Paul S. Frenette - US grants

The overall objective of the proposal is to evaluate the adhesive mechanisms mediating the in vivo trafficking of hematopoietic and endothelial progenitor cells. While the emigration of mature leukocytes to areas of inflammation is well characterized, little is known on the trafficking of hematopoietic progenitor cells (HPC). There is growing evidence suggesting that hematopoietic and endothelial progenitor cells have a common origin and that circulating endothelial progenitor cells (EPC) may contribute to neovascularization in vivo. Using a new technique of intravital examination of the bone marrow (BM) microvasculature, colony-forming unit assays, and competitive reconstitution experiments, we propose to investigate the adhesion pathways regulating the homing of HPCs to and their egress from the murine BM. We will also examine using intravital microscopy the differential adhesion mechanisms used by bone marrow, placental and mobilized CD34+ HPCs to interact with the bone marrow microvasculature of immunodeficient mice. Furthermore, we propose to assess the contribution of blood-borne EPCs in neovascularization during tumor development and wound healing, and to dissect the adhesion pathways regulating the recruitment of circulating EPCs in these neovessels using novel intravital murine models. These experiments should shed further light into the mechanisms mediating the trafficking of hematopoietic and endothelial progenitors in normal physiology and certain pathologic situations. A greater knowledge in these areas may lead to better ways to deliver HPCS modified by gene therapy to the bone marrow, to improve the collection of mobilized HPCs, and may impact diseases where new blood vessel formation is critical, such as cancer, wound healing and ischemic vascular illnesses.

DESCRIPTION (provided by applicant): The overall objective of this proposal is to understand the adhesive mechanisms mediating vaso-occlusion in sickle cell disease. Although several studies have documented roles for a number of adhesion molecules in sickle cell adhesion in vitro, there is very little in vivo data. The recent development of a mouse strain that exclusively expresses human globins provides us with the opportunity to study the adhesive mechanisms in vivo using intravital microscopy. Our preliminary studies indicate that cytokine-induced inflammation produces severe vasoocclusion in post-capillary venules in the cremaster muscle of sickle cell mice. Our results indicate that adherent leukocytes play a key role in this process since they can bind circulating sickle cell erythrocytes (SS RBCs) and initiate venular occlusion. Endothelial selectin-deficient mice, which display defects in leukocyte recruitment in venules, show few SS RBC-leukocyte interactions and no vasooclusion. In this proposal, we wish to further investigate the hypothesis that vasooclusion in sickle cell disease is initiated by the adhesive interaction between SS RBCs and adherent leukocytes. In specific aim I, we will identify the type of leukocyte (mononuclear vs polymorphonuclear) that interacts with SS RBCs in vivo, and we will further evaluate the roles of leukocyte and endothelial adhesion molecules in vasoocclusion using adhesion molecule knockout mice and inhibitory antibodies; we will also develop a model to investigate vasoocclusion in the bone marrow microvasculature, an important target in sickle cell disease. In specific aim II, we will develop adhesion assays to dissect the molecular mechanisms mediating SS RBC-leukocyte interactions and, in parallel, we will conduct intravital experiments to confirm putative adhesion pathways. In specific aim III, we propose to evaluate the in vivo functions of three adhesion molecules previously shown to participate in sickle cell adhesion (von Willebrand factor, beta3 integrins and thrombospondin), using adhesion molecule knockout mice and sickle cell animals. The proposed studies should shed new light on the in vivo mechanisms of sickle cell vasoocclusion and may lead to novel ways to treat sickle cell crises and other complications of this disease.

DESCRIPTION (provided by applicant): The selectin family of adhesion molecules plays key role in the rolling and adhesion of leukocytes in systemic venules. Previous studies have shown that sickle erythrocytes can interact with P-selectin expressed on activated endothelium. Other studies have revealed that the adhesion of leukocytes in venules of sickle cell mice played a major role in the vascular occlusion of sickle cell disease (SCD) and that sickle mice lacking both P-and E-selectin genes were protected from sickle vasoocclusion. Selectins bind to glycoconjugated ligands on leukocytes that contain sialyl Lewis X (sLex), a sialylated and fucosylated tetrasaccharide. The synthesis of selectin ligands requires the expression of several glycosyltransferases that modify the carbohydrate composition of specific polypeptide or lipid, allowing high-affinity selectin binding. The role of alpha (1,3)fucose has been well demonstrated using mice lacking leukocyte fucosyltransferases (FucTs). Mice lacking FucTVII, in particular, showed dramatic reductions in the expression of ligands for all three selectins, suggesting that FucTs may represent a useful target for therapeutic intervention. Alpha (1,3)FucTs catalyze the formation of an alpha anomeric glycosidic bond between carbon 1 of the fucose and carbon 3 of N-acetylglucosamine. We have developed an ELISA-based assay to evaluate rapidly the FucT activity in cell lysates. Herein, we propose to format this assay for high throughput screening (HTS) for small molecular weight compounds that inhibit alpha (1,3)FucT activity. In this assay, the neoglycoprotein 3'sialyl-Nacetyllactosamine oligosaccharide acceptor will be fucosylated by HL60 cell lysates as a source of leukocyte FucT activity and newly synthesized sialyl Lewis X will be detected specifically by the HECA-452 antibody followed by a peroxidase-conjugated antibody. In Specific Aim 1, we propose to test the reproducibility and robustness of the FucT assay in 384-well plates for HTS. Specific Aim 2 will validate the FucT assay using the statistical parameter Z' and with a small collection of compounds. In Specific Aim 3, we propose to initiate the primary HTS, and identify "hits" that can inhibit leukocyte FucT activity in both cell lysates and live myeloid cells. We will perform in vitro and in vivo counter-screening studies to assess further the efficacy and specificity of selected "hits" or lead compounds. These studies may pave the way to important progress in the treatment or prevention of vasoocclusive episodes in sickle cell disease.

