Heme-stress activated NRF2 skews fate trajectories of bone marrow cells from dendritic cells towards red pulp-like macrophages in hemolytic anemia.

Heme is an erythrocyte-derived toxin that drives disease progression in hemolytic anemias, such as sickle cell disease. During hemolysis, specialized bone marrow-derived macrophages with a high heme-metabolism capacity orchestrate disease adaptation by removing damaged erythrocytes and heme-protein complexes from the blood and supporting iron recycling for erythropoiesis. Since chronic heme-stress is noxious for macrophages, erythrophagocytes in the spleen are continuously replenished from bone marrow-derived progenitors. Here, we hypothesized that adaptation to heme stress progressively shifts differentiation trajectories of bone marrow progenitors to expand the capacity of heme-handling monocyte-derived macrophages at the expense of the homeostatic generation of dendritic cells, which emerge from shared myeloid precursors. This heme-induced redirection of differentiation trajectories may contribute to hemolysis-induced secondary immunodeficiency. We performed single-cell RNA-sequencing with directional RNA velocity analysis of GM-CSF-supplemented mouse bone marrow cultures to assess myeloid differentiation under heme stress. We found that heme-activated NRF2 signaling shifted the differentiation of bone marrow cells towards antioxidant, iron-recycling macrophages, suppressing the generation of dendritic cells in heme-exposed bone marrow cultures. Heme eliminated the capacity of GM-CSF-supplemented bone marrow cultures to activate antigen-specific CD4 T cells. The generation of functionally competent dendritic cells was restored by NRF2 loss. The heme-induced phenotype of macrophage expansion with concurrent dendritic cell depletion was reproduced in hemolytic mice with sickle cell disease and spherocytosis and associated with reduced dendritic cell functions in the spleen. Our data provide a novel mechanistic underpinning of hemolytic stress as a driver of hyposplenism-related secondary immunodeficiency.

Cerebrospinal fluid hemoglobin drives subarachnoid hemorrhage-related secondary brain injury.

Secondary brain injury after aneurysmal subarachnoid hemorrhage (SAH-SBI) contributes to poor outcomes in patients after rupture of an intracranial aneurysm. The lack of diagnostic biomarkers and novel drug targets represent an unmet need. The aim of this study was to investigate the clinical and pathophysiological association between cerebrospinal fluid hemoglobin (CSF-Hb) and SAH-SBI. In a cohort of 47 patients, we collected daily CSF-samples within 14 days after aneurysm rupture. There was very strong evidence for a positive association between spectrophotometrically determined CSF-Hb and SAH-SBI. The accuracy of CSF-Hb to monitor for SAH-SBI markedly exceeded that of established methods (AUC: 0.89 [0.85-0.92]). Temporal proteome analysis revealed erythrolysis accompanied by an adaptive macrophage response as the two dominant biological processes in the CSF-space after aneurysm rupture. Ex-vivo experiments on the vasoconstrictive and oxidative potential of Hb revealed critical inflection points overlapping CSF-Hb thresholds in patients with SAH-SBI. Selective depletion and in-solution neutralization by haptoglobin or hemopexin efficiently attenuated the vasoconstrictive and lipid peroxidation activities of CSF-Hb. Collectively, the clinical association between high CSF-Hb levels and SAH-SBI, the underlying pathophysiological rationale, and the favorable effects of haptoglobin and hemopexin in ex-vivo experiments position CSF-Hb as a highly attractive biomarker and potential drug target.

Acute Hemolysis and Heme Suppress Anti-CD40 Antibody-Induced Necro-Inflammatory Liver Disease.

