Myeloperoxidase induces monocyte migration and activation after acute myocardial infarction.

Introduction: Myocardial infarction (MI) is a significant contributor to morbidity and mortality worldwide. Many individuals who survive the acute event continue to experience heart failure (HF), with inflammatory and healing processes post-MI playing a pivotal role. Polymorphonuclear neutrophils (PMN) and monocytes infiltrate the infarcted area, where PMN release high amounts of the heme enzyme myeloperoxidase (MPO). MPO has numerous inflammatory properties and MPO plasma levels are correlated with prognosis and severity of MI. While studies have focused on MPO inhibition and controlling PMN infiltration into the infarcted tissue, less is known on MPO's role in monocyte function. Methods and results: Here, we combined human data with mouse and cell studies to examine the role of MPO on monocyte activation and migration. We revealed a correlation between plasma MPO levels and monocyte activation in a patient study. Using a mouse model of MI, we demonstrated that MPO deficiency led to an increase in splenic monocytes and a decrease in cardiac monocytes compared to wildtype mice (WT). In vitro studies further showed that MPO induces monocyte migration, with upregulation of the chemokine receptor CCR2 and upregulation of inflammatory pathways identified as underlying mechanisms. Conclusion: Taken together, we identify MPO as a pro-inflammatory mediator of splenic monocyte recruitment and activation post-MI and provide mechanistic insight for novel therapeutic strategies after ischemic injury.

Myeloperoxidase is a critical mediator of anthracycline-induced cardiomyopathy.

Cardiotoxicity is a major complication of anthracycline therapy that negatively impacts prognosis. Effective pharmacotherapies for prevention of anthracycline-induced cardiomyopathy (AICM) are currently lacking. Increased plasma levels of the neutrophil-derived enzyme myeloperoxidase (MPO) predict occurrence of AICM in humans. We hypothesized that MPO release causally contributes to AICM. Mice intravenously injected with the anthracycline doxorubicin (DOX) exhibited higher neutrophil counts and MPO levels in the circulation and cardiac tissue compared to saline (NaCl)-treated controls. Neutrophil-like HL-60 cells exhibited increased MPO release upon exposition to DOX. DOX induced extensive nitrosative stress in cardiac tissue alongside with increased carbonylation of sarcomeric proteins in wildtype but not in Mpo-/- mice. Accordingly, co-treatment of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with DOX and MPO aggravated loss of hiPSC-CM-contractility compared to DOX treatment alone. DOX-treated animals exhibited pronounced cardiac apoptosis and inflammation, which was attenuated in MPO-deficient animals. Finally, genetic MPO deficiency and pharmacological MPO inhibition protected mice from the development of AICM. The anticancer efficacy of DOX was unaffected by MPO deficiency. Herein we identify MPO as a critical mediator of AICM. We demonstrate that DOX induces cardiac neutrophil infiltration and release of MPO, which directly impairs cardiac contractility through promoting oxidation of sarcomeric proteins, cardiac inflammation and cardiomyocyte apoptosis. MPO thus emerges as a promising pharmacological target for prevention of AICM.

Limited plasticity of monocyte fate and function associated with epigenetic scripting at the level of progenitors.

Myeloid cell heterogeneity is known, but whether it is cell-intrinsic or environmentally-directed remains unclear. Here, an inducible/reversible system pausing myeloid differentiation allowed the definition of clone-specific functions that clustered monocytes into subsets with distinctive molecular features. These subsets were orthogonal to the classical/nonclassical categorization and had inherent, restricted characteristics that did not shift under homeostasis, after irradiation, or with infectious stress. Rather, their functional fate was constrained by chromatin accessibility established at or before the granulocyte-monocyte or monocyte-dendritic progenitor level. Subsets of primary monocytes had differential ability to control distinct infectious agents in vivo. Therefore, monocytes are a heterogeneous population of functionally restricted subtypes defined by the epigenome of their progenitors that are differentially selected by physiologic challenges with limited plasticity to transition from one subset to another.

