Protective role of host extracellular acid ceramidase in periodontal lesions

Objectives: Acid ceramidase (aCDase) is actively secreted extracellularly by macrophages under the physiological conditions where it participates in hydrolysis of extracellular pro-inflammatory ceramide-lipids. We demonstrated that aCDase possesses anti-inflammatory effects in periodontal lesions. Although the synthesis of ceramides by bacteria is not common, Porphyromonas gingivalis produces a unique dihydroceramide lipid termed phosphoglycerol dihydroceramide (PGDHC) that also promotes osteoclastogensis. The possible protective role of host aCDase in periodontal bone loss remains unknown.
Methods: Gingival crevicular fluid (GCF) samples were collected from adult periodontitis patients (ten patients with periodontitis; six – healthy controls) were subject to aCDase ELISA. RAW264.7 and mouse primary osteoclast precursors were stimulated with RANKL in the presence or absence of 1) live P. gingivalis (33277), 2) PGDHC isolated from P. gingivalis, 3) P. gingivalis-LPS, 4) recombinant aCDase and 5) D-NMAPPD (aCDase inhibitor) followed by TRAP-staining (Day-5) . The expressions of genes associated with osteoclast differentiation and activation, including, OC-STAMP, Cathepsin B, and TRAP, were evaluated after 24 and 48h of lipid treatment by q-PCR and flow cytometry, respectively.
Results: The level of aCDase in GCF was significantly lower in periodontitis patients than that of healthy patients, indicating that the extracellular levels of aCDase is diminished in periodontal lesions. Furthermore, extracellular release of aCDase from osteoclast precursors was significantly suppressed by the stimulation with live P. gingivalis in vitro. Exposure of RANKL-stimulated osteoclast precursors to PGDHC promoted the development of TRAP+ osteoclasts accompanied by elevated expression of OC-STAMP, Cathepsin B, and TRAP mRNAs which were, then, inhibited by recombinant aCDase. No significant effect of recombinant aCDase was observed in RANKL-stimulated osteoclast precursors exposed to P. gingivalis-LPS. Finally, D-NMAPPD-mediated inhibition of extracellular aCDase release from RAW264.7 cells significantly promoted PGDHC’s effect to upregulate RANKL-mediated osteoclastogenesis.
Conclusions: The present study demonstrated for the first time the protective effect of host extracellular aCDase in osteoclastogenesis induced by P. gingivalis-PGDHC.