Regulatory T cells are expanded by Teriparatide treatment in humans and mediate intermittent PTH ‐induced bone anabolism in mice
2017; Springer Nature; Volume: 19; Issue: 1 Linguagem: Inglês
10.15252/embr.201744421
ISSN1469-3178
AutoresMingcan Yu, Patrizia D’Amelio, Abdul Malik Tyagi, Chiara Vaccaro, Jau‐Yi Li, Emory Hsu, Ilaria Buondonno, Francesca Sassi, Jon Adams, M. Neale Weitzmann, Richard J. DiPaolo, Roberto Pacifici,
Tópico(s)Cancer Cells and Metastasis
ResumoArticle20 November 2017free access Transparent process Regulatory T cells are expanded by Teriparatide treatment in humans and mediate intermittent PTH-induced bone anabolism in mice Mingcan Yu Division of Endocrinology, Metabolism and Lipids, Department of Medicine, Emory University, Atlanta, GA, USA Search for more papers by this author Patrizia D'Amelio Gerontology Section, Department of Medical Sciences, University of Torino, Torino, Italy Search for more papers by this author Abdul Malik Tyagi Division of Endocrinology, Metabolism and Lipids, Department of Medicine, Emory University, Atlanta, GA, USA Search for more papers by this author Chiara Vaccaro Division of Endocrinology, Metabolism and Lipids, Department of Medicine, Emory University, Atlanta, GA, USA Search for more papers by this author Jau-Yi Li Division of Endocrinology, Metabolism and Lipids, Department of Medicine, Emory University, Atlanta, GA, USA Search for more papers by this author Emory Hsu Division of Endocrinology, Metabolism and Lipids, Department of Medicine, Emory University, Atlanta, GA, USA Search for more papers by this author Ilaria Buondonno Gerontology Section, Department of Medical Sciences, University of Torino, Torino, Italy Search for more papers by this author Francesca Sassi Gerontology Section, Department of Medical Sciences, University of Torino, Torino, Italy Search for more papers by this author Jonathan Adams Division of Endocrinology, Metabolism and Lipids, Department of Medicine, Emory University, Atlanta, GA, USA Search for more papers by this author M Neale Weitzmann Division of Endocrinology, Metabolism and Lipids, Department of Medicine, Emory University, Atlanta, GA, USA Atlanta Department of Veterans Affairs Medical Center, Decatur, GA, USA Search for more papers by this author Richard DiPaolo Department of Molecular Microbiology & Immunology, Saint Louis University School of Medicine, St. Louis, MO, USA Search for more papers by this author Roberto Pacifici Corresponding Author [email protected] orcid.org/0000-0001-6077-8250 Division of Endocrinology, Metabolism and Lipids, Department of Medicine, Emory University, Atlanta, GA, USA Immunology and Molecular Pathogenesis Program, Emory University, Atlanta, GA, USA Search for more papers by this author Mingcan Yu Division of Endocrinology, Metabolism and Lipids, Department of Medicine, Emory University, Atlanta, GA, USA Search for more papers by this author Patrizia D'Amelio Gerontology Section, Department of Medical Sciences, University of Torino, Torino, Italy Search for more papers by this author Abdul Malik Tyagi Division of Endocrinology, Metabolism and Lipids, Department of Medicine, Emory University, Atlanta, GA, USA Search for more papers by this author Chiara Vaccaro Division of Endocrinology, Metabolism and Lipids, Department of Medicine, Emory University, Atlanta, GA, USA Search for more papers by this author Jau-Yi Li Division of Endocrinology, Metabolism and Lipids, Department of Medicine, Emory University, Atlanta, GA, USA Search for more papers by this author Emory Hsu Division of Endocrinology, Metabolism and Lipids, Department of Medicine, Emory University, Atlanta, GA, USA Search for more papers by this author Ilaria Buondonno Gerontology Section, Department of Medical Sciences, University of Torino, Torino, Italy Search for more papers by this author Francesca Sassi Gerontology Section, Department of Medical Sciences, University of Torino, Torino, Italy Search for more papers by this author Jonathan Adams Division of Endocrinology, Metabolism and Lipids, Department of Medicine, Emory University, Atlanta, GA, USA Search for more papers by this author M Neale Weitzmann Division of Endocrinology, Metabolism and Lipids, Department of Medicine, Emory