Heme Overload Research Articles

TLR7 signaling drives development of a unique population of hemophagocytic macrophages associated with anemia

Abstract Toll-like receptor (TLR) 7 activity is associated with myeloid expansion in the autoimmune disease Systemic Lupus Erythematosus (SLE), yet how this contributes to severe anemia, often seen in SLE complication, is unclear. Transcriptional and functional assays indicate that TLR7 signaling in the common myeloid progenitor (CMP) preferentially skews lineage choice towards a hemophagocytic macrophage. Transgenic mice that overexpress Tlr7 and develop SLE-like disease exhibited increased hemophagocytic macrophages and progressively developed anemia. Interestingly, in these mice, we observed not only conventional hemophagocytic macrophage populations known as red pulp macrophages (RPM), but also an inflammation-induced hemophagocytic macrophage population (HPM). Unlike RPM that are primarily yolk sac-derived and require the transcription factor Spi-C for their development, we have found that HPM are monocyte-derived and do not require Spi-C. Additionally, TLR7 drives an expansion of hemophagocytic CD11bhi bone marrow macrophages (BMM) that are not found in the absence of TLR7 signaling. Although it has been shown that pathological conditions including excess heme overload can drive monocytes to differentiate to hemophagocytic macrophages, this is the first account of inflammation driving this process. These findings uncover a novel mechanism by which TLR7 signaling participates in hemophagocytic macrophage differentiation from monocytes and may account for one of the mechanisms driving anemia during chronic inflammation and chronic autoimmune diseases such as SLE.

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.

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.

Alteration of heme metabolism in a cellular model of Diamond-Blackfan anemia.

Diamond-Blackfan anemia (DBA) is a congenital pure red cell aplasia often associated with skeletal malformations. Mutations in ribosomal protein coding genes, mainly in RPS19, account for the majority of DBA cases. The molecular mechanisms underlying DBA pathogenesis are still not completely understood. Alternative spliced isoforms of FLVCR1 (feline leukemia virus subgroup C receptor 1) transcript coding for non-functional proteins have been reported in some DBA patients. Consistently, a phenotype very close to DBA has been described in animal models of FLVCR1 deficiency. FLVCR1 gene codes for two proteins: the plasma membrane heme exporter FLVCR1a and the mitochondrial heme exporter FLVCR1b. The coordinated expression of both FLVCR1 isoforms regulates an intracellular heme pool, necessary for proper expansion and differentiation of erythroid precursors. Here, we investigate the role of FLVCR1 isoforms in a cellular model of DBA. RPS19-downregulated TF1 cells show reduced FLVCR1a and FLVCR1b mRNA levels associated with heme overload. The downregulation of FLVCR1 isoforms affects cell cycle progression and apoptosis in differentiating K562 cells, a phenotype similar to DBA. Taken together, these data suggest that alteration of heme metabolism could play a role in the pathogenesis of DBA.

Hemopexin Therapy Improves Cardiovascular Function by Preventing Heme-Induced Endothelial Toxicity in Mouse Models of Hemolytic Diseases

Hemolytic diseases are characterized by enhanced intravascular hemolysis resulting in heme-catalyzed reactive oxygen species generation, which leads to endothelial dysfunction and oxidative damage. Hemopexin (Hx) is a plasma heme scavenger able to prevent endothelial damage and tissue congestion in a model of heme overload. Here, we tested whether Hx could be used as a therapeutic tool to counteract heme toxic effects on the cardiovascular system in hemolytic diseases. By using a model of heme overload in Hx-null mice, we demonstrated that heme excess in plasma, if not bound to Hx, promoted the production of reactive oxygen species and the induction of adhesion molecules and caused the reduction of nitric oxide availability. Then, we used β-thalassemia and sickle cell disease mice as models of hemolytic diseases to evaluate the efficacy of an Hx-based therapy in the treatment of vascular dysfunction related to heme overload. Our data demonstrated that Hx prevented heme-iron loading in the cardiovascular system, thus limiting the production of reactive oxygen species, the induction of adhesion molecules, and the oxidative inactivation of nitric oxide synthase/nitric oxide, and promoted heme recovery and detoxification by the liver mainly through the induction of heme oxygenase activity. Moreover, we showed that in sickle cell disease mice, endothelial activation and oxidation were associated with increased blood pressure and altered cardiac function, and the administration of exogenous Hx was found to almost completely normalize these parameters. Hemopexin treatment is a promising novel therapy to protect against heme-induced cardiovascular dysfunction in hemolytic disorders.

