RESUMO
The cardiovascular complications of atherosclerosis are thought to arise from an inflammatory response to the accumulation of cholesterol-rich lipoproteins in the arterial wall. The positive outcome of CANTOS (Canakinumab Anti-inflammatory Thrombosis Outcome Study) provided key evidence to support this concept and suggested that inflammasomes and IL-1ß are important inflammatory mediators in human atherosclerotic cardiovascular diseases (ACVD). In specific settings NLRP3 or AIM2 inflammasomes can induce inflammatory responses in the arterial wall and promote the formation of unstable atherosclerotic plaques. Clonal hematopoiesis (CH) has recently emerged as a major independent risk factor for ACVD. CH mutations arise during ageing and commonly involves variants in genes mediating epigenetic modifications (TET2, DNMT3A, ASXL1) or cytokine signaling (JAK2). Accumulating evidence points to the role of inflammasomes in the progression of CH-induced ACVD events and has shed light on the regulatory pathways and possible therapeutic approaches that specifically target inflammasomes in atherosclerosis. Epigenetic dynamics play a vital role in regulating the generation and activation of inflammasome components by causing changes in DNA methylation patterns and chromatin assembly. This review examines the genetic and epigenetic regulation of inflammasomes, the intersection of macrophage cholesterol accumulation with inflammasome activation and their roles in atherosclerosis. Understanding the involvement of inflammasomes in atherosclerosis pathogenesis may lead to customized treatments that reduce the burden of ACVD.
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The deposition of cholesterol-rich lipoproteins in the arterial wall triggers macrophage inflammatory responses, which promote atherosclerosis. The NLRP3 inflammasome aggravates atherosclerosis; however, cellular mechanisms connecting macrophage cholesterol accumulation to inflammasome activation are poorly understood. We investigated the mechanisms of NLRP3 inflammasome activation in cholesterol-loaded macrophages and in atherosclerosis-prone Ldlr-/- mice with defects in macrophage cholesterol efflux. We found that accumulation of cholesterol in macrophages treated with modified LDL or cholesterol crystals, or in macrophages defective in the cholesterol efflux promoting transporters ABCA1 and ABCG1, leads to activation of NLRP3 inflammasomes as a result of increased cholesterol trafficking from the plasma membrane to the ER, via Aster-B. In turn, the accumulation of cholesterol in the ER activates the inositol triphosphate-3 receptor, CaMKII/JNK, and induces NLRP3 deubiquitylation by BRCC3. An NLRP3 deubiquitylation inhibitor or deficiency of Abro1, an essential scaffolding protein in the BRCC3-containing cytosolic complex, suppressed inflammasome activation, neutrophil extracellular trap formation (NETosis), and atherosclerosis in vivo. These results identify a link between the trafficking of cholesterol to the ER, NLRP3 deubiquitylation, inflammasome activation, and atherosclerosis.
Assuntos
Aterosclerose , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Colesterol , Retículo Endoplasmático , Proteína 3 que Contém Domínio de Pirina da Família NLR , Animais , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Aterosclerose/metabolismo , Aterosclerose/patologia , Camundongos , Colesterol/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Retículo Endoplasmático/metabolismo , Inflamassomos/metabolismo , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Transporte Biológico , Camundongos KnockoutRESUMO
Clonal hematopoiesis (CH) is an independent risk factor for atherosclerotic cardiovascular disease. Murine models of CH suggest a central role of inflammasomes and IL-1ß in accelerated atherosclerosis and plaque destabilization. Here we show using single-cell RNA sequencing in human carotid plaques that inflammasome components are enriched in macrophages, while the receptor for IL-1ß is enriched in fibroblasts and smooth muscle cells (SMCs). To address the role of inflammatory crosstalk in features of plaque destabilization, we conducted SMC fate mapping in Ldlr-/- mice modeling Jak2VF or Tet2 CH treated with IL-1ß antibodies. Unexpectedly, this treatment minimally affected SMC differentiation, leading instead to a prominent expansion of fibroblast-like cells. Depletion of fibroblasts from mice treated with IL-1ß antibody resulted in thinner fibrous caps. Conversely, genetic inactivation of Jak2VF during plaque regression promoted fibroblast accumulation and fibrous cap thickening. Our studies suggest that suppression of inflammasomes promotes plaque stabilization by recruiting fibroblast-like cells to the fibrous cap.
