title: FTY720 inhibited proinflammatory cytokine release and osteoclastogenesis induced by Aggregatibacter actinomycetemcomitans.
abstract: BACKGROUND
  Periodontitis is a bacteria-driven inflammatory bone loss disease. Previous studies showed that the oral pathogen Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) stimulated the generation of sphingosine 1 phosphate (S1P). In addition, S1P signaling regulated the migration of osteoclast precursors and affected osteoclastogenesis. Furthermore, treatment with FTY720 (also called fingolimod, a modulator of multiple S1P receptors) alleviated osteoporosis and suppressed arthritis in animals. This study determined the effect of FTY720 on proinflammatory cytokine production and osteoclastogenesis in murine bone marrow cells with or without A. actinomycetemcomitans stimulation.
  
  
  METHODS
  Murine bone marrow-derived monocytes and macrophages (BMMs) were treated with vehicle ethanol or FTY720, and were either unstimulated or stimulated for 0.5 to 6 h with A. actinomycetemcomitans. The protein levels of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α in the media of BMMs were quantified by enzyme-linked immunosorbent assay (ELISA). Protein expressions, including phosphorylated phosphoinositide 3-kinase (p-PI3K), p-Akt, p-extracellular signal-regulated kinase (p-ERK), PI3K, Akt, and ERK were evaluated by Western blot. In addition, murine bone marrow-derived pre-osteoclasts were treated with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL) for three days. Then the cells were treated with either vehicle or FTY720 and were either unstimulated or stimulated with A. actinomycetemcomitans for 4 to 24 h. Control cells were treated with M-CSF alone with or without bacterial stimulation. Osteoclasts were stained by tartrate-resistant acid phosphatase (TRAP) staining. The mRNA levels of osteoclastogenic factors, including nuclear factor of activated T-cells cytoplasmic calcineurin-dependent 1 (Nfatc1), cathepsin K (Ctsk), acid phosphatase 5 (Acp5), osteoclast-associated receptor (Oscar), and RANKL were quantified by quantitative real-time polymerase chain reaction (PCR).
  
  
  RESULTS
  FTY720 dose-dependently inhibited IL-1β, IL-6, and TNF-α protein levels induced by A. actinomycetemcomitans in BMMs compared with controls. Additionally, FTY720 attenuated p-PI3K, p-Akt, and p-ERK expressions induced by A. actinomycetemcomitans. Furthermore, FTY720 suppressed osteoclastogenesis in bone marrow-derived pre-osteoclasts with or without bacterial stimulation and reduced the mRNA levels of Nfatc1, Ctsk, Acp5, and Oscar, but not RANKL in bone marrow-derived pre-osteoclasts.
  
  
  CONCLUSIONS
  FTY720 inhibited proinflammatory cytokine production and suppressed osteoclastogenesis, supporting FTY720 as a potential therapy for inflammatory bone loss diseases.
authors.affiliation: Department of Oral Health Sciences and the Center for Oral Health Research, Medical University of South Carolina, 173 Ashley Avenue, Charleston, SC, 29425, USA. yuho@musc.edu.
authors.forename: Hong
authors.initials: H
authors.lastname: Yu
authors.affiliation: Department of Oral Health Sciences and the Center for Oral Health Research, Medical University of South Carolina, 173 Ashley Avenue, Charleston, SC, 29425, USA. herbertb@musc.edu.
authors.forename: Bethany A
authors.initials: BA
authors.lastname: Herbert
authors.affiliation: Department of Oral Health Sciences and the Center for Oral Health Research, Medical University of South Carolina, 173 Ashley Avenue, Charleston, SC, 29425, USA. valeriom@musc.edu.
authors.forename: Michael
authors.initials: M
authors.lastname: Valerio
authors.affiliation: Clemson University, Clemson, SC, 29634, USA. leighyarborough@gmail.com.
authors.forename: Leigh
authors.initials: L
authors.lastname: Yarborough
authors.affiliation: Clemson University, Clemson, SC, 29634, USA. lhsu@g.clemson.edu.
authors.forename: Li-Chien
authors.initials: LC
authors.lastname: Hsu
authors.affiliation: Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, 173 Ashley Avenue, Charleston, SC, 29425, USA. argravek@musc.edu.
authors.forename: Kelley M
authors.initials: KM
authors.lastname: Argraves
mesh_terms: D016976:Aggregatibacter actinomycetemcomitans / Q000502:physiology*; D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology; D016207:Cytokines / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D000068876:Fingolimod Hydrochloride / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects; D018836:Inflammation Mediators / Q000378:metabolism*; D053583:Interleukin-1beta / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism / Q000382:microbiology; D008297:Male; D008810:Mice, Inbred C57BL; D010010:Osteoclasts / Q000187:drug effects / Q000378:metabolism* / Q000382:microbiology* / Q000473:pathology; D010012:Osteogenesis / Q000187:drug effects*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism
chemical_list: D016207:Cytokines; D048049:Extracellular Signal-Regulated MAP Kinases; D000068876:Fingolimod Hydrochloride; D018836:Inflammation Mediators; D053583:Interleukin-1beta; D015850:Interleukin-6; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D012333:RNA, Messenger