IFN-γ stimulates osteoclast formation and bone loss in vivo via antigen-driven T cell activation

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IFN-γ stimulates osteoclast formation and bone loss in vivo via antigen-driven T cell activation

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  Research article 122  The Journal of Clinical Investigation http://www.jci.org    Volume 117   Number 1    January 2007 IFN- γ  stimulates osteoclast formation and bone loss in vivo via antigen-driven T cell activation  Yuhao Gao, 1  Francesco Grassi, 1  Michaela Robbie Ryan, 1  Masakazu Terauchi, 1  Karen Page, 1   Xiaoying Yang, 1  M. Neale Weitzmann, 1  and Roberto Pacifici 1,2 1 Division of Endocrinology, Metabolism and Lipids, Department of Medicine, and 2 Immunology and Molecular Pathogenesis Program, Emory University, Atlanta, Georgia, USA. T cell–produced cytokines play a pivotal role in the bone loss caused by inflammation, infection, and estrogen deficiency. IFN- γ  is a major product of activated T helper cells that can function as a pro- or antiresorptive cytokine, but the reason why IFN- γ  has variable effects in bone is unknown. Here we show that IFN- γ  blunts osteoclast formation through direct targeting of osteoclast precursors but indirectly stimulates osteoclast for-mation and promotes bone resorption by stimulating antigen-dependent T cell activation and T cell secretion of the osteoclastogenic factors RANKL and TNF- α . Analysis of the in vivo effects of IFN- γ  in 3 mouse models of bone loss — ovariectomy, LPS injection, and inflammation via silencing of TGF- β  signaling in T cells — reveals that the net effect of IFN- γ  in these conditions is that of stimulating bone resorption and bone loss. In sum-mary, IFN- γ  has both direct anti-osteoclastogenic and indirect pro-osteoclastogenic properties in vivo. Under conditions of estrogen deficiency, infection, and inflammation, the net balance of these 2 opposing forces is biased toward bone resorption. Inhibition of IFN- γ  signaling may thus represent a novel strategy to simultane-ously reduce inflammation and bone loss in common forms of osteoporosis. Introduction Physiological osteoclast renewal is regulated by the key osteo-clastogenic cytokines M-CSF and receptor activator of NF- κ B ligand (RANKL). However, under pathological conditions, such as those occurring during inflammation, infection, and estrogen deficiency, bone resorption is significantly stimulated due to dys-regulated production of additional pro- and anti-osteoclastogenic factors, including IFN- γ , a central mediator of adaptive immunity. Estrogen deficiency, infection by LPS-producing bacteria such as occurs in periodontitis, and inflammatory diseases like RA are all characterized by a state of immune activation, leading to elevated production of IFN- γ  by Th1 cells (1–5).Substantial evidence demonstrates that IFN- γ  strongly sup-presses osteoclastogenesis in vitro (6, 7). However, other studies have shown that IFN- γ  enhances osteoclast generation in cultures of peripheral blood from osteopetrotic patients, in part by nor-malizing superoxide production (8). Additional studies revealed that preexposure of osteoclast precursors to RANKL renders them resistant to the inhibitory effects of IFN- γ  by inducing terminal differentiation (9). Furthermore, IFN- γ –producing human Th1 cells, but not IFN- γ –negative T cells, were found to directly induce the differentiation of human macrophages into osteoclasts via expression of RANKL (10).The effects of IFN- γ  in vivo are equally controversial. Silencing of IFN- γ  receptor (IFN- γ R) signaling led to a more rapid onset of collagen-induced arthritis and bone resorption (11). Furthermore, IFN- γ  was found to decrease serum calcium and osteoclastic bone resorption in vivo in nude mice (12, 13), suggesting that IFN- γ  is a bone-sparing cytokine in vivo. In