Section The main hypothesis of this competing renewal is based on new exciting data that further supports the pivotal role of the complement cascade (ComC) and other elements of innate immunity in the mobilization of hematopoietic stem/progenitor cells (HSPCs). The ComC is activated by the i) classical, ii) mannan-binding lectin (Mbl), and iii) alternative pathways, and, as we already reported, mice that lack the common distal component of the ComC (C5?/? mice), are poor mobilizers. To our surprise, however, we found that mice that do not activate the classical ComC (C1q?/? mice) are normal mobilizers. This ?discrepancy? is now explained by our latest results that show the pivotal involvement of the Mbl-dependent ComC pathway and not the classical pathway in mobilization. We also recently found that two stress-induced enzymes, heme oxygenase 1 (HO-1) and inducible nitric oxide synthetase (iNOS), are negative regulators of cell migration. Based on these findings, we have assembled a proposal that, in a comprehensive way, addresses the involvement of elements of innate immunity (ComC, Gr-1+ granulocytes/monocytes, naturally occurring antibodies) in the mobilization of HSPCs. In three interrelated specific aims, we address the crucial: i) inititation, ii) amplification, and execution phases of mobilization.
Specific Aim 1. The initiation phase of mobilization is explained by Mbl-mediated ComC activation. We have maintained that the classical ComC activation pathway plays a crucial role in triggering mobilization. However, as we discovered recently, it is not the classical but the Mbl pathway that is crucial in this process. The main target cells for mobilizing agents are Gr-1+ cells, which, in addition to proteolytic and lipolytic enzymes, also secrete ROS and danger-associated molecular pattern molecules (DAMPs). While proteolyic and lipolytic enzymes perturb the retention of HSPCs in bone marrow (BM) niches, ROS-exposed neoepitope antigens bound by naturally occurring IgM antibodies and DAMPs released from Gr-1+ cells are recognized by the circulating pattern recognition receptor (PRR) Mbl, which, via mannan-binding lectin serine proteases (MASPs), activates the ComC and coagulation cascade (CoaC).
Specific Aim 2. The amplification phase of mobilization - the generation of C5 convertase. Since C5 cleavage is crucial for egress of HSPCs from BM, the main function of this phase is generation of C5 convertase activity. This enzyme is generated as a result of C3 cleavage, which provides elements of classical C5 convertase, and additionally by thrombin, which has C5 ?convertase-like? activity. The amplification phase is also modulated by a spontaneous amplification loop of C3 activation and by possible involvement of an alternative pathway of ComC activation and will be studied in detail in this aim.
Specific Aim 3. Execution phase of ComC-mediated mobilization. Our recent results show that egress of HSPCs from BM is negatively controlled by HO-1 and iNOS, which are negative regulators of cell migration. We will focus on the molecular mechanisms of this phenomenon. It is expected that these results will be translated into the development of better mobilization protocols. 1
Section In this proposal, we will focus on the role of a major component of innate immunity, namely, the complement cascade, in the trafficking of hematopoietic stem/progenitor cells. This application will allow us to better understand this process and will lead to the development of novel therapeutic strategies to treat patients with leukemia and inborn defects of hematopoiesis by employing hematopoietic stem cell transplants. 2
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