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Research ArticleVascular biology
Open Access | 
10.1172/JCI182939
1Division of Molecular Medicine, Department of Medicine, and
2Division of Cardiology, Department of Medicine, Columbia University, New York, New York, USA.
3Institute of Molecular Vascular Medicine, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany.
4German Center for Cardiovascular Research (DZHK), partner site Munich Heart Alliance, Germany.
5Department of Medicine, Karolinska Institute, Stockholm, Sweden.
6German Center for Neurodegenerative Diseases (DZNE), Munich, Germany.
7Metabolic Biochemistry, Biomedical Center, Faculty of Medicine, Ludwig-Maximilians University, Munich, Germany.
8Munich Cluster for Systems Neurology (SyNergy), Munich, Germany.
Address correspondence to: Wenli Liu or Alan R. Tall, Department of Medicine, Columbia University, 630W 168TH P&S building 8-401, New York, New York, 10032, USA. Phone: 212.305.3263; Email: wl2608@cumc.columbia.edu (WL); Phone: 212.305.9418. Email: art1@cumc.columbia.edu (ART).
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1Division of Molecular Medicine, Department of Medicine, and
2Division of Cardiology, Department of Medicine, Columbia University, New York, New York, USA.
3Institute of Molecular Vascular Medicine, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany.
4German Center for Cardiovascular Research (DZHK), partner site Munich Heart Alliance, Germany.
5Department of Medicine, Karolinska Institute, Stockholm, Sweden.
6German Center for Neurodegenerative Diseases (DZNE), Munich, Germany.
7Metabolic Biochemistry, Biomedical Center, Faculty of Medicine, Ludwig-Maximilians University, Munich, Germany.
8Munich Cluster for Systems Neurology (SyNergy), Munich, Germany.
Address correspondence to: Wenli Liu or Alan R. Tall, Department of Medicine, Columbia University, 630W 168TH P&S building 8-401, New York, New York, 10032, USA. Phone: 212.305.3263; Email: wl2608@cumc.columbia.edu (WL); Phone: 212.305.9418. Email: art1@cumc.columbia.edu (ART).
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1Division of Molecular Medicine, Department of Medicine, and
2Division of Cardiology, Department of Medicine, Columbia University, New York, New York, USA.
3Institute of Molecular Vascular Medicine, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany.
4German Center for Cardiovascular Research (DZHK), partner site Munich Heart Alliance, Germany.
5Department of Medicine, Karolinska Institute, Stockholm, Sweden.
6German Center for Neurodegenerative Diseases (DZNE), Munich, Germany.
7Metabolic Biochemistry, Biomedical Center, Faculty of Medicine, Ludwig-Maximilians University, Munich, Germany.
8Munich Cluster for Systems Neurology (SyNergy), Munich, Germany.
Address correspondence to: Wenli Liu or Alan R. Tall, Department of Medicine, Columbia University, 630W 168TH P&S building 8-401, New York, New York, 10032, USA. Phone: 212.305.3263; Email: wl2608@cumc.columbia.edu (WL); Phone: 212.305.9418. Email: art1@cumc.columbia.edu (ART).
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1Division of Molecular Medicine, Department of Medicine, and
2Division of Cardiology, Department of Medicine, Columbia University, New York, New York, USA.
3Institute of Molecular Vascular Medicine, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany.
4German Center for Cardiovascular Research (DZHK), partner site Munich Heart Alliance, Germany.
5Department of Medicine, Karolinska Institute, Stockholm, Sweden.
6German Center for Neurodegenerative Diseases (DZNE), Munich, Germany.
7Metabolic Biochemistry, Biomedical Center, Faculty of Medicine, Ludwig-Maximilians University, Munich, Germany.
8Munich Cluster for Systems Neurology (SyNergy), Munich, Germany.
Address correspondence to: Wenli Liu or Alan R. Tall, Department of Medicine, Columbia University, 630W 168TH P&S building 8-401, New York, New York, 10032, USA. Phone: 212.305.3263; Email: wl2608@cumc.columbia.edu (WL); Phone: 212.305.9418. Email: art1@cumc.columbia.edu (ART).
