Defective Efferocytosis Research Articles

Regulatory T Cells Promote Macrophage Efferocytosis during Inflammation Resolution

Regulatory T (Treg) cell responses and apoptotic cell clearance (efferocytosis) represent critical arms of the inflammation resolution response. We sought to determine whether these processes might be linked through Treg-cell-mediated enhancement of efferocytosis. In zymosan-induced peritonitis and lipopolysaccharide-inducedlung injury, Treg cells increased early in resolution, and Treg cell depletion decreased efferocytosis. In advanced atherosclerosis, where defective efferocytosis drives disease progression, Treg cell expansion improved efferocytosis. Mechanisticstudies revealed the following sequence: (1)Treg cells secreted interleukin-13 (IL-13), which stimulated IL-10 production in macrophages; (2) autocrine-paracrine signaling by IL-10 induced Vav1 in macrophages; and (3) Vav1 activated Rac1 to promote apoptotic cell engulfment. In summary, Treg cells promote macrophage efferocytosis during inflammation resolution via a transcellular signaling pathway that enhances apoptotic cell internalization. These findings suggest an expanded role of Treg cells in inflammation resolution and provide a mechanistic basis for Treg-cell-enhancement strategies for non-resolving inflammatory diseases.

Abstract 040: Accelerated Atherosclerosis and Thrombosis in Jak2v617f Mice

The mechanisms driving increased athero-thrombotic risk in individuals with JAK2V617F positive clonal hematopoiesis or myeloproliferative neoplasms are poorly understood. This study was to assess atherosclerosis and thrombosis and associated mechanisms in hypercholesterolemic mice with hematopoietic JAK2V617F expression. Irradiated Ldlr -/- mice were transplanted with bone marrow from WT or JAK2V617F mice and fed a high fat high cholesterol diet (WD). After 7 weeks of WD JAK2V617F mice showed increased hematopoiesis, platelet activation, accelerated thrombosis and increased atherosclerosis. Early atherosclerotic lesions showed increased neutrophils, correlating with lesion size and in association with increased rolling and adhesion of neutrophils on endothelial cells in carotid artery. After 12 weeks of WD JAK2V617F lesions were slightly larger than controls but showed additional complexity, with increased necrotic core area, prominent iron deposition and co-staining of erythrocytes and macrophages suggesting erythrophagocytosis. JAK2V617F erythrocytes were more susceptible to phagocytosis by WT macrophages and showed decreased surface expression of CD47, a “don’t eat me” signal. Human JAK2V617F erythrocytes were also more susceptible to erythrophagocytosis. JAK2V617F macrophages displayed increased expression of pro-inflammatory cytokines and chemokines, increased p38 map kinase signaling and increased cleavage and reduced levels of MerTK, a key molecule mediating phagocytosis of apoptotic cells (efferocytosis) in atherosclerotic lesions. Erythrophagocytosis also suppressed efferocytosis. As a result, JAK2V617F lesional efferocytosis was reduced in advanced lesions. In conclusion, JAK2V617F promotes early lesion formation, arterial thrombosis and increased complexity in advanced atherosclerosis. In addition to increasing hematopoiesis and neutrophil infiltration in early lesions, JAK2V617F caused cellular defects in erythrocytes and macrophages, leading to increased erythrophagocytosis but defective efferocytosis. These changes appear to promote accumulation of iron and increased necrotic core formation which, together with exacerbated pro-inflammatory responses, may contribute to plaque instability.

Abstract 637: Genome-wide Crispr Screen in Primary Macrophages for Identification of a Regulatory Network for Macrophage Efferocytosis

