Defective Efferocytosis Research Articles

Macrophage efferocytosis in health and disease.

Creating cellular homeostasis within a defined tissue typically relates to the processes of apoptosis and efferocytosis. A great example here is cell debris that must be removed to prevent unwanted inflammatory responses and then reduce autoimmunity. In view of that, defective efferocytosis is often assumed to be responsible for the improper clearance of apoptotic cells (ACs). This predicament triggers off inflammationand even results in disease development. Any disruption of phagocytic receptors, molecules as bridging groups, or signaling routes can also inhibit macrophage efferocytosisand lead to the impaired clearance of the apoptotic body. In this line, macrophages as professional phagocytic cells take the lead in the efferocytosis process. As well, insufficiency in macrophage efferocytosis facilitates the spread of a wide variety of diseases, including neurodegenerative diseases,kidney problems, types of cancer, asthma, and the like. Establishing the functions of macrophages in this respect can be thus useful in the treatment of many diseases. Against this background, this review aimed to recapitulate the knowledge about the mechanisms related to macrophage polarization under physiological or pathological conditions, and shed light on its interaction with efferocytosis.

Walnut inclusion in a palm oil-based atherogenic diet promotes traits predicting stable atheroma plaque in Apoe-deficient mice.

The lower rates of cardiovascular disease in Southern Europe could be partially explained by the low prevalence of lipid-rich atheroma plaques. Consumption of certain foods affects the progression and severity of atherosclerosis. We investigated whether the isocaloric inclusion of walnuts within an atherogenic diet prevents phenotypes predicting unstable atheroma plaque in a mouse model of accelerated atherosclerosis. Apolipoprotein E-deficient male mice (10-week-old) were randomized to receive a control diet (9.6% of energy as fat, n = 14), a palm oil-based high-fat diet (43% of energy as fat, n = 15), or an isocaloric diet in which part of palm oil was replaced by walnuts in a dose equivalent to 30 g/day in humans (n = 14). All diets contained 0.2% cholesterol. After 15 weeks of intervention, there were no differences in size and extension in aortic atherosclerosis among groups. Compared to control diet, palm oil-diet induced features predicting unstable atheroma plaque (higher lipid content, necrosis, and calcification), and more advanced lesions (Stary score). Walnut inclusion attenuated these features. Palm oil-based diet also boosted inflammatory aortic storm (increased expression of chemokines, cytokines, inflammasome components, and M1 macrophage phenotype markers) and promoted defective efferocytosis. Such response was not observed in the walnut group. The walnut group's differential activation of nuclear factor kappa B (NF-κB; downregulated) and Nrf2 (upregulated) in the atherosclerotic lesion could explain these findings. The isocaloric inclusion of walnuts in an unhealthy high-fat diet promotes traits predicting stable advanced atheroma plaque in mid-life mice. This contributes novel evidence for the benefits of walnuts, even in an unhealthy dietary environment.

Cytoskeletal orchestration of glucose uptake in Sertoli cell to support efferocytosis of apoptotic germ cells

Efferocytosis of non-viable germ cells by Sertoli cells (SCs) constitutes a sentinel for testis homeostasis, yet how SCs signal for the metabolic and cytoskeletal adaption to this energetically costly process remains unexplored. Spectrin is membrane-associated periodic skeleton assembled into an actin-spectrin-based cytoskeletal structure with an interaction with glucose transporter Glut1. The contribution of spectrin to glucose uptake and efferocytosis is unknown. In this study, we identified a cross-regulation between glucose metabolism and efferocytosis in SCs. Pharmacological or genetic inhibition of glucose uptake or glycolysis compromises efferocytosis activity. We further found that βII-spectrin is a hitherto unappreciated regulator of glucose metabolism and cytoskeletal architecture. βII-spectrin deficiency impairs glucose uptake and lactate production in SCs. Moreover, a defective assembly of cytoskeleton and a loss of blood-testis barrier integrity are also featured by SCs deficient in βII-spectrin. The disruption in glucose metabolism and cytoskeletal organization synergistically lead to a defective efferocytosis. In vivo siRNA-mediated targeting of βII-spectrin in testis causes an obvious morphological aberration in seminiferous epithelium with the presence of exfoliated germ cells and multinucleated giant cells. Importantly, a decrease in expression of αII/βII-spectrin was observed in testes of Adjudin-induced infertility model. By exploring the functional relevance of βII-spectrin to the metabolic and cytoskeletal regulation of efferocytosis, our study proposes a potential link between βII-spectrin deregulation and male infertility.

PCSK9 attenuates efferocytosis in endothelial cells and promotes vascular aging.

