Systems Biology

Cells in complex organisms undergo frequent functional changes, but few methods allow comprehensive longitudinal profiling of living cells. Here we introduce scission-accelerated fluorophore exchange (SAFE), a method for multiplexed temporospatial imaging of living cells with immunofluorescence. SAFE uses a rapid bioorthogonal click chemistry to remove immunofluorescent signals from the surface of labeled cells, cycling the nanomolar-concentration reagents in seconds and enabling multiple rounds of staining of the same samples. It is non-toxic and functional in both dispersed cells and intact living tissues. We demonstrate multiparameter (n >= 14), non-disruptive imaging of murine peripheral blood mononuclear and bone marrow cells to profile cellular differentiation. We also show longitudinal multiplexed imaging of bone marrow progenitor cells as they develop into neutrophils over 6 days and real-time multiplexed cycling of living mouse hepatic tissues. We anticipate that SAFE will find broad utility for investigating physiologic dynamics in living systems. Live cells and tissues are imaged over long time periods using a fast, non-toxic click chemistry.

Interactions between the immune and central nervous systems strongly influence brain health. Although the blood-brain barrier restricts this crosstalk, we now know that meningeal gateways through brain border tissues facilitate intersystem communication. Cerebrospinal fluid (CSF), which interfaces with the glymphatic system and thereby drains the brain’s interstitial and perivascular spaces, facilitates outward signaling beyond the blood-brain barrier. In the present study, we report that CSF can exit into the skull bone marrow. Fluorescent tracers injected into the cisterna magna of mice migrate along perivascular spaces of dural blood vessels and then travel through hundreds of sub-millimeter skull channels into the calvarial marrow. During meningitis, bacteria hijack this route to invade the skull’s hematopoietic niches and initiate cranial hematopoiesis ahead of remote tibial sites. As skull channels also directly provide leukocytes to meninges, the privileged sampling of brain-derived danger signals in CSF by regional marrow may have broad implications for inflammatory neurological disorders. This manuscript describes a new cerebral spinal fluid exit route via hundreds of skull channels, with the cranial bone marrow as a destination. In meningitis, bacteria hijack this path and alert hematopoietic stem cells residing in the skull marrow.

Communication within the glial cell ecosystem is essential for neuronal and brain health(1-3). The influence of glial cells on the accumulation and clearance of beta-amyloid (A beta) and neurofibrillary tau in the brains of individuals with Alzheimer’s disease (AD) is poorly understood, despite growing awareness that these are therapeutically important interactions(4,5). Here we show, in humans and mice, that astrocyte-sourced interleukin-3 (IL-3) programs microglia to ameliorate the pathology of AD. Upon recognition of A beta deposits, microglia increase their expression of IL-3R alpha-the specific receptor for IL-3 (also known as CD123)-making them responsive to IL-3. Astrocytes constitutively produce IL-3, which elicits transcriptional, morphological, and functional programming of microglia to endow them with an acute immune response program, enhanced motility, and the capacity to cluster and clear aggregates of A beta and tau. These changes restrict AD pathology and cognitive decline. Our findings identify IL-3 as a key mediator of astrocyte-microglia cross-talk and a node for therapeutic intervention in AD.

BACKGROUND Recurrent myocardial infarction (MI) is common in patients with coronary artery disease and is associated with high mortality. Long-term reprogramming of myeloid progenitors occurs in response to inflammatory stimuli and alters the organism’s response to secondary inflammatory challenges. OBJECTIVES This study examined the effect of recurrent MI on bone marrow response and cardiac inflammation. METHODS The investigators developed a surgical mouse model in which 2 subsequent MIs affected different left ventricular regions in the same mouse. Recurrent MI was induced by ligating the left circumflex artery followed by the left anterior descending coronary artery branch. The study characterized the resulting ischemia by whole-heart fluorescent coronary angiography after optical organ clearing and by cardiac magnetic resonance imaging. RESULTS A first MI-induced bone marrow "memory" via a drculating signal, reducing hematopoietic maintenance factor expression in bone marrow macrophages. This dampened the organism’s reaction to subsequent events. Despite a similar extent of injury according to troponin levels, recurrent MI caused reduced emergency hematopoiesis and less leukocytosis than a first MI. Consequently, fewer leukocytes migrated to the ischemic myocardium. The hematopoietic response to lipopolysaccharide was also mitigated after a previous ML The increase of white blood count in 28 patients was lower after recurrent MI compared with their first MI. CONCLUSIONS The data suggested that hematopoietic and innate immune responses are shaped by a preceding MI. (C) 2020 by the American College of Cardiology Foundation.

