Erythrophagocyte

Phagocyte reprogramming and heme-suppressed immunity in hemolytic anemias

Upon hemolytic stress, receptor-mediated clearance of cell-free hemoglobin, phagocytosis of damaged erythrocytes and sequestration of RBC toxins represent a key homeostatic function of the mononuclear phagocytes of the reticuloendothelial system limiting hemoglobin and heme toxicities. Through heme-oxygenase 1 (Hmox1), macrophages degrade heme and provide the iron source for the erythropoiesis. We have demonstrated that heme-exposure transforms bone marrow (BM)-derived inflammatory macrophages into anti-inflammatory erythrophagocytes. Erythrophagocytes display high expression of genes related to heme-degradation, antioxidant metabolism, and iron-handling, supporting their homeostatic function in erythropoiesis and protection against hemolysis-driven pathologies. Simultaneously, heme-exposure profoundly suppressed MHC-class II expression and induced tolerance against inflammatory activation by endogenous (e.g., CD40 ligation) and foreign (e.g., TLR agonists) immune-activation signals. This anti-inflammatory macrophage polarization, driven by heme, could be an essential mechanism by which the immune system is regulated to prevent overactivation against self-antigens in patients with acute and chronic hemolysis or in a wound. Over the last few years, we have invested strong efforts to establish experimental models (e.g., different genetic models of hemolysis) and analytic tools in the field of system biology (e.g., single-cell RNAseq analysis of macrophage populations in tissues) to investigate this process in vivo.

Pathophysiology of erythrophagocytes.

(adapted from Buehler PW et al., 2020, Ingoglia G et al., 2020 and Pfefferlé M. et al. 2020).

Readings

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

Abstract

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.

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

Abstract

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.

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

Abstract

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.