Clearance of red blood cells and hemoproteins is a key metabolic function of macrophages during hemolytic disorders and following tissue injury. Through this archetypical phagocytic function, heme is detoxified and iron is recycled to support erythropoiesis. Reciprocal interaction of heme metabolism and inflammatory macrophage functions may modify disease outcomes in a broad range of clinical conditions. We hypothesized that acute hemolysis and heme induce acute anti-inflammatory signals in liver macrophages. Using a macrophage-driven model of sterile liver inflammation, we showed that phenylhydrazine (PHZ)-mediated acute erythrophagocytosis blocked the anti-CD40 antibody-induced pathway of macrophage activation. This process attenuated the inflammatory cytokine release syndrome and necrotizing hepatitis induced by anti-CD40 antibody treatment of mice. We further established that administration of heme-albumin complexes specifically delivered heme to liver macrophages and replicated the anti-inflammatory effect of hemolysis. The anti-inflammatory heme-signal was induced in macrophages by an increased intracellular concentration of the porphyrin independently of iron. Overall, our work suggests that induction of heme-signaling strongly suppresses inflammatory macrophage function, providing protection against sterile liver inflammation.

Hemolysis transforms liver macrophages into antiinflammatory erythrophagocytes.

During hemolysis, macrophages in the liver phagocytose damaged erythrocytes to prevent the toxic effects of cell-free hemoglobin and heme. It remains unclear how this homeostatic process modulates phagocyte functions in inflammatory diseases. Using a genetic mouse model of spherocytosis and single-cell RNA sequencing, we found that erythrophagocytosis skewed liver macrophages into an antiinflammatory phenotype that we defined as MarcohiHmoxhiMHC class IIlo erythrophagocytes. This phenotype transformation profoundly mitigated disease expression in a model of an anti-CD40-induced hyperinflammatory syndrome with necrotic hepatitis and in a nonalcoholic steatohepatitis model, representing 2 macrophage-driven sterile inflammatory diseases. We reproduced the antiinflammatory erythrophagocyte transformation in vitro by heme exposure of mouse and human macrophages, yielding a distinctive transcriptional signature that segregated heme-polarized from M1- and M2-polarized cells. Mapping transposase-accessible chromatin in single cells by sequencing defined the transcription factor NFE2L2/NRF2 as a critical driver of erythrophagocytes, and Nfe2l2/Nrf2 deficiency restored heme-suppressed inflammation. Our findings point to a pathway that regulates macrophage functions to link erythrocyte homeostasis with innate immunity.

Line-selective macrophage activation with an anti-CD40 antibody drives a hemophagocytic syndrome in mice.

Hemophagocytic syndromes comprise a cluster of hyperinflammatory disorders, including hemophagocytic lymphohistiocytosis and macrophage activation syndrome. Overwhelming macrophage activation has long been considered a final common pathway in the pathophysiology of hemophagocytic syndromes leading to the characteristic cytokine storm, laboratory abnormalities, and organ injuries that define the clinical spectrum of the disease. So far, it is unknown whether primary macrophage activation alone can induce the disease phenotype. In this study, we established a novel mouse model of a hemophagocytic syndrome by treating mice with an agonistic anti-CD40 antibody (Ab). The response in wild-type mice is characterized by a cytokine storm, associated with hyperferritinemia, high soluble CD25, erythrophagocytosis, secondary endothelial activation with multiple organ vaso-occlusion, necrotizing hepatitis, and variable cytopenias. The disease is dependent on a tumor necrosis factor-α-interferon-γ-driven amplification loop. After macrophage depletion with clodronate liposomes or in mice with a macrophage-selective deletion of the CD40 gene (CD40flox/flox/LysMCre), the disease was abolished. These data provide a new preclinical model of a hemophagocytic syndrome and reinforce the key pathophysiological role of macrophages.

Determining the Optimal Normalization Factor of Different Target Arteries for ex vivo Vascular Function Experiments: A New Standardized Procedure.

