Phagocytosis Of Red Blood Cells Research Articles

Autoimmune haemolytic anaemias.

Adult autoimmune haemolytic anaemias (AIHAs) include different subtypes of a rare autoimmune disease in which autoantibodies targeting autoantigens expressed on the membrane of autologous red blood cells (RBCs) are produced, leading to their accelerated destruction. In the presence of haemolytic anaemia, the direct antiglobulin test is the cornerstone of AIHA diagnosis. AIHAs are classified according to the isotype and the thermal optimum of the autoantibody into warm (wAIHAs), cold and mixed AIHAs. wAIHAs, the most frequent type of AIHAs, are associated with underlying conditions in ~50% of cases. In wAIHA, IgG autoantibody reacts with autologous RBCs at 37 °C, leading to antibody-dependent cell-mediated cytotoxicity and increased phagocytosis of RBCs in the spleen. Cold AIHAs include cold agglutinin disease (CAD) and cold agglutinin syndrome (CAS) when there is an underlying condition. CAD and cold agglutinin syndrome are IgM cold antibody-driven AIHAs characterized by classical complement pathway-mediated haemolysis. The management of wAIHAs has long been based around corticosteroids and splenectomy and on symptomatic measures and non-specific cytotoxic agents for CAD. Rituximab and the development of complement inhibitors, such as the anti-C1s antibody sutimlimab, have changed the therapeutic landscape of AIHAs, and new promising targeted therapies are under investigation.

Biomechanics of phagocytosis of red blood cells by macrophages in the human spleen

The clearance of senescent and altered red blood cells (RBCs) in the red pulp of the human spleen involves sequential processes of prefiltration, filtration, and postfiltration. While prior work has elucidated the mechanisms underlying the first two processes, biomechanical processes driving the postfiltration phagocytosis of RBCs retained at interendothelial slits (IES) are still poorly understood. We present here a unique computational model of macrophages to study the role of cell biomechanics in modulating the kinetics of phagocytosis of aged and diseased RBCs retained in the spleen. After validating the macrophage model using in vitro phagocytosis experiments, we employ it to probe the mechanisms underlying the kinetics of phagocytosis of mechanically altered RBCs, such as heated RBCs and abnormal RBCs in hereditary spherocytosis (HS) and sickle cell disease (SCD). Our simulations show pronounced deformation of the flexible and healthy RBCs in contrast to minimal shape changes in altered RBCs. Simulations also show that less deformable RBCs are engulfed faster and at lower adhesive strength than flexible RBCs, consistent with our experimental measurements. This efficient sensing and engulfment by macrophages of stiff RBCs retained at IES are expected to temper splenic congestion, a common pathogenic process in malaria, HS, and SCD. Altogether, our combined computational and in vitro experimental studies suggest that mechanical alterations of retained RBCs may suffice to enhance their phagocytosis, thereby adapting the kinetics of their elimination to the kinetics of their mechanical retention, an equilibrium essential for adequately cleaning the splenic filter to preserve its function.

Clearance of pathogenic erythrocytes is maintained despite spleen dysfunction in children with sickle cell disease.

In children with sickle cell disease (SCD), splenectomy is immediately beneficial for acute sequestration crises and hypersplenism (ASSC/HyS) but portends a long-term risk of asplenia-related complications. We retrieved peripheral and splenic red blood cells (RBCs) from 17 SCD children/teenagers undergoing partial splenectomy for ASSC/HyS, 12 adult subjects without RBC-related disease undergoing splenectomy (controls), five human spleens perfused ex vivo with HbSS- and HbAA-RBC, and quantified abnormal RBC by microscopy, spleen-mimetic RBC filtration, and adhesion assays. Spleens were analyzed by immunohistochemistry and transmission electron microscopy (TEM). In circulating blood of SCD and control subjects, dysmorphic (elongated/spherocytic) RBCs were <2%, while proportions of pocked-RBC were 4.3-fold higher in SCD children than in controls. Compared to controls, splenic RBCs were more frequently dysmorphic (29.3% vs. 0.4%), stiffer (42.2% vs. 12.4%), and adherent (206 vs. 22 adherent RBC/area) in SCD subjects. By TEM, both polymer-containing and homogenous RBC contributed to spleen congestion, resulting in 3.8-fold higher RBC population density in SCD spleens than in control spleens, predominantly in the cords. Perfused spleens with normal function displayed similar congestion and retention of dysmorphic RBC as SCD spleens. The population density of active macrophages was similar in SCD and control spleens, with a relative deficit in phagocytosis of polymer-containing RBC. Despite the existence of hyposplenism, splenectomy in SCD children removes an organ that still efficiently filters out potentially pathogenic altered RBC. Innovative treatments allowing fine-tuned reduction of RBC retention would alleviate spleen congestion, the major pathogenic process in ASSC/HyS, while preserving spleen protective functions for the future.