University, Atlanta, GA, USA Atlanta Department of Veterans Affairs Medical Center, Decatur, GA, USA Search for more papers by this author Richard DiPaolo Department of Molecular Microbiology & Immunology, Saint Louis University School of Medicine, St. Louis, MO, USA Search for more papers by this author Roberto Pacifici Corresponding Author [email protected] orcid.org/0000-0001-6077-8250 Division of Endocrinology, Metabolism and Lipids, Department of Medicine, Emory University, Atlanta, GA, USA Immunology and Molecular Pathogenesis Program, Emory University, Atlanta, GA, USA Search for more papers by this author Author Information Mingcan Yu1, Patrizia D'Amelio2, Abdul Malik Tyagi1, Chiara Vaccaro1, Jau-Yi Li1, Emory Hsu1, Ilaria Buondonno2, Francesca Sassi2, Jonathan Adams1, M Neale Weitzmann1,3, Richard DiPaolo4 and Roberto Pacifici *,1,5 1Division of Endocrinology, Metabolism and Lipids, Department of Medicine, Emory University, Atlanta, GA, USA 2Gerontology Section, Department of Medical Sciences, University of Torino, Torino, Italy 3Atlanta Department of Veterans Affairs Medical Center, Decatur, GA, USA 4Department of Molecular Microbiology & Immunology, Saint Louis University School of Medicine, St. Louis, MO, USA 5Immunology and Molecular Pathogenesis Program, Emory University, Atlanta, GA, USA *Corresponding author. Tel: +1 404 712 8420; Fax: +1 404 727 1300; E-mail: [email protected] EMBO Rep (2018)19:156-171https://doi.org/10.15252/embr.201744421 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Teriparatide is a bone anabolic treatment for osteoporosis, modeled in animals by intermittent PTH (iPTH) administration, but the cellular and molecular mechanisms of action of iPTH are largely unknown. Here, we show that Teriparatide and iPTH cause a ~two-threefold increase in the number of regulatory T cells (Tregs) in humans and mice. Attesting in vivo relevance, blockade of the Treg increase in mice prevents the increase in bone formation and trabecular bone volume and structure induced by iPTH. Therefore, increasing the number of Tregs is a pivotal mechanism by which iPTH exerts its bone anabolic activity. Increasing Tregs pharmacologically may represent a novel bone anabolic therapy, while iPTH-induced Treg increase may find applications in inflammatory conditions and transplant medicine. Synopsis Parathyroid hormone (PTH) causes a ~two–threefold increase in the number of regulatory T cells (Tregs) in humans and mice, an effect which is required for PTH to exert its bone anabolic activity. Parathyroid hormone (PTH) increases the number of regulatory T cells (Tregs) in humans and mice. Blockade of the PTH-induced increase in Tregs prevents iPTH-induced bone anabolism. Introduction Primary hyperparathyroidism is a common cause of bone loss and fractures due to the continuous production of high levels of parathyroid hormone (PTH) by the parathyroid glands 12. By contrast, when PTH is injected daily, a regimen known as intermittent PTH (iPTH) treatment, the hormone increases bone volume and strength due to a stimulation of bone formation tempered by a more moderate increase in resorption 34. As a result, intermittent treatment with the 1–34 fragment of PTH is an FDA-approved treatment modality for postmenopausal osteoporosis (PMO) 5. PTH acts by binding to the PTH/PTHrP receptor PPR, which is expressed in all osteoblastic cells, including stromal cells (SCs), osteoblasts, and osteocytes 678910. Moreover, PPR is expressed in conventional CD4+ and CD8+ T cells 11 and macrophages 12. iPTH stimulates bone formation by increasing osteoblast formation and life span. Activation of Wnt signaling in osteocytes and osteoblasts is one of the proposed mechanisms by which iPTH stimulates bone formation 41314. Wnt signaling activation is achieved through multiple mechanisms including Wnt ligand-independent activation of Wnt coreceptors 15, blunted osteocytic production of the Wnt inhibitor sclerostin 161718, and decreased production by osteoblasts of the Wnt inhibitor Dkk1 19. While osteocytes and their production of sclerostin are critical for the activity of iPTH, part of this hormone activity is sclerostin independent 20 and mediated by T cells 21, a cell lineage that potentiates the anabolic activity