The standardization of resistance vessel preparation is crucial to compare physiologic vascular reactivity under different experimental conditions. Here, we describe a generalizable experimental setup for ex vivo vascular function experiments and their mathematical basis. Porcine basilar arteries and chicken common carotid arteries were isolated post mortem via standardized surgical approaches. The inner circumference of these vessels with a passive wall tension corresponding to 100 mm Hg (IC100) as well as the circumference at which the active force production of the vessel is maximal (IC1) were determined systematically. The IC1/IC100 ratio (also referred to as factor k), a value that is believed to be constant for a defined vessel type in one species, was calculated by a novel mathematical approach. Here, we present an easy-to-use toolbox for the systematic and computer-based calculation of factor k and simplified optimal pre-stretching of any vascular segments for wire myography experiments.

Haptoglobin administration into the subarachnoid space prevents hemoglobin-induced cerebral vasospasm.

Delayed ischemic neurological deficit (DIND) is a major driver of adverse outcomes in patients with aneurysmal subarachnoid hemorrhage (aSAH), defining an unmet need for therapeutic development. Cell-free hemoglobin that is released from erythrocytes into the cerebrospinal fluid (CSF) is suggested to cause vasoconstriction and neuronal toxicity, and correlates with the occurrence of DIND. Cell-free hemoglobin in the CSF of patients with aSAH disrupted dilatory NO signaling ex vivo in cerebral arteries, which shifted vascular tone balance from dilation to constriction. We found that selective removal of hemoglobin from patient CSF with a haptoglobin-affinity column or its sequestration in a soluble hemoglobin-haptoglobin complex was sufficient to restore physiological vascular responses. In a sheep model, administration of haptoglobin into the CSF inhibited hemoglobin-induced cerebral vasospasm and preserved vascular NO signaling. We identified 2 pathways of hemoglobin delocalization from CSF into the brain parenchyma and into the NO-sensitive compartment of small cerebral arteries. Both pathways were critical for hemoglobin toxicity and were interrupted by the large hemoglobin-haptoglobin complex that inhibited spatial requirements for hemoglobin reactions with NO in tissues. Collectively, our data show that compartmentalization of hemoglobin by haptoglobin provides a novel framework for innovation aimed at reducing hemoglobin-driven neurological damage after subarachnoid bleeding.

Heart rate elevations during early sepsis predict death in fluid-resuscitated rats with fecal peritonitis.

BACKGROUND: In sepsis, early outcome prediction would allow investigation of both adaptive mechanisms underlying survival and maladaptive mechanisms resulting in death. The aim of this study was to test whether early changes in heart rate monitored by telemetry could predict outcome in a long-term rat model of fecal peritonitis. METHODS: Male Wistar rats (n = 24) were instrumented with a central venous line for administration of fluids, antibiotics and analgesics. A telemetry transmitter continuously collected electrocardiogram signals. Sepsis was induced by intraperitoneal injection of fecal slurry, and the animals were observed for 48 h. Additional animals underwent arterial cannulation at baseline (n = 9), 4 h (n = 16), or 24 h (n = 6) for physiology and laboratory measurements. RESULTS: 48-h mortality was 33% (8/24), with all deaths occurring between 4 and 22 h. Septic animals were characterized by lethargy, fever, tachycardia, positive blood cultures, and elevated cytokine (IL-1, IL-6, TNF alpha) levels. An increase in heart rate ≥ 50 bpm during the first 4 h of sepsis predicted death with sensitivity and specificity of 88% (p = 0.001). CONCLUSIONS: In this long-term rat sepsis model, prognostication could be made early by telemetry-monitored changes in heart rate. This model enables the study of underlying mechanisms and the assessment of any differential effects of novel therapies in predicted survivors or non-survivors.

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.

Phenotype-specific recombinant haptoglobin polymers co-expressed with C1r-like protein as optimized hemoglobin-binding therapeutics.