Ferroptosis inhibitor improves outcome after early and delayed treatment in mild spinal cord injury

We show that redox active iron can induce a regulated form of non-apoptotic cell death and tissue damage called ferroptosis that can contribute to secondary damage and functional loss in the acute and chronic periods after spinal cord injury (SCI) in young, adult, female mice. Phagocytosis of red blood cells at sites of hemorrhage is the main source of iron derived from hemoglobin after SCI. Expression of hemeoxygenase-1 that induces release of iron from heme, is increased in spinal cord macrophages 7 days after injury. While iron is stored safely in ferritin in the injured spinal cord, it can, however, be released by NCOA4-mediated shuttling of ferritin to autophagosomes for degradation (ferritinophagy). This leads to the release of redox active iron that can cause free radical damage. Expression of NCOA4 is increased after SCI, mainly in macrophages. Increase in the ratio of redox active ferrous (Fe2+) to ferric iron (Fe3+) is also detected after SCI by capillary electrophoresis inductively coupled mass spectrometry. These changes are accompanied by other hallmarks of ferroptosis, i.e., deficiency in various elements of the antioxidant glutathione (GSH) pathway. We also detect increases in enzymes that repair membrane lipids (ACSL4 and LPCAT3) and thus promote on-going ferroptosis. These changes are associated with increased levels of 4-hydroxynonenal (4-HNE), a toxic lipid peroxidation product. Mice with mild SCI (30 kdyne force) treated with the ferroptosis inhibitor (UAMC-3203-HCL) either early or delayed times after injury showed improvement in locomotor recovery and secondary damage. Cerebrospinal fluid and serum samples from human SCI cases show evidence of increased iron storage (ferritin), and other iron related molecules, and reduction in GSH. Collectively, these data suggest that ferroptosis contributes to secondary damage after SCI and highlights the possible use of ferroptosis inhibitors to treat SCI.

Metabolic Reprogramming of Human Macrophages by Sickle Cell Hemoglobin

BACKGROUND: Sickle cell disease is caused by a single nucleotide mutation in the β-globin gene, resulting in the substitution of valine for glutamic acid, polymerization of hemoglobin and sickling of red blood cells (RBCs) under low oxygen conditions causing chronic hemolysis, and organs damage. SCA is characterized by the presence of cell-free plasma hemoglobin. Organ damage is associated with chronic inflammation and pro-inflammatory macrophages phenotype. Typically, anti-inflammatory response is elicited by the phagocytosis of senescent RBCs and cell-free hemoglobin. We have demonstrated that treatment of human macrophages with sickle cell hemoglobin (HbS) leads to lysosome accumulation, autophagy activation, and pro-inflammatory phenotype. The propensity of mutant HbS to polymerize under low pH may promote lysosomal HbS polymer accumulation, hindering degradation, and inducing macrophage differentiation towards a pro-inflammatory state. The differentiation of macrophages into specific phenotypes is associated with significant metabolic reprogramming. HYPOTHESIS: We hypothesize that the uptake of HbS by human macrophages induces their metabolic reprogramming via mitochondrial remodeling. METHODS: Human THP-1 cells were differentiated into macrophages with PMA and treated with either purified HbS or normal hemoglobin (HbA). Mitochondria were detected with MitoTrackerTM Green FM dye, and mitochondrial ROS (mtROS) level was detected by Mito SOX Red Kit. Transmission electron microscopy imaging was performed at Talos 200X transmission electron microscope. EPView (Olympus) software was used for the structure analysis. Total RNA was isolated by Trizol and sequenced on an Illumina® NextSeq 500. Comparisons between treatments were conducted using Gene Set Enrichment Analysis (GSEA). RESULTS: Immunofluorescent staining demonstrated accumulation of mitochondria after treatment with HbS compared to untreated (ctrl) or HbA-treated macrophages. Electron microscopy revealed an increase in the number and size of mitochondria. In 25 random microphotographs of cytoplasm, we detected 51 mitochondria in control, 79 in HbA treated and, 96 in HbS treated macrophages. The mitochondria volume and cristae size were significantly increased in macrophages treated with HbS. There was no increase in mtROS production in HbS treatment compared to ctrl. As expected, HbA treatment reduced mtROS production. The mitochondrial gene set (242 genes) was the second most enriched GSEA set in HbS-treated compared to HbA-treated macrophages. The larger mitochondrial size may be associated with a reduction of fission or an increase in fusion. We did not find differences in the expression of mitochondrial fission and fusion genes between HbS and HbA-treated macrophages. The increase in the mitochondrial size and cristae size may also be associated with increased oxidative phosphorylation. We found significant enrichment of genes responsible for oxidative phosphorylation in the HbS-treated macrophages. We did not find significant differences in the expression of glycolysis genes. CONCLUSION: Treatment of human macrophages with HbS significantly increases the number and volume of mitochondria, size of the cristae, and oxidative phosphorylation without upregulation of mtROS production and reduction of glycolysis. This work was supported by NIH Research Grants SC1HL150685, P50HL118006, and R01HL125005. We thank Drs. Castle Raley and Keith Crandall, George Washington University SPH Genomics Core, for sequencing and guidance in data analysis; Dr. Christine Brantner, GW Nanofabrication and Imaging Center, for TEM imaging. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Signaling-biophysical modeling unravels mechanistic control of red blood cell phagocytosis by macrophages in sickle cell disease.