of iPTH in trabecular bone 112022. Accordingly, iPTH fails to stimulate bone formation and increase bone mass in T-cell null mice 11. By contrast, the effects of iPTH in cortical bone are completely T cell independent 112022, likely due to the fact that T cells have no contacts with periosteal surfaces and have limited capacity to communicate with osteocytes. Among the T cells required for iPTH to exert its full anabolic activity are bone marrow (BM) CD8+ T cells 11. CD8+ T cells express higher levels of the receptor PPR than CD4+ T cells 112022. Moreover, BM CD8+ T cells, but not CD4+ T cells, respond to iPTH by releasing Wnt10b 112022, an osteogenic Wnt ligand that activates Wnt signaling in osteoblastic cells 23. Regulatory T cells (Tregs) are a suppressive population of predominantly CD4+ T cells that play a critical role in maintaining immune tolerance and immune homeostasis. Tregs are comprised of thymus-derived Tregs (tTregs, also known as nTregs) and peripherally derived Tregs (pTregs, also known as iTregs) 24. Tregs are defined by the expression of the transcription factor Foxp3 and the ability to block inflammatory diseases and maintain immune homeostasis and tolerance 25. Accordingly, defects in Treg numbers and/or activity have been implicated in several chronic inflammatory diseases. Moreover, Tregs have furthermore been found blunt bone resorption 2627, prevent ovariectomy-induced bone loss 28, and regulate osteoclast formation 262930. In vitro, conventional CD4+ T cells differentiate into Tregs by TCR stimulation under the influence of TGFβ and IL-2 313233. Recently, IGF-1 has been recognized as an additional inducer of Tregs 3435. Since iPTH increases TGFβ and IGF-1 production in bone 363738, it is likely that iPTH may induce and/or expand BM Tregs. This study was designed to investigate the effects of iPTH on Treg formation and activity in humans and mice, and to determine whether Tregs play a role in the bone anabolic activity of iPTH in mice. We report that treatment with iPTH increases the number of Tregs in humans and mice. In rodents, an increase in the number of Tregs is required for iPTH to exert its bone anabolic activity. Results Teriparatide treatment increases the number of Tregs in human peripheral blood The PTH fragment Teriparatide is the only approved bone anabolic treatment for osteoporosis but its intricate mechanism of action remains largely unknown. Among the pleiotropic effects of PTH is the capacity to increase the production of TGFβ1 and IGF-1 by human osteoblasts 394041, factors which induce Treg differentiation. To investigate whether Teriparatide regulates the number of Tregs in humans, 40 Italian women afflicted by PMO of similar age and years since menopause were enrolled in a 6-month-long prospective clinical trial. Twenty of the 40 women were treated with calcium and vitamin D (control treatment), while the remaining 20 women were treated with calcium, vitamin D, and human PTH 1-34 (Teriparatide), a treatment modality referred to hereafter as Teriparatide treatment. The baseline demographic characteristics of the study population and the serum levels of calcium, PTH, and 25-hydroxy vitamin D are shown in Table 1. Peripheral blood mononuclear cells (PBMCs) were obtained at baseline and 3 and 6 months of treatment. Analysis by flow cytometry revealed that Teriparatide treatment increased the absolute and relative number of Tregs (CD4+CD25+Foxp3+ cells) in human PBMC at 3 and 6 months of treatment, compared to baseline (Fig 1A and B). By contrast, treatment with calcium and vitamin D did not alter the number of Tregs during the 6 months of the study. As a result, both at 3 and 6 months the absolute and relative number of Tregs in PBMC was higher in women treated with Teriparatide than in those in the calcium and vitamin D control group. Table 1. Demographic and clinical data of patients with postmenopausal osteoporosis treated with calcium and vitamin D, or calcium and vitamin D and teriparatide Control Teriparatide P n 20 20 Age 68.5 ± 1.8 69.7 ± 1.6 0.535 Years since menopause 18.4 ± 2.1 20.8 ± 1.6 0.232 Ca (mg/dl) [8.8–10.4 mg/dl] 9.6 ± 0.1 9.5 ± 0.1 0.851 Serum P (mg/dl) [2.5–4.48 mg/dl] 3.5 ± 0.2 3.4 ± 0.1 0.608 PTH (pg/ml) [10–65 pg/ml] 