Aims: Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a serine protease that binds to low-density lipoprotein receptors. Efferocytosis is the process by which phagocytes remove apoptotic cells. Both PCSK9 and efferocytosis play important roles in regulating redox biology and inflammation, the key factors contributing to vascular aging. This study was designed to investigate the impact of PCSK9 on efferocytosis in endothelial cells (ECs) and its implications in vascular aging. Methods and Results: Studies were performed in primary human aortic ECs (HAECs) and primary mouse aortic ECs (MAECs) isolated from male wild-type (WT) and PCSK9-/- mice, and in young and aged mice treated with saline or the PCSK9 inhibitor Pep2-8. Our findings include that recombinant PCSK9 protein induces defective efferocytosis and aging marker senescence-associated-β-galactosidase (SA-β-gal) expression in ECs, while PCSK9-/- restores efferocytosis and inhibits SA-β-gal activity. Further studies in aged mice showed that endothelial deficiency of MerTK, a critical receptor for efferocytosis that allows phagocytes to detect the presence of apoptotic cells, may be an indicator of vascular dysfunction in the aortic arch. Pep2-8 treatment markedly restored efferocytosis in endothelium from the aged mice. A proteomics study in the aortic arch from aged mice revealed that Pep2-8 administration significantly downregulates expression of NOX4, MAPK subunits, NF-κB, and secretion of pro-inflammatory cytokines, all known to promote vascular aging. Immunofluorescent staining showed that Pep2-8 administration upregulates expression of eNOS and downregulates expression of pro-IL-1β, NF-κB and p22phox compared to saline treated group. Conclusions: These findings provide initial evidence for the ability of aortic ECs to accomplish efferocytosis and argue for a role of PCSK9 in attenuating EC efferocytosis, thereby leading to vascular dysfunction and acceleration in vascular aging.

Axl receptor induces efferocytosis, dampens M1 macrophage responses and promotes heart pathology in Trypanosoma cruzi infection

Adaptive immunity controls Trypanosoma cruzi infection, but the protozoan parasite persists and causes Chagas disease. T cells undergo apoptosis, and the efferocytosis of apoptotic cells might suppress macrophages and exacerbate parasite infection. Nonetheless, the receptors involved in the efferocytosis of apoptotic lymphocytes during infection remain unknow. Macrophages phagocytose apoptotic cells by using the TAM (Tyro3, Axl, Mer) family of receptors. To address how the efferocytosis of apoptotic cells affects macrophage-mediated immunity, we employ here Axl receptor- and Mer receptor-deficient mouse strains. In bone marrow-derived macrophages (BMDMs), both Axl and Mer receptors play a role in the efferocytosis of proapoptotic T cells from T. cruzi-infected mice. Moreover, treatment with a TAM receptor inhibitor blocks efferocytosis and upregulates M1 hallmarks induced by immune T cells from infected mice. Remarkably, the use of Axl−/− but not Mer−/− macrophages increases T-cell-induced M1 responses, such as nitric oxide production and control of parasite infection. Furthermore, infected Axl−/− mice show reduced peak parasitemia, defective efferocytosis, improved M1 responses, and ameliorated cardiac inflammation and fibrosis. Therefore, Axl induces efferocytosis, disrupts M1 responses, and promotes parasite infection and pathology in experimental Chagas disease. Axl stands as a potential host-direct target for switching macrophage phenotypes in infectious diseases.

Heme and Iron Aggravate Tissue Damage in Sickle Cell Disease By Driving Macrophage Functional Reprogramming through the Coordinated Suppression of Efferocytosis and Mitochondrial Metabolism

