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. 2019 Jan;19(1):414-422.
doi: 10.3892/mmr.2018.9662. Epub 2018 Nov 15.

Streptolysin O derived from Streptococcus pyogenes inhibits RANKL‑induced osteoclastogenesis through the NF‑κB signaling pathway

Jin Yi  1 , Ruohui Tang  1 , Jing Yang  1 , Yueqi Chen  2 , Jun Fei  1
Affiliations

Streptolysin O derived from Streptococcus pyogenes inhibits RANKL‑induced osteoclastogenesis through the NF‑κB signaling pathway

Jin Yi et al. Mol Med Rep. 2019 Jan.

Abstract

Streptococcus pyogenes (GAS) is a clinically significant bacterial strain that causes bacterial arthritis, osteomyelitis and implant infections. Infection complications can lead to serious bone destruction. Osteoclasts, the only type of cell with bone resorption function, participate in this process. Streptolysin O (SLO) is produced by almost all clinical Streptococcus pyogenes isolates. However, the role of SLO in bone infection caused by GAS had not been previously examined. The current study was performed to define the effects of SLO on receptor activator of NF‑κB ligand‑stimulated osteoclast differentiation in vitro. Results demonstrated that SLO decreased the phosphorylation of p65 and NF‑κB inhibitor α, suppressed c‑FOS and nuclear factor of activated T‑cells cytoplasmic 1, and downregulated the expression of osteoclast marker genes. SLO also induced apoptosis of mature osteoclasts. The results suggested that SLO blocked osteoclast activation during GAS infection. These findings may prove useful in the development of novel strategies for treating GAS‑associated bone infectious diseases.

Keywords: GAS; SLO; bone infection; osteoclast; NF-κB.

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Figures

Figure 1.
Figure 1.
SLO toxicity evaluation in vitro. RAW 264.7 cells were cultured in medium containing 10% fetal bovine serum with the indicated dosages of SLO for (A) 24 h and (B) 72 h. Cell viability was measured by Cell Counting kit-8 assay. **P<0.01 vs. 0 µg/ml SLO. SLO, streptolysin O.
Figure 2.
Figure 2.
SLO inhibits osteoclast differentiation. (A) Raw 264.7 cells were stimulated by different concentrations of SLO and 50 ng/ml M-CSF and 50 ng/ml RANKL for 3 days. Cells were then fixed and stained for TRAP (magnification, ×40). TRAP-positive cells with ≥3 nuclei were considered to be osteoclasts. (B) Quantification of osteoclast number in each well. The date in the figures represents the mean ± standard deviation. **P<0.01 vs. RANKL group. TRAP, tartrate-resistant acid phosphatase; RANKL, receptor activator of NF-κB ligand; SLO, streptolysin O; N.D., no data (no TRAP-positive cells detected).
Figure 3.
Figure 3.
Osteoclast fusion and bone resorption assay. (A) Actin cytoskeleton and focal adhesion staining of RAW264.7 cells. F-actin was detected using TRITC-conjugated phalloidin (green). Vinculin was revealed using vinculin monoclonal antibody (red). Nuclei were revealed with DAPI (blue). Monochrome images of TRITC-conjugated phalloidin, vinculin monoclonal antibody vinculin and DAPI were merged (magnification, ×40). (B) Average number of nuclei in each group (mean ± standard deviation of 3 fields of view). (C) Raw 264.7 cells were seeded into osteo assay surface 96-well plates and cultured in medium containing 50 ng/ml RANKL and 50 ng/ml M-CSF with different dosages of SLO for 5 days. Bone resorption area was observed under a light microscope (magnification, ×40). (D) The area of resorption pits formed in the SLO-treated group was significantly lower than that of the control group (3 fields of view). **P<0.01 vs. RANKL group. TRITC, tetramethylrhodamine; RANKL, receptor activator of NF-κB ligand; SLO, streptolysin O; M-CSF, macrophage colony-stimulating factor; N.D., no data (no resorption pits detected).
Figure 4.
Figure 4.
RT-qPCR analysis of mRNA during SLO stimulation. Raw 264.7 cells were cultured with M-CSF (50 ng/ml) and RANKL (50 ng/ml), with or without SLO. The expression of TRAP, CTR, MMP9, ATP6v0d2, DC-STAMP and integrinβ3 were analyzed by RT-qPCR. The results were normalized to the expression of GAPDH. The levels of these mRNAs were significantly reduced with SLO treatment (particularly 2.5 µg/ml SLO) compared to the control group. *P<0.05 and **P<0.01 vs. RANKL group. RT-qPCR, reverse transcription-quantitative polymerase chain reaction; RANKL, receptor activator of NF-κB ligand; M-CSF, macrophage colony-stimulating factor; SLO, streptolysin O; TRAP, tartrate-resistant acid phosphatase; CTR, calcitonin receptor; MMP9, matrix metalloproteinase-9; ATP6v0d2, ATPase H+ transporting V0 subunit d2; DC-STAMP, dendritic cell-specific transmembrane protein.
Figure 5.
Figure 5.
Western blot analysis of protein expression during SLO stimulation. (A) RAW 264.7 cells were stimulated with RANKL for the indicated durations in the presence or absence of 2.5 µg/ml SLO. The effects of RANKL and RANKL + SLO are presented. (B) Raw 264.7 cells were treated with 1 and 2.5 µg/ml for 72 h. The protein expression of c-FOS and NFATc1 were significantly reduced in the experimental group. RANKL, receptor activator of NF-κB ligand; SLO, streptolysin O; p-, phospho-; IκBα, NF-κB inhibitor α; c-FOS, Fos proto-oncogene; NFATc1, nuclear factor of activated T cells cytoplasmic 1.
Figure 6.
Figure 6.
Osteoclast apoptosis assay. Raw 264.7 cells were cultured with 50 ng/ml M-CSF and 50 ng/ml RANKL with 1 or 2.5 µg/ml SLO for 72 h. (A and B) The cells then were stained with Annexin-V-FITC and PI fort 15 min in the dark for flow cytometry. *P<0.05 vs. RANKL group. (C) Western blotting was performed to analyze the protein expression of Bax, Bcl-2 and cleaved caspase-3. RANKL, receptor activator of NF-κB ligand; SLO, streptolysin O; PI, propidium iodide; FITC, fluorescein isothiocyanate; Bcl-2, BCL2 apoptosis regulator; Bax, BCL2 associated X apoptosis regulator.
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