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1Division of Molecular Medicine, Department of Medicine, and
2Division of Cardiology, Department of Medicine, Columbia University, New York, New York, USA.
3Institute of Molecular Vascular Medicine, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany.
4German Center for Cardiovascular Research (DZHK), partner site Munich Heart Alliance, Germany.
5Department of Medicine, Karolinska Institute, Stockholm, Sweden.
6German Center for Neurodegenerative Diseases (DZNE), Munich, Germany.
7Metabolic Biochemistry, Biomedical Center, Faculty of Medicine, Ludwig-Maximilians University, Munich, Germany.
8Munich Cluster for Systems Neurology (SyNergy), Munich, Germany.
Address correspondence to: Wenli Liu or Alan R. Tall, Department of Medicine, Columbia University, 630W 168TH P&S building 8-401, New York, New York, 10032, USA. Phone: 212.305.3263; Email: wl2608@cumc.columbia.edu (WL); Phone: 212.305.9418. Email: art1@cumc.columbia.edu (ART).
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1Division of Molecular Medicine, Department of Medicine, and
2Division of Cardiology, Department of Medicine, Columbia University, New York, New York, USA.
3Institute of Molecular Vascular Medicine, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany.
4German Center for Cardiovascular Research (DZHK), partner site Munich Heart Alliance, Germany.
5Department of Medicine, Karolinska Institute, Stockholm, Sweden.
6German Center for Neurodegenerative Diseases (DZNE), Munich, Germany.
7Metabolic Biochemistry, Biomedical Center, Faculty of Medicine, Ludwig-Maximilians University, Munich, Germany.
8Munich Cluster for Systems Neurology (SyNergy), Munich, Germany.
Address correspondence to: Wenli Liu or Alan R. Tall, Department of Medicine, Columbia University, 630W 168TH P&S building 8-401, New York, New York, 10032, USA. Phone: 212.305.3263; Email: wl2608@cumc.columbia.edu (WL); Phone: 212.305.9418. Email: art1@cumc.columbia.edu (ART).
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1Division of Molecular Medicine, Department of Medicine, and
2Division of Cardiology, Department of Medicine, Columbia University, New York, New York, USA.
3Institute of Molecular Vascular Medicine, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany.
4German Center for Cardiovascular Research (DZHK), partner site Munich Heart Alliance, Germany.
5Department of Medicine, Karolinska Institute, Stockholm, Sweden.
6German Center for Neurodegenerative Diseases (DZNE), Munich, Germany.
7Metabolic Biochemistry, Biomedical Center, Faculty of Medicine, Ludwig-Maximilians University, Munich, Germany.
8Munich Cluster for Systems Neurology (SyNergy), Munich, Germany.
Address correspondence to: Wenli Liu or Alan R. Tall, Department of Medicine, Columbia University, 630W 168TH P&S building 8-401, New York, New York, 10032, USA. Phone: 212.305.3263; Email: wl2608@cumc.columbia.edu (WL); Phone: 212.305.9418. Email: art1@cumc.columbia.edu (ART).
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1Division of Molecular Medicine, Department of Medicine, and
2Division of Cardiology, Department of Medicine, Columbia University, New York, New York, USA.
3Institute of Molecular Vascular Medicine, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany.
4German Center for Cardiovascular Research (DZHK), partner site Munich Heart Alliance, Germany.
5Department of Medicine, Karolinska Institute, Stockholm, Sweden.
6German Center for Neurodegenerative Diseases (DZNE), Munich, Germany.
7Metabolic Biochemistry, Biomedical Center, Faculty of Medicine, Ludwig-Maximilians University, Munich, Germany.
8Munich Cluster for Systems Neurology (SyNergy), Munich, Germany.
