Impact of Titanium-mediated Oxidative Stress on LPS/TLR4 Signaling

项目总结 种植体周围炎是一种导致种植体周软组织和骨骼破坏的炎症性疾病，至今仍是一种 牙种植体存活的主要障碍是缺乏有效的治疗策略。直到 最近，口腔细菌被认为是引起牙周种植体周围炎的主要因素。 疾病。然而，种植体周围疾病的研究已将重点转向了解口腔免疫机制。 它们感知口腔中的外部提示，并与口腔微生物群保持动态平衡。一口井- 已建立的口服免疫监测机制是Toll样受体(TLR)对内毒素的感知 内毒素(LPs)是一种内毒素，也是革兰氏阴性菌的组成部分，广泛存在于口腔中。监管 内毒素耐受是维持口腔内环境平衡的重要机制。 TLR4激活触发信号级联，涉及包括免疫细胞在内的多种细胞功能 招募，反应氧物种(ROS)的产生，以中和入侵的微生物，以及促炎 细胞因子的分泌。脂多糖/TLR4轴在几个阶段受到多个负调控因子的严密控制 TLR4信号级联反应可减轻导致宿主组织破坏的失控炎症。在.期间 种植体周围炎性疾病进展，过量的ROS水平，即氧化应激，这会破坏 细胞过程包括对内毒素/TLR4信号的调节。抑制TLR4负性调节器是 推测TLR4在种植体周围炎中过度表达，从而增加对内毒素的敏感性。钛 (Ti)牙种植体表面的微粒溶解可在邻近的种植体周围组织中积聚，以及 钛颗粒浓度的增加与种植体周围炎有关。然而，钛颗粒在金属中的作用 介导性种植体周围疾病的机制仍有待阐明。在这项拟议的研究中，种植体衍生的影响 探讨了钛颗粒(ITips)对过量ROS产生和随后的内毒素/TLR4轴的失调的影响。 在目标1中，包含钛颗粒的人类种植体周围感染组织的转录组分析是通过 RNA测序，同时专注于TLR4信号、ROS产生、它们的调节因子和 促炎细胞因子通过免疫组织化学进行评估。在目标2中，人体周围组织中的体外钛颗粒- 植入组织的特征是产生形态相似的iTiP，然后评估其诱导效果 单核细胞来源的免疫细胞体外产生ROS的研究。在目标3中，iTips诱导ROS的能力 通过基因表达评估脂多糖/TLR4信号、ROS生成和促炎状态的失调 体外研究和成像流式细胞术以及体内小鼠口腔粘膜模型。这项奖学金将帮助 应聘者从事生物材料、免疫学和口腔领域的独立研究工作 微生物组研究，重点是内毒素/TLR4信号，以确定种植体周围潜在的治疗靶点 炎症性疾病治疗。该项目的成功完成将通过以下方式填补关键的知识空白 阐明iTips在驱动种植体周围炎中的作用及其与氧化应激和TLR4信号的相互作用。

PROJECT SUMMARY Peri-implantitis, an inflammatory disease resulting in destruction of peri-implant soft tissue and bone, remains a major obstacle to dental implant survival due to a lack of effective treatment strategies for this disease. Until recently, oral bacteria were considered a primary factor triggering peri-implantitis as observed for periodontal diseases. However, peri-implant disease research has shifted focus to understanding oral immune mechanisms which sense external cues in the oral cavity and maintain homeostasis with the oral microbiome. One well- established mechanism of oral immune surveillance is Toll-like receptor (TLR) sensing of lipopolysaccharide (LPS), an endotoxin and component of gram-negative bacteria, ubiquitous in the oral cavity. The regulation of the LPS/TLR4 axis, known as endotoxin tolerance, is an important mechanism in maintaining oral homeostasis. TLR4 activation triggers signaling cascades involved with multiple cellular functions including immune cell recruitment, reaction oxygen species (ROS) generation to neutralize invading microbes, and proinflammatory cytokine secretion. The LPS/TLR4 axis is tightly controlled by multiple negative regulators at several stages of TLR4 signaling cascades which mitigate uncontrolled inflammation resulting in host tissue destruction. During peri-implant inflammatory disease progression, excess ROS levels, i.e., oxidative stress, occurs, which disrupts cellular processes including regulation of LPS/TLR4 signaling. Suppression of TLR4 negative regulators is hypothesized to drive TLR4 overexpression in peri-implantitis, thereby increasing sensitivity to LPS. Titanium (Ti) particle dissolution from a dental implant surface can accumulate in adjacent peri-implant tissue, and increasing concentrations of Ti particles are associated with peri-implantitis. However, the role of Ti particles in mediating peri-implant disease remains to be elucidated. In this proposed study, the impact of implant-derived Ti particles (iTiPs) on excess ROS production and subsequent dysregulation of the LPS/TLR4 axis is explored. In Aim 1, transcriptome analysis of human peri-implantitis-affected tissue containing Ti particles is performed via RNA sequencing, while focused gene translation study for TLR4 signaling, ROS production, their regulators, and proinflammation cytokines is assessed via immunohistochemistry. In Aim 2, ex vivo Ti particles in human peri- implant tissue are characterized to generate morphologically similar iTiPs, which are then assessed for induction of ROS production in monocyte-derived immune cells in vitro. In Aim 3, the ability of iTiPs-induced ROS to dysregulate LPS/TLR4 signaling, ROS generation, and proinflammatory status is evaluated via gene expression studies and imaging flow cytometry in vitro and a murine oral mucosal model in vivo. This fellowship will help the applicant pursue a career as an independent investigator in the field of biomaterials, immunology, and oral microbiome studies with a focus on LPS/TLR4 signaling to identify potential therapeutic targets for peri-implant inflammatory disease treatment. Successful completion of this project will fill a crucial knowledge gap by elucidating the role of iTiPs and its interaction with oxidative stress and TLR4 signaling in driving peri-implantitis.