Silencing of CCR2 in myocarditis.

BACKGROUND: Myocarditis is characterized by inflammatory cell infiltration of the heart and subsequent deterioration of cardiac function. Monocytes are the most prominent population of accumulating leucocytes. We investigated whether in vivo administration of nanoparticle-encapsulated siRNA targeting chemokine (C-C motif) receptor 2 (CCR2)-a chemokine receptor crucial for leucocyte migration in humans and mice--reduces inflammation in autoimmune myocarditis. METHODS AND RESULTS: In myocardium of patients with myocarditis, CCL2 mRNA levels and CCR2(+) cells increased (P < 0.05), motivating us to pursue CCR2 silencing. Flow cytometric analysis showed that siRNA silencing of CCR2 (siCCR2) reduced the number of Ly6C(high) monocytes in hearts of mice with acute autoimmune myocarditis by 69% (P < 0.05), corroborated by histological assessment. The nanoparticle-delivered siRNA was not only active in monocytes but also in bone marrow haematopoietic progenitor cells. Treatment with siCCR2 reduced the migration of bone marrow granulocyte macrophage progenitors into the blood. Cellular magnetic resonance imaging (MRI) after injection of macrophage-avid magnetic nanoparticles detected myocarditis and therapeutic effects of RNAi non-invasively. Mice with acute myocarditis showed enhanced macrophage MRI contrast, which was prevented by siCCR2 (P < 0.05). Follow-up MRI volumetry revealed that siCCR2 treatment improved ejection fraction (P < 0.05 vs. control siRNA-treated mice). CONCLUSION: This study highlights the importance of CCR2 in the pathogenesis of myocarditis. In addition, we show that siCCR2 affects leucocyte progenitor trafficking. The data also point to a novel therapeutic strategy for the treatment of myocarditis.

Modified mRNA directs the fate of heart progenitor cells and induces vascular regeneration after myocardial infarction.

In a cell-free approach to regenerative therapeutics, transient application of paracrine factors in vivo could be used to alter the behavior and fate of progenitor cells to achieve sustained clinical benefits. Here we show that intramyocardial injection of synthetic modified RNA (modRNA) encoding human vascular endothelial growth factor-A (VEGF-A) results in the expansion and directed differentiation of endogenous heart progenitors in a mouse myocardial infarction model. VEGF-A modRNA markedly improved heart function and enhanced long-term survival of recipients. This improvement was in part due to mobilization of epicardial progenitor cells and redirection of their differentiation toward cardiovascular cell types. Direct in vivo comparison with DNA vectors and temporal control with VEGF inhibitors revealed the greatly increased efficacy of pulse-like delivery of VEGF-A. Our results suggest that modRNA is a versatile approach for expressing paracrine factors as cell fate switches to control progenitor cell fate and thereby enhance long-term organ repair.

Local proliferation dominates lesional macrophage accumulation in atherosclerosis.

During the inflammatory response that drives atherogenesis, macrophages accumulate progressively in the expanding arterial wall. The observation that circulating monocytes give rise to lesional macrophages has reinforced the concept that monocyte infiltration dictates macrophage buildup. Recent work has indicated, however, that macrophage accumulation does not depend on monocyte recruitment in some inflammatory contexts. We therefore revisited the mechanism underlying macrophage accumulation in atherosclerosis. In murine atherosclerotic lesions, we found that macrophages turn over rapidly, after 4 weeks. Replenishment of macrophages in these experimental atheromata depends predominantly on local macrophage proliferation rather than monocyte influx. The microenvironment orchestrates macrophage proliferation through the involvement of scavenger receptor A (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.

Angiotensin II drives the production of tumor-promoting macrophages.

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 overproduction 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 S1P(1) 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.

Implantable microenvironments to attract hematopoietic stem/cancer cells.

The environments that harbor hematopoietic stem and progenitor cells are critical to explore for a better understanding of hematopoiesis during health and disease. These compartments often are inaccessible for controlled and rapid experimentation, thus limiting studies to the evaluation of conventional cell culture and transgenic animal models. Here we describe the manufacture and image-guided monitoring of an engineered microenvironment with user-defined properties that recruits hematopoietic progenitors into the implant. Using intravital imaging and fluorescence molecular tomography, we show in real time that the cell homing and retention process is efficient and durable for short- and long-term engraftment studies. Our results indicate that bone marrow stromal cells, precoated on the implant, accelerate the formation of new sinusoidal blood vessels with vascular integrity at the microcapillary level that enhances the recruitment hematopoietic progenitor cells to the site. This implantable construct can serve as a tool enabling the study of hematopoiesis.

Myocardial infarction accelerates atherosclerosis.

During progression of atherosclerosis, myeloid cells destabilize lipid-rich plaques in the arterial wall and cause their 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 ischaemic 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. Seeking the source of surplus monocytes in plaques, we found that myocardial infarction liberated haematopoietic stem and progenitor cells from bone marrow niches via sympathetic nervous system signalling. 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.

Sensitive and direct detection of circulating tumor cells by multimarker µ-nuclear magnetic resonance.

