Dendritic Cells and Periodontal Disease

描述（由申请人提供）：树突状细胞（DC）可能在牙周病的发病机制中发挥核心作用，参与将细菌刺激与牙周骨质流失联系起来的事件。它们可能通过产生调节适应性和先天免疫反应的因子来实现这一目标。此外，据报道，未成熟的dc具有破骨细胞前体的功能。体外dc实验的初步数据表明，转录因子FOXO1对LPS上调IL-1、TNF、IL-6和IL-12等多种细胞因子以及下调抗炎细胞因子IL-10至关重要。此外，Akt通过减少DC中FOXO1的核定位和限制炎症细胞因子的产生，在这一过程中发挥重要作用。因此，我们提出FOXO1在上调dc的炎症反应中起关键作用，Akt可阻止高炎症反应。这些发现为目前的提议提供了基础，我们将在体内测试fox01 - akt轴调节DC细胞因子表达的假设。此外，我们将确定这种调节途径是否对刺激对牙周病原体牙龈卟啉卟啉菌的适应性免疫反应至关重要，以及它是否在细菌诱导的骨质流失中起重要作用。为了删除dc中的fox01，我们将使用Cre-lox方法。我们已经将fox01小鼠与在CD11c启动子控制下表达Cre重组酶的小鼠杂交。与同窝对照小鼠（CD11cCre-/FOXO1L/L）相比，从实验小鼠（CD11cCre+/FOXO1L/L）分离的DC在LPS刺激下表现出细胞因子表达的降低。我们将通过在颅骨模型中注射牙龈假单胞菌来检查宿主-细菌在体内的相互作用。由于小鼠对牙龈卟啉单胞菌是幼稚的，我们可以检查不存在适应性免疫反应时的反应，并将其与通过牙龈卟啉单胞菌预先免疫激活适应性免疫反应的小鼠进行比较。通过流式细胞术，我们将详细分析FOXO1缺失对产生系统性和局部适应性免疫反应的影响。下面的颅骨也将进行组织学检查，以研究对破骨细胞发生和骨吸收的后续影响。我们将在实验小鼠（CD11cCre+/FOXO1L/L）和对照小鼠（CD11cCre-/FOXO1L/L）中建立牙龈假单胞菌口腔灌胃模型，以确定dc中FOXO1缺失是否调节宿主对牙龈假单胞菌的反应、破骨细胞的发生和牙周病的进展。使用相同的两种体内模型的实验将确定Akt是否对预防DC的高炎症反应是必要的。这些实验将检查实验小鼠（CD11cCre+/FOXO1L/L）和对照小鼠（CD11cCre-/FOXO1L/L）。在Aim 3中，我们将在体外研究fox01 - akt轴调控选定靶基因（IL-1、TNF、IL-6和IL-12）表达的机制。

DESCRIPTION (provided by applicant): Dendritic cells (DC) may play a central role in the pathogenesis of periodontal disease by participating in events that link bacterial stimulation to periodontal bone loss. They may accomplish this by the production of factors that regulate the adaptive and innate immune responses. Furthermore, immature DCs have been reported to function as osteoclasts precursors. Preliminary Data examining DCs in vitro establish that the transcription factor FOXO1 is essential for LPS up-regulation of several cytokines, IL-1, TNF, IL-6, and IL-12 and down-regulation of the anti-inflammatory cytokine IL-10. Moreover, Akt plays an important role in this process by reducing FOXO1 nuclear localization and limiting inflammatory cytokine production in DC. Thus, we propose that FOXO1 is critical in upregulating an inflammatory response in DCs and that a hyper- inflammatory response is prevented by Akt. These findings serve as the basis for the current proposal in which we will test in vivo the hypothesis that the FOXO1-Akt axis regulates DC cytokine expression. Moreover, we will determine whether this regulatory pathway is essential for stimulating the adaptive immune response to the periodontal pathogen, P. gingivalis, and whether it plays a significant role in bacteria induced bone loss. To delete FOXO1 in DCs we will use the Cre-lox approach. We have already bred floxed FOXO1 mice with mice that express Cre recombinase under control of the CD11c promoter. DC isolated from the resulting experimental (CD11cCre+/FOXO1L/L) mice exhibit reduced cytokine expression stimulated by LPS compared to littermate control mice (CD11cCre-/FOXO1L/L). We will examine host-bacteria interactions in vivo by injecting P. gingivalis in the calvarial model. Because mice are naive to P. gingivalis we can examine the response when the adaptive immune response is not present and compare it to mice in which the adaptive immune response is activated by pre-immunization with P. gingivalis. By flow cytometry we will have a detailed analysis on the impact of FOXO1 deletion in generating a systemic and local adaptive immune response. The underlying calvarial bone will also be examined histologically to investigate the subsequent effect on osteoclastogenesis and bone resorption. The oral gavage model of applying P. gingivalis to the oral cavity will be studied in experimental (CD11cCre+/FOXO1L/L) and control mice (CD11cCre-/FOXO1L/L ) to determine whether FOXO1 deletion in DCs modulates the host response to P. gingivalis, osteoclastogenesis and periodontal disease progression. Experiments using the same two in vivo models will determine whether Akt is necessary to prevent a hyperinflammatory response in DC. These experiments will examine experimental (CD11cCre+/FOXO1L/L) and control (CD11cCre-/FOXO1L/L) mice. In Aim 3 the mechanisms by which the FOXO1-Akt axis regulates selected target gene expression (IL-1, TNF, IL-6, and IL-12) will be examined in vitro.