[unreadable] DESCRIPTION (provided by applicant): Selectins and their glycoconjugated ligands play key role in the rolling and adhesion of leukocytes in systemic venules. While leukocyte adhesion in systemic venules represents an essential line of defense against infections, dysregulated or excessive leukocyte recruitment can cause tissue damage and contribute to the pathology of several inflammatory diseases including ischemia-reperfusion injuries, autoimmune and allergic diseases, atherosclerosis, thrombosis, and sickle cell disease. The synthesis of selectin ligands require the expression of several glycosyltransferases that modify the carbohydrate composition of specific polypeptide or lipid, allowing high-affinity selectin binding. The role of a (1,3)fucose has been clearly demonstrated using mice lacking leukocyte fucosyltransferases (FucT). In particular, mice deficient in FucTVII showed dramatic reductions in the expression of ligands for all three selectins, suggesting that FucTs may represent a useful target for therapeutic intervention. a(1,3)FucTs catalyze the formation of alpha anomeric glycosidic bond between carbon 1 of the fucose and carbon 3 of N-acetylglucosamine. We have developed an ELISA-based assay to evaluate rapidly the FucT activity from cell lysates. Herein, we propose to format this assay for high throughput screening (HTS) for small molecular weight compounds that inhibit leukocyte FucT activity. In this assay, the neoglycoprotein 3'sialyl-N-acetyllactosarhine oligosaccharide acceptor will be fucosylated by FucT activity derived from HL60 cell lysates and newly synthesized sialyl Lewis X will be detected specifically by the HECA-452 antibody followed by a peroxidase-conjugated antibody. In Specific Aim 1, we propose to optimize the FucT assay for HTS in 384-well plates. Specific Aim 2 will validate the FucT assay using the statistical parameter Z' and with a small collection of compounds. In Specific Aim 3, we propose to initiate HTS and identify "hits" that can inhibit leukocyte FucT activity in both cell lysates and live myeloid cells. We will perform in vitro and in vivo counter-screening studies to assess further the efficacy and specificity of selected "hits" or lead compounds. These studies may pave the way to important progress in the development of new drugs for the treatment or prevention of vasoocclusive episodes in sickle cell disease, and in other inflammatory diseases. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Hematopoietic stem and progenitor cells (HSPC), attracted by the chemokine CXCL12, reside in specific niches in the bone marrow (BM). The long-term objective of our studies is to understand how stem cells egress from the BM into the circulation. This critical process, often referred to as "mobilization", underlies modern clinical stem cell transplantation. Our preliminary studies suggest that signals of the sympathetic nervous system are required for mobilization induced by granulocyte colony-stimulating factor (G-CSF), the most commonly used agent in the clinic. Pharmacological or genetic ablation of adrenergic neurotransmission indicates that norepinephrine (NE) signaling controls G-CSF-induced osteoblast suppression, bone CXCL12 downregulation and HSPC mobilization. In this grant application, we propose to evaluate further the role of adrenergic signals in HSPC mobilization using gain- and loss-of-function mouse models and we will investigate the molecular and cellular basis of collaborating signals. In Specific Aim I, we will evaluate the influence of hyperadrenergic states in HSPC mobilization. We will use strenuous exercise as a physiological model, and mice lacking the NE transporter (Slc6a2-/-) or alpha2A/c adrenergic receptors as genetic models. In Specific Aim II, we will assess the expression of adrenergic receptors in purified osteoblast and osteoclasts and determine their role in HSPC mobilization using knockout animals (loss-of function models). In Specific Aim III, we will characterize G-CSF-induced depletion in bone NE using [3H]NE release and uptake in vivo using radioactive decay methods, and in vitro using SH-SY5Y cells and catecholaminergic neurons derived from embryonic stem cells. In the final Aim, we will test whether the release of active TGF-beta in the stem cell niche contributes to osteoblast suppression and mobilization. We will use in vitro culture systems and Tgfbr2(fi/fl) mice expressing Cre under the MX or Col1a1 promoters, which will delete TGF-beta in an inducible manner or specifically in the osteoblasts, respectively. A function of the sympathetic nervous system in the attraction of stem cells to their niche may explain the conspicuous variability in mobilization efficiencies among normal peripheral blood stem cell donors. In addition, these studies may reveal novel strategies, based on the modulation of the sympathetic outflow to the stem cell niche, to increase the efficiency of HSC harvests for stem cell-based therapeutics. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The long-term objective of this proposal is to characterize the adhesion mechanisms mediating vaso-occlusion (VOC) in sickle cell disease. We have previously shown using intravital microscopy in sickle cell mice that sickle red blood cells (RBCs) interact with adherent leukocytes (WBCs) in inflamed venules in vivo. The interactions between RBCs and WBCs appear to be biologically important since they occur in both wild-type and sickle cell mice, and their numbers in sickle cell mouse venules correlate with reduced microvascular blood flow velocities and shorter survival during intravital microscopy. In addition, inhibition of RBC-WBC interactions protects sickle cell mice from VOC. Recent studies using a novel digital multichannel widefield fluorescence microscopy system has identified polymorphonuclear neutrophils (PMNs) as the major leukocyte subset capturing circulating RBCs. E-selectin-mediated signaling plays a critical role in enabling activated PMNs to capture RBCs. Other studies from the PI's laboratory have revealed that all E-selectin ligand (ESL) activity on PMN is conferred by three glycoproteins, PSGL-1, CD44 and ESL-1. Here, we propose to study further the cellular and molecular mechanisms mediating VOC in sickle cell mice. In Specific Aim 1, we will identify which ESL induces leukocyte activation and the capture of circulating RBCs. We will use genetic deletion (Cd44 / , Selplg / , or mice lacking both) and RNA interference (short hairpin RNAi targeting ESL-1) introduced by lentiviral transduction of hematopoietic stem cells (HSCs). In Specific Aim 2, we will define the microdomains and molecular determinants on leukocytes that mediate the capture of circulating sickle RBCs using high-speed digital videomicroscopy. Preliminary experiments in C57BL/6 mice suggest that receptors that cluster to the leading edge of adherent leukocytes mediate the capture of RBCs, and that expression of the b2 integrin Mac-1 is critical. In Specific Aim 3, we will investigate the role of the leukocyte fucosyltransferase (Fut7), required for the synthesis of all selectin ligands, as therapeutic target for sickle cell VOC using RNAi downregulation of Fut7 expression in HSCs followed by generation of radiation chimeras, and using sickle cell mice deficient in Fut7. In the last Specific Aim, we will use the animal models of leukocyte adhesion defects that we have generated to evaluate the long-term impact of leukocyte adhesion deficits on chronic hemolysis, and on the function and pathology of target organs. The proposed studies will provide new mechanistic insights into the role of leukocytes in sickle cell vascular occlusion and may lead to new therapeutic options to prevent or treat this debilitating illness.

