Publications

Key Publications

Hemorrhage-activated NRF2 in tumor-associated macrophages drives cancer growth, invasion, and immunotherapy resistance

Schaer DJ, Schulthess-Lutz N, Baselgia L, Hansen K, Buzzi RM, Humar R, Dürst E, Vallelian F. (2024).  

Journal of Clinical Investigation.  

https://doi.org/10.1172/JCI174528

Microscopic hemorrhage is a common aspect of cancers, yet its potential role as an independent factor influencing both cancer progression and therapeutic response is largely ignored. Recognizing the essential function of macrophages in red blood cell disposal, we explored a pathway that connects intratumoral hemorrhage with the formation of cancer-promoting tumor-associated macrophages (TAMs). Using spatial transcriptomics, we found that NRF2-activated myeloid cells possessing characteristics of procancerous TAMs tend to cluster in perinecrotic hemorrhagic tumor regions. These cells resembled antiinflammatory erythrophagocytic macrophages. We identified heme, a red blood cell metabolite, as a pivotal microenvironmental factor steering macrophages toward protumorigenic activities. Single-cell RNA-Seq and functional assays of TAMs in 3D cell culture spheroids revealed how elevated intracellular heme signals via the transcription factor NRF2 to induce cancer-promoting TAMs. These TAMs stabilized epithelial-mesenchymal transition, enhancing cancer invasiveness and metastatic potential. Additionally, NRF2-activated macrophages exhibited resistance to reprogramming by IFN-γ and anti-CD40 antibodies, reducing their tumoricidal capacity. Furthermore, MC38 colon adenocarcinoma–bearing mice with NRF2 constitutively activated in leukocytes were resistant to anti-CD40 immunotherapy. Overall, our findings emphasize hemorrhage-activated NRF2 in TAMs as a driver of cancer progression, suggesting that targeting this pathway could offer new strategies to enhance cancer immunity and overcome therapy resistance.

Antibody-induced erythrophagocyte reprogramming of Kupffer cells prevents anti-CD40 cancer immunotherapy-associated liver toxicity

Pfefferlé M*, Dubach IL*, Buzzi RM, Dürst E, Schulthess-Lutz N, Baselgia L, Hansen K, Imhof L, Koernig S, Le Roy D, Roger T, Humar R, Schaer DJ, Vallelian F. (2023).  

Journal of ImmunoTherapy of Cancer.  

http://dx.doi.org/10.1136/jitc-2022-005718

Background Agonistic anti-CD40 monoclonal antibodies (mAbs) have emerged as promising immunotherapeutic compounds with impressive antitumor effects in mouse models. However, preclinical and clinical studies faced dose-limiting toxicities mediated by necroinflammatory liver disease. An effective prophylactic treatment for liver immune-related adverse events that does not suppress specific antitumor immunity remains to be found.   Methods We used different mouse models and time-resolved single-cell RNA-sequencing to characterize the pathogenesis of anti-CD40 mAb induced liver toxicity. Subsequently, we developed an antibody-based treatment protocol to selectively target red blood cells (RBCs) for erythrophagocytosis in the liver, inducing an anti-inflammatory liver macrophage reprogramming.   Results We discovered that CD40 signaling in Clec4f+ Kupffer cells is the non-redundant trigger of anti-CD40 mAb-induced liver toxicity. Taking advantage of the highly specific functionality of liver macrophages to clear antibody-tagged RBCs from the blood, we hypothesized that controlled erythrophagocytosis and the linked anti-inflammatory signaling by the endogenous metabolite heme could be exploited to reprogram liver macrophages selectively. Repeated low-dose administration of a recombinant murine Ter119 antibody directed RBCs for selective phagocytosis in the liver and skewed the phenotype of liver macrophages into a Hmoxhigh/Marcohigh/MHCIIlow anti-inflammatory phenotype. This unique mode of action prevented necroinflammatory liver disease following high-dose administration of anti-CD40 mAbs. In contrast, extrahepatic inflammation, antigen-specific immunity, and antitumor activity remained unaffected in Ter119 treated animals.   Conclusions Our study offers a targeted approach to uncouple CD40-augmented antitumor immunity in peripheral tissues from harmful inflammatoxicity in the liver.

A model to visualize the fate of iron after intracranial hemorrhage using isotopic tracers and elemental bioimaging

Bücker P*, Buzzi RM*, Akeret K, Mosberger L, Richter H, Sperling M, Hugelshofer M, Schaer DJ, Karst U. (2023).

