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FTY720 inhibited proinflammatory cytokine release and osteoclastogenesis induced by Aggregatibacter actinomycetemcomitans.

Yu H, Herbert BA, Valerio M, Yarborough L, Hsu LC, Argraves KM - Lipids Health Dis (2015)

Bottom Line: This study determined the effect of FTY720 on proinflammatory cytokine production and osteoclastogenesis in murine bone marrow cells with or without A. actinomycetemcomitans stimulation.Osteoclasts were stained by tartrate-resistant acid phosphatase (TRAP) staining.Additionally, FTY720 attenuated p-PI3K, p-Akt, and p-ERK expressions induced by A. actinomycetemcomitans.

View Article: PubMed Central - PubMed

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.

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.

No MeSH data available.


Related in: MedlinePlus

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)
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Fig3: 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)

Mentions: Osteoclasts originate from the fusion of monocytes and macrophages [27]. It has been recognized that phosphoinositides signaling controls the activation of Nfatc1 and influences osteoclastogenesis [28]. Furthermore, proinflammatory cytokines promote the differentiation of osteoclasts [1]. Since FTY720 significantly inhibited PI3K signaling and attenuated proinflammatory cytokine expression induced by A. actinomycetemcomitans, we hypothesized that FTY720 could further inhibit osteoclastogenesis. To test our hypothesis, murine bone marrow cells were treated with M-CSF (50 μg/mL) for two days to allow bone marrow progenitor cells to differentiate into bone marrow-derived pre-osteoclasts. To determine if FTY720 could inhibit osteoclastogenesis induced by RANKL, bone marrow-derived pre-osteoclasts were treated with M-CSF (50 μg/mL) and RANKL (100 ng/mL) for three days; then the media were changed with fresh media containing M-CSF (50 μg/mL) and RANKL (100 ng/mL). Cells were treated with FTY720 (2 μM) or vehicle (ethanol) for 24 h (Fig. 3a). Additionally, to determine if FTY720 could attenuate osteoclastogenesis induced by A. actinomycetemcomitans, bone marrow-derived pre-osteoclasts were treated with M-CSF (50 μg/mL) and RANKL (100 ng/mL) for three days. To reduce the background of osteoclastogenesis induced by RANKL, the media were changed with media containing only M-CSF (50 μg/mL) without RANKL. The cells were treated for 30 min with vehicle or FTY720 (2 μM). Then the cells were either unstimulated or stimulated for 24 h with A. actinomycetemcomitans (0.5 CFU/cell), in the presence of FTY720 or vehicle (Fig. 3a). Control cells were treated with media containing only M-CSF (50 μg/mL) with or without bacterial stimulation. Osteoclasts were detected by tartrate-resistant acid phosphatase (TRAP) staining 24 h after FTY720 or vehicle treatment. As shown in Fig. 3b, there were no TRAP+ osteoclasts in cells treated with only M-CSF with or without bacterial stimulation. There were many TRAP+ multinucleated osteoclasts in cells treated with vehicle in the presence of both M-CSF and RANKL with or without bacterial stimulation. In contrast, FTY720 (2 μM) treatment decreased both size and number of TRAP+ multinucleated osteoclasts compared with vehicle groups. Quantification of the number of osteoclasts showed that there was a 1.9-fold increase of the number of osteoclasts in cells treated with M-CSF and RANKL stimulated with A. actinomycetemcomitans compared with cells treated with M-CSF and RANKL without bacterial stimulation (Fig. 3c). FTY720 treatment reduced the number of osteoclasts by 53.2 % in cells treated with M-CSF and RANKL without bacterial stimulation, and decreased the number of osteoclasts by 64.3 % in cells treated with M-CSF and RANKL with bacterial stimulation, as compared with vehicle controls, respectively (Fig. 3c). Quantification of total area of osteoclasts per image revealed that FTY720 reduced the area of osteoclasts by 74.2 % in cells treated with M-CSF and RANKL without bacterial stimulation, and FTY720 decreased the area of osteoclasts by 71.4 % in cells treated with M-CSF and RANKL with bacterial stimulation (Fig. 3d). FTY720 (2 μM) treatment for 24 h did not induce cell death in bone marrow-derived pre-osteoclast (Fig. 3e). These data demonstrated that FTY720 suppressed osteoclastogenesis in bone marrow-derived pre-osteoclasts treated with M-CSF and RANKL with or without bacterial stimulation.Fig. 3


FTY720 inhibited proinflammatory cytokine release and osteoclastogenesis induced by Aggregatibacter actinomycetemcomitans.