BACKGROUND: Preclinical studies have evaluated haptoglobin (Hp) polymers from pooled human plasma as a therapeutic protein to attenuate toxic effects of cell-free hemoglobin (Hb). Proof of concept studies have demonstrated efficacy of Hp in hemolysis associated with transfusion and sickle cell anemia. However, phenotype-specific Hp products might be desirable to exploit phenotype specific activities of Hp 1-1 versus Hp 2-2, offering opportunities for recombinant therapeutics. Prohaptoglobin (proHp) is the primary translation product of the Hp mRNA. ProHp is proteolytically cleaved by complement C1r subcomponent-like protein (C1r-LP) in the endoplasmic reticulum. Two main allelic Hp variants, HP1 and HP2 exist. The larger HP2 is considered to be the ancestor variant of all human Hp alleles and is characterized by an α2-chain, which contains an extra cysteine residue that pairs with additional α-chains generating multimers with molecular weights of 200-900 kDa. The two human HP1 alleles (HP1F and HP1S) differ by a two-amino-acid substitution polymorphism within the α-chain and are derived from HP2 by recurring exon deletions. RESULTS: In the present study, we describe a process for the production of recombinant phenotype specific Hp polymers in mammalian FS293F cells. This approach demonstrates that efficient expression of mature and fully functional protein products requires co-expression of active C1r-LP. The functional characterization of our proteins, which included monomer/polymer distribution, binding affinities as well as NO-sparing and antioxidant functions, demonstrated that C1r-LP-processed recombinant Hp demonstrates equal protective functions as plasma derived Hp in vitro as well as in animal studies. CONCLUSIONS: We present a recombinant production process for fully functional phenotype-specific Hp therapeutics. The proposed process could accelerate the development of Hb scavengers to treat patients with cell-free Hb associated disease states, such as sickle cell disease and other hemolytic conditions.

Haptoglobin Preserves Vascular Nitric Oxide Signaling during Hemolysis.

RATIONALE: Hemolysis occurs not only in conditions such as sickle cell disease and malaria but also during transfusion of stored blood, extracorporeal circulation, and sepsis. Cell-free Hb depletes nitric oxide (NO) in the vasculature, causing vasoconstriction and eventually cardiovascular complications. We hypothesize that Hb-binding proteins may preserve vascular NO signaling during hemolysis. OBJECTIVES: Characterization of an archetypical function by which Hb scavenger proteins could preserve NO signaling during hemolysis. METHODS: We investigated NO reaction kinetics, effects on arterial NO signaling, and tissue distribution of cell-free Hb and its scavenger protein complexes. MEASUREMENTS AND MAIN RESULTS: Extravascular translocation of cell-free Hb into interstitial spaces, including the vascular smooth muscle cell layer of rat and pig coronary arteries, promotes vascular NO resistance. This critical disease process is blocked by haptoglobin. Haptoglobin does not change NO dioxygenation rates of Hb; rather, the large size of the Hb:haptoglobin complex prevents Hb extravasation, which uncouples NO/Hb interaction and vasoconstriction. Size-selective compartmentalization of Hb functions as a substitute for red blood cells after hemolysis and preserves NO signaling in the vasculature. We found that evolutionarily and structurally unrelated Hb-binding proteins, such as PIT54 found in avian species, functionally converged with haptoglobin to protect NO signaling by sequestering cell-free Hb in large protein complexes. CONCLUSIONS: Sequential compartmentalization of Hb by erythrocytes and scavenger protein complexes is an archetypical mechanism, which may have supported coevolution of hemolysis and normal vascular function. Therapeutic supplementation of Hb scavengers may restore vascular NO signaling and attenuate disease complications in patients with hemolysis.

Different target specificities of haptoglobin and hemopexin define a sequential protection system against vascular hemoglobin toxicity.