Introduction: Sickle cell disease (SCD) is hallmarked by an underlying chronic inflammatory status, which is in part driven by pro-inflammatory macrophages. Macrophages are highly plastic immune cells which integrate and respond to a variety of signals they are exposed to in the surrounding microenvironment. One of these stimuli is represented by heme released upon hemolysis, which acts as damage-associated molecular pattern to stimulate macrophage inflammatory phenotypic switching through TLR4 signaling and ROS production (Vinchi et al., Blood 2016). Heme-induced pro-inflammatory activation program in macrophages promotes sterile inflammation and aggravates hepatic fibrosis in SCD, contributing to the inflammatory landscape and organ damage typically associated with this disease. While previous studies addressed heme ability to induce inflammatory cytokine production in macrophages, how heme alters cell functional properties remains unexplored. Macrophage functions such as immune cell recruitment ability and apoptotic cell clearance are relevant in the context of SCD, where tissue damage and cell apoptosis occur frequently due to vaso-occlusive episodes, hypoxia and ischemic injury. Methods: Here we analyzed macrophage response to apoptotic stimuli in vivo, in mouse models of heme overload and SCD, as well as in vitro, in bone-marrow-derived macrophages (BMDMs), to unveil the impact of hemolysis on macrophage functional properties. Results: Our results demonstrate that, in addition to inflammatory activation, heme severely alters macrophage functional response to apoptotic cell damage by exacerbating their immune cell recruitment ability and impairing their efferocytic capacity. We show that exposure to heme excess drives defective efferocytosis of apoptotic neutrophils in BMDMs and mouse model of heme overload. This is fully recapitulated in SCD mice, where limited efferocytosis contributes to impaired apoptotic cell clearance and exacerbated tissue damage. Mechanistically, altered efferocytosis depends on heme-driven activation of TLR4 signaling pathway and the suppression of the nuclear transcription factor PPARγ and its coactivator PGC1α, which lead to reduced expression of efferocytic receptors and impaired mitochondrial dynamics and functions. This results in limited recognition/engulfment of apoptotic cells and decreased shift to aerobic mitochondrial fatty acid β-oxidation and anti-inflammatory IL-4 and IL-10 release, with consequent inhibition of continual efferocytosis, inflammation resolution and tissue repair. Heme-driven changes are associated with cell metabolic skewing towards glycolysis and pentose phosphate pathway and reduced reliance on mitochondrial fatty acid oxidation and TCA cycle. Macrophages pre-loaded with fatty acids showed decreased oxygen consumption rate and ATP production after heme exposure, indicating that heme-altered mitochondrial dynamics impair lipid catabolism via limited mitochondrial oxidative phosphorylation. Overall, heme induces macrophage phenotypic and functional reprogramming through the coordinated suppression of efferocytosis, via efferocytosis receptor downregulation, and fatty acid β-oxidation, via altered mitochondrial dynamics. We further demonstrate that heme-impaired efferocytosis and tissue damage promote autoimmunity through anti-nuclear antibody production against 'self' secondary necrotic cells, potentially explaining the increasing evidence of autoimmune diseases in SCD patients. Finally, hemopexin-mediated heme scavenging and anti-inflammatory IL-4 treatment improve tissue damage in hemolytic mice through macrophage rewiring. Defective efferocytosis is reproduced in vitro by macrophage exposure to SCD patients' plasma and rescued by heme scavenging via haptoglobin/hemopexin and PPARγ/PGC1α modulation via PPARα agonist or IL-4. Conclusion: Our data indicate that the therapeutic improvement of heme-altered macrophage functional properties via heme scavenging or PPARγ/PGC1α modulation significantly promotes the resolution of inflammation and ameliorates tissue damage associated with SCD pathophysiology. Thus, macrophage functional rewiring offers potentially valuable therapeutic strategies for SCD patients to improve tissue damage resolution upon vaso-occlusive crisis and ischemic events and prevent autoimmune conditions.

Abstract 10435: Blood Flow Patterns Regulate Efferocytosis in Endothelia Cells

Introduction: Flowing blood generates a frictional force called shear stress that plays an important role in endothelial dysfunction and atherosclerosis. The disturbed flow (d-flow) pattern causes the preferential localization of atherosclerotic lesions, while regions with steady laminar flow (s-flow) are protected against atherosclerosis. Efferocytosis is a process by which apoptotic tissue is recognized for engulfment by phagocytic cells. Defective efferocytosis promotes advanced atherosclerosis. Hypothesis: We hypothesized that blood flow patterns regulate EC efferocytosis and subsequent endothelial dysfunction and contribute to the development of atherosclerosis. Methods: Primary human aortic ECs (HAECs) were exposed to s-flow and d-flow in a step-flow channel, and the changes of efferocytosis receptors in ECs and endothelial dysfunction were investigated. To investigate whether defective efferocytosis plays a role in endothelial dysfunction, HAECs were transfected with a control plasmid or MerTK CRISPR/Cas9 knockout plasmid. Results and Conclusions: Our results showed that efferocytosis regulators, such as Gas6, Pros1, and MerTK, were upregulated under s-flow conditions while inhibited under d-flow conditions compared to static conditions. Our data also showed that MerTK knockout decreased expression of VE-cadherin and CD31, while increased expression of Snail, Slug, and Twist1 as well as α-SMA, N-cadherin, calponin, and FSP1. Of note, VE-cadherin is a strictly endothelial-specific adhesion molecule located at junctions between ECs. As expected, our data showed that MerTK knockout markedly inhibited VE-cadherin expression, indicating that defective efferocytosis increases endothelial barrier dysfunction. This indicates that MerTK-mediated defective efferocytosis in ECs may play an important role in arterial areas exposed to d-flow where prone to atherosclerosis.

Defective efferocytosis of vascular cells in heart disease.

The efficient phagocytic clearance of dying cells and apoptotic cells is one of the processes that is essential for the maintenance of physiologic tissue function and homeostasis, which is termed “efferocytosis.” Under normal conditions, “find me” and “eat me” signals are released by apoptotic cells to stimulate the engulfment and efferocytosis of apoptotic cells. In contrast, abnormal efferocytosis is related to chronic and non-resolving inflammatory diseases such as atherosclerosis. In the initial steps of atherosclerotic lesion development, monocyte-derived macrophages display efficient efferocytosis that restricts plaque progression; however, this capacity is reduced in more advanced lesions. Macrophage reprogramming as a result of the accumulation of apoptotic cells and augmented inflammation accounts for this diminishment of efferocytosis. Furthermore, defective efferocytosis plays an important role in necrotic core formation, which triggers plaque rupture and acute thrombotic cardiovascular events. Recent publications have focused on the essential role of macrophage efferocytosis in cardiac pathophysiology and have pointed toward new therapeutic strategies to modulate macrophage efferocytosis for cardiac tissue repair. In this review, we discuss the molecular and cellular mechanisms that regulate efferocytosis in vascular cells, including macrophages and other phagocytic cells and detail how efferocytosis-related molecules contribute to the maintenance of vascular hemostasis and how defective efferocytosis leads to the formation and progression of atherosclerotic plaques.

