FTY720 inhibited proinflammatory cytokine release and osteoclastogenesis induced by Aggregatibacter actinomycetemcomitans

Research

Hong Yu^1*, Bethany A. Herbert¹, Michael Valerio¹, Leigh Yarborough², Li-Chien Hsu² and Kelley M. Argraves³

• *
  Corresponding author: Hong Yu [email protected]

Author Affiliations

¹ Department of Oral Health Sciences and the Center for Oral Health Research, Medical University of South Carolina, 173 Ashley Avenue, Charleston 29425, SC, USA

² Clemson University, Clemson 29634, SC, USA

³ Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, 173 Ashley Avenue, Charleston 29425, SC, USA

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Lipids in Health and Disease 2015, 14:66 
doi:10.1186/s12944-015-0057-7

Published: 4 July 2015

Abstract (provisional)

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)-1?, IL-6, and
tumor necrosis factor (TNF)-? 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-1?, IL-6, and TNF-? 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. Conclusion FTY720 inhibited proinflammatory
cytokine production and suppressed osteoclastogenesis, supporting FTY720 as a potential
therapy for inflammatory bone loss diseases.