Metallomics.   

https://doi.org/10.1093/mtomcs/mfac057

Hemoglobin-iron is a red blood cell toxin contributing to secondary brain injury after intracranial bleeding. We present a model to visualize an intracerebral hematoma and secondary hemoglobin-iron distribution by detecting 58Fe-labeled hemoglobin (Hb) with laser ablation-inductively coupled plasma-mass spectrometry on mouse brain cryosections after stereotactic whole blood injection for different time periods. The generation of 58Fe-enriched blood and decisive steps in the acute hemorrhage formation and evolution were evaluated. The model allows visualization and quantification of 58Fe with high spatial resolution and striking signal-to-noise ratio. Script-based evaluation of the delocalization depth revealed ongoing 58Fe delocalization in the brain even 6 days after hematoma induction. Collectively, the model can quantify the distribution of Hb-derived iron post-bleeding, providing a methodological framework to study the pathophysiological basis of cell-free Hb toxicity in hemorrhagic stroke.

Erythrophagocytes in hemolytic anemia, wound healing, and cancer

Humar R, Schaer DJ, Vallelian F (2022).

Trends in Molecular Medicine.

https://doi.org/10.1016/j.molmed.2022.08.005

Hemolysis is a ubiquitous pathology defined as premature red blood cell destruction within the circulation or local tissues. One of the most archetypal functions of macrophages is phagocytosis of damaged or extravasated red blood cells, preventing the extracellular release of toxic hemoglobin and heme. Upon erythrophagocytosis, spiking intracellular heme concentrations drive macrophage transformation into erythrophagocytes, leveraging antioxidative and iron recycling capacities to defend against hemolytic stress. This unique phenotype transformation is coordinated by a regulatory network comprising the transcription factors BACH1, SPI-C, NRF2, and ATF1. Erythrophagocytes negatively regulate inflammation and immunity and may modulate disease-specific outcomes in hemolytic anemia, wound healing, atherosclerosis, and cancer. In this opinion article, we outline the known and presumed functions of erythrophagocytes and their implications for therapeutic innovation and research.

Graphical_Abstract_CDD_2022_right

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

Vallelian F, Buzzi RM, Pfefferlé M, Yalamanoglu A, Dubach IL, Wassmer A, Gentinetta T, Hansen K, Humar R, Schulthess N, Schaer CA, Schaer DJ. (2022).

Cell Death & Differentiation.

https://doi.org/10.1038/s41418-022-00932-1

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.

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Spatial transcriptome analysis defines heme as a hemopexin-targetable inflammatoxin in the brain

Buzzi RM, Akeret K, Schwendinger N, Klohs J, Vallelian F, Hugelshofer M, Schaer DJ. (2022).

Free Radical Biology and Medicine.

https://doi.org/10.1016/j.freeradbiomed.2021.11.011

After intracranial hemorrhage, heme is released from cell-free hemoglobin. This red blood cell component may drive secondary brain injury at the hematoma‒brain interface. This study aimed to generate a spatially resolved map of transcriptome-wide gene expression changes in the heme-exposed brain and to define the potential therapeutic activity of the heme-binding protein, hemopexin. We stereotactically injected saline, heme, or heme‒hemopexin into the striatum of C57BL/6J mice. After 24 h, we elucidated the two-dimensional spatial transcriptome by sequencing 21760 tissue-covered features, at a mean transcript coverage of 3849 genes per feature. In parallel, we studied the extravasation of systemically administered fluorescein isothiocyanate labeled (FITC)-dextran, magnetic resonance imaging features indicative of focal edema and perfusion, and neurological functions as translational correlates of heme toxicity. We defined a cerebral heme-response signature by performing bidimensional differential gene expression analysis, based on unsupervised clustering and manual segmentation of sequenced features. Heme exerted a consistent and dose-dependent proinflammatory activity in the brain, which occurred at minimal exposures, below the toxicity threshold for the induction of vascular leakage. We found dose-dependent regional divergence of proinflammatory heme signaling pathways, consistent with reactive astrocytosis and microglial activation. Co-injection of heme with hemopexin attenuated heme-induced gene expression changes and preserved the homeostatic microglia signature. Hemopexin also prevented heme-induced disruption of the blood‒brain barrier and radiological and functional signals of heme injury in the brain. In conclusion, we defined heme as a potent inflammatoxin that may drive secondary brain injury after intracerebral hemorrhage. Co-administration of hemopexin attenuated the heme-derived toxic effects on a molecular, cellular, and functional level, suggesting a translational therapeutic strategy.