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ABSTRACT: JAK2 V617F (JAK2VF) clonal hematopoiesis (CH) has been associated with atherothrombotic cardiovascular disease (CVD). We assessed the impact of Jak2VF CH on arterial thrombosis and explored the underlying mechanisms. A meta-analysis of 3 large cohort studies confirmed the association of JAK2VF with CVD and with platelet counts and adjusted mean platelet volume (MPV). In mice, 20% or 1.5% Jak2VF CH accelerated arterial thrombosis and increased platelet activation. Megakaryocytes in Jak2VF CH showed elevated proplatelet formation and release, increasing prothrombogenic reticulated platelet counts. Gp1ba-Cre-mediated expression of Jak2VF in platelets (VFGp1ba) increased platelet counts to a similar level as in 20% Jak2VF CH mice while having no effect on leukocyte counts. Like Jak2VF CH mice, VFGp1ba mice showed enhanced platelet activation and accelerated arterial thrombosis. In Jak2VF CH, both Jak2VF and wild-type (WT) platelets showed increased activation, suggesting cross talk between mutant and WT platelets. Jak2VF platelets showed twofold to threefold upregulation of COX-1 and COX-2, particularly in young platelets, with elevated cPLA2 activation and thromboxane A2 production. Compared with controls, conditioned media from activated Jak2VF platelets induced greater activation of WT platelets that was reversed by a thromboxane receptor antagonist. Low-dose aspirin ameliorated carotid artery thrombosis in VFGp1ba and Jak2VF CH mice but not in WT control mice. This study shows accelerated arterial thrombosis and platelet activation in Jak2VF CH with a major role of increased reticulated Jak2VF platelets, which mediate thromboxane cross talk with WT platelets and suggests a potential beneficial effect of aspirin in JAK2VF CH.
Assuntos
Hematopoiese Clonal , Trombose , Animais , Humanos , Camundongos , Aspirina/farmacologia , Aspirina/uso terapêutico , Plaquetas/metabolismo , Camundongos Knockout , Ativação Plaquetária , Trombose/genética , Trombose/metabolismoRESUMO
Increased eosinophil counts are associated with cardiovascular disease and may be an independent predictor of major cardiovascular events. However, the causality and underlying mechanisms are poorly understood. GWAS have shown an association of a common LNK variant (R262W, T allele) with eosinophilia and atherothrombotic disorders. LNK(TT) reduces LNK function and Lnk-deficient mice display accelerated atherosclerosis and thrombosis. This study was undertaken to assess the role of eosinophils in arterial thrombosis in mice with hematopoietic Lnk deficiency. Hematopoietic Lnk deficiency increased circulating and activated eosinophils, JAK/STAT signaling in eosinophils and carotid arterial thrombosis with increased eosinophil abundance and extracellular trap formation (EETosis) in thrombi. Depletion of eosinophils by anti-Siglec-F antibody or by the ∆dbIGata1 mutation eliminated eosinophils in thrombi and markedly reduced thrombosis in mice with hematopoietic Lnk deficiency but not in control mice. Eosinophil depletion reduced neutrophil abundance and NETosis in thrombi without altering circulating neutrophil counts. To assess the role of Lnk specifically in eosinophils, we crossed Lnkf/f mice with eoCre mice. Lnk∆eos mice displayed isolated eosinophilia, increased eosinophil activation and accelerated arterial thrombosis associated with increased EETosis and NETosis in thrombi. DNase I infusion abolished EETs and NETs in thrombi and reversed the accelerated thrombosis. Human iPSC-derived LNK(TT) eosinophils showed increased activation and EETosis relative to isogenic LNK(CC) eosinophils, demonstrating human relevance. These studies show a direct link between eosinophilia, EETosis and atherothrombosis in hematopoietic Lnk deficiency and an essential role of eosinophil LNK in suppression of arterial thrombosis.