Rapid, automated, point-of-care cellular diagnosis of cancer remains difficult in remote settings due to lack of specialists and medical infrastructure. To address the need for same-day diagnosis, we developed an automated image cytometry system (CytoPAN) that allows rapid breast cancer diagnosis of scant cellular specimens obtained by fine needle aspiration (FNA) of palpable mass lesions. The system is devoid of moving parts for stable operations, harnesses optimized antibody kits for multiplexed analysis, and offers a user-friendly interface with automated analysis for rapid diagnoses. Through extensive optimization and validation using cell lines and mouse models, we established breast cancer diagnosis and receptor subtyping in 1 hour using as few as 50 harvested cells. In a prospective patient cohort study (n = 68), we showed that the diagnostic accuracy was 100% for cancer detection and the receptor subtyping accuracy was 96% for human epidermal growth factor receptor 2 and 93% for hormonal receptors (ER/PR), two key biomarkers associated with breast cancer. A combination of FNA and CytoPAN offers faster, less invasive cancer diagnoses than the current standard (core biopsy and histopathology). This approach should enable the ability to more rapidly diagnose breast cancer in global and remote settings.

Background. Extracellular vesicles (EV) are shed by tumor cells but little is known about their individual molecular phenotypes and heterogeneity. While exosomes have received considerable attention, much less is known about larger microvesicles. Here we profile single microvesicles (MV) and exosomes from glioblastoma (GB) cells and MV from the plasma of patients. Methods. EV secreted from mouse glioma GL261 and human primary GBM8 cell lines as well as from the plasma of 8 patients with diagnoses of GB and 2 healthy controls were isolated and processed for single vesicle analysis. EV were immobilized on glass slides and the heterogeneity of vesicle and tumor markers were analyzed at the single vesicle level. Results. We show that (i) MV are abundant, (ii) only a minority of MV expresses putative MV markers, and (iii) MV share tetraspanin biomarkers previously thought to be diagnostic of exosomes. Using MV capture and staining techniques that allow differentiation of host cell and GB-derived MV we further demonstrate that (i) tumoral MV often present as <10% of all MV in GB patient plasma, and (ii) there is extensive heterogeneity in tumor marker expression in these tumor-derived MV. Conclusion. These results indicate that single MV analysis is likely necessary to identify rare tumoral MV populations and the single vesicle analytical technique used here can be applied to both MV and exosome fractions without the need for their separation from each other. These studies form the basis for using single EV analyses for cancer diagnostics.

Innate immune cells recruited to inflammatory sites have short life spans and originate from the marrow, which is distributed throughout the long and flat bones. While bone marrow production and release of leukocyte increases after stroke, it is currently unknown whether its activity rises homogeneously throughout the entire hematopoietic system. To address this question, we employed spectrally resolved in vivo cell labeling in the murine skull and tibia. We show that in murine models of stroke and aseptic meningitis, skull bone marrow-derived neutrophils are more likely to migrate to the adjacent brain tissue than cells that reside in the tibia. Confocal microscopy of the skull-dura interface revealed myeloid cell migration through microscopic vascular channels crossing the inner skull cortex. These observations point to a direct local interaction between the brain and the skull bone marrow through the meninges.

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 F-18-FDGPET/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.

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 alpha V beta 3 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.

Here we present the first generation of two-photon beta cell specific in vivo imaging probes based on GLP1R targeting peptides. Among the three compounds of potential interest, we found quite unexpectedly that a squarine-rotaxane conjugate (2PEx-647) had near ideal in vivo imaging characteristics.