Myeloid leukocytes' diverse effects on cardiovascular and systemic inflammation in chronic kidney disease.

Chronic kidney disease's prevalence rises globally. Whereas dialysis treatment replaces the kidney's filtering function and prolongs life, dreaded consequences in remote organs develop inevitably over time. Even milder reductions in kidney function not requiring replacement therapy associate with bacterial infections, cardiovascular and heart valve disease, which markedly limit prognosis in these patients. The array of complications is diverse and engages a wide gamut of cellular and molecular mechanisms. The innate immune system is profoundly and systemically altered in chronic kidney disease and, as a unifying element, partakes in many of the disease's complications. As such, a derailed immune system fuels cardiovascular disease progression but also elevates the propensity for serious bacterial infections. Recent data further point towards a role in developing calcific aortic valve stenosis. Here, we delineate the current state of knowledge on how chronic kidney disease affects innate immunity in cardiovascular organs and on a systemic level. We review the role of circulating myeloid cells, monocytes and neutrophils, resident macrophages, dendritic cells, ligands, and cellular pathways that are activated or suppressed when renal function is chronically impaired. Finally, we discuss myeloid cells' varying responses to uremia from a systems immunology perspective.

Bone marrow endothelial dysfunction promotes myeloid cell expansion in cardiovascular disease.

Abnormal hematopoiesis advances cardiovascular disease by generating excess inflammatory leukocytes that attack the arteries and the heart. The bone marrow niche regulates hematopoietic stem cell proliferation and hence the systemic leukocyte pool, but whether cardiovascular disease affects the hematopoietic organ's microvasculature is unknown. Here we show that hypertension, atherosclerosis and myocardial infarction (MI) instigate endothelial dysfunction, leakage, vascular fibrosis and angiogenesis in the bone marrow, altogether leading to overproduction of inflammatory myeloid cells and systemic leukocytosis. Limiting angiogenesis with endothelial deletion of Vegfr2 (encoding vascular endothelial growth factor (VEGF) receptor 2) curbed emergency hematopoiesis after MI. We noted that bone marrow endothelial cells assumed inflammatory transcriptional phenotypes in all examined stages of cardiovascular disease. Endothelial deletion of Il6 or Vcan (encoding versican), genes shown to be highly expressed in mice with atherosclerosis or MI, reduced hematopoiesis and systemic myeloid cell numbers in these conditions. Our findings establish that cardiovascular disease remodels the vascular bone marrow niche, stimulating hematopoiesis and production of inflammatory leukocytes.

Increased stem cell proliferation in atherosclerosis accelerates clonal hematopoiesis.

Clonal hematopoiesis, a condition in which individual hematopoietic stem cell clones generate a disproportionate fraction of blood leukocytes, correlates with higher risk for cardiovascular disease. The mechanisms behind this association are incompletely understood. Here, we show that hematopoietic stem cell division rates are increased in mice and humans with atherosclerosis. Mathematical analysis demonstrates that increased stem cell proliferation expedites somatic evolution and expansion of clones with driver mutations. The experimentally determined division rate elevation in atherosclerosis patients is sufficient to produce a 3.5-fold increased risk of clonal hematopoiesis by age 70. We confirm the accuracy of our theoretical framework in mouse models of atherosclerosis and sleep fragmentation by showing that expansion of competitively transplanted Tet2-/- cells is accelerated under conditions of chronically elevated hematopoietic activity. Hence, increased hematopoietic stem cell proliferation is an important factor contributing to the association between cardiovascular disease and clonal hematopoiesis.

Immunotherapeutic Strategies in Cancer and Atherosclerosis-Two Sides of the Same Coin.