Modular Platform for the Development of Recombinant Hemoglobin Scavenger Biotherapeutics
Buzzi RM, Owczarek CM, Akeret K, Tester A, Pereira N, Butcher R, Brügger-Verdon V, Hardy MP, Illi M, Wassmer A, Vallelian F, Humar R, Hugelshofer M, Buehler PW, Gentinetta T, Schaer DJ. (2021).

Molecular Pharmaceutics.
https://doi.org/10.1021/acs.molpharmaceut.1c00433

Cell-free hemoglobin (Hb) is a driver of disease progression in conditions with intravascular or localized hemolysis. Genetic and acquired anemias or emergency medical conditions such as aneurysmal subarachnoid hemorrhage involve tissue Hb exposure. Haptoglobin (Hp) captures Hb in an irreversible protein complex and prevents its pathophysiological contributions to vascular nitric oxide depletion and tissue oxidation. Preclinical proof-of-concept studies suggest that human plasma-derived Hp is a promising therapeutic candidate for several Hb-driven diseases. Optimizing the efficacy and safety of Hb-targeting biotherapeutics may require structural and functional modifications for specific indications. Improved Hp variants could be designed to achieve the desired tissue distribution, metabolism, and elimination to target hemolytic disease states effectively. However, it is critical to ensure that these modifications maintain the function of Hp. Using transient mammalian gene expression of Hp combined with co-transfection of the pro-haptoglobin processing protease C1r-LP, we established a platform for generating recombinant Hp-variants. We designed an Hpβ-scaffold, which was expressed in this system at high levels as a monomeric unit (mini-Hp) while maintaining the key protective functions of Hp. We then used this Hpβ-scaffold as the basis to develop an initial proof-of-concept Hp fusion protein using human serum albumin as the fusion partner. Next, a hemopexin-Hp fusion protein with bispecific heme and Hb detoxification capacity was generated. Further, we developed a Hb scavenger devoid of CD163 scavenger receptor binding. The functions of these proteins were then characterized for Hb and heme-binding, binding of the Hp-Hb complexes with the clearance receptor CD163, antioxidant properties, and vascular nitric oxide sparing capacity. Our platform is designed to support the generation of innovative Hb scavenger biotherapeutics with novel modes of action and potentially improved formulation characteristics, function, and pharmacokinetics.

Cerebrospinal fluid hemoglobin drives subarachnoid hemorrhage-related secondary brain injury
Akeret K, Buzzi RM, Schaer CA, Thomson BR, Vallelian F, Wang S, Willms J, Sebök M, Held U, Deuel JW, Humar R, Regli L, Keller E, Hugelshofer M, Schaer DJ. (2021).

Journal of Cerebral Blood Flow & Metabolism.
https://doi.org/10.1177/0271678X211020629

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.

Hemolysis transforms liver macrophages into anti-inflammatory erythrophagocytes
Pfefferlé M, Ingoglia G, Schaer CA, Yalamanoglu A, Buzzi RM, Dubach IL, Tan G, López-Cano EY, Schulthess N, Hansen K, Humar R, Schaer DJ, Vallelian F. (2020). 

The Journal of Clinical Investigation.
https://doi.org/10.1172/JCI137282

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 a unique anti-inflammatory phenotype that we defined as Marcohigh/Hmoxhigh/MHC-class IIlow erythrophagocytes. This phenotype transformation profoundly mitigated disease expression in a model of an anti-CD40-induced hyperinflammatory syndrome with necrotic hepatitis and in a non-alcoholic steatohepatitis model, representing two macrophage-driven sterile inflammatory diseases. We reproduced the anti-inflammatory 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 (scATAC-seq) 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.

Haptoglobin Therapeutics and Compartmentalization of Cell-Free Hemoglobin Toxicity
Buehler PW, Humar R, Schaer DJ. (2020).

