Search

Phagocytic clearance of dying cells, termed efferocytosis, is essential for maintaining tissue homeostasis, yet our understanding of efferocytosis regulation remains incomplete. Here we perform a FACS-based, genome-wide CRISPR knockout screen in primary mouse macrophages to search for novel regulators of efferocytosis. The results show that Wdfy3 knockout in macrophages specifically impairs uptake, but not binding, of apoptotic cells due to defective actin disassembly. Additionally, WDFY3 interacts with GABARAP, thus facilitating LC3 lipidation and subsequent lysosomal acidification to permit the degradation of apoptotic cell components. Mechanistically, while the C-terminus of WDFY3 is sufficient to rescue the impaired degradation induced by Wdfy3 knockout, full-length WDFY3 is required to reconstitute the uptake of apoptotic cells. Finally, WDFY3 is also required for efficient efferocytosis in vivo in mice and in vitro in primary human macrophages. This work thus expands our knowledge of the mechanisms of macrophage efferocytosis, as well as supports genome-wide CRISPR screen as a platform for interrogating complex functional phenotypes in primary macrophages.

Incomplete understanding of how hepatosteatosis transitions to fibrotic non-alcoholic steatohepatitis (NASH) has limited therapeutic options. Two molecules that are elevated in hepatocytes in human NASH liver are cholesterol, whose mechanistic link to NASH remains incompletely understood, and TAZ, a transcriptional regulator that promotes fibrosis but whose mechanism of increase in NASH is unknown. We now show that increased hepatocyte cholesterol upregulates TAZ and promotes fibrotic NASH. ASTER-B/C-mediated internalization of plasma membrane cholesterol activates soluble adenylyl cyclase (sAC; ADCY10), triggering a calcium-RhoA-mediated pathway that suppresses β-TrCP/proteasome-mediated TAZ degradation. In mice fed with a cholesterol-rich NASH-inducing diet, hepatocyte-specific silencing of ASTER-B/C, sAC, or RhoA decreased TAZ and ameliorated fibrotic NASH. The cholesterol-TAZ pathway is present in primary human hepatocytes, and associations among liver cholesterol, TAZ, and RhoA in human NASH liver are consistent with the pathway. Thus, hepatocyte cholesterol contributes to fibrotic NASH by increasing TAZ, suggesting new targets for therapeutic intervention.

The interplay between myeloid cells and T-lymphocytes is critical to the regulation of host defense and inflammation resolution. Dysregulation of this interaction can contribute to the development of chronic inflammatory diseases. Important among these diseases is atherosclerosis, which refers to focal lesions in the arterial intima driven by elevated apolipoprotein B-containing lipoproteins, notably low-density lipoprotein (LDL), and characterized by the formation of a plaque composed of inflammatory immune cells, a collection of dead cells and lipids called the necrotic core, and a fibrous cap. As the disease progresses, the necrotic core expands, and the fibrous cap becomes thin, which increases the risk of plaque rupture or erosion. Plaque rupture leads to a rapid thrombotic response that can give rise to heart attack, stroke, or sudden death. With marked lowering of circulating LDL, however, plaques become more stable and cardiac risk is lowered--a process known as atherosclerosis regression. A critical aspect of both atherosclerosis progression and regression is the crosstalk between innate (myeloid cells) and adaptive (T-lymphocytes) immune cells. Myeloid cells are specialized at clearing apoptotic cells by a process called efferocytosis, which is necessary for inflammation resolution. In advanced disease, efferocytosis is impaired, leading to secondary necrosis of apoptotic cells, inflammation, and, most importantly, defective tissue resolution. In regression, efferocytosis is reawakened aiding in inflammation resolution and plaque stabilization. Here, we will explore how efferocytosing myeloid cells could affect T-cell function and vice versa through antigen presentation, secreted factors, and cell-cell contacts and how this cellular crosstalk may contribute to the progression or regression of atherosclerosis. [ABSTRACT FROM AUTHOR]

Fibrinolysis is initiated by tissue-type plasminogen activator (tPA) and inhibited by plasminogen activator inhibitor 1 (PAI-1). In obese humans, plasma PAI-1 and tPA proteins are increased, but PAI-1 dominates, leading to reduced fibrinolysis and thrombosis. To understand tPA-PAI-1 regulation in obesity, we focused on hepatocytes, a functionally important source of tPA and PAI-1 that sense obesity-induced metabolic stress. We showed that obese mice, like humans, had reduced fibrinolysis and increased plasma PAI-1 and tPA, due largely to their increased hepatocyte expression. A decrease in the PAI-1 (SERPINE1) gene corepressor Rev-Erba increased PAI-1, which then increased the tPA gene PLAT via a PAI-1/LRP1/PKA/p-CREB1 pathway. This pathway was partially counterbalanced by increased DACH1, a PLAT-negative regulator. We focused on the PAI-1/PLAT pathway, which mitigates the reduction in fibrinolysis in obesity. Thus, silencing hepatocyte PAI-1, CREB1, or tPA in obese mice lowered plasma tPA and further impaired fibrinolysis. The PAI-1/PLAT pathway was present in primary human hepatocytes, and associations among PAI-1, tPA, and PLAT in livers from obese and lean humans were consistent with these findings. Knowledge of PAI-1 and tPA regulation in hepatocytes in obesity may suggest therapeutic strategies for improving fibrinolysis and lowering the risk of thrombosis in this setting., Introduction Obesity, which has now reached epidemic proportions, increases risks for arterial, venous, and microvascular thrombosis, including coronary thrombosis (1, 2), stroke (3), deep vein thrombosis (4, 5), pulmonary embolism [...]

