Fluorescence microscopy tensor imaging representations for large-scale dataset analysis

Understanding complex biological systems requires the system-wide characterization of cellular and molecular features. Recent advances in optical imaging technologies and chemical tissue clearing have facilitated the acquisition of whole-organ imaging datasets, but automated tools for their quantitative analysis and visualization are still lacking. We have here developed a visualization technique capable of providing whole-organ tensor imaging representations of local regional descriptors based on fluorescence data acquisition. This method enables rapid, multiscale, analysis and virtualization of large-volume, high-resolution complex biological data while generating 3D tractographic representations. Using the murine heart as a model, our method allowed us to analyze and interrogate the cardiac microvasculature and the tissue resident macrophage distribution and better infer and delineate the underlying structural network in unprecedented detail

Targeted delivery to inflammatory monocytes for efficient RNAi-mediated immuno-intervention in auto-immune arthritis

Poster presentationInternational audienc

Chronic variable stress activates hematopoietic stem cells

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

Monocyte-Directed RNAi Targeting CCR2 Improves Infarct Healing in Atherosclerosis-Prone Mice

Background—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. Here, we used nanoparticle-facilitated silencing of CCR2, the chemokine receptor that governs inflammatory Ly-6Chigh monocyte subset traffic, to reduce infarct inflammation in apolipoprotein E–deficient (apoE−/−) mice after MI. We used dual-target positron emission tomography/magnetic resonance imaging 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 cross-linking noninvasively, we developed a fluorine-18–labeled positron emission tomography agent (18F-FXIII). Recruitment of MPO-rich inflammatory leukocytes was imaged with a molecular magnetic resonance imaging sensor of MPO activity (MPO-Gd). Positron emission tomography/magnetic resonance imaging detected anti-inflammatory effects of intravenous nanoparticle-facilitated siRNA therapy (75% decrease of MPO-Gd signal; P<0.05), whereas 18F-FXIII positron emission tomography reflected unimpeded matrix cross-linking 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). Conclusion—CCR2-targeted RNAi reduced recruitment of Ly-6Chigh monocytes, attenuated infarct inflammation, and curbed post-MI left ventricular remodeling.National Heart, Lung, and Blood InstituteUnited States. Dept. of Health and Human Services (contract No. HHSN268201000044C)National Institutes of Health (U.S.) (grant R01-HL096576)National Institutes of Health (U.S.) (grant R01-HL095629)National Institutes of Health (U.S.) (grant T32-HL094301)Deutsche Forschungsgemeinschaft (HE-6382/1-1

Macrophages retain hematopoietic stem cells in the spleen via VCAM-1

Splenic myelopoiesis provides a steady flow of leukocytes to inflamed tissues, and leukocytosis correlates with cardiovascular mortality. Yet regulation of hematopoietic stem cell (HSC) activity in the spleen is incompletely understood. Here, we show that red pulp vascular cell adhesion molecule 1 (VCAM-1)[superscript +] macrophages are essential to extramedullary myelopoiesis because these macrophages use the adhesion molecule VCAM-1 to retain HSCs in the spleen. Nanoparticle-enabled in vivo RNAi silencing of the receptor for macrophage colony stimulation factor (M-CSFR) blocked splenic macrophage maturation, reduced splenic VCAM-1 expression and compromised splenic HSC retention. Both, depleting macrophages in CD169 iDTR mice or silencing VCAM-1 in macrophages released HSCs from the spleen. When we silenced either VCAM-1 or M-CSFR in mice with myocardial infarction or in ApoE[superscript −/−] mice with atherosclerosis, nanoparticle-enabled in vivo RNAi mitigated blood leukocytosis, limited inflammation in the ischemic heart, and reduced myeloid cell numbers in atherosclerotic plaques

Targeting Interleukin-1β 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-1β, increase bone marrow hematopoietic stem cell proliferation after MI. Data obtained in bone marrow reconstitution experiments reveal that interleukin-1β 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-1β suppresses these effects. Anti-interleukin-1β 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

Myocardial Infarction Accelerates Atherosclerosis

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

IL-6-Dependent PGE2 Secretion by Mesenchymal Stem Cells Inhibits Local Inflammation in Experimental Arthritis

