MMP-8 as a novel therapeutic target in sepsis

描述(由申请人提供)：感染性休克是成人和儿童的主要公共卫生问题，因此需要开发新的靶点和治疗策略。基于以基因芯片为中心的临床研究和以动物为中心的后续实验，我们已确定基质金属多肽酶-8(MMP8)是脓毒症病理的候选靶点。基质金属蛋白酶-8最为人所知的是一种中性粒细胞产物，它在细胞外基质转化过程中裂解1型胶原。然而，现在已经很明显，基质金属蛋白酶-8也调节急性炎症，并可能在脓毒症的病理中发挥作用。这一建议的主要假设是，基质金属蛋白酶-8的表达和活性在脓毒症的病理过程中起主要作用。我们现在提议通过四个相辅相成的具体目标来检验这一假设。在特定的目标1中，我们将测试这一假设，即抑制基质金属蛋白酶-8的活性可以改善与脓毒症病理相关的多个终点。这个目标将使用基质金属蛋白酶-8缺失的动物和一种药物抑制基质金属蛋白酶-8的活性。在特定的目标2中，我们将检验发育年龄影响基质金属蛋白酶-8在脓毒症中的作用的假设。这个目标将使用一种适合于一周大的小鼠的脓毒症模型，也将使用MMP8基因缺失的小鼠和一种MMP8活性的药物抑制物。在特定的目标3中，我们将检验骨髓来源的细胞是脓毒症中基质金属蛋白酶-8基因调控的主要来源的假设。为了达到这一目的，体内实验将涉及将MMP-8缺失的骨髓过继转移到野生型小鼠，反之亦然。为了达到这一目的，体外实验将系统地阐明导致中性粒细胞和单核细胞重新表达基质金属蛋白酶-8基因的信号机制。在特定的目标4中，我们将检验这一假设，即1型胶原降解产物参与了基质金属蛋白酶-8调节炎症的机制。为了达到这一目的，体外实验将测试从1型胶原涂层组织培养板中提取的条件培养液，经活化的基质金属蛋白酶-8处理后，激活巨噬细胞的能力。为了达到这个目的，体内实验将测量具有1型胶原突变的小鼠对脓毒症的反应，这种突变使其对基质金属蛋白酶-8的切割具有抵抗力。这一建议的主要成果包括建立一个新的脓毒症治疗靶点，阐明发育如何影响MMP-8在脓毒症中的作用，阐明脓毒症中基质金属蛋白酶-8的主要细胞来源，全面了解炎症过程中参与基质金属蛋白酶-8表达的信号机制，以及建立依赖于基质金属蛋白酶-8的1型胶原降解产物作为先天免疫系统的危险相关分子模式。

DESCRIPTION (provided by applicant): Septic shock is a major public health problem in both adults and children, and consequently there is a need to develop novel targets and therapeutic strategies. Based on microarray-centered clinical studies and follow up animal-centered experiments, we have indentified matrix metallopeptidase-8 (MMP-8) as a candidate target in sepsis pathology. MMP-8 is best known as a neutrophil product that cleaves collagen type 1 during extra-cellular matrix turnover. However, it has now become evident that MMP-8 also modulates acute inflammation and may play a role in sepsis pathology. The overarching hypothesis of this proposal is that MMP-8 expression and activity play a major role in sepsis pathology. We are now proposing to test this hypothesis via four complementary Specific Aims. In Specific Aim 1 we will test the hypothesis that inhibition of MMP-8 activity improves multiple endpoints relevant to sepsis pathology. This Aim will use MMP-8 null animals and a pharmacologic inhibitor of MMP-8 activity. In Specific Aim 2 we will test the hypothesis that developmental age influences the role of MMP-8 in sepsis. This Aim will use a sepsis model adaptable to 1 week old mouse pups, and will also make use of MMP-8 null mice and a pharmacologic inhibitor of MMP-8 activity. In Specific Aim 3 we will test the hypothesis that bone marrow-derived cells are the main source of MMP-8 gene regulation in sepsis. The in vivo experiments for this Aim will involve adoptive transfer of MMP-8 null bone marrow to wild-type mice, and vice versa. The in vitro experiments for this Aim will systematically elucidate the signaling mechanisms that lead to de novo MMP-8 gene expression in neutrophils and monocytes. In Specific Aim 4 we will test the hypothesis that collagen type 1 degradation products are involved in the mechanism by which MMP-8 regulates inflammation. The in vitro experiments for this Aim will test the ability of conditioned media, derived from collagen type 1 coated tissue culture plates treated with activated MMP-8, to activate macrophages. The in vivo experiments for this Aim will measure the response to sepsis in mice having a mutation of collagen type 1, which renders it resistant to cleavage by MMP-8. The major deliverables of this proposal include the establishment of MMP-8 as a novel therapeutic target in sepsis, the elucidation of how development influences the role of MMP-8 in sepsis, the elucidation of the major cellular source of MMP-8 in sepsis, a comprehensive understanding of the signaling mechanisms involved in MMP-8 expression during inflammation, and the establishment of MMP-8-dependent collagen type 1 degradation products as danger associated molecular patterns for the innate immune system.