 37.8±0.16, 12.3±10.2, respectively, compared to 142.9±20.6 mg/dL in CON, p<0.05; N≥10), while SHS presented similar levels to those of CON (189.9±38.8 mg/dL, N=5). In contrast, somewhat surprisingly, Hpx levels varied considerably in each patient group, with a significant depletion only observed in the SHS group (12.2±0.3, 15.9±5.4, 15.0±3.8, 6.0±1.5, 1.5±0.4, 17.8±5.0mg/dL for CON, SCA, SCAHU, HS, SHS, PNH, respectively; P<0.05, SHS compared to CON, N≥10). As such, plasma heme was slightly lower in PNH than in SCA, in association with very high plasma LDH, suggesting that either IVH is slightly less severe or that Hb/heme scavenging is more efficient in the PNH population studied than in SCA. While EHV may be abolished by splenectomy in HS, intermediate heme levels may suggest that some IVH, or reduced heme catabolism, may still occur in some of these patients. Interestingly, depleted Hp stores were coupled with maintained (or even elevated) Hpx levels in SCA and PNH, indicating that hemolytic processes may stimulate Hpx production, potentially via a heme-oxygenase-1 (HO-1)-dependent pathway. Furthermore, significantly diminished levels of Hpx in SHS suggest a role for the spleen in Hpx maintenance, possibly due to the importance of this organ for HO-1 activity. In conclusion, while parameters suggestive of hemolysis can be observed in SCA, HS and PNH, specific profiles of hemolytic markers and heme catabolism indicate that hemolytic events may be modulated differently in these diseases. DisclosuresConran:Bayer AG: Research Funding. Almeida:Jassen & Cilag: Other: Currently employed with.

A Novel Role for P-Selectin in the Pathogenesis of Acute Chest Syndrome

Acute chest syndrome (ACS) is a potentially fatal lung complication of sickle cell disease (SCD). There are currently no mechanistic based therapies for ACS and it continues to be a leading cause of death in SCD. Recent studies have identified independent links between extracellular heme with ACS, and with vaso-occlusive crisis (VOC) a common prodrome of ACS. Our experimental ACS model involves intravenous injection of purified hemin typically 35 µmoles/kg, which raises total plasma heme to a clinically relevant pathophysiologic level equivalent to ~0.5g/dl of hemoglobin, and causes respiratory failure exclusively in transgenic SCD mice of both the Townes and Berkeley strains (Ghosh et al., J Clin Invest, 2013). We have determined using time-lapse video microscopy and electrical cell impedance that hemin rapidly disrupts the pulmonary endothelial barrier in vitro. Hemopexin blocks hemin-induced pulmonary endothelial barrier disruption in vitro and respiratory failure in hemin-challenged SS mice. Importantly, genetic polymorphisms in the gene encoding heme oxygenase-1 the rate-limiting heme degradation enzyme are associated with ACS risk in the Cooperative Study of Sickle Cell Disease and the Silent Infarct Transfusion cohorts. These human data and our mechanistic results in SS mice support the heme hypothesis for ACS pathogenesis. P-selectin is a cell adhesion molecule involved in vascular inflammation. It is normally sequestered inside quiescent endothelium. However, recent evidence indicates extracellular heme activates the release of P-selectin unto the endothelial surface wall. Thus, in this study, we tested the hypothesis that P-selectin promotes ACS development. Infusion of a function blocking anti-P-selectin antibody protected SS mice (n=3) from hemin-induced ACS while all control SS mice (n=3) pretreated with IgG before the hemin challenge died (p=0.03) with severe hypoxemia and postmortem evidence of alveolar flooding. The apparent requirement for P-selectin in heme-induced lethal acute lung injury was confirmed by a 100% survival of P-selectin-/- (n=5) and 100% lethality of congenic P-selectin+/+ (n=5) mice challenged with hemin infusions (p=0.005). To identify the cell population involved in this disease process we generated bone marrow chimeric C57BL/6 mice lacking P-selectin in hematopoietic (P-selectinPLT-/-) and non-hematopoietic (P-selectinEC-/-) compartments. Seventy-five percent of P-selectinEC-/- mice were protected while all P-selectinPLT-/- congenic controls succumbed to hemin (n=3-4). The severity of lung injury in the P-selectinPLT-/- C57BL/6 mice was reflected by severe hypoxemia (SpO2: 82.75±2.14%) and a significantly higher lung wet/dry weight ratio compared to the ratio of the P-selectinEC-/- mouse lungs (p<0.05). Next, we transplanted congenic P-selectin-/- and P-selectin+/+ mice with SS mouse bone marrow to generate chimeric SS mice lacking endothelial P-selectin or expressing endothelial P-selectin respectively. Induction of ACS resulted in 40% lethality in the SS/P-selectinEC-/- mice (n=7) and 100% lethality in SS/P-selectinEC+/+ mice (n=7; p<0.01). The development of respiratory failure in the hemin modelinvolves de novo heme release from acute intravascular hemolysis. In this study, we found that SS/P-selectinEC-/- mice cleared ~70% of the hemin bolus within 30 min, which attenuated intravascular hemolysis and damped de novo heme release, while total plasma heme increased over 2-fold in the SS/P-selectinEC+/+ mice during the same time interval. Together these results demonstrate a critical intermediary role for P-selectin in the ACS triggered by acute intravascular hemolysis and elevated extracellular heme. In addition, we provide proof-of-principle in mice that antagonists of P-selectin can prevent and potentially treat ACS. DisclosuresNo relevant conflicts of interest to declare.

