Glucocorticoids Regulate Bone Marrow B Lymphopoiesis After Stroke.

RATIONALE: After a stroke, patients frequently experience altered systemic immunity resulting in peripheral immunosuppression and higher susceptibility to infections, which is at least partly attributed to lymphopenia. The mechanisms that profoundly change the systemic leukocyte repertoire after stroke are incompletely understood. Emerging evidence indicates that stroke alters hematopoietic output of the bone marrow. OBJECTIVE: To explore the mechanisms that lead to defects of B lymphopoiesis after ischemic stroke. METHODS AND RESULTS: We here report that ischemic stroke triggers brain-bone marrow communication via hormonal long-range signals that regulate hematopoietic B lineage decisions. Bone marrow fluorescence-activated cell sorter analyses and serial intravital microscopy indicate that transient middle cerebral artery occlusion in mice arrests B-cell development beginning at the pro-B-cell stage. This phenotype was not rescued in Myd88-/- and TLR4-/- mice with disrupted TLR (Toll-like receptor) signaling or after blockage of peripheral sympathetic nerves. Mechanistically, we identified stroke-induced glucocorticoid release as the main instigator of B lymphopoiesis defects. B-cell lineage-specific deletion of the GR (glucocorticoid receptor) in CD19-Cre loxP Nr3c1 mice attenuated lymphocytopenia after transient middle cerebral artery. In 20 patients with acute stroke, increased cortisol levels inversely correlated with blood lymphocyte numbers. CONCLUSIONS: Our data demonstrate that the hypothalamic-pituitary-adrenal axis mediates B lymphopoiesis defects after ischemic stroke.

Resident and Monocyte-Derived Macrophages in Cardiovascular Disease.

Macrophages are ubiquitous cells that reside in all major tissues. Counter to long-held beliefs, we now know that resident macrophages in many organs are seeded during embryonic development and self-renew independently from blood monocytes. Under inflammatory conditions, those tissue macrophages are joined and sometimes replaced by recruited monocyte-derived macrophages. Macrophage function in steady state and disease depends on not only their developmental origin but also the tissue environment. Here, we discuss the ontogeny, function, and interplay of tissue-resident and monocyte-derived macrophages in various organs contributing to the development and progression of cardiovascular disease.

Imaging the Vascular Bone Marrow Niche During Inflammatory Stress.

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

New molecular probes of vascular inflammation.

New molecular imaging approaches featuring the assessment of inflammatory processes in the vascular wall on top of existing anatomic and functional vessel imaging procedures could emerge as decisive tools for the understanding and prevention of cardiovascular events. In this respect imaging approaches addressing specific molecular and cellular targets in atherosclerosis are of high interest. This review summarizes the rationale and current status of nuclear imaging probes which possess high translational potential.

Calpain-mediated ataxin-3 cleavage in the molecular pathogenesis of spinocerebellar ataxia type 3 (SCA3).

Spinocerebellar ataxia type 3 (SCA3) is pathologically characterized by the formation of intranuclear aggregates which contain ataxin-3, the mutated protein in SCA3, in a specific subtype of neurons. It has been proposed that ataxin-3 is cleaved by proteolytic enzymes, in particular by calpains and caspases, eventually leading to the formation of aggregates. In our study, we examined the ability of calpains to cleave ataxin-3 in vitro and in vivo. We demonstrated in cell culture and mouse brain homogenates that cleavage of overexpressed ataxin-3 by calpains and in particular by calpain-2 occur and that polyglutamine expanded ataxin-3 is more sensitive to calpain degradation. Based on these results, we investigated the influence of calpains on the pathogenesis of SCA3 in vivo. For this purpose, we enhanced calpain activity in a SCA3 transgenic mouse model by knocking out the endogenous calpain inhibitor calpastatin. Double-mutant mice demonstrated an aggravated neurological phenotype with an increased number of nuclear aggregates and accelerated neurodegeneration in the cerebellum. This study confirms the critical importance of calcium-dependent calpain-type proteases in the pathogenesis of SCA3 and suggests that the manipulation of the ataxin-3 cleavage pathway and the regulation of intracellular calcium homeostasis may represent novel targets for therapeutic intervention in SCA3.

Bone marrow adipocytes fuel emergency hematopoiesis after myocardial infarction.

After myocardial infarction (MI), emergency hematopoiesis produces inflammatory myeloid cells that accelerate atherosclerosis and promote heart failure. Since the balance between glycolysis and mitochondrial metabolism regulates hematopoietic stem cell homeostasis, metabolic cues may influence emergency myelopoiesis. Here, we show in humans and female mice that hematopoietic progenitor cells increase fatty acid metabolism after MI. Blockade of fatty acid oxidation by deleting carnitine palmitoyltransferase (Cpt1A) in hematopoietic cells of Vav1Cre/+Cpt1Afl/fl mice limited hematopoietic progenitor proliferation and myeloid cell expansion after MI. We also observed reduced bone marrow adiposity in humans, pigs and mice following MI. Inhibiting lipolysis in adipocytes using AdipoqCreERT2Atglfl/fl mice or local depletion of bone marrow adipocytes in AdipoqCreERT2iDTR mice also curbed emergency hematopoiesis. Furthermore, systemic and regional sympathectomy prevented bone marrow adipocyte shrinkage after MI. These data establish a critical role for fatty acid metabolism in post-MI emergency hematopoiesis.