Identifying circulating tumor cells (CTCs) with greater sensitivity could facilitate early detection of cancer and rapid assessment of treatment response. Most current technologies use EpCAM expression as a CTC identifier. However, given that a significant fraction of cancer patients have low or even absent EpCAM levels, there is a need for better detection methods. Here, we hypothesize that a multimarker strategy combined with direct sensing of CTC in whole blood would increase the detection of CTC in patients. Accordingly, molecular profiling of biopsies from a patient cohort revealed a four-marker set (EpCAM, HER-2, EGFR, and MUC-1) capable of effectively differentiating cancer cells from normal host cells. Using a point-of-care micro-nuclear magnetic resonance (µNMR) system, we consequently show that this multimarker combination readily detects individual CTC directly in whole blood without the need for primary purification. We also confirm these results in a comparative trial of patients with ovarian cancer. This platform could potentially benefit a broad range of applications in clinical oncology.

Intact B7-H3 signaling promotes allograft prolongation through preferential suppression of Th1 effector responses.

Ligands of the B7 family provide both positive and negative costimulatory signals to the CD28 family of receptors on T lymphocytes, the balance of which determines the immune response. B7-H3 is a member of the B7 family whose function in T-cell activation has been the subject of some controversy: in autoimmunity and tumor immunity, it has been described as both costimulatory and coinhibitory, while in transplantation, B7-H3 signaling is thought to contribute to graft rejection. However, we now demonstrate results to the contrary. Signaling through a putative B7-H3 receptor prolonged allograft survival in a fully MHC-mismatched cardiac model and promoted a shift toward a Th2 milieu; conversely, B7-H3 blockade, achieved by use of a blocking antibody, resulted in accelerated rejection, an effect associated with enhanced IFN-γ production. Finally, graft prolongation achieved by CTLA4 Ig was shortened both by B7-H3 blockade and the absence of recipient B7-H3. These findings suggest a coinhibitory role for B7-H3. However, experience with other CD28/B7 family members suggests that immune redundancy plays a crucial role in determining the functions of various pathways. Given the abundance of conflicting data, it is plausible that, under differing conditions, B7-H3 may have both positive and negative costimulatory functions.

In vivo imaging of drug-induced mitochondrial outer membrane permeabilization at single-cell resolution.

Observing drug responses in the tumor microenvironment in vivo can be technically challenging. As a result, cellular responses to molecularly targeted cancer drugs are often studied in cell culture, which does not accurately represent the behavior of cancer cells growing in vivo. Using high-resolution microscopy and fluorescently labeled genetic reporters for apoptosis, we developed an approach to visualize drug-induced cell death at single-cell resolution in vivo. Stable expression of the mitochondrial intermembrane protein IMS-RP was established in human breast and pancreatic cancer cells. Image analysis was then used to quantify release of IMS-RP into the cytoplasm upon apoptosis and irreversible mitochondrial permeabilization. Both breast and pancreatic cancer cells showed higher basal apoptotic rates in vivo than in culture. To study drug-induced apoptosis, we exposed tumor cells to navitoclax (ABT-263), an inhibitor of Bcl-2, Bcl-xL, and Bcl-w, both in vitro and in vivo. Although the tumors responded to Bcl-2 inhibition in vivo, inducing apoptosis in around 20% of cancer cells, the observed response was much higher in cell culture. Together, our findings show an imaging technique that can be used to directly visualize cell death within the tumor microenvironment in response to drug treatment.

Origins of tumor-associated macrophages and neutrophils.

Tumor-associated macrophages (TAMs) and tumor-associated neutrophils (TANs) can control cancer growth and exist in almost all solid neoplasms. The cells are known to descend from immature monocytic and granulocytic cells, respectively, which are produced in the bone marrow. However, the spleen is also a recently identified reservoir of monocytes, which can play a significant role in the inflammatory response that follows acute injury. Here, we evaluated the role of the splenic reservoir in a genetic mouse model of lung adenocarcinoma driven by activation of oncogenic Kras and inactivation of p53. We found that high numbers of TAM and TAN precursors physically relocated from the spleen to the tumor stroma, and that recruitment of tumor-promoting spleen-derived TAMs required signaling of the chemokine receptor CCR2. Also, removal of the spleen, either before or after tumor initiation, reduced TAM and TAN responses significantly and delayed tumor growth. The mechanism by which the spleen was able to maintain its reservoir capacity throughout tumor progression involved, in part, local accumulation in the splenic red pulp of typically rare extramedullary hematopoietic stem and progenitor cells, notably granulocyte and macrophage progenitors, which produced CD11b(+) Ly-6C(hi) monocytic and CD11b(+) Ly-6G(hi) granulocytic cells locally. Splenic granulocyte and macrophage progenitors and their descendants were likewise identified in clinical specimens. The present study sheds light on the origins of TAMs and TANs, and positions the spleen as an important extramedullary site, which can continuously supply growing tumors with these cells.

Innate response activator B cells protect against microbial sepsis.

Recognition and clearance of a bacterial infection are a fundamental properties 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 granulocyte-macrophage colony-stimulating factor. 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.