Analgesic Agents; Analgesic Drugs; Analgesic Preparation; Analgesics; Anodynes; Antinociceptive Agents; Antinociceptive Drugs; Blood Diseases; Blood Vessels; Blood capillaries; Blood flow; Blood leukocyte; CRISP; Capillaries; Capillary; Capillary, Unspecified; Clinical; Clinical Research; Clinical Study; Clinical Treatment; Clinical Trials; Clinical Trials, Unspecified; Computer Retrieval of Information on Scientific Projects Database; Cremaster Muscle; Disease; Disorder; Dose; Endothelium; Funding; Grant; Hb SS disease; HbSS disease; Hematologic Diseases; Hematological Disease; Hematological Disorder; Hemoglobin S Disease; Hemoglobin sickle cell disease; Hemoglobin sickle cell disorder; IGIV; IV Immunoglobulins; IVIG; Immune Globulin, Intravenous; Immune globulin IV; Immunoglobulins, Intravenous; Institution; Intravenous Antibodies; Intravenous IG; Intravenous Immunoglobulins; Investigators; Leukocytes; Mammals, Mice; Marrow leukocyte; Medical center; Mice; Molecular; Murine; Mus; NIH; National Institutes of Health; National Institutes of Health (U.S.); Other Therapy; Patients; Phase; Play; Recurrence; Recurrent; Research; Research Personnel; Research Resources; Researchers; Resources; Reticuloendothelial System, Leukocytes; Role; Sickle Cell; Sickle Cell Anemia; Source; Structure of cremaster muscle; Structure of venule; Supportive Therapy; Supportive care; United States National Institutes of Health; Venules; White Blood Cells; White Cell; analgesia; blood disorder; capillary; clinical investigation; cremaster muscle; disease/disorder; drepanocyte; improved; mouse model; sickle RBC; sickle cell disease; sickle disease; sickle erythrocyte; sickle red blood cell; sicklemia; sickling; social role; trial regimen; trial treatment; vascular; venule; white blood cell; white blood corpuscle

