Atherosclerotic cardiovascular disease (CVD), including myocardial infarction and stroke, is the leading cause of death worldwide. Major risk factors for CVD include dyslipoproteinemia, smoking, hypertension, obesity, and diabetes. However, despite optimal pharmacological treatment of these risk factors, a considerable residual CVD risk remains. Evidence is rapidly accumulating that chronic low-grade inflammation of the vascular wall is a key pathophysiological component of atherosclerosis, and that treatment with anti-inflammatory drugs, such as canakinumab and colchicine, additionally lowers future CVD risk (Nidorf et al., 2020; Ridker et al., 2017; Tardif et al., 2019). More detailed knowledge of this inflammatory process and the role of individual immune cells would allow the development of more specific and safe anti-inflammatory therapies.

Within atherosclerotic plaques, monocyte-derived macrophages are the most abundant immune cells, which play a key role in initiation, progression, and destabilization of these plaques, although the mechanisms driving the persistent inflammatory activation are poorly understood (Moore et al., 2013). In patients with CVD or risk factors for CVD, circulating leukocytes show differences in composition and individual cell phenotypes, compared to healthy individuals. Isolated monocytes from patients with CVD are characterized by an increased cytokine production capacity and higher glycolytic metabolism (Bekkering et al., 2016; Elsenberg et al., 2013; Shirai et al., 2016). This also holds true for patients with increased CVD risk due to familial hypercholesterolemia (Bekkering et al., 2019). In addition, higher circulating levels of granulocytes are associated with an increased CVD risk in a general population study (Fani et al., 2020).

Experimental studies in animal models of atherosclerosis have revealed reprogramming of hematopoietic stem and progenitor cells (HSPCs) in the bone marrow as a cause of these changes in circulating innate immune cells under conditions that predispose to CVD, including hyperlipidemia (Christ et al., 2018), and stress (Heidt et al., 2014). Western-type diet feeding of low-density lipoprotein receptor deficient (Ldlr-/-) mice induces inflammatory activation of monocytes by epigenetic reprogramming of HSPCs which persists in normocholesterolemic conditions (Christ et al., 2018). This resembles the finding of persistent functional and transcriptional hyperresponsive monocytes in patients with dyslipidemia despite cholesterol-lowering treatment (Bekkering et al., 2019). In a retrospective analysis of bone marrow samples from cancer patients, HSPCs had a higher proliferative potential in patients with known atherosclerotic CVD than in control patients (van der Valk et al., 2016). A detailed assessment, however, of HSPC composition and function in patients with atherosclerosis is lacking.

Here we performed a comprehensive study in patients with and without coronary artery disease (CAD). We obtained circulating leukocytes and HSPCs and assessed composition with flow cytometry, as well as cytokine production capacity, metabolism, and the transcriptional profile. In addition, we performed 2’-deoxy-2’-[¹⁸F]fluoro-D-glucose positron-emission-tomography ([¹⁸F]FDG PET/CT) scanning to detect bone marrow and splenic activity. We report that bone marrow mononuclear cells in patients with atherosclerosis are characterized by enhanced cytokine production capacity and an increased metabolic rate. Flow cytometry and RNAseq analysis revealed inflammatory transcriptional reprogramming and myeloid skewing. These results show that in patients with atherosclerosis, activation of the innate immune system occurs at the level of bone marrow myeloid progenitors, which adds exciting opportunities for novel treatment strategies.

The role of activated innate immune cells in the development of atherosclerotic plaques is well-established. In the current paper we add an important dimension to this pathophysiological framework by showing for the first time that inflammatory reprogramming of innate immune cells in humans with coronary artery disease occurs at the level of HSPCs in the bone marrow. In patients with chronic CAD due to severe coronary atherosclerosis, isolated bone marrow mononuclear cells showed a higher cytokine production capacity and increased metabolic rate compared to individuals without coronary atherosclerosis. Transcriptional signatures of HSPCs were enriched for neutrophil and monocyte related pathways, indicating that the HSPCs are primed to differentiate into an inflammatory myeloid lineage. These findings addinclude an additional layer of inflammatory regulation in atherogenesis which potentially offers novel targets for pharmacological strategies to prevent or treat atherosclerotic CVD.