Address correspondence to: Wenli Liu or Alan R. Tall, Department of Medicine, Columbia University, 630W 168TH P&S building 8-401, New York, New York, 10032, USA. Phone: 212.305.3263; Email: wl2608@cumc.columbia.edu (WL); Phone: 212.305.9418. Email: art1@cumc.columbia.edu (ART).
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1Division of Molecular Medicine, Department of Medicine, and
2Division of Cardiology, Department of Medicine, Columbia University, New York, New York, USA.
3Institute of Molecular Vascular Medicine, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany.
4German Center for Cardiovascular Research (DZHK), partner site Munich Heart Alliance, Germany.
5Department of Medicine, Karolinska Institute, Stockholm, Sweden.
6German Center for Neurodegenerative Diseases (DZNE), Munich, Germany.
7Metabolic Biochemistry, Biomedical Center, Faculty of Medicine, Ludwig-Maximilians University, Munich, Germany.
8Munich Cluster for Systems Neurology (SyNergy), Munich, Germany.
Address correspondence to: Wenli Liu or Alan R. Tall, Department of Medicine, Columbia University, 630W 168TH P&S building 8-401, New York, New York, 10032, USA. Phone: 212.305.3263; Email: wl2608@cumc.columbia.edu (WL); Phone: 212.305.9418. Email: art1@cumc.columbia.edu (ART).
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1Division of Molecular Medicine, Department of Medicine, and
2Division of Cardiology, Department of Medicine, Columbia University, New York, New York, USA.
3Institute of Molecular Vascular Medicine, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany.
4German Center for Cardiovascular Research (DZHK), partner site Munich Heart Alliance, Germany.
5Department of Medicine, Karolinska Institute, Stockholm, Sweden.
6German Center for Neurodegenerative Diseases (DZNE), Munich, Germany.
7Metabolic Biochemistry, Biomedical Center, Faculty of Medicine, Ludwig-Maximilians University, Munich, Germany.
8Munich Cluster for Systems Neurology (SyNergy), Munich, Germany.
Address correspondence to: Wenli Liu or Alan R. Tall, Department of Medicine, Columbia University, 630W 168TH P&S building 8-401, New York, New York, 10032, USA. Phone: 212.305.3263; Email: wl2608@cumc.columbia.edu (WL); Phone: 212.305.9418. Email: art1@cumc.columbia.edu (ART).
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1Division of Molecular Medicine, Department of Medicine, and
2Division of Cardiology, Department of Medicine, Columbia University, New York, New York, USA.
3Institute of Molecular Vascular Medicine, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany.
4German Center for Cardiovascular Research (DZHK), partner site Munich Heart Alliance, Germany.
5Department of Medicine, Karolinska Institute, Stockholm, Sweden.
6German Center for Neurodegenerative Diseases (DZNE), Munich, Germany.
7Metabolic Biochemistry, Biomedical Center, Faculty of Medicine, Ludwig-Maximilians University, Munich, Germany.
8Munich Cluster for Systems Neurology (SyNergy), Munich, Germany.
Address correspondence to: Wenli Liu or Alan R. Tall, Department of Medicine, Columbia University, 630W 168TH P&S building 8-401, New York, New York, 10032, USA. Phone: 212.305.3263; Email: wl2608@cumc.columbia.edu (WL); Phone: 212.305.9418. Email: art1@cumc.columbia.edu (ART).
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1Division of Molecular Medicine, Department of Medicine, and
2Division of Cardiology, Department of Medicine, Columbia University, New York, New York, USA.
3Institute of Molecular Vascular Medicine, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany.
4German Center for Cardiovascular Research (DZHK), partner site Munich Heart Alliance, Germany.
5Department of Medicine, Karolinska Institute, Stockholm, Sweden.
6German Center for Neurodegenerative Diseases (DZNE), Munich, Germany.
7Metabolic Biochemistry, Biomedical Center, Faculty of Medicine, Ludwig-Maximilians University, Munich, Germany.
8Munich Cluster for Systems Neurology (SyNergy), Munich, Germany.
