Obstructive sleep apnea (OSA) is characterized by recurrent airway obstruction, leading to intermittent hypoxia (IH) and sleep fragmentation. OSA is highly prevalent, affecting approximately 1 billion people worldwide and has been associated with increased cardiovascular risk. However, the underlying mechanisms remain largely unknown. With the recent discovery of venous endothelial cells (vECs) in human atherosclerotic plaque, we hypothesized that unbiased profiling of vECs from healthy controls and patients with OSA would provide novel insights into mechanisms of OSA-mediated cardiovascular risk. Brachial vein ECs were harvested from 14 otherwise healthy patients with OSA (6F; mean±SD age 44±9y, body mass index [BMI] 35±7kg/m 2 , apnea-hypopnea index [AHI] 33 ±18/h) and 13 healthy controls (6F; mean±SD age 41±16y, BMI 28±4kg/m 2 , AHI 1.8 ±1.3/h) and subjected to single-nucleus RNA sequencing. Contactin-associated protein-like 3 ( CNTNAP3 ), a transmembrane protein involved in cell adhesion and intercellular communication, was downregulated in vECs from OSA patients compared with controls (log 2 FC=-0.89,P adj =5.76x10 -14 ). Notably, an inverse relationship between CNTNAP3 and AHI during non-rapid eye movement sleep has been found in GWAS, suggesting enrichment for reduced expression CNTNAP3 variants in blood vessels in OSA. Gene Set Enrichment Analysis reveals that patients with OSA have reduced cell junction assembly (NES=-1.6,P adj =3.5x10 -5 ) and organization (NES=-1.44,P adj =6.9x10 -5 ), blood vessel morphogenesis (NES=-1.40,P adj =0.001), and nitric oxide metabolic process (NES=-1.68,P adj =0.013). Venous-specific genes - FLRT2 (log 2 FC=-1.34,P adj =2.09x10 -15 ) and ARHGAP6 (log 2 FC=-0.71,P adj =4.99x10 -9 ) were downregulated in OSA patients compared with controls. Reduced expression of FLRT2, which has a key role in EC junction formation, was confirmed by qPCR in patients with OSA. Compared with normoxia, IH reduced FLRT2 expression by 40% in cultured vECs, suggesting that IH associated with transient airway obstructions in OSA impairs EC junction integrity in these patients. Consistently, we observed a vEC cluster enriched with these venous-specific genes in publicly available single-cell RNA sequencing datasets from human carotid atherosclerotic plaques. Our finding suggests that dysfunctional vECs that fail to maintain blood vessel integrity could play a role in IH-mediated atherosclerotic plaque instability, leading to increased cardiovascular risk in patients with OSA.

The Jak2V617F (Jak2VF) mutation is an important cause of both clonal hematopoiesis of indeterminate potential (CHIP) and myeloproliferative neoplasms (MPNs). Mouse models of Jak2VF CHIP and MPN show accelerated atherosclerosis progression, driven by macrophage inflammasome activation. We undertook the present study to assess the hypothesis that ongoing inflammation would impede atherosclerosis resolution in Jak2VF mice. Chimeric Jak2VF/WT or control WT/WT bone marrow was transplanted into Ldlr-/- mice and, following 13-16 weeks of western diet-induced atherosclerosis progression, cholesterol was lowered either moderately (to 200-300 mg/dl) or markedly (to 100 mg/dl). With moderate cholesterol lowering, there was impaired resolution of lesions in Jak2VF MPN mice compared to controls. However, with marked cholesterol lowering, progression of lesions was halted in both Jak2VF MPN and control mice while macrophage burden was decreased and lesional collagen was increased similarly in Jak2VF MPN and control mice. Two mechanisms of low-density lipoprotein (LDL) lowering-induced suppression of inflammation in plaques were implicated: 1) reversal of increased proliferation, DNA damage and absent in melanoma 2 (AIM2) inflammasome activation specifically in Jak2VF macrophages and 2) markedly increased macrophage triggering receptor expressed on myeloid cells 2 (TREM2), c-Myc expressing macrophages in both Jak2VF and control mice. In summary, aggressive LDL lowering reverses inflammasome activation and induces pro-resolving changes in macrophages in Jak2VF MPN, halting atherosclerosis progression and promoting features of plaque stabilization. These findings suggest that aggressive LDL cholesterol lowering could reverse atherosclerotic cardiovascular disease risk in individuals with JAK2VF CHIP or MPN.