Free hemoglobin (Hb) triggered vascular damage occurs in many hemolytic diseases, such as sickle cell disease, with an unmet need for specific therapeutic interventions. Based on clinical observations the Hb and heme scavenger proteins haptoglobin (Hp) and hemopexin (Hx) have been characterized as a sequential defense system with Hp as the primary protector and Hx as a backup when all Hp is depleted during more severe intravascular hemolysis. In this study we present a mechanistic rationale for this paradigm based on a combined biochemical and cell biological approach directed at understanding the unique roles of Hp and Hx in Hb detoxification. Using a novel in vitro model of Hb triggered endothelial damage, which recapitulates the well-characterized pathophysiologic sequence of oxyHb(Fe(2+)) transformation to ferric Hb(Fe(3+)), free heme transfer from ferric Hb(Fe(3+)) to lipoprotein and subsequent oxidative reactions in the lipophilic phase. The accumulation of toxic lipid peroxidation products liberated during oxidation reactions ultimately lead to endothelial damage characterized by a specific gene expression pattern with reduced cellular ATP and monolayer disintegration. Quantitative analysis of key chemical and biological parameters allowed us to precisely define the mechanisms and concentrations required for Hp and Hx to prevent this toxicity. In the case of Hp we defined an exponential relationship between Hp availability relative to oxyHb(Fe(2+)) and related protective activity. This exponential relationship demonstrates that large Hp quantities are required to prevent Hb toxicity. In contrast, the linear relationship between Hx concentration and protection defines a highly efficient backup scavenger system during conditions of large excess of free oxyHb(Fe(2+)) that occurs when all Hp is consumed. The diverse protective function of Hp and Hx in this model can be explained by the different target specificities of the two proteins.

Integrative proteome and transcriptome analysis of extramedullary erythropoiesis and its reversal by transferrin treatment in a mouse model of beta-thalassemia.

Beta-thalassemia results from mutations of the ß-hemoglobin (Hbb) gene and reduced functional Hbb synthesis. Excess α-Hb causes globin chain aggregation, oxidation, cytoskeletal damage, and increased red blood cell clearance. These events result in anemia, altered iron homeostasis, and expansion of extramedullary erythropoiesis. Serum transferrin (Tf) is suggested to be an important regulator of erythropoiesis in murine models of thalassemia. The present study was conducted to establish a quantitative proteomic and transcriptomic analysis of transferrin-modulated extramedullary erythropoiesis in the spleen of wild type and thalassemic Hbb(th3/+) mice. Our LC-MS/MS protein analysis and mRNA sequencing data provide quantitative expression estimates of 1590 proteins and 24,581 transcripts of the murine spleen and characterize key processes of erythropoiesis and RBC homeostasis such as the whole heme synthesis pathway as well as critical components of the red blood cell antioxidant systems and the proliferative cell cycling pathway. The data confirm that Tf treatment of nontransfused Hbb(th3/+) mice induces a systematic correction of these processes at a molecular level. Tf treatment of Hbb(th3/+) mice for 60 days leads to a complete molecular restoration of the normal murine spleen phenotype. These findings support further investigation of plasma-derived Tf as a treatment for thalassemia.

Chloroquine interference with hemoglobin endocytic trafficking suppresses adaptive heme and iron homeostasis in macrophages: the paradox of an antimalarial agent.

The CD163 scavenger receptor pathway for Hb:Hp complexes is an essential mechanism of protection against the toxicity of extracellular hemoglobin (Hb), which can accumulate in the vasculature and within tissues during hemolysis. Chloroquine is a lysosomotropic agent, which has been extensively used as an antimalarial drug in the past, before parasite resistance started to limit its efficacy in most parts of the world. More recent use of chloroquine is related to its immunomodulatory activity in patients with autoimmune diseases, which may also involve hemolytic disease components. In this study we examined the effects of chloroquine on the human Hb clearance pathway. For this purpose we developed a new mass-spectrometry-based method to specifically quantify intracellular Hb peptides within the endosomal-lysosomal compartment by single reaction monitoring (SRM). We found that chloroquine exposure impairs trafficking of Hb:Hp complexes through the endosomal-lysosomal compartment after internalization by CD163. Relative quantification of intracellular Hb peptides by SRM confirmed that chloroquine blocked cellular Hb:Hp catabolism. This effect suppressed the cellular heme-oxygenase-1 (HO-1) response and shifted macrophage iron homeostasis towards inappropriately high expression of the transferrin receptor with concurrent inhibition of ferroportin expression. A functional deficiency of Hb detoxification and heme-iron recycling may therefore be an adverse consequence of chloroquine treatment during hemolysis.