Our findings align with previous studies in animal models in atherosclerosis that show increased myelopoiesis in specific conditions that promote CVD. Accumulation of cholesterol in HSPC increases proliferation and mobilization from the bone marrow and a differentiation bias towards the myeloid lineage, which is associated with accelerated atherosclerosis (Yvan-Charvet et al., 2010). In an animal model of diabetes, higher circulating numbers of inflammatory Ly6-C^hi monocytes and neutrophils are present, which is due to increased myelopoiesis. In this model, the increased myelopoiesis is driven by the increased expression of S100A8 and S100A9 in neutrophils (Nagareddy et al., 2013). Interestingly, the expression of one of the S100 proteins (S100A11) was also increased in the HSCs in patients with CAD in our study. Chronic psychological stress and disturbed sleep have also been reported to increase myelopoiesis, which is associated with elevated circulating levels of neutrophils and Ly6C^high monocytes and augmented atherosclerosis compared with control mice (Heidt et al., 2014; McAlpine et al., 2019).

Since we did not include patients with specific cardiovascular risk factors, but rather with established symptomatic coronary atherosclerosis, we cannot conclude which factors are responsible for the HSPC reprogramming in our patients. BMI and blood pressure were slightly higher in the patients compared to the controls. Cholesterol concentrations were lower at the moment of inclusion in our study, but might well have been higher previously, since the patients also used more statins. We do not have information about stress and sleep patterns. Another mechanism that might contribute to the HSPC functional reprogramming is trained immunity. Trained immunity describes the phenomenon that brief stimulation of innate immune cells leads to the development of a long-term hyperresponsive phenotype. This is mediated by profound intracellular metabolic and epigenetic reprogramming and occurs both at the level of mature circulating monocytes as well as at the level of their bone marrow progenitors (Netea et al., 2020). Christ et al have recently described that a 4-week period of Western-type diet feeding in atherosclerosis prone Ldlr-/-mice induced a functional, transcriptional and epigenetic reprogramming of circulating myeloid cells and their bone marrow progenitors, which persisted for at least for weeks after switching back to a chow diet (Christ et al., 2018). This was associated with increased circulating concentrations of inflammatory monocytes and granulocytes. Functional enrichment analysis of GMPs showed overrepresentation of TNF and Toll-like receptor signaling pathways, which align with our observations. In contrast, although monocytic signature genes were enriched, most of the granulocytic signature genes were downregulated, including S100A8 and S100A9, in GMPs isolated from WD-fed as compared to CD-fed mice (Christ et al., 2018). Patients with hypercholesterolemia also have circulating monocytes with augmented cytokine production capacity, increased glycolytic metabolism, and enrichment of activating histone modifications, which persist for three months despite cholesterol lowering with statin therapy. Circulating monocyte and neutrophil numbers, however, were not increased in these patients (Bekkering et al., 2019). In our current patient cohort, it remains to be established whether trained immunity is present since we did not assess epigenetic markers in the HSPCs.

The observation that absolute numbers of myeloid progenitors and circulating monocytes and neutrophils are not increased, despite functional and transcriptional inflammatory programming, fits with the effects of alternative inducers of trained immunity in humans. Vaccination with the Bacille Calmette-Guérin (BCG) vaccine induces trained immunity in humans in vivo (Arts et al., 2018). This is accompanied by an increased cytokine production capacity of bone marrow mononuclear cells, comparable to the result in patients with CAD in the current study (Cirovic et al., 2020). Also, 90 days post-BCG administration, HSPCs showed myeloid skewing with activation of neutrophil-associated pathways and pathways associated with regulation of immune responses, comparable to our patients. This functional and transcriptional reprogramming was accompanied by an increase in numbers of circulating myeloid cells in large cohorts of infants vaccinated with BCG (Cirovic et al., 2020).