Red blood cell (RBC) aging manifests through progressive changes in cell morphology, rigidity, and expression of membrane proteins. To maintain the quality of circulating blood, splenic macrophages detect the biochemical signals and biophysical changes of RBCs and selectively clear them through erythrophagocytosis. In sickle cell disease (SCD), RBCs display alterations affecting their interaction with macrophages, leading to aberrant phagocytosis that may cause life-threatening spleen sequestration crises. To illuminate the mechanistic control of RBC engulfment by macrophages in SCD, we integrate a system biology model of RBC-macrophage signaling interactions with a biophysical model of macrophage engulfment, as well as in vitro phagocytosis experiments using the spleen-on-a-chip technology. Our modeling framework accurately predicts the phagocytosis dynamics of RBCs under different disease conditions, reveals patterns distinguishing normal and sickle RBCs, and identifies molecular targets including Src homology 2 domain-containing protein tyrosine phosphatase-1 (SHP1) and cluster of differentiation 47 (CD47)/signal regulatory protein α (SIRPα) as therapeutic targets to facilitate the controlled clearance of sickle RBCs in the spleen.

Ineffective Erythropoiesis Negatively Impacts the Erythroblastic Island in Sickle Cell Disease

Sickle cell disease (SCD) is a genetic recessive disorder, characterized by painful episodes of vaso-occlusion, chronic hemolytic anemia, and progressive organ failure. It is one of the most common and severe monogenic diseases worldwide. SCD is caused by a point mutation in the β-globin gene, leading to the expression of an abnormal hemoglobin (HbS) that polymerizes under hypoxic conditions, driving red blood cells (RBC) sickling. Peripheral RBCs are extensively studied in SCD, as hemolysis is the major contributor to anemia, but little is known about erythroid differentiation in this pathology. We recently demonstrated the occurrence of ineffective erythropoiesis in the bone marrow (BM) of SCD patients, characterized by the death of a significant proportion of erythroblasts between the polychromatic and the orthochromatic stages. We hypothesize that this increased cell death could negatively impact the central macrophages of the erythroblastic islands (EBIs) as they would engulf high numbers of hemoglobin-rich apoptotic erythroblasts and undergo ferroptosis because of excess iron. To investigate our hypothesis, we used an in vitro system whereby CD14 + monocytes were isolated from peripheral blood and cultured in media supplemented with M-CSF and dexamethasone. This led them to differentiate towards anti-inflammatory tissue macrophages, with an “EBI-like” phenotype, expressing markers such as CD16, CD163, CD169, CD206. Phagocytosis of high numbers of RBCs by “EBI-like” macrophages led to increased cell death, reactive oxygen species, and lipid peroxidation, and to the upregulation of genes involved in ferroptosis pathways such as HO-1 and SLC7A11. This was circumvented by the ferroptosis inhibitors ferrostatin-1 and liproxtatin-1. Furthermore, phagocytosis of apoptotic in vitro-generated SCD erythroblasts also induced ferroptosis in EBI-like macrophages, with increased cell death and lipid peroxidation in a time-dependent manner. Interestingly, while this was the case with hemoglobin-rich, terminally differentiating erythroblasts, phagocytosis of cells without hemoglobin, such as K562 or GPA - progenitor cells did not affect cell viability, indicating that the observed ferroptosis was due to the iron content of the internalized cells. We investigated the effect of increased RBC phagocytosis on the phenotype of surviving macrophages and found an M2 to M1 shift indicating a switch to a pro-inflammatory phenotype under the condition of elevated levels of intracellular iron and oxidative stress. We challenged these findings in vivo using the Townes humanized SCD mouse model. Data on 4-month-old mice showed the expected switch in erythroid production from steady-state erythropoiesis in the BM to a stress erythropoiesis in the spleen. This was associated with a decrease in the number of EBI macrophages (F4/80 HighVCAM+CD169+Ly6G-) in the BM of SS mice when compared to AS and AA mice, and an expansion of red pulp macrophages (RPMs) F4/80 HighCD169+VCAM+. Using a flow cytometry-based assay we found high lipid peroxidation levels in BM EBI macrophages of SS mice as compared to AA, suggesting occurrence of ferroptosis in these cells. Our study suggests that IE in SCD may contribute to anemia, directly by cell death of differentiating erythroblasts, and indirectly by disrupting the erythroblastic island or by inducing its collapse following ferroptosis of the central macrophage. Our work with the Townes mouse model and BM samples from SCD patients will help to elucidate this hypothesis.