The development and clinical approval of immunotherapies has revolutionized cancer therapy. Although the role of adaptive immunity in atherogenesis is now well-established and several immunomodulatory strategies have proven beneficial in preclinical studies, anti-atherosclerotic immunotherapies available for clinical application are not available. Considering that adaptive immune responses are critically involved in both carcinogenesis and atherogenesis, immunotherapeutic approaches for the treatment of cancer and atherosclerosis may exert undesirable but also desirable side effects on the other condition, respectively. For example, the high antineoplastic efficacy of immune checkpoint inhibitors, which enhance effector immune responses against tumor cells by blocking co-inhibitory molecules, was recently shown to be constrained by substantial proatherogenic properties. In this review, we outline the specific role of immune responses in the development of cancer and atherosclerosis. Furthermore, we delineate how current cancer immunotherapies affect atherogenesis and discuss whether anti-atherosclerotic immunotherapies may similarly have an impact on carcinogenesis.

Nanoparticle-encapsulated siRNAs for gene silencing in the haematopoietic stem-cell niche.

Bone-marrow endothelial cells in the haematopoietic stem-cell niche form a network of blood vessels that regulates blood-cell traffic as well as the maintenance and function of haematopoietic stem and progenitor cells. Here, we report the design and in vivo performance of systemically injected lipid-polymer nanoparticles encapsulating small interfering RNA (siRNA), for the silencing of genes in bone-marrow endothelial cells. In mice, nanoparticles encapsulating siRNA sequences targeting the proteins stromal-derived factor 1 (Sdf1) or monocyte chemotactic protein 1 (Mcp1) enhanced (when silencing Sdf1) or inhibited (when silencing Mcp1) the release of stem and progenitor cells and of leukocytes from the bone marrow. In a mouse model of myocardial infarction, nanoparticle-mediated inhibition of cell release from the haematopoietic niche via Mcp1 silencing reduced leukocytes in the diseased heart, improved healing after infarction and attenuated heart failure. Nanoparticle-mediated RNA interference in the haematopoietic niche could be used to investigate haematopoietic processes for therapeutic applications in cancer, infection and cardiovascular disease.

Imaging Cardiovascular and Lung Macrophages With the Positron Emission Tomography Sensor 64Cu-Macrin in Mice, Rabbits, and Pigs.

BACKGROUND: Macrophages, innate immune cells that reside in all organs, defend the host against infection and injury. In the heart and vasculature, inflammatory macrophages also enhance tissue damage and propel cardiovascular diseases. METHODS: We here use in vivo positron emission tomography (PET) imaging, flow cytometry, and confocal microscopy to evaluate quantitative noninvasive assessment of cardiac, arterial, and pulmonary macrophages using the nanotracer 64Cu-Macrin-a 20-nm spherical dextran nanoparticle assembled from nontoxic polyglucose. RESULTS: PET imaging using 64Cu-Macrin faithfully reported accumulation of macrophages in the heart and lung of mice with myocardial infarction, sepsis, or pneumonia. Flow cytometry and confocal microscopy detected the near-infrared fluorescent version of the nanoparticle (VT680Macrin) primarily in tissue macrophages. In 5-day-old mice, 64Cu-Macrin PET imaging quantified physiologically more numerous cardiac macrophages. Upon intravenous administration of 64Cu-Macrin in rabbits and pigs, we detected heightened macrophage numbers in the infarcted myocardium, inflamed lung regions, and atherosclerotic plaques using a clinical PET/magnetic resonance imaging scanner. Toxicity studies in rats and human dosimetry estimates suggest that 64Cu-Macrin is safe for use in humans. CONCLUSIONS: Taken together, these results indicate 64Cu-Macrin could serve as a facile PET nanotracer to survey spatiotemporal macrophage dynamics during various physiological and pathological conditions. 64Cu-Macrin PET imaging could stage inflammatory cardiovascular disease activity, assist disease management, and serve as an imaging biomarker for emerging macrophage-targeted therapeutics.

Bone Marrow Endothelial Cells Regulate Myelopoiesis in Diabetes Mellitus.