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BACKGROUND: Clonal hematopoiesis (CH) has emerged as an independent risk factor for atherosclerotic cardiovascular disease, with activation of macrophage inflammasomes as a potential underlying mechanism. The NLRP3 (NLR family pyrin domain containing 3) inflammasome has a key role in promoting atherosclerosis in mouse models of Tet2 CH, whereas inhibition of the inflammasome product interleukin-1ß appeared to particularly benefit patients with TET2 CH in CANTOS (Cardiovascular Risk Reduction Study [Reduction in Recurrent Major CV Disease Events]). TET2 is an epigenetic modifier that decreases promoter methylation. However, the mechanisms underlying macrophage NLRP3 inflammasome activation in TET2 (Tet methylcytosine dioxygenase 2) deficiency and potential links with epigenetic modifications are poorly understood. METHODS: We used cholesterol-loaded TET2-deficient murine and embryonic stem cell-derived isogenic human macrophages to evaluate mechanisms of NLRP3 inflammasome activation in vitro and hypercholesterolemic Ldlr-/- mice modeling TET2 CH to assess the role of NLRP3 inflammasome activation in atherosclerosis. RESULTS: Tet2 deficiency in murine macrophages acted synergistically with cholesterol loading in cell culture and with hypercholesterolemia in vivo to increase JNK1 (c-Jun N-terminal kinase 1) phosphorylation and NLRP3 inflammasome activation. The mechanism of JNK (c-Jun N-terminal kinase) activation in TET2 deficiency was increased promoter methylation and decreased expression of the JNK-inactivating dual-specificity phosphatase Dusp10. Active Tet1-deadCas9-targeted editing of Dusp10 promoter methylation abolished cholesterol-induced inflammasome activation in Tet2-deficient macrophages. Increased JNK1 signaling led to NLRP3 deubiquitylation and activation by the deubiquitinase BRCC3 (BRCA1/BRCA2-containing complex subunit 3). Accelerated atherosclerosis and neutrophil extracellular trap formation (NETosis) in Tet2 CH mice were reversed by holomycin, a BRCC3 deubiquitinase inhibitor, and also by hematopoietic deficiency of Abro1, an essential scaffolding protein in the BRCC3-containing cytosolic complex. Human TET2-/- macrophages displayed increased JNK1 and NLRP3 inflammasome activation, especially after cholesterol loading, with reversal by holomycin treatment, indicating human relevance. CONCLUSIONS: Hypercholesterolemia and TET2 deficiency converge on a common pathway of NLRP3 inflammasome activation mediated by JNK1 activation and BRCC3-mediated NLRP3 deubiquitylation, with potential therapeutic implications for the prevention of cardiovascular disease in TET2 CH.
Assuntos
Aterosclerose , Doenças Cardiovasculares , Dioxigenases , Hipercolesterolemia , Animais , Humanos , Camundongos , Aterosclerose/metabolismo , Colesterol/metabolismo , Hematopoiese Clonal , Enzimas Desubiquitinantes , Proteínas de Ligação a DNA/genética , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismoRESUMO
Clonal hematopoiesis (CH) increases the risk of atherosclerotic cardiovascular disease possibly due to increased plaque inflammation. Human studies suggest that limitation of interleukin-6 (IL-6) signaling could be beneficial in people with large CH clones, particularly in TET2 CH. Here we show that IL-6 receptor antibody treatment reverses the atherosclerosis promoted by Tet2 CH, with reduction of monocytosis, lesional macrophage burden and macrophage colony-stimulating factor 1 receptor (CSF1R) expression. IL-6 induces expression of Csf1r in Tet2-deficient macrophages through enhanced STAT3 binding to its promoter. In mouse and human Tet2-deficient macrophages, IL-6 increases CSF1R expression and enhances macrophage survival. Treatment with the CSF1R inhibitor PLX3397 reversed accelerated atherosclerosis in Tet2 CH mice. Our study demonstrates the causality of IL-6 signaling in Tet2 CH accelerated atherosclerosis, identifies IL-6-induced CSF1R expression as a critical mechanism and supports blockade of IL-6 signaling as a potential therapy for CH-driven cardiovascular disease.