Impact of Intravascular Hemolysis in Malaria on Liver Dysfunction: INVOLVEMENT OF HEPATIC FREE HEME OVERLOAD, NF-κB ACTIVATION, AND NEUTROPHIL INFILTRATION

We have investigated the impact of persistent intravascular hemolysis on liver dysfunction using the mouse malaria model. Intravascular hemolysis showed a positive correlation with liver damage along with the increased accumulation of free heme and reactive oxidants in liver. Hepatocytes overinduced heme oxygenase-1 (HO-1) to catabolize free heme in building up defense against this pro-oxidant milieu. However, in a condition of persistent free heme overload in malaria, the overactivity of HO-1 resulted in continuous transient generation of free iron to favor production of reactive oxidants as evident from 2',7'-dichlorofluorescein fluorescence studies. Electrophoretic mobility shift assay documented the activation of NF-κB, which in turn up-regulated intercellular adhesion molecule 1 as evident from chromatin immunoprecipitation studies. NF-κB activation also induced vascular cell adhesion molecule 1, keratinocyte chemoattractant, and macrophage inflammatory protein 2, which favored neutrophil extravasation and adhesion in liver. The infiltration of neutrophils correlated positively with the severity of hemolysis, and neutrophil depletion significantly prevented liver damage. The data further documented the elevation of serum TNFα in infected mice, and the treatment of anti-TNFα antibodies also significantly prevented neutrophil infiltration and liver injury. Deferoxamine, which chelates iron, interacts with free heme and bears antioxidant properties that prevented oxidative stress, NF-κB activation, neutrophil infiltration, hepatocyte apoptosis, and liver damage. Furthermore, the administration of N-acetylcysteine also prevented NF-κB activation, neutrophil infiltration, hepatocyte apoptosis, and liver damage. Thus, hepatic free heme accumulation, TNFα release, oxidative stress, and NF-κB activation established a link to favor neutrophil infiltration in inducing liver damage during hemolytic conditions in malaria.

Lon Peptidase 1 (LONP1)-dependent Breakdown of Mitochondrial 5-Aminolevulinic Acid Synthase Protein by Heme in Human Liver Cells

5-Aminolevulinic acid synthase (ALAS-1) is the first rate controlling enzyme that controls cellular heme biosynthesis. Negative feedback regulation of ALAS-1 by the end product heme is well documented and provides the foundation for heme treatment of acute porphyrias, a group of diseases caused by genetic defects in the heme biosynthesis pathway and exacerbated by controlled up-regulation of ALAS-1. Heme is known to affect ALAS-1 activity by repressing gene transcription, accelerating mRNA degradation, and impeding pre-ALAS-1 mitochondrial translocation. In the current study, we examined the effect of heme on the rate of mature ALAS-1 protein turnover in human cells and tissues and explored the mediator involved in this new regulatory mechanism. We found that heme and other metalloporphyrins such as CoPP and CrPP decreased mitochondrial ALAS-1 protein through proteolysis. This degradative effect cannot be emulated by iron or free protoporphyrin, two major chemical components of the heme ring, and is independent of oxidative stress. Down-regulating the activity of mitochondrial LONP1, an ATP-dependent protease that controls the selective turnover of mitochondrial matrix proteins, with potent inhibitors and specific siRNA diminished the negative effect of heme on mitochondrial ALAS-1. Therefore, our data support the existence of a conserved heme feedback regulatory mechanism that functions on the mature form of ALAS-1 protein through the activity of a mitochondrial proteolytic system.