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (04) Clinical Research, and the specific Challenge Topic 04-HL-103 Assess the role of leukocyte interaction with platelets, erythrocytes, and endothelium in the pathogenesis of heart, lung, and blood diseases. Transfusion-related acute lung injury (TRALI), the main cause of transfusion-related morbidity and mortality reported to the FDA, is a clinical syndrome in which acute lung injury occurs within 6 hours of a blood transfusion. Although its pathophysiology remains unclear, antibodies and/or bioactive lipids activating polymorphonuclear neutrophils (PMNs) have been suggested to initiate the syndrome in patients with a predisposing pro-inflammatory event. Our preliminary studies suggest that platelet-leukocyte interactions play a key role in the disease. More specifically, endothelial signals emerging from the engagement of E- selectin with E-selectin ligand-1 (ESL-1) expressed on PMNs, induced the activation of the integrin Mac-1 at the leading edge of crawling PMNs, allowing the capture of platelets which, in turn, led to injury through the production of reactive oxygen species (ROS). The present challenge is to conduct pre-clinical studies and develop novel methods to assay this type of delayed PMN activation. The proposed translational studies will lead to a clinical trial testing the safety and efficacy of novel antagonists against adhesion molecules involved in TRALI. In Specific Aim 1, we evaluate the efficacy of GMI-1070, novel selectin antagonist, in an established mouse model of TRALI. In Specific Aim 2, we will develop a humanized model of TRALI in which mice harbor circulating human PMNs by transplantation of cord blood-derived hematopoietic stem and progenitor cells in immunodeficient NOD/SCID mice deficient in the IL-2r common gamma chain. We will induce TRALI by injection of an anti-HNA-2a, which was previously shown to produce lung injury in a rat ex vivo model. We will evaluate using multichannel fluorescence intravital microcopy (MFIM) whether the administration of this antibody induces platelet-leukocyte interactions and ROS generation. We will determine the effect of GMI-1070 administration on lung injury, and platelet-leukocyte interactions by MFIM. In Specific Aim 3, we will develop a clinically relevant biological marker of the secondary wave of PMN activation using flow-based imaging of platelet complexes with the polarized leading edge of PMNs. This assay will provide a novel biological end-point to assess PMN activation and the efficacy of anti-inflammatory drugs in clinical trials. PUBLIC HEALTH RELEVANCE: Preliminary studies suggest that platelet-leukocyte interactions play a key role in the pathogenesis of TRALI. In this proposal, we will perform translational studies and develop new tools in preparation of a clinical trial to treat and/or prevent TRALI.

DESCRIPTION (provided by applicant): The regulation of leukocyte infiltration into tissues is a crucial parameter of the immune response. Circadian manifestations of vascular diseases, including ischemic vasculopathies and sickle cell disease, have been well documented. Although diurnal variations in blood leukocyte counts have been reported, whether leukocyte recruitment is influenced by circadian rhythms is unclear. In addition, the identification of the cell subsets involved and the mechanisms that regulate the circadian oscillations in leukocyte behavior is not understood. Greater understanding in this area will help to unravel the physiological and pathophysiological relevance of these endogenous rhythms in vascular biology. We have recently found that hematopoietic stem cells are released from the bone marrow (BM) through circadian regulation by rhythmic signals delivered locally in the BM microenvironment by the sympathetic nervous system (SNS) via the b3 adrenergic receptor (Mendez-Ferrer et al. Nature, 2008). Our preliminary studies using real-time multichannel fluorescence intravital microscopy (MFIM) have revealed that leukocyte-endothelial cell interactions are increased at night in mice resulting in enhanced leukocyte recruitment in tissues. Furthermore, using chemical sympathectomy and surgical denervation, we have found that the fluctuations of leukocyte recruitment were abolished in mice with an impaired SNS and were dependent on the fluctuations of endothelial selectins in the BM. These studies have led us to hypothesize that the circadian fluctuations in leukocyte recruitment to peripheral tissues are regulated by oscillations in endothelial cell adhesion molecule expression that is controlled by the SNS. This hypothesis will be tested in three Specific Aims. In Specific Aim 1, we will identify which leukocyte populations exhibit circadian fluctuations in the BM, cremaster muscle and dermal tissues under homeostatic conditions using brightfield and MFIM techniques for the real-time in vivo evaluation of leukocyte-endothelial interactions, with whole-mount ex vivo immunofluorescence imaging, and flow cytometry analyses. In Specific Aim 2, we will define the mechanisms regulating the circadian oscillations in leukocyte recruitment. We will identify the promigratory molecules and assess the mechanisms implementing these circadian rhythms, focusing on the role of the SNS and adrenergic receptors using surgical, pharmacological and genetic approaches. In Specific Aim 3, we will investigate whether circadian rhythms of leukocyte trafficking influence the inflammatory response in models of acute and chronic inflammation. The results generated from this proposal will enhance our basic understanding of this important biological phenomenon, and will likely identify novel chronotherapeutic targets for interventions in inflammatory diseases. (End of Abstract)