KLF10 deficiency in CD4+ T cells promotes atherosclerosis progression by altering macrophage dynamics

Background and aimsAccumulating evidence supports a critical role for CD4+ T cells as drivers and modifiers of the chronic inflammatory response in atherosclerosis. Effector T cells have pro-atherogenic properties, whereas CD4+ regulatory T cells (Tregs) exert suppressive activity in atherosclerosis through increased secretion of inhibitory cytokines such as transforming growth factor-β or interleukin-10. In addition, Tregs have been shown to suppress inflammatory macrophages and promote the resolution of atherosclerosis plaques. Impaired Treg numbers and function have been associated with atherosclerosis plaque development. However, the underlying mechanisms remain unclear. Methods and resultsHere, we investigated a cell-autonomous role of a transcription factor, Krüppel-like factor 10 (KLF10), in CD4+ T cells in regulating atherosclerosis progression. Using CD4+ T-cell-specific KLF10 knockout (TKO) mice, we identified exaggerated plaque progression due to defects in immunosuppressive functions of Tregs on macrophages. TKO mice exhibited increased lesion size as well as higher CD4+ T cells and macrophage content compared to WT mice. TKO plaques also showed increased necrotic cores along with defective macrophage efferocytosis. In contrast, adoptive cellular therapy using WT Tregs abrogated the accelerated lesion progression and deleterious effects in TKO mice. Intriguingly, RNA-seq analyses of TKO lesions revealed increased chemotaxis and cell proliferation, and reduced phagocytosis compared to WT lesions. Mechanistically, TKO-Tregs impaired the efferocytosis capacity of macrophages in vitro and promoted a pro-inflammatory macrophage phenotype via increased IFN-γ and decreased TGF-β secretion. ConclusionsTaken together, these findings establish a critical role for KLF10 in regulating CD4+ Treg-macrophage interactions and atherosclerosis.

S269: MACROPHAGE FUNCTIONAL AND METABOLIC REWIRING THROUGH PPAR/PGC1 MODULATION IMPROVES HEME/IRON-DRIVEN DEFECTIVE EFFEROCYTOSIS AND PROMOTES TISSUE DAMAGE RESOLUTION IN SICKLE CELL DISEASE

Background: Sickle cell disease (SCD) is hallmarked by an underlying chronic inflammatory status, which is contributed by pro-inflammatory macrophages. Macrophages are highly plastic immune cells which integrate and respond to a variety of signals they are exposed to in the surrounding microenvironment. One of these stimuli is represented by heme, which is released from oxidized hemoglobin upon hemolysis and acts as damage-associated molecular pattern to stimulate macrophage pro-inflammatory phenotypic switching through TLR4 signaling activation and ROS production. Heme-induced M1 pro-inflammatory activation program in macrophages promotes sterile inflammation and aggravates hepatic fibrosis in SCD, contributing to organ damage typically associated with this disease. Aims: While previous studies addressed heme ability to induce inflammatory cytokine production in macrophages, how heme alters cell functional properties and whether this exacerbates tissue injury remain unexplored. Macrophage functions as immune cell recruitment ability and apoptotic cell clearance are relevant in the context of SCD, where tissue damage and cell apoptosis occur frequently due to vaso-occlusive episodes, hypoxia and ischemic injury. Methods: Here we analyzed macrophage response to apoptotic stimuli in vivo, in mouse models of heme overload and SCD, as well as in vitro, in bone-marrow-derived macrophages, to unveil the impact of hemolysis on macrophage functional properties. Results: Our results demonstrate that, in addition to inflammatory activation, heme strongly alters macrophage functional response to apoptotic cell damage by exacerbating their immune cell recruitment ability and impairing their efferocytic capacity. We show that exposure to heme and iron excess drives defective efferocytosis of apoptotic neutrophils in vitro, in cultured bone-marrow-derived macrophages, and in vivo, in mouse model of heme overload. This is fully recapitulated in SCD mice, where limited efferocytosis contributes to impaired apoptotic cell clearance and exacerbated tissue damage. Mechanistically, altered efferocytosis depends on heme-driven activation of TLR4 signaling pathway and the suppression of the transcription factor PPARγ and its coactivator PGC1α. These changes lead to reduced expression of efferocytic receptors and impaired mitochondrial biogenesis as well as mitochondrial dysfunction, and is associated with metabolic skewing. This results in limited recognition and engulfment of apoptotic cells and decreased shift to aerobic mitochondrial fatty acid β-oxidation and anti-inflammatory IL-4/10 release, with consequent inhibition of continual efferocytosis, inflammation resolution and tissue repair. We further demonstrate that impaired phagocytic capacity and tissue damage are improved by hemopexin-mediated heme scavenging and anti-inflammatory IL-4 treatment in SCD mice through phenotypic and functional macrophage rewiring. Interestingly, defective efferocytosis is reproduced in vitro by macrophage exposure to SCD patients’ plasma and rescued by hemoglobin/heme scavenging via haptoglobin and hemopexin and PPARγ/PGC1α modulation via PPARγ agonist or IL-4. Summary/Conclusion: Our data indicate that the therapeutic improvement of heme-altered macrophage functional properties via heme scavenging or PPARγ/PGC1α modulation promotes the resolution of inflammation and ameliorates tissue damage associated with SCD pathophysiology. Thus, macrophage functional rewiring offers potentially valuable therapeutic strategies for SCD patients to improve tissue damage resolution upon vaso-occlusive crisis and ischemic events.