A Novel Role for Progesterone Receptor Membrane Component 1 (PGRMC1): A Partner and Regulator of Ferrochelatase.

Heme is an iron-containing cofactor essential for multiple cellular processes and fundamental activities such as oxygen transport. To better understand the means by which heme synthesis is regulated during erythropoiesis, affinity purification coupled with mass spectrometry (MS) was performed to identify putative protein partners interacting with ferrochelatase (FECH), the terminal enzyme in the heme biosynthetic pathway. Both progesterone receptor membrane component 1 (PGRMC1) and progesterone receptor membrane component 2 (PGRMC2) were identified in these experiments. These interactions were validated by reciprocal affinity purification followed by MS analysis and immunoblotting. The interaction between PGRMC1 and FECH was confirmed in vitro and in HEK 293T cells, a non-erythroid cell line. When cells that are recognized models for erythroid differentiation were treated with a small molecule inhibitor of PGRMC1, AG-205, there was an observed decrease in the level of hemoglobinization relative to that of untreated cells. In vitro heme transfer experiments showed that purified PGRMC1 was able to donate heme to apo-cytochrome b5. In the presence of PGRMC1, in vitro measured FECH activity decreased in a dose-dependent manner. Interactions between FECH and PGRMC1 were strongest for the conformation of FECH associated with product release, suggesting that PGRMC1 may regulate FECH activity by controlling heme release. Overall, the data illustrate a role for PGRMC1 in regulating heme synthesis via interactions with FECH and suggest that PGRMC1 may be a heme chaperone or sensor.

Differential heme release from various hemoglobin redox states and the upregulation of cellular heme oxygenase-1.

Despite advances in our understanding of the oxidative pathways mediated by free hemoglobin (Hb), the precise contribution of its highly reactive redox forms to tissue and organ toxicities remains ambiguous. Heme, a key degradation byproduct of Hb oxidation, has recently been recognized as a damage‐associated molecular pattern (DAMP) molecule, able to trigger inflammatory responses. Equally damaging is the interaction of the highly redox active forms of Hb with other biological molecules. We determined the kinetics of heme loss from individual Hb redox states—ferrous (Fe2+), ferric (Fe3+), and ferryl (Fe4+)—using two different heme receptor proteins: hemopexin (Hxp), a naturally occurring heme scavenger in plasma, and a double mutant (H64Y/V86F), apomyoglobin (ApoMb), which avidly binds heme released from Hb. We show for the first time that ferric Hb (Fe3+) loses heme at rates substantially higher than that of ferryl Hb (Fe4+). This was also supported by a higher expression of heme oxygenase‐1 (HO‐1) when ferric Hb was added to cultured lung alveolar epithelial cells (E10). The reported cytotoxicity of Hb may therefore be attributed to a combination of accelerated heme loss from the ferric form and protein radical formation associated with ferryl Hb. Targeted therapeutic interventions can therefore be designed to curb specific oxidative pathways of Hb in hemolytic anemias and when Hb is used as an oxygen‐carrying therapeutic.

Altered heme-mediated modulation of dendritic cell function in sickle cell alloimmunization.

