Novel hematopoietic effects of C3 cleavage fragments

描述（由申请方提供）：我们实验室的初步数据确定了补体（C）系统在调节骨髓（BM）中造血干/祖细胞（HSPC）的SDF-1-CXCR 4轴依赖性归巢/滞留/动员中的一种新的和先前未被充分认识的作用。我们发现C蛋白在BM中合成，并且C激活提供了BM损伤的最早信号之一，在通过放疗/化疗或G-CSF诱导的动员进行移植预处理期间。我们报道了HSPC表达功能性C3 aR和iC 3b-受体CR 3，并且虽然HSPC的C3 a和desArgCSa刺激增强了HSPC对SDF-1梯度的反应性/使HSPC对SDF-1梯度的反应性增敏，但iC 3b在BM基质上的沉积束缚了早期CR 3阳性造血祖细胞，导致其滞留在BM中。提出了四个目标，以更好地阐明在应激情况下C3在造血中的作用：1。HSPC在骨髓中归巢/植入中C3 a-C3 aR轴的分子方面。初步研究指出C3 a-C3 aR轴在HSPC在骨髓中的归巢/保留中起关键作用。我们将重点确定对这些过程至关重要的HSPC中C3 aR信号传导的分子机制，并测试在移植前用C3切割片段引发HSPC是否增强wt小鼠中长期再增殖HSPC的植入和NOD/SCID小鼠中人脐带血HSPC的植入。2.阻断C3 a-C3 aR轴作为动员干细胞的新策略。我们报道了小分子C3 aR拮抗剂SB 2900157增强和加速G-CSF诱导的动员。作为这项工作的继续，我们将研究动员细胞的表型，并测试它们在移植到同基因小鼠后是否有效地植入。3. C3片段作为SDF-1-CXCR 4轴的调节剂。HSPC对SDF-1的C3 a、desArgCSa和iC 3b引发趋化性。这一目标将定义这一现象的分子机制，并探讨假设C3切割片段促进CXCR 4转移到膜脂筏，增加其对SDF-1的反应性。4. C3和免疫缺陷小鼠的动员受损。我们观察到免疫缺陷RAG 2-/-和SCID小鼠显示HSPC动员受损，其在输注免疫球蛋白后恢复。我们将检验这一缺陷与缺乏天然存在的C级联激活IgM抗体有关的假设。

DESCRIPTION (provided by applicant): The preliminary data from our laboratory established a novel and previously underappreciated role of the complement (C) system in modulating SDF-1-CXCR4 axis-dependent homing/retention/mobilization of hematopoietic stem/progenitor cells (HSPC) in bone marrow (BM). We found that C proteins are synthesized in BM and C activation provides one of the earliest signals of BM injury e.g., during conditioning for transplant by irradiation/chemotherapy or G-CSF-induced mobilization. We reported that HSPC express functional C3aR and the iC3b-receptor, CR3, and while C3a and desArgCSa stimulation of HSPC enhances/sensitizes responsiveness of HSPC to SDF-1 gradients, deposition of iC3b on BM stroma tethers early CR3-positive hematopoietic progenitors causing retention in the BM. Four aims are proposed to better elucidate the role of C3 in hematopoiesis during stress situations: 1. The molecular aspects of the C3a-C3aR axis in homing/engraftment of HSPC in bone marrow. Preliminary research points to a crucial role for the C3a-C3aR axis in homing/retention of HSPC in the bone marrow. We will focus on determining the molecular mechanisms of C3aR signaling in HSPC crucial for these processes and test whether priming of HSPC before transplantation with C3 cleavage fragments enhances engraftment of long-term repopulating HSPC in wt mice and human cord blood HSPC engraftment in NOD/SCID mice. 2. Blockade of the C3a-C3aR axis as a new strategy to mobilize stem cells. We reported that the small molecular C3aR antagonist SB2900157 enhances and accelerates G-CSF-induced mobilization. As a continuation of this work we will study the phenotype of mobilized cells and test whether they engraft efficiently after transplantation into syngeneic mice. 3. C3 fragments as modulators of the SDF-1-CXCR4 axis. C3a, desArgCSa and iC3b prime chemotaxis of HSPC to SDF-1. This aim will define the molecular mechanism of this phenomenon and explore the hypothesis that C3 cleavage fragments promote a shift of CXCR4 into membrane lipid rafts which increases their responsiveness to SDF-1. 4. C3 and impaired mobilization in immunodeficient mice. We have observed that immunodeficient RAG2-/- and SCID mice show impaired mobilization of HSPC, which is restored after infusion of immunoglobulins. We will test the hypothesis that this defect is related to the lack of naturally occurring C cascade-activating IgM antibodies.