Glucocorticoid treatment skews human monocyte differentiation into a hemoglobin-clearance phenotype with enhanced heme-iron recycling and antioxidant capacity.

Glucocorticoids are used extensively to treat autoimmune hemolytic anemias. Some beneficial effects of glucocorticoid pulse therapy have also been reported in sickle cell disease and paroxysmal nocturnal hemoglobinuria. Based on established concepts of hemoglobin (Hb) toxicity and physiologic Hb scavenger systems, we evaluated whether glucocorticoids could support an adaptive response to extracellular Hb independently of their immunosuppressive activities. Using global proteome and transcriptome analysis with mass-spectrometry (isobaric tag for relative and absolute quantitation and liquid chromatography-mass spectrometry) and gene-array experiments, we found that glucocorticoid treatment in vitro and in patients on glucocorticoid-pulse therapy polarized monocytes into a M2/alternatively activated phenotype with high Hb-scavenger receptor (CD163) expression and enhanced Hb-clearance and detoxification capability. Monocytes concurrently exposed to the interactive activity of glucocorticoids and extracellular Hb were characterized by high expression of a group of antioxidant enzymes known to be regulated by the conserved oxidative response transcription factor nuclear factor E2-related factor. Further, suppressed transferrin receptor, together with high ferroportin expression, pointed to a shift in iron homeostasis directed toward an increased cellular export of heme-derived iron. Therefore, stimulating Hb-endocytosis by CD163 and enhancing antioxidative homeostasis and iron recycling may be an essential activity of glucocorticoids that helps alleviate the adverse effects of extracellular Hb.

Heme carrier protein (HCP-1) spatially interacts with the CD163 hemoglobin uptake pathway and is a target of inflammatory macrophage activation.

Macrophages constitute the major cellular compartment for hemoglobin (Hb) degradation and subsequent recycling of heme-iron to erythropoiesis. Dysregulation of macrophage iron and heme metabolism is a major pathophysiologic determinant of anemia of chronic disease. In this study, we show that the heme transporter heme carrier protein 1 (HCP-1) is expressed in human macrophages. Within early endosomes, HCP-1 colocalizes with endocytosed Hb-haptoglobin (Hp) complexes, which are taken up via the CD163 scavenger receptor pathway. Hb-Hp passes the divalent metal transporter 1B/HCP-1-positive endosomal compartment on its route from the cell surface to lysosomes. HCP-1 mRNA and protein expression are down-regulated by stimulation of macrophages with various TLR agonists and IFN-gamma. The profound suppression of HCP-1 expression by inflammatory macrophage activation parallels the regulation of the iron exporter ferroportin. In contrast, dexamethasone enhanced HCP-1 expression significantly. Given the spatial relationship, we propose that the Hb scavenger receptor CD163 and HCP-1 constitute a linked pathway for Hb catabolism and heme-iron recycling in human macrophages.

CD163-expressing monocytes constitute an endotoxin-sensitive Hb clearance compartment within the vascular system.