Transfusions are the main treatment for patients with sickle cell disease. However, alloimmunization remains a major life-threatening complication for these patients, but the mechanism underlying pathogenesis of alloimmunization is not known. Given the chronic hemolytic state characteristic of sickle cell disease, resulting in release of free heme and activation of inflammatory cascades, we tested the hypothesis that anti-inflammatory response to heme is compromised in alloimmunized sickle patients, increasing their risk of alloimmunization. Heme-exposed monocyte-derived dendritic cells from both non-alloimmunized sickle patients and healthy donors inhibited priming of pro-inflammatory CD4(+) type 1 T cells, and exhibited significantly reduced levels of the maturation marker CD83. In contrast, in alloimmunized patients, heme did not reverse priming of pro-inflammatory CD4(+) cells by monocyte-derived dendritic cells or their maturation. Furthermore, heme dampened NF-κB activation in non-alloimmunized, but not in alloimmunized monocyte-derived dendritic cells. Heme-mediated CD83 inhibition depended on Toll-like receptor 4 but not heme oxygenase 1. These data suggest that extracellular heme limits CD83 expression on dendritic cells in non-alloimmunized sickle patients through a Toll-like receptor 4-mediated pathway, involving NF-κB, resulting in dampening of pro-inflammatory responses, but that in alloimmunized patients this pathway is defective. This opens up the possibility of developing new therapeutic strategies to prevent sickle cell alloimmunization.

Structural basis for haem piracy from host haemopexin by Haemophilus influenzae.

Haemophilus influenzae is an obligate human commensal/pathogen that requires haem for survival and can acquire it from several host haemoproteins, including haemopexin. The haem transport system from haem-haemopexin consists of HxuC, a haem receptor, and the two-partner-secretion system HxuB/HxuA. HxuA, which is exposed at the cell surface, is strictly required for haem acquisition from haemopexin. HxuA forms complexes with haem-haemopexin, leading to haem release and its capture by HxuC. The key question is how HxuA liberates haem from haemopexin. Here, we solve crystal structures of HxuA alone, and HxuA in complex with the N-terminal domain of haemopexin. A rational basis for the release of haem from haem-haemopexin is derived from both in vivo and in vitro studies. HxuA acts as a wedge that destabilizes the two-domains structure of haemopexin with a mobile loop on HxuA that favours haem ejection by redirecting key residues in the haem-binding pocket of haemopexin.

Heme changes HIF-α, eNOS and nitrite production in HUVECs after simvastatin, HU, and ascorbic acid therapies

The sickle cell disease (SCD) is a hemolytic genetic anemia characterized by free heme and hemoglobin release into intravascular spaces, with endothelial activation. Heme is a proinflammatory molecule able to directly activate vascular endothelium, thus, endothelial dysfunction and vascular disease are major chronic events described in SCD. The aim of this study was to evaluate the production of endothelial nitric oxide synthase (eNOS), nitrite and hypoxia inducible factor alpha (HIF-α) in HUVECs (human umbilical vein endothelial cells) activated by heme in response to simvastatin, hydroxyurea (HU), and ascorbic acid therapies. eNOS and HIF-α production were evaluated by ELISA and nitrite was measured by the Griess technique. The production of HIF-α increased when the cells were stimulated by heme (p<0.01), while treatment with HU and simvastatin reduced the production (p<0.01), and treatment with ascorbic acid increased HIF-1a production by the cells (p<0.01). Heme increased eNOS production, (p<0.01) but showed a heterogeneous pattern, and the lowest concentrations of all the treatments reduced the enzyme production (p<0.01). The nitrite production by HUVECs was enhanced by stimulation with heme (p<0.001) and was reduced by treatment with HU (p<0.001), ascorbic acid (p<0.001) and simvastatin (p<0.01). In summary, our results suggest that the hemolytic vascular microenvironment in SCD requires different therapeutic approaches to promote clinical improvement, and that a combination of therapies may be a viable strategy for treating patients.