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Clonal hematopoiesis of indeterminate potential (CHIP) is associated with an increased risk of cardiovascular diseases (CVDs), putatively via inflammasome activation. We pursued an inflammatory gene modifier scan for CHIP-associated CVD risk among 424,651 UK Biobank participants. We identified CHIP using whole-exome sequencing data of blood DNA and modeled as a composite, considering all driver genes together, as well as separately for common drivers (DNMT3A, TET2, ASXL1, and JAK2). We developed predicted gene expression scores for 26 inflammasome-related genes and assessed how they modify CHIP-associated CVD risk. We identified IL1RAP as a potential key molecule for CHIP-associated CVD risk across genes and increased AIM2 gene expression leading to heightened JAK2- and ASXL1-associated CVD risk. We show that CRISPR-induced Asxl1-mutated murine macrophages had a particularly heightened inflammatory response to AIM2 agonism, associated with an increased DNA damage response, as well as increased IL-10 secretion, mirroring a CVD-protective effect of IL10 expression in ASXL1 CHIP. Our study supports the role of inflammasomes in CHIP-associated CVD and provides evidence to support gene-specific strategies to address CHIP-associated CVD risk.
Assuntos
Doenças Cardiovasculares , Humanos , Animais , Camundongos , Doenças Cardiovasculares/genética , Hematopoiese Clonal/genética , Fatores de Risco , Inflamassomos/genética , Hematopoese/genética , Inflamação/genética , Inflamação/complicações , Fatores de Risco de Doenças Cardíacas , MutaçãoRESUMO
The CANTOS (Canakinumab Anti-inflammatory Thrombosis Outcome Study) and colchicine trials suggest an important role of inflammasomes and their major product IL-1ß (interleukin 1ß) in human atherosclerotic cardiovascular disease. Moreover, studies in mouse models indicate a causal role of inflammasomes and IL-1ß in atherosclerosis. However, recent studies have led to a more granular view of the role of inflammasomes in atherosclerosis. Studies in hyperlipidemic mouse models suggest that prominent activation of the NLRP3 inflammasome requires a second hit such as defective cholesterol efflux, defective DNA repair, clonal hematopoiesis or diabetes. Similarly in humans some mutations promoting clonal hematopoiesis increase coronary artery disease risk in part by promoting inflammasome activation. Recent studies in mice and humans point to a wider role of the AIM2 (absent in melanoma 2) inflammasome in promoting cardiovascular disease including in some forms of clonal hematopoiesis and diabetes. These developments suggest a precision medicine approach in which treatments targeting inflammasomes or IL-1ß might be best employed in clinical settings involving increased inflammasome activation.
Assuntos
Aterosclerose , Doenças Cardiovasculares , Trombose , Camundongos , Humanos , Animais , Inflamassomos/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Aterosclerose/genética , Interleucina-1betaRESUMO
Serum apolipoprotein C3 (APOC3) predicts incident cardiovascular events in people with type 1 diabetes, and silencing of APOC3 prevents both lesion initiation and advanced lesion necrotic core expansion in a mouse model of type 1 diabetes. APOC3 acts by slowing the clearance of triglyceride-rich lipoproteins, but lipid-free APOC3 has recently been reported to activate an inflammasome pathway in monocytes. We therefore investigated the contribution of hematopoietic inflammasome pathways to atherosclerosis in mouse models of type 1 diabetes. LDL receptor-deficient diabetes mouse models were transplanted with bone marrow from donors deficient in NOD, LRR and pyrin domain-containing protein 3 (NLRP3), absent in melanoma 2 (AIM2) or gasdermin D (GSDMD), an inflammasome-induced executor of pyroptotic cell death. Mice with diabetes exhibited inflammasome activation and consistently, increased plasma interleukin-1ß (IL-1ß) and IL-18. Hematopoietic deletions of NLRP3, AIM2, or GSDMD caused smaller atherosclerotic lesions in diabetic mice. The increased lesion necrotic core size in diabetic mice was independent of macrophage pyroptosis because hematopoietic GSDMD deficiency failed to prevent necrotic core expansion in advanced lesions. Our findings demonstrate that AIM2 and NLRP3 inflammasomes contribute to atherogenesis in diabetes and suggest that necrotic core expansion is independent of macrophage pyroptosis. ARTICLE HIGHLIGHTS: The contribution of hematopoietic cell inflammasome activation to atherosclerosis associated with type 1 diabetes is unknown. The goal of this study was to address whether hematopoietic NOD, LRR, and pyrin domain-containing protein 3 (NLRP3), absent in melanoma 2 (AIM2) inflammasomes, or the pyroptosis executioner gasdermin D (GSDMD) contributes to atherosclerosis in mouse models of type 1 diabetes. Diabetic mice exhibited increased inflammasome activation, with hematopoietic deletions of NLRP3, AIM2, or GSDMD causing smaller atherosclerotic lesions in diabetic mice, but the increased lesion necrotic core size in diabetic mice was independent of macrophage pyroptosis. Further studies on whether inflammasome activation contributes to cardiovascular complications in people with type 1 diabetes are warranted.