Defective macrophage efferocytosis drives important diseases including atherosclerosis, whereas enhancing efferocytosis has potential therapeutic benefits. A systematic approach to identify regulators of efferocytosis in an unbiased manner holds promise for novel molecular mechanisms and targets. This study establishes an experimental framework using a novel CRISPR knockout screen to systematically map the regulatory networks of macrophage efferocytosis. We infected bone marrow-derived macrophages (BMDM) isolated from Cas9 transgenic mice with a pooled, genome-wide lentiviral library with 78,637 gRNAs, and induced efferocytosis by fluorescently-labeled apoptotic Jurkat cells. We used FACS to isolate BMDM that engulfed multiple apoptotic cells (enriched in gRNAs targeting negative regulators) or did not engulf apoptotic cells (enriched in gRNAs targeting positive regulators), and determined gRNA abundance by deep sequencing. The top-ranked 1,000 genes analyzed by two published methods MAGeCK and PBNPA showed substantial overlap (53% for positive regulators, 36% for negative regulators). We recovered many of the known regulators. e.g., Actr2 , Actr3 and Arpc3 , key components of the Arp2/3 complex regulating actin polymerization, were ranked 2, 8 and 4; Atp6v0b and Atp6v0c , subunits of vacuolar ATPase responsible for acidification of intracellular organelles, were ranked 22 and 27. Cd36 , a well-known efferocytosis receptor, was ranked 5. The top ranked positive regulators were enriched in GO terms “positive regulation of response to stimulus”, “Fcγ receptor signaling pathway” and “phagocytosis” (FDR-adjusted P = 2.19E-14, 1.10E-12 and 4.08E-11, respectively). Individual gRNAs verified a number of top hits from the pooled screen. In summary, we have developed a functional screen that has defined number of genes essential for macrophage efferocytosis. By focusing on a functional phenotype beyond cell viability and growth, we illustrate the versatility of CRISPR screens and provide a general strategy for systematically identifying gene of interest and uncovering novel regulators of complex macrophage functions.

Abstract 028: Neutrophils Aged Under Oxidative Stress Impede Vascular Repair

Objectives: The link between chronic inflammation and poor cardiovascular outcomes has long been appreciated, but the regulation of immune cellular targets to improve cardiovascular health remains a challenge. Mounting evidence suggests that neutrophils, previously considered acute first responder cells, play critical role in chronic inflammation. Following systemic surveillance of tissues, patrolling neutrophils age in the circulation and traffic to the bone marrow, where they undergo macrophage-mediated clearance. Here, we assessed how chronic inflammation and oxidative stress modify neutrophil trafficking and function during ischemia. Methods: Neutrophils from the Townes humanized sickle cell (SS) mice - surrogate for chronic inflammation and oxidative stress - were compared to neutrophils from MerTK deficient mice (MerKO), which age in the circulation owing to defective efferocytosis. Hind limb ischemia (HLI) was used as model of vascular injury. LASER Doppler perfusion imaging (LDPI) measured perfusion recovery after HLI. Results: The proportion of CXCR4 Hi CD62L lo aged neutrophils in circulation was markedly elevated in the SS (34 ± 4%) and MerKO mice (38 ± 4%) when compared to wild-type mice (7 ± 1.2%). However, in contrast with MerKO neutrophils, aged neutrophils from SS showed impaired phagocytosis, increased capacity to produce reactive oxygen species, and pronounced release of extracellular traps (NETs). In response to HLI, neutrophils accumulated in the MerKO and SS mice, but perfusion recovery on LDPI was only impaired in the SS mice, suggesting differential role of neutrophils aged by oxidative stress. Importantly, the phenotype of SS mice was reversed by treatment with the anti-oxidant N-acetyl cysteine (NAC), which had no impact on perfusion recovery in MerKO mice. Conclusion: Although SS and MerKO mice demonstrated elevated numbers of aged neutrophils, our data suggest that chronic inflammation and oxidative stress modifies the function of aged CXCR4 Hi CD62L lo neutrophils to impede vascular repair after ischemia. This specialized subset of neutrophils may be targeted to improve vascular health without compromising overall anti-microbial host defenses

Efferocytosis in atherosclerotic lesions: Malfunctioning regulatory pathways and control mechanisms.

Atherosclerosis is a dynamic and progressive inflammatory process in the intimal layer of large and medium-sized arteries, and it is the major contributor to the atherosclerotic cardiovascular disease (ACVD), the leading cause of death worldwide. In an atherosclerotic plaque, phagocytosis of apoptotic cells occurs through an intricate process designated efferocytosis. Defective efferocytosis has emerged as a causal factor in the etiopathogenesis of atherosclerosis and its progression into overt ACVD. Both specialized phagocytes (macrophages and dendritic cells) and non-specialized cells with phagocytic capabilities (smooth muscle and endothelial cells) are involved in the efferocytotic process. Moreover, several signaling and regulatory molecules are involved in the different steps of efferocytosis, and they include "Find-Me" signals (lysophosphatidylcholine), "Eat-Me" signals [phosphatidylserine, Mer tyrosine kinase (MerTK), and milk fat globule-EGF factor 8], and "Don't Eat-Me" signals [cluster of differentiation 47 (CD47)]. Regulation of efferocytosis is in a close nexus with inflammation, the key component in atherosclerosis. The predominance of pro-inflammatory and anti-inflammatory molecules plays a crucial role in lesion progression and regression, respectively. Polarization of macrophages towards the M1 phenotype causes them to secrete proinflammatory cytokines, while polarization towards the M2 phenotype causes them to secrete of anti-inflammatory cytokines, including interleukin-10 and transforming growth factor β, so tending to shift the balance towards resolution of the inflammation. Dysfunction of any regulatory signal may cause expansion of the necrotic core of an atherosclerotic plaque with ensuing conversion of the plaque into an unstable plaque with an increased susceptibility to rupture and to atherothrombotic complication. In this review we aim at elucidating the determinant factors and pathways of efferocytosis which can be considered as potential novel targets when striving to develop more personalized and efficient treatment regimens for patients with ACVD.