Inflammasome activation (IA) has been identified as a potential therapeutic target in atherosclerotic cardiovascular disease (CVD). Clonal hematopoiesis (CH), involving somatic mutations in genes driving hematopoiesis, has emerged as an independent CVD risk factor. JAK2V617F (JAK2VF) is a CH variant that is increasingly recognized as a cause of myocardial infarction, ischemic stroke, and thoracic aortic aneurysm in humans. JAK2VF has been shown to promote atherosclerosis via increased macrophage IA. Ninjurin-1 (Ninj1) is a transmembrane protein that mediates plasma membrane rupture (PMR) downstream of IA and other programmed cell death. Ninj1-mediated PMR has been shown to promote aortic aneurysm and liver injury, but its role in atherosclerosis remains poorly understood. By generating a chimeric mouse model with and without hematopoietic Ninj1-deficency, we identified an atheroprotective role of Ninj1 in low allele burden Jak2VF CH but not control mice. Jak2VF exacerbates atherosclerosis by increasing lesion area (50x10 4 vs 40x10 4 mm 2 , p=0.04) and Ninj1-deficiency further worsened Jak2VF-mediated lesion progression by increased lesion area (64x10 4 vs 50x10 4 mm 2 , p=0.002) and necrotic core area (20x10 4 vs 13x10 4 mm 2 , p=0.006). Single-cell RNA sequencing analysis of the aortic intimal-medial layer from different chimeric mice revealed a landscape that was enriched with inflammatory myeloid cells under Jak2VF conditions, which was further exacerbated by Ninj1-deficiency. The deletion of Ninj1 increased the accumulation of inflammatory macrophages enriched with lipid transport (p=0.004) and iron ion transport (p=0.045) genes. Consistently, immunohistochemical staining of Jak2VF;Ninj1 -/- aortic root revealed increased cleaved-gasdermin D (p=0.04), a marker of pyroptosis, and transferrin receptor (p=0.03), a marker of ferroptosis. A similar pro-inflammatory macrophage population was identified in integrated single-cell RNA sequencing analysis of human carotid lesions. The population is also enriched with lipid transport (p=0.018) and iron ion transport genes (p=0.046). Our findings suggest that Ninj1-deficiency promotes atherosclerosis progression under Jak2VF conditions by sustaining pro-inflammatory cell death pathways, like pyroptosis and ferroptosis, and that therapeutic targeting of Ninj1 could potentially increase CVD risk.

Clonal hematopoiesis (CH) increases inflammasome-linked atherosclerosis, but the mechanisms by which CH mutant cells transmit inflammatory signals to nonmutant cells are largely unknown. To address this question, we transplanted 1.5% Jak2 V617F ( Jak2 VF ) bone marrow (BM) cells with 98.5% WT BM cells into hyperlipidemic Ldlr-/- mice. Low-allele-burden (LAB) mice showed accelerated atherosclerosis with increased features of plaque instability, decreased levels of the macrophage phagocytic receptors c-Mer tyrosine kinase (MERTK) and triggering receptor expressed on myeloid cells 2 (TREM2), and increased neutrophil extracellulartraps (NETs). These changes were reversed when Jak2VFBM was transplanted with Il1r1-/- BM. LAB mice with noncleavable MERTK in WT BM showed improvements in necrotic core and fibrous cap formation and reduced NETs. An agonistic TREM2 antibody (4D9) markedly increased fibrous caps in both control and LAB mice, eliminating the difference between the groups. Mechanistically, 4D9 increased TREM2+PDGFB+ macrophages and PDGF receptor-alpha+ fibroblast-like cells in the cap region. TREM2 and PDGFB mRNA levels were positively correlated in human carotid plaques and coexpressed in macrophages. In summary, low frequencies of Jak2VF mutations promoted atherosclerosis via IL-1 signaling from Jak2VFto WT macrophages and neutrophils, promoting cleavage of phagocytic receptors and features of plaque instability. Therapeutic approaches that stabilize MERTK or TREM2 could promote plaque stabilization, especially in CH- and inflammasome-driven atherosclerosis.

The process of arterial atherosclerosis is characterised by accumulation of lipids and fibrous material with accompanying inflammation. As plaques progress, they restrict blood flow and cause rupture, which results in life threatening organ ischemia and dysfunction. Although extensively studied, a clear understanding of plaque heterogeneity and mechanisms that trigger their destabilization remains elusive. Our study reveals the molecular microarchitecture of human carotid artery plaques, using bulk and single-cell RNA sequencing combined with single-cell spatial transcriptomics, for which we present optimized cell segmentation algorithms. We identified distinct plaque morphologies linked to different cell type compositions, impacting early and advanced lesion formation, as well as destabilization. Spatial transcriptomics enabled us to further determine an inflammatory smooth muscle cell subtype, localize regions of neovascularization, and assign hotspots for macrophage activity within distinct cellular neighbourhoods across lesions. For different macrophage substates, we propose gradual and locally contained transdifferentiation of subluminal inflammatory HMOX1+ macrophages into a lipid-handling TREM2+ phenotype within border zones of the fibrous cap and necrotic core. Our findings provide insight into the complex heterogeneity of human atherosclerosis by unravelling location and proximity of different mural and immune cell substates involved in plaque progression and vulnerability.