Towards Optimized Bioavailability of 99mTc-Labeled Barbiturates for Non-invasive Imaging of Matrix Metalloproteinase Activity.

INTRODUCTION: Dysregulated activity of matrix metalloproteinases (MMPs) drives a variety of pathophysiological conditions. Non-invasive imaging of MMP activity in vivo promises diagnostic and prognostic value. However, current targeting strategies by small molecules are typically limited with respect to the bioavailability of the labeled MMP binders in vivo. To this end, we here introduce and compare three chemical modifications of a recently developed barbiturate-based radiotracer with respect to bioavailability and potential to image MMP activity in vivo. METHODS: Barbiturate-based MMP inhibitors with an identical targeting unit but varying hydrophilicity were synthesized, labeled with technetium-99m, and evaluated in vitro and in vivo. Biodistribution and radiotracer elimination were determined in C57/BL6 mice by serial SPECT imaging. MMP activity was imaged in a MMP-positive subcutaneous xenograft model of human K1 papillary thyroid tumors. In vivo data were validated by scintillation counting, autoradiography, and MMP immunohistochemistry. RESULTS: We prepared three new 99mTc-labeled MMP inhibitors, bearing either a glycine ([99mTc]MEA39), lysine ([99mTc]MEA61), or the ligand HYNIC with the ionic co-ligand TPPTS ([99mTc]MEA223) yielding gradually increasing hydrophilicity. [99mTc]MEA39 and [99mTc]MEA61 were rapidly eliminated via hepatobiliary pathways. In contrast, [99mTc]MEA223 showed delayed in vivo clearance and primary renal elimination. In a thyroid tumor xenograft model, only [99mTc]MEA223 exhibited a high tumor-to-blood ratio that could easily be delineated in SPECT images. CONCLUSION: Introduction of HYNIC/TPPTS into the barbiturate lead structure ([99mTc]MEA223) results in delayed renal elimination and allows non-invasive MMP imaging with high signal-to-noise ratios in a papillary thyroid tumor xenograft model.

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

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

POU5F1P1, a putative cancer susceptibility gene, is overexpressed in prostatic carcinoma.

BACKGROUND: Association between genetic variants located on human chromosome 8q24.21 with an increased risk for prostatic carcinoma has been well established. POU5F1P1, a processed pseudogene homologous to the pluripotency factor OCT4, is the only sequence with coding capacity in this region. The objective of this study was to investigate the POU5F1P1 expression in prostatic carcinoma and carcinoma surrounding prostatic tissue. METHODS: RT-PCR and real-time PCR was used to measure the expression of POU5F1P1 relative to the expression of HPRT1 in cell lines, prostatic carcinoma and carcinoma surrounding prostatic tissue. The structure of the POU5F1P1 mRNA and the promoter sequence were elucidated by 5'-RACE experiments. The POU5F1P1 protein was shown with immunohistochemistry on prostate tissue. RESULTS: POU5F1P1 was found to be the only member of the POU5F1 family to be expressed in prostate with over-expression in prostatic carcinoma compared to surrounding prostatic tissue probably because of an increased density of expressing cells. The POU5F1P1 expression is driven by a variety of promoter structures scattered over a genomic region of 860 kB. CONCLUSIONS: The over-expression of POU5F1P1 in prostatic carcinoma in addition to its genomic location and the putative function of its gene product render POU5F1P1 a good candidate to harbour functional genetic variants which modulate prostatic cancer susceptibility.

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

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

In vivo imaging biomarkers of neuroinflammation in the development and assessment of stroke therapies - towards clinical translation.

Modulation of the inflammatory microenvironment after stroke opens a new avenue for the development of novel neurorestorative therapies in stroke. Understanding the spatio-temporal profile of (neuro-)inflammatory imaging biomarkers in detail thereby represents a crucial factor in the development and application of immunomodulatory therapies. The early integration of quantitative molecular imaging biomarkers in stroke drug development may provide key information about (i) early diagnosis and follow-up, (ii) spatio-temporal drug-target engagement (pharmacodynamic biomarker), (iii) differentiation of responders and non-responders in the patient cohort (inclusion/exclusion criteria; predictive biomarkers), and (iv) the mechanism of action. The use of targeted imaging biomarkers for may thus allow clinicians to decipher the profile of patient-specific inflammatory activity and the development of patient-tailored strategies for immunomodulatory and neuro-restorative therapies in stroke. Here, we highlight the recent developments in preclinical and clinical molecular imaging biomarkers of neuroinflammation (endothelial markers, microglia, MMPs, cell labeling, future developments) in stroke and outline how imaging biomarkers can be used in overcoming current translational roadblocks and attrition in order to advance new immunomodulatory compounds within the clinical pipeline.

