Novel hematopoietic effects of C5 cleavage fragment

描述（由申请方提供）：造血干祖细胞（HSPC）的药理学动员和移植的清髓性预处理均可激活骨髓（BM）中的补体级联（CC），在上一个资助期，我们重点关注第三补体成分（C3）在HSPC动员和归巢中的作用。在这种竞争性更新中，我们将重点关注位于C3下游的第五补体成分（C5）。C5裂解导致产生强效过敏毒素C5 a和desArgC 5a，并产生非溶解性和溶解性C5 b-C9，称为膜攻击复合物（MAC）。我们最近发表的数据支持C5 a/C5 b-C9在HSPC贩运中的关键作用。特别地，C5缺陷型小鼠是响应于粒细胞集落刺激因子（G-CSF）的不良动员者，并且在用野生型（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的远端步骤的激活和C5 a和C5 b-C9（MAC）的释放是HSPC动员和归巢的重要调节剂。为了解决这一假设，我们提出：具体目标1。C5缺陷小鼠动员缺陷的分子基础。基于我们观察到C5缺陷型小鼠是较差的G-CSF动员剂，我们将解决C5裂解是否i）昼夜HSPC动员所需，ii）由其他药剂诱导的HSPC动员和iii）C5活化和S1 P血浆水平是否与患者中的动员功效相关。我们还将通过在对C5 b-C9（MAC）溶解高度敏感的CD 55/CD 59缺陷小鼠中进行动员研究，重点关注红细胞释放的S1 P在动员中的作用。最后，我们将重点关注C5 b-C9诱导的S1 P释放在溶血综合征患者动员中的作用。具体目标2。C5缺陷小鼠归巢缺陷的分子基础。我们发现CC在致死性照射的小鼠中被激活，并且C5缺陷的小鼠与WT BM细胞移植不良。为了更清楚地阐明CC的远端部分（C5 a/C5 b-C9）在归巢/植入中的作用，我们将研究通过化疗进行移植的预处理是否也激活BM中的CC，并研究C5 a/C5 b-C9对i）BM中的HSPC化学引诱物，ii）BM内皮上HSPC的粘附/拴系分子，和ii）促进归巢的因子分泌的影响。具体目标3。C5 a/C5 b-C9诱导的BM基质衍生的凯萨林菌素（LL-37）作为有效的归巢致敏因子。我们发现，LL-37，像C3 a一样，是SDF-1的一个非常强的启动因子，其在以C5 a/C5 b-C9依赖性方式进行移植后在BM基质中上调。在免疫缺陷小鼠模型中，我们将测试HSPC在移植前短时间暴露于LL-37是否增加人BM和脐带血（UCB）来源的HSPC的归巢。我们还将讨论LL-37是否也引发HSPCs对其他归巢因子的反应，并关注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.