Study On The Effects Of S.aureus,SpA,PGN And LTA On Osteoclast Differentiation And Its Molecular Mechanism

Objectives:To investigate the effects of Staphylococrcus aureus(S.aureus)and its cell wall proteins-SpA,PGN and LTA on osteoclast differentiation and bone resorption,and to explore the underlying molecular mechanisms of osteoclast differentiation induced by S.aureus,SpA,PGN and LTA.Methods:1.MTT assay.RAW 264.7 cells were plated in 96-well plates,and then the cells were treated with live S.aureus(multiplicity of infections(MOIs)of 5:1,10:1,20:1 and 40:1 CFU per cell),inactivated S.aureus(MOIs of 10:1,20:1 and 40:1 CFU per cell),S.aureus filtrate(concentrations of 5,10 and 20?g/ml),SpA(concentrations of 50,100,200,400,800 and 1600ng/ml),PGN(concentrations of 100,200 and 400 ng/ml)and LTA(concentrations of 100,200 and 400ng/ml)for 1,3 and 5 days respectively.At the indicated days,the MTT assay was used to detect the optical density(OD)values of every well in 96-well plates to assess the effects of various stimuli on the proliferation of RAW264.7 cells.2.Tartrate resistant acid phosphatase staining(TRAP staining).RAW 264.7 cells were seeded in 96-well plates,then the cells were treated with different multiplicity of infections of live S.aureus(5-40:1 CFU per cell),inactivated S.aureus(10-40:1 CFU per cell),and with different concentrations of S.aureus filtrate(5-20?g/ml),SpA(50-800ng/ml),PGN(100-400ng/ml)and LTA(100-400ng/ml)for 5 days.After culturing for total 5 days,the cells were fixed and stained using TRAP staining kit according to the manufacturer's instructions.TRAP-positive and multi-nucleated(?3 nuclei)cells in each experimental groups were counted as osteoclast-like cells.In order to verify the role of NF-?B signaling pathway,JSH-23(20?M),a transcription inhibitor of NF-?B,was added one hour ahead of stimuli,and worked for 25 hours,all these experiments were repeated.3.Bone resorption assay.RAW 264.7 cells were seeded in 24-well Corning(?)Osteo Assay Surface Multiple Well Plates,and then the cells were treated with different multiplicity of infections of live S.aureus(5-40:1 CFU per cell),inactivated S.aureus(10-40:1 CFU per cell),and with different concentrations of S.aureus filtrate(5-20?g/ml),SpA(50-800ng/ml),PGN(100-400ng/ml)and LTA(100-400ng/ml)for 5 days.After culture of 5 days,the mediums and cells were removed,and the ratio of accumulative area of resorption pits to the visual field area was analyzed with ImagePro Plus 6.0 software.In order to verify the role of NF-?B signaling pathway,JSH-23(20?M)was added one hour ahead of stimuli,and all these experiments were repeated.4.Real-time quantitative reverse transcription polymerase chain reaction(RT-PCR)assay.RAW 264.7 cells were seeded in 6-well cell Plates,then the cells were treated with live S.aureus(MOIs 5-40:1 CFU per cell),inactivated S.aureus(MOIs 10-40:1 CFU per cell),S.aureus filtrate(5-20?g/ml)and SpA(50-800ng/ml)for 5 days.After that,the cells were harvested,and the expression of osteoclast specific genes——TRAP,MMP-9,cathepsin K,calcitonin receptors and Atp6v0d2 were measured by RT-PCR assay.In order to verify the role of NF-?B signaling pathway,JSH-23(20?M)was added one hour ahead of stimuli,and all these experiments were repeated.5.Western blot assay.RAW 264.7 cells were cultured in 25 cm2 cell culture flasks,when the cells covering the bottom of the flasks were about 80%,the cells were stimulated with S.aureus(MOI 20:1 CFU per cell),SpA(400ng/ml),PGN(200ng/ml)and LTA(200ng/ml)for 0,5,10,20,40,60 minutes.At the indicated time point,the cells were harvested and used to detected the expression of ERK1/2,p38,JNK,NF-?B p65,I?B-?,Akt,and the phosphorylated form of ERK1/2,p38,JNK,NF-?B p65 and Akt.For NFATcl measurement,RAW264.7 cells were seeded in 6-well plates,and treated with S.aureus,SpA,PGN and LTA for 0,1,2 and 3 days respectively,at the indicated time point,the cells were harvested for detecting the expression of NFATcl.In order to verify the relationship between NF-?B and NFATcl,JSH-23(20?M)was added one hour ahead of stimuli,and all these experiments were repeated.6.Enzyme-linked immunosorbent assay(ELISA).RAW 264.7 cells were plated in 24-well Plates,and the cells were stimulted with live S.aureus(MOIs 5-40:1 CFU per cell),SpA(50-800ng/ml),PGN(100-400ng/ml)and LTA(100-400ng/ml)for 1,2,3 days respectively.At the