42.8 ± 9.3 46.8 ± 4.1 0.272 25OH vitamin D (ng/ml) [20–100 ng/ml] 30.2 ± 3.1 27.3 ± 2.7 0.997 Data are shown as mean ± SEM, and P values were calculated by unpaired t-test. Values in squared parenthesis denote normal range. Figure 1. Teriparatide treatment in humans increases the absolute and relative number of Tregs in peripheral blood and TGFβ expression by Tregs Relative frequency of Tregs in PBMC at 3 and 6 months of treatment. n = 20 patients per group. Absolute frequency of Tregs in PBMC at 3 and 6 months of treatment. n = 20 patients per group. mRNA levels of TGFβ1 in peripheral blood CD4+CD25+ T cells at 3 and 6 months of treatment. n = 9 patients per group. Relative frequency of Tregs in cultures of peripheral blood CD4+CD25+ T cells stimulated with vehicle or PTH. mRNA levels of TGFβ1 in peripheral blood CD4+CD25+ T cells stimulated with vehicle or PTH. Data information: All data are expressed as mean ± SEM. All data were normally distributed according to the Shapiro–Wilk normality test and analyzed by two-way ANOVA for repeated measures. *P < 0.05, **P < 0.01, and ***P < 0.001 compared to baseline. #P < 0.05 and ##P < 0.01, compared to Ca + D group. Download figure Download PowerPoint TGFβ1 not only is a critical inducer of Treg differentiation, but is also an important product of Tregs that contributes to suppress effector T cells in vivo 2542. Production of TGFβ1 by Tregs is therefore an indicator of Treg function. To determine if Teriparatide regulates the function of Tregs, we measured the level of TGFβ1 mRNA in sorted peripheral blood CD4+CD25+ T cells from the last 18 women enrolled in the trial. We selected this cell population because most CD4+CD25+ T cells are Foxp3+ Tregs, while measurements of TGFβ1 mRNA in Tregs sorted by Foxp3 staining is not feasible due to the loss of cell viability caused by intracellular staining. We found that the level of TGFβ1 mRNA in this Treg-enriched population was higher in the Teriparatide-treated group than in the control group (Fig 1C). Post hoc analysis showed that TGFβ1 mRNA levels were higher at 3 months than at baseline in the Teriparatide-treated group. Moreover, at 6 months TGFβ1 mRNA levels were higher in the Teriparatide-treated group than in the control group. Together, these findings demonstrate that Teriparatide treatment expands the number and the activity of circulating human Tregs. To determine whether Teriparatide targets Tregs directly or indirectly, human peripheral blood CD4+CD25+ T cells were stimulated in vitro with anti-CD3 Ab and IL-2 for 6 days. Vehicle or Teriparatide were added every 2 days for 1 or 24 h. Analysis by flow cytometry revealed that Teriparatide did not increase the relative number of Tregs (CD4+CD25+Foxp3+ cells) in cultures of peripheral blood CD4+CD25+ T cells (Fig 1D), suggesting that Teriparatide regulates the number of Tregs via indirect mechanisms. We also found that in vitro stimulation with Teriparatide does not increase the expression of TGFβ mRNA in sorted peripheral blood CD4+CD25+ T cells (Fig 1E), confirming that Teriparatide does not directly targets Tregs. iPTH treatment in mice expands the pool of BM Tregs by increasing Treg differentiation As in human cells, in vitro PTH stimulation increases in production of TGFβ1 and IGF-1 by murine osteoblasts 363738. However, the effects of iPTH treatment on the production of these factors are largely unknown. To investigate this matter, 6-week-old mice were treated with vehicle or iPTH for 2 weeks. BM was then harvested and cultured for 24 h. ELISAs revealed that iPTH significantly increases the levels of TGFβ1 and IGF-1 in the whole BM culture media (Appendix Fig S1A and B), suggesting that iPTH may regulate Treg differentiation. To investigate the effect of iPTH treatment on the number of BM Tregs, 6-week-old mice were treated with vehicle or iPTH for 1, 2, or 4 weeks. BM was then harvested and stained for TCRβ, CD4, and Foxp3. iPTH treatment increased the relative and absolute number of BM Tregs (TCRβ+CD4+Foxp3+ cells) during the entire study period (Fig 2A and B). The increase in absolute number of BM Tregs was already significant at 1 week of iPTH treatment, peaked at 2 weeks, and remained significantly increased at 4 weeks of