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.

Conclusion: FTY720 inhibited proinflammatory cytokine production and suppressed osteoclastogenesis, supporting FTY720 as a potential therapy for inflammatory bone loss diseases.

Fig. 1

FTY720 dose-dependently inhibited IL-1β, IL-6, and TNF-α expressions induced by A. actinomycetemcomitans in BMMs. Murine BMMs were treated with vehicle (ethanol) or FTY720 (2 to 8 μM) for 30 min. Then the cells were either unstimulated or stimulated for 6 h with A. actinomycetemcomitans (Aa) (1.5 CFU/cell). a IL-1β, (b) IL-6, and (c) TNF-α protein levels in the cell culture media of BMMs were analyzed by ELISA. d Cell viability was tested in BMMs treated with vehicle or FTY720 (2 to 8 μM) for 8 h. Data are expressed as mean ± SEM (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001)

Fig. 2

FTY720 attenuated p-PI3K, p-Akt, and p-ERK expressions induced by A. actinomycetemcomitans in BMMs. Murine BMMs were treated with vehicle (ethanol) or FTY720 (8 μM) for 30 min. Then the cells were either unstimulated or stimulated with A. actinomycetemcomitans (Aa) (1.5 CFU/cell) for 30 to 120 min. a P-PI3K, PI3K, p-Akt, Akt, p-ERK, and ERK expressions were evaluated by Western blot. b P-PI3K protein density, (c) P-Akt protein density, and (d) P-ERK protein density were analyzed by Quantity One Software and normalized by total protein expression, respectively. Data are expressed as mean ± SEM (n = 3, *p < 0.05, *** p < 0.001)

Fig. 3

FTY720 suppressed osteoclastogenesis in bone marrow-derived pre-osteoclasts treated with or without A. actinomycetemcomitans (Aa) stimulation. a Bone marrow (BM) cells were treated as described in Methods. b Representative images show TRAP-stained cells with or without A. actinomycetemcomitans stimulation. Pictures were taken at 100× magnification. c Number of TRAP⁺ multinucleated (more than 3 nuclei) osteoclasts/well (96-well) were quantified. d Total areas for osteoclasts/image were quantified. e Cell viability was tested in bone marrow-derived pre-osteoclasts treated with vehicle or FTY720 (2 μM) for 24 h. Data are expressed as mean ± SEM (n = 4, ***p < 0.001)

Fig. 4

FTY720 significantly decreased Nfatc1, Ctsk, Acp5, and Oscar expressions in bone marrow-derived pre-osteoclasts with or without A. actinomycetemcomitans (Aa) stimulation. Bone marrow-derived pre-osteoclasts were treated as described in Methods. Cells were treated with vehicle (ethanol) or FTY720 (2 μM) for 30 min. Then the cells were either unstimulated or stimulated with A. actinomycetemcomitans (Aa) (0.5 CFU/cell) in the presence of vehicle or FTY720 for 4 h (a-d) or for 24 h (e-h). a, e Nfatc1 mRNA, (b, f) Ctsk mRNA, (c, g) Acp5 mRNA, and (d, h) Oscar mRNA levels were normalized by an endogenous control GAPDH expression and expressed as fold change compared with control group. Data are expressed as mean ± SEM (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001)