During progression of atherosclerosis, myeloid cells destabilize lipid-rich plaque in the arterial wall and cause its rupture, thus triggering myocardial infarction and stroke. Survivors of acute coronary syndromes have a high risk of recurrent events for unknown reasons. Here we show that the systemic response to ischemic injury aggravates chronic atherosclerosis. After myocardial infarction or stroke, apoE−/− mice developed larger atherosclerotic lesions with a more advanced morphology. This disease acceleration persisted over many weeks and was associated with markedly increased monocyte recruitment. When seeking the source of surplus monocytes in plaque, we found that myocardial infarction liberated hematopoietic stem and progenitor cells from bone marrow niches via sympathetic nervous system signaling. The progenitors then seeded the spleen yielding a sustained boost in monocyte production. These observations provide new mechanistic insight into atherogenesis and provide a novel therapeutic opportunity to mitigate disease progression.
Atherosclerotic lesions are believed to grow via the recruitment of bone marrow-derived monocytes. Among the known murine monocyte subsets, Ly-6Chigh monocytes are inflammatory, accumulate in lesions preferentially, and differentiate. Here we hypothesized that the bone marrow outsources the production of Ly-6Chigh monocytes during atherosclerosis.
Methods and Results
Using murine models of atherosclerosis and fate-mapping approaches, we show that hematopoietic stem and progenitor cells (HSPC) progressively relocate from the bone marrow to the splenic red pulp where they encounter GM-CSF and IL-3, clonally expand, and differentiate to Ly-6Chigh monocytes. Monocytes born in such extramedullary niches intravasate, circulate, and accumulate abundantly in atheromata. Upon lesional infiltration, Ly-6Chigh monocytes secrete inflammatory cytokines, reactive oxygen species, and proteases. Eventually, they ingest lipids and become foam cells.
Our findings indicate that extramedullary sites supplement the bone marrow’s hematopoietic function by producing circulating inflammatory cells that infiltrate atherosclerotic lesions.
Atherosclerosis; Imaging; Immune System; Immunology; Macrophage
editorials; atherosclerosis; folate; folate receptor; immunotoxins; macrophages
Atherosclerosis is characterized by the progressive accumulation of lipids and leukocytes in the arterial wall. Leukocytes such as macrophages accumulate oxidized lipoproteins in the growing atheromata and give rise to foam cells, which can then contribute to a lesion’s necrotic core. Lipids and leukocytes interact also in other important ways. In experimental models, systemic hypercholesterolemia is associated with severe neutrophilia and monocytosis. Recent evidence indicates that cholesterol sensing pathways control the proliferation of hematopoietic stem cell progenitors. Here we review some of the studies that are forging this particular link between metabolism and inflammation and propose several strategies that could target this axis for the treatment of cardiovascular disease.
hypercholesterolemia; atherosclerosis; hematopoietic stem and progenitor cells; monocytes; neutrophils
Exposure to psychosocial stress is a risk factor for many diseases, including atherosclerosis1,2. While incompletely understood, interaction between the psyche and the immune system provides one potential mechanism linking stress and disease inception and progression. Known crosstalk between the brain and immune system includes the hypothalamic–pituitary–adrenal axis, which centrally drives glucocorticoid production in the adrenal cortex, and the sympathetic–adrenal–medullary axis, which controls stress–induced catecholamine release in support of the fight–or–flight reflex3,4. It remains unknown however if chronic stress changes hematopoietic stem cell activity. Here we show that stress increases proliferation of these most primitive progenitors, giving rise to higher levels of disease–promoting inflammatory leukocytes. We found that chronic stress induced monocytosis and neutrophilia in humans. While investigating the source of leukocytosis in mice, we discovered that stress activates upstream hematopoietic stem cells. Sympathetic nerve fibers release surplus noradrenaline, which uses the β3 adrenergic receptor to signal bone marrow niche cells to decrease CXCL12 levels. Consequently, elevated hematopoietic stem cell proliferation increases output of neutrophils and inflammatory monocytes. When atherosclerosis–prone ApoE−/− mice encounter chronic stress, accelerated hematopoiesis promotes plaque features associated with vulnerable lesions that cause myocardial infarction and stroke in humans.
