Inhibition Effect Of Estrogen On Osteoclast Differentiation And Bone Resorption Through CD4~+CD25~+Foxp3~+ Regulatory T Cells

The immune and skeletal system share a lot of regulatory factors including cytokines and its receptors,signaling molecules and transcription factors;both of the bone and immune cells are in the same micro-environment,bone marrow.Various factors produced and released during immune responses markedly affect bone cells and bone metabolism.Communication between the immune and the skeletal systems is observed in physiological status,but more obviously in autoimmune and other inflammatory diseases.This interrelationship leads to Arron and Choi to propose a concept 'osteoimmunology' to describe the interaction between immune system and bone metabolism.Osteoclasts play pivotal roles in bone remodeling and some diseases as Postmenopausal osteoporosis(PMO),rheumatoid arthritis(RA) and periodontal disease.Osteoclasts are multinucleated cells that derive from hematopoietic progenitors in the bone marrow.Receptor activator of nuclear factor kappaB ligand (RANKL) and macrophage colony-stimulating factor(M-CSF) induces the differentiation of hematopoietic progenitors into mature osteoclasts.In RA and inflammatory bowel disease,activated T cells were observed to express RANKL and promote osteoclastogenesis,which links the activation of the immune system and osteoclastogenesis,bone resorption.The present study is to elucidate the mechanisms underlying modulation of CD4⁺CD25⁺Foxp3⁺Regulatory T Cells on Osteoclast differentiation and bone resorption.Partâ… Suppressive regulation of IL-10 and TGF-β1 derived from regulatory T cells on the osteoclast differentiation and bone resorptionObjective Regulatory T cells play an immune regulatory role by secreting such cytokines as IL-10 and TGF-β1.The aim of this study is to investigate the effect of IL-10 and TGF-β1 derived from regulatory T cells on the osteoclast differentiation and bone resorption.Methods Human bone marrow cells were isolated from embryos terminated in 13 and 16 weeks gestation fro non-medicine reason with mifepristone and misoprostol, and maintained in MEM alpha supplemented with 50ng/ml M-CSF and RANKL. After treated with different of concentration of IL-10 or TGF-β1,the BMCs were used for differentiation assay by tartrate-resistant acid phosphatase(TRAP)-staining. Bone resorption was performed in a resorption pit assay using ivory dentine slices with Image-Pro plus 6.0.Results IL-10 or TGF-β1 of 50ng/ml concentration is able to significantly inhibit the osteoclast differentiation and pit formation on dentine slices(P＜0.05),and the inhibition is in a dosage-dependent manner.The combined treatment of IL-10 and TGF-β1 to BMCs exhibit synergistic effect on osteoclastogenesis and bone resorption at a lower concentration,10ng/ml(P＜0.01).Conclusion These results above indicate that IL-10 and/or TGF-β1,efficiency cytokines of the regulatory T cells,suppress osteoclastogenesis and bone resorption mediated by the osteoclasts.Partâ…¡Impact of the CD4⁺CD25⁺Foxp3⁺regulatory T cells on the osteoclast differentiation and bone resorptionObjective The study is to investigate impact and mechanism of the CD4⁺CD25⁺ Foxp3⁺ regulatory T cells on the differentiation of OC from bone marrow cells and bone resorption.Methods The CD4⁺CD25⁺ Foxp3⁺ regulatory T cells were isolated and purified from the PBMCs of healthy adults with MACS.To Study the effect of Tregs on the differentiation of OC from bone marrow cells and bone resorption,the Tregs and BMCs were in direct co-culture or indirect co-culture by transwell in different ratios from 10:50 to 1:50.The IL-10 and TGF-β1 production in the co-culture supematant was determined by ELISA.Moreover,the cytokine-blocking experiments were performed with anti-IL-10 and anti-TGFβ1 antibodies.Results The CD4+^CD25⁺ Foxp3⁺ regulatory T cells inhibit the differentiation of OC from bone marrow cells in a dose-dependent manner(P＜0.05).Pit formation is also inhibited by Tregs(P＜0.05).There is no difference of the suppressive effect between direct and indirect co-culture(P＞0.05).Further study indicates that the inhibition depends on cytokine excretion.IL-10 and TGF-β1 production is up-regulated with the increasing ratio ofTregs:BMCs.After treated with anti-IL-10 and anti-TGFβ1 antibodies,the suppression of Tregs to OC differentiation and bone resorption is reversed completely.Conclusion These data suggest that the CD4⁺CD25⁺ Foxp3⁺ regulatory T cells can suppress osteoclast differentiation and bone resorption by way of secretion of IL-10 and TGF-β1.Partâ…¢17-beta estradiol induces CD4⁺CD25⁺Foxp3⁺ regulatory T cells to secret IL-10 for suppression of OC differentiation and bone resorptionObjective The study is to explore the regulation of 17-beta estradiol in the OC differentiation and function mediated by Tregs.Methods Tregs and BMCs were co-cultured at ratio 2:50 with respective BMCs or Tregs as control.The co-culture was treated respectively by different concentrations of E2 from 10^-7 to10^-10 mol/L for 48 hours with dissolvent as control.Thereafter,the supernatant was collected to determine the levels of IL-10 and TGF-β1 with ELISA.After further culture of 7 and 10 days,the TRAP positive cells and bone resorption lacuna area were calculated,respectively.Results The OC differentiation and bone resorption were suppressed efficiently by E2 at the concentration of 10^-7-10^-9mol/L in the BMCs and Tregs/BMCs(P＜0.05). And the inhibition of E2 on OC differentiation and bone resorption showed a dose-dependent manner.When E2 reached 10^-10 mol/L,it had no significant effect (P＞0.05).IL-10 and TGF-β1 expression was up-regulated after adding Tregs(P＜0.05). After treated with E2,the IL-10 production in the Tregs/BMCs and Tregs supernatant shows a pronounced increase(P＜0.01),but TGF-β1 had no significant change (P＞0.05).In addition,the secretion of IL-10 increases with the increasing concentration of E2.Conclusion 17-beta estradiol has suppressive effects on OC differentiation and bone resorption.E2 at physiological dosage enhances IL-10 secretion by Tregs,and inhibits OC differentiation and bone resorption.IL-10 is likely involved in the regulation of E2 on bone metabolism via the interaction of Tregs to osteoclast,which may act as potential target for the therapeutics of PMO.