eThe total cellular lipids of Porphyromas gingivalis, a known periodontal pathogen, were previously shown to promote dendritic cell activation and inhibition of osteoblasts through engagement of Toll-like receptor 2 (TLR2). The purpose of the present investigation was to fractionate all lipids of P. gingivalis and define which lipid classes account for the TLR2 engagement, based on both in vitro human cell assays and in vivo studies in mice. Specific serine-containing lipids of P. gingivalis, called lipid 654 and lipid 430, were identified in specific high-performance liquid chromatography fractions as the TLR2-activating lipids. The structures of these lipids were defined using tandem mass spectrometry and nuclear magnetic resonance methods. T oll-like receptors (TLRs) represent a diverse family of molecules that play a critical role in activating the innate immune system in response to pathogens (1, 2). Toll-like receptor 2 (TLR2) recognizes diverse molecular structures of microbial cell wall origin, including lipoteichoic acid, lipoproteins, peptidoglycan from Gram-positive bacteria, lipoarabinomannan from mycobacteria, and zymosan from yeast cell walls. TLR2 is reported to be activated by many other microbial products, including phenol-soluble modulins (3) and Porphyromonas gingivalis lipoprotein (4), lipopolysaccharide (LPS) (5-7), and fimbriae (8-10). However, two recent reports have questioned the extent to which lipoprotein, LPS, or fimbriae mediate TLR2 engagement by P. gingivalis (11,12).We previously reported that the total lipid extract of P. gingivalis promotes activation of mouse dendritic cells and inhibits osteoblast-mediated bone deposition through engagement of TLR2 (13,14). These effects were attributed to the dominant phosphorylated dihydroceramide lipids of P. gingivalis, in particular, phosphoethanolamine dihydroceramides. These studies reported engagement of TLR2 only in vitro in mouse cells. Recent reports have demonstrated TLR2-dependent periodontal bone loss in mice following oral infection with P. gingivalis (15,16). Most recently, cell adhesion mediated through the expression of fimbriae by P. gingivalis has been implicated in promoting of TLR2-dependent oral bone loss (17). In contrast, two recent reports indicated that the capacity of fimbriae to engage TLR2 is dependent on the presence of a contaminating factor that is susceptible to hydrolysis by lipoprotein lipase (11,18).In addition to effects on mouse cells, the phosphorylated dihydroceramide lipids of P. gingivalis have been shown to promote proinflammatory responses in human fibroblasts and to cause disruption of human fibroblast adherence/vitality in culture (19). However, it is not clear whether these effects require engagement of TLR2. Since the total lipid extract of P. gingivalis has been shown to activate TLR2 in mice and in mouse cells, the primary purpose of this investigation was to further identify and characterize the specific lipid classes of P. gingivalis that are responsible for engagement of TLR2 and, specifica...

Multiple sclerosis (MS) is an autoimmune disease of unknown etiology. Infectious agents have been suggested to have a role as environmental factors in MS, but this concept remains controversial. Recently, gastrointestinal commensal bacteria have been implicated in the pathogenesis of autoimmune diseases, but mechanisms underlying the relationship of human systemic autoimmunity with the commensal microbiome have yet to be identified. Consistent with the lack of understanding of pathogenic mechanisms and relevant environmental factors in MS, no blood biomarkers have been identified that distinguish MS patients from healthy individuals. We recently identified a unique gastrointestinal and oral bacteria-derived lipodipeptide, Lipid 654, which is produced by commensal bacteria and functions as a human and mouse Toll-like receptor 2 ligand. Using multiple-reaction-monitoring mass spectrometry, a critical approach in targeted lipidomics, we now report that Lipid 654 can be recovered in the serum of healthy individuals. Most interestingly, we find that Lipid 654 is expressed at significantly lower levels in the serum of patients with MS compared with both healthy individuals and patients with Alzheimer's disease. These results thus identify for the first time a potential mechanism relating the gastrointestinal and oral commensal microbiome to a human systemic autoimmune disease. In addition, these results also identify a potential etiologic environmental factor and novel clinically relevant serum biomarker for MS.

Multiple reaction monitoring-MS analysis of lipid extracts from human carotid endarterectomy and carotid artery samples from young individuals consistently demonstrated the presence of bacterial serine dipeptide lipid classes, including Lipid 654, an agonist for human and mouse Toll-like receptor (TLR)2, and Lipid 430, the deacylated product of Lipid 654. The relative levels of Lipid 654 and Lipid 430 were also determined in common oral and intestinal bacteria from the phylum Bacteroidetes and human serum and brain samples from healthy adults. The median Lipid 430/Lipid 654 ratio observed in carotid endarterectomy samples was significantly higher than the median ratio in lipid extracts of common oral and intestinal Bacteroidetes bacteria, and serum and brain samples from healthy subjects. More importantly, the median Lipid 430/Lipid 654 ratio was significantly elevated in carotid endarterectomies when compared with control artery samples. Our results indicate that deacylation of Lipid 654 to Lipid 430 likely occurs in diseased artery walls due to phospholipase A2 enzyme activity. These results suggest that commensal Bacteriodetes bacteria of the gut and the oral cavity may contribute to the pathogenesis of TLR2-dependent atherosclerosis through serine dipeptide lipid deposition and metabolism in artery walls.

Porphyromonas gingivalis is a periodontal pathogen strongly associated with loss of attachment and supporting bone for teeth. We have previously shown that the total lipid extract of P. gingivalis inhibits osteoblast differentiation through engagement of Toll-like receptor 2 (TLR2) and that serine dipeptide lipids of P. gingivalis engage both mouse and human TLR2. The purpose of the present investigation was to determine whether these serine lipids inhibit osteoblast differentiation in vitro and in vivo and whether TLR2 engagement is involved. Osteoblasts were obtained from calvaria of wild type or TLR2 knockout mouse pups that also express the Col2.3GFP transgene. Two classes of serine dipeptide lipids, termed Lipid 654 and Lipid 430, were tested. Osteoblast differentiation was monitored by cell GFP fluorescence and osteoblast gene expression and osteoblast function was monitored as von Kossa stained mineral deposits. Osteoblast differentiation and function were evaluated in calvarial cell cultures maintained for 21 days. Lipid 654 significantly inhibited GFP expression, osteoblast gene expression and mineral nodule formation and this inhibition was dependent on TLR2 engagement. Lipid 430 also significantly inhibited GFP expression, osteoblast gene expression and mineral nodule formation but these effects were only partially attributed to engagement of TLR2. More importantly, Lipid 430 stimulated TNF-α and RANKL gene expression in wild type cells but not in TLR2 knockout cells. Finally, osteoblast cultures were observed to hydrolyze Lipid 654 to Lipid 430 and this likely occurs through elevated PLA2 activity in the cultured cells. In conclusion, our results show that serine dipeptide lipids of P. gingivalis inhibit osteoblast differentiation and function at least in part through engagement of TLR2. The Lipid 430 serine class also increased the expression of genes that could increase osteoclast activity. We conclude that Lipid 654 and Lipid 430 have the potential to promote TLR2-dependent bone loss as is reported in experimental periodontitis following oral infection with P. gingivalis. These results also support the conclusion that serine dipeptide lipids are involved in alveolar bone loss in chronic periodontitis.

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