treatment. By contrast, iPTH did not increase the number of splenic, thymic, and intestinal Tregs (Appendix Fig S2A–E). Figure 2. iPTH treatment increases the number of BM Tregs and Treg differentiationTo determine the effect of treatment on the number of Tregs, BM TCRβ+CD4+Foxp3+ T cells were counted by flow cytometry following 1, 2, and 4 weeks of treatment with vehicle or iPTH. To assess Treg differentiation, conventional CD4+ T cells (TCRβ+CD4+eGFP−) from B6.Foxp3.eGFP reporter mice were transferred into TCRβ−/− mice. Recipient mice were treated with vehicle or iPTH for 1, 2, or 4 weeks starting 2 weeks after the T-cell transfer. The number of BM TCRβ+CD4+eGFP+ cells was then determined by flow cytometry. Relative frequency of BM Tregs at 1, 2, and 4 weeks of treatment. n = 10–32 mice per group. Absolute frequency of Tregs at 1, 2, and 4 weeks of treatment. n = 10–32 mice per group. Relative frequency of eGFP+ Tregs at 1, 2, and 4 weeks of treatment n = 10 mice per group. Absolute frequency of eGFP+ Tregs at 1, 2, and 4 weeks of treatment n = 10 mice per group. Data information: Data are expressed as mean ± SEM. All data were normally distributed according to the Shapiro–Wilk normality test and analyzed by unpaired t-tests. ***P < 0.001, and ****P < 0.0001 compared to the corresponding vehicle. Download figure Download PowerPoint iPTH could expand the pool of BM Tregs via multiple mechanisms including increasing the differentiation of conventional CD4+ T cells into Tregs or the proliferation of Tregs within the BM. To gain mechanistic insights, we determined the effects of iPTH on Treg differentiation, which is defined as the induction of Foxp3 expression in CD4+Foxp3− T cells 25. For this purpose, we made use of B6.Foxp3.eGFP reporter mice, a strain in which eGFP expression is co-expressed with Foxp3 and restricted to CD4+ T cells. Conventional CD4+ T cells (CD4+eGFP−) were FACS-sorted from the spleens of B6.Foxp3.eGFP reporter mice and transferred into TCRβ−/− mice, a strain lacking αβ T cells. After 2 weeks, a length of time sufficient for the engraftment and expansion of donor T cells, recipient mice were treated with vehicle or iPTH for 1–4 weeks. We then determined the number of CD4+eGFP+ cells in the BM by flow cytometry. Treatment with iPTH increased the relative and the absolute number of CD4+eGFP+ cell in the BM at 1, 2, and 4 weeks of treatment (Fig 2C and D), demonstrating that iPTH increases the differentiation of BM Tregs. Additional studies that used BrdU incorporation to measure proliferation revealed that iPTH treatment for 1, 2, or 4 weeks does not increase BM Treg proliferation (Appendix Fig S2F). In an additional set of experiments, Tregs (CD4+eGFP+ cells) were FACS-sorted from the spleens of B6.Foxp3.eGFP reporter mice and transferred into TCRβ−/− mice. Recipient mice were treated with vehicle or iPTH for 2 weeks, starting the day of the Treg transfer. This design was selected to minimize the confounding effect of the partial loss of Foxp3 expression by CD4+eGFP+ cells, which may occur after Treg transfer into lymphopenic host mice 43. These studies revealed that iPTH does not affect the relative and the absolute number of CD4+eGFP+ cells residing in the BM and the spleen (Appendix Fig S2G–J). These findings, together with a lack of an effect of iPTH on Treg proliferation, indicate that iPTH does not alter the homing of Tregs to the spleen and the BM. In addition to increasing the BM levels of TGFβ and IGF-1 363738, iPTH enhances the sensitivity of conventional CD4+ cells to TGFβ. This was disclosed by experiments in which unstimulated splenic CD4+CD25− cells purified from iPTH-treated mice were found to express lower levels of the negative regulator of TGFβ signaling SMAD7 as compared to CD4+CD25− cells from vehicle-treated mice (Fig 3A). To ascertain the functional relevance of this finding, splenic CD4+CD25− cells from vehicle- or iPTH-treated mice were stimulated in vitro with CD3/CD28 Ab, IL-2, and TGFβ at 2.5 ng/ml for 72 h to induce their differentiation into Tregs 44. Measurements of phosphorylated SMAD2 and SMAD3 (pSMAD2 and pSMAD3) at the end of the culture period revealed higher concentrations of pSMAD2 and pSMAD3 (Fig 3B) in cells from iPTH-treated mice