contrast, observations in humans and rodents suggest that IFN- γ  promotes bone resorption and causes bone loss in a variety of pathological conditions. For exam-ple, IFN- γ  has been reported to be efficacious in the treatment of osteopetrosis through restoration of osteoclast formation and bone resorption, in both humans (14) and rodents (15). Addition of recombinant IFN- γ  (rIFN- γ ) rescues the defect in osteoclasto-genesis in peripheral white blood cells from malignant osteopetro-sis patients in vitro (8). Systemic administration of rIFN- γ  causes loss of bone volume in rats (16, 17). Moreover, mice lacking IFN- γ  production are protected against infection-induced alveolar bone loss (18), and IFN- γ  receptor –/–  (  IFN- γ  R  –/– ) mice fail to undergo ovariectomy-induced (ovx-induced) bone loss (2). Furthermore, IFN- γ  production correlates positively with tissue destruction in erosive tuberculoid leprosy, and bone resorption in psoriatic arthritis (19, 20) increases in parallel with bacterial LPS-induced bone resorption in mice (21) and positively modulates actinobacil-lus actinomycetemcomitans–specific RANKL +  CD4 +  Th cell–medi-ated alveolar bone destruction in vivo (22). Further support for the hypothesis that IFN- γ  does not inhibit bone resorption in vivo was provided by the outcome of randomized controlled trials which have shown that IFN- γ  does not prevent bone loss in patients with RA (23, 24) or the bone-wasting effect of cyclosporin A (16).IFN- γ  has been shown to directly block osteoclast formation by inducing the rapid degradation of the RANK adapter protein TNF receptor–associated factor 6 (TRAF6), resulting in strong inhibition of the RANKL-induced activation of NF- κ B and c-Jun N-terminal kinase (25). IFN- γ  also downregulates the RANKL sig-naling pathway by antagonizing RANKL-stimulated cathepsin K (7, 26, 27) and by upregulating cathepsin S and cathepsin L gene Nonstandard abbreviations used:  BMD, bone mineral density; BMM, BM macro-phage; BV/TV, trabecular bone volume; CIITA , class II transactivator  ; CM, conditioned medium; CTX, C-terminal telopeptide; DXA, dual x-ray absorptiometry; IFN- γ R, IFN- γ  receptor; ovx, ovariectomy, ovariectomized; RANKL, receptor activator of NF- κ B ligand; rIFN- γ , recombinant IFN- γ ; TRAF6, TNF receptor–associated factor 6; TRAP, tartrate-resistant acid phosphatase. Conflict of interest:  The authors have declared that no conflict of interest exists. Citation for this article:    J. Clin. Invest.   117 :122–132 (2007). doi:10.1172/JCI30074.  research article  The Journal of Clinical Investigation   http://www.jci.org Volume 117 Number 1 January 2007 123 expression in preosteoclastic cells (26). Furthermore, IFN- γ  induc-es superoxide production in osteoclasts, inducing apoptosis of osteoclast progenitors and suppressing osteoclast activity (28, 29). However, IFN- γ  is the physiologic inducer of MHC class II expres-sion and thus of antigen presentation (30–32). As a result, IFN- γ  leads to T cell activation and T cell secretion of the osteoclasto-genic factors RANKL and TNF- α  (2). Together, these data suggest that IFN- γ  inhibits osteoclast formation through direct targeting of maturing osteoclasts, while it promotes osteoclastogenesis indi-rectly, by stimulating T cell activation.In this study, we investigated the hypothesis that the net effect of IFN- γ  on bone resorption in vivo represents the balance of its direct and indirect activities. We report that IFN- γ  promotes osteo-clast formation through stimulation of antigen-dependent T cell activation and show that under conditions of estrogen deficiency, infection, and direct T cell activation by suppression of TGF- β  sig-naling, the net effect of IFN- γ  is that of inducing bone resorption and bone loss. Results  IFN- γ  blocks osteoclastogenesis via direct targeting of maturing osteoclast  precursors but stimulates osteoclast