In response to lung infection, pleural innate response activator B cells produce GM-CSF–dependent IgM and ensure a frontline defense against bacterial invasion.
Pneumonia is a major cause of mortality worldwide and a serious problem in critical care medicine, but the immunophysiological processes that confer either protection or morbidity are not completely understood. We show that in response to lung infection, B1a B cells migrate from the pleural space to the lung parenchyma to secrete polyreactive emergency immunoglobulin M (IgM). The process requires innate response activator (IRA) B cells, a transitional B1a-derived inflammatory subset which controls IgM production via autocrine granulocyte/macrophage colony-stimulating factor (GM-CSF) signaling. The strategic location of these cells, coupled with the capacity to produce GM-CSF–dependent IgM, ensures effective early frontline defense against bacteria invading the lungs. The study describes a previously unrecognized GM-CSF-IgM axis and positions IRA B cells as orchestrators of protective IgM immunity.
Healing of myocardial infarction (MI) requires monocytes/macrophages. These mononuclear phagocytes likely degrade released macromolecules and aid in scavenging of dead cardiomyocytes, while mediating aspects of granulation tissue formation and remodeling. The mechanisms that orchestrate such divergent functions remain unknown. In view of the heightened appreciation of the heterogeneity of circulating monocytes, we investigated whether distinct monocyte subsets contribute in specific ways to myocardial ischemic injury in mouse MI. We identify two distinct phases of monocyte participation after MI and propose a model that reconciles the divergent properties of these cells in healing. Infarcted hearts modulate their chemokine expression profile over time, and they sequentially and actively recruit Ly-6Chi and -6Clo monocytes via CCR2 and CX3CR1, respectively. Ly-6Chi monocytes dominate early (phase I) and exhibit phagocytic, proteolytic, and inflammatory functions. Ly-6Clo monocytes dominate later (phase II), have attenuated inflammatory properties, and express vascular–endothelial growth factor. Consequently, Ly-6Chi monocytes digest damaged tissue, whereas Ly-6Clo monocytes promote healing via myofibroblast accumulation, angiogenesis, and deposition of collagen. MI in atherosclerotic mice with chronic Ly-6Chi monocytosis results in impaired healing, underscoring the need for a balanced and coordinated response. These observations provide novel mechanistic insights into the cellular and molecular events that regulate the response to ischemic injury and identify new therapeutic targets that can influence healing and ventricular remodeling after MI.
Only a few decades ago, students of the pathophysiology of cardiovascular disease paid little heed to the involvement of inflammation and immunity. Multiple lines of evidence now point to the participation of innate and adaptive immunity and inflammatory signaling in a variety of cardiovascular conditions. Hence, interest has burgeoned in this intersection. This review will focus on the contribution of innate immunity to both acute injury to the heart muscle itself, notably myocardial infarction, and to chronic inflammation in the artery wall, namely atherosclerosis, the cause of most myocardial infarctions. Our discussion of the operation of innate immunity in cardiovascular diseases will focus on functions of the mononuclear phagocytes with special attention to emerging data regarding the participation of different functional subsets of these cells in cardiovascular pathophysiology.
myocardial infarction; atherosclerosis; acute coronary syndromes; inflammation; mononuclear phagocytes; innate immunity
Monocytes and macrophages are innate immune cells that reside and accumulate in the healthy and injured heart. The cells and their subsets pursue distinct functions in steady state and disease, and their tenure may range between hours to months. Some subsets are highly inflammatory, while others support tissue repair. This review discusses current concepts of lineage relationships and systems’ cross talk, highlights open questions, and describes tools for studying monocyte and macrophage subsets in the murine and human heart.