DESCRIPTION (provided by applicant): HSCs inhabit specific niches that regulate their commitment, survival, proliferation and differentiation. However the cellular constituents of the HSC niche remain unclear. Several lines of evidence have suggested a role for osteoblasts in providing a specific site where HSCs are maintained in a quiescent state. Other data have suggested that HSCs are predominantly localized near sinusoidal vessels in the bone marrow and spleen. We have recently found that the trafficking, both enforced and homeostatic, is regulated by signals from the sympathetic nervous system (SNS). Searching for the cellular stromal target of the SNS, we have found using a mouse expressing the green fluorescence protein (GFP) under the Nestin gene promoter, that GFP expressing cells formed the HSC niche. Our preliminary data suggest that the vast majority of CD150+CD48- HSCs and SNS-fibers are localized near Nestin+ cells, and that these cells express high levels of key genes involved in stem cell maintenance. Upon culturing, sorted Nestin+ cells rapidly differentiate into mesenchymal lineages. Moreover, we show using novel culture methods that sorted Nestin+ cells can self-renew in vitro or in vivo when grown as spheres (termed "mesenspheres") in non-adherent dishes or attached to ceramic cubes implanted subcutaneously. We thus hypothesize that Nestin+ cells are bona fide mesenchymal stem cells which are tightly regulated by the SNS, and which form the HSC niche in both medullary and extra-medullary sites. We will explore this hypothesis with our collaborators in three Specific Aims. In Specific Aim I, we will evaluate the spatial location and relationships of HSCs with Nestin+ cells in the aorta-gonad-mesonephros (AGM) region and the fetal liver. We will assess the impact of Nestin+ cell depletion on hematopoiesis at these stages. We will study a subset of Nestin+ niche cells expressing the early osteoblast gene Osterix using promoter-driven doxycycline-induced expression. We will evaluate differential gene expression in sorted fetal liver and bone marrow-derived Nestin+ cells to identify novel niche regulators of HSC proliferation. In Specific Aim II, we will characterize the spatial localization and mobilization behavior of quiescent HSCs within the bone marrow using novel imaging models using mCherry red expressing Nestin+ cells and GFP label-retaining HSCs. In Specific Aim III, we will assess the number and function of Nestin+ niche cells in pathological models of myelofibrosis (bone marrow attrition) and sickle cell disease (bone marrow expansion). The analyses proposed in these pathologically relevant models will lay the groundwork for studies to define core universal niche genes whose function extends beyond anatomical or developmental confines. PUBLIC HEALTH RELEVANCE: The spatial localization and cellular constituents forming the hematopoietic stem cell (HSC) niche are unclear, with studies suggesting that HSC localizes either near osteoblasts or the vasculature. Preliminary studies supporting this proposal suggest that bona fide mesenchymal stem cells, peri-vascular and isolatable by Nestin expression, form a unique HSC niche in the bone marrow. This proposal explores the function of this candidate niche cell during the normal development and pathological states of bone marrow attrition and expansion.

DESCRIPTION (provided by applicant): The trafficking of HSCs / progenitors from the bone marrow (BM) to the bloodstream represents the basis of modern transplantation procedures. We have characterized in the past funding cycle the mechanisms and biological impact of signals from the sympathetic nervous system (SNS) on HSC / progenitor egress from the BM. We have recently discovered that HSCs are released under state-state conditions in a circadian manner through rhythmic release of noradrenaline emanating from SNS nerve terminals in the BM whose signals are transmitted into stromal cells through the b3 adrenergic receptor. In preliminary studies, we have identified that the cellular target of the SNS in the BM is a bona fide self-renewing mesenchymal stem cell (MSC) marked by Nestin expression. In addition, we have found that these Nestin+ cells are anatomically and functionally associated with CD150+ CD48- Lin- HSCs near blood vessels of the BM. These putative Nestin+ niche cells express high levels of core genes regulating HSC retention (Cxcl12, Kit ligand, Vcam-1, Angiopoietin-1), which are downregulated by the administration of the HSC mobilizer granulocyte colony- stimulating factor (G-CSF) or b3 adrenergic receptor (Adrb3) activation. Further, our preliminary studies suggest that BM monocytes / macrophages (MO/MV) exert the opposite effect, enhancing the expression of HSC retention signals. Based on our progress and these preliminary data, we have hypothesized that the BM niche is constituted of the two stem cells, mesenchymal and hematopoietic, inhabiting its space, and is regulated by opposing signals from the SNS and macrophages. This hypothesis will be tested with three Specific Aims. In Specific Aim 1, we will evaluate the circadian influence on HSC / progenitor homing to BM. We will assess the effect of local denervation, the role of adrenergic receptors, and the function of Nestin+ niche cells. Elucidation of optimal circadian time and the mechanisms entraining oscillations in homing efficiencies may have profound impact on transplantation biology. In Specific Aim 2, we will assess whether chemotherapy-induced neurotoxicity alters HSC niche function. Our preliminary studies suggest that a lesion to the SNS impairs the regeneration of the niche and recovery of hematopoiesis following a stressful challenge such as stem cell transplantation or 5-fluorouracyl (5FU) administration. We will assess the effect of the neuropathy on HSC / progenitor mobilization and niche function in a clinically relevant model (cisplatin therapy), and evaluate the effect of neuroprotection on HSC niche function. In Specific Aim 3, we will determine the role of the MO/MV lineage in the regulation of Nestin+ niche cells and HSC / progenitor retention. Our preliminary studies indicate that MO/MV cells contribute to HSC / progenitor retention in the BM by promoting the expression of key stem cell niche genes by Nestin+ cells (Cxcl12, Kit ligand, Angiopoietin-1, Vcam-1) that retain HSC in the BM microenvironment. We have developed an in vitro system to identify the molecular mechanism mediating the crosstalk between MO/MV cells and Nestin+ niche cells. These studies may lead to the identification of a novel pathway regulating the stem cell niche and the trafficking of HSCs. PUBLIC HEALTH RELEVANCE: Studies conducted under this project have revealed that the bone marrow niche consists of the paring of the two stem cells, mesenchymal and hematopoietic, that inhabit the marrow. Here, we will explore further the hypothesis that the retention of hematopoietic stem and progenitor cells in the niche is regulated by differing signals from the sympathetic nervous system and macrophages.