BACKGROUND: Diabetes mellitus is a prevalent public health problem that affects about one-third of the US population and leads to serious vascular complications with increased risk for coronary artery disease. How bone marrow hematopoiesis contributes to diabetes mellitus complications is incompletely understood. We investigated the role of bone marrow endothelial cells in diabetic regulation of inflammatory myeloid cell production. METHODS: In 3 types of mouse diabetes mellitus, including streptozotocin, high-fat diet, and genetic induction using leptin-receptor-deficient db/db mice, we assayed leukocytes, hematopoietic stem and progenitor cells (HSPC). In addition, we investigated bone marrow endothelial cells with flow cytometry and expression profiling. RESULTS: In diabetes mellitus, we observed enhanced proliferation of HSPC leading to augmented circulating myeloid cell numbers. Analysis of bone marrow niche cells revealed that endothelial cells in diabetic mice expressed less Cxcl12, a retention factor promoting HSPC quiescence. Transcriptome-wide analysis of bone marrow endothelial cells demonstrated enrichment of genes involved in epithelial growth factor receptor (Egfr) signaling in mice with diet-induced diabetes mellitus. To explore whether endothelial Egfr plays a functional role in myelopoiesis, we generated mice with endothelial-specific deletion of Egfr (Cdh5CreEgfrfl/fl). We found enhanced HSPC proliferation and increased myeloid cell production in Cdh5CreEgfrfl/fl mice compared with wild-type mice with diabetes mellitus. Disrupted Egfr signaling in endothelial cells decreased their expression of the HSPC retention factor angiopoietin-1. We tested the functional relevance of these findings for wound healing and atherosclerosis, both implicated in complications of diabetes mellitus. Inflammatory myeloid cells accumulated more in skin wounds of diabetic Cdh5CreEgfrfl/fl mice, significantly delaying wound closure. Atherosclerosis was accelerated in Cdh5CreEgfrfl/fl mice, leading to larger and more inflamed atherosclerotic lesions in the aorta. CONCLUSIONS: In diabetes mellitus, bone marrow endothelial cells participate in the dysregulation of bone marrow hematopoiesis. Diabetes mellitus reduces endothelial production of Cxcl12, a quiescence-promoting niche factor that reduces stem cell proliferation. We describe a previously unknown counterregulatory pathway, in which protective endothelial Egfr signaling curbs HSPC proliferation and myeloid cell production.

Exercise reduces inflammatory cell production and cardiovascular inflammation via instruction of hematopoietic progenitor cells.

A sedentary lifestyle, chronic inflammation and leukocytosis increase atherosclerosis; however, it remains unclear whether regular physical activity influences leukocyte production. Here we show that voluntary running decreases hematopoietic activity in mice. Exercise protects mice and humans with atherosclerosis from chronic leukocytosis but does not compromise emergency hematopoiesis in mice. Mechanistically, exercise diminishes leptin production in adipose tissue, augmenting quiescence-promoting hematopoietic niche factors in leptin-receptor-positive stromal bone marrow cells. Induced deletion of the leptin receptor in Prrx1-creERT2; Leprfl/fl mice reveals that leptin's effect on bone marrow niche cells regulates hematopoietic stem and progenitor cell (HSPC) proliferation and leukocyte production, as well as cardiovascular inflammation and outcomes. Whereas running wheel withdrawal quickly reverses leptin levels, the impact of exercise on leukocyte production and on the HSPC epigenome and transcriptome persists for several weeks. Together, these data show that physical activity alters HSPCs via modulation of their niche, reducing hematopoietic output of inflammatory leukocytes.

Tissue-Specific Macrophage Responses to Remote Injury Impact the Outcome of Subsequent Local Immune Challenge.