Bone marrow-derived macrophages from a murine model of Sjögren's syndrome demonstrate an aberrant, inflammatory response to apoptotic cells

Sjögren's syndrome (SjS) is a female-dominated autoimmune disease involving lymphocytic infiltration of the exocrine glands. We have previously demonstrated cleavage of the TAM (Tyro3, Axl, Mer) receptor Mer is enhanced in SjS, leading to defective efferocytosis. Mer also plays a role in modulating phagocyte inflammatory response to apoptotic cells. Here we investigated the SjS macrophage response to apoptotic cells (AC). Bone marrow-derived macrophages (BMDMs) from SjS-susceptible (SjSs) C57BL/6.NOD-Aec1Aec2 mice and C57BL/6 (B6) controls were treated with either AC or CpG-oligodeoxynucleotides. RNA was collected from macrophages and bulk sequencing was performed to analyze transcripts. Cytokine expression was confirmed by Bio-plex. RT-qPCR was used to determine toll-like receptor (TLR) 7 and 9 involvement in BMDM inflammatory response to apoptotic cells. SjSS BMDMs exhibited a distinct transcriptional profile involving upregulation of a broad array of inflammatory genes that were not elevated in B6 BMDMs by AC. Inhibition of TLR 7 and 9 was found to limit the inflammatory response of SjSS BMDMs to ACs. ACs elicit an inflammatory reaction in SjSS BMDMs distinct from that observed in B6 BMDMs. This discovery of aberrant macrophage behavior in SjS in conjunction with previously described efferocytosis defects suggests an expanded role for macrophages in SjS, where uncleared dead cells stimulate an inflammatory response through macrophage TLRs recruiting lymphocytes, participating in co-stimulation and establishing an environment conducive to autoimmunity.

Macrophage-produced VEGFC is induced by efferocytosis to ameliorate cardiac injury and inflammation.

Clearance of dying cells by efferocytosis is necessary for cardiac repair after myocardial infarction (MI). Recent reports have suggested a protective role for vascular endothelial growth factor C (VEGFC) during acute cardiac lymphangiogenesis after MI. Here, we report that defective efferocytosis by macrophages after experimental MI led to a reduction in cardiac lymphangiogenesis and Vegfc expression. Cell-intrinsic evidence for efferocytic induction of Vegfc was revealed after adding apoptotic cells to cultured primary macrophages, which subsequently triggered Vegfc transcription and VEGFC secretion. Similarly, cardiac macrophages elevated Vegfc expression levels after MI, and mice deficient for myeloid Vegfc exhibited impaired ventricular contractility, adverse tissue remodeling, and reduced lymphangiogenesis. These results were observed in mouse models of permanent coronary occlusion and clinically relevant ischemia and reperfusion. Interestingly, myeloid Vegfc deficiency also led to increases in acute infarct size, prior to the amplitude of the acute cardiac lymphangiogenesis response. RNA-Seq and cardiac flow cytometry revealed that myeloid Vegfc deficiency was also characterized by a defective inflammatory response, and macrophage-produced VEGFC was directly effective at suppressing proinflammatory macrophage activation. Taken together, our findings indicate that cardiac macrophages promote healing through the promotion of myocardial lymphangiogenesis and the suppression of inflammatory cytokines.

Macrophage ALDH2 (Aldehyde Dehydrogenase 2) Stabilizing Rac2 Is Required for Efferocytosis Internalization and Reduction of Atherosclerosis Development.