Address correspondence to: Wenli Liu or Alan R. Tall, Department of Medicine, Columbia University, 630W 168TH P&S building 8-401, New York, New York, 10032, USA. Phone: 212.305.3263; Email: wl2608@cumc.columbia.edu (WL); Phone: 212.305.9418. Email: art1@cumc.columbia.edu (ART).
Find articles by Schlepckow, K. in: JCI | PubMed | Google Scholar
1Division of Molecular Medicine, Department of Medicine, and
2Division of Cardiology, Department of Medicine, Columbia University, New York, New York, USA.
3Institute of Molecular Vascular Medicine, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany.
4German Center for Cardiovascular Research (DZHK), partner site Munich Heart Alliance, Germany.
5Department of Medicine, Karolinska Institute, Stockholm, Sweden.
6German Center for Neurodegenerative Diseases (DZNE), Munich, Germany.
7Metabolic Biochemistry, Biomedical Center, Faculty of Medicine, Ludwig-Maximilians University, Munich, Germany.
8Munich Cluster for Systems Neurology (SyNergy), Munich, Germany.
Address correspondence to: Wenli Liu or Alan R. Tall, Department of Medicine, Columbia University, 630W 168TH P&S building 8-401, New York, New York, 10032, USA. Phone: 212.305.3263; Email: wl2608@cumc.columbia.edu (WL); Phone: 212.305.9418. Email: art1@cumc.columbia.edu (ART).
Find articles by Haass, C. in: JCI | PubMed | Google Scholar
1Division of Molecular Medicine, Department of Medicine, and
2Division of Cardiology, Department of Medicine, Columbia University, New York, New York, USA.
3Institute of Molecular Vascular Medicine, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany.
4German Center for Cardiovascular Research (DZHK), partner site Munich Heart Alliance, Germany.
5Department of Medicine, Karolinska Institute, Stockholm, Sweden.
6German Center for Neurodegenerative Diseases (DZNE), Munich, Germany.
7Metabolic Biochemistry, Biomedical Center, Faculty of Medicine, Ludwig-Maximilians University, Munich, Germany.
8Munich Cluster for Systems Neurology (SyNergy), Munich, Germany.
Address correspondence to: Wenli Liu or Alan R. Tall, Department of Medicine, Columbia University, 630W 168TH P&S building 8-401, New York, New York, 10032, USA. Phone: 212.305.3263; Email: wl2608@cumc.columbia.edu (WL); Phone: 212.305.9418. Email: art1@cumc.columbia.edu (ART).
Find articles by Wang, N. in: JCI | PubMed | Google Scholar
1Division of Molecular Medicine, Department of Medicine, and
2Division of Cardiology, Department of Medicine, Columbia University, New York, New York, USA.
3Institute of Molecular Vascular Medicine, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany.
4German Center for Cardiovascular Research (DZHK), partner site Munich Heart Alliance, Germany.
5Department of Medicine, Karolinska Institute, Stockholm, Sweden.
6German Center for Neurodegenerative Diseases (DZNE), Munich, Germany.
7Metabolic Biochemistry, Biomedical Center, Faculty of Medicine, Ludwig-Maximilians University, Munich, Germany.
8Munich Cluster for Systems Neurology (SyNergy), Munich, Germany.
Address correspondence to: Wenli Liu or Alan R. Tall, Department of Medicine, Columbia University, 630W 168TH P&S building 8-401, New York, New York, 10032, USA. Phone: 212.305.3263; Email: wl2608@cumc.columbia.edu (WL); Phone: 212.305.9418. Email: art1@cumc.columbia.edu (ART).
