Generation of Cre/lox Mice for Inducible Deletion of PKC-epsilon in the Immune System

项目总结 在一个由11种异构体组成的家族中，蛋白激酶C-epsilon(PKC-)具有独特的结合和调节特性 不能由其他PKC补偿。它参与神经、血管和伤口的愈合，如 全球pkc-基因敲除小鼠的研究证实了这一点。值得注意的是，全球KO具有潜在的免疫力 阻止纯合子繁殖和pkc-KO小鼠的缺陷会屈服于它们野生清除的感染 键入对应物。由于几乎每种疾病都有免疫/炎症成分，来自体内的结果 对KO小鼠的研究必须在免疫受损的未知影响的背景下进行解释 系统。因此，对研究PKC-在疾病中的作用的“清洁”小鼠的需求尚未得到满足， 其中PKC-可以以组织特异性的方式被删除。即，PKC-loxP/loxP(flx‘d)小鼠。这 应用程序将产生一种PKC-牙线状小鼠，当它与组织选择性Cre杂交时，将专门 删除所选单元格中的pkc-。重要的是，pkc-基因修饰的小鼠将拥有完整的免疫 当与选择的Cre杂交时，将产生仅在组织中缺乏PKC-的后代 兴趣。pkc-小鼠可以被认为是一种门槛品系，为其他研究人员提供了一个工具 在他们选择的模型中研究PKC-，独立于全球记录的免疫缺陷 淘汰赛。我们将把它们交叉到表达lox-Stop-lox ZsGreen和LysM-Cre的报告小鼠。由此产生的 小鼠将在巨噬细胞(M？)和中性粒细胞中选择性地删除PKC-，这也将表达 记者ZsGreen报道。我们的初步数据表明，来自PKC-小鼠的M？积累了更多和更大的脂肪 并产生更多的肿瘤坏死因子-，但较少的决议1，以响应脂肪摄取或免疫复合体。我们 假设PKC-在M？中的表达将减缓动脉粥样硬化的发展。在体内，我们将检查 用PKC-ef/fLysM-Cre/±小鼠建立AAV8-PCSK9动脉粥样硬化模型中M？PKC-的作用AAV8- PCSK9是一种功能增强的病毒，表达一种导致高胆固醇血症和动脉粥样硬化的突变体 在老鼠身上。突变的PCSK9基因在高胆固醇血症患者中表达，带来翻译 与模型的相关性。WT和PKC-f/fLysM-Cre/±小鼠的主动脉根部斑块将被计分 斑块稳定性适用于人类疾病。成像将被用来量化数量和定位 斑块M？、它们的偏振状态(M1、M2和Mox标记的免疫荧光)和脂肪含量(油 红O染色)。此外，还将对血清中细胞因子和脂质介质的水平进行量化。体外研究 将确定PKC-在泡沫细胞形成中起作用的具体步骤，并将定义细胞因子的特征 和由WT和PKC-产生的脂质介质f/fLysM-Cre/±M？ 凋亡性细胞。总之，我们发现pkc-是一种新的调节脂质滞留和 动脉粥样硬化环境中的炎症。这些研究将确定与动脉粥样硬化相关的途径， 由巨噬细胞PKC-调节，这些通路可以在未来的R01中详细研究。

PROJECT SUMMARY In a family of 11 isoforms, protein kinase C-epsilon (PKC-) has unique binding and regulatory properties that cannot be compensated for by other PKCs. It is involved in neurological, vascular, and wound healing as evidenced from studies in the global PKC- knockout mouse. Notably, the global KO has underlying immune defects that prevent homozygous breeding and PKC- KO mice succumb to infections cleared by their wild type counterparts. As virtually every disease has an immune/inflammatory component, the results from in vivo studies with the KO mouse must be interpreted in the context of the unknown effects of the impaired immune system. Thus, there is an unmet need for a “cleaner” mouse in which to study the role of PKC- in disease, one in which PKC- can be deleted in a tissue specific manner. That is, a PKC-loxP/loxP (flox’d) mouse. This application will generate a PKC- floxed mouse, which, when crossed to a tissue-selective Cre, will specifically delete PKC- in the cells of choice. The significance is that PKC- flox’d mice will have an intact immune system that, when crossed to a Cre of choice, will produce offspring lacking PKC- only in the tissue of interest. The PKC- flox’d mouse can be considered a gateway strain, providing a tool for other investigators to study PKC- in their model of choice independently of the immune defects documented for the global knockout. We will cross them to reporter mice expressing lox-STOP-lox ZsGreen and LysM-Cre. The resulting mouse will have PKC- selectively deleted in macrophages (MØ) and neutrophils, which will also express the ZsGreen reporter. Our preliminary data indicate that MØ from PKC- mice accumulate more, and larger, lipid droplets and produce more TNF-, but less ResolvinD1, in response to lipid feeding or immune complexes. We hypothesize that PKC- expression in MØ will slow atherosclerosis development. In vivo, we will examine the role of MØ PKC- in the AAV8-PCSK9 model of atherosclerosis, using PKC-ef/fLysM-Cre+/± mice. AAV8- PCSK9 is a gain of function virus expressing a mutant that produces hypercholesterolemia and atherosclerosis in mice. The mutant PCSK9 gene is expressed in humans with hypercholesterolemia, bringing translational relevance to the model. Aortic root plaques from WT and PKC-f/fLysM-Cre+/± mice will be scored for metrics of plaque stability applied to the human disease. Imaging will be used to quantify the number and localization of plaque MØ, their polarization state (immunofluorescence for markers of M1, M2, and Mox) and lipid content (Oil Red O staining). Additionally, serum levels of cytokines and lipid mediators will be quantified. In vitro studies will identify specific steps in foam cell formation in which PKC- acts and will define the signature of cytokines and lipid mediators produced by WT and PKC-f/fLysM-Cre+/± MØ in response to immune complexes and apoptotic cells. In conclusion, we have identified PKC- as a novel player in regulating lipid retention and inflammation in an atherogenic environment. These studies will identify atherosclerosis-relevant pathways that are regulated by macrophage PKC-, pathways that can be examined in detail in a future R01.