Novel hematopoietic effects of C5 cleavage fragment

描述(申请人提供)：造血干细胞(HSPC)的药理学动员和用于移植的清髓条件都激活了骨髓(BM)中的补体级联(CC)，在之前的资助期间，我们重点研究了第三补体成分(C3)在HSPC动员和归巢中的作用。在这次竞争性更新中，我们将重点关注位于C3下游的第五个补体组件(C5)。C5裂解导致产生强有力的过敏性毒素C5a和desArgC5a，并产生非裂解和裂解C5b-C9，称为膜攻击复合体(MAC)。我们最近公布的数据支持C5a/C5b-C9在HSPC贩运中的关键作用。特别是，C5缺陷小鼠对粒细胞集落刺激因子(G-CSF)的反应很差，在与野生型(WT)BM细胞移植后表现出延迟的造血重建。基于这些发现，这种竞争性更新的中心假设是，补体级联远端步骤的激活和C5a和C5b-C9(MAC)的释放是HSPC动员和归巢的重要调节因素。为了解决这一假设，我们提出：特定目标1。C5缺陷小鼠动员缺陷的分子基础。根据我们的观察，C5缺乏的小鼠是糟糕的G-CSF动员者，我们将讨论C5裂解是否是日常HSPC动员所必需的，ii)其他因素诱导的HSPC动员，以及III)C5激活和S1P血浆水平是否与患者的动员效果相关。我们还将通过对对C5b-C9(MAC)裂解高度敏感的CD55/CD59缺陷小鼠进行动员研究，重点研究红细胞释放的S1P在动员中的作用。最后，我们将重点介绍C5b-C9诱导的S1P释放在溶血综合征患者动员中的作用。具体目的2.C5缺陷小鼠归巢缺陷的分子基础。我们发现，在致死性照射的小鼠中，CC被激活，C5缺乏的小鼠与WT BM细胞的移植很差。为了进一步阐明CC的远端部分(C5a/C5b-C9)在归巢/植入中的作用，我们将研究化疗条件下的移植是否也激活了骨髓中的CC，并研究了C5a/C5b-C9对骨髓中HSPC趋化因子表达的影响，ii)HSPC在骨髓内皮细胞上的黏附/拴系分子，以及ii)促进归巢因子的分泌。具体目的3.C5a/C5b-C9诱导的骨髓间质来源的趋化因子(LL-37)作为一种有效的归巢敏感因子。我们发现，像C3a一样，IL-37是SDF-1的一个非常强的启动因子，在C5a/C5b-C9依赖的条件移植后，SDF-1在骨髓基质中上调。在免疫缺陷的小鼠模型中，我们将测试移植前短期暴露于LL-37的HSPC是否会增加人骨髓和脐带血来源的HSPC的归巢。我们还将讨论IL-37是否也启动了HSPC对其他归巢因子的反应，并专注于这些现象的分子机制。

DESCRIPTION (provided by applicant): 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.