Myocardial infarction, stroke, and sepsis trigger systemic inflammation and organism-wide complications that are difficult to manage. Here, we examined the contribution of macrophages residing in vital organs to the systemic response after these injuries. We generated a comprehensive catalog of changes in macrophage number, origin, and gene expression in the heart, brain, liver, kidney, and lung of mice with myocardial infarction, stroke, or sepsis. Predominantly fueled by heightened local proliferation, tissue macrophage numbers increased systemically. Macrophages in the same organ responded similarly to different injuries by altering expression of tissue-specific gene sets. Preceding myocardial infarction improved survival of subsequent pneumonia due to enhanced bacterial clearance, which was caused by IFNÉ£ priming of alveolar macrophages. Conversely, EGF receptor signaling in macrophages exacerbated inflammatory lung injury. Our data suggest that local injury activates macrophages in remote organs and that targeting macrophages could improve resilience against systemic complications following myocardial infarction, stroke, and sepsis.

Sleep modulates haematopoiesis and protects against atherosclerosis.

Sleep is integral to life1. Although insufficient or disrupted sleep increases the risk of multiple pathological conditions, including cardiovascular disease2, we know little about the cellular and molecular mechanisms by which sleep maintains cardiovascular health. Here we report that sleep regulates haematopoiesis and protects against atherosclerosis in mice. We show that mice subjected to sleep fragmentation produce more Ly-6Chigh monocytes, develop larger atherosclerotic lesions and produce less hypocretin-a stimulatory and wake-promoting neuropeptide-in the lateral hypothalamus. Hypocretin controls myelopoiesis by restricting the production of CSF1 by hypocretin-receptor-expressing pre-neutrophils in the bone marrow. Whereas hypocretin-null and haematopoietic hypocretin-receptor-null mice develop monocytosis and accelerated atherosclerosis, sleep-fragmented mice with either haematopoietic CSF1 deficiency or hypocretin supplementation have reduced numbers of circulating monocytes and smaller atherosclerotic lesions. Together, these results identify a neuro-immune axis that links sleep to haematopoiesis and atherosclerosis.

Imaging the Vascular Bone Marrow Niche During Inflammatory Stress.

RATIONALE: Inflammatory stress induced by exposure to bacterial lipopolysaccharide causes hematopoietic stem cell expansion in the bone marrow niche, generating a cellular immune response. As an integral component of the hematopoietic stem cell niche, the bone marrow vasculature regulates the production and release of blood leukocytes, which protect the host against infection but also fuel inflammatory diseases. OBJECTIVE: We aimed to develop imaging tools to explore vascular changes in the bone marrow niche during acute inflammation. METHODS AND RESULTS: Using the TLR (Toll-like receptor) ligand lipopolysaccharide as a prototypical danger signal, we applied multiparametric, multimodality and multiscale imaging to characterize how the bone marrow vasculature adapts when hematopoiesis boosts leukocyte supply. In response to lipopolysaccharide, ex vivo flow cytometry and histology showed vascular changes to the bone marrow niche. Specifically, proliferating endothelial cells gave rise to new vasculature in the bone marrow during hypoxic conditions. We studied these vascular changes with complementary intravital microscopy and positron emission tomography/magnetic resonance imaging. Fluorescence and positron emission tomography integrin αVß3 imaging signal increased during lipopolysaccharide-induced vascular remodeling. Vascular leakiness, quantified by albumin-based in vivo microscopy and magnetic resonance imaging, rose when neutrophils departed and hematopoietic stem and progenitor cells proliferated more vigorously. CONCLUSIONS: Introducing a tool set to image bone marrow either with cellular resolution or noninvasively within the entire skeleton, this work sheds light on angiogenic responses that accompany emergency hematopoiesis. Understanding and monitoring bone marrow vasculature may provide a key to unlock therapeutic targets regulating systemic inflammation.

E-Selectin Inhibition Mitigates Splenic HSC Activation and Myelopoiesis in Hypercholesterolemic Mice With Myocardial Infarction.