Numerical assessment of the impact of hemozoin on the dynamics of a within-host malaria model

Phagocytosis of infected red blood cells by macrophages can lead to the elimination of malaria parasites. However, the phagocytic function of macrophages can be diminished when they ingest the malarial pigment called hemozoin. In this study, we consider an in-host malaria model which assesses the impact of the ingestion or uptake of hemozoin by macrophages on the dynamics of malaria in a human host. It is shown that the model undergoes the backward bifurcation phenomenon. This phenomenon is induced by the suppression or inhibition of the phagocytic function of macrophages resulting from their interaction with hemozoin. More so, it is shown numerically that the model can undergo a Hopf bifurcation with periodic solutions appearing in all compartments of the model when the suppression rate of the phagocytic function of macrophages by hemozoin is sufficiently small.

Erythrophagocytosis Mediated By Circulating Monocytes Is Induced By Distinct Mechanisms Depending on the Disease

Stress erythropoiesis (SE) occurs when the steady state erythropoiesis is no longer sufficient to sustain the number of healthy red blood cells (RBC) needed in the circulation. There is an extension of this activity to extramedullary sites, contributing to the increase in cell production in the erythroblastic island (EBI) niche, and altered or damaged RBCs are then cleared by macrophages. Some diseases have a state of chronic SE. Sickle cell anemia (SCA) affects multiple aspects of RBCs, leading to recurrent vaso-occlusive crisis, hemolytic anemia, and inflammation. Polycythemia Vera (PV), an acquired neoplastic disease with mutations in JAK2 kinase, is characterized by the overproduction of RBCs, increasing blood viscosity, and resulting in a higher risk of thrombotic events. We previously showed that circulating monocytes (MC) of both diseases have a phenotype similar to the EBIs macrophages, with higher expression of CD169, CD163, CD206 and VCAM-1, and also participate in the phagocytosis of RBCs, function typically carried out by macrophages. Therefore, the aim of this study was to determine whether the increased phagocytic activity found in these patients was due to the alterations on the MCs phenotype or if it was an influence of the RBCs, as well as investigate the consequences to the iron metabolism after phagocytosis. MCs from peripheral blood were isolated and incubated for 2 hours with previously stained RBCs, to allow the phagocytosis to happen. After this period, the remaining RBC were lysed and the antibodies anti-intracellular heme-oxigenase-1 (HO-1) and anti-membrane ferroportin (FPN) were used for flow cytometry analysis. The statistical analysis was done by paired t-student test (P&amp;lt;0.05). When exposed to SCA RBCs, MCs from SCA patients and healthy controls (HC) show higher phagocytic capability, than when exposed to HC RBCs (MC HC 22.9±8 vs 9.4±1.1, **P=0,0099; MC SCA 33.6±11.4 vs 16.4±11, **P=0,0054; respectively). The same happens to PV and HC MCs: when exposed to PV RBCs: they show higher phagocytosis compared to the presence of HC RBCs (MC HC 9.3±4.4 vs 2.8±4.7, **P=0.0065; MC PV 21.3±6.1 vs 11±6.8, *P=0.0146; respectively). Investigating the role of MCs in this activity, we compared SCA, PV and HC MCs in the presence of patients RBCs. SCA MCs internalized RBCs similarly to HC MCs (P=0.0699); on the other hand, PV MCs presented higher phagocytic capability when compared to HC MCs (**P=0.0022). In all groups, the expressions of both HO-1 and FPN were higher in those MCs that participated in the phagocytosis than in those that didn't contribute to this function (HO-1: MC SCA 91.9±4 vs 65.9±16.8, **P=0.0098, MC PV 92.1±5.5 vs 61.7±15.5, ***P=0.0007; FPN: MC SCA 81.8±13.8 vs 66.5±15.7, ***P=0.0003, MC PV 70.9±17 vs 53.2±16.5, **P=0.0013 respectively). This indicates an increase in the ability to degrade heme and export iron post-phagocytosis. The results suggest that RBCs from patients with SCA are the ones responsible for the increase in the phagocytic activity by MCs, indicating that RBCs clearance is not an innate function of SCA MCs, even though they are active participants in the circulation. For PV, both RBCs and MCs seem to contribute to the increase of this process in the circulation of patients. The higher expression of molecules related to the iron metabolism suggests a participation of circulating MCs to the iron disponibility, helping with the maintenance of stock for the erythrocytic production. Even though SCA and PV have different pathophysiology, they both present SE and the circulating MCs from both groups show an important participation in the clearance of RBCs, either by an intrinsic function of MCs or a direct influence of changes in the RBCs, suggesting the important contribution of MCs to avoid oxidative stress and help with iron disponibility for erythropoiesis during the course of SE.

Curcumin as a Stabilizer of Macrophage Polarization during Plasmodium Infection.

