Osteocytes-bacteria interaction via NOD1 as a mechanism of bone loss in periodontitis

Project Summary/Abstract Periodontal disease (PD) is a prevalent disorder affecting nearly half of US adults. Bacterially induced PD is a leading cause of tooth loss. Around 20% of the patients do not respond to current treatment, and PD remains a global health problem. In order to develop novel strategies to prevent tooth loss caused by PD, an improved understanding of the molecular mechanism of alveolar bone loss is needed. Osteocytes are the most abundant bone cells embedded in the bone matrix. Osteocytes orchestrate bone resorption by osteoclasts and bone formation by osteoblasts, playing a vital role in maintaining bone homeostasis and the progression of skeletal diseases. Recently, we and others have demonstrated that osteocytes are a crucial source of RANKL for bone loss in PD. Additionally, we have shown that bacterial stimulation of osteocytes induces RANKL and significant bone loss, indicating bacterial detection by osteocytes could be a mechanism of PD bone loss. Nucleotide- binding oligomerization domain containing 1 (NOD1) is an intracellular pattern recognition receptor that selectively recognizes specific types of peptidoglycan fragments primarily found in gram-negative bacteria. Periodontal pathogenic bacteria, including Porphyromonas gingivalis (Pg), Fusobacterium nucleatum (Fn), and Aggregatibacter actinomycetemcomitans (Aa), activate NOD1 signaling and induce inflammatory responses in host cells. Additionally, NOD1-deficient mice are protected from inflammation and osteoclastic bone resorption in the mouse ligature-induced periodontitis (LIP), revealing that NOD1 is a regulator of host responses in PD and that LIP is a beneficial model to study the impact of NOD1 on PD bone loss. Recently, we discovered that osteocytes increased Rankl expression in response to NOD1 stimulation, suggesting that the detection of periodontal pathogens by osteocyte NOD1 could be a key mechanism of bone loss in PD. However, it remains unknown whether periodontal pathogens can directly activate osteocyte NOD1 and whether osteocyte NOD1 is a driver of bone loss in vivo. We hypothesize that osteocyte NOD1 senses peptidoglycan fragments that are derived from periodontopathic bacteria to induce RANKL in PD, resulting in bone loss. To test our hypothesis, we propose the following specific aims. Aim 1: Identify whether osteocytes use NOD1 to recognize periodontal pathogens for their activation and RANKL induction. We will stimulate osteocytes with periodontopathic bacteria, Pg, Fn, and Aa, with or without genetic or pharmacological inhibition of NOD1, and analyze RANKL expression. Aim 2: Determine whether the lack of NOD1 in osteocytes protects alveolar bone from mouse LIP. We will delete NOD1 in osteocytes (Dmp1-Cre Nod1fl/fl) and challenge the mice with LIP to determine the role of osteocyte NOD1 in LIP bone loss. The outcome of this R03 will be the vital foundation for the future R01, where we will investigate the mechanism of NOD1-induced Rankl transcription in osteocytes, the potential of systemic or local administration of NOD1-specific inhibitors as a therapeutic approach for PD bone loss, and the characteristics of peptidoglycan fragments from periodontal pathogens and mechanisms for their production.