In the patient group with CAD, the increased cytokine production capacity of the bone marrow mononuclear cell fraction was accompanied by an upregulation of both glycolytic metabolism as well as oxygen consumption by oxidative phosphorylation. It is important to realize that the bone marrow mononuclear fraction also contains large amounts of mature monocytes, so we cannot conclude on the metabolic landscape of HSPCs and specific progenitor subtypes. Isolated monocytes and monocyte-derived macrophages from patients with atherosclerosis are characterized by increased glycolysis, which is coupled to the hyperresponsiveness in terms of cytokine release (Bekkering et al., 2016; Shirai et al., 2016). Also, HSPCs of mice after induction of trained immunity with β-glucan have an upregulation of the glycolytic pathway (Mitroulis et al., 2018).

Analysis of the differentially upregulated genes transcriptome of the flow-sorted HSCs and GMPs showed significant enrichment for neutrophil activation pathways. This further supports a role for neutrophils in the pathophysiology of atherosclerotic CVD, which is suggested by various recent findings in the literature. Although neutrophils are the most abundant circulating white blood cell type, neutrophils only recently received attention in the context of cardiovascular inflammation. A large epidemiological study shows that circulating granulocyte counts are strongly associated with the occurrence of CVD (Fani et al., 2020). During atherogenesis, platelet-derived chemokines, such as CC-chemokine ligand 5, promote neutrophil activation and recruitment (Silvestre-Roig et al., 2020). At the luminal side, these activated neutrophils secrete granule proteins, including cathepsin G, which further promotes myeloid cell recruitment. Several actions further fuel atherosclerotic plaque formation, including secretion of reactive oxygen species and myeloperoxidase, which mediates oxidation of LDL, promoting foam cell formation (Silvestre-Roig et al., 2020). Also, neutrophils can form neutrophil extracellular traps (NETs) (Döring et al., 2020) and they can secrete pro-inflammatory microvesicles, which further fuel vascular wall inflammation and atherosclerosis (Gomez et al., 2020). Interestingly, a recent study used plasma proteomics analysis to explore the mechanism by which colchicine lowers cardiovascular risk in patients with CAD and showed mainly a downregulation of neutrophil activation pathways (Opstal et al., 2020).

Analysis of the individual significantly differentially expressed genes in the HSPCs showed that the glycolytic enzyme phosphofructokinase (PFKP) was downregulated in patients with CAD. Quiescent HSCs depend mainly on anaerobic glycolysis to sustain survival, quiescence, and retention within the bone marrow (Takubo et al., 2010). Once stimulated to divide, HSCs start to activate OXPHOS to meet metabolic demands of proliferation and differentiation (Karigane and Takubo, 2017). Therefore, the downregulation of PFKP might be related to the activation of HSCs. This finding is in contrast to the higher extracellular acidification rate of the bone marrow mononuclear cells which points to the activation of glycolysis. We speculate that this finding is dominated by the inflammatory activation of bone marrow monocytes, which constitute a large percentage of the bone marrow mononuclear cells. The chemokine receptor CCR2 showed significant upregulation in the HSPCs of the patients with CAD. CCR2 is critical for monocyte egress from the bone marrow and for the recruitment of circulating monocytes to the arterial wall (Tsou et al., 2007). The upregulation of CCR2 in our study might contribute to the increased circulating HSC percentage in patients with CAD. In mice, increased levels of circulating HSPCs are found after myocardial infarction that migrate to the spleen for extramedullary hematopoiesis (Dutta et al., 2012). In humans, splenic extramedullary hematopoiesis can occur in response to extreme physiologic stress (e.g. sepsis or hematologic malignancies), but has not been described yet during the development of CVD. Indirect evidence shows that splenic activity in humans is increased after myocardial infarction assessed with PET/CT scanning (van der Valk et al., 2016), suggesting increased immune cell activation. In our study, we observed a correlation between splenic activity and CCR2 expression on circulating HSPCs which might suggest that the spleen performs a role in human atherosclerosis.