Malaria is a parasitic infection responsible for high morbidity and mortality rates worldwide. During the disease, phagocytosis of infected red blood cells by the macrophages induces the production of reactive oxygen (ROS) and nitrogen species (RNS), culminating in parasite death. Curcumin (CUR) is a bioactive compound that has been demonstrated to reduce the production of pro-inflammatory cytokines and chemokines produced by macrophages but to reduce parasitemia in infected mice. Hence, the main purpose of this study is to investigate whether curcumin may interfere with macrophage function and polarization after Plasmodium berghei infection in vitro. In our findings, non-polarized macrophage (M0), classically activated (M1), and alternatively activated (M2) phenotypes showed significantly increased phagocytosis of infected red blood cells (iRBCs) when compared to phagocytosis of uninfected red blood cells (RBCs) 3 h after infection. After 24 h, M1 macrophages exposed to RBCs + CUR showed greater elimination capacity when compared to macrophages exposed to iRBCs + CUR, suggesting the interference of curcumin with the microbicidal activity. Additionally, curcumin increased the phagocytic activity of macrophages when used in non-inflammatory conditions (M0) and reduced the inducible nitric oxide synthase (iNOS) and arginase activities in all macrophage phenotypes infected (M0, M1, and M2), suggesting interference in arginine availability by curcumin and balance promotion in macrophage polarization in neutral phenotype (M0). These results support the view of curcumin treatment in malaria as an adjuvant, promoting a balance between pro- and anti-inflammatory responses for a better clinical outcome.

The effects of the differentiated macrophages by dexamethasone on the immune responses

The characteristics of M2 macrophages suggest immunotherapeutic approaches for inducing immunological tolerance. The current study aimed to evaluate the effect of Dexamethasone (Dex) treatment on monocytes polarization and its impact on immune responses. The monocytes were extracted from the rat's blood samples. The effects of Dex concentration and treatment duration on monocyte viability, phagocytosis of rabbit red blood cell (RRBC) antigens, and cytokine gene expression were evaluated using MTT, ELISA, and Real-Time PCR analysis, respectively. The monocytes treated with Dex were injected into the rats as an autograft. The effects of the grafted cells were assessed on immune responses, monocyte differentiation, and pathological lesions, in comparison to the control groups.Treatment of monocytes with 10-5 M of Dex for 48 h increased the expression of IL-10 and TGF-β genes, while reducing the expression of TNF-α and IL-1 genes. The monocytes treated with antigen and Dex showed higher CD206 gene expression compared to CD80. The cells that were treated with Dex had the highest concentration of antigens after five days. Administration of the grafted cells to the animals has some significant effects on innate immune responses and no impact on pathological lesions. The group that received cells treated with Dex and antigen experienced a significant decrease in anti-RBC antibody titers. Additionally, there was a significant difference in the expression of cytokine genes and M2 differentiation markers among the groups that were evaluated. The effects of Dex on the viability and differentiation of monocytes depend on the dosage, timing, and duration of the treatment.

Pharmacological Prevention of Ectopic Erythrophagocytosis by Cilostazol Mitigates Ferroptosis in NASH.

Hepatic iron overload (HIO) is a hallmark of nonalcoholic fatty liver disease (NAFLD) with a poor prognosis. Recently, the role of hepatic erythrophagocytosis in NAFLD is emerging as a cause of HIO. We undertook various assays using human NAFLD patient pathology samples and an in vivo nonalcoholic steatohepatitis (NASH) mouse model named STAMTM. To make the in vitro conditions comparable to those of the in vivo NASH model, red blood cells (RBCs) and platelets were suspended and subjected to metabolic and inflammatory stresses. An insert-coculture system, in which activated THP-1 cells and RBCs are separated from HepG2 cells by a porous membrane, was also employed. Through various analyses in this study, the effect of cilostazol was examined. The NAFLD activity score, including steatosis, ballooning degeneration, inflammation, and fibrosis, was increased in STAMTM mice. Importantly, hemolysis occurred in the serum of STAMTM mice. Although cilostazol did not improve lipid or glucose profiles, it ameliorated hepatic steatosis and inflammation in STAMTM mice. Platelets (PLTs) played an important role in increasing erythrophagocytosis in the NASH liver. Upregulated erythrophagocytosis drives cells into ferroptosis, resulting in liver cell death. Cilostazol inhibited the augmentation of PLT and RBC accumulation. Cilostazol prevented the PLT-induced increase in ectopic erythrophagocytosis in in vivo and in vitro NASH models. Cilostazol attenuated ferroptosis of hepatocytes and phagocytosis of RBCs by THP-1 cells. Augmentation of hepatic erythrophagocytosis by activated platelets in NASH exacerbates HIO. Cilostazol prevents ectopic erythrophagocytosis, mitigating HIO-mediated ferroptosis in NASH models.

