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

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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.