Effects Of Triptolide On OC Differentiation And Bone Resorption In Tregs - OC Co - Culture System

1 BackgroundRheumatoid arthritis (RA) is autoimmune disease characterized by chronic and progressive multiple joint inflammations. Destruction of articular cartilage and bone is one of main pathogenic mechanisms of RA which often leads to deformation and functional disability. Tripterygium Wilfordii Glycosides (TWG), with very potent anti-inflammatory and immunosuppressive effects which has been one of major medicine clinically used to treat RA. Triptolide (TP) has turned out to be the major active component of TWG. Previous studies have also found that triptolide could lower the arthritis scores and prevent the bone destruction of collagen-induced arthritis (CIA) mice or rat. Osteoclasts (OC) play an important role in bone destruction of RA. The number and function of regulatory T cells (Tregs) is linked to Ihe development of RA. Recent studies have suggested that Tregs have an effect on osteoclast differentiation and resorption function. In this study, using Tregs and osteoclast co-culture system, we investigated the effect of triptolide on the Tregs-mediated regulation of osteoclasts differentiation and bone resorption.2 ObjectiveTo investigate the effect of triptolide on osteoclast differentiation and bone resorption in co-culture system of Tregs with osteoclast, and indicating the therapeutic efficiency of triptolide in preventing bone destruction of RA.3 Methods3.1 The effect of triptolide on osteoclasts differentiation and bone resorptionBone marrow cells were collected from the bilateral tibias and femus of C57BL/6 mice. After 24 hours, non-adherent cells were pre-induced with macrophage colony-stimulating factor (M-CSF). After another 3 days, the adherent cells were cultured in the presence of receptor of nuclear factor-κB ligand (RANKL) and with different concentration of triptolide (0,2.5,5,10, 20,40 nmol/L). On the 4th day, CCK-8 method was used to identify if triptolide had toxicity on osteoclasts. Tartrate-resistant acid phosphatase (TRAP) activity was measured to compare the activity of osteoclasts. Cells were stained by TRAP to compare the number of osteoclasts. On the 9th day, the bone slices were stained by toluidine blue and quantified the resorption area by Leica Qwin system.3.2 Co-culture system of Tregs with osteoclastsPooled cell suspensions were obtained from spleens of C57BL/6 mice. CD4+CD25" T cells were negative selected by magnetic cell sorting after removing the red blood cells. The freshly isolated CD4+CD25" T cells were plated in a round 96-well plate that had been pre-coated with anti-CD3 and supplemented with anti-CD28, IL-2 and TGF-β for induction. On the 7th day, CD4+CD25+regulatory T cells were then purified by using magnetic cell sorting again and analyzed on a flow cytometer to measure Foxp3 expression.Tregs and osteoclasts were co-cultured by different ratios (1:50,2:50, or 10:50) in the direct co-culture and indirect co-culture by Transwell inserts and compare with osteoclasts alone group as control group. Osteoclasts were cultured as 3.1, cells were cultured in the presence of RANKL and with different concentration of Tregs for co-culture. After being co-cultured for 24 hours, the supernatants were collected tested the level of IL-10 and TGF-β 1 by ELISA assay. On the 5th day, the cells were stained by TRAP to determine the number of osteoclasts. On the 12th day, the resorbed surfaces of the bone slices were observed by staining with toluidine blue and calculated by Leica Qwin image analysis software.3.3 The effect of triptolide on osteoclast differentiation and bone resorption in TregrOC co-culture systemThe activated Tregs were cultured with different concentration of triptolide (0,2.5,5,10,20, 40 nmol/L). After 24 hours, CCK-8 method was used to identify if triptolide had toxicity on Tregs. Based on the results of 3.1, the optimal dose of triptolide was determined to this experiment. According to the results of 3.2, a co-culture system comprised of Tregs and osteoclastss at a ratio of 2:50 by direct cell-to-cell contact was determined to this experiment.Moreover, the activated Tregs were cultured with osteoclasts in the presence of anti-IL10 and anti-TGFβ1 neutralizing monoclonal antibodies to determine the mechanism of Tregs to