Generation of a potent anti-PD-L1-CD47 bispecific antibody with a strong therapeutic and safety profile for cancer immunotherapy

Cell surface molecules PD-L1 and CD47 are potent inhibitors of adaptive and innate anti-cancer immunity. We sought to generate a safe, therapeutic, bispecific antibody specifically targeting, and blocking both PD-L1 and CD47 inhibitory activity. Novel anti-PDL-1 and anti-CD47 antibodies with favorable inhibitory activity, were humanized and constructed into a unique bi-specific antibody intended for clinical use. Previous pre-clinical and clinical studies using anti-CD47 antibodies indicated anemia and thrombocytopenia as potential risks. QL401 is a PD-L1 x CD47 bispecific antibody engineered to reduce effect on red blood cells while retaining potent phagocytic activation of macrophages in vitro and delayed tumor growth in vivo. QL401 comprises three functional components: a PD-L1 binding Fab arm, a CD47 binding scFv arm, and a human IgG4 backbone. The PD-L1 binding arm provides both tumor targeting and blocking of PD-1 for reactivating T cells. The CD47 arm blocks the binding of SIRPα, while the IgG4 Fc retains Fc gamma receptor binding to provide a phagocytic signal. In preclinical efficacy studies, QL401 potently blocked SIRPα to promote phagocytosis of tumor cells with sub-nanomolar potency. In vivo efficacy studies in mouse xenograft tumor models showed QL401 to be comparable or superior to PD-L1 or CD47 monoclonal antibodies alone or in combination. In vitro safety evaluation of QL401 showed significantly reduced binding and phagocytosis of red blood cells, in contrast to CD47 monoclonal antibodies. In addition, QL401 did not induce hemagglutination. In non-human primates, QL401 was well tolerated up to 100 mg/kg without reduction of red blood cells or platelets below the normal range. QL401 is presently in a human phase I safety study.

Abstract 6357: Generation of a mutant SIRPa fusion protein with highly-improved affinity and favorable safety profile

Abstract Background/Purpose: CD47 has been validated to be expressed on various tumor types including acute myeloid leukemia, myelodysplastic syndrome and other hematologic or solid tumors. As primary ‘don’t eat me’ signal, high expression of CD47 on tumor cells interacts with SIRPα negatively regulating the phagocytosis level. However, poor safety profile as well as insufficient combinatory effect remain to be the issues limiting the clinical outcomes of CD47-targeting molecules. Herein, we used rational design strategies to generate SCR9168 with potent affinity improvement, and maintained favorable safety profiles in different animal species, including cynomolgus monkeys. Procedures: Mutagenesis was introduced to the residues critical for CD47/SIRPα binding interface. Affinity was measured by SPR analysis. In vitro activities were determined by biochemical- or cell-based binding, blocking and antibody-dependent cellular phagocytosis (ADCP) assays. The efficacy in vivo was assessed with the OE19 xenograft model. Pharmacokinetics (PK) and safety were monitored in both mice and cynomolgus monkeys. Results: The affinity of SCR9168 achieves 178 pM for human CD47 which is nearly 50-fold increase in comparison with wild-type SIRPα. Enhanced affinity has also been confirmed in binding to OE19 or DLD1 cell lines, as well as in blocking human SIRPα binding to Raji cells. SCR9168 remains strong binding potency to monkey, mouse or rat CD47 which allows the PK and safety assessment using these animal species. The combined effects of SCR9168 and multiple therapeutic antibodies targeting tumor-associated antigens (TAAs) have been evaluated with ADCP assays using human monocyte-derived macrophages. SCR9168 markedly increases the ratio of phagocytic cells combining with antibodies, such as cetuximab. However, due to the inert function of Fc fragment, no phagocytosis of red blood cells or platelets was caused after the incubation in vitro. In addition, SCR9168 treatment in combination with trastuzumab leads to significantly improved suppression of tumor growth in a dose-dependent manner. Moreover, administration of two doses at day 1 and day 11 in cynomolgus monkeys results in no toxicity events related to SCR9168 based on the data from hematological analysis. Conclusion: SCR9168 demonstrates the best-in-class potential among SIRPα mutein molecules. It elicits improved and dose-dependent efficacy in phagocytosis or tumor suppression combining with therapeutic antibodies, such as trastuzumab or cetuximab. Favorable safety profile with no phagocytosis of RBC or platelets in vitro as well as no hematological toxicity observation in cynomolgus monkeys allows broader dose range exploration in early clinical phase. SCR9168 is currently in development stage and IND enabling will be expected in the end of 2023. Citation Format: Yingying Hu, Yayuan Fu, Lei Liu, Wenjing Li, Liting Xue, Zhiyong Yu, Yun Zhang, Meijuan Gao, Yixin Tan, Fudong Wang, Yadan Wu, Jie Li, Zhenzhen Li, Feng Zhou, Wenqing Yang, Zhuoxiao Cao, Renhong Tang. Generation of a mutant SIRPa fusion protein with highly-improved affinity and favorable safety profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6357.

Inhibition of the LRRC8A channel promotes microglia/macrophage phagocytosis and improves outcomes after intracerebral hemorrhagic stroke.

Promoting microglial/macrophage (M/Mφ) phagocytosis accelerates hematoma clearance and improves the prognosis of intracerebral hemorrhagic stroke (ICH). Cation channels such as Piezo1 modulate bacterial clearance by regulating M/Mφ. Whether LRRC8A, an anion channel, affects M/Mφ erythrophagocytosis and functional recovery after ICH was investigated here. We found that LRRC8A is highly expressed on M/Mφ in the perihematomal region of ICH mice. Conditional knockout of Lrrc8a in M/Mφ or treatment with an LRRC8A channel blocker accelerated hematoma clearance, reduced neuronal death, and improved functional recovery after ICH. Mechanistically, the LRRC8A channel inhibition promoted M/Mφ phagocytosis by activating AMP-activated protein kinase (AMPK), thereby inducing nuclear translocation of nuclear factor-erythroid 2 related factor 2 (Nrf2) and increasing Cd36 transcription. Our findings illuminate the regulation of M/Mφ phagocytosis by the LRRC8A channel via the AMPK-Nrf2-CD36 pathway after ICH, suggesting that LRRC8A is a potential target for hematoma clearance in ICH treatment.