Background:Clinical studies show that the most common single-point mutation in humans, ALDH2 (aldehyde dehydrogenase 2) rs671 mutation, is a risk factor for the development and poor prognosis of atherosclerotic cardiovascular diseases, but the underlying mechanism remains unclear. Apoptotic cells are phagocytosed and eliminated by macrophage efferocytosis during atherosclerosis, and enhancement of arterial macrophage efferocytosis reduces atherosclerosis development.Methods:Plaque areas, necrotic core size, apoptosis, and efferocytosis in aortic lesions were investigated in APOE−/− mice with bone marrow transplanted from APOE−/−ALDH2−/− and APOE−/− mice. RNA-seq, proteomics, and immunoprecipitation experiments were used to screen and validate signaling pathways affected by ALDH2. Efferocytosis and protein levels were verified in human macrophages from wild-type and rs671 mutation populations.Results:We found that transplanting bone marrow from APOE−/−ALDH2−/− to APOE−/− mice significantly increased atherosclerosis plaques compared with transplanting bone marrow from APOE−/− to APOE−/− mice. In addition to defective efferocytosis in plaques of APOE−/− mice bone marrow transplanted from APOE−/−ALDH2−/− mice in vivo, macrophages from ALDH2−/− mice also showed significantly impaired efferocytotic activity in vitro. Subsequent RNA-seq, proteomics, and immunoprecipitation experiments showed that wild-type ALDH2 directly interacted with Rac2 and attenuated its degradation due to decreasing the K48-linked polyubiquitination of lysine 123 in Rac2, whereas the rs671 mutant markedly destabilized Rac2. Furthermore, Rac2 played a more crucial role than other Rho GTPases in the internalization process in which Rac2 was up-regulated, activated, and clustered into dots. Overexpression of wild-type ALDH2 in ALDH2−/− macrophages, rather than the rs671 mutant, rescued Rac2 degradation and defective efferocytosis. More importantly, ALDH2 rs671 in human macrophages dampened the apoptotic cells induced upregulation of Rac2 and subsequent efferocytosis.Conclusions:Our study has uncovered a pivotal role of the ALDH2-Rac2 axis in mediating efferocytosis during atherosclerosis, highlighting a potential therapeutic strategy in cardiovascular diseases, especially for ALDH2 rs671 mutation carriers.

PM2.5 induce the defective efferocytosis and promote atherosclerosis via HIF-1α activation in macrophage

Epidemiological studies demonstrate that fine particulate matter (PM2.5) promotes the development of atherosclerosis. However, the mechanism insight of PM2.5-induced atherosclerosis is still lacking. The aim of this study was to explore the biological effects of hypoxia-inducible factor 1α (HIF-1α) on PM2.5-triggered atherosclerosis. The vascular stiffness, carotid intima-media thickness (CIMT), lipid and atherosclerotic lesion were increased when von Hippel-Lindau (VHL)-null mice were exposed to PM2.5. Yet, knockout of HIF-1α markedly decreased the PM2.5-triggered atherosclerotic lesion. We firstly performed microarray analysis in PM2.5-treated bone morrow-derived macrophages (BMDMs), which showed that PM2.5 significantly changed the genes expression patterns and affected biological processes such as phagocytosis, apoptotic cell clearance, cellular response to hypoxia, apoptotic process and inflammatory response. Moreover, the data showed knockout of HIF-1α remarkably relieved PM2.5-induced defective efferocytosis. Mechanistically, PM2.5 inhibited the level of genes and proteins of efferocytosis receptor c-Mer tyrosine kinase (MerTK), especially in VHL-null BMDMs. In addition, PM2.5 increased the genes and proteins of a disintegrin and metallopeptidase domain 17 (ADAM17), which caused the MerTK cleavage to form soluble MerTK (sMer) in plasma and cellular supernatant. The sMer was significantly up-regulated in plasma of VHL-null PM2.5-exposed mice. Moreover, PM2.5 could induce defective efferocytosis and activate inflammatory response through MerTK/IFNAR1/STAT1 signaling pathway in macrophages. Our results demonstrate that PM2.5 could induce defective efferocytosis and inflammation by activating HIF-1α in macrophages, ultimately resulting in accelerating atherosclerotic lesion formation and development. Our data suggest HIF-1α in macrophages might be a potential target for PM2.5-related atherosclerosis.

Importance of Efferocytosis in COVID-19 Mortality.

COVID-19 is a generally benign coronavirus disease that can spread rapidly, except for those with a group of risk factors. Since the pathogenesis responsible for the severity of the disease has not been clearly revealed, effective treatment alternatives has not been developed. The hallmark of the SARS-CoV-2-infected cells is apoptosis. Apoptotic cells are cleared through a sterile process defined as efferocytosis by professional and nonprofessional phagocytic cells. The disease would be rapidly brought under control in the organism that can achieve effective efferocytosis, which is also a kind of innate immune response. In the risk group, the efferocytic process is defective. With the addition of the apoptotic cell load associated with SARS-COV-2 infection, failure to achieve efferocytosis of dying cells can initiate secondary necrosis, which is a highly destructive process. Uncontrolled inflammation and coagulation abnormalities caused by secondary necrosis reason in various organ failures, lung in particular, which are responsible for the poor prognosis. Following the short and simplified information, this opinion paper aims to present possible treatment options that can control the severity of COVID-19 by detailing the mechanisms that can cause defective efferocytosis.