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Aterosclerose , Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1 , Melanoma , Camundongos , Animais , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Piroptose/fisiologia , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Experimental/complicações , Gasderminas , Camundongos Endogâmicos NOD , Necrose , Proteínas de TransporteAssuntos
Hematopoiese Clonal , AVC Isquêmico , Humanos , Hematopoiese Clonal/genética , Hematopoese/genética , MutaçãoRESUMO
AIMS: Neutrophil extracellular trap formation (NETosis) increases atherosclerotic plaque vulnerability and athero-thrombosis. However, mechanisms promoting NETosis during atherogenesis are poorly understood. We have shown that cholesterol accumulation due to myeloid cell deficiency of the cholesterol transporters ATP Binding Cassette A1 and G1 (ABCA1/G1) promotes NLRP3 inflammasome activation in macrophages and neutrophils and induces prominent NETosis in atherosclerotic plaques. We investigated whether NETosis is a cell-intrinsic effect in neutrophils or is mediated indirectly by cellular crosstalk from macrophages to neutrophils involving IL-1ß. METHODS AND RESULTS: We generated mice with neutrophil or macrophage-specific Abca1/g1 deficiency (S100A8CreAbca1fl/flAbcg1fl/fl or CX3CR1CreAbca1fl/flAbcg1fl/fl mice, respectively), and transplanted their bone marrow into low-density lipoprotein receptor knockout mice. We then fed the mice a cholesterol-rich diet. Macrophage, but not neutrophil Abca1/g1 deficiency activated inflammasomes in macrophages and neutrophils, reflected by caspase-1 cleavage, and induced NETosis in plaques. NETosis was suppressed by administering an interleukin (IL)-1ß neutralizing antibody. The extent of NETosis in plaques correlated strongly with the degree of neutrophil accumulation, irrespective of blood neutrophil counts, and neutrophil accumulation was decreased by IL-1ß antagonism. In vitro, IL-1ß or media transferred from Abca1/g1-deficient macrophages increased NETosis in both control and Abca1/Abcg1 deficient neutrophils. This cell-extrinsic effect of IL-1ß on NETosis was blocked by an NLRP3 inhibitor. CONCLUSION: These studies establish a new link between inflammasome-mediated IL-1ß production in macrophages and NETosis in atherosclerotic plaques. Macrophage-derived IL-1ß appears to increase NETosis both by increasing neutrophil recruitment to plaques and by promoting neutrophil NLRP3 inflammasome activation.
Assuntos
Placa Aterosclerótica , Camundongos , Animais , Placa Aterosclerótica/metabolismo , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Macrófagos/metabolismo , Colesterol/metabolismo , Camundongos KnockoutRESUMO
Clonal hematopoiesis arises from somatic mutations that provide a fitness advantage to hematopoietic stem cells and the outgrowth of clones of blood cells. Clonal hematopoiesis commonly involves mutations in genes that are involved in epigenetic modifications, signaling and DNA damage repair. Clonal hematopoiesis has emerged as a major independent risk factor in atherosclerotic cardiovascular disease, thrombosis and heart failure. Studies in mouse models of clonal hematopoiesis have shown an increase in atherosclerosis, thrombosis and heart failure, involving increased myeloid cell inflammatory responses and inflammasome activation. Although increased inflammatory responses have emerged as a common underlying principle, some recent studies indicate mutation-specific effects. The discovery of the association of clonal hematopoiesis with cardiovascular disease and the recent demonstration of benefit of anti-inflammatory treatments in human cardiovascular disease converge to suggest that anti-inflammatory treatments should be directed to individuals with clonal hematopoiesis. Such treatments could target specific inflammasomes, common downstream mediators such as IL-1ß and IL-6, or mutations linked to clonal hematopoiesis.
