Serine Lipids of Porphyromonas gingivalis Are Human and Mouse Toll-Like Receptor 2 Ligands

Abstract

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...

TLR Tolerance as a Treatment for Central Nervous System Autoimmunity

“…L654 stimulates cells through TLR2, does not stimulate through TLR4, and fails to stimulate in the absence of TLR2 (16). These findings led us to ask why a TLR ligand, normally thought to be proinflammatory, would be lower in the setting of an autoimmune disease such as MS.…”

“…Purified Pam2CSK 4 (Pam2Cys), purified lipoteichoic acid (LTA), oligodeoxynucleotide (ODN) 1668, and R848 were obtained from InvivoGen (San Diego, CA). L654 was derived from Porphyromonas gingivalis (American Type Culture Collection 33277 type strain) and purified, as previously described (16).…”

Periodontitis: from microbial immune subversion to systemic inflammation

“…FimA fimbriae also induce TLR2-dependent expression of endothelial adhesion molecules (ICAM-1, VCAM-1 and E-selectin) and chemokines (MCP-1 and CXCL8) involved in leukocyte recruitment, as well as TLR2 inside-out signalling that activates leukocyte integrins that mediate transendothelial migration 6,113,114 . However, additional TLR2 ligands of P. gingivalis , including serine lipids and lipoproteins 115,116 , probably also participate in these pro-atherogenic activities.…”

Biomarkers in multiple sclerosis

“…species, but not by human cells (122,123). There was insufficient sample size to determine an impact on disease progression or treatment from bacterial lipopeptides, however this raises the intriguing possibility that certain commensal micro-organisms may be playing a critical role in MS, that lipids from those bacteria may be seeding the blood stream, and that they may represent novel biomarkers for MS. Further studies will be required to confirm these findings and determine if they are predictive of disease severity and progression.…”