Hemopexin therapy reverts heme-induced proinflammatory phenotypic switching of macrophages in a mouse model of sickle cell disease

AbstractHemolytic diseases, such as sickle cell anemia and thalassemia, are characterized by enhanced release of hemoglobin and heme into the circulation, heme-iron loading of reticulo-endothelial system macrophages, and chronic inflammation. Here we show that in addition to activating the vascular endothelium, hemoglobin and heme excess alters the macrophage phenotype in sickle cell disease. We demonstrate that exposure of cultured macrophages to hemolytic aged red blood cells, heme, or iron causes their functional phenotypic change toward a proinflammatory state. In addition, hemolysis and macrophage heme/iron accumulation in a mouse model of sickle disease trigger similar proinflammatory phenotypic alterations in hepatic macrophages. On the mechanistic level, this critically depends on reactive oxygen species production and activation of the Toll-like receptor 4 signaling pathway. We further demonstrate that the heme scavenger hemopexin protects reticulo-endothelial macrophages from heme overload in heme-loaded Hx-null mice and reduces production of cytokines and reactive oxygen species. Importantly, in sickle mice, the administration of human exogenous hemopexin attenuates the inflammatory phenotype of macrophages. Taken together, our data suggest that therapeutic administration of hemopexin is beneficial to counteract heme-driven macrophage-mediated inflammation and its pathophysiologic consequences in sickle cell disease.

Heme acquisition mechanisms of Porphyromonas gingivalis - strategies used in a polymicrobial community in a heme-limited host environment.

Porphyromonas gingivalis, a main etiologic agent and key pathogen responsible for initiation and progression of chronic periodontitis requires heme as a source of iron and protoporphyrin IX for its survival and the ability to establish an infection. Porphyromonas gingivalis is able to accumulate a defensive cell-surface heme-containing pigment in the form of μ-oxo bisheme. The main sources of heme for P.gingivalis invivo are hemoproteins present in saliva, gingival crevicular fluid, and erythrocytes. To acquire heme, P.gingivalis uses several mechanisms. Among them, the best characterized are those employing hemagglutinins, hemolysins, and gingipains (Kgp, RgpA, RgpB), TonB-dependent outer-membrane receptors (HmuR, HusB, IhtA), and hemophore-like proteins (HmuY, HusA). Proteins involved in intracellular heme transport, storage, and processing are less well characterized (e.g. PgDps). Importantly, P.gingivalis may also use the heme acquisition systems of other bacteria to fulfill its own heme requirements. Porphyromonas gingivalis displays a novel paradigm for heme acquisition from hemoglobin, whereby the Fe(II)-containing oxyhemoglobin molecule must first be oxidized to methemoglobin to facilitate heme release. This process not only involves P.gingivalis arginine- and lysine-specific gingipains, but other proteases (e.g. interpain A from Prevotella intermedia) or pyocyanin produced by Pseudomonas aeruginosa. Porphyromonas gingivalis is then able to fully proteolyze the more susceptible methemoglobin substrate to release free heme or to wrest heme from it directly through the use of the HmuY hemophore.

Diverse Nrf2 Activators Coordinated to Cobalt Carbonyls Induce Heme Oxygenase-1 and Release Carbon Monoxide in Vitro and in Vivo

The Nrf2/heme oxygenase-1 (HO-1) axis affords significant protection against oxidative stress and cellular damage. We synthesized a series of cobalt-based hybrid molecules (HYCOs) that combine an Nrf2 inducer with a releaser of carbon monoxide (CO), an anti-inflammatory product of HO-1. Two HYCOs markedly increased Nrf2/HO-1 expression, liberated CO and exerted anti-inflammatory activity in vitro. HYCOs also up-regulated tissue HO-1 and delivered CO in blood after administration in vivo, supporting their potential use against inflammatory conditions.

The Pathophysiology of Heme in the Brain.

Heme is essential for the survival of most organisms, despite the fact of being potentially toxic. This dual effect is due to the ability of the iron (Fe) atom contained within the protoporphyrin ring of the heme molecule to participate in redox reactions and exchange electrons with a variety of substrates. Therefore, the pro-oxidant reactivity of heme needs to be kept under control, an effect achieved by its incorporation into the heme pockets of hemoproteins, i.e. proteins required to exert vital biological functions in which heme acts as prosthetic group. The release of heme from hemoproteins and the participation of Fe in the Fenton reaction lead to the generation of unfettered oxidative stress and programmed cell death. Although further investigations would be required to elucidate the regulation of heme in the brain, this molecule appears to be critically involved in the pathogenesis of different neurodegenerative diseases, as heme accumulation or deficiency is associated with impaired brain activity and neuronal death. Thus, the aim of this review is to provide an overview on the importance of heme in the brain and the pathophysiologic consequences associated with its accumulation.