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Published November 12, 2024 - More info
Published in Volume 135, Issue 1 on January 2, 2025
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Abstract
Clonal hematopoiesis (CH) is a condition in which hematopoietic stem cells (HSCs) acquire mutations seen in leukemia. While individuals with CH generally do not show signs of hematologic disease, the condition becomes more common with age and correlates with age-related diseases, especially cardiovascular disease (CVD). JAK2 mutations in HSCs can lead to CH and correlate with atherosclerosis, but the condition has been difficult to study because of challenges modeling the mutant cells at very low frequency. In this issue of the JCI, Liu et al. developed a low-allele-burden (LAB) mouse model in which a small number of bone marrow cells carrying the Jak2VF mutation were transplanted into mice predisposed to hyperlipidemia. Along with recapitulating features of plaque development, the authors identified the phagocytic receptors MERTK and TREM2 in WT cells as downstream of the inflammatory cytokine IL-1. These findings provide potential targets for preventing or treating patients at risk for CH-associated CVD.
Authors
× AbstractClonal 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% Jak2V617F (Jak2VF) 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 extracellular traps (NETs). These changes were reversed when Jak2VF BM 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-α+ 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 Jak2VF to 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.
Graphical Abstract
Introduction
Although LDL-lowering treatments have been proven effective, atherosclerotic cardiovascular disease (CVD) remains the leading cause of morbidity and mortality in the United States, accounting for 28% of all deaths (1). Despite achieving considerable reductions in LDL cholesterol in clinical studies (2–4), a significant residual cardiovascular risk remains, highlighting the need for potential treatment alternatives. Recent clinical trials utilizing antiinflammatory therapies, especially IL-1β antibodies (5) or colchicine (6), have demonstrated a decrease in CVD events. However, these treatments were associated with an increased risk of infections. IL-1β antibodies remain unapproved for CVD indications, and although colchicine was recently approved by the FDA, its therapeutic potential may be limited (7). This indicates an urgent need for additional antiinflammatory treatments targeted for patients with high levels of inflammatory risk, identified either by biomarkers or by genetic factors.
Clonal hematopoiesis (CH) has recently been identified as an important genetic risk factor for CVD (8). This condition results from somatic mutations in leukemogenic genes, providing a survival advantage to hematopoietic stem cells and causing the clonal expansion of blood cells (9, 10). While CH is associated with an increased risk of hematological malignancy, it is much more commonly associated with coronary heart disease, premature myocardial infarction, thrombotic stroke, and heart failure (8, 11, 12). CH of indeterminate potential (CHIP) is identified by the existence of a leukemogenic gene mutation exhibiting a variant allele frequency (VAF) of no less than 2% in peripheral blood, without clinical indications of hematological malignancy (13). Targeted and deeper DNA sequencing methods reveal higher frequencies of CH mutations (14). CVD risk associated with TET2 and DNMT3A CHIP increases with VAF and is only significantly increased at a VAF of greater than 10% (15); however, detection sensitivity and accuracy may have been limited (16). Targeted sequencing has shown progression of heart failure in CH at a VAF of less than 5% (17, 18). An increase in incident coronary heart disease events in CH at a VAF of 0.5%–2% has recently been reported in East Asian cohorts (19).
JAK2V617F (JAK2VF) increases hematopoietic cytokine signaling and is a frequent driver mutation in myeloproliferative neoplasms (MPNs) (20). JAK2VF CH, while less common than DNMT3A, TET2, or ASXL1 CH, shows the strongest association with CVD. In a high-risk population, the increased CVD risk for JAK2VF was 12-fold (8). In a recent meta-analysis of relatively healthy individuals in the United Kingdom Biobank, Mass General Brigham Biobank, and all of the US Biobank, CH overall significantly increased CVD risk by approximately 1.1-fold, whereas the increased risk for JAK2VF was 2-fold. The increased CVD risk was also found in individuals without overt MPN or altered blood cell counts (13, 21). While formerly considered rare, Jak2VF has been detected in 3.1% of a general European population using digital droplet PCR (22) and associates with thrombotic risk at low allele burden (LAB) (2%) (22, 23). In the European population, the JAK2VF mutation was found in 11% of patients with ischemic stroke versus 4% in matched controls (24). JAK2VF is present at similar frequencies in men and women and in different ethnic groups in the US population (24, 25). Thus, JAK2VF is emerging as an important CVD risk factor. However, the effect of low VAF JAK2VF on atherosclerosis remains poorly understood.