heart failure; myocardial infarction; healing; monocyte; macrophage
During the inflammatory response that drives atherogenesis, macrophages accumulate progressively in the expanding arterial wall1,2. The observation that circulating monocytes give rise to lesional macrophages3–9 has reinforced the concept that monocyte infiltration dictates macrophage build-up. Recent work indicates, however, that macrophages do not depend on monocytes in some inflammatory contexts10. We therefore revisited the mechanism of macrophage accumulation in atherosclerosis. We show that murine atherosclerotic lesions experience a surprisingly rapid, 4-week, cell turnover. Replenishment of macrophages in these experimental atheromata depends predominantly on local macrophage proliferation rather than monocyte influx. The microenvironment orchestrates macrophage proliferation via the involvement of scavenger receptor (SR)-A. Our study reveals macrophage proliferation as a key event in atherosclerosis and identifies macrophage self-renewal as a therapeutic target for cardiovascular disease.
Macrophages frequently infiltrate tumors and can enhance cancer growth, yet the origins of the macrophage response are not well understood. Here we address molecular mechanisms of macrophage production in a conditional mouse model of lung adenocarcinoma. We report that over-production of the peptide hormone Angiotensin II (AngII) in tumor-bearing mice amplifies self-renewing hematopoietic stem cells (HSCs) and macrophage progenitors. The process occurred in the spleen but not the bone marrow, and was independent of hemodynamic changes. The effects of AngII required direct hormone ligation on HSCs, depended on S1P1 signaling, and allowed the extramedullary tissue to supply new tumor-associated macrophages throughout cancer progression. Conversely, blocking AngII production prevented cancer-induced HSC and macrophage progenitor amplification and thus restrained the macrophage response at its source. These findings indicate that AngII acts upstream of a potent macrophage amplification program and that tumors can remotely exploit the hormone’s pathway to stimulate cancer-promoting immunity.
Cardiovascular diseases claim more lives worldwide than any other. Etiologically, the dominant trajectory involves atherosclerosis, a chronic inflammatory process of lipid-rich lesion growth in the vascular wall that can cause life-threatening myocardial infarction (MI). Those who survive MI can develop congestive heart failure, a chronic condition of inadequate pump activity that is frequently fatal. Leukocytes – white blood cells – are important participants at the various stages of cardiovascular disease progression and complication. This review will discuss leukocyte function in atherosclerosis, myocardial infarction, and heart failure.
Macrophages (MΦ) predominate among the inflammatory cells in rejecting allografts. These innate immune cells, in addition to allospecific T cells, can damage cardiomyocytes directly.
Methods and Results
We explored if sensitive PET/CT imaging of MΦ-avid nanoparticles detects rejection of heart allografts in mice. In addition, we employed the imaging method to follow the immunomodulatory impact of angiotensin converting enzyme inhibititor therapy (ACEi) on myeloid cells in allografts. Dextran nanoparticles were derivatized with the PET isotope copper-64 and imaged seven days after transplantation. C57/BL6 recipients of BALB/c allografts displayed robust PET signal (standard uptake value allograft, 2.8±0.3; isograft control, 1.7±0.2; p<0.05). Autoradiography and scintillation counting confirmed the in vivo findings. We then imaged the effects of ACEi (5mg/kg Enalapril). ACEi significantly decreased nanoparticle signal (p<0.05). Histology and flow cytometry showed a reduced number of myeloid cells in the graft, blood and lymph nodes, as well as diminished antigen presentation (p<0.05 versus untreated allografts). ACEi also significantly prolonged allograft survival (12 versus 7 days, p<0.0001).
Nanoparticle MΦ PET-CT detects heart transplant rejection and predicts organ survival by reporting on myeloid cells.
heart transplantation; macrophages; imaging; PET/CT
Macrophages are central regulators of disease progression in both atherosclerosis and myocardial infarction. In atherosclerosis, macrophages are the dominant leukocyte population that influences lesional development. In myocardial infarction, which is caused by atherosclerosis, macrophages accumulate readily and play important roles in inflammation and healing. Molecular imaging has grown considerably as a field and can reveal biological process at the molecular, cellular, and tissue levels. Here we explore how various imaging modalities, from intravital microscopy in mice to organ-level imaging in patients, are contributing to our understanding of macrophages and their progenitors in cardiovascular disease.