Mechanisms and Consequences of Defective Efferocytosis in Atherosclerosis.

Efficient clearance of apoptotic cells, termed efferocytosis, critically regulates normal homeostasis whereas defective uptake of apoptotic cells results in chronic and non-resolving inflammatory diseases, such as advanced atherosclerosis. Monocyte-derived macrophages recruited into developing atherosclerotic lesions initially display efficient efferocytosis and temper inflammatory responses, processes that restrict plaque progression. However, during the course of plaque development, macrophages undergo cellular reprogramming that reduces efferocytic capacity, which results in post-apoptotic necrosis of apoptotic cells and inflammation. Furthermore, defective efferocytosis in advanced atherosclerosis is a major driver of necrotic core formation, which can trigger plaque rupture and acute thrombotic cardiovascular events. In this review, we discuss the molecular and cellular mechanisms that regulate efferocytosis, how efferocytosis promotes the resolution of inflammation, and how defective efferocytosis leads to the formation of clinically dangerous atherosclerotic plaques.

Sonodynamic therapy-induced foam cells apoptosis activates the phagocytic PPARγ-LXRα-ABCA1/ABCG1 pathway and promotes cholesterol efflux in advanced plaque.

In advanced atherosclerotic plaques, defective efferocytosis of apoptotic foam cells and decreased cholesterol efflux contribute to lesion progression. In our previous study, we demonstrated that 5-aminolevulinic acid (ALA)-mediated sonodynamic therapy (SDT) could induce foam cells apoptosis via the mitochondrial-caspase pathway. In the current research, we sought to explore ALA-SDT-induced apoptosis of phagocytes and the effects of cholesterol efflux and efferocytosis in advanced apoE-/- mice plaque.Methods: apoE-/- mice fed western diet were treated with ALA-SDT and sacrificed at day 1, day 3, day 7 and day 28 post treatment. THP-1 macrophage-derived foam cells were treated with ALA-SDT. 5 hours later, the supernatant was collected and added to fresh foam cells (phagocytes). Then, the lipid area, efferocytosis, cholesterol efflux, anti-inflammatory reactions and PPARγ-LXRα-ABCA1/ABCG1 pathway were detected in plaque in vivo and in phagocytes in vitro.Results: We found that ALA-SDT induced foam cells apoptosis coupled with efferocytosis and upregulation of Mer tyrosine kinase (MerTK) both in vivo and in vitro. The lipid content in plaque decreased as early as 1 day after ALA-SDT and this tendency persisted until 28 days. The enhancement of phagocytes cholesterol efflux was accompanied by an approximately 40% decrease in free cholesterol and a 24% decrease in total cholesterol in vitro. More importantly, anti-inflammatory factors such as TGFβ and IL-10 were upregulated by ALA-SDT treatment. Finally, we found that PPARγ-LXRα-ABCA1/ABCG1 pathway was activated both in vivo and in vitro by ALA-SDT, which could be blocked by PPARγ siRNA.Conclusions: Activation of PPARγ-LXRα-ABCA1/ABCG1 pathway induced by ALA-SDT treatment engages a virtuous cycle that enhances efferocytosis, cholesterol efflux and anti-inflammatory reactions in advanced plaque in vivo and in phagocytes in vitro.