Trends in Molecular Medicine.

https://doi.org/10.1016/j.molmed.2020.02.004

Lysis of red blood cells in the circulation or within confined anatomical spaces accompanies many disease conditions. Translocation of hemoglobin dimers across tissue barriers is the key initiation step of Hb toxicity, which facilitates chemical Hb reactions in vulnerable tissue compartments. The biochemical drivers of Hb toxicity are oxidative reactions and reactions that consume the vasodilator nitric oxide, causing vasoconstriction. Haptoglobin binds Hb-dimers within a complex too large to translocate across tissue barriers. An auxiliary function of the Hb–haptoglobin complex changes the structural conformation of the heme-pocket, blocking heme release and providing antioxidative protection. Haptoglobin compartmentalization of cell-free Hb provides opportunities for drug development in disease areas such as sickle cell anemia, sepsis, blood transfusion, and subarachnoid hemorrhage. Hemolysis and accumulation of cell-free hemoglobin (Hb) in the circulation or in confined tissue compartments such as the subarachnoid space is an important driver of disease. Haptoglobin is the Hb binding and clearance protein in human plasma and an efficient antagonist of Hb toxicity resulting from physiological red blood cell turnover. However, endogenous concentrations of haptoglobin are insufficient to provide protection against Hb-driven disease processes in conditions such as sickle cell anemia, sepsis, transfusion reactions, medical-device associated hemolysis, or after a subarachnoid hemorrhage. As a result, there is increasing interest in developing haptoglobin therapeutics to target ‘toxic’ cell-free Hb exposures. Here, we discuss key concepts of Hb toxicity and provide a perspective on the use of haptoglobin as a therapeutic protein.

Haptoglobin administration into the subarachnoid space prevents hemoglobin-induced cerebral vasospasm
Michael Hugelshofer, Raphael M. Buzzi, Christian A. Schaer, Henning Richter, Kevin Akeret, Vania Anagnostakou, Leila Mahmoudi, Raphael Vaccani, Florence Vallelian, Jeremy W. Deuel, Peter W. Kronen, Zsolt Kulcsar, Luca Regli, Jin Hyen Baek, Ivan S. Pires, Andre F. Palmer, Matthias Dennler, Rok Humar, Paul W. Buehler, Patrick R. Kircher, Emanuela Keller, Dominik J. Schaer. (2019).

The Journal of clinical investigation, 129(12).
https://doi.org/10.1172/JCI130630

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.

Cell-Free Oxyhemoglobin in Cerebrospinal Fluid After Aneurysmal Subarachnoid Hemorrhage: Biomarker and Potential Therapeutic Target
Hugelshofer M, Sikorski CM, Seule M, Deuel J, Muroi CI, Seboek M, Akeret K, Buzzi R, Regli L, Schaer DJ, Keller E. (2018).

World Neurosurgery, 120, e660-e666.
https://doi.org/10.1016/j.wneu.2018.08.141

Background Aneurysmal subarachnoid hemorrhage (aSAH) is often complicated by the occurrence of delayed ischemic neurologic deficits (DIND), which impairs the clinical outcome of patients. The release of oxyhemoglobin (oxyHb) from lysing erythrocytes into cerebrospinal fluid (CSF) may critically contribute to the development of DIND.  [ … ]

Haptoglobin Preserves Vascular Nitric Oxide Signaling during Hemolysis
Christian A. Schaer, Jeremy W. Deuel, Daniela Schildknecht, Leila Mahmoudi, Ines Garcia-Rubio, Catherine Owczarek, Stefan Schauer, Reinhard Kissner, Uddyalok Banerjee, Andre F. Palmer, Donat R. Spahn, David C. Irwin, Florence Vallelian, Paul W. Buehler, Dominik J. Schaer. (2015).

American journal of respiratory and critical care medicine, 193(10), 1111-1122.

https://doi.org/10.1164/rccm.201510-2058OC

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.

Line-selective macrophage activation with an anti-CD40 antibody drives a hemophagocytic syndrome in mice
Ingoglia G, Yalamanoglu A, Pfefferlé M, Dubach IL, Schaer CA, Valkova K, Hansen K, Schulthess N, Humar R, Schaer DJ, Vallelian F. (2020). 

Blood advances, 4(12), 2751-2761.
https://doi.org/10.1182/bloodadvances.2020001624

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.

Sequestration of extracellular hemoglobin within a haptoglobin complex decreases its hypertensive and oxidative effects in dogs and guinea pigs
Boretti FS, Buehler PW, D’Agnillo F, Kluge K, Glaus T, Butt OI, Jia Y, Goede J, Pereira CP, Maggiorini M, Schoedon G, Alayash AI, Schaer DJ. (2009).