From many perspectives, cardiovascular diseases and cancers are fundamentally different. On the one hand, atherosclerosis is a disease of lipid accumulation driven by diet and lifestyle, whereas cancer is an attack “from within” driven by mutations. Nevertheless, studies over the last 20 years have forced us to re-evaluate such a view. We are learning that, among other factors, the immune system is indispensable to the development and progression of both diseases. Its components are not only reactive but can also orchestrate both tumor and atherosclerotic lesion growth. In this Viewpoint, we explore how monocytes, which are key constituents of the immune system, forge links between cardiovascular diseases and cancers.
Monocytes are frequently described as bone marrow-derived precursors of macrophages. Although many studies support this view, we now appreciate that monocytes neither develop exclusively in the bone marrow nor give rise to all macrophages and dendritic cells. In addition to differentiating to specific leukocyte populations, monocytes, as monocytes, are functionally and ontogenically heterogeneous. In this review we will focus on the development and activity of monocytes and their subsets in mice (Ly-6Chigh/low) and humans (CD14+/dim/− CD16+/−) in the context of atherosclerosis and its complications.
monocyte subsets; monocyte heterogeneity; Ly-6C; CD14; CD16; macrophage; dendritic cells; atherosclerosis; myocardial infarction; cardiovascular disease; extramedullary hematopoeisis; inflammation
Exaggerated and prolonged inflammation after myocardial infarction (MI) accelerates left ventricular remodeling. Inflammatory pathways may present a therapeutic target to prevent post-MI heart failure. However, the appropriate magnitude and timing of interventions are largely unknown, in part because noninvasive monitoring tools are lacking. We here employed nanoparticle-facilitated silencing of CCR2, the chemokine receptor that governs inflammatory Ly-6Chigh monocyte subset traffic, to reduce infarct inflammation in apoE−/− mice after MI. We used dual target PET/MRI of transglutaminase factor XIII (FXIII) and myeloperoxidase (MPO) activity to monitor how monocyte subset-targeted RNAi altered infarct inflammation and healing.
Methods and Results
Flow cytometry, gene expression analysis and histology revealed reduced monocyte numbers and enhanced resolution of inflammation in infarcted hearts of apoE−/− mice that were treated with nanoparticle-encapsulated siRNA. To follow extracellular matrix crosslinking non-invasively, we developed a fluorine-18 labeled PET agent (18F-FXIII). Recruitment of MPO-rich inflammatory leukocytes was imaged using a molecular MRI sensor of MPO activity (MPO-Gd). PET/MRI detected anti-inflammatory effects of intravenous nanoparticle-facilitated siRNA therapy (75% decrease of MPO-Gd signal, p<0.05) while 18F-FXIII PET reflected unimpeded matrix crosslinking in the infarct. Silencing of CCR2 during the first week after MI improved ejection fraction on day 21 after MI from 29 to 35% (p<0.05).
CCR2 targeted RNAi reduced recruitment of Ly-6Chigh monocytes, attenuated infarct inflammation and curbed post-MI left ventricular remodeling.
myocardial infarction; remodeling; monocytes; RNAi; PET/MRI
Myeloid cell content in atherosclerotic plaques associates with rupture and thrombosis. Thus, imaging of lesional monocyte and macrophages (Mo/Mϕ) could serve as a biomarker of disease progression and therapeutic intervention.
To noninvasively assess plaque inflammation with dextran nanoparticle-facilitated hybrid PET/MR imaging.