Mitochondrial Fission Promotes the Continued Clearance of Apoptotic Cells by Macrophages

Clearance of apoptotic cells (ACs) by phagocytes (efferocytosis) prevents post-apoptotic necrosis and dampens inflammation. Defective efferocytosis drives important diseases, including atherosclerosis. For efficient efferocytosis, phagocytes must be able to internalize multiple ACs. We show here that uptake of multiple ACs by macrophages requires dynamin-related protein 1 (Drp1)-mediated mitochondrial fission, which is triggered by AC uptake. When mitochondrial fission is disabled, AC-induced increase in cytosolic calcium is blunted owing to mitochondrial calcium sequestration, and calcium-dependent phagosome formation around secondarily encountered ACs is impaired. These defects can be corrected by silencing the mitochondrial calcium uniporter (MCU). Mice lacking myeloid Drp1 showed defective efferocytosis and its pathologic consequences in the thymus after dexamethasone treatment and in advanced atherosclerotic lesions in fat-fed Ldlr-/- mice. Thus, mitochondrial fission in response to AC uptake is a critical process that enables macrophages to clear multiple ACs and to avoid the pathologic consequences of defective efferocytosis invivo.

Boosting efferocytosis in alveolar space using BCG vaccine to protect host against influenza pneumonia.

Efferocytosis by alveolar phagocytes (APs) is pivotal in maintenance of lung homeostasis. Increased efferocytosis by APs results in protection against lethal acute lung injury due to pulmonary infections whereas defective efferocytosis by APs results in chronic lung inflammation. In this report, we show that pulmonary delivery of Bacillus Calmette-Guerin (BCG) significantly enhances efferocytosis by APs. Increased efferocytosis by APs maintains lung homeostasis and protects mice against lethal influenza pneumonia. Intranasally treated wild type C57Bl/6 (WT) mice with BCG showed significant increase in APs efferocytosis in vivo compared to their PBS-treated counterparts. All BCG-treated WT mice survived lethal influenza A virus (IAV) infection whereas all PBS-treated mice succumbed. BCG-induced resistance was abrogated by depleting AP prior to IAV infection. BCG treatment increased uptake, and digestion/removal of apoptotic cells by APs. BCG significantly increased the expression of TIM4 on APs and increased expression of Rab5 and Rab7. We demonstrated that increased efferocytosis by APs through pulmonary delivery of BCG initiated rapid clearance of apoptotic cells from the alveolar space, maintained lung homeostasis, reduced inflammation and protected host against lethal IAV pneumonia.

MicroRNA-33 Regulates Macrophage Autophagy in Atherosclerosis.

Defective autophagy in macrophages leads to pathological processes that contribute to atherosclerosis, including impaired cholesterol metabolism and defective efferocytosis. Autophagy promotes the degradation of cytoplasmic components in lysosomes and plays a key role in the catabolism of stored lipids to maintain cellular homeostasis. microRNA-33 (miR-33) is a post-transcriptional regulator of genes involved in cholesterol homeostasis, yet the complete mechanisms by which miR-33 controls lipid metabolism are unknown. We investigated whether miR-33 targeting of autophagy contributes to its regulation of cholesterol homeostasis and atherogenesis. Using coherent anti-Stokes Raman scattering microscopy, we show that miR-33 drives lipid droplet accumulation in macrophages, suggesting decreased lipolysis. Inhibition of neutral and lysosomal hydrolysis pathways revealed that miR-33 reduced cholesterol mobilization by a lysosomal-dependent mechanism, implicating repression of autophagy. Indeed, we show that miR-33 targets key autophagy regulators and effectors in macrophages to reduce lipid droplet catabolism, an essential process to generate free cholesterol for efflux. Notably, miR-33 regulation of autophagy lies upstream of its known effects on ABCA1 (ATP-binding cassette transporter A1)-dependent cholesterol efflux, as miR-33 inhibitors fail to increase efflux upon genetic or chemical inhibition of autophagy. Furthermore, we find that miR-33 inhibits apoptotic cell clearance via an autophagy-dependent mechanism. Macrophages treated with anti-miR-33 show increased efferocytosis, lysosomal biogenesis, and degradation of apoptotic material. Finally, we show that treating atherosclerotic Ldlr-/- mice with anti-miR-33 restores defective autophagy in macrophage foam cells and plaques and promotes apoptotic cell clearance to reduce plaque necrosis. Collectively, these data provide insight into the mechanisms by which miR-33 regulates cellular cholesterol homeostasis and atherosclerosis.

Diminished airway macrophage expression of the Axl receptor tyrosine kinase is associated with defective efferocytosis in asthma

Diminished airway macrophage expression of the Axl receptor tyrosine kinase is associated with defective efferocytosis in asthma

The walking dead: macrophage inflammation and death in atherosclerosis.