ObjectiveLipid-loaded macrophage-derived foam cells populate atherosclerotic lesions and produce many pro-inflammatory and plaque-destabilizing factors. An excessive accumulation of extensively oxidized low-density lipoprotein (OxLDL) or free cholesterol (FC), both of which are believed to be major lipid components of macrophages in advanced lesions, rapidly induces apoptosis in macrophages. Indeed, there is evidence of macrophage death in lesions, but how the surviving macrophages avoid death induced by OxLDL, FC, and other factors is not known.Methods and resultsMinimally oxidized LDL (mmLDL), which is an early product of progressive LDL oxidation in atherosclerotic lesions, countered OxLDL-induced or FC-induced apoptosis and stimulated macrophage survival both in cell culture and in vivo. DNA fragmentation and caspase-3 activity in OxLDL-treated peritoneal macrophages were significantly reduced by coincubation with mmLDL. In a separate set of experiments, mmLDL significantly reduced annexin V binding to macrophages in which apoptosis was induced by FC loading. In both cellular models, mmLDL activated a pro-survival PI3K/Akt signaling pathway, and PI3K inhibitors, wortmannin and LY294002, eliminated the pro-survival effect of mmLDL. Immunohistochemical examination demonstrated phospho-Akt in murine atherosclerotic lesions.ConclusionsMinimally oxidized LDL, an early form of oxidized LDL in atherosclerotic lesions, may contribute to prolonged survival of macrophage foam cells in lesions via a PI3K/Akt-dependent mechanism.

Non-resolving inflammation drives the development of clinically dangerous atherosclerotic lesions by promoting sustained plaque inflammation, large necrotic cores, thin fibrous caps, and thrombosis. Resolution of inflammation is not merely a passive return to homeostasis, but rather an active process mediated by specific molecules, including fatty acid-derived specialized pro-resolving mediators (SPMs). In advanced atherosclerosis, there is an imbalance between levels of SPMs and proinflammatory lipid mediators, which results in sustained leukocyte influx into lesions, inflammatory macrophage polarization, and impaired efferocytosis. In animal models of advanced atherosclerosis, restoration of SPMs limits plaque progression by suppressing inflammation, enhancing efferocytosis, and promoting an increase in collagen cap thickness. This Review discusses the roles of non-resolving inflammation in atherosclerosis and highlights the unique therapeutic potential of SPMs in blocking the progression of clinically dangerous plaques., Introduction Atherosclerosis is a lipoprotein-driven, non-resolving chronic inflammatory disease of the arterial subendothelial space (intima) and involves activation of both the innate and adaptive branches of the host immune system. [...]

Emerging data suggest that hypercholesterolemia has stimulatory effects on adaptive immunity and that these effects can promote atherosclerosis and perhaps other inflammatory diseases. However, research in this area has relied primarily on inbred strains of mice whose adaptive immune system can differ substantially from that of humans. Moreover, the genetically induced hypercholesterolemia in these models typically results in plasma cholesterol levels that are much higher than those in most humans. To overcome these obstacles, we studied human immune system- reconstituted mice (hu-mice) rendered hypercholesterolemic by treatment with adeno-associated virus 8-proprotein convertase subtilisin/kexin type 9 (AAV8-PCSK9) and a high-fat/high-cholesterol Western-type diet (WD). These mice had a high percentage of human T cells and moderate hypercholesterolemia. Compared with hu-mice that had lower plasma cholesterol, the PCSK9-WD mice developed a T cell-mediated inflammatory response in the lung and liver. Human [CD4.sup.+] and [CD8.sup.+] T cells bearing an effector memory phenotype were significantly elevated in the blood, spleen, and lungs of PCSK9-WD hu-mice, whereas splenic and circulating regulatory T cells were reduced. These data show that moderately high plasma cholesterol can disrupt human T cell homeostasis in vivo. This process may not only exacerbate atherosclerosis, but also contribute to T cell-mediated inflammatory diseases in the hypercholesterolemia setting., Introduction Cardiovascular disease (CVD) remains the number one cause of death in the Western world. Dyslipidemia, the elevation of low-density lipoprotein cholesterol (LDL-C), is a critical risk factor for developing [...]