Epithelial cells are generally considered to be static relative to their neighbours. Basal cells in pseudostratified epithelia display a single long cytoplasmic process that can cross the tight junction barrier to reach the lumen. Using in vivo microscopy to visualize the epididymis, a model system for the study of pseudostratified epithelia, we report here the surprising discovery that these basal cell projections-which we call axiopodia-periodically extend and retract over time. We found that axiopodia extensions and retractions follow an oscillatory pattern. This movement, which we refer to as periodic axial motility (PAM), is controlled by c-Src and MEK1/2-ERK1/2. Therapeutic inhibition of tyrosine kinase activity induces a retraction of these projections. Such unexpected cell motility may reflect a novel mechanism by which specialized epithelial cells sample the luminal environment.

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.

The ability to isolate pure pancreatic beta-cells would greatly aid multiple areas of diabetes research. We developed a fluorescent exendin-4-like neopeptide conjugate for the rapid purification and isolation of functional mouse pancreatic beta-cells. By targeting the glucagon-like peptide-1 receptor with the fluorescent conjugate, beta-cells could be quickly isolated by flow cytometry and were >99% insulin positive. These studies were confirmed by immunostaining, microscopy and gene expression profiling on isolated cells. Gene expression profiling studies of cytofluorometrically sorted beta-cells from 4 and 12 week old NOD mice provided new insights into the genetic programs at play of different stages of type-1 diabetes development. The described isolation method should have broad applicability to the beta-cell field.

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.

Exposure to psychosocial stress is a risk factor for many diseases, including atherosclerosis(1,2). Although incompletely understood, interaction between the psyche and the immune system provides one potential mechanism linking stress and disease inception and progression. Known cross-talk 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 reflex(3,4). It remains unknown, however, whether chronic stress changes hematopoietic stem cell activity. Here we show that stress increases proliferation of these most primitive hematopoietic 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. Under conditions of chronic variable stress in mice, sympathetic nerve fibers released surplus noradrenaline, which signaled bone marrow niche cells to decrease CXCL12 levels through the beta(3)-adrenergic receptor. Consequently, hematopoietic stem cell proliferation was elevated, leading to an increased output of neutrophils and inflammatory monocytes. When atherosclerosis-prone Apoe(-/-) mice were subjected to chronic stress, accelerated hematopoiesis promoted plaque features associated with vulnerable lesions that cause myocardial infarction and stroke in humans.

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. Cancer Res; 72(12); 2949-56. (C)2012 AACR.

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 micro-capillary level that enhances the recruitment hematopoietic progenitor cells to the site. This implantable construct can serve as a tool enabling the study of hematopoiesis.

New approaches that allow precise spatiotemporal control of gene expression in model organisms at the single cell level are necessary to better dissect the role of specific genes and cell populations in development, disease, and therapy. Here, we describe a new optochemogenetic switch (OCG switch) to control CreER/loxP-mediated recombination via photoactivatable ("caged") tamoxifen analogues in individual cells in cell culture, organoid culture, and in vivo in adult mice. This approach opens opportunities to more fully exploit existing CreER transgenic mouse strains to achieve more precise temporal- and location-specific regulation of genetic events and gene expression.

Cell division (mitosis) is a tightly controlled process that is coordinated and regulated by a network of proteins localized in the nucleus. The key stages of mitosis are centrosome maturation, chromosome condensation, nuclear envelope breakdown, centrosome separation, bipolar spindle formation, chromosome separation, and finally cytokinesis. Aurora kinase A (AKA) belongs to the Aurora kinase family of serine/threonine kinases, which have been shown toplaycriticalrolesinmitoticprogression. Duringmitosis, AKA localizes to centrosomes during late S to early G2 phase. As the cell proceeds to metaphase, AKA localizes to the microtubules and near the spindle poles, where it remains until anaphase when it migrates to some extent to the spindle midzone. Finally, during cytokinesis, AKA localizes to the midbody. Whilst localized to these specific cellular regions, AKA interacts with and phosphorylates several intracellular targets, including p53, MBD3, and BRCA1, each of which are critical mediators of malignant transformation. The unique stage-specific nuclear and intracellular locations of AKA during mitosis thus make it an interesting imaging target