To highlight recent studies that describe novel inflammatory and signaling mechanisms that regulate macrophage death in atherosclerosis. Macrophages contribute to all stages of atherosclerosis. The traditional dogma states that in homeostatic conditions, macrophages undergo apoptosis and are efficiently phagocytosed to be cleared by a process called efferocytosis. In advanced atherosclerosis, however, defective efferocytosis results in secondary necrosis of these uncleared apoptotic cells, which ultimately contributes to the formation of the characteristic necrotic core and the vulnerable plaque. Here, we outline the different types of lesional macrophage death: apoptosis, autophagic and the newly defined necroptosis (i.e. a type of programmed necrosis). Recent discoveries demonstrate that macrophage necroptosis directly contributes to necrotic core formation and plaque instability. Further, promoting the resolution of inflammation using preresolving mediators has been shown to enhance efferocytosis and decrease plaque vulnerability. Finally, the canonical 'don't eat me' signal CD47 has recently been described as playing an important role in atherosclerotic lesion progression by impairing efficient efferocytosis. Although we have made significant strides in improving our understanding of cell death and clearance mechanisms in atherosclerosis, there still remains unanswered questions as to how these pathways can be harnessed using therapeutics to promote lesion regression and disease stability. Improving our understanding of the mechanisms that regulate macrophage death in atherosclerosis, in particular apoptosis, necroptosis and efferocytosis, will provide novel therapeutic opportunities to resolve atherosclerosis and promote plaque stability.

Nonantibiotic macrolides restore airway macrophage phagocytic function with potential anti-inflammatory effects in chronic lung diseases.

We reported defective efferocytosis associated with cigarette smoking and/or airway inflammation in chronic lung diseases, including chronic obstructive pulmonary disease, severe asthma, and childhood bronchiectasis. We also showed defects in phagocytosis of nontypeable Haemophilus influenzae (NTHi), a common colonizer of the lower airway in these diseases. These defects could be substantially overcome with low-dose azithromycin; however, chronic use may induce bacterial resistance. The aim of the present study was therefore to investigate two novel macrolides-2'-desoxy-9-(S)-erythromycylamine (GS-459755) and azithromycin-based 2'-desoxy molecule (GS-560660)-with significantly diminished antibiotic activity against Staphylococcus aureus, Streptococcus pneumonia, Moraxella catarrhalis, and H. influenzae We tested their effects on efferocytosis, phagocytosis of NTHi, cell viability, receptors involved in recognition of apoptotic cells and/or NTHi (flow cytometry), secreted and cleaved intracellular IL-1β (cytometric bead array, immunofluorescence/confocal microscopy), and nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) using primary alveolar macrophages and THP-1 macrophages ± 10% cigarette smoke extract. Dose-response experiments showed optimal prophagocytic effects of GS-459755 and GS-560660 at concentrations of 0.5-1 µg/ml compared with our findings with azithromycin. Both macrolides significantly improved phagocytosis of apoptotic cells and NTHi (e.g., increases in efferocytosis and phagocytosis of NTHi: GS-459755, 23 and 22.5%, P = 0.043; GS-560660, 23.5 and 22%, P = 0.043, respectively). Macrophage viability remained >85% following 24 h exposure to either macrolide at concentrations up to 20 µg/ml. Secreted and intracellular-cleaved IL-1β was decreased with both macrolides with no significant changes in recognition molecules c-mer proto-oncogene tyrosine kinase; scavenger receptor class A, member 1; Toll-like receptor 2/4; or CD36. Particulate cytoplasmic immunofluorescence of NLRP3 inflammasome was also reduced significantly. We conclude that GS-459755 and GS-560660 may be useful for reducing airway inflammation in chronic lung diseases without inducing bacterial resistance.

"Attack of the Clones": Commonalities Between Cancer and Atherosclerosis.

