title: Periodontal inflammation and alveolar bone loss induced by Aggregatibacter actinomycetemcomitans is attenuated in sphingosine kinase 1-deficient mice.
abstract: OBJECTIVE
  Sphingosine-1-phosphate (S1P) 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 Aggregatibacter actinomycetemcomitans. S1P 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 SK1 deficiency on S1P generation, proinflammatory cytokine production, chemotaxis of monocytes and macrophages, and periodontitis induced by A. actinomycetemcomitans.
  
  
  METHODS
  Murine bone marrow-derived monocytes and macrophages (BMMs) from SK1 knockout (KO) mice or wild-type (WT) mice were either untreated or exposed to A. actinomycetemcomitans. The mRNA levels of SK1, SK2 and intracellular sphingolipid levels were quantified. In addition, murine WT BMMs were treated with vehicle, S1P, 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 E2, IL-1β, IL-6 and TNF-α were quantified in the cell media of SK1 KO BMMs or WT BMMs with or without bacterial stimulation. Furthermore, a transwell migration assay was performed and the number of migrated WT BMMs in the presence of vehicle, bacteria-stimulated media, with or without S1P was quantified. Finally, in vivo studies were performed on SK1 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
  SK1 mRNA levels significantly increased after A. actinomycetemcomitans stimulation in murine WT BMMs, but were undetectable in SK1 KO BMMs. Deficiency of SK1 in murine BMMs resulted in decreased S1P generation induced by A. actinomycetemcomitans as compared with WT BMMs. Additionally, low levels of S1P (≤ 1 μM) did not have a significant impact on the mRNA production of COX-2, IL-1β, IL-6 and TNF in murine BMMs with or without the presence of A. actinomycetemcomitans. There were no significant differences in prostaglandin E2 , IL-1β, IL-6 and TNF-α protein levels in the media between SK1 KO BMMs and WT BMMs with or without bacterial stimulation. Importantly, low levels of S1P (≤ 1 μM) dose-dependently promoted the chemotaxis of BMMs. The bacteria-stimulated media derived from SK1 BMMs significantly reduced the chemotaxis response compared with WT control. Finally, SK1 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 SK1 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.
  
  
  CONCLUSIONS
  Our studies support that SK1 and S1P play an important role in the inflammatory bone loss response induced by the oral pathogen A. actinomycetemcomitans. Reducing S1P generation by inhibiting SK1 has the potential as a novel therapeutic strategy for periodontitis and other inflammatory bone loss diseases.
authors.affiliation: Department of Oral Health Sciences and the Center for Oral Health Research, Medical University of South Carolina, Charleston, SC, USA.
authors.forename: H
authors.initials: H
authors.lastname: Yu
authors.affiliation: Department of Comparative Medicine, Medical University of South Carolina, Charleston, SC, USA.
authors.forename: C
authors.initials: C
authors.lastname: Sun
authors.affiliation: Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston, SC, USA.
authors.forename: K M
authors.initials: KM
authors.lastname: Argraves
mesh_terms: D016976:Aggregatibacter actinomycetemcomitans; D016301:Alveolar Bone Loss; D000818:Animals; D007249:Inflammation; D051379:Mice; D010518:Periodontitis; D014409:Tumor Necrosis Factor-alpha; D055114:X-Ray Microtomography
chemical_list: D014409:Tumor Necrosis Factor-alpha
keywords: Aggregatibacter actinomycetemcomitans; cytokine; osteoclast; periodontal disease; sphingosine kinase; sphingosine-1-phosphate