Hemoglobin (Hb) is released into the circulation during intravascular hemolysis and exerts toxic effects through oxidative damage and NO scavenging. According to the traditional concept of Hb clearance, free Hb is bound to the plasma protein haptoglobin (Hp), and the Hb-Hp complexes are cleared by liver and spleen macrophages via the Hb scavenger receptor CD163. Using a novel whole blood assay, we demonstrate that clearance of Hb-Hp is also mediated by CD14(high)/CD64(high) peripheral blood monocytes, which express CD163. Hb-Hp uptake by these cells is Ca(2+)-dependent and is abrogated by the addition of CD163-blocking antibodies. Accordingly, LPS treatment reduces monocyte surface CD163 and impairs Hb-Hp uptake. Monocytes likely mediate Hp-Hb uptake in vivo, as a high expression of the heme breakdown enzyme heme oxygenase-1 was observed in CD163(+) monocytes but not in other leukocyte populations obtained from healthy blood donors. We propose that CD163-mediated Hb-Hp uptake by peripheral blood monocytes constitutes an Hb-Hp clearance pathway, which acts at the site of intravascular hemolysis to reduce Hb-Hp circulation time and toxicity. Disruption of monocyte Hb-Hp clearance may increase Hb-Hp toxicity and contribute to the pathogenesis of systemic inflammatory diseases associated with reduced monocyte CD163 expression.

Hemophagocytic macrophages constitute a major compartment of heme oxygenase expression in sepsis.

OBJECTIVES: Uncontrolled macrophage activation with hemophagocytosis is a distinctive feature of hemophagocytic syndromes (HPS). We examined whether lympho-histiocytic infiltration of the bone marrow and liver, as well as hemo-/erythrophagocytosis also occurs during sepsis and whether this process could account for the increased production of anti-inflammatory heme-oxygenase (HO-1) products observed during sepsis. METHODS: Hemophagocytosis and expression of CD163, HO-1, ferritin as well as CD8 and granzyme-B were examined in post-mortem bone marrow samples from 28 patients with sepsis and from eight control patients. RESULTS: Comparison of samples from non-septic patients with samples from patients with fatal sepsis revealed that the latter group displayed dense lympho-histiocytic bone marrow infiltration with CD163(+)/HO-1(+)/ferritin(+) macrophages as well as with CD8(+) and granzyme-B(+) T-cells. Hemophagocytosis with prominent phagocytosis of erythroid cells was readily apparent in septic patients, implying that this process is a likely stimulus for the up-regulation of macrophage HO-1 expression. CONCLUSIONS: Lympho-histiocytic activation with hemophagocytosis is a shared pathophysiologic mechanism in HPS and sepsis. Furthermore, the association of hemophagocytosis with an increase in HO-1 expression may indicate a novel role for this apparently futile process as a negative regulator of inflammation.

Constitutive endocytosis of CD163 mediates hemoglobin-heme uptake and determines the noninflammatory and protective transcriptional response of macrophages to hemoglobin.

Heme toxicity contributes to the pathogenesis of chronic inflammatory diseases, atherosclerosis, and hemolysis associated vasculopathy. Macrophage clearance of cell free hemoglobin (Hb) is thus an essential homeostatic function of these cells. We examined the transcriptional response of human PBMC derived macrophages to Hb by gene array analysis. The observed noninflammatory macrophage response was characterized by induction of an antioxidative and antiinflammatory gene expression pattern with most prominent induction of the inducible heme oxygenase (HO-1). The metabolically active Hb-CD163-HO-1 pathway resulted in synthesis of ferritin-1 of the antioxidative and antiinflammatory end products linked to heme breakdown by HO-1. This response was mediated by the Hb scavenger receptor CD163 and heme and was not related to Hb mediated depletion of reduced glutathione. In contrast to other cellular responses induced by CD163, there was no role of protein phosphorylation dependent CD163 signaling in the protective macrophage response to Hb. Instead, CD163 acted as an Hb transporter, which undergoes constitutive and ligand independent internalization and recycling between the cell surface and early endosomes. The expression of CD163 and HO-1 in macrophages of neovascularized atherosclerotic lesions suggests that the pathway described herein is active in vivo. Noninflammatory Hb clearance and intimately linked HO-1 expression may provide the long sought-after explanation for the antiinflammatory activity associated with CD163-positive macrophages.