as compared to those from control mice, suggesting that conventional CD4+ T cells from iPTH-treated mice have a higher sensitivity to TGFβ. To confirm this hypothesis, splenic CD4+CD25− cells were purified from vehicle- or iPTH-treated mice and then cultured in vitro for 72 h with anti CD3/CD28 Ab, IL-2, and increasing doses of TGFβ (0.1–5 ng/ml). At each dose of TGFβ, cultures of CD4+CD25− T cells from iPTH-treated mice yielded a greater percentage of Foxp3+ Tregs than those from vehicle-treated mice (Fig 3C). Together, these findings indicate that CD4+ T cells from iPTH-treated mice possess a greater sensitivity to TGFβ, which results in enhanced differentiation of CD4+ T cells into Tregs. Figure 3. iPTH treatment increases the sensitivity of conventional CD4+ T cells to TGFβ Western blotting analysis of SMAD7 levels in unstimulated splenic conventional CD4+ cells. Data are from 1 representative experiment of a total of four experiments. R.I., relative intensity. Western blotting analysis of pSMAD2 and pSMAD3 levels in splenic conventional CD4+ T cells stimulated with anti-CD3/CD28 Ab, IL-2, and recombinant TGFβ1 (rTGFβ1) at 2.5 ng/ml for 72 h to induce their differentiation into Tregs. Data are from one representative experiment of a total of four experiments. R.I., relative intensity. Relative frequency (mean ± SEM) of Foxp3+ Tregs in cultures of conventional CD4+ T cells stimulated with anti-CD3/CD28 Ab, IL-2, and increasing doses of rTGFβ1. Splenic conventional CD4+ T cells were obtained after 1, 2, and 4 weeks of treatment with vehicle or iPTH. n = 10 mice per group. Data were analyzed by two-way ANOVA and post hoc tests applying the Bonferroni correction for multiple comparisons. **P < 0.01 and ****P < 0.0001 compared to vehicle. Download figure Download PowerPoint An increase in the number of Tregs is required for iPTH to induce bone anabolism in mice A direct means to investigate the contribution of Tregs to the anabolic activity of iPTH is to assess the effects of iPTH in a model in which the increase in the frequency of Tregs is prevented. The surface marker CD25 is expressed at high levels by most CD4+ Foxp3+ Tregs 45. Accordingly, treatment with anti-CD25 Abs capable of deleting CD25hi is used to partially deplete Tregs in vivo 4647. We thus treated 6-week-old mice with vehicle or iPTH (days 1–28) plus four injections (days −2, 0, 5, and 7) of isotype control Ab or the anti-CD25 Ab PC61 4647. We found CD25hi to be expressed by CD4+ T cells and by a negligible fraction of CD8+ T cells (Appendix Fig S3A). Anti-CD25 Ab decreased the frequency of CD25hi CD4+ T cells but not that of CD25lo CD4+ T cells. As previously reported 48, we also found that treatment with anti-CD25 Ab decreased the number of CD25hiFoxp3+CD4+ T cells, but not the number of CD25loFoxP3−CD4+ T cells (Appendix Fig S3B). In addition, treatment with anti-CD25 Ab did not decrease the percentage of BM of conventional CD4+ T cells (TCRβ+CD4+CD25+Foxp3− cells) and that of CD8+ T cells (TCRβ+CD8+CD25+ cells) (Appendix Fig S3C–F). Together, these findings demonstrate that anti-CD25 Ab specifically depletes Tregs. At sacrifice control mice treated with anti-CD25 Ab had ~37% fewer Tregs than mice treated with irrelevant (Irr.) Ab (Fig 4A and B). Moreover, treatment with anti-CD25 Ab prevented the increase in BM Tregs induced by iPTH. The partial depletion of Tregs induced by anti-CD25 Ab did not increase the production of inflammatory and lineage-specific cytokines in the BM. In fact, in both the vehicle- and iPTH-treated groups, BM cells from mice treated with anti-CD25 Ab expressed similar levels of TNF, IL-17A, IL-6, IL-4, and IL-13 mRNAs to those from mice treated with Irr. Ab (Appendix Fig S4). Moreover, iPTH lowered the mRNA levels of IFNγ, in both the Irr. Ab and the anti-CD25 Ab groups but did not affect the other cytokines. Since inflammatory cytokines blunt bone formation 49, these findings indicate treatment with anti-CD25 Ab does not alter the bone anabolic activity of iPTH by inducing inflammation. Analysis by in vitro μCT of femurs harvested at sacrifice revealed that iPTH induced a significant increase in trabecular bone volume (BV/TV) in mice treated with Irr. Ab (Fig 4C and D), but not in those treated