Imaging, myeloid precursor immortalization, and genome editing for defining mechanisms of leukocyte recruitment in vivo.

Recruitment of leukocytes from the blood to sites of inflammation poses a promising target for new diagnostic and therapeutic approaches. We aimed to develop a novel method to non-invasively analyze molecular mechanisms of leukocyte migration in pre-clinical models of inflammation in vivo. Methods: We used the ER-HoxB8 system to transiently immortalize murine myeloid precursors from wildtype and CD18- as well as MRP14-deficient mice. A VLA4α-/- cell line was generated by CRISPR/Cas9-mediated gene editing. We analyzed the migration of wildtype and knockout leukocytes in vivo by optical and nuclear imaging in mice with irritant contact dermatitis, cutaneous granuloma, experimental arthritis and myocardial infarction. Results: Transient immortalization, gene editing and in vivo imaging can be combined to analyze migratory mechanisms of murine leukocytes, even for gene deletions resulting in lethal phenotypes in mice. We reliably confirmed known migratory defects of leukocytes deficient for the adhesion molecules CD18 or VLA4α. Also, using our new method we identified a new role of the most abundant calcium-binding proteins in phagocytes and major alarmins in many inflammatory diseases, MRP8 and MRP14, for transmigration in vivo. Conclusion: We provide a combinatorial approach to rapidly characterize molecular mechanisms of leukocyte recruitment in vivo, with the potential to aid in identification of diagnostic and therapeutic targets in inflammatory pathologies.

Combined PET Imaging of the Inflammatory Tumor Microenvironment Identifies Margins of Unique Radiotracer Uptake.

The tumor microenvironment is highly heterogeneous. For gliomas, the tumor-associated inflammatory response is pivotal to support growth and invasion. Factors of glioma growth, inflammation, and invasion, such as the translocator protein (TSPO) and matrix metalloproteinases (MMP), may serve as specific imaging biomarkers of the glioma microenvironment. In this study, noninvasive imaging by PET with [18F]DPA-714 (TSPO) and [18F]BR-351 (MMP) was used for the assessment of localization and quantification of the expression of TSPO and MMP. Imaging was performed in addition to established clinical imaging biomarker of active tumor volume ([18F]FET) in conjunction with MRI. We hypothesized that each imaging biomarker revealed distinct areas of the heterogeneous glioma tissue in a mouse model of human glioma. Tracers were found to be increased 1.4- to 1.7-fold, with [18F]FET showing the biggest volume as depicted by a thresholding-based, volumes of interest analysis. Tumor areas, which could not be detected by a single tracer and/or MRI parameter alone, were measured. Specific compartments of [18F]DPA-714 (14%) and [18F]BR-351 (11%) volumes along the tumor rim could be identified. [18F]DPA-714 (TSPO) and [18F]BR-351 (MMP) matched with histology. Glioma-associated microglia/macrophages (GAM) were identified as TSPO and MMP sources. Multitracer and multimodal molecular imaging approaches may allow us to gain important insights into glioma-associated inflammation (GAM, MMP). Moreover, this noninvasive technique enables characterization of the glioma microenvironment with respect to the disease-driving cellular compartments at the various disease stages. Cancer Res; 77(8); 1831-41. ©2017 AACR.

Imaging matrix metalloproteinase activity in multiple sclerosis as a specific marker of leukocyte penetration of the blood-brain barrier.

The enzymes gelatinase A/matrix metalloproteinase-2 (MMP-2) and gelatinase B/MMP-9 are essential for induction of neuroinflammatory symptoms in experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis (MS); in the absence of these enzymes, the disease does not develop. We therefore investigated the cellular sources and relative contributions of MMP-2 and MMP-9 to disease at early stages of EAE induction. We demonstrated that MMP-9 from an immune cell source is required in EAE for initial infiltration of leukocytes into the central nervous system and that MMP-9 activity is a reliable marker of leukocyte penetration of the blood-brain barrier. We then developed a molecular imaging method to visualize MMP activity in the brain using fluorescent- and radioactive-labeled MMP inhibitors (MMPis) in EAE animals and used the radioactive MMP ligand for positron emission tomography (PET) imaging of MMP activity in patients with MS. In contrast to traditional T1-gadolinium contrast-enhanced MRI, MMPi-PET enabled tracking of MMP activity as a unique feature of early lesions and ongoing leukocyte infiltration. MMPi-PET therefore allows monitoring of the early steps of MS development and provides a sensitive, noninvasive means of following lesion formation and resolution in murine EAE and human MS.