Methods and Results
Using clinically approved building blocks, we systematically developed 13nm polymeric nanoparticles consisting of crosslinked short chain dextrans which were modified with desferoxamine for zirconium-89 radiolabeling (89Zr-DNP) and a near infrared fluorochrome (VT680) for microscopic and cellular validation. Flow cytometry of cells isolated from excised aortas showed DNP uptake predominantly in Mo/Mϕ (76.7%) and lower signal originating from other leukocytes such as neutrophils and lymphocytes (11.8% and 0.7%, p<0.05 versus Mo/Mϕ). DNP colocalized with the myeloid cell marker CD11b on immunohistochemistry. PET/MRI revealed high uptake of 89Zr-DNP in the aortic root of ApoE−/− mice (standard uptake value, ApoE−/− mice versus wild type controls, 1.9±0.28 versus 1.3±0.03, p<0.05), corroborated by ex vivo scintillation counting and autoradiography. Therapeutic silencing of the monocyte-recruiting receptor CCR2 with siRNA decreased 89Zr-DNP plaque signal (p<0.05) and inflammatory gene expression (p<0.05).
Hybrid PET/MR imaging with a 13nm DNP enables noninvasive assessment of inflammation in experimental atherosclerotic plaques and reports on therapeutic efficacy of anti-inflammatory therapy.
PET/MRI; inflammation; atherosclerosis; molecular imaging; nanoparticles
Acute rupture of vulnerable plaques frequently leads to myocardial infarction and stroke. Within the last decades, several cellular and molecular players have been identified that promote atherosclerotic lesion formation, maturation and plaque rupture. It is now widely recognized that inflammation of the vessel wall and distinct leukocyte subsets are involved throughout all phases of atherosclerotic lesion development. The mechanisms that render a stable plaque unstable and prone to rupture, however, remain unknown and the identification of the vulnerable plaque remains a major challenge in cardiovascular medicine. Imaging technologies used in the clinic offer minimal information about the underlying biology and potential risk for rupture. New imaging technologies are therefore being developed, and in the preclinical setting have enabled new and dynamic insights into the vessel wall for a better understanding of this complex disease. Molecular imaging has the potential to track biological processes, such as the activity of cellular and molecular biomarkers in vivo and over time. Similarly, novel imaging technologies specifically detect effects of therapies that aim to stabilize vulnerable plaques and silence vascular inflammation. Here we will review the potential of established and new molecular imaging technologies in the setting of atherosclerosis, and discuss the cumbersome steps required for translating molecular imaging approaches into the clinic.
Molecular imaging; Inflammation; Atherosclerosis
IL-1b signaling augments continued splenic monocyte supply during acute inflammation.
Monocytes (Mo) and macrophages (MΦ) are emerging therapeutic targets in malignant, cardiovascular, and autoimmune disorders. Targeting of Mo/MΦ and their effector functions without compromising innate immunity’s critical defense mechanisms first requires addressing gaps in knowledge about the life cycle of these cells. Here we studied the source, tissue kinetics, and clearance of Mo/MΦ in murine myocardial infarction, a model of acute inflammation after ischemic injury. We found that a) Mo tissue residence time was surprisingly short (20 h); b) Mo recruitment rates were consistently high even days after initiation of inflammation; c) the sustained need of newly made Mo was fostered by extramedullary monocytopoiesis in the spleen; d) splenic monocytopoiesis was regulated by IL-1β; and e) the balance of cell recruitment and local death shifted during resolution of inflammation. Depending on the experimental approach, we measured a 24 h Mo/MΦ exit rate from infarct tissue between 5 and 13% of the tissue cell population. Exited cells were most numerous in the blood, liver, and spleen. Abrogation of extramedullary monocytopoiesis proved deleterious for infarct healing and accelerated the evolution of heart failure. We also detected rapid Mo kinetics in mice with stroke. These findings expand our knowledge of Mo/MΦ flux in acute inflammation and provide the groundwork for novel anti-inflammatory strategies for treating heart failure.