Although macrophages play a key role in the progression of atherosclerosis, distinct macrophage populations that express phagocytic receptors may have antiinflammatory, pro-resolving properties (26). For example, c-Mer tyrosine kinase (MERTK), an efferocytosis receptor, reduces necrotic core formation and increases fibrous caps in advanced plaques (27). griggering receptor expressed on myeloid cells 2 (TREM2) is a lipid/lipoprotein-activated phagocytic molecule that promotes microglial or macrophage viability and expression of genes involved in efferocytosis, lysosomal catabolism, and cholesterol efflux (28, 29). Trem2hi macrophages take up modified LDL, become foam cells with low expression of inflammatory genes, and have a potential beneficial role in atherogenesis (30–33). The goals of the present study were to assess atherogenesis in a hyperlipidemic, LAB model of Jak2VF, then to define the mechanisms of accelerated atherosclerosis, especially those involving IL-1β–mediated crosstalk from mutant to WT myeloid cells that led to reduced levels of MERTK and TREM2. We showed that interventions that stabilized these receptors produced improvements in features of plaque stability, establishing causation and suggesting alternative therapeutic interventions.
ResultsLAB Jak2VF CH increases atherosclerosis and features of plaque instability. We recently developed a LAB model of Jak2VF CH by mixing 1.5% Jak2VF bone marrow (BM) with 98.5% WT BM and transplantation into LDL receptor–deficient (Ldlr–/–) mice. These mice displayed accelerated FeCl3-stimulated carotid artery thrombosis (25). To assess the effect of LAB Jak2VF CH on atherosclerosis, we transplanted 1.5% Jak2VF mixed with 98.5% GFP+ WT BM cells into lethally irradiated Ldlr–/– female mice (Figure 1A). After recovery, mice were fed a Western diet (WD) for 16 weeks to promote advanced atherosclerotic lesions (Figure 1A). Similar to an earlier study (25), we found that blood cell counts were unchanged, and there was no expansion of Jak2VF alleles within WBCs, neutrophils, monocytes, or monocyte subsets (Supplemental Figure 1, A and B; supplemental material available online with this article; https://doi.org/10.1172/JCI182939DS1). In striking contrast to higher VAF mice (34), plasma cholesterol levels were identical between the groups (Figure 1B), probably reflecting the lack of splenomegaly that accelerates LDL degradation in MPNs (35). The atherosclerotic lesion area in the LAB Jak2VF CH group was increased by approximately 50% and the necrotic core area by nearly 2-fold (Figure 1C), with size effects similar to those seen in the 20% Jak2VF CH mice (34). Moreover, the area of the fibrous cap, which is considered a protective structure in human plaques (36), was decreased in the LAB mice (Figure 1D), while there was comparable lesional collagen content between the groups (Figure 1D). We observed increased macrophage staining areas in lesions, including in both GFP+ (WT) and GFP– (Jak2VF) macrophages, in LAB Jak2VF CH mice compared with controls, along with an increased ratio of mutant/WT macrophages in the lesions (Figure 1, E and F). We also observed a moderate expansion of GFP– (Jak2VF) cells in LAB mice compared with controls, but they still only represented approximately 7% of total macrophages in the lesions (Figure 1F). Studies have shown increased neutrophil extracellular traps (NETs) in lesions as a result of aggravated inflammation (37–39). As assessed by costaining of citrullinated histones (3H-Cit) and myeloperoxidase (MPO), we found increased NETs in the LAB Jak2VF CH lesions compared with control mice (Figure 1G). These data showed a marked increase in atherosclerosis with features of plaque destabilization in LAB Jak2VF CH mice, involving both mutant and WT macrophages. Since a minor population of Jak2VF macrophages was inducing these overall changes, there was likely major inflammatory crosstalk from mutant to WT cells in lesions.
