Cardiac macrophages promote diastolic dysfunction.

Macrophages populate the healthy myocardium and, depending on their phenotype, may contribute to tissue homeostasis or disease. Their origin and role in diastolic dysfunction, a hallmark of cardiac aging and heart failure with preserved ejection fraction, remain unclear. Here we show that cardiac macrophages expand in humans and mice with diastolic dysfunction, which in mice was induced by either hypertension or advanced age. A higher murine myocardial macrophage density results from monocyte recruitment and increased hematopoiesis in bone marrow and spleen. In humans, we observed a parallel constellation of hematopoietic activation: circulating myeloid cells are more frequent, and splenic 18F-FDG PET/CT imaging signal correlates with echocardiographic indices of diastolic dysfunction. While diastolic dysfunction develops, cardiac macrophages produce IL-10, activate fibroblasts, and stimulate collagen deposition, leading to impaired myocardial relaxation and increased myocardial stiffness. Deletion of IL-10 in macrophages improves diastolic function. These data imply expansion and phenotypic changes of cardiac macrophages as therapeutic targets for cardiac fibrosis leading to diastolic dysfunction.

Osteoblasts remotely supply lung tumors with cancer-promoting SiglecFhigh neutrophils.

Bone marrow-derived myeloid cells can accumulate within tumors and foster cancer outgrowth. Local immune-neoplastic interactions have been intensively investigated, but the contribution of the systemic host environment to tumor growth remains poorly understood. Here, we show in mice and cancer patients (n = 70) that lung adenocarcinomas increase bone stromal activity in the absence of bone metastasis. Animal studies reveal that the cancer-induced bone phenotype involves bone-resident osteocalcin-expressing (Ocn+) osteoblastic cells. These cells promote cancer by remotely supplying a distinct subset of tumor-infiltrating SiglecFhigh neutrophils, which exhibit cancer-promoting properties. Experimentally reducing Ocn+ cell numbers suppresses the neutrophil response and lung tumor outgrowth. These observations posit osteoblasts as remote regulators of lung cancer and identify SiglecFhigh neutrophils as myeloid cell effectors of the osteoblast-driven protumoral response.

The infarcted myocardium solicits GM-CSF for the detrimental oversupply of inflammatory leukocytes.

Myocardial infarction (MI) elicits massive inflammatory leukocyte recruitment to the heart. Here, we hypothesized that excessive leukocyte invasion leads to heart failure and death during acute myocardial ischemia. We found that shortly and transiently after onset of ischemia, human and mouse cardiac fibroblasts produce granulocyte/macrophage colony-stimulating factor (GM-CSF) that acts locally and distally to generate and recruit inflammatory and proteolytic cells. In the heart, fibroblast-derived GM-CSF alerts its neighboring myeloid cells to attract neutrophils and monocytes. The growth factor also reaches the bone marrow, where it stimulates a distinct myeloid-biased progenitor subset. Consequently, hearts of mice deficient in either GM-CSF or its receptor recruit fewer leukocytes and function relatively well, whereas mice producing GM-CSF can succumb from left ventricular rupture, a complication mitigated by anti-GM-CSF therapy. These results identify GM-CSF as both a key contributor to the pathogenesis of MI and a potential therapeutic target, bolstering the idea that GM-CSF is a major orchestrator of the leukocyte supply chain during inflammation.

Polyglucose nanoparticles with renal elimination and macrophage avidity facilitate PET imaging in ischaemic heart disease.

Tissue macrophage numbers vary during health versus disease. Abundant inflammatory macrophages destruct tissues, leading to atherosclerosis, myocardial infarction and heart failure. Emerging therapeutic options create interest in monitoring macrophages in patients. Here we describe positron emission tomography (PET) imaging with 18F-Macroflor, a modified polyglucose nanoparticle with high avidity for macrophages. Due to its small size, Macroflor is excreted renally, a prerequisite for imaging with the isotope flourine-18. The particle's short blood half-life, measured in three species, including a primate, enables macrophage imaging in inflamed cardiovascular tissues. Macroflor enriches in cardiac and plaque macrophages, thereby increasing PET signal in murine infarcts and both mouse and rabbit atherosclerotic plaques. In PET/magnetic resonance imaging (MRI) experiments, Macroflor PET imaging detects changes in macrophage population size while molecular MRI reports on increasing or resolving inflammation. These data suggest that Macroflor PET/MRI could be a clinical tool to non-invasively monitor macrophage biology.

Surface biotinylation of cytotoxic T lymphocytes for in vivo tracking of tumor immunotherapy in murine models.