Ralph van Furth and Zanvil A. Cohn proposed, in their classic 1968 paper, that bone marrow-dwelling promonocytes differentiate to monocytes, which then intravasate, circulate and, upon tissue entry, differentiate to sessile macrophages. Since then, understanding of the macrophage family relationship has undergone substantial enhancement and occasional revision. It is currently recognized that, in addition to their role in the bone marrow, hematopoietic progenitors circulate and give rise to their descendants in extramedullary niches. Monocytes, of which there are several subsets, are not merely circulating macrophage precursors, but participate in the immune response in their own right. Macrophages are highly heterogeneous and, recent studies indicate, can arise in the absence of a monocyte intermediate. These spatial and developmental relationships reveal a complex interactive network and underscore the importance of context in evaluating biological systems. The observations have significant implications for how we image, target and treat disease.
hematopoietic progenitors; monocytes; macrophages
Inflammatory monocytes -- but not the non-inflammatory subset -- depend on the chemokine receptor CCR2 for distribution to injured tissue and stimulate disease progression. Precise therapeutic targeting of this inflammatory monocyte subset could spare innate immunity's essential functions for maintenance of homeostasis and thus limit unwanted effects. Here we developed siRNA nanoparticles targeting CCR2 expression in inflammatory monocytes. We identified an optimized lipid nanoparticle and silencing siRNA sequence that when administered systemically, had rapid blood clearance, accumulated in spleen and bone marrow and showed high cellular localization of fluorescently tagged siRNA inside monocytes. Efficient degradation of CCR2 mRNA in monocytes prevented their accumulation in sites of inflammation. Specifically, the treatment attenuated their number in atherosclerotic plaques, reduced infarct size following coronary artery occlusion, prolonged normoglycemia in diabetic mice after pancreatic islet transplantation and resulted in reduced tumor volumes and lower numbers of tumor-associated macrophages. Taken together, siRNA nanoparticle-mediated CCR2 gene silencing in leukocytes selectively modulates functions of innate immune cell subtypes and may allow for the development of specific anti-inflammatory therapy.
Monocytes serve as a central defense system against infection and injury but can also promote pathological inflammatory responses. Considering the evidence that monocytes exist in at least two subsets committed to divergent functions, we investigated whether distinct factors regulate the balance between monocyte subset responses in vivo. We identified a microRNA (miRNA), miR-146a, which is differentially regulated both in mouse (Ly-6Chi/Ly-6Clo) and human (CD14hi/CD14loCD16+) monocyte subsets. The single miRNA controlled the amplitude of the Ly-6Chi monocyte response during inflammatory challenge whereas it did not affect Ly-6Clo cells. miR-146a–mediated regulation was cell-intrinsic and depended on Relb, a member of the non-canonical NF-κB/Rel family, which we identified as a direct miR-146a target. These observations not only provide novel mechanistic insights into the molecular events that regulate responses mediated by committed monocyte precursor populations but also identify novel targets to manipulate Ly-6Chi monocyte responses while sparing Ly-6Clo monocyte activity.
Coagulase-positive Staphylococcus aureus (S. aureus) is the major causal pathogen of acute endocarditis, a rapidly progressing, destructive infection of the heart valves. Bacterial colonization occurs at sites of endothelial damage, where (together with fibrin and platelets) it initiates the formation of abnormal growths known as vegetations. Here we report that an engineered analog of prothrombin detected S. aureus in endocarditic vegetations via noninvasive fluorescence or PET imaging. These prothrombin derivatives bound to staphylocoagulase and intercalated into growing bacterial vegetations. We also present evidence for bacterial quorum sensing in the regulation of staphylocoagulase expression by S. aureus. Staphylocoagulase expression was limited to the growing edge of mature vegetations, where it was exposed to the host and co-localized with the imaging probe. When endocarditis was induced with an S. aureus strain with genetic deletion of coagulases, survival of mice improved, highlighting the role of staphylocoagulase as a virulence factor.
endocarditis; staphylocoagulase; prothrombin; noninvasive imaging; von Willebrand factor binding protein
Recognition and clearance of bacterial infection is a fundamental property of innate immunity. Here we describe an effector B cell population that protects against microbial sepsis. Innate response activator (IRA)-B cells are phenotypically and functionally distinct, develop and diverge from B1a B cells, depend on pattern recognition receptors, and produce GM-CSF. Specific deletion of IRA-B cell activity impairs bacterial clearance, elicits a cytokine storm, and precipitates septic shock. These observations enrich our understanding of innate immunity, position IRA-B cells as gatekeepers of bacterial infection, and identify new treatment avenues for infectious diseases.