Figure 1Accelerated atherogenesis and plaque instability in LAB Jak2VF CH mice. (A) Experimental design. (B) Plasma cholesterol (CHO) levels. (C) Representative H&E staining and quantification of the lesion and necrotic core areas in aortic root sections. Necrotic core regions are indicated by dotted lines. Scale bar: 200 μm. Original magnification, ×100. (D) Aortic root sections were stained with Masson’s trichrome for the fibrous cap (outlined by dashed lines) and collagen content area and then quantified as the ratio of the total lesion area. Scale bar: 100 μm. (E and F) Representative immunofluorescence staining of macrophages (anti-Mac2) and GFP and quantification of the absolute Mac2+ area, the GFP–Mac2+ (Mx1-Cre or Jak2VF macrophages) area, and the GFP+Mac2+ area (WT macrophages), as well as the GFP–Mac2+ area as the percentage of the total Mac2+ area in aortic root sections. Scale bar: 250 μm. Original magnification, ×50. (G) Representative immunofluorescence staining for H3cit and MPO and quantification of the double-positive area (NETosis) as a percentage of the lesion area. Scale bar: 200 μm. Original magnification, ×200. (H) Representative immunofluorescence staining for IL-1β and anti-Mac2 (macrophages) and quantification of the fluorescence intensity of IL-1β were performed and normalized to the lesion area. Scale bar: 50 μm. Original magnification, ×100. Data are presented as the mean ± SEM. n = 15 (B–G), n = 13 (H). *P < 0.05 and **P < 0.01, by unpaired, 2-tailed Student’s t test.
Next, we attempted to identify inflammatory cytokines responsible for the crosstalk. IL-1β is an apical cytokine that plays an important role in human CVD (5), and we have shown that IL-1β and AIM2 inflammasome–induced pyroptosis promote features of plaque instability in higher VAF Jak2VF CH mice (34). Immunostaining revealed an increased abundance of IL-1β in LAB mice compared with control mice (Figure 1H). We noted an increase in Il1b mRNA expression in isolated aortic macrophages from LAB mice in both GFP+ and GFP– cells (Supplemental Figure 1C), consistent with IL-1 induction of its own expression (40) in both Jak2VF and WT macrophages. A survey of other potential inflammatory mediators showed trends for increased Il6, Tnfa, and Ifng expression in macrophages from LAB lesions compared with control lesions (Supplemental Figure 1D). Immunostaining confirmed a significant increase of IL-6 staining, which may be increased downstream of IL-1 signaling (41, 42) in LAB mice, but not of TNF-α or IFN-γ staining (Supplemental Figure 1, E–G).
Inflammatory crosstalk is mediated through IL-1 receptor, type 1–dependent signaling. To test the hypothesis that inflammatory crosstalk from mutant to WT cells is mediated through IL-1 signaling, we used IL-1 receptor 1 deficiency (Il1r1–/–) in non-Jak2VF BM cells in the LAB mice (Figure 2A). Donor BM in this cohort included control or Jak2VF (GFP+) BM (1.5%, tagged with GFP to better identify mutant cells) mixed with WT or Il1r1–/– BM (98.5%, GFP–) that was transplanted into irradiated Ldlr–/– mice (Figure 2A). After 6 weeks of recovery from BM transplantation (BMT), mice were placed on the WD for 16 weeks. At the end of WD feeding, the 4 groups had similar circulating WBCs, RBCs, and platelets (Supplemental Figure 2A). We also observed comparable GFP+ WBCs among groups as well as body weight, spleen weight, and liver weight (Supplemental Figure 2, B and C).