Efferocytosis and Its Role in Inflammatory Disorders.

Efferocytosis is the effective clearance of apoptotic cells by professional and non-professional phagocytes. The process is mechanically different from other forms of phagocytosis and involves the localization, binding, internalization, and degradation of apoptotic cells. Defective efferocytosis has been demonstrated to associate with the pathogenesis of various inflammatory disorders. In the current review, we summarize recent findings with regard to efferocytosis networks and discuss the relationship between efferocytosis and different immune cell populations, as well as describe how efferocytosis helps resolve inflammatory response and modulate immune balance. Our knowledge so far about efferocytosis suggests that it may be a useful target in the treatment of numerous inflammatory diseases.

3127 – ENHANCING RESOLUTION PATHWAYS LIMITS INFLAMMATION AND MITIGATES BONE MARROW FAILURE IN MURINE SEVERE APLASTIC ANEMIA

Severe aplastic anemia (SAA) is an acquired bone marrow failure (BMF) disease typically treated with immunosuppressive therapies or transplantation. Disease is driven by uncontrolled inflammation, including elevated interferon gamma (IFNγ), and the loss of hematopoietic stem and progenitor cells (HSC/HSPCs). In a mouse model of SAA, we observed robust cell death and progressive accumulation of necrotic cells in the BM. In health, inflammation resolves via efficient clearance of dead and damaged cells initiating reparative programs and limiting inflammatory signals. We hypothesized that delayed/impaired resolution hinged on defective efferocytosis and contributed to the pathophysiology of SAA. Indeed, efferocytosis was impaired in the BM of mice with SAA. To test whether failed clearance of apoptotic cells was due to reduced expression of, or cleavage of, efferocytic receptors we analyzed their expression of by flow cytometry. Unexpectedly, BM monocytes and macrophages in SAA exhibited a significant increase in Mer tyrosine kinase (MerTK) expression. Mechanistically, high MerTK expression required cell autonomous IFNγ signaling, and occurred in conjunction with increased expression of CD47 and signal regulatory protein alpha (SIRPα), an ITIM-containing inhibitory receptor that prevents phagocytosis via CD47 engagement. To test whether the CD47-SIRPα axis contributed to SAA pathogenesis, we treated mice with a blocking antibody to SIRPα. SAA mice treated with anti-SIPRα antibodies had increased HSCs and platelets, as compared to controls, along with reduced inflammatory cytokines and reduced necrotic cells in the BM. Mice expressing mutant Mertk, lacking the cleavage site, also exhibited reduced SAA pathogenesis. Together, our findings support a novel role for impaired efferocytosis and delayed inflammation resolution in the pathophysiology of SAA. Severe aplastic anemia (SAA) is an acquired bone marrow failure (BMF) disease typically treated with immunosuppressive therapies or transplantation. Disease is driven by uncontrolled inflammation, including elevated interferon gamma (IFNγ), and the loss of hematopoietic stem and progenitor cells (HSC/HSPCs). In a mouse model of SAA, we observed robust cell death and progressive accumulation of necrotic cells in the BM. In health, inflammation resolves via efficient clearance of dead and damaged cells initiating reparative programs and limiting inflammatory signals. We hypothesized that delayed/impaired resolution hinged on defective efferocytosis and contributed to the pathophysiology of SAA. Indeed, efferocytosis was impaired in the BM of mice with SAA. To test whether failed clearance of apoptotic cells was due to reduced expression of, or cleavage of, efferocytic receptors we analyzed their expression of by flow cytometry. Unexpectedly, BM monocytes and macrophages in SAA exhibited a significant increase in Mer tyrosine kinase (MerTK) expression. Mechanistically, high MerTK expression required cell autonomous IFNγ signaling, and occurred in conjunction with increased expression of CD47 and signal regulatory protein alpha (SIRPα), an ITIM-containing inhibitory receptor that prevents phagocytosis via CD47 engagement. To test whether the CD47-SIRPα axis contributed to SAA pathogenesis, we treated mice with a blocking antibody to SIRPα. SAA mice treated with anti-SIPRα antibodies had increased HSCs and platelets, as compared to controls, along with reduced inflammatory cytokines and reduced necrotic cells in the BM. Mice expressing mutant Mertk, lacking the cleavage site, also exhibited reduced SAA pathogenesis. Together, our findings support a novel role for impaired efferocytosis and delayed inflammation resolution in the pathophysiology of SAA.