formation through upregulation of anti- gen presentation . To investigate the direct effects of IFN- γ  on osteo-clast formation, macrophages were purified by positive immuno-magnetic selection from either the BM or the spleen of intact WT mice and cultured with optimal amounts of RANKL and M-CSF and increasing doses of rIFN- γ . RANKL equally stimulated the dif-ferentiation of BM and splenic macrophages into osteoclasts, and IFN- γ  dose-dependently inhibited osteoclastogenesis, with signifi-cant suppression occurring at 1 ng/ml and complete suppression at 100 ng/ml of rIFN- γ  (Figure 1A). These findings confirm ear-lier reports that IFN- γ  has direct anti-osteoclastogenic effects on RANKL-induced osteoclastogenesis in vitro (7, 25–27).Since IFN- γ  is a potent inducer of MHC class II expression and antigen presentation in macrophages, IFN- γ  may induce T cell secretion of osteoclastogenic factors by promoting antigen-depen-dent T cell activation. To test this hypothesis, BM macrophages (BMMs) were used as APCs (herein designated as APC-BMMs) and were purified from WT mice and pretreated with rIFN- γ  for 72 hours to upregulate MHC class II expression. After removal of rIFN- γ  by washing, these cells were cocultured for 72 hours with the avian antigen OVA and T cells from OT-II mice, a strain with T cells that harbor a transgenic T cell receptor responsive exclu-sively to OVA. In this model, OVA-derived peptides presented by  APCs to T cells induce T cell activation and T cell cytokine secre-tion. Measurements of cytokine levels in the conditioned medium (CM) revealed that pretreatment of APC-BMMs with rIFN- γ  led to a significant increase in TNF- α , RANKL, and IFN- γ  production, as compared with pretreatment of APC-BMMs with vehicle (Figure 1B). To demonstrate that T cells were a major source of the secret-ed cytokines, T cells were purified by positive immunomagnetic selection at the end of the coculture and analyzed by real-time RT-PCR. The data show that pretreatment of APC-BMMs with rIFN- γ  significantly increased the T cell expression of TNF- α , IFN- γ , and RANKL mRNA (Figure 1C).In order to investigate the indirect pro-osteoclastogenic effect of IFN- γ  in the absence of its direct anti-osteoclastogenic activity, we utilized early osteoclast precursors (macrophages) that were derived from  IFN- γ  R  –/–  mice. The preosteoclasts and osteoclasts formed from these cells are consequently insensitive to IFN- γ  and thus resistant to the direct anti-osteoclastogenic effect of IFN- γ . Osteoclastogenesis was initiated by addition to osteoclast precur-sors from  IFN- γ  R  –/–  mice of CM derived from T cells that had been activated in vitro by WT APCs, in the presence or absence of IFN- γ . Under these conditions, osteoclast formation reflects the capac-ity of IFN- γ  to stimulate antigen-induced cytokine production by T cells. We found that the number of osteoclasts produced in response to CM from T cells that had been activated in vitro by WT  APCs, in the presence of IFN- γ , was 2-fold higher than that induced by CM generated in the absence of IFN- γ . When the same experi-ment was repeated using osteoclast precursors from WT mice, rIFN- γ –pretreated APCs and unstimulated APCs induced equal osteoclast formation (Figure 1D). These findings suggest that under these conditions, the indirect pro-osteoclastogenic effect of IFN- γ  is neutralized by the direct anti-osteoclastogenic activ-ity of the IFN- γ  secreted by activated T cells. Together, these data demonstrate that IFN- γ  represses osteoclastogenesis by directly repressing the differentiation of macrophages into osteoclasts but indirectly stimulates osteoclast formation through stimulation of antigen presentation.We have previously reported that ovx increases MHC class II expression in macrophages and monocyte APC activity (2). We took advantage of this phenomenon to further