Figure 2Accelerated atherogenesis in LAB mice is reversed by IL-1R1 deficiency in non-Jak2VF cells. (A) Experimental design. (B) Representative H&E staining and quantification of the lesion and necrotic core areas in aortic root sections. Necrotic core regions are indicated by dashed lines. Scale bar: 200 μm. Original magnification, ×100. (C and D) Aortic root sections were stained with Masson’s trichrome for the fibrous cap and collagen content area and quantified as the ratio of total lesion area. Scale bar: 100 μm. Original magnification, ×200. (E and F) Representative immunofluorescence staining for MERTK (E) or TREM2 (F) and anti-Mac2 (macrophages). The fluorescence intensity of MERTK (E) and TREM2 (F) was quantified and normalized by the lesional macrophage area. Scale bars: 50 μm. Original magnification, ×200. Data are presented as the mean ± SEM. n = 20 (control), n = 19 (LAB VF), n = 19 (control/II1r–/–), n = 18 (LAB VF/II1r–/–) (B and D); n = 18 (control), n = 18 (LAB VF), n = 19 (control/II1r–/–), n = 16 (LAB VF/II1r–/–) (E); n = 18 (control), n = 17 (LAB VF),n = 17 ( control/II1r–/–), n = 16 (LAB VF/II1r–/–) (F). *P < 0.05, **P < 0.01, and ***P < 0.001, by 2-way ANOVA.
Deficiency of IL-1R1 in non-Jak2VF cells reversed the increase in plaque size and necrotic core area in LAB Jak2VF mice but did not affect these parameters in control mice (Figure 2B). The reduced fibrous cap in LAB Jak2VF mice was partially reversed by IL-1R deficiency (Figure 2C). Consistent with prior reports (43), IL-1R deficiency reduced fibrous cap formation in control mice lacking the Jak2VF mutation (Figure 2, C and D). Collagen content showed no significant change in LAB Jak2VF mice with or without IL-1R deficiency (Figure 2, C and D).
Impaired efferocytosis has been shown to promote necrotic core formation and decrease fibrous cap thickness in advanced atherosclerotic plaques (44–46). We thus assessed the expression of the phagocytic receptors MERTK and TREM2, which have both been implicated in efferocytosis and necrotic core formation in atherosclerosis (27, 47). Immunostaining revealed an approximate 30% reduction in receptor levels in lesions of LAB mice (Figure 2, E and F). However, this change was reversed by IL-1R1 deficiency in non-Jak2VF cells, leading to a significant approximately 1.4-fold increase in MERTK levels and a significant approximately 1.3-fold increase in TREM2 levels (Figure 2, E and F). The role of these receptors in plaque development was further assessed with in vivo models (see below).
IL-1–mediated crosstalk in myeloid cells induces pyroptosis and NETosis. IL-1R1 deficiency reversed the increase in macrophages in LAB mice for both GFP+ and GFP– macrophages and decreased GFP+ macrophages as a percentage of total macrophages in plaques (Figure 3A), indicating that IL-1 signaling in both Jak2VF-mutant and non-Jak2VF-mutant macrophages was increasing their content in plaques. We found that when IL-1R1 was absent in WT cells, elevated NETosis in the LAB lesions was abolished (Figure 3, B and C). To assess pyroptosis, we used an antibody to detect the N-terminal, cleaved form of gasdermin D (Cl-GSDMD), the mediator of pyroptosis downstream of inflammasomes (48). The specificity of this antibody was verified by Western blotting (WB) and by lack of staining in the lesions of Gsdmd-deficient mice (Supplemental Figure 2, D and E). However, WB showed that, while the antibody mainly recognized the cleaved form of GSDMD, there was a faint band corresponding to full-length GSDMD (Supplemental Figure 2D). To assess in vivo specificity, we compared immunostaining by this antibody with that of an antibody recognizing full-length GSDMD. In sections from 4 different mice, there was no overlap in staining of the 2 antibodies, suggesting specificity of the Cl-GSDMD antibody (a representative section is shown in Supplemental Figure 2F). The full-length antibody stained primarily in the cellular area and the Cl-GSDMD antibody predominantly in the necrotic core region. This suggests that the Cl-GSDMD antibody mainly recognized cleaved GSDMD in necrotic regions where pyroptosis had occurred. Cl-GSDMD staining was significantly increased in LAB mice, and this appeared to be especially the case in areas of NETosis (Figure 3, B and D). Further characterization revealed that Cl-GSDMD was largely expressed in GFP– neutrophils with lower expression in GFP– macrophages
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