Abstract 2894: QL401, a dual PD-L1/CD47 blocker effective against both solid and hematological malignancies with improved blood safety

Abstract Upregulation of CD47, a “Do Not Eat Me” signal, is observed in nearly all solid and hematological malignancies. Engagement of CD47 on tumor cells with SIRPα on macrophages inhibits phagocytosis of tumor cells. Anti-CD47 antibodies block the CD47 - SIRPα engagement and reactivate phagocytosis of tumor cells by macrophages. Yet the ubiquitous expression of CD47 on normal cells, including red blood cells, presents a therapeutic challenge. Systemic targeting of CD47, by either anti-CD47 monoclonal antibodies or SIRPα-Fc fusion proteins, yielded only moderate clinical benefit due to severe adverse side effects, mainly anemia. QL401 is PD-L1 x CD47 bispecific antibody engineered to reduce binding to red blood cells while retaining potent phagocytic activation of macrophages in vitro and delayed tumor growth in vivo. QL401 comprises three functional components: a PD-L1 binding Fab arm, a CD47 binding scFv arm, and a human IgG4 backbone. The PD-L1 binding arm provides both tumor targeting and blocking of PD-1 for reactivating T cells. The CD47 arm blocks the binding of SIRPα, the “Do Not Eat Me” signal, while the IgG4 Fc retains Fc gamma receptor binding to provide a phagocytic “Eat Me” signal. Therefore, QL401 is multi-functional antibody that re-actives both the innate and adaptive immune response. In preclinical efficacy studies, QL401 potently blocked SIRPα to promote phagocytosis of tumor cells with sub-nanomolar potency. In vivo efficacy studies in mouse models of solid and hematological tumors showed QL401 to be comparable or superior to PD-L1 or CD47 monoclonal antibodies alone and in combination. In vitro safety evaluation of Q401 showed significantly reduced binding and phagocytosis of red blood cells, in contrast to CD47 monoclonal antibodies. In addition, Q401 did not induce hemagglutination. In non-human primates, QL401 was well tolerated up to 100 mg/kg without reduction of red blood cells below normal range. A phase 1 dose escalation and expansion study is expected to start Q1 2022 to evaluate the safety, tolerability, and early efficacy of QL401. Citation Format: Irene Tang, Lauren Schwimmer, Shenda Gu, Wei Wei Prior, Hieu V. Tran, Allan Chan, Anna McClain, Shihao Chen, Chuanzeng Cao, Chunyan Sun, Meimei Si, Guijiang Wang. QL401, a dual PD-L1/CD47 blocker effective against both solid and hematological malignancies with improved blood safety [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2894.

Developmental intracerebral hemorrhage induces microglial heterogeneity

Microglia arise from embryonic yolk sac and colonize the brain parenchyma, followed by the maturation of microglia-specific gene expression. Single-cell transcriptional analysis has revealed that microglia are transcriptionally heterogeneous. However, the environmental factors that induce microglial heterogeneity are largely unknown. Here we found that developmental intracerebral hemorrhage induces microglial heterogeneity. In neonatal mice, we found that a portion of microglia phagocytose the red blood cells (RBCs) and expressed Hmox1, which encodes Heme Oxygenase 1, significantly higher than non-RBC-phagocytic microglia. To examine the effect of RBC phagocytosis on the transcriptional property of microglia, we labeled Hmox1-expressing microglia with red fluorescent protein by developing a transgenic mouse line, finding that microglia that underwent RBC phagocytosis expressed genes typical for yolk sac microglia in the second postnatal week. Thus, this study reveals that neonatal environmental factors induce microglial heterogeneity.