The hallmark of type 1 diabetes is autoimmune destruction of the insulin-producing beta-cells of the pancreatic islets. Autoimmune diabetes has been difficult to study or treat because it is not usually diagnosed until substantial beta-cell loss has already occurred. Imaging agents that permit noninvasive visualization of changes in beta-cell mass remain a high-priority goal. We report on the development and testing of a near-infrared fluorescent beta-cell imaging agent. Based on the amino acid sequence of exendin-4, we created a neopeptide via introduction of an unnatural amino acid at the K(12) position, which could subsequently be conjugated to fluorophores via bioorthogonal copper-catalyzed click-chemistry. Cell assays confirmed that the resulting fluorescent probe (E4(x12)-VT750) had a high binding affinity (similar to 3 nM). Its in vivo properties were evaluated using high-resolution intravital imaging, histology, whole-pancreas visualization, and endoscopic imaging. According to intravital microscopy, the probe rapidly bound to beta-cells and, as demonstrated by confocal microscopy, it was internalized. Histology of the whole pancreas showed a close correspondence between fluorescence and insulin staining, and there was an excellent correlation between imaging signals and beta-cell mass in mice treated with streptozotocin, a beta-cell toxin. Individual islets could also be visualized by endoscopic imaging. In short, E4(x12)-VT750 showed strong and selective binding to glucose-like peptide-1 receptors and permitted accurate measurement of beta-cell mass in both diabetic and nondiabetic mice. This near-infrared imaging probe, as well as future radioisotope-labeled versions of it, should prove to be important tools for monitoring diabetes, progression, and treatment in both experimental and clinical contexts.

Background The vertebrate limb is a classical model for understanding patterning of three-dimensional structures during embryonic development Although decades of research have elucidated the tissue and molecular interactions within the limb bud required for patterning and morphogenesis of the limb, the cellular and molecular events that shape the limb bud itself have remained largely unknown Results We show that the mesenchymal cells of the early limb bud are not disorganized within the ectoderm as previously thought but are instead highly organized and polarized Using time-lapse video microscopy, we demonstrate that cells move and divide according to this orientation The combination of oriented cell divisions and movements drives the proximaldistal elongation of the limb bud necessary to set the stage for subsequent morphogenesis These cellular events are regulated by the combined activities of the WNT and FGF pathways We show that WNT5A/JNK is necessary for the proper orientation of cell movements and cell division In contrast, the FGF/MAPK signaling pathway, emanating from the apical ectodermal ridge, does not regulate cell orientation in the limb bud but instead establishes a gradient of cell velocity enabling continuous rearrangement of the cells at the distal tip of the limb Conclusions Together these data shed light on the cellular basis of vertebrate limb bud morphogenesis and uncover new layers to the sequential signaling pathways acting during vertebrate limb development

Eosinophils are multifunctional leukocytes that degrade and remodel tissue extracellular matrix through production of proteolytic enzymes, release of proinflammatory factors to initiate and propagate inflammatory responses, and direct activation of mucus secretion and smooth muscle cell constriction. Thus, eosinophils are central effector cells during allergic airway inflammation and an important clinical therapeutic target. Here we describe the use of an injectable MMP-targeted optical sensor that specifically and quantitatively resolves eosinophil activity in the lungs of mice with experimental allergic airway inflammation. Through the use of real-time molecular imaging methods, we report the visualization of eosinophil. responses in vivo and at different scales. Eosinophil responses were seen at single-cell resolution in conducting airways using near-infrared fluorescence fiberoptic bronchoscopy, in lung parenchyma using intravital microscopy, and in the whole body using fluorescence-mediated molecular tomography. Using these real-time imaging methods, we confirmed the immunosuppressive effects of the glucocorticoid drug dexamethasone in the mouse model of allergic airway inflammation and identified a viridin-derived prodrug that potently inhibited the accumulation and enzyme activity of eosinophils in the lungs. The combination of sensitive enzyme-targeted sensors with noninvasive molecular imaging approaches permitted evaluation of airway inflammation severity and was used as a model to rapidly screen for new drug effects. Both fluorescence-mediated tomography and fiberoptic bronchoscopy techniques have the potential to be translated into the clinic.