The Heme Scavenger Hemopexin Reverts Heme-Driven Pro-Inflammatory Phenotypic Switching of Macrophages in Sickle Cell Disease

Hemolytic diseases, such as sickle cell anemia and thalassemia, are characterized by enhanced release of hemoglobin and heme into the circulation, heme-iron loading of reticulo-endothelial system macrophages as well as chronic inflammation. Here we demonstrate that in addition to activating the vascular endothelium, hemoglobin and heme alter the macrophage phenotype in sickle cell disease.We show that exposure of cultured macrophages to hemolytic aged red blood cells, heme or iron causes their functional phenotypic change towards a pro-inflammatory phenotype, with increased expression levels of inflammatory markers such as IL-6 (P<0.001), TNFα (P<0.0001), IL-1β (P<0.001) MHCII (P<0.001), CD86 (P<0.05) and CD14 (P<0.0001) and decreased expression levels of anti-inflammatory markers such as IL-10 (P<0.0001), CD206 (P<0.0001), Arginase-1 and Ym1. Interestingly, hemolysis and macrophage heme/iron accumulation in a mouse model of sickle cell disease triggers similar pro-inflammatory phenotypic alterations in hepatic macrophages, increasing the expression levels of IL-6 (P<0.0001), MHCII (P<0.05), CD86 (P<0.0001) and iNOS (P<0.01). On the mechanistic level, heme-induced pro-inflammatory phenotype switching of macrophages critically depends on ROS production and activation of the TLR4 signaling pathway.We further demonstrate that the heme scavenger hemopexin protects reticulo-endothelial macrophages from heme overload in heme-loaded Hx-null mice (P<0.05) and reduces production of cytokines (IL-6, TNFα: P<0.01) and reactive oxygen species (P<0.001). Importantly, in sickle mice the administration of human exogenous hemopexin attenuates the inflammatory phenotype of macrophages, by decreasing the expression of IL-6 (P<0.05), MHCII (P<0.01), CD86 (P<0.001) and iNOS (P<0.05).Taken together, our data suggest that therapeutical administration of hemopexin is beneficial to counteract heme-driven macrophage-mediated inflammation in sickle cell disease. DisclosuresBrinkman:CSL Behring: Employment. Zuercher:CSL Behring: Employment.

Dysregulation of the haem-haemopexin axis is associated with severe malaria in a case-control study of Ugandan children.

Background Malaria is associated with haemolysis and the release of plasma haem. Plasma haem can cause endothelial injury and organ dysfunction, and is normally scavenged by haemopexin to limit toxicity. It was hypothesized that dysregulation of the haem-haemopexin pathway contributes to severe and fatal malaria infections.MethodsPlasma levels of haemin (oxidized haem), haemopexin, haptoglobin, and haemoglobin were quantified in a case–control study of Ugandan children with Plasmodium falciparum malaria. Levels at presentation were compared in children with uncomplicated malaria (UM; n = 29), severe malarial anaemia (SMA; n = 27) or cerebral malaria (CM; n = 31), and evaluated for utility in predicting fatal (n = 19) vs non-fatal (n = 39) outcomes in severe disease. A causal role for haemopexin was assessed in a pre-clinical model of experimental cerebral malaria (ECM), following disruption of mouse haemopexin gene (hpx). Analysis was done using Kruskall Wallis tests, Mann–Whitney tests, log-rank tests for survival, and repeated measures ANOVA.ResultsIn Ugandan children presenting with P. falciparum malaria, haemin levels were higher and haemopexin levels were lower in SMA and CM compared to children with UM (haemin, p < 0.01; haemopexin, p < 0.0001). Among all cases of severe malaria, elevated levels of haemin and cell-free haemoglobin at presentation were associated with subsequent mortality (p < 0.05). Compared to ECM-resistant BALB/c mice, susceptible C57BL/6 mice had lower circulating levels of haemopexin (p < 0.01), and targeted deletion of the haemopexin gene, hpx, resulted in increased mortality compared to their wild type littermates (p < 0.05).ConclusionsThese data indicate that plasma levels of haemin and haemopexin measured at presentation correlate with malaria severity and levels of haemin and cell-free haemoglobin predict outcome in paediatric severe malaria. Mechanistic studies in the ECM model support a causal role for the haem-haemopexin axis in ECM pathobiology.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-1028-1) contains supplementary material, which is available to authorized users.