Currently, there is no stable and flexible method to label and track cytotoxic T lymphocytes (CTLs) in vivo in CTL immunotherapy. We aimed to evaluate whether the sulfo-hydroxysuccinimide (NHS)-biotin-streptavidin (SA) platform could chemically modify the cell surface of CTLs for in vivo tracking. CD8+ T lymphocytes were labeled with sulfo-NHS-biotin under different conditions and then incubated with SA-Alexa647. Labeling efficiency was proportional to sulfo-NHS-biotin concentration. CD8+ T lymphocytes could be labeled with higher efficiency with sulfo-NHS-biotin in DPBS than in RPMI (P < 0.05). Incubation temperature was not a key factor. CTLs maintained sufficient labeling for at least 72 h (P < 0.05), without altering cell viability. After co-culturing labeled CTLs with mouse glioma stem cells (GSCs) engineered to present biotin on their surface, targeting CTLs could specifically target biotin-presenting GSCs and inhibited cell proliferation (P < 0.01) and tumor spheres formation. In a biotin-presenting GSC brain tumor model, targeting CTLs could be detected in biotin-presenting gliomas in mouse brains but not in the non-tumor-bearing contralateral hemispheres (P < 0.05). In vivo fluorescent molecular tomography imaging in a subcutaneous U87 mouse model confirmed that targeting CTLs homed in on the biotin-presenting U87 tumors but not the control U87 tumors. PET imaging with 89Zr-deferoxamine-biotin and SA showed a rapid clearance of the PET signal over 24 h in the control tumor, while only minimally decreased in the targeted tumor. Thus, sulfo-NHS-biotin-SA labeling is an efficient method to noninvasively track the migration of adoptive transferred CTLs and does not alter CTL viability or interfere with CTL-mediated cytotoxic activity.

Proliferation and Recruitment Contribute to Myocardial Macrophage Expansion in Chronic Heart Failure.

RATIONALE: Macrophages reside in the healthy myocardium, participate in ischemic heart disease, and modulate myocardial infarction (MI) healing. Their origin and roles in post-MI remodeling of nonischemic remote myocardium, however, remain unclear. OBJECTIVE: This study investigated the number, origin, phenotype, and function of remote cardiac macrophages residing in the nonischemic myocardium in mice with chronic heart failure after coronary ligation. METHODS AND RESULTS: Eight weeks post MI, fate mapping and flow cytometry revealed that a 2.9-fold increase in remote macrophages results from both increased local macrophage proliferation and monocyte recruitment. Heart failure produced by extensive MI, through activation of the sympathetic nervous system, expanded medullary and extramedullary hematopoiesis. Circulating Ly6C(high) monocytes rose from 64±5 to 108±9 per microliter of blood (P<0.05). Cardiac monocyte recruitment declined in Ccr2(-/-) mice, reducing macrophage numbers in the failing myocardium. Mechanical strain of primary murine and human macrophage cultures promoted cell cycle entry, suggesting that the increased wall tension in post-MI heart failure stimulates local macrophage proliferation. Strained cells activated the mitogen-activated protein kinase pathway, whereas specific inhibitors of this pathway reduced macrophage proliferation in strained cell cultures and in the failing myocardium (P<0.05). Steady-state cardiac macrophages, monocyte-derived macrophages, and locally sourced macrophages isolated from failing myocardium expressed different genes in a pattern distinct from the M1/M2 macrophage polarization paradigm. In vivo silencing of endothelial cell adhesion molecules curbed post-MI monocyte recruitment to the remote myocardium and preserved ejection fraction (27.4±2.4 versus 19.1±2%; P<0.05). CONCLUSIONS: Myocardial failure is influenced by an altered myeloid cell repertoire.

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.

RNAi targeting multiple cell adhesion molecules reduces immune cell recruitment and vascular inflammation after myocardial infarction.

Myocardial infarction (MI) leads to a systemic surge of vascular inflammation in mice and humans, resulting in secondary ischemic complications and high mortality. We show that, in ApoE(-/-) mice with coronary ligation, increased sympathetic tone up-regulates not only hematopoietic leukocyte production but also plaque endothelial expression of adhesion molecules. To counteract the resulting arterial leukocyte recruitment, we developed nanoparticle-based RNA interference (RNAi) that effectively silences five key adhesion molecules. Simultaneously encapsulating small interfering RNA (siRNA)-targeting intercellular cell adhesion molecules 1 and 2 (Icam1 and Icam2), vascular cell adhesion molecule 1 (Vcam1), and E- and P-selectins (Sele and Selp) into polymeric endothelial-avid nanoparticles reduced post-MI neutrophil and monocyte recruitment into atherosclerotic lesions and decreased matrix-degrading plaque protease activity. Five-gene combination RNAi also curtailed leukocyte recruitment to ischemic myocardium. Therefore, targeted multigene silencing may prevent complications after acute MI.

Targeting Interleukin-1ß Reduces Leukocyte Production After Acute Myocardial Infarction.