Involvement of the heme oxygenase–carbon monoxide–cGMP pathway in the nociception induced by acute painful stimulus in rats

Heme oxygenase–carbon monoxide–cGMP (HO–CO–cGMP) pathway has been reported to be involved in peripheral and spinal modulation of inflammatory pain. However, the involvement of this pathway in the modulation of acute painful stimulus in the absence of inflammation remains unknown. Thus, we evaluated the involvement of the HO–CO–cGMP pathway in nociception by means the of analgesia index (AI) in the tail flick test. Rats underwent surgery for implantation of unilateral guide cannula directed toward the lateral ventricle and after the recovery period (5–7days) were subjected to the measures of baseline tail flick test. Animals were divided into groups to assess the effect of intracerebroventricular administration (i.c.v.) of the following compounds: ZnDPBG (HO inhibitor) or vehicle (Na2CO3), heme-lysinate (substrate overload) or vehicle (l-lysine), or the selective inhibitor of soluble guanilate cyclase ODQ or vehicle (DMSO 1%) following the administration of heme-lysinate or vehicle. Heme overload increased AI, indicating an antinociceptive role of the pathway. This response was attenuated by i.c.v. pretreatment with the HO inhibitor ZnDPBG. In addition, this effect was dependent on cGMP activity, since the pretreatment with ODQ blocked the increase in the AI. Because CO produces most of its actions via cGMP, these data strongly imply that CO is the HO product involved in the antinociceptive response. This modulation seems to be phasic rather than tonic, since i.c.v. treatment with ZnDPBG or ODQ did not alter the AI. Therefore, we provide evidence consistent with the notion that HO–CO–cGMP pathway plays a key phasic antinociceptive role modulating noninflammatory acute pain.

Hemopexin Prevents Endothelial Damage and Liver Congestion in a Mouse Model of Heme Overload

Intravascular hemolysis results in the release of massive amounts of hemoglobin and heme into plasma, where they are rapidly bound by haptoglobin and hemopexin, respectively. Data from haptoglobin and hemopexin knockout mice have shown that both proteins protect from renal damage after phenylhydrazine-induced hemolysis, whereas double-mutant mice were especially prone to liver damage. However, the specific role of hemopexin remains elusive because of the difficulty in discriminating between hemoglobin and heme recovery. To study the specific role of hemopexin in intravascular hemolysis, we established a mouse model of heme overload. Under these conditions, both endothelial activation and vascular permeability were significantly higher in hemopexin-null mice compared with wild-type controls. Vascular permeability was particularly altered in the liver, where congestion in the centrolobular area was believed to be associated with oxidative stress and inflammation. Liver damage in hemopexin- null mice may be prevented by induction of heme oxygenase-1 before heme overload. Furthermore, heme-treated hemopexin-null mice exhibited hyperbilirubinemia, prolonged heme oxygenase-1 expression, excessive heme metabolism, and lack of H-ferritin induction in the liver compared with heme-treated wild-type controls. Moreover, these mutant mice metabolize an excess of heme in the kidney. These studies highlight the importance of hemopexin in heme detoxification, thus suggesting that drugs mimicking hemopexin activity might be useful to prevent endothelial damage in patients suffering from hemolytic disorders.

Hemopexin: structure, function, and regulation.

Hemopexin (HPX) is the plasma protein with the highest binding affinity to heme among known proteins. It is mainly expressed in liver, and belongs to acute phase reactants, the synthesis of which is induced after inflammation. Heme is potentially highly toxic because of its ability to intercalate into lipid membrane and to produce hydroxyl radicals. The binding strength between heme and HPX, and the presence of a specific heme-HPX receptor able to catabolize the complex and to induce intracellular antioxidant activities, suggest that hemopexin is the major vehicle for the transportation of heme in the plasma, thus preventing heme-mediated oxidative stress and heme-bound iron loss. In this review, we discuss the experimental data that support this view and show that the most important physiological role of HPX is to act as an antioxidant after blood heme overload, rather than to participate in iron metabolism. Particular attention is also put on the structure of the protein and on its regulation during the acute phase reaction.

Heme oxygenase and ferroxidase activities in chronically hemodialysed patients 60

Hemodialysis may induce hemolysis, thereby heme overload, release of iron and further increase of the peroxidative reactions in the blood of patients. We studied the activity of the rate limiting enzyme of heme degradation, heme oxygenase (HOX) and that of ferroxidase (FOX), together with the level of plasma lipid peroxides (expressed as MDA) in 11 adolescent patients and in 12 aged matched controls.