Yu H, Herbert BA, Valerio M, Yarborough L, Hsu LC, Argraves KM - Lipids Health Dis (2015)

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)
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Related In: Results  -  Collection

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Fig3: 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)
Mentions: Osteoclasts originate from the fusion of monocytes and macrophages [27]. It has been recognized that phosphoinositides signaling controls the activation of Nfatc1 and influences osteoclastogenesis [28]. Furthermore, proinflammatory cytokines promote the differentiation of osteoclasts [1]. Since FTY720 significantly inhibited PI3K signaling and attenuated proinflammatory cytokine expression induced by A. actinomycetemcomitans, we hypothesized that FTY720 could further inhibit osteoclastogenesis. To test our hypothesis, murine bone marrow cells were treated with M-CSF (50 μg/mL) for two days to allow bone marrow progenitor cells to differentiate into bone marrow-derived pre-osteoclasts. To determine if FTY720 could inhibit osteoclastogenesis induced by RANKL, bone marrow-derived pre-osteoclasts were treated with M-CSF (50 μg/mL) and RANKL (100 ng/mL) for three days; then the media were changed with fresh media containing M-CSF (50 μg/mL) and RANKL (100 ng/mL). Cells were treated with FTY720 (2 μM) or vehicle (ethanol) for 24 h (Fig. 3a). Additionally, to determine if FTY720 could attenuate osteoclastogenesis induced by A. actinomycetemcomitans, bone marrow-derived pre-osteoclasts were treated with M-CSF (50 μg/mL) and RANKL (100 ng/mL) for three days. To reduce the background of osteoclastogenesis induced by RANKL, the media were changed with media containing only M-CSF (50 μg/mL) without RANKL. The cells were treated for 30 min with vehicle or FTY720 (2 μM). Then the cells were either unstimulated or stimulated for 24 h with A. actinomycetemcomitans (0.5 CFU/cell), in the presence of FTY720 or vehicle (Fig. 3a). Control cells were treated with media containing only M-CSF (50 μg/mL) with or without bacterial stimulation. Osteoclasts were detected by tartrate-resistant acid phosphatase (TRAP) staining 24 h after FTY720 or vehicle treatment. As shown in Fig. 3b, there were no TRAP+ osteoclasts in cells treated with only M-CSF with or without bacterial stimulation. There were many TRAP+ multinucleated osteoclasts in cells treated with vehicle in the presence of both M-CSF and RANKL with or without bacterial stimulation. In contrast, FTY720 (2 μM) treatment decreased both size and number of TRAP+ multinucleated osteoclasts compared with vehicle groups. Quantification of the number of osteoclasts showed that there was a 1.9-fold increase of the number of osteoclasts in cells treated with M-CSF and RANKL stimulated with A. actinomycetemcomitans compared with cells treated with M-CSF and RANKL without bacterial stimulation (Fig. 3c). FTY720 treatment reduced the number of osteoclasts by 53.2 % in cells treated with M-CSF and RANKL without bacterial stimulation, and decreased the number of osteoclasts by 64.3 % in cells treated with M-CSF and RANKL with bacterial stimulation, as compared with vehicle controls, respectively (Fig. 3c). Quantification of total area of osteoclasts per image revealed that FTY720 reduced the area of osteoclasts by 74.2 % in cells treated with M-CSF and RANKL without bacterial stimulation, and FTY720 decreased the area of osteoclasts by 71.4 % in cells treated with M-CSF and RANKL with bacterial stimulation (Fig. 3d). FTY720 (2 μM) treatment for 24 h did not induce cell death in bone marrow-derived pre-osteoclast (Fig. 3e). These data demonstrated that FTY720 suppressed osteoclastogenesis in bone marrow-derived pre-osteoclasts treated with M-CSF and RANKL with or without bacterial stimulation.Fig. 3

Bottom Line: This study determined the effect of FTY720 on proinflammatory cytokine production and osteoclastogenesis in murine bone marrow cells with or without A. actinomycetemcomitans stimulation.Osteoclasts were stained by tartrate-resistant acid phosphatase (TRAP) staining.Additionally, FTY720 attenuated p-PI3K, p-Akt, and p-ERK expressions induced by A. actinomycetemcomitans.

View Article: PubMed Central - PubMed

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.

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.

No MeSH data available.


Related in: MedlinePlus