BACKGROUND: Myocardial infarction (MI) is an ischemic wound that recruits millions of leukocytes. MI-associated blood leukocytosis correlates inversely with patient survival, yet the signals driving heightened leukocyte production after MI remain incompletely understood. METHODS AND RESULTS: With the use of parabiosis surgery, this study shows that soluble danger signals, among them interleukin-1ß, increase bone marrow hematopoietic stem cell proliferation after MI. Data obtained in bone marrow reconstitution experiments reveal that interleukin-1ß enhances hematopoietic stem cell proliferation by both direct actions on hematopoietic cells and through modulation of the bone marrow's hematopoietic microenvironment. An antibody that neutralizes interleukin-1ß suppresses these effects. Anti-interleukin-1ß treatment dampens the post-MI increase in hematopoietic stem cell proliferation. Consequently, decreased leukocyte numbers in the blood and infarct reduce inflammation and diminish post-MI heart failure in ApoE(-/-) mice with atherosclerosis. CONCLUSIONS: The presented insight into post-MI bone marrow activation identifies a mechanistic target for muting inflammation in the ischemically damaged heart.

Lp-PLA2 Antagonizes Left Ventricular Healing After Myocardial Infarction by Impairing the Appearance of Reparative Macrophages.

BACKGROUND: Healing after myocardial infarction (MI) involves the biphasic accumulation of inflammatory Ly-6C(high) and reparative Ly-6C(low) monocytes/macrophages. Excessive inflammation disrupts the balance between the 2 phases, impairs infarct healing, and contributes to left ventricle remodeling and heart failure. Lipoprotein-associated phospholipase A2 (Lp-PLA2), a member of the phospholipase A2 family of enzymes, produced predominantly by leukocytes, participates in host defenses and disease. Elevated Lp-PLA2 levels associate with increased risk of cardiovascular events across diverse patient populations, but the mechanisms by which the enzyme elicits its effects remain unclear. This study tested the role of Lp-PLA2 in healing after MI. METHODS AND RESULTS: In response to MI, Lp-PLA2 levels markedly increased in the circulation. To test the functional importance of Lp-PLA2, we generated chimeric mice whose bone marrow-derived leukocytes were Lp-PLA2-deficient (bmLp-PLA2 (-/-)). Compared with wild-type controls, bmLp-PLA2 (-/-) mice subjected to MI had lower serum levels of inflammatory cytokines tumor necrosis factor-α, interleukin (IL)-1ß, and IL-6, and decreased number of circulating inflammatory myeloid cells. Accordingly, bmLp-PLA2 (-/-) mice developed smaller and less inflamed infarcts with reduced numbers of infiltrating neutrophils and inflammatory Ly-6C(high) monocytes. During the later, reparative phase, infarcts of bmLp-PLA2 (-/-) mice contained Ly-6C(low) macrophages with a skewed M2-prone gene expression signature, increased collagen deposition, fewer inflammatory cells, and improved indices of angiogenesis. Consequently, the hearts of bmLp-PLA2 (-/-) mice healed more efficiently, as determined by improved left ventricle remodeling and ejection fraction. CONCLUSIONS: Lp-PLA2 augments the inflammatory response after MI and antagonizes healing by disrupting the balance between inflammation and repair, providing a rationale for focused study of ventricular function and heart failure after targeting this enzyme acutely in MI.

In vivo silencing of the transcription factor IRF5 reprograms the macrophage phenotype and improves infarct healing.

OBJECTIVES: The aim of this study was to test whether silencing of the transcription factor interferon regulatory factor 5 (IRF5) in cardiac macrophages improves infarct healing and attenuates post-myocardial infarction (MI) remodeling. BACKGROUND: In healing wounds, the M1 toward M2 macrophage phenotype transition supports resolution of inflammation and tissue repair. Persistence of inflammatory M1 macrophages may derail healing and compromise organ functions. The transcription factor IRF5 up-regulates genes associated with M1 macrophages. METHODS: Here we used nanoparticle-delivered small interfering ribonucleic acid (siRNA) to silence IRF5 in macrophages residing in MIs and in surgically-induced skin wounds in mice. RESULTS: Infarct macrophages expressed high levels of IRF5 during the early inflammatory wound-healing stages (day 4 after coronary ligation), whereas expression of the transcription factor decreased during the resolution of inflammation (day 8). Following in vitro screening, we identified an siRNA sequence that, when delivered by nanoparticles to wound macrophages, efficiently suppressed expression of IRF5 in vivo. Reduction of IRF5 expression, a factor that regulates macrophage polarization, reduced expression of inflammatory M1 macrophage markers, supported resolution of inflammation, accelerated cutaneous and infarct healing, and attenuated development of post-MI heart failure after coronary ligation as measured by protease targeted fluorescence molecular tomography-computed tomography imaging and cardiac magnetic resonance imaging (p < 0.05). CONCLUSIONS: This work identified a new therapeutic avenue to augment resolution of inflammation in healing infarcts by macrophage phenotype manipulation. This therapeutic concept may be used to attenuate post-MI remodeling and heart failure.