Abstract 11646: ATVB Outstanding Research Award : WDFY3 is Required for the Efficient Degradation of Engulfed Apoptotic Cells by Macrophages During Efferocytosis

Introduction: Defective clearance of apoptotic cells (ACs) by macrophages (efferocytosis) contributes to unresolved inflammation. Our recent genome-wide CRISPR screen has discovered WDFY3 as a novel regulator required for the efficient uptake of ACs during efferocytosis. WDFY3 is well known as a scaffold protein facilitating the autophagic degradation of aggregated proteins in neurons. Yet, if and how WDFY3 may also be required for the efficient degradation of engulfed ACs by macrophages have not been characterized. Hypothesis: WDFY3 is responsible for the efficient degradation of ACs by interacting with other autophagic proteins to facilitate phagosome-lysosome fusion, a process known as LC3-associated phagocytosis (LAP). Methods: Bone marrow-derived macrophages (BMDMs), peritoneal macrophages (PMs), and mice with myeloid-specific Wdfy3 knockout (LysMCre +/- Wdfy3 fl/fl ) or transgenic overexpression of human WDFY3 ( hWDFY3 Tg ) were used for in vitro and in vivo assays. Results: Wdfy3 knockout BMDMs and PMs showed impaired lysosomal acidification and degradation of engulfed ACs. Mechanistically, Wdfy3 knockout impedes efficient LC3 lipidation upon AC engulfment, the last step required for phagosome-lysosome fusion and subsequent lysosomal degradation. Pull-down assay supports that WDFY3 interacts with GABARAP, but not LC3B, to mediate LC3 lipidation, supporting the direct involvement of WDFY3 protein in the process. At the transcriptomic level, RNA-seq and pathway analyses further revealed that gene sets of ROS/RNS production by phagocytes, fatty acid oxidation, and autophagosome were enriched in differentially expressed genes downregulated in Wdfy3 knockout BMDM, supporting the secondary effects of WDFY3-deficiency on the mRNA expression of genes and pathways essential for cargo degradation. Intriguingly, BMDMs and PMs from hWDFY3 Tg mice showed enhanced AC uptake and degradation both in vitro and in vivo , suggesting that WDFY3 has a wide dynamic range in its regulatory capacity. Conclusion: We discovered a novel role of WDFY3 in the degradation of ACs during macrophage efferocytosis. Enhancing WDFY3 may represent a therapeutic approach to promote the resolution of inflammation in diseases due to defective efferocytosis.

Efferocytosis in multisystem diseases (Review).

Efferocytosis, the phagocytosis of apoptotic cells performed by both specialized phagocytes (such as macrophages) and non-specialized phagocytes (such as epithelial cells), is involved in tissue repair and homeostasis. Effective efferocytosis prevents secondary necrosis, terminates inflammatory responses, promotes self-tolerance and activates pro-resolving pathways to maintain homeostasis. When efferocytosis is impaired, apoptotic cells that could not be cleared in time aggregate, resulting in the necrosis of apoptotic cells and release of pro-inflammatory factors. In addition, defective efferocytosis inhibits the intracellular cholesterol reverse transportation pathways, which may lead to atherosclerosis, lung damage, non-alcoholic fatty liver disease and neurodegenerative diseases. The uncleared apoptotic cells can also release autoantigens, which can cause autoimmune diseases. Cancer cells escape from phagocytosis via efferocytosis. Therefore, new treatment strategies for diseases related to defective efferocytosis are proposed. This review illustrated the mechanisms of efferocytosis in multisystem diseases and organismal homeostasis and the pathophysiological consequences of defective efferocytosis. Several drugs and treatments available to enhance efferocytosis are also mentioned in the review, serving as new evidence for clinical application.

The role of calmodulin and calmodulin-dependent protein kinases in the pathogenesis of atherosclerosis.

Atherosclerosis is a chronic inflammatory disease that triggers severe thrombotic cardiovascular events, such as stroke and myocardial infarction. In atherosclerotic processes, both macrophages and vascular smooth muscle cells (VSMCs) are essential cell components in atheromata formation through proinflammatory cytokine secretion, defective efferocytosis, cell migration, and proliferation, primarily controlled by Ca2+-dependent signaling. Calmodulin (CaM), as a versatile Ca2+ sensor in diverse cell types, regulates a broad spectrum of Ca2+-dependent cell functions through the actions of downstream protein kinases. Thus, this review focuses on discussing how CaM and CaM-dependent kinases (CaMKs) regulate the functions of macrophages and VSMCs in atherosclerotic plaque development based on literature from open databases. A central theme in this review is a summary of the mechanisms and consequences underlying CaMK-mediated macrophage inflammation and apoptosis, which are the key processes in necrotic core formation in atherosclerosis. Another central theme is addressing the role of CaM and CaMK-dependent pathways in phenotypic modulation, migration, and proliferation of VSMCs in atherosclerotic progression. A complete understanding of CaM and CaMK-controlled individual processes involving macrophages and VSMCs in atherogenesis might provide helpful information for developing potential therapeutic targets and strategies.