The Journal of clinical investigation, 119(8), 2271-2280.
https://doi.org/10.1172/JCI39115

Release of hemoglobin (Hb) into the circulation is a central pathophysiologic event that contributes to morbidity and mortality in chronic hemolytic anemias and severe malaria. These toxicities arise from Hb-mediated vasoactivity, possibly due to NO scavenging and localized tissue oxidative processes. Currently, there is no established treatment that targets circulating extracellular Hb. Here, we assessed the role of haptoglobin (Hp), the primary scavenger of Hb in the circulation, in limiting the toxicity of cell-free Hb infusion. Using a canine model, we found that glucocorticoid stimulation of endogenous Hp synthesis prevented Hb-induced hemodynamic responses. Furthermore, guinea pigs administered exogenous Hp displayed decreased Hb-induced hypertension and oxidative toxicity to extravascular environments, such as the proximal tubules of the kidney. The ability of Hp to both attenuate hypertensive responses during Hb exposure and prevent peroxidative toxicity in extravascular compartments was dependent on Hb-Hp complex formation, which likely acts through sequestration of Hb rather than modulation of its NO- and O2-binding characteristics. Our data therefore suggest that therapies involving supplementation of endogenous Hb scavengers may be able to treat complications of acute and chronic hemolysis, as well as counter the adverse effects associated with Hb-based oxygen therapeutics.

Haptoglobin preserves the CD163 hemoglobin scavenger pathway by shielding hemoglobin from peroxidative modification.
Buehler PW, Abraham B, Vallelian F, Linnemayr C, Pereira CP, Cipollo JF, Jia Y, Mikolajczyk M, Boretti FS, Schoedon G, Alayash AI, Schaer DJ. (2009).

Blood (2009) 113 (11): 2578–2586.

https://doi.org/10.1182/blood-2008-08-174466

Detoxification and clearance of extracellular hemoglobin (Hb) have been attributed to its removal by the CD163 scavenger receptor pathway. However, even low-level hydrogen peroxide (H2O2) exposure irreversibly modifies Hb and severely impairs Hb endocytosis by CD163. We show here that when Hb is bound to the high-affinity Hb scavenger protein haptoglobin (Hp), the complex protects Hb from structural modification by preventing α-globin cross-links and oxidations of amino acids in critical regions of the β-globin chain (eg, Trp15, Cys93, and Cys112). As a result of this structural stabilization, H2O2-exposed Hb-Hp binds to CD163 with the same affinity as nonoxidized complex. Endocytosis and lysosomal translocation of oxidized Hb-Hp by CD163-expressing cells were found to be as efficient as with nonoxidized complex. Hp complex formation did not alter Hb’s ability to consume added H2O2 by redox cycling, suggesting that within the complex the oxidative radical burden is shifted to Hp. We provide structural and functional evidence that Hp protects Hb when oxidatively challenged with H2O2 preserving CD163-mediated Hb clearance under oxidative stress conditions. In addition, our data provide in vivo evidence that unbound Hb is oxidatively modified within extravascular compartments consistent with our in vitro findings.

CD163 is the macrophage scavenger receptor for native and chemically modified hemoglobins in the absence of haptoglobin.
Schaer DJ, Schaer CA, Buehler PW, Boykins RA, Schoedon G, Alayash AI, Schaffner A. (2006).

Blood (2006) 107 (1): 373–380.

https://doi.org/10.1182/blood-2005-03-1014

CD163 mediates the internalization of hemoglobin-haptoglobin (Hb-Hp) complexes by macrophages. Because Hp binding capacity is exhausted during severe hemolysis, an Hp-independent Hb-clearance pathway is presumed to exist. We demonstrate that Hb interacts efficiently with CD163 in the absence of Hp. Not only is Hb internalized into an endosomal compartment by CD163 as a result of active receptor-dependent endocytosis; it also inhibits the uptake of Hb-Hp complexes, suggesting a common receptor-binding site. Free Hb further induces heme oxygenase mRNA expression in CD163+ HEK293 cells, but not in CD163– cells. Additional evidence for Hp-independent Hb-CD163 interaction is provided by the demonstration that CD163 mediates the uptake of αα-DBBF crosslinked Hb, a chemically modified Hb that forms minimal Hp complexes. Moreover, certain modifications to Hb, such as polymerization or the attachment of specific functional groups (3 lysyl residues) to the β-Cys93 can reduce or enhance this pathway of uptake. In human macrophages, Hp-complex formation critically enhances Hb uptake at low (1 μg/mL), but not at high (greater than 100 μg/mL), ligand concentrations, lending support for a concentration-dependent biphasic model of macrophage Hb-clearance. These results identify CD163 as a scavenger receptor for native Hb and small-molecular-weight Hb-based blood substitutes after Hp depletion.

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