indicated time point,the culture supernatants were collected and used for detecting the levels of TNF-?,IL-6 and IL-1?.Results:1.The results of MTT assay.live S.aureus with MOIs of 5:1 and 10:1 CFU per cell showed no obvious cytotoxicity to RAW 264.7 cells,but live S.aureus with MOIs of 20:1 and 40:1 CFU per cell inhibited the proliferation activity of RAW264.7 cells.On the first day after SpA stimulation,SpA promoted cell proliferation under the concentrations of 100-400ng/ml,and SpA had no cytotoxicity to RAW264.7 cells at the concentrations of 50ng/ml and 800ng/ml,but SpA inhibited the proliferation activity of RAW264.7cells at the concentration 1600ng/ml.On the days 2 and 3 after SpA stimulation,SpA had no cytotoxicity to RAW264.7 cells below the concentration of 800ng/ml,but SpA inhibited the proliferation activity of RAW264.7cells at the concentration of 1600ng/ml.While the inactivated S.aureus,S.aureus filtrate,PGN and LTA used in experiments,showed no obvious cytotoxicity to RAW 264.7 cells.2.The results of TRAP staining.Live S.aureus,inactivated S.aureus,S.aureus filtrate,SpA,PGN and LTA significantly promoted the formation of osteoclast-like cells in a dose-dependent manner,with the increase of stimuli dose,the number of osteoclast like cells increased gradually.However,when treated with JSH-23,the formation of osteoclast-like cells induced by stimuli mentioned above was obviously inhibited.In addition,SpA promoted the formation of osteoclast-like.cells in the presence of RANKL,and the number of osteoclast-like cells was higher than that without RANKL at the same concentration.3.The results of bone resorption assay.Live S.aureus,inactivated S.aureus,S.aureus filtrate,SpA,PGN and LTA significantly promoted the formation of resorption pits in a dose-dependent manner,and with the increase of stimuli dose,the area of resorption pits increased gradually.However,when treated with JSH-23,the area of resorption pits induced by stimuli mentioned above was obviously inhibited.In addition,SpA promoted the formation of resorption pits in the presence of RANKL,and the area of resorption pits was higher than that without RANKL at the same concentration.4.The results of RT-PCR assay.Live S.aureus,inactivated S.aureus,S.aureus filtrate and SpA significantly promoted the expression of osteoclast specific genes in a dose-dependent manner,and with the increase of stimuli dose,the expression of osteoclast specific genes increased gradually.However,when treated with JSH-23,the expression of osteoclast specific genes induced by stimuli mentioned above was obviously inhibited.In addition,SpA promoted the expression of osteoclast specific genes in the presence of RANKL,and the expression of osteoclast specific genes was higher than that without RANKL at the same concentration.5.The results of western blot assay.The degradation of I?B?occurred at 10min after S.aureus and SpA stimulation,while the phosphorylation of NF-?B rose markedly at 5 and 10min.Under the stimulation of PGN,the degradation of I?B? occurred at 5 and 10min,while the phosphorylation of NF-?B rose markedly at 5 and 10min.And under the stimulation of LTA,the degradation of I?B? occurred at 5 and 10min,while the phosphorylation of NF-?B rose markedly at 10min.In addition,the other proteins in the signaling pathways showed no obvious change when treated with S.aureus,SpA,PGN and LTA.Moreover,S.aureus,SpA,PGN and LTA significantly promoted the expression of NFATcl on days 2 and 3,but JSH-23 inhibited the increased expression of NFATcl induced by S.aureus,SpA,PGN and LTA.6.The results of ELISA assay.S.aureus induced a robust production of-TNF-a,IL-la and IL-6 in a time-and dose-dependent way on days 2 and 3.While SpA,PGN and LTA induced a robust production of IL-6 in a time-and concentration-dependent manner on days 2 and 3,but the expression of TNF-a and IL-la showed no significant increase.Conclusions:1.Staphylococcus aureus can promote the formation of osteoclast differentiation and promote bone resorption,which is the combined action of its cell wall compounds and the secretion of active molecules.2.SpA,PGN and LTA have the effects of promoting osteoclast differentiation and promoting bone resorption.3.NF-kappa B signaling pathway plays an important role in osteoclast differentiation and bone resorption induced by Staphylococcus aureus,SpA,PGN and LTA.