Our study has several limitations. First, the sample size is relatively small. However, this sample size needs to be considered in the context of the invasive and complex nature of the study, and this sample size usually allows to detect relevant differences (Cirovic et al., 2020; van der Heijden et al., 2020). The sample size might also be an explanation for the lack of a significant difference in FDG uptake in the large arteries, which has been shown previously in patients with CVD. (Tarkin et al., 2014; van Wijk et al., 2014). Another possible explanation for our results is that our patients had severe mature coronary atherosclerotic plaques with calcium deposits, which are known to have less FDG uptake than uncalcified arterial plaques at the early stage of atherosclerosis (Fernández-Friera et al., 2019). In addition, lipid-lowering therapy is effective in reducing vascular wall inflammation (Pirro et al., 2019). Second, we did not explore epigenetic programs of the HSPCs. As such, we cannot conclude whether the mechanism of trained immunity contributes to our findings. This should be the focus of future studies. Third, in a study on symptomatic patients with CVD, it is inevitable to have differences in medication use compared to the healthy control group.

To summarize, our study is the first to prospectively assess HSPC phenotype in patients with CAD and is unique in providing a comprehensive phenotype by the integration of powerful tools such as RNAseq on flow-sorted HSPC populations, functional parameters, Seahorse respiratory, and PET imaging of vascular wall and hematopoietic activity. These results show that in patients with CAD, activation of the innate immune system occurs at the level of bone marrow myeloid progenitors, which adds exciting opportunities for novel treatment strategies.

RNA-seq data have been deposited in the ArrayExpress database at EMBL-EBI (https://www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-9399.

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Author details

1. Marlies P Noz

   Department of Internal Medicine and Radboud Institute for Molecular Life Science (RIMLS), Radboud University Medical Center, Nijmegen, Netherlands

   Contribution

   Data curation, Formal analysis, Investigation, Visualization, Writing - original draft, Project administration

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-2299-8313

2. Siroon Bekkering

   Department of Internal Medicine and Radboud Institute for Molecular Life Science (RIMLS), Radboud University Medical Center, Nijmegen, Netherlands

   Contribution

   Conceptualization, Formal analysis, Supervision, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-1149-466X

3. Laszlo Groh

   Department of Internal Medicine and Radboud Institute for Molecular Life Science (RIMLS), Radboud University Medical Center, Nijmegen, Netherlands

   Contribution

   Formal analysis, Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

4. Tim MJ Nielen

   Department of Cardiology, Canisius Wilhelmina Hospital, Nijmegen, Netherlands

   Contribution

   Formal analysis, Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7762-5912

5. Evert JP Lamfers

   Department of Cardiology, Canisius Wilhelmina Hospital, Nijmegen, Netherlands

   Contribution

   Formal analysis, Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-5582-3720

6. Andreas Schlitzer

   Quantitative Systems Biology, Life & Medical Sciences Institute, University of Bonn, Single Cell Genomics and Epigenomics Unit at the German Center for Neurodegenerative Diseases and the University of Bonn, Bonn, Germany

   Contribution

   Formal analysis, Supervision, Writing - review and editing

   Competing interests

   No competing interests declared

7. Saloua El Messaoudi

   Department of Cardiology, Radboud University Medical Center, Nijmegen, Netherlands

   Contribution

   Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

8. Niels van Royen

   Department of Cardiology, Radboud University Medical Center, Nijmegen, Netherlands

   Contribution

   Supervision, Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

9. Erik HJPG Huys

   Department of Laboratory Medicine – Laboratory for Haematology, Radboud University Medical Center, Nijmegen, Netherlands

   Contribution

   Formal analysis, Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

10. Frank WMB Preijers

    Department of Laboratory Medicine – Laboratory for Haematology, Radboud University Medical Center, Nijmegen, Netherlands