investigate the indirect effects of IFN- γ  on osteoclast formation. Thus, APC-BMMs from WT sham-operated and ovx mice were cocultured with OVA and T cells from OT-II mice. T cell CM was then collected 72 hours after culture, added to a new batch of osteoclast precursors from intact WT mice, and cultured for 7 days with M-CSF and RANKL in the presence or absence of neutralizing anti–IFN- γ  Ab (IFN- γ  Ab). Osteoclast formation in the samples treated with IFN- γ  Ab reflect-ed the impact of stimulated IFN- γ –driven antigen presentation by macrophages without the direct inhibitory effect of the cytokine. Under these conditions, osteoclast formation was approximately 3-fold higher in samples containing APC-BMM CM from ovx mice compared with those with APC-BMM CM from sham-operated mice (Figure 1E). In the absence of IFN- γ  Ab, osteoclast formation was only 2-fold higher in samples with APC-BMMs from ovx mice than those with APC-BMMs from sham-operated mice, reflecting the direct inhibitory effect of IFN- γ . These data confirm that IFN- γ  directly suppresses osteoclast formation. However, under conditions of estrogen deficiency, the overall balance between the stimulatory effect of antigen presentation on T cell activation and the direct sup-pressive effect of IFN- γ  is a net increase in osteoclast formation.To further investigate the mechanism by which IFN- γ  regulates osteoclast formation, T cells were activated in vitro with PMA and ionomycin and cultured for 3 days. T cell CM was added to BMMs along with optimal amounts of RANKL and M-CSF, and the mix-ture cultured for an additional 3-day period with and without Abs against IFN- γ , TNF- α , and RANKL to induce the differentiation of BMMs into TRAP +  mononuclear osteoclast precursors in the absence of the direct effects of the neutralized cytokines. At the end of the culture period, the proliferation of the nascent osteoclasts was measured by thymidine incorporation; their rate of apoptosis by intracellular caspase-3 activity; and their expression of TRAP mRNA, a marker of osteoclast differentiation, by real-time PCR.These experiments revealed that in conditions in which the direct effect of IFN- γ  is neutralized, cytokines released by acti- vated T cells increase both the proliferation (Figure 1F) and the survival of osteoclast precursors (Figure 1G), while they have no effect on the rate of their differentiation (data not shown). Acti-  research article 124  The Journal of Clinical Investigation   http://www.jci.org Volume 117 Number 1 January 2007  vated T cells stimulate the proliferation of osteoclast precursors by secreting TNF and RANK, as the stimulatory effect of T cell CM was abolished by the neutralization of these cytokines. The capacity of IFN- γ –neutralized T cell CM to blunt the apoptosis of maturing osteoclasts was further enhanced by anti–TNF- α  Ab and reversed by neutralization of RANKL, demonstrating that IFN- γ  and TNF- α  increase preosteoclast apoptosis while RANKL suppresses it. Taken together, the data demonstrate that activated T cells stimulate the proliferation and survival of maturing osteo-clasts through TNF and RANKL, while inhibiting these processes through the direct activity of IFN- γ .  IFN- γ  causes bone loss in T cell–replete but not T cell–deficient mice . Having established the direct and indirect effects of IFN- γ  in  vitro, we verified our findings in vivo by examining the effects of treatment with rIFN- γ  in nude mice, a strain in which the indirect effects of IFN- γ  are abrogated by the lack of T cells. Thus, 16-week-old WT nude mice and nude mice subjected to adoptive transfer of WT T cells 2 weeks earlier were injected i.p. twice a week for 3 weeks with rIFN- γ  (10 6  IU/kg). Bone mineral density (BMD) was measured by dual x-ray absorptiometry (DXA), a technique that provided a combined assessment of cortical and trabecular bone, at baseline and at 