Dendritic Cells and Periodontal Disease

描述（由申请人提供）：树突状细胞（DC）可能通过参与将细菌刺激与牙周骨丢失联系起来的事件，在牙周病的发病机制中发挥核心作用。它们可以通过产生调节适应性和先天性免疫反应的因子来实现这一点。此外，据报道，未成熟的DC作为破骨细胞前体发挥作用。体外检测DC的初步数据确定转录因子FOXO 1对于LPS上调几种细胞因子IL-1、TNF、IL-6和IL-12以及下调抗炎细胞因子IL-10是必需的。此外，Akt通过减少FOXO 1核定位和限制DC中炎性细胞因子的产生在此过程中起重要作用。因此，我们提出FOXO 1在上调DC中的炎症反应中是关键的，并且Akt阻止了过度炎症反应。这些发现作为目前建议的基础，我们将在体内测试FOXO 1-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是刺激牙周病原体牙龈卟啉单胞菌的适应性免疫反应所必需的，以及它是否在细菌诱导的骨丢失中起重要作用。为了删除DC中的FOXO 1，我们将使用Cre-lox方法。我们已经将floxed FOXO 1小鼠与在CD 11 c启动子控制下表达Cre重组酶的小鼠进行了繁殖。与同窝对照小鼠（CD 11 cCre-/FOXO 1 L/L）相比，从所得实验小鼠（CD 11 cCre +/FOXO 1 L/L）中分离的DC表现出LPS刺激的细胞因子表达降低。我们将通过在颅骨模型中注射牙龈卟啉单胞菌来检查体内宿主-细菌的相互作用。因为小鼠对牙龈卟啉单胞菌是幼稚的，所以我们可以检查适应性免疫应答不存在时的应答，并将其与通过用牙龈卟啉单胞菌预免疫激活适应性免疫应答的小鼠进行比较。通过流式细胞术，我们将详细分析FOXO 1缺失对产生全身和局部适应性免疫应答的影响。还将对下方颅骨进行组织学检查，以研究对破骨细胞生成和骨吸收的后续影响。将在实验小鼠（CD 11 cCre +/FOXO 1 L/L）和对照小鼠（CD 11 cCre-/FOXO 1 L/L）中研究将牙龈卟啉单胞菌应用于口腔的经口灌胃模型，以确定DC中的FOXO 1缺失是否调节宿主对牙龈卟啉单胞菌的反应、破骨细胞生成和牙周疾病进展。使用相同的两种体内模型的实验将确定Akt是否是防止DC中的过度炎症反应所必需的。这些实验将检查实验（CD 11 cCre +/FOXO 1 L/L）和对照（CD 11 cCre-/FOXO 1 L/L）小鼠。在目的3中，将在体外检查FOXO 1-Akt轴调节所选靶基因表达（IL-1、TNF、IL-6和IL-12）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.