DESCRIPTION (provided by applicant): Under this project, we have previously found using intravital microcopy imaging in a humanized mouse model of sickle cell disease (SCD) that adherent leukocytes recruited in inflamed venules, played a direct role in VOC by interacting with circulating erythrocytes (RBC). We have determined that RBCs interact specifically with adherent polymorphonuclear neutrophils (PMNs) in vivo. We have identified E-selectin ligand-1 as a major adhesion receptor sending signals that contribute to activate the beta2 integrin Mac-1 specifically at the leading edge of crawling PMNs. Neutrophil Mac-1 captures circulating RBC and thus contribute to VOC. Our preliminary studies suggest that VOC is mediated by a subset of PMNs (senescent phenotype) which we propose to characterize. Other exciting preliminary studies suggest that neural signals regulate the expression of adhesion molecules on venular endothelial cells through circadian adrenergic signals and that these signals can modulate the inflammatory response and the clearance of senescent PMNs. In Specific Aim 1, we will test whether senescent PMNs promote heterotypic interactions and acute VOC in SCD. We will dissect the contribution of PMN aging vs. activation on their ability to promote in vivo heterotypi RBC-WBC interactions following ex vivo aging and stimulation. We will evaluate gain- and loss-of function model systems that promote or reduce senescent PMN clearance in the bone marrow or survival in the circulation. Preliminary data indicate that circadian adrenergic signals are locally delivered by nerves of the sympathetic nervous system (SNS). However, SNS nerves innervate arterioles, but not venules where leukocytes are recruited, raising the intriguing question as to how these signals are transmitted to venular endothelial cells. In Specific Aim 2, we will define how SNS nerves control endothelial cell function in venules. We will track in vivo signal transduction by imaging calcium waves using GCaMP3 mice following direct neural stimulation of the genitofemoral nerve or pericytes by micropipette stimulation in collaboration with Dr. David Spray (Einstein). We will identify genetically the cellular and molecular basis by specific deletion of connexins or beta2 adrenergic receptors (Adrb2fl/fl) in pericytes or endothelial cells, and we will investigate the functional consequences of tissue-specific deletions on circadian leukocyte recruitment in healthy wild-type mice and in SCD mice. In addition to the SNS, our preliminary data also suggest a role for parasympathetic nervous system (PNS) in leukocyte recruitment. We will investigate in Specific Aim 3 the role of PNS (cholinergic) signals in leukocyte adhesion and sickle cell vaso-occlusion. We will explore in collaboration with Dr. Kevin Tracey (Feinstein Institute for Medical Research), the influence of vagal stimulation and nicotinic receptor signaling. We will also evaluate the role of the type 1 muscarinic receptor (Chrm1) using pharmacologic and genetic analyses since our preliminary studies suggest a role for this receptor in leukocyte trafficking. These studies will provide new insight on the mechanisms regulating sickle cell VOC and lead to novel ways to target inflammation.