Neutrophil activation and nucleosomes as markers of systemic inflammation in paroxysmal nocturnal hemoglobinuria: effects of eculizumab

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by complement-mediated hemolysis and a high risk of life-threatening venous and arterial thrombosis. Uncontrolled complement activation and the release of cell-free heme may result in systemic inflammation, neutrophil activation, and the release of procoagulant neutrophilic proteases. Eculizumab, an antibody to complement factor C5, inhibits hemolysis and reduces thrombotic risk. To study neutrophil activation and nucleosome levels in relation to thrombosis in PNH patients before and during treatment with eculizumab. In 51 untreated PNH patients, including 20 patients before and after commencing eculizumab treatment, we have assessed neutrophil activation by measuring elastase-α1 -antitrypsin (EA) complexes and circulating nucleosomes, as established markers for systemic inflammation and cell death. Nucleosomes (median; range; 95% confidence interval [CI]), but not EA complexes, were higher in PNH patients with a history of thrombosis (16; 7-264; 0.3-94UmL(-1) , n=12) than in those without (6; 6-35; 7-11U mL(-1) , n=39) or controls (8; 6-23; 7-12UmL(-1) , n=17). EA complexes, but not nucleosomes, decreased promptly and markedly upon eculizumab treatment. EA complexes (estimated marginal means; 95% CI) remained low at ≥12weeks (50; 34-67) compared with baseline (12; -6 to 29). The increased nucleosome levels in PNH patients with a history of thrombosis suggest systemic inflammation and/or cell death. Neutrophil activation markers did not differ between patients with and without a history of thrombosis and healthy controls. Interestingly, basal neutrophil activation in PNH patients significantly decreases on treatment with eculizumab, indicating that neutrophil activation is C5a driven.

The free heme concentration in healthy human erythrocytes

Heme, the prosthetic group of hemoglobin, may be released from its host due to an intrinsic instability of hemoglobin and accumulate in the erythrocytes. Free heme is in the form of hematin (Fe3+ protoporphyrin IX OH) and follows several pathways of biochemical toxicity to tissues, cells, and organelles since it catalyzes the production of reactive oxygen species. To determine concentration of soluble free heme in human erythrocytes, we develop a new method. We lyse the red blood cells and isolate free heme from hemoglobin by dialysis. We use the heme to reconstitute horseradish peroxidase (HRP) from an excess of the apoenzyme and determine the HRP reaction rate from the evolution of the emitted luminescence. We find that in a population of five healthy adults the average free heme concentration in the erythrocytes is 21±2μM, ca. 100× higher than previously determined. Tests suggest that the lower previous value was due to the use of elevated concentrations of NaCl, which drive hematin precipitation and re-association with apoglobin. We show that the found hematin concentration is significantly higher than estimates based on equilibrium release and the known hematin dimerization. The factors that lead to enhanced heme release remain an open question.

Kaposi Sarcoma Herpesvirus Induces HO-1 during De Novo Infection of Endothelial Cells via Viral miRNA-Dependent and -Independent Mechanisms.

ABSTRACTKaposi sarcoma (KS) herpesvirus (KSHV) infection of endothelial cells (EC) is associated with strong induction of heme oxygenase-1 (HO-1), a stress-inducible host gene that encodes the rate-limiting enzyme responsible for heme catabolism. KS is an angioproliferative tumor characterized by the proliferation of KSHV-infected spindle cells, and HO-1 is highly expressed in such cells. HO-1 converts the pro-oxidant, proinflammatory heme molecule into metabolites with antioxidant, anti-inflammatory, and proliferative activities. Previously published work has shown that KSHV-infected EC in vitro proliferate in response to free heme in a HO-1-dependent manner, thus implicating virus-enhanced HO-1 activity in KS tumorigenesis. The present study investigated the molecular mechanisms underlying KSHV induction of HO-1 in lymphatic EC (LEC), which are the likely spindle cell precursors. In a time course analysis of KSHV-infected cells, HO-1 expression displays biphasic kinetics characterized by an early transient induction that is followed by a more sustained upregulation coincident with the establishment of viral latency. A viral microRNA miR-K12-11 deletion mutant of KSHV was found to be defective for induction of HO-1 during latency. A potential mechanism for this phenotype was provided by BACH1, a cellular HO-1 transcriptional repressor targeted by miR-K12-11. In fact, in KSHV-infected LEC, the BACH1 message level is reduced, BACH1 subcellular localization is altered, and miR-K12-11 mediates the inverse regulation of HO-1 and BACH1 during viral latency. Interestingly, the data indicate that neither miR-K12-11 nor de novo KSHV gene expression is required for the burst of HO-1 expression observed at early times postinfection, which suggests that additional virion components promote this phenotype.