Many studies have noted similarities between atherosclerosis and cancer including pronounced cellular plasticity, clonal expansion of cellular subtypes, increased DNA mutations, defective efferocytosis pathways, and an important role for proto-oncogenes in disease development. Although it is clear that these 2 diseases have disparate causes, noting the parallels between atherosclerosis and cancer may help us identify unique, targeted therapeutic strategies. Early articles described atheromas as benign neoplasms of the blood vessel comprised primarily of fibrous smooth muscle cells (SMCs). However, additional studies soon noted the presence of macrophages and other cell types, thereby promoting controversy as to which cells actually give rise to the atherosclerotic plaque. Much of this controversy results from the promiscuity of the so-called lineage-specific markers.1 For example, bone marrow–derived cells can migrate into plaques and begin to express certain SMC markers,2 whereas SMCs can upregulate macrophage-specific markers such as galectin-3, CD11b, and F4/80 as they migrate into the plaque.3,4 In addition, recent lineage tracing studies suggest that endothelial cells can acquire mesenchymal cell characteristics, and some adventitial cells may have the capability of forming SMC-like cells. Collectively, these studies demonstrate the impressive capacity of many vascular cell types to dedifferentiate or transdifferentiate in response to the atherosclerotic environment and suggest that plaques may arise from multiple vascular and circulating cell types. Clearly, cellular plasticity is a major theme in atherosclerotic plaque development—as it is in cancer—and methods to reprogram proatherosclerotic cells could have tremendous therapeutic potential. The clonal evolution theory of cancer development posits that certain tumors arise from a single cell that expands in response to a series of acquired mutations. In 1973, Benditt and Benditt5 performed X-inactivation studies on human atherosclerotic lesions and found evidence that atherosclerotic plaques could be derived from a single cell that underwent monoclonal expansion, …

MerTK receptor cleavage promotes plaque necrosis and defective resolution in atherosclerosis.

Atherothrombotic vascular disease is often triggered by a distinct type of atherosclerotic lesion that displays features of impaired inflammation resolution, notably a necrotic core and thinning of a protective fibrous cap that overlies the core. A key cause of plaque necrosis is defective clearance of apoptotic cells, or efferocytosis, by lesional macrophages, but the mechanisms underlying defective efferocytosis and its possible links to impaired resolution in atherosclerosis are incompletely understood. Here, we provide evidence that proteolytic cleavage of the macrophage efferocytosis receptor c-Mer tyrosine kinase (MerTK) reduces efferocytosis and promotes plaque necrosis and defective resolution. In human carotid plaques, MerTK cleavage correlated with plaque necrosis and the presence of ischemic symptoms. Moreover, in fat-fed LDL receptor-deficient (Ldlr-/-) mice whose myeloid cells expressed a cleavage-resistant variant of MerTK, atherosclerotic lesions exhibited higher macrophage MerTK, lower levels of the cleavage product soluble Mer, improved efferocytosis, smaller necrotic cores, thicker fibrous caps, and increased ratio of proresolving versus proinflammatory lipid mediators. These findings provide a plausible molecular-cellular mechanism that contributes to defective efferocytosis, plaque necrosis, and impaired resolution during the progression of atherosclerosis.

Deficiency of AXL in Bone Marrow-Derived Cells Does Not Affect Advanced Atherosclerotic Lesion Progression.

AXL, a member of the TAM (Tyro3, Axl, MerTK) family of receptors, plays important roles in cell survival, clearance of dead cells (efferocytosis), and suppression of inflammation, which are processes that critically influence atherosclerosis progression. Whereas MerTK deficiency promotes defective efferocytosis, inflammation, and plaque necrosis in advanced murine atherosclerosis, the role of Axl in advanced atherosclerosis progression is not known. Towards this end, bone marrow cells from Axl−/− or wild-type mice were transplanted into lethally irradiated Ldlr−/− mice. These chimeric mice were then fed the Western-type diet (WD) for 17 weeks. We demonstrate that lesional macrophages in WT mice express Axl but that Axl deficiency in bone marrow-derived cells does not affect lesion size, cellularity, necrosis, or inflammatory parameters in advanced atherosclerotic plaques. Moreover, apoptosis of lesional cells was unaffected, and we found no evidence of defective lesional efferocytosis. In contrast to previously reported findings with MerTK deficiency, hematopoietic cell-Axl deficiency in WD-fed Ldlr−/− mice does not affect the progression of advanced atherosclerosis or lesional processes associated with TAM receptor signaling. These findings suggest a heretofore unappreciated TAM receptor hierarchy in advanced atherosclerosis.