DESCRIPTION (provided by applicant): The notion that a specific bone marrow (BM) microenvironment promotes the differentiation of lineage- committed blood precursors was suggested 50 years ago by Marcel Bessis where he described in seminal studies the erythroblastic island, a structure made of a central macrophage (M) surrounded by erythroblasts at varying stages of differentiation. Although studies using in vitro reconstitution analyses have clearly suggested a role for marrow-derived M¿ in erythropoiesis, there is currently no in vivo evidence that M¿ are required or even play a role in postnatal erythropoiesis. A major reason for the deficit of in vivo knowledge stems from the poorly defined nature of BM M¿ and, until recently, the lack of specific genetic models. We have defined subsets of BM mononuclear phagocytes and showed that CD169+ M¿ regulated the hematopoietic stem cell niche. Our preliminary studies also suggest that BM CD169+ M¿ promote erythropoiesis since their selective in vivo depletion compromised erythropoietic recovery after 5-fluorouracil- or phenylhydrazine-induced anemia. Additionally, in a genetic model of polycythemia vera (PV) from transgenic overexpression of mutated JAK2V617F, we have found that M¿ depletion normalized the hematocrit, suggesting that erythropoiesis in PV, unexpectedly, depends on signals from the microenvironment. Based on these preliminary results, we propose to evaluate further the molecular basis and function of BM M¿ in healthy and diseased erythropoiesis. In Specific Aim 1, we will define in vivo molecular mechanisms regulating the BM erythroid niche (erythroblastic island). We will characterize CD169+ erythroblastic islands in collaboration with Drs M. Narla (NYBC) and J. Chasis (Berkeley) and get new insight on central M¿ by flow cytometry and transcriptional profiling collaboration with Dr. M. Merad (Mt. Sinai). We will generate macrophage-specific genetic models for candidate M¿ receptors previously suggested to play a role in the erythroblastic island (Vcam1, Itgav, and Maea). In Specific Aim 2, we will examine the contribution of bone marrow M¿ in chronically diseased erythron. We will analyze models of erythropoietic stress such as sickle cell disease and thalassemia, the latter in collaboration with Dr. S. Rivella (Cornell). We will then evaluate the impact of macrophage in transgenic JAK2V617F models in collaboration with Dr. J. Zhao (Oklahoma) and Tony Green (Cambridge) using M¿-specific CD169-DTR depletion and M¿-specific genetic models generated in the first aim. In Specific Aim 3, we will examine the potential therapeutic benefit of expanding the central macrophage in the erythropoietic recovery after myeloablation. We will use transgenic models (Csf1 overexpression in collaboration with Dr. R. Stanley, Einstein) and pharmacological approaches (long-acting IL-4 complexes). These studies will define for the first time the in vivo roles of BM macrophages in erythropoiesis and will likely lead to novel ways to regulate erythropoiesis in diseases characterized by erythron expansion. (End of Abstract)

DESCRIPTION (provided by applicant): This R01 project has investigated the adhesion mechanisms mediating progenitor homing to bone marrow (BM) and uncovered the role of the sympathetic nervous system (SNS) in regulating homeostatic circadian and enforced release of hematopoietic stem cell (HSC) from BM. We have identified Nestin+ cells as candidate niche cell that are regulated by SNS nerve fibers. In preliminary studies for this renewal application, we have developed a novel approach combining whole-mount imaging confocal immunofluorescence with computational analyses to assess significant associations between stromal BM structures and HSCs. We have uncovered distinct subsets of Nestin+ cells: the pericytic Nes-GFPbright NG2+ LepR- cells are exclusively associated with arterioles, whereas reticular Nes-GFPdim NG2- LepR+ cells are found near sinusoids. We have found that the most quiescent HSCs are significantly associated with arterioles whereas less quiescent HSCs are found away from arterioles. Further preliminary studies suggest that an HSC progeny, the megakaryocyte (Mk) can feed back to the HSC and regulate quiescence. In Aim 1, we will assess the differential functions of arteriolar and sinusoidal niches in HSC maintenance using conditional deletion of CXCL12 or SCF. In Aim 2, we will investigate the role of the Mk niche in HSC maintenance, focusing on the chemokine CXCL4 as putative contributor, and explore the hypothesis that the Mk niche may represent a reserve for Mk-biased HSCs. Finally, we propose in Aim 3 to dissect further how neural signals link the brain and BM by addressing two different aspects of communication between neural signals and BM. In the first subaim, we will investigate using transgenic and optogenetic tools how the signals from SNS nerves which are tightly associated with arteriolar niche, can reach the sinusoidal niche which is not innervated. I the second subaim, we will explore how the brain communicates with BM to release HSCs based on our preliminary data implicating M1R signals originating from the brain. We will test the idea that a hormonal factor links the brain to the BM using parabiosis and candidate hormone analyses. These innovative studies will shed new insight into HSC regulation by the microenvironment.

PROJECT SUMMARY: The bone marrow is the major site of hematopoiesis where all blood cells emerge from the regulated differentiation of hematopoietic stem cells (HSCs). Studies from the applicant?s laboratory have uncovered key functions for innervation of the sympathetic nervous system (SNS) in the egress of HSCs from marrow. SNS nerves are required to entrain the circadian release of HSCs and also the circadian recruitment of mature leukocytes to the periphery. In addition, the bone marrow is also innervated by peptidergic sensory fibers whose functions in hematopoiesis remain unclear. In this application, we propose a 3-year experimental plan that will advance our knowledge on the neuroanatomy and neurophysiology of the bone marrow. In Specific Aim 1, we will characterize the functions of sensory nerves in the marrow. We will define the functional neural circuits using immunofluorescence imaging and transneuronal viral tract tracing, and evaluate the interplay of signals between sensory and SNS fibers using pharmacological and genetic model systems. In Specific Aim 2, we will identify the intercellular transduction pathways that relay SNS nerve signals in bone marrow. We will identify the stromal cell types that receive adrenergic signals mediating ROS oscillations which may represent an important link for the propagation of neural signals. Specific Aim 3 will harness endogenous neural circuits to improve hematopoietic regeneration. We will establish selective bone marrow DREADDs using adenoviral transduction that will lead to organ-specific neural activation to enhance regeneration following genotoxic insults such as ionizing irradiation or chemotherapy. Manipulation of endogenous neural circuits may indeed provide a useful future strategy to accelerate hematopoietic regeneration. !