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We assessed the cross-sectional and longitudinal relationships of high-density lipoprotein (HDL)-mediated cholesterol mass efflux capacity (CMEC) with coronary artery calcium (CAC) score and CAC density. CMEC was measured in 1626 Multi-Ethnic Study of Atherosclerosis (MESA) participants in samples obtained between 2000 and 2002 as part of two nested case-control studies, one with cases of incident cardiovascular disease and the other with cases of carotid plaque progression by ultrasound. Cardiac CT examinations for the presence of CAC were performed at baseline and at two additional examinations. CAC scores (Agatston and volume) and density scores (for those with positive CAC) were calculated. Multivariable linear regression modeling per SD increment of CMEC was used to estimate the associations of CMEC with each of these CAC measures. We found no association between higher CMEC and either lower CAC scores or a higher CAC density. We also found no association between higher CMEC and progression of any of these CAC measures. These findings suggest that HDL-mediated cholesterol efflux may be associated with cardiovascular risk via mechanisms unrelated to burden of calcified plaque.
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Aterosclerose , Doença da Artéria Coronariana , Placa Aterosclerótica , Humanos , Cálcio , HDL-Colesterol , Vasos Coronários/diagnóstico por imagem , Estudos Transversais , Placa Aterosclerótica/diagnóstico por imagem , Fatores de RiscoRESUMO
Elevated hematocrit is associated with cardiovascular risk; however, the causality and mechanisms are unclear. The JAK2V617F (Jak2VF) mutation increases cardiovascular risk in myeloproliferative disorders and in clonal hematopoiesis. Jak2VF mice with elevated WBCs, platelets, and RBCs display accelerated atherosclerosis and macrophage erythrophagocytosis. To investigate whether selective erythroid Jak2VF expression promotes atherosclerosis, we developed hyperlipidemic erythropoietin receptor Cre mice that express Jak2VF in the erythroid lineage (VFEpoR mice). VFEpoR mice without elevated blood cell counts showed increased atherosclerotic plaque necrosis, erythrophagocytosis, and ferroptosis. Selective induction of erythrocytosis with low-dose erythropoietin further exacerbated atherosclerosis with prominent ferroptosis, lipid peroxidation, and endothelial damage. VFEpoR RBCs had reduced antioxidant defenses and increased lipid hydroperoxides. Phagocytosis of human or murine WT or JAK2VF RBCs by WT macrophages induced ferroptosis, which was prevented by the ferroptosis inhibitor liproxstatin-1. Liproxstatin-1 reversed increased atherosclerosis, lipid peroxidation, ferroptosis, and endothelial damage in VFEpoR mice and in Jak2VF chimeric mice simulating clonal hematopoiesis, but had no impact in controls. Erythroid lineage Jak2VF expression led to qualitative and quantitative defects in RBCs that exacerbated atherosclerosis. Phagocytosis of RBCs by plaque macrophages promoted ferroptosis, suggesting a therapeutic target for reducing RBC-mediated cardiovascular risk.
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Aterosclerose , Ferroptose , Linfo-Histiocitose Hemofagocítica , Placa Aterosclerótica , Animais , Aterosclerose/genética , Aterosclerose/metabolismo , Linhagem da Célula , Macrófagos/metabolismo , Camundongos , Fagocitose , Placa Aterosclerótica/metabolismoRESUMO
Efferocytosis, the clearance of apoptotic cells (ACs) by macrophages, is critical for tissue resolution, with defects driving many diseases. Mechanisms of efferocytosis-mediated resolution are incompletely understood. Here, we show that AC-derived methionine regulates resolution through epigenetic repression of the extracellular signal-regulated kinase 1/2 (ERK1/2) phosphatase Dusp4. We focus on two key efferocytosis-induced pro-resolving mediators, prostaglandin E2 (PGE2) and transforming growth factor beta 1 (TGF-ß1), and show that efferocytosis induces prostaglandin-endoperoxide synthase 2/cyclooxygenase 2 (Ptgs2/COX2), leading to PGE2 synthesis and PGE2-mediated induction of TGF-ß1. ERK1/2 phosphorylation/activation by AC-activated CD36 is necessary for Ptgs2 induction, but this is insufficient owing to an ERK-DUSP4 negative feedback pathway that lowers phospho-ERK. However, subsequent AC engulfment and phagolysosomal degradation lead to Dusp4 repression, enabling enhanced p-ERK and induction of the Ptgs2-PGE2-TGF-ß1 pathway. Mechanistically, AC-derived methionine is converted to S-adenosylmethionine, which is used by DNA methyltransferase-3A (DNMT3A) to methylate Dusp4. Bone-marrow DNMT3A deletion in mice blocks COX2/PGE2, TGF-ß1, and resolution in sterile peritonitis, apoptosis-induced thymus injury and atherosclerosis. Knowledge of how macrophages use AC-cargo and epigenetics to induce resolution provides mechanistic insight and therapeutic options for diseases driven by impaired resolution.