Abstract 14224: JAK2V617F Red Blood Cells Promote Atherosclerosis and Thrombosis

Elevated hematocrit is associated with cardiovascular risk; however, the causality and mechanisms are unclear. The JAK2V617F (JAK2VF) mutation markedly increases cardiovascular risk both in myeloproliferative disorders with elevated blood cell counts and in the much more prevalent clonal hematopoiesis of indeterminate potential with normal blood cell counts. Jak2VF mouse models with elevated white blood cells (WBC), platelets and red blood cells (RBCs) display accelerated atherosclerosis. However, whether Jak2VF RBCs promote atherogenesis and thrombosis independently from increased WBC and platelets remains uncertain. We used Erythropoietin Receptor (EpoR) Cre recombinase to generate a conditional knock-in of murine Jak2VF (VFEpoR) in the RBC lineage. Hyperlipidemic VFEpoR mice without elevated blood cell counts showed accelerated arterial thrombosis and increased atherosclerotic plaque necrosis. Induction of erythrocytosis with low dose erythropoietin (EPO) without concomitant leukocytosis or thrombocytosis further worsened thrombosis and atherosclerosis. VFEpoR atherosclerotic lesions featured endothelial barrier breakdown, RBC infiltration, iron deposition, lipid peroxidation, macrophage erythrophagocytosis and macrophage ferroptosis. VFEpoR RBCs had reduced antioxidant defenses and contained increased ROS and lipid hydroperoxides. Phagocytosis of murine or human WT or Jak2 VF RBCs by WT macrophages induced lipid peroxidation and ferroptotic death that was prevented by the ferroptosis inhibitor Liproxstatin-1. Ferroptosis inhibition reversed increased atherosclerosis and necrotic core formation in VFEpoR mice but had no impact in controls. These studies demonstrate that both qualitative and quantitative defects in Jak2VF RBCs accelerate atherosclerosis and thrombosis. Phagocytosis of WT or Jak2VF RBCs by macrophages leads to ferroptotic cell death, suggesting ferroptosis as a new therapeutic target to reduce RBC-mediated cardiovascular risk.

PPAR-γ activation enhances myelination and neurological recovery in premature rabbits with intraventricular hemorrhage

Intraventricular hemorrhage (IVH) results in periventricular inflammation, hypomyelination of the white matter, and hydrocephalus in premature infants. No effective therapy exists to prevent these disorders. Peroxisome proliferator activated receptor-γ (PPAR-γ) agonists reduce inflammation, alleviate free radical generation, and enhance microglial phagocytosis, promoting clearance of debris and red blood cells. We hypothesized that activation of PPAR-γ would enhance myelination, reduce hydrocephalus, and promote neurological recovery in newborns with IVH. These hypotheses were tested in a preterm rabbit model of IVH; autopsy brain samples from premature infants with and without IVH were analyzed. We found that IVH augmented PPAR-γ expression in microglia of both preterm human infants and rabbit kits. The treatment with PPAR-γ agonist or PPAR-γ overexpression by adenovirus delivery further elevated PPAR-γ levels in microglia, reduced proinflammatory cytokines, increased microglial phagocytosis, and improved oligodendrocyte progenitor cell (OPC) maturation in kits with IVH. Transcriptomic analyses of OPCs identified previously unrecognized PPAR-γ-induced genes for purinergic signaling, cyclic adenosine monophosphate generation, and antioxidant production, which would reprogram these progenitors toward promoting myelination. RNA-sequencing analyses of microglia revealed PPAR-γ-triggered down-regulation of several proinflammatory genes and transcripts having roles in Parkinson's disease and amyotrophic lateral sclerosis, contributing to neurological recovery in kits with IVH. Accordingly, PPAR-γ activation enhanced myelination and neurological function in kits with IVH. This also enhanced microglial phagocytosis of red blood cells but did not reduce hydrocephalus. Treatment with PPAR-γ agonist might enhance myelination and neurological recovery in premature infants with IVH.

Lipopolysaccharide Preconditioning Augments Phagocytosis of Malaria-Parasitized Red Blood Cells by Bone Marrow-Derived Macrophages in the Liver, Thereby Increasing the Murine Survival after Plasmodium yoelii Infection.

Malaria remains a grave concern for humans, as effective medical countermeasures for Plasmodium infection have yet to be developed. Phagocytic clearance of parasitized red blood cells (pRBCs) by macrophages is an important front-line innate host defense against Plasmodium infection. We previously showed that repeated injections of low-dose lipopolysaccharide (LPS) prior to bacterial infection, called LPS preconditioning, strongly augmented phagocytic/bactericidal activity in murine macrophages. However, whether LPS preconditioning prevents murine Plasmodium infection is unclear. We investigated the protective effects of LPS preconditioning against lethal murine Plasmodium infection, focusing on CD11bhigh F4/80low liver macrophages, which are increased by LPS preconditioning. Mice were subjected to LPS preconditioning by intraperitoneal injections of low-dose LPS for 3 consecutive days, and 24 h later, they were intravenously infected with pRBCs of Plasmodium yoelii 17XL. LPS preconditioning markedly increased the murine survival and reduced parasitemia, while it did not reduce tumor necrosis factor (TNF) secretions, only delaying the peak of plasma gamma interferon (IFN-γ) after Plasmodium infection in mice. An in vitro phagocytic clearance assay of pRBCs showed that the CD11bhigh F4/80low liver macrophages, but not spleen macrophages, in the LPS-preconditioned mice had significantly augmented phagocytic activity against pRBCs. The adoptive transfer of CD11bhigh F4/80low liver macrophages from LPS-preconditioned mice to control mice significantly improved survival after Plasmodium infection. We conclude that LPS preconditioning stimulated CD11bhigh F4/80low liver macrophages to augment the phagocytic clearance of pRBCs, which may play a central role in resistance against Plasmodium infection.