OBJECTIVE: Atherosclerosis is a chronic disease characterized by lipid accumulation in the arterial wall. After myocardial infarction (MI), atherosclerotic plaques are infiltrated by inflammatory myeloid cells that aggravate the disease and increase the risk of secondary myocardial ischemia. Splenic myelopoiesis provides a steady flow of myeloid cells to inflamed atherosclerotic lesions after MI. Therefore, targeting myeloid cell production in the spleen could ameliorate increased atherosclerotic plaque inflammation after MI. APPROACH AND RESULTS: Here we show that MI increases splenic myelopoiesis by driving hematopoietic stem and progenitor cells into the cell cycle. In an atherosclerotic mouse model, E-selectin inhibition decreased hematopoietic stem and progenitor cell proliferation in the spleen after MI. This led to reduced extramedullary myelopoiesis and decreased myeloid cell accumulation in atherosclerotic lesions. Finally, we observed stable atherosclerotic plaque features, including smaller plaque size, reduced necrotic core area, and thicker fibrous cap after E-selectin inhibition. CONCLUSIONS: Inhibiting E-selectin attenuated inflammation in atherosclerotic plaques, likely by reducing leukocyte recruitment into plaques and by mitigating hematopoietic stem and progenitor cell activation in the spleen of mice with MI.

Myeloid-Specific Deletion of Diacylglycerol Lipase α Inhibits Atherogenesis in ApoE-Deficient Mice.

BACKGROUND: The endocannabinoid 2-arachidonoylglycerol (2-AG) is a known modulator of inflammation. Despite its high concentration in vascular tissue, the role of 2-AG in atherogenesis has not yet been examined. METHODS: ApoE-deficient mice were sublethally irradiated and reconstituted with bone marrow from mice with a myeloid-specific knockout of the 2-AG synthesising enzyme diacylglycerol lipase α (Dagla) or control bone marrow with an intact 2-AG biosynthesis. After a cholesterol-rich diet for 8 weeks, plaque size and plaque morphology were examined in chimeric mice. Circulating inflammatory cells were assessed by flow cytometry. Aortic tissue and plasma levels of endocannabinoids were measured using liquid chromatography-multiple reaction monitoring. RESULTS: Mice with Dagla-deficient bone marrow and circulating myeloid cells showed a significantly reduced plaque burden compared to controls. The reduction in plaque size was accompanied by a significantly diminished accumulation of both neutrophil granulocytes and macrophages in atherosclerotic lesions of Dagla-deficient mice. Moreover, CB2 expression and the amount of oxidised LDL within atherosclerotic lesions was significantly reduced. FACS analyses revealed that levels of circulating inflammatory cells were unaltered in Dagla-deficient mice. CONCLUSIONS: Myeloid synthesis of the endocannabinoid 2-AG appears to promote vascular inflammation and atherogenesis. Thus, myeloid-specific disruption of 2-AG synthesis may represent a potential novel therapeutic strategy against atherosclerosis.

Myocardial Infarction Activates CCR2(+) Hematopoietic Stem and Progenitor Cells.

Following myocardial infarction (MI), myeloid cells derived from the hematopoietic system drive a sharp increase in systemic leukocyte levels that correlates closely with mortality. The origin of these myeloid cells, and the response of hematopoietic stem and progenitor cells (HSPCs) to MI, however, is unclear. Here, we identify a CCR2(+)CD150(+)CD48(-) LSK hematopoietic subset as the most upstream contributor to emergency myelopoiesis after ischemic organ injury. This subset has 4-fold higher proliferation rates than CCR2(-)CD150(+)CD48(-) LSK cells, displays a myeloid differentiation bias, and dominates the migratory HSPC population. We further demonstrate that the myeloid translocation gene 16 (Mtg16) regulates CCR2(+) HSPC emergence. Mtg16(-/-) mice have decreased levels of systemic monocytes and infarct-associated macrophages and display compromised tissue healing and post-MI heart failure. Together, these data provide insights into regulation of emergency hematopoiesis after ischemic injury and identify potential therapeutic targets to modulate leukocyte output after MI.