osteoclasts. Cells were divided into 6 groups:osteoclasts alone group, Tregs+ osteoclasts+triptolide group, Tregs+osteoclasts+anti-IL10+anti-TGFβ1 group, osteoclasts +triptolide group, Tregs+osteoclasts+triptolide group, Tregs+osteoclasts+triptolide group+anti-IL10+anti-TGFβ1 group. Osteoclasts were cultured in 24-well plate as above 3.1, when cells were cultured in the presence of receptor of RANKL, we added the activated Tregs, triptolide or neutralizing monoclonal antibodies at same time. After 24 hours, the supernatants of each group were collected to test the level of IL-10 and TGF-β1 by ELISA assay. After being co-cultured for 5 days, the cells were stained by TRAP to determine the number of osteoclasts. After 12 days, the resorbed surfaces of the bone slices were observed by staining with toluidine blue and calculated by Leica Qwin image analysis software.4 Results4.1 The effect of triptolide on osteoclasts differentiation and bone resorptionCompare with control group (triptolide 0 nmol/L), TRAP activity of osteoclasts were down-regulated with the increasing of boldine concentration.10 nmol/L,20 nmol/L and 40 nmol/L triptolide can significantly inhibit TRAP activity of osteoclasts (P<0.05, P<0.01). Meantime, the number of osteoclasts and bone resorption were significantly decreased in a dosage-dependent manner, (P<0.05, P<0.01). But the groups of 20nmol/L and 40nmol/L triptolide showed toxicity to osteoclasts.4.2 Co-culture system of Tregs with osteoclastsAnalysis by flow cytometry revealed that the CD4+CD25+T cells were 98.81% pure and that the CD25+Foxp3+T cells were 96.49% pure to confirm the identity of these Tregs.Compare with osteoclasts alone group, when the ratio of Tregs:osteoclasts was 2:50, the production of IL-10 and TGF-β1 was both up-regulated in co-culture systems (P<0.01). The osteoclasts differentiation and bone resorption were also efficiently suppressed. When the ratio approached 10:50, the production of IL-10 and TGF-β1 was both increased more significantly (P<0.01). Osteoclast formation and the resorbed surfaces of the bone slices were rarely observed.There was no significant difference between the cell-contact and Transwell groups. So we chose a 2:50 co-culture ratio of Tregs to osteoclasts for further research. Moreover, when a combination of anti-IL 10 and anti-TGF β1 neutralizing antibodies was involved in co-culture, the inhibitory effect of Tregs on osteoclasts differentiation and function has no significant difference.4.3 The effect of triptolide on osteoclast differentiation and bone resorption in Tregs-OC co-culture systemCompared with control group (triptolide 0 nmol/L), the groups of 40 nmol/L triptolide showed toxicity to Tregs. However,20 nmol/L and 40 nmol/L triptolide had toxicity to osteoclasts. Therefore, we chose lOnmol/L triptolide for further experiments. Compared with osteoclasts alone group, osteoclasts number and bone resorption of Tregs+osteoclasts group were significantly decreased (P<0.01). Compared with Tregs+osteoclasts group, osteoclasts number and bone resorption of Tregs+osteoclasts+triptolide group were significantly decreased (P<0.05), Compared with Tregs alone group, the level of IL-10 and TGF-β1 in Tregs+osteoclasts group were significantly increased (P<0.05), and the production of IL-10 and TGF-β1 in Tregs+triptolide group were also significantly increased (P<0.05, P<0.01). Compared with Tregs+osteoclasts group, the level of IL-10 and TGF-β1 in Tregs+ osteoclasts+triptolide group were both significantly increased (P<0.05, P<0.01). When the co-culture systems were supplement with neutralizing monoclonal antibodies, compared with Tregs+osteoclasts group, osteoclasts number and bone resorption of Tregs+osteoclasts+ anti-IL 10+anti-TGF β1 group were significantly increased (P<0.01). Compared with Tregs +osteoclasts+triptolide group, osteoclasts number and bone resorption of Tregs+osteoclasts +triptolide group+anti-IL 10+anti-TGF β1 group were also significantly increased (P< 0.01).5 ConclusionTriptolide inhibits osteoclasts differentiation and bone resorption function in culture of osteoclasts alone, and further inhibits osteoclasts in co-culture system of Tregs with osteoclasts. Furthermore, we found that one of the mechanisms is that triptolide enhances the level of IL-10 and TGF-β1 secreted by Tregs in co-culture system.