    Contribution

    Conceptualization, Formal analysis, Supervision, Writing - review and editing

    Competing interests

    No competing interests declared

11. Esther MM Smeets

    Department of Radiology and Nuclear Medicine, Radboud University Medical Center, Nijmegen, Netherlands

    Contribution

    Data curation, Formal analysis, Writing - review and editing

    Competing interests

    No competing interests declared

12. Erik HJG Aarntzen

    Department of Radiology and Nuclear Medicine, Radboud University Medical Center, Nijmegen, Netherlands

    Contribution

    Conceptualization, Supervision, Writing - review and editing

    Competing interests

    No competing interests declared

13. Bowen Zhang

    Department of Computational Biology for Individualised Infection Medicine, Centre for Individualised Infection Medicine (CiiM) & TWINCORE, joint ventures between the Helmholtz-Centre for Infection Research (HZI) and the Hannover Medical School (MHH), Hannover, Germany

    Contribution

    Data curation, Software, Formal analysis, Visualization, Writing - original draft, Writing - review and editing

    Competing interests

    No competing interests declared

14. Yang Li

    1. Department of Internal Medicine and Radboud Institute for Molecular Life Science (RIMLS), Radboud University Medical Center, Nijmegen, Netherlands
    2. Department of Computational Biology for Individualised Infection Medicine, Centre for Individualised Infection Medicine (CiiM) & TWINCORE, joint ventures between the Helmholtz-Centre for Infection Research (HZI) and the Hannover Medical School (MHH), Hannover, Germany

    Contribution

    Conceptualization, Data curation, Supervision, Methodology, Writing - review and editing

    Competing interests

    No competing interests declared

15. Manita EJ Bremmers

    Department of Haematology, Radboud University Medical Center, Nijmegen, Netherlands

    Contribution

    Investigation, Methodology, Writing - review and editing

    Competing interests

    No competing interests declared

16. Walter JFM van der Velden

    Department of Haematology, Radboud University Medical Center, Nijmegen, Netherlands

    Contribution

    Supervision, Methodology, Writing - review and editing

    Competing interests

    No competing interests declared

17. Harry Dolstra

    Department of Laboratory Medicine – Laboratory for Haematology, Radboud University Medical Center, Nijmegen, Netherlands

    Contribution

    Supervision, Methodology, Writing - review and editing

    Competing interests

    No competing interests declared

18. Leo AB Joosten

    1. Department of Internal Medicine and Radboud Institute for Molecular Life Science (RIMLS), Radboud University Medical Center, Nijmegen, Netherlands
    2. Department of Medical Genetics, Iuliu Hatieganu University of Medicine and Pharmacy, Cluj-Napoca, Romania

    Contribution

    Conceptualization, Supervision, Funding acquisition, Writing - review and editing

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0001-6166-9830

19. Marc E Gomes

    Department of Cardiology, Canisius Wilhelmina Hospital, Nijmegen, Netherlands

    Contribution

    Conceptualization, Supervision, Writing - review and editing

    Competing interests

    No competing interests declared

20. Mihai G Netea

    1. Department of Internal Medicine and Radboud Institute for Molecular Life Science (RIMLS), Radboud University Medical Center, Nijmegen, Netherlands
    2. Department for Genomics & Immunoregulation, Life and Medical Sciences Institute (LIMES), University of Bonn, Bonn, Germany

    Contribution

    Conceptualization, Supervision, Funding acquisition, Writing - review and editing

    Competing interests

    No competing interests declared

21. Niels P Riksen

    Department of Internal Medicine and Radboud Institute for Molecular Life Science (RIMLS), Radboud University Medical Center, Nijmegen, Netherlands

    Contribution

    Conceptualization, Resources, Supervision, Funding acquisition, Investigation, Methodology, Writing - original draft, Project administration, Writing - review and editing

    For correspondence

    niels.riksen@radboudumc.nl

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0001-9197-8124

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