Efferocytosis, the process through which apoptotic cells (ACs) are cleared through actin-mediated engulfment by macrophages, prevents secondary necrosis, suppresses inflammation, and promotes resolution. Impaired efferocytosis drives the formation of clinically dangerous necrotic atherosclerotic plaques, the underlying etiology of coronary artery disease (CAD). An intron of the gene encoding PHACTR1 contains rs9349379 (A>G), a common variant associated with CAD. As PHACTR1 is an actin-binding protein, we reasoned that if the rs9349379 risk allele G causes lower PHACTR1 expression in macrophages, it might link the risk allele to CAD via impaired efferocytosis. We show here that rs9349379-G/G was associated with lower levels of PHACTR1 and impaired efferocytosis in human monocyte-derived macrophages and human atherosclerotic lesional macrophages compared with rs9349379-A/A. Silencing PHACTR1 in human and mouse macrophages compromised AC engulfment, and Western diet-fed [Ldlr.sup.-/-] mice in which hematopoietic Phactr1 was genetically targeted showed impaired lesional efferocytosis, increased plaque necrosis, and thinner fibrous caps--all signs of vulnerable plaques in humans. Mechanistically, PHACTR1 prevented dephosphorylation of myosin light chain (MLC), which was necessary for AC engulfment. In summary, rs9349379-G lowered PHACTR1, which, by lowering phospho-MLC, compromised efferocytosis. Thus, rs9349379-G may contribute to CAD risk, at least in part, by impairing atherosclerotic lesional macrophage efferocytosis., Introduction Despite the availability of safe and effective risk-reducing therapies, coronary artery disease (CAD) remains the leading cause of morbidity and mortality in the United States and other developed countries [...]

Atherosclerosis is the underlying etiology of cardiovascular disease, the leading cause of death worldwide. Atherosclerosis is a heterogeneous disease in which only a small fraction of lesions lead to heart attack, stroke, or sudden cardiac death. A distinct type of plaque containing large necrotic cores with thin fibrous caps often precipitates these acute events. Here, we show that [Ca.sup.2+]/calmodulin-dependent protein kinase y (CaMKII[gamma]) in macrophages plays a major role in the development of necrotic, thin-capped plaques. Macrophages in necrotic and symptomatic atherosclerotic plaques in humans as well as advanced atherosclerotic lesions in mice demonstrated activation of CaMKII. Western diet-fed LDL receptor-deficient ([Ldlr.sup.-/-]) mice with myeloid-specific deletion of CaMKII had smaller necrotic cores with concomitantly thicker collagen caps. These lesions demonstrated evidence of enhanced efferocytosis, which was associated with increased expression of the macrophage efferocytosis receptor MerTK. Mechanistic studies revealed that CaMKII[gamma]- deficient macrophages and atherosclerotic lesions lacking myeloid CaMKII[gamma] had increased expression of the transcription factor ATF6. We determined that ATF6 induces liver X receptor-[alpha] (LXR[alpha]), an Mertk-inducing transcription factor, and that increased MerTK expression and efferocytosis in CaMKII[gamma]deficient macrophages is dependent on LXR[alpha]. These findings identify a macrophage CaMKII[gamma]/ATF6/LXR[alpha]/MerTK pathway as a key factor in the development of necrotic atherosclerotic plaques., Introduction Despite major medical advances in the past 50 years, cardiovascular disease remains the major cause of morbidity and mortality worldwide (1). Ischemic cardiovascular disease is caused by atherosclerosis, a [...]

Tyro3, AXL, and MerTK (TAM) receptors are activated in macrophages in response to tissue injury and as such have been proposed as therapeutic targets to promote inflammation resolution during sterile wound healing, including myocardial infarction. Although the role of MerTK in cardioprotection is well characterized, the unique role of the other structurally similar TAMs, and particularly AXL, in clinically relevant models of myocardial ischemia/reperfusion infarction (IRI) is comparatively unknown. Utilizing complementary approaches, validated by flow cytometric analysis of human and murine macrophage subsets and conditional genetic loss and gain of function, we uncover a maladaptive role for myeloid AXL during IRI in the heart. Cross signaling between AXL and TLR4 in cardiac macrophages directed a switch to glycolytic metabolism and secretion of proinflammatory IL-1[beta], leading to increased intramyocardial inflammation, adverse ventricular remodeling, and impaired contractile function. AXL functioned independently of cardioprotective MerTK to reduce the efficacy of cardiac repair, but like MerTK, was proteolytically cleaved. Administration of a selective small molecule AXL inhibitor alone improved cardiac healing, which was further enhanced in combination with blockade of MerTK cleavage. These data support further exploration of macrophage TAM receptors as therapeutic targets for myocardial infarction., Introduction Myocardial infarction (MI) is a leading cause of heart failure and is associated with an increased risk of death. Intramyocardial inflammation arising after cardiomyocyte cell death from reperfused infarction [...]

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.sup.-/-]) 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., Introduction Atherothrombotic vascular disease is the leading cause of death in the industrialized world. While the majority of atherosclerotic lesions are clinically silent, acute cardiovascular events can be triggered by [...]