Periodontal inflammation and alveolar bone loss induced by A ggregatibacter actinomycetemcomitans is attenuated in sphingosine kinase 1‐deficient mice

Background and Objective Sphingosine‐1‐phosphate ( S 1 P ) is a bioactive sphingolipid, which is generated by activation of sphingosine kinase ( SK ) 1 and/or 2 in most mammalian cells with various stimuli, including the oral pathogen A ggregatibacter actinomycetemcomitans . S 1 P signaling has been shown to regulate the migration of monocytes and macrophages (osteoclast precursors) from the circulation to bone tissues and affect bone homeostasis. We aimed to determine the effects of SK 1 deficiency on S 1 P generation, proinflammatory cytokine production, chemotaxis of monocytes and macrophages, and periodontitis induced by A . actinomycetemcomitans . Material and Methods Murine bone marrow‐derived monocytes and macrophages ( BMM s) from SK 1 knockout ( KO ) mice or wild‐type ( WT ) mice were either untreated or exposed to A . actinomycetemcomitans . The mRNA levels of SK 1, SK 2 and intracellular sphingolipid levels were quantified. In addition, murine WT BMM s were treated with vehicle, S 1 P , with or without A . actinomycetemcomitans and the mRNA levels of cyclooxygenase 2 (COX‐2), interleukin ( IL )‐1β, IL ‐6 and tumor necrosis factor ( TNF ) were quantified. The protein levels of prostaglandin E 2 , IL ‐1β, IL ‐6 and TNF ‐α were quantified in the cell media of SK 1 KO BMM s or WT BMM s with or without bacterial stimulation. Furthermore, a transwell migration assay was performed and the number of migrated WT BMM s in the presence of vehicle, bacteria‐stimulated media, with or without S 1 P was quantified. Finally, in vivo studies were performed on SK 1 KO and WT mice by injecting either phosphate‐buffered saline or A . actinomycetemcomitans in the periodontal tissues. The mice maxillae were scanned by micro‐computed tomography, and alveolar bone volume was analyzed. The number of periodontal leukocytes and osteoclasts were quantified in maxillary tissue sections. Results SK 1 mRNA levels significantly increased after A . actinomycetemcomitans stimulation in murine WT BMM s, but were undetectable in SK 1 KO BMM s. Deficiency of SK 1 in murine BMM s resulted in decreased S 1 P generation induced by A . actinomycetemcomitans as compared with WT BMM s. Additionally, low levels of S 1 P (≤ 1 μ m ) did not have a significant impact on the mRNA production of COX‐2, IL ‐1β, IL ‐6 and TNF in murine BMM s with or without the presence of A . actinomycetemcomitans . There were no significant differences in prostaglandin E 2 , IL ‐1β, IL ‐6 and TNF ‐α protein levels in the media between SK 1 KO BMM s and WT BMM s with or without bacterial stimulation. Importantly, low levels of S 1 P (≤ 1 μ m ) dose‐dependently promoted the chemotaxis of BMM s. The bacteria‐stimulated media derived from SK 1 BMM s significantly reduced the chemotaxis response compared with WT control. Finally, SK 1 KO mice showed significantly attenuated alveolar bone loss stimulated by A . actinomycetemcomitans compared with WT mice treated with A . actinomycetemcomitans . Histological analysis of periodontal tissue sections revealed that SK 1 KO mice treated with A . actinomycetemcomitans significantly reduced the number of infiltrated periodontal leukocytes and mature osteoclasts attached on the alveolar bone compared with WT mice. Conclusion Our studies support that SK 1 and S 1 P play an important role in the inflammatory bone loss response induced by the oral pathogen A . actinomycetemcomitans . Reducing S 1 P generation by inhibiting SK 1 has the potential as a novel therapeutic strategy for periodontitis and other inflammatory bone loss diseases.