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

项目摘要 种植体周围炎是一种导致种植体周围软组织和骨破坏的炎性疾病， 由于缺乏有效的治疗策略，这种疾病是种植牙生存的主要障碍。直到 最近，口腔细菌被认为是引发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经将重点转移到了解口腔免疫机制 其感知口腔中的外部线索并维持口腔微生物组的体内平衡。一口井- 口服免疫监视的既定机制是Toll样受体（TLR）对脂多糖的感应 (LPS)是一种内毒素，是革兰氏阴性菌的组成部分，在口腔中普遍存在。调控 LPS/TLR 4轴，称为内毒素耐受性，是维持口腔内稳态的重要机制。 TLR 4激活触发涉及多种细胞功能的信号级联，包括免疫细胞 募集，反应氧（ROS）的产生，以中和入侵的微生物，和促炎 细胞因子分泌LPS/TLR 4轴在几个阶段受到多个负调节因子的严格控制。 TLR 4信号级联，其减轻导致宿主组织破坏的不受控制的炎症。期间 植入物周围炎性疾病进展，过量的ROS水平，即，氧化应激会发生， 细胞过程，包括调节LPS/TLR 4信号传导。TLR 4负调节因子的抑制是 假设在种植体周围炎中驱动TLR 4过表达，从而增加对LPS的敏感性。钛 (Ti)从牙科植入物表面溶解的颗粒可积聚在邻近的植入物周围组织中，并且 Ti颗粒浓度的增加与种植体周围炎有关。然而，Ti颗粒在 介导的植入物周围疾病仍有待阐明。在这项拟议的研究中， 探索了Ti颗粒（iTiP）对过量ROS产生和随后LPS/TLR 4轴的失调的影响。 在目标1中，通过以下方法对含有Ti颗粒的人类种植体周围炎影响组织进行转录组分析： RNA测序，同时专注于TLR 4信号传导、ROS产生、其调节因子和 通过免疫组织化学评估促炎细胞因子。在目标2中，离体Ti颗粒在人骨组织中的分布。 表征植入组织以产生形态学上相似iTiP，然后评估其诱导 体外单核细胞衍生的免疫细胞中ROS的产生。在目标3中，iTiPs诱导的ROS对细胞的增殖能力增强。 通过基因表达评估LPS/TLR 4信号传导、ROS产生和促炎状态的失调 研究和成像流式细胞术在体外和小鼠口腔粘膜模型在体内。该奖学金将帮助 申请人在生物材料，免疫学和口腔医学领域担任独立研究员 微生物组研究，重点关注LPS/TLR 4信号传导，以确定植入物周围的潜在治疗靶点 炎症性疾病治疗。该项目的成功完成将填补一个关键的知识空白， 阐明iTiPs的作用及其与氧化应激和TLR 4信号传导在驱动种植体周围炎中的相互作用。

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.