The structure of haemoglobin bound to the haemoglobin receptor IsdH from Staphylococcus aureus shows disruption of the native α-globin haem pocket.

Staphylococcus aureus is a common and serious cause of infection in humans. The bacterium expresses a cell-surface receptor that binds to, and strips haem from, human haemoglobin (Hb). The binding interface has previously been identified; however, the structural changes that promote haem release from haemoglobin were unknown. Here, the structure of the receptor-Hb complex is reported at 2.6 Å resolution, which reveals a conformational change in the α-globin F helix that disrupts the haem-pocket structure and alters the Hb quaternary interactions. These features suggest potential mechanisms by which the S. aureus Hb receptor induces haem release from Hb.

Cigarette smokers develop structurally modified hemoglobin: a possible way of increasing oxidative stress

Context: Increased levels of free radicals and various reactive species along with reduced antioxidant defence system are the major threat to erythrocyte in tobacco smokers. Thus, the hemoglobin (Hb) within the erythrocyte is very prone to oxidative damage. Earlier reports suggest that cigarette smoking is related with the glutathionylation and formation of adducts of Hb.Objective: We have highlighted the possible changes in secondary and tertiary structures of Hb in cigarette smokers and its physiological consequences.Material and method: Twenty smokers and 18 non-smokers, aged 25–35 years were volunteered in this study. We used flow cytometry for measuring intracellular reactive oxygen species (IcROS). The purified Hb was subjected to different spectrophotometric, fluoremetric and circular dichroic (CD) analysis. The hydrophobicity, thermal stability, heme release and oxidation of purified Hb were also studied.Result: We observed that the IcROS was also higher in cigarette smokers than non-smokers. The data of intrinsic fluorescence, synchronous fluorescence and tryptophan quenching studies showed that the microenvironments of β37 tryptophan and tyrosine residues of Hb were moved toward more hydrophobic region in cigarette smokers. Increased hydrophobicity and thermal stability furthermore indicated more compactness of smokers' hemoglobin. From CD spectra, we confirmed an overall modification of the secondary and tertiary structures of hemoglobin in smokers. Both auto- and co-oxidation rates of purified Hb were found to be higher in cigarette smokers.Discussion and conclusion: We conclude that the modified Hb in cigarette smokers may further enhance the oxidative insult within the cell.

Allosteric regulation of the Plasmodium falciparum cysteine protease falcipain-2 by heme

During the erythrocytic cycle of Plasmodium falciparum malaria parasites break down host hemoglobin, resulting in the release of free heme (ferriprotoporphyrin IX). Heme is a generator of free radicals that cause oxidative stress, but it is detoxified by crystallization into hemozoin inside the food vacuole. We evaluated the interaction of heme and heme analogues with falcipain-2, a P. falciparum food vacuole cysteine protease that plays a key role in hemoglobin digestion. Heme bound to falcipain-2 with a 1:1 stoichiometry, and heme inhibited falcipain-2 activity against both human hemoglobin and chromogenic peptide substrates through a noncompetitive-like mechanism. A series of porphyrin analogues was screened for inhibition of falcipain-2, demonstrating a minor contribution of iron to heme–falcipain-2 interaction, and revealing dependence on both propionic and vinyl groups for inhibition of falcipain-2 by heme. Docking and molecular dynamics simulation unveiled a novel, inducible heme-binding moiety in falcipain-2 adjacent to the catalytic site. Kinetic data suggested that the noncompetitive-like inhibition was substrate inhibition induced by heme. Collectively these data suggest that binding of heme to falcipain-2 may limit the accumulation of free heme in the parasite food vacuole, providing a means of heme detoxification in addition to crystallization into hemozoin.