Both pharmacological mobilization of hematopoietic stem progenitor cells (HSPCs) and myeloablative conditioning for transplantation activate the complement cascade (CC) in bone marrow (BM), and in the previous funding period we focused on the role of the third complement component (C3) in mobilization and homing of HSPCs. In this competing renewal we will focus on the fifth complement component (C5), which is located downstream from C3. C5 cleavage leads to generation of the potent anaphylatoxins C5a and desArgC5a and generation of non-lytic and lytic C5b-C9, known as the membrane attack complex (MAC). Our recently published data support a crucial role for C5a/C5b-C9 in trafficking of HSPCs. In particular, C5-deficient mice are poor mobilizers in response to granulocyte colony stimulating factor (G-CSF) and exhibit delayed hematopoietic reconstitution after transplantation with wild type (WT) BM cells. Based on these findings, the central hypothesis of this competing renewal is that the activation of distal steps of the complement cascade and release of C5a and C5b-C9 (MAC) are important regulators of mobilization and homing of HSPCs. To address this hypothesis, we propose:
Specific Aim 1. The molecular basis of the mobilization defect in C5-deficient mice. Based on our observation that C5-deficient mice are poor G-CSF mobilizers, we will address whether C5 cleavage is i) required for circadian HSPC mobilization, ii) mobilization of HSPC induced by other agents and iii) whether C5 activation and S1P plasma level correlates with mobilization efficacy in patients. We will also focus on the role of erythrocyte-released S1P in mobilization by performing mobilization studies in CD55/CD59-deficient mice that are highly sensitive to C5b-C9 (MAC) lysis. Finally, we will focus on the role of C5b-C9-induced release of S1P in mobilization observed in patients with hemolytic syndromes.
Specific Aim 2. The molecular basis of the homing defect in C5-deficient mice. We found that the CC is activated in lethally irradiated mice and that C5-deficient mice engraft poorly with WT BM cells. To shed more light on the role of the distal part of the CC (C5a/C5b-C9) in homing/engraftment, we will study whether conditioning for transplantation by chemotherapy also activates the CC in BM and study the effect of C5a/C5b-C9 on expression of i) HSPC chemoattractants in BM, ii) adhesion/tethering molecules for HSPCs on BM endothelium, and ii) on secretion of factors facilitating homing.
Specific Aim 3. The C5a/C5b-C9-induced BM stroma-derived cathelicidin (LL-37) as a potent homing sensitizing factor. We found that LL-37, like C3a, is a very strong priming factor for SDF-1 that becomes upregulated in BM stroma after conditioning for transplantation in a C5a/C5b-C9-dependent manner. In an immunodeficient mouse model, we will test whether short exposure of HSPCs to LL-37 before transplantation increases homing of human BM- and umbilical cord blood (UCB)-derived HSPCs. We will also address whether LL-37 also primes the responsiveness of HSPCs to other homing factors and focus on the molecular mechanism of these phenomena.

Public Health Relevance

Section In this competing renewal, we will focus on the role of a major component of innate immunity, namely, activation of the complement cascade in trafficking of hematopoietic stem/progenitor cells. This application will allow us to better understand this process, as well as lead to the development of novel therapeutic strategies to treat patients with leukemias and inborn defects of hematopoiesis by transplantation of hematopoietic stem cells.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Special Emphasis Panel (ZRG1)
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Bishop, Terry Rogers
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University of Louisville
Internal Medicine/Medicine
Schools of Medicine
United States
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Schneider, Gabriela; Sellers, Zachariah Payne; Ratajczak, Mariusz Z (2016) Parentally imprinted genes regulate hematopoiesis-new evidence from the Dlk1-Gtl2 locus. Stem Cell Investig 3:29
Borkowska, Sylwia; Suszynska, Malwina; Ratajczak, Janina et al. (2016) Evidence of a Pivotal Role for the Distal Part of the Complement Cascade in the Diurnal Release of Hematopoietic Stem Cells Into Peripheral Blood. Cell Transplant 25:275-82
Abdelbaset-Ismail, Ahmed; Borkowska, Sylwia; Janowska-Wieczorek, Anna et al. (2016) Novel evidence that pituitary gonadotropins directly stimulate human leukemic cells-studies of myeloid cell lines and primary patient AML and CML cells. Oncotarget 7:3033-46
Poniewierska-Baran, Agata; Schneider, Gabriela; Sun, Wenyue et al. (2016) Human rhabdomyosarcoma cells express functional pituitary and gonadal sex hormone receptors: Therapeutic implications. Int J Oncol 48:1815-24
Adamiak, M; Poniewierska-Baran, A; Borkowska, S et al. (2016) Evidence that a lipolytic enzyme-hematopoietic-specific phospholipase C-β2-promotes mobilization of hematopoietic stem cells by decreasing their lipid raft-mediated bone marrow retention and increasing the promobilizing effects of granulocytes. Leukemia 30:919-28
Ratajczak, Mariusz Z; Suszynska, Malwina (2016) Emerging Strategies to Enhance Homing and Engraftment of Hematopoietic Stem Cells. Stem Cell Rev 12:121-8
Adamiak, M; Abdelbaset-Ismail, A; Kucia, M et al. (2016) Toll-like receptor signaling-deficient mice are easy mobilizers: evidence that TLR signaling prevents mobilization of hematopoietic stem/progenitor cells in HO-1-dependent manner. Leukemia 30:2416-2419
Marycz, Krzysztof; Mierzejewska, Katarzyna; Åšmieszek, Agnieszka et al. (2016) Endurance Exercise Mobilizes Developmentally Early Stem Cells into Peripheral Blood and Increases Their Number in Bone Marrow: Implications for Tissue Regeneration. Stem Cells Int 2016:5756901
Ratajczak, Mariusz Z; Ratajczak, Janina (2016) Horizontal transfer of RNA and proteins between cells by extracellular microvesicles: 14 years later. Clin Transl Med 5:7
Abdelbaset-Ismail, Ahmed; Suszynska, Malwina; Borkowska, Sylwia et al. (2016) Human haematopoietic stem/progenitor cells express several functional sex hormone receptors. J Cell Mol Med 20:134-46

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