Reduced DNA methylation of sphingosine‐1 phosphate receptor 5 in alveolar macrophages in COPD: A potential link to failed efferocytosis

We previously showed that alveolar macrophages from COPD patients are defective in their ability to phagocytose apoptotic cells ('efferocytosis') and that this defect is potentially linked to the sphingosine-1 phosphate (S1P) system, in particular the sphingosine-1 phosphate receptor 5 (S1PR5). In alveolar macrophages from COPD patients, S1PR5 mRNA expression levels increased and were correlated with both lung function and efferocytosis. However, it us unknown whether these changes are under epigenetic control via DNA methylation or whether DNA methylation directly modulates macrophage function. Bisulfite sequencing was used to assess DNA methylation levels at CpG islands associated with genes encoding selected S1P system components, including sphingosine kinase 1 (SPHK1), S1PR1 and S1PR5, in alveolar macrophages from 20 COPD patients, 7 healthy smokers and 10 healthy non/ex-smokers) by methyl quantitative real-time PCR (methyl qPCR). The effect of the DNA methyltransferase inhibitor, 5-azacytidine on the efferocytosis capacity of THP-1 macrophages was assessed using flow cytometry. Among the S1P system genes examined, S1PR5 was the single target that showed significant changes in DNA methylation between patient groups. Alveolar macrophages isolated from COPD patients showed lower methylation levels in the same region compared to macrophages from non/ex-smokers. in vitro studies using THP-1 macrophages showed that DNA demethylation with 5-azacytidine increased the efferocytosis capacity and dose-dependently rescued the cells from the cigarette smoke-induced defect in efferocytosis. Macrophage function can be modulated epigenetically. Reduced methylation may underlie the increased expression of the S1PR5 gene in alveolar macrophages and associated defective efferocytosis in COPD.

MafB and the role of macrophage apoptosis in atherosclerosis: A time to kill, a time to heal

MafB and the role of macrophage apoptosis in atherosclerosis: A time to kill, a time to heal

Inhibition of transglutaminase 2 reduces efferocytosis in human macrophages: Role of CD14 and SR-AI receptors

Background and aimsTransglutaminase 2 (TGM2), a member of the transglutaminase family of enzymes, is a multifunctional protein involved in numerous events spanning from cell differentiation, to signal transduction, apoptosis, and wound healing. It is expressed in a variety of cells, macrophages included. Macrophage TGM2 promotes the clearance of apoptotic cells (efferocytosis) and emerging evidence suggests that defective efferocytosis contributes to the consequences of inflammation-associated diseases, including atherosclerotic lesion progression and its sequelae. Of interest, active TGM2 identified in human atherosclerotic lesions plays critical roles in plaque stability through effects on matrix cross-linking and TGFβ activity. This study explores the mechanisms by which TGM2 controls efferocytosis in human macrophages. Methods and resultsHerein we show that TGM2 increases progressively during monocyte differentiation towards macrophages and controls their efferocytic potential as well as morphology and viability. Two experimental approaches that took advantage of the inhibition of TGM2 activity and protein silencing give proof that TGM2 reduction significantly impairs macrophage efferocytosis. Among the mechanisms involved we highlighted a role of the receptors CD14 and SR-AI whose levels were markedly reduced by TGM2 inhibition. Conversely, CD36 receptor and αvβ3 integrin levels were not influenced. Of note, lipid accumulation and IL-10 secretion were reduced in macrophages displaying defective efferocytosis. ConclusionOverall, our data define a crucial role of TGM2 activity during macrophage differentiation via mechanisms involving CD14 and SR-AI receptors and show that TGM2 inhibition triggers a pro-inflammatory phenotype.

Mechanisms of MicroRNAs in Atherosclerosis.

The maladaptation of endothelial cells to disturbed flow at arterial bifurcations increases permeability for lipoproteins. Additional injury by chemically modified lipoproteins disrupts the continuous repair of maladapted endothelial cells and triggers intimal macrophage accumulation. Macrophages remove modified lipoproteins from the extracellular space until the cholesterol overload leads to macrophage death and insufficient efferocytosis. This macrophage failure promotes the progression to advanced lesions by formation of a lipid-rich necrotic core, which may rupture and cause myocardial infarction and stroke. In this article, we summarize the fundamental roles of microRNAs (miRNAs) in the regulation of endothelial maladaptation and macrophage failure during atherosclerosis. We describe how miRNAs coordinate the mutual interaction between chronic endothelial repair and endothelial senescence and mechanistically link the regulation of macrophage cholesterol homeostasis with defective efferocytosis. Lastly, we discuss how miRNAs may challenge and extend current theories about atherosclerosis.