Rapid monocyte kinetics in acute myocardial infarction are sustained by extramedullary monocytopoiesis.

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-1ß; 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.

PET/MRI of inflammation in myocardial infarction.

OBJECTIVES: The aim of this study was to explore post-myocardial infarction (MI) myocardial inflammation. BACKGROUND: Innate immune cells are centrally involved in infarct healing and are emerging therapeutic targets in cardiovascular disease; however, clinical tools to assess their presence in tissue are scarce. Furthermore, it is currently not known if the nonischemic remote zone recruits monocytes. METHODS: Acute inflammation was followed in mice with coronary ligation by 18-fluorodeoxyglucose ((18)FDG) positron emission tomography/magnetic resonance imaging, fluorescence-activated cell sorting, polymerase chain reaction, and histology. RESULTS: Gd-DTPA-enhanced infarcts showed high (18)FDG uptake on day 5 after MI. Cell depletion and isolation data confirmed that this largely reflected inflammation; CD11b(+) cells had 4-fold higher (18)FDG uptake than the infarct tissue from which they were isolated (p < 0.01). Surprisingly, there was considerable monocyte recruitment in the remote myocardium (approximately 10(4)/mg of myocardium, 5.6-fold increase; p < 0.01), a finding mirrored by macrophage infiltration in the remote myocardium of patients with acute MI. Temporal kinetics of cell recruitment were slower than in the infarct, with peak numbers on day 10 after ischemia. Quantitative polymerase chain reaction showed a robust increase of recruiting adhesion molecules and chemokines in the remote myocardium (e.g., 12-fold increase of monocyte chemoattractant protein-1), although levels were always lower than in the infarct. Finally, matrix metalloproteinase activity was significantly increased in noninfarcted myocardium, suggesting that monocyte recruitment to the remote zone may contribute to post-MI dilation. CONCLUSIONS: This study shed light on the innate inflammatory response in remote myocardium after MI.

Extramedullary hematopoiesis generates Ly-6C(high) monocytes that infiltrate atherosclerotic lesions.

BACKGROUND: Atherosclerotic lesions are believed to grow via the recruitment of bone marrow-derived monocytes. Among the known murine monocyte subsets, Ly-6C(high) monocytes are inflammatory, accumulate in lesions preferentially, and differentiate. Here, we hypothesized that the bone marrow outsources the production of Ly-6C(high) monocytes during atherosclerosis. METHODS AND RESULTS: Using murine models of atherosclerosis and fate-mapping approaches, we show that hematopoietic stem and progenitor cells progressively relocate from the bone marrow to the splenic red pulp, where they encounter granulocyte macrophage colony-stimulating factor and interleukin-3, clonally expand, and differentiate to Ly-6C(high) monocytes. Monocytes born in such extramedullary niches intravasate, circulate, and accumulate abundantly in atheromata. On lesional infiltration, Ly-6C(high) monocytes secrete inflammatory cytokines, reactive oxygen species, and proteases. Eventually, they ingest lipids and become foam cells. CONCLUSIONS: Our findings indicate that extramedullary sites supplement the hematopoietic function of the bone marrow by producing circulating inflammatory cells that infiltrate atherosclerotic lesions.

Nanoparticle-mediated measurement of target-drug binding in cancer cells.

Responses to molecularly targeted therapies can be highly variable and depend on mutations, fluctuations in target protein levels in individual cells, and drug delivery. The ability to rapidly quantitate drug response in cells harvested from patients in a point-of-care setting would have far reaching implications. Capitalizing on recent developments with miniaturized NMR technologies, we have developed a magnetic nanoparticle-based approach to directly measure both target expression and drug binding in scant human cells. The method involves covalent conjugation of the small-molecule drug to a magnetic nanoparticle that is then used as a read-out for target expression and drug-binding affinity. Using poly(ADP-ribose) polymerase (PARP) inhibition as a model system, we developed an approach to distinguish differential expression of PARP in scant cells with excellent correlation to gold standards, the ability to mimic drug pharmacodynamics ex vivo through competitive target-drug binding, and the potential to perform such measurements in clinical samples.

Therapeutic siRNA silencing in inflammatory monocytes in mice.

Excessive and prolonged activity of inflammatory monocytes is a hallmark of many diseases with an inflammatory component. In such conditions, precise targeting of these cells could be therapeutically beneficial while sparing many essential functions of the innate immune system, thus limiting unwanted effects. Inflammatory monocytes-but not the noninflammatory subset-depend on the chemokine receptor CCR2 for localization to injured tissue. Here we present an optimized lipid nanoparticle and a CCR2-silencing short interfering RNA that, when administered systemically in mice, show rapid blood clearance, accumulate in spleen and bone marrow, and localize to monocytes. Efficient degradation of CCR2 mRNA in monocytes prevents their accumulation in sites of inflammation. Specifically, the treatment attenuates their number in atherosclerotic plaques, reduces infarct size after coronary artery occlusion, prolongs normoglycemia in diabetic mice after pancreatic islet transplantation, and results in reduced tumor volumes and lower numbers of tumor-associated macrophages.