Background:Inflammasome activation promotes atherosclerosis in clonal hematopoiesis (CH). Active inflammasomes secrete both IL-1β and IL-18. Plasma IL-18 levels are elevated in Jak2 VF CH. Genetic deficiency of IL-18 has been shown to reduce atherosclerosis in non-CH murine models. However, whether IL-18 inhibition promotes atherosclerosis in control or Jak2 VF CH is unknown. Methods and results:Ldlr -/- mice were transplanted with bone marrow (BM) from Mx1-cre Jak2 VF (20%) and wild-type (80%) mice or with control BM, fed a Western-type diet (WTD) for 8, 10 or 16 weeks and administered control or IL-18 IgG from 4 weeks onwards. IL-18 antibody treatment increased plaque collagen content and cap thickness. Unexpectedly, IL-18 antibody treatment increased the size of early lesions and promoted formation of advanced lesions with large necrotic cores in Jak2 VF CH mice. IL-18 antibody treatment was associated with diminished interferon (IFN)-γ and AIM2 levels and reduced macrophage pyroptosis especially in Jak2 VF CH mice. However, IL-18 antibodies increased cleaved Caspase-3 and TUNEL + macrophages (indicating increased apoptosis) and reduced efferocytosis. Sc-RNA-seq analysis showed that IL-18 antibody treatment reduced expression of MHC class II genes, a marker of IFN-γ signaling, and of genes mediating efferocytosis ( Mertk and Axl) , in resident-like macrophage subpopulations in Jak2 VF CH mice. Consistently, IFN-γ injection increased Axl and Mertk expression in resident peritoneal macrophages. Conclusions:Despite improvements in collagen and fibrous cap thickness in Jak2 VF CH mice, IL-18 antibody treatment increased advanced necrotic lesions, reflecting a shift from pyroptotic to apoptotic cell death coupled with defective efferocytosis, events which were coordinated by reduced IFN-γ signaling. These findings indicate a mixed atherosclerosis phenotype resulting from IL-18 inhibition, advocating for alternative therapeutic strategies. Inhibition of IL-18 has been considered as a novel therapeutic approach to reduce atherosclerosis and stabilize atherosclerotic plaques. We show that IL-18 antibodies have adverse effects on atherosclerotic lesional necrosis, calling this approach into question. Highlights:Inflammasome activation produces active IL-1 and IL-18 and worsens atherosclerosis in clonal hematopoiesis (CH) however the contribution of IL-18 is unknown. Antibody inhibition of IL-18 increased plaque collagen but also increased early lesion area and late lesions with large necrotic cores in Jak2 VF CH mice. There was a reversal of AIM2 inflammasome activation but a switch to apoptosis which along with reduced efferocytosis increased necrosisThese events appeared to be coordinated by reduced IFN-γ which increased collagen but also decreased expression of efferocytotic genes. Our studies call into question whether inhibition of IL-18 would stabilize plaques in CH.

Background:Vascular smooth muscle cells (VSMCs) play a central role in atherosclerosis by undergoing phenotypic modulation from a quiescent, contractile state to a range of synthetic phenotypes, including fibroblast-like, macrophage-like, and lipid-laden foam cell-like states. However, a comprehensive multimodal characterization and understanding of the transcriptional programs driving these transitions remain incomplete. Methods:To comprehensively define the phenotypic diversity of VSMCs during atherosclerosis progression, we performed in-depth profiling using cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) and bulk RNA sequencing in a VSMC lineage-tracing atherosclerotic mouse model. Insights from these datasets guided the design of targeted in vitro experiments to investigate candidate regulatory mechanisms. Results:Single-cell multi-omics revealed extensive cellular heterogeneity within atherosclerotic plaques, including a rare population of VSMC-derived macrophage-like cells, whose presence was confirmed by histological analysis. These studies also identified a substantial population of VSMC-derived foam cells, comprising approximately 70% of all foam cells in the lesions. These cells exhibited activation of gene programs associated with lipid metabolism, proliferation, and tumor-like features. The transcription factor Bhlhe40 emerged as a key regulator of this phenotypic transition, with elevated expression in VSMC-derived foam cells during disease progression. Functional knockdown of Bhlhe40 suppressed VSMC phenotypic switching and foam cell characteristics, underscoring its potential role as a driver of VSMC modulation. Conclusions:These findings advance our understanding of VSMC phenotypic modulation in atherosclerosis and highlight Bhlhe40 as a key regulator of this process. Elucidating the mechanisms governing VSMC plasticity may offer new therapeutic opportunities to reduce cardiovascular risk by targeting disease-driving cellular transitions.