BACKGROUND: Based on their capacity to suppress immune responses, multipotent mesenchymal stromal cells (MSC) are intensively studied for various clinical applications. Although it has been shown in vitro that the immunomodulatory effect of MSCs mainly occurs through the secretion of soluble mediators, the mechanism is still not completely understood. The aim of the present study was to better understand the mechanisms underlying the suppressive effect of MSCs in vivo, using cells isolated from mice deficient in the production of inducible nitric oxide synthase (iNOS) or interleukin (IL)-6 in the murine model of collagen-induced arthritis. PRINCIPAL FINDINGS: In the present study, we show that primary murine MSCs from various strains of mice or isolated from mice deficient for iNOS or IL-6 exhibit different immunosuppressive potential. The immunomodulatory function of MSCs was mainly attributed to IL-6-dependent secretion of prostaglandin E2 (PGE2) with a minor role for NO. To address the role of these molecules in vivo, we used the collagen-induced arthritis as an experimental model of immune-mediated disorder. MSCs effectively inhibited collagen-induced inflammation during a narrow therapeutic window. In contrast to wild type MSCs, IL-6-deficient MSCs and to a lesser extent iNOS-deficient MSCs were not able to reduce the clinical signs of arthritis. Finally, we show that, independently of NO or IL-6 secretion or Treg cell induction, MSCs modulate the host response by inducing a switch to a Th2 immune response. SIGNIFICANCE: Our data indicate that mscs mediate their immunosuppressive effect via two modes of action: locally, they reduce inflammation through the secretion of anti-proliferative mediators, such as NO and mainly PGE2, and systemically they switch the host response from a Th1/Th17 towards a Th2 immune profile

Development of RNAi-based anti-inflammatory strategies in experimental arthritis

La polyarthrite rhumatoïde (PR) est le plus fréquent des rhumatismes inflammatoires et représente un problème de santé publique majeur. A l'heure actuelle, les biothérapies anti-TNF sous forme de protéines recombinantes constituent une avancée considérable dans le traitement de la polyarthrite rhumatoïde (PR). Néanmoins, il convient de développer des approches thérapeutiques alternatives pour traiter les 40% de patients non-répondeurs ainsi queceux qui échappent à plusieurs années de traitement. La recherche de nouvelles cibles thérapeutiques est indispensable pour proposer des approches alternatives à ces biothérapies. Par ailleurs, les techniques de transfert de gène offrent une alternative thérapeutique possible pour pallier aux limitations des biothérapies actuelles, à condition de les adapter aux contraintes du tissu cible de la PR, les articulations. Les projets ont consisté à développer et valider dans des modèles expérimentaux d'arthrite de nouvelles stratégies anti-inflammatoires basées sur l'utilisation de l'ARN interférence comme outil thérapeutique. En effet, la possibilité d'interférer au niveau des mécanismes responsables de l'expression des protéines,la régulation de la stabilité des ARNm et de l'efficacité de la machinerie traductionnelle, présente un intérêt thérapeutique supérieur aux biothérapies actuelles basées sur l'inhibition des protéines sécrétées (anticorps ou récepteurs solubles) mais nécessite cependant de posséder un vecteur qui transduit efficacement les cellules productrices de la molécule ciblée.Rheumatoid arthritis (RA) is the most frequent chronic inflammatory systemicautoimmune disease that remains a major medical challenge as the exact causes of the disease are not completely elucidated. The principal treatment strategies arebased on the inhibition of TNF-α, one of the major inflammatory cytokine in RA.Although risk and benefit analyses are in favour of the use of monoclonal antibodiesagainst TNF-α, the most currently used biotherapy, they are not devoid from multipleside effects. The search for new therapeutic targets is essential to proposealternative approaches to non responders to such biotherapies. The possibility to interfere in the mechanisms responsible for regulating mRNA stability andeffectiveness of the translational machinery also present a therapeutic benefitsuperior to current biologic therapies based on inhibition secreted proteins(antibodies or soluble receptors). Such approach however requires developingvectors that efficiently transduced the specific cell type producing the targeted gene.Projects of my PhD fellowship have included both the development of gene therapyvehicles for RNAi-based intervention in experimental mouse models of arthritis andevaluation of novel candidate genes for alternative anti-inflammatory therapy in RA