Porphyromonas gingivalis is a periodontal pathogen implicated in the initiation and progression of chronic periodontitis in adults. We recently demonstrated that this organism produces two novel classes of serine lipids that inhibit bone cell function and activate macrophages to release important cytokines. At least one of these serine lipids also mediates its effects on mouse bone cells and macrophages through engagement of the innate immune system, specifically through Toll Receptor 2 (TLR2). We also have recently discovered a new class of glycine lipids in P. gingivalis that also engage TLR2. The glycine class called Lipid 342 engages TLR2 but contains only one acyl chain, indicating that it is the smallest TLR2 agonist yet described. Understanding how glycine lipids promote TLR2-dependent cellular effects is relevant specifically to the reported effects of P. gingivalis on periodontal bone loss in experimental animals. P. gingivalis glycine lipids are unusual in that they can be produced from serine lipids when exposed to primary cultures of mouse bone marrow macrophages and Lipid 342 can be produced from another glycine lipid, Lipid 567, when treated with phospholipase A2 (PLA2). Of note, chronic inflammation is associated with increased expression of PLA2 which results in elevated prostaglandin levels within chronically inflamed tissues. This application proposes to quantify the uptake and hydrolysis of serine and glycine lipids in human gingival cells including epithelial, fibrobast and macrophages. Next, we will evaluate the capacity of these lipid classes to engage TLR2 and its co-receptor in primary cultures of the human gingival cells. Finally, we will evaluate the capacity of these lipid classes to promote osteoclast formation from human macrophages. Human macrophages will be differentiated from peripheral blood monocytes by treatment with M-CSF and osteoclast formation will be evaluated after treatment with the glycine of serine lipid classes. We will also treat M-CSF differentiated macrophages with the various classes of glycine and serine lipids and evaluate the capacity of macrophage culture supernatants to promote osteoclast formation. The experiments summarized in this proposal will clarify how P. gingivalis promotes TLR2-dependent bone loss in periodontal diseases.