Older men with loss of the Y chromosome are more susceptible to heart failure but the responsible genes have not been identified. A study now shows that loss of a single Y chromosome gene in bone marrow cells induces heart failure by switching cardiac macrophages from an inflammatory to a fibrogenic pattern of gene activity.

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-1β 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.

Introduction: Enhancing macrophage efferocytosis represents a potential therapeutic strategy for residual risk reduction in atherosclerotic cardiovascular disease. Our genome-wide CRISPR screening discovered Pdcd6ip as a top-ranked negative regulator of efferocytosis. PDCD6IP, also known as ALIX, is an accessory protein of ESCRT machinery, regulating multivesicular body formation and cytokinesis. Yet, how PDCD6IP may act as a molecular break on macrophage efferocytosis has not been characterized. Goals: Determine if knockout of Pdcd6ip enhances macrophage efferocytosis and protects against atherosclerosis pathogenesis. Methods: In vitro efferocytosis assays were performed in bone marrow-derived macrophages (BMDMs) and human monocyte-derived macrophages (HMDM). In vivo studies involved Ldlr -/- recipient mice with bone marrow transplantation (BMT) of WT and Pdcd6ip -/- BMs. Results: Pdcd6ip -/- induces cytokinesis arrest in proliferating BMDMs, resulting in an increased formation of binuclear polyploid BMDMs. Binuclear BMDMs have a remarkable capacity to engulf multiple apoptotic cells (ACs), termed continuing efferocytosis. While both mononuclear and binuclear Pdcd6ip -/- BMDMs demonstrate enhanced efferocytosis in primary efferocytosis, binuclear Pdcd6ip -/- BMDMs show enhanced continuing efferocytosis compared to binuclear WT BMDMs. Pdcd6ip -/- BMDMs have increased phagolysosomal acidification of the engulfed ACs, implicating proper degradation. Pdcd6ip -/- did not alter the engulfment of live cells, beads, or zymosan, supporting specific regulation of AC clearance. We hypothesize that Pdcd6ip -/- will be protective in atherosclerosis. Indeed, BMT of Pdcd6ip -/- BMs into Ldlr -/- mice led to increased atherosclerotic plaque stability after 22 weeks of Western diet feeding, characterized by decreased necrotic core area and increased fibrous cap thickness, without affecting the overall lesion area. Knockdown of PDCD6IP in HMDMs also led to an increased formation of binuclear HMDMs and enhanced continuing efferocytosis. Conclusion: We discovered a new role of PDCD6IP as a molecular break of macrophage efferocytosis. Inhibiting macrophage PDCD6IP may represent a therapeutic opportunity in atherosclerosis.

Clonal hematopoiesis (CH) is an independent risk factor for atherosclerotic cardiovascular disease. Murine models of CH suggest a central role of inflammasomes and IL-1β 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-1β 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-1β 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-1β 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. Fidler et al. show that anti-IL-1β treatment of atherosclerotic Ldlr-null mice with clonal expansion of Tet2-null or Jak2VF hematopoietic stem cells promotes the recruitment of fibroblast-like cells to the plaque fibrous cap, leading to cap thickening and increased plaque stability. Conversely, the removal of fibroblast-like cells during atherosclerosis progression results in reduced fibrous cap formation in mice receiving anti-IL-1β antibody treatment.

ABSTRACT: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. Genome-wide association studies 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 ΔdbIGata1 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. LnkΔeos 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 neutrophil extracellular traps (NETs) in thrombi and reversed the accelerated thrombosis. Human induced pluripotent stem cell-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.

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 trafficking of cholesterol to the ER, NLRP3 deubiquitylation, inflammasome activation and atherosclerosis.

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-1β 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.

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-1b (IL-1b) 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.

The CANTOS (Canakinumab Anti-inflammatory Thrombosis Outcome Study) and colchicine trials suggest an important role of inflammasomes and their major product IL-1β (interleukin 1β) in human atherosclerotic cardiovascular disease. Moreover, studies in mouse models indicate a causal role of inflammasomes and IL-1β 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-1β might be best employed in clinical settings involving increased inflammasome activation.

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-1β.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)-1β 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-1β antagonism. In vitro, IL-1β or media transferred from Abca1/g1-deficient macrophages increased NETosis in both control and Abca1/Abcg1 deficient neutrophils. This cell-extrinsic effect of IL-1β on NETosis was blocked by an NLRP3 inhibitor.CONCLUSION:These studies establish a new link between inflammasome-mediated IL-1β production in macrophages and NETosis in atherosclerotic plaques. Macrophage-derived IL-1β appears to increase NETosis both by increasing neutrophil recruitment to plaques and by promoting neutrophil NLRP3 inflammasome activation.