PROJECT SUMMARY: In this project, we have discovered using intravital microcopy imaging in a humanized mouse model of sickle cell disease (SCD) that activated neutrophils play a direct role in vaso-occlusive crisis (VOC) by interacting with circulating erythrocytes (sRBC). We have described heterogeneity in the ability of neutrophils to capture sRBC and recently ascribed it to their chronological aging in the circulation and the exposure to the microbiota. Indeed, microbiota depletion markedly reduces aged neutrophil counts, and improves the acute and chronic SCD complications. Preliminary data using a model of psychological stress, a known VOC trigger, suggest that stress- induced exacerbation of VOC also depends on the microbiota and aged neutrophil generation promoted by the IL-17A/G-CSF pathway. In this funding period, we propose to investigate the innovative hypothesis that the microbiota critically regulates SCD activity. In Specific Aim 1, we will evaluate how the microbiota modulates psychological stress-induced sickle cell VOC. We will assess the mechanisms of stress signals linking the brain to the immune response, focusing on neural (sympathetic nervous system) and stress hormones (glucocorticoids). We will identify the source of IL-17A elicited by stress, evaluate its function using IL-17A- deficient mice, and the mechanisms by which the microbiota activates the IL-17A/G-CSF pathway. In Specific Aim 2, we will define the role of neutrophils and microbiota in chronic sickle cell-induced end-organ damage. We have found that depletion of the microbiota markedly improved the chronic organ damage in SCD mice. Whether the microbiota mediates organ damage through interactions with leukocytes or other targets is unclear. We will investigate the role of neutrophils in organ damage in G-CSF-deficient mice which are neutropenic. We will investigate whether the microbiota signals to hematopoietic or non-hematopoietic cells using conditional Myd88- deletion. Since our preliminary data suggest that microbiota depletion significantly reduces iron deposition in tissues, we will investigate the role of iron chelation therapy in SCD mice. In collaboration with Dr. Craig Branch, we will monitor the impact of microbiota depletion by T2* magnetic resonance imaging and test the potential of siderophore probiotics. In Specific Aim 3, we will develop new strategies to harness the microbiota for SCD treatment. We will characterize the differential effect of antibiotics using 16S rDNA sequencing. We will manipulate the microbiota with probiotics and investigate specifically the role of segmented filamentous bacteria (SFB), as they are known to induce IL-17A/G-CSF, using fecal transplantations in vancomycin-treated or germ- free SCD mice. Since hydroxyurea (HU) has antimicrobial activity that can significantly alter the microbiome, we will evaluate its effect on gut microbiota by 16S rDNA sequencing and determine using fecal transplant the contribution of HU-modified microbiota in disease activity. These studies will provide insight on a new mechanism regulating SCD manifestations and lead to novel ways to target inflammation in this disease.

PROJECT SUMMARY: Mammalian aging is associated with reduced tissue regeneration due to declining function of tissue-specific stem cells. In the blood system, hematopoietic stem cell (HSC) aging is accompanied by an expansion of myeloid-biased HSCs with reduced self-renewal functions. Aging of HSCs and their niches likely contributes to aged-related hematologic malignancies such a myelodysplastic syndromes or acute myelogenous leukemia. Whether the aging microenvironment drives the phenotypic features of aging HSCs, however, remains unclear. In the healthy bone marrow of young mice, our prior studies show that niches cells containing mesenchymal stem cell (MSC) activity, marked by Nestin expression, are innervated and regulated by the sympathetic nervous system (SNS). Preliminary data supporting this grant application have revealed marked alterations in the vasculature of aged bone marrow, associated with loss of SNS innervation, and increased Nestin+ cell numbers. In addition, we find that the generation of reactive oxygen species (ROS) in endothelial and perivascular cells oscillates in a circadian manner, and that these oscillations are impaired in aged bone marrow. Aged bone marrow endothelial cells also exhibit significant alterations in glucose metabolism, that are regulated by SNS-derived signals. Based on these preliminary data, we propose to explore the hypothesis that the loss of SNS nerve signals contributes to the hematopoietic aging phenotype via regulation of the vascular- associated niche cells. This hypothesis will be tested in three aims wherein: Specific Aim 1 will investigate the function of ?-adrenergic signals in the aging HSC niche using pharmacological and genetic approaches to modulate the sympathetic tone. In Specific Aim 2, we will investigate how SNS-enabled circadian rhythms and ROS homeostasis alter MSCs and niche function to regulate niche structure and HSC aging phenotypes. Specific Aim 3 will investigate the role of SNS innervation in age-related alterations of vascular metabolism in the HSC niche. The proposed studies will shed new light on the contributions of the aging niche on HSC function, and will help to devise new therapeutic strategies to prevent or improve the course of age-associated hematopoietic diseases.#