with anti-CD25 Ab. Trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular space (Tb.Sp), which are indices of trabecular structure, were altered by iPTH in mice treated with Irr. Ab, but not in those treated with anti-CD25 Ab (Appendix Fig S5A–C). By contrast, iPTH increases cortical volume (Ct.Vo) (Fig 4C and E) and cortical thickness (Ct.Th) (Appendix Fig S5D) in both groups of mice, confirming that T cells are not implicated in the mechanism by which iPTH increases cortical volume. Together, these findings demonstrate that the anabolic effects of iPTH in trabecular bone are dependent on increased numbers of Tregs. Figure 4. Depletion of Tregs by treatment with anti-CD25 Ab prevents the bone anabolic activity of iPTH A, B. Relative and absolute frequency of BM Tregs. C. Images of representative three-dimensional μCT reconstructions of examined femurs from each group. D. Femoral trabecular bone volume (BV/TV) as measured by μCT scanning. E. Femoral cortical bone volume (Ct.Vo) by μCT scanning F. Images are representative sections displaying the calcein double-fluorescence labeling. Original magnification 20×. Scale bar represents 300 μm. G. Mineral apposition rate (MAR). H. Bone formation rate per mm bone surface (BFR/BS). I. The number of osteoblasts per mm bone surface (N.Ob/BS). J. The percentage of bone surface covered by osteoblasts (Ob.S/BS). K. The images show tartrate-resistant acid phosphatase (TRAP)-stained sections of the distal femur. Original magnification 40×. Scale bar represents 300 μm. L. The number of osteoclasts per mm bone surface (N.Oc/BS). M. The percentage of bone surface covered by osteoclasts (Oc.S/BS). N. Serum levels of osteocalcin (OCN), a marker of bone formation. O. Serum levels of type 1 cross-linked C-telopeptide (CTX), a marker of resorption. Data information: n = 10–25 mice per group. Data are expressed as mean ± SEM. All data were normally distributed according to the Shapiro–Wilk normality test and analyzed by two-way ANOVA and post hoc tests applying the Bonferroni correction for multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 compared to the indicated group. Download figure Download PowerPoint Analysis of femoral cancellous bone by histomorphometry revealed that iPTH increased the dynamic indices of bone formation mineral apposition rate (MAR) and bone formation rate (BFR/BS) in Treg-replete mice but not in those treated with anti-CD25 Ab (Fig 4F–H). Moreover, iPTH increased two static indices of bone formation, the number of osteoblasts per bone surface (N.Ob/BS) (Fig 4I) and the percentage of surfaces covered by osteoblasts (Ob.S/BS) (Fig 4J) in Treg-replete mice but not in those treated with anti-CD25 Ab. The finding that treatment with anti-CD25 Ab did not decrease bone formation in vehicle-treated mice provides evidence that partial Treg depletion does not cause a nonspecific inhibitory effect of on bone formation. Two indices of bone resorption, the number of OCs per bone surface (N.Oc/BS) and the percentage of surfaces covered by OCs (Oc.S/BS), were not affected by iPTH (Fig 4K–M) in both control and Treg-depleted groups. However, N.Oc/BS was higher in mice treated with iPTH and anti-CD25 Ab, as compared to those treated with iPTH and Irr. Ab, suggesting that Treg depletion may stimulate bone resorption. Measurements of serum levels of osteocalcin, a marker for bone formation, revealed that iPTH increased bone formation in mice treated with Irr. Ab but not in those treated with anti-CD25 Ab (Fig 4N). Serum CTX, a marker for bone resorption, also increased significantly in response to iPTH in mice treated with Irr. Ab but not in those injected with anti-CD25 Ab (Fig 4O). Moreover, mice treated with anti-CD25 Ab had higher CTX levels than those treated with Irr. Ab, confirming that Treg depletion is associated with an increase in bone resorption. The differences between the CTX data and histomorphometric indices of bone resorption are explained by the fact that CTX reflects cortical and trabecular bone resorption, while the histomorphometric analysis was limited to the trabecular compartment. To determine the role of Tregs in mediating the effects of iPTH on osteoblastogene
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