Novel hematopoietic effects of C3 cleavage fragments

描述(申请人提供)：我们实验室的初步数据确立了补体(C)系统在调节SDF-1-CXCR4轴依赖的归巢/保留/动员骨髓(BM)中的造血干/祖细胞(HSPC)中的新的、以前未被认识的作用。我们发现，在骨髓中合成C蛋白，C蛋白的激活提供了骨髓损伤的最早信号之一，例如，在接受放/化疗或G-CSF诱导动员的移植条件下。我们报道了HSPC表达功能性C3aR和iC3b受体CR3，当HSPC的C3a和desArgCSa刺激增强/敏化HSPC对SDF-1梯度的反应性时，iC3b在骨髓基质上的沉积束缚了早期CR3阳性的造血祖细胞，导致骨髓滞留。为了更好地阐明C3在应激状态下的造血作用，提出了四个目标：1.C3a-C3aR轴在HSPC在骨髓中归巢/植入中的分子方面。初步研究表明，C3a-C3aR轴在HSPC在骨髓中的归巢/滞留中起着至关重要的作用。我们将着重于确定在这些过程中至关重要的HSPC中C3aR信号的分子机制，并测试在移植前将HSPC与C3裂解片段一起预充是否能促进长期重新植入的HSPC在wt小鼠中的植入以及人脐血HSPC在NOD/SCID小鼠中的植入。2.阻断C3a-C3aR轴作为动员干细胞的新策略。我们报道了小分子C3aR拮抗剂SB2900157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.作为SDF-1-CXCR4轴调节器的C3片段。HSPC的C3a、desArgCSa和iC3b对SDF-1有趋化作用。这一目的将明确这一现象的分子机制，并探索C3裂解片段促进CXCR4进入膜脂筏，从而增强其对SDF-1的反应性的假说。4.补体C3与免疫缺陷小鼠的运动障碍。我们观察到免疫缺陷的RAG2-/-和SCID小鼠表现出HSPC动员受损，而HSPC在注射免疫球蛋白后可以恢复。我们将检验这样的假设，即这种缺陷与缺乏自然产生的C级联激活的IgM抗体有关。

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.