Inhibiting Periodontitis by Targeting Cathepsin K and Attenuating TLR Signaling

描述（由申请人提供）：本申请的长期目标是通过靶向组织蛋白酶K （Ctsk）和减弱toll样受体（TLR）信号来开发治疗牙周炎的新疗法。牙周炎是人类最常见的炎症性疾病之一，它会导致牙周组织和牙槽骨的破坏，最终导致牙齿脱落。据估计，大多数30岁以上的成年人都患有牙周骨质流失。越来越多的证据表明，慢性牙周炎症是心血管疾病、糖尿病、动脉粥样硬化和关节炎等多种病理疾病的重要危险因素。因此，牙周炎是一个重要的公共卫生问题，特别是对老年人。因此，仍然迫切需要开发更好的治疗和预防策略，以显著减少炎症、骨质流失和牙周炎的全身后果。目前缺乏高效的治疗方法可能主要是由于对牙周炎发病机制的不完全了解。令人惊讶的是，尽管tlr对宿主防御和炎症性疾病至关重要，但tlr在牙周炎发病机制中的作用仍然很大程度上未知。我们发现腺相关病毒（AAV） Ctsk shRNAi （AAV- shrna -Ctsk）介导的沉默可以预防牙髓疾病小鼠模型中的骨质流失和炎症。此外，据报道，在类风湿性关节炎大鼠模型中，Ctsk是TLR9信号通路所必需的。值得注意的是，我们的初步数据显示，在牙周炎小鼠模型中，组织蛋白酶K基因敲除和AAV-shRNA-Ctsk介导的沉默可显著防止骨质流失和炎症。总的来说，这些研究强烈表明Ctsk是一个主要的“骨免疫基因”，可以靶向控制炎症和骨质流失，并且Ctsk可能是TLRs信号传导的关键调节因子。基于我们和其他研究者的研究，我们假设靶向Ctsk通过抑制TLRs信号传导抑制牙周炎细菌感染引起的炎症和骨质流失。为了验证我们的假设，提出了三个具体目标。我们将在Aim 1中通过对感染牙周病原体的Ctsk-/-、Ctsk+/-和Ctsk+/+小鼠的比较分析，确定Ctsk在TLRs信号介导的牙周炎诱导的免疫反应和骨吸收中的功能作用。我们将在Aim 2中确定AAV-shRNA-Ctsk的治疗潜力，通过减弱TLRs信号来减少体内牙周炎的进展和严重程度。我们将在Aim 3中使用树突状细胞、成纤维细胞和巨噬细胞来表征Ctsk介导病原体和炎症介质诱导的小鼠牙周炎模型中TLRs信号的机制。为了实现这一研究目标，我们建立了一个多学科的研究团队（包括分子遗传学家、牙医科学家、动物模型专家和免疫学家）。本研究不仅将提高我们对牙周炎发病机制的基本认识，而且将有助于设计新的治疗方法。

DESCRIPTION (provided by applicant): The long-term objective of this proposal is to develop novel therapeutics for periodontitis by targeting cathepsin K (Ctsk) and attenuating Toll-like receptor (TLR) signaling. Periodontitis is one of the most common inflammatory diseases in humans that results in the destruction of periodontal tissues and alveolar bone, which ultimately leads to teeth loss. It is estimated that majority of adults over the age of 30 suffer from periodontal bone loss. Growing evidence suggests that chronic periodontal inflammation is an important risk factor for several pathological disorders including cardiovascular disease, diabetes, atherosclerosis and arthritis. Thus, periodontitis is a significant public health concern particularly to aged people. Consequently, there is still an urgent need for developing better treatments and preventative strategies that can dramatically reduce the inflammation, bone loss, and systemic ramifications of periodontitis. The current lack of highly effective therapies may be largely due to incomplete knowledge of the mechanism of periodontitis pathogenesis. Surprisingly, although TLRs are critical for host defense and inflammatory diseases, the role of TLRs in the pathogenesis of periodontitis remains largely unknown. We found that adeno-associated virus (AAV) Ctsk shRNAi (AAV-shRNA-Ctsk) mediated silencing prevents both bone loss and inflammation in a mouse model of endodontic disease. Moreover, it was reported that Ctsk is required for TLR9 signaling in a rat model of rheumatoid arthritis. Notably, our preliminary data showed that Cathepsin K gene knockout and AAV Ctsk shRNA (AAV-shRNA-Ctsk) mediated silencing dramatically prevents both bone loss and inflammation in a mouse model of periodontitis. Collectively, these studies strongly indicate that Ctsk is a major "osteoimmune gene" that can be targeted to control both inflammation and bone loss, and that Ctsk may be a key regulator of TLRs signaling. Based on our studies and those of others, we hypothesize that targeting Ctsk inhibits inflammation and bone loss caused by bacteria infection in periodontitis through attenuation of TLRs signaling. Three specific aims are proposed to test our hypothesis. We will define the functional role of Ctsk in the TLRs signaling-mediated immune response and bone resorption induced by periodontitis through comparative analysis of Ctsk-/-, Ctsk+/-, and Ctsk+/+ mice infected with periodontal pathogens in Aim 1. We will determine the therapeutic potential of AAV-shRNA-Ctsk as a means to reduce the progression and severity of periodontitis in vivo by attenuating TLRs signaling in Aim 2. We will characterize the mechanism by which Ctsk mediates TLRs signaling induced by pathogens and inflammatory mediators in a mouse periodontitis model using dendritic cells, fibroblasts and macrophages in Aim 3. A multidisciplinary research team (i.e. a molecular geneticist, dentist scientists, animal model experts and an immunologist) has been established to achieve the research goal. This study will not only improve our understanding mechanism of basic knowledge of the pathogenesis of periodontitis, but it will facilitate the design of novel therapeutic approaches fo this disease.