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DNA Metiltransferase 3A/metabolismo , Metionina , Fator de Crescimento Transformador beta1 , Animais , Apoptose , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Macrófagos/metabolismo , Metionina/metabolismo , Camundongos , Prostaglandinas E/metabolismo , Fator de Crescimento Transformador beta1/metabolismoRESUMO
BACKGROUND: Cholesterol loaded macrophage foam cells are a prominent feature of atherosclerotic plaques. Single-cell RNA sequencing has identified foam cells as TREM2 (triggering receptor expressed on myeloid cells 2) positive populations with low expression of inflammatory genes, resembling the TREM2 positive microglia of neurodegenerative diseases. Cholesterol loading of macrophages in vitro results in activation of LXR (liver X receptor) transcription factors and suppression of inflammatory genes. METHODS: To test the hypothesis that LXRs mediate anti-inflammatory effects in Trem2 expressing atherosclerotic plaque foam cells, we performed RNA profiling on plaque cells from hypercholesterolemic mice with myeloid LXR deficiency. RESULTS: Myeloid LXR deficiency led to a dramatic increase in atherosclerosis with increased monocyte entry, foam cell formation, and plaque inflammation. Bulk cell-RNA profiling of plaque myeloid cells showed prominent upregulation of inflammatory mediators including oxidative, chemokine, and chemotactic genes. Single-cell RNA sequencing revealed increased numbers of foamy TREM2-expressing macrophages; however, these cells had reduced expression of the Trem2 gene expression module, including phagocytic and cholesterol efflux genes, and had switched to a proinflammatory and proliferative phenotype. Expression of Trem2 was suppressed by inflammatory signals but not directly affected by LXR activation in bone marrow-derived macrophages. CONCLUSIONS: Our current studies reveal the key role of macrophage LXRs in promoting the Trem2 gene expression program and in suppressing inflammation in foam cells of atherosclerotic plaques.
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Aterosclerose , Placa Aterosclerótica , Animais , Aterosclerose/genética , Aterosclerose/metabolismo , Colesterol/metabolismo , Células Espumosas/metabolismo , Expressão Gênica , Inflamação/genética , Inflamação/metabolismo , Receptores X do Fígado/genética , Receptores X do Fígado/metabolismo , Macrófagos/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Placa Aterosclerótica/metabolismo , RNA , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismoRESUMO
Despite the success of LDL-lowering drugs in reducing cardiovascular disease (CVD), there remains a large burden of residual disease due in part to persistent dyslipidemia characterized by elevated levels of triglyceride-rich lipoproteins (TRLs) and reduced levels of HDL. This form of dyslipidemia is increasing globally as a result of the rising prevalence of obesity and metabolic syndrome. Accumulating evidence suggests that impaired hepatic clearance of cholesterol-rich TRL remnants leads to their accumulation in arteries, promoting foam cell formation and inflammation. Low levels of HDL may associate with reduced cholesterol efflux from foam cells, aggravating atherosclerosis. While fibrates and fish oils reduce TRL, they have not been uniformly successful in reducing CVD, and there is a large unmet need for new approaches to reduce remnants and CVD. Rare genetic variants that lower triglyceride levels via activation of lipolysis and associate with reduced CVD suggest new approaches to treating dyslipidemia. Apolipoprotein C3 (APOC3) and angiopoietin-like 3 (ANGPTL3) have emerged as targets for inhibition by antibody, antisense, or RNAi approaches. Inhibition of either molecule lowers TRL but respectively raises or lowers HDL levels. Large clinical trials of such agents in patients with high CVD risk and elevated levels of TRL will be required to demonstrate efficacy of these approaches.