3 weeks after initial rIFN- γ  injection. rIFN- γ  treatment led to a significant decrease in spine BMD in WT and T cell–replete nude mice, but not in T cell–deficient nude mice (Fig-ure 2A). Consistent with the BMD data, treatment with rIFN- γ  also caused a significant increase in the serum level of C-terminal telopeptides (CTXs), a marker of bone resorption, in WT mice and T cell–reconstituted nude mice, but not in T cell–deficient nude mice (Figure 2B). The serum level of osteocalcin, a marker of bone formation, did not change in response to in vivo rIFN- γ  treatment in any of the groups of mice, indicating that the chang-es in BMD induced by IFN- γ  were not due to blunted bone forma-tion (Figure 2C). T cell production of TNF- α , RANKL, and IFN- γ  was increased by rIFN- γ  in the T cell–replete groups (Figures 2, D–F). Due to the absence of T cells in nude mice, cytokine levels fell below detection in this group. These findings demonstrate that T cells are critical to the mechanism by which IFN- γ  stimu-lates bone resorption in vivo. Figure 1 IFN- γ  directly suppresses and indirectly stimulates osteoclastogenesis in vitro.   ( A ) rIFN- γ  suppresses osteoclastogenesis induced by RANKL (50 ng/ml) and M-CSF (10 ng/ml) in macrophages purified from BM and spleen. # P  < 0.01 compared with vehicle-treated controls. ( B ) Cytokine levels in CM derived from cocultures of IFN- γ –pretreated WT macrophages and OT-II T cells were measured using ELISA. * P  < 0.05 compared with vehicle-treated controls. ( C ) The expression of T cell cytokine mRNA was measured by real-time RT-PCR in T cells cocultured with rIFN- γ – pretreated macrophages. # P  < 0.01 compared with vehicle-pretreated controls. ( D ) The ability of CM from cocultures of rIFN- γ –pretreated macrophages and OT-II T cells to enhance M-CSF– and RANKL-stimulated osteoclastogenesis was examined in macrophages from WT and IFN- γ R  –/–   mice. * P  < 0.05 compared with vehicle-treated controls. ( E ) The ability of CM from coculture of macrophages from sham-operated or ovx WT mice and OT-II T cells to induce osteoclastogenesis was examined in macrophages from WT mice in the presence of M-CSF and RANKL with or without neutralizing IFN- γ  Ab (10 μ g/ml ). * P  < 0.05 compared with vehicle-treated controls. The proliferation ( F ) and rate of apoptosis ( G ) of maturing osteoclasts were determined by [ 3 H]thymidine incorporation and intracellular caspase-3 activity in 3-day cultures of RANKL- and M-CSF–stimulated BMMs cultured in the presence of activated T cell CM and Abs directed against IFN- γ , TNF, and RANKL. * P  < 0.05 compared with irrelevant IgG-treated controls. All data are expressed as mean ± SD.  research article  The Journal of Clinical Investigation   http://www.jci.org Volume 117 Number 1 January 2007 125  Effects of IFN- γ  in ovx mice . Since the pattern of T cell activation  varies in different pathological conditions, we next compared the effects of IFN- γ  on bone homeostasis in estrogen deficiency and LPS-induced inflammation, 2 conditions that cause bone loss through T cell–dependent mechanisms (3, 4, 33, 34). We also examined an experimental model of autoimmune disease induced by specifically ablating TGF- β  signaling in T cells (35).To investigate the role of IFN- γ  in the bone loss induced by estrogen deficiency, WT and  IFN- γ –/–  mice were sham operated or subjected to ovx at the age of 16 weeks and sacrificed 4 weeks later. In vivo measurements of spine BMD at baseline and 4 weeks after surgery by DXA revealed that ovx caused a significant bone loss in WT but not in  IFN- γ –/–  mice (Figure 3A), suggesting that  IFN- γ –/–  mice are protected against the overall loss of cortical and trabecular bone induced by ovx. μ CT analysis of femurs obtained at sacrifice revealed that ovx caused a loss of trabecular bone  volume (BV/TV) in both WT mice and  IFN- γ –/–  mice (Figure 3B). However, the difference between sham-operated and ovx mice was larger in WT mice (21%) than in  IFN- γ –/–  mice (14%) and only reached statistical significance in WT mice. Together, these find-ings indicate that  IFN- γ –/–  mice are partially protected against the loss of trabecular bone induced by ovx. At 4 weeks from surgery, the levels of serum CTX and osteocalcin were higher in WT ovx mice than in WT sham-operated controls (Figure 3, C and D). In contrast,  IFN- γ –/–  sham-operated and ovx mice had similar levels of both CTX and osteocalcin. Flow cytometric analysis revealed that ovx increases the percentage of CD4 +  T cells expressing the early activation marker CD69 in WT but failed to stimulated T cell activation in  IFN- γ –/–  mice (Figure 3E). This data are consis-tent with previous reports demonstrating that ovx leads to sig-nificant activation and expansion of TNF-producing T cell popu-lations (34, 36) and that IFN- γ  plays a causal role in the bone loss induced by estrogen deficiency (2).  Effects of IFN- γ  in LPS-induced bone loss . To investigate the contri-bution of IFN- γ  to LPS-induced bone resorption in vivo, WT and  IFN- γ –/–  mice of 10 weeks of age were injected subcutaneously with LPS (25 mg/kg once a week for 3 weeks) and BMD measured at baseline and weekly thereafter. LPS injection led to a significant reduction in spine BMD in both WT and  IFN- γ –/–  mice (Figure 4A), although in  IFN- γ –/–  mice, bone loss plateaued at 2 weeks, while in WT mice there was a progressive bone loss for the 3 weeks of follow-up. As a result, the bone loss at 3 weeks was significantly greater in WT ( ~ 16.5%) than in  IFN- γ –/–  mice ( ~ 10.5%). μ CT analysis of femur trabecular bone in samples harvested at 3 weeks showed (Figure 4B) that LPS caused a decrease in BV/TV in both WT ( ~ 30%) and  IFN- γ –/–  mice ( ~ 23%). Although there was a trend toward less bone loss in  IFN- γ –/–  mice, the difference between the 2 groups was not significant. In contrast to the effects on bone volume, LPS caused a larger increase in CTX levels in WT than in  IFN- γ –/–  mice (Figure 4C). However, in all groups of mice, LPS had no significant effects on serum osteocalcin (Figure 4D). As expected  IFN- γ –/–  mice had lower basal APC activity and a neg-ligible response to LPS. In contrast, WT mice had a higher APC activity at baseline, and LPS caused a further 2-fold increase in  APC activity (Figure 4E). LPS treatment also resulted in an approxi-mately 3-fold and approximately 1.5-fold increase in the percentage of activated (CD69 + ) CD4 +  T cells in WT mice and  IFN- γ –/–  mice, respectively (Figure 4F). Taken together, these data suggest that LPS induces bone loss primarily via IFN- γ –independent mechanisms, although an IFN- γ –dependent mechanism plays a contributory role. The IFN- γ –dependent bone loss results from LPS stimulation of antigen presentation and the resulting T cell activation. Silencing IFN- γ  production reduces the bone loss induced by the disruption of TGF- β  signaling in T cells . To further investigate the contribution of the indirect stimulatory effects of IFN- γ  on bone resorption in vivo, we made use of a conditional transgenic mouse that overexpresses a dominant-negative form of TGF- β  type II receptor ( CD4dnTGF  β  IIR   mouse) exclusively in T cells. TGF- β  signaling in T cells markedly represses T cell activation. In the absence of the repressive effect of TGF- β , the T cells of this transgenic secrete increased amounts Figure 2 Systemic administration of rIFN- γ  stimulates bone resorption in WT mice and nude mice reconstituted with WT T cells (Nude + T), but not in T cell–deficient nude mice. rIFN- γ  (1 ×  10 6  IU/kg body weight) was injected subcutaneously twice a week into 16-week-old WT mice, T cell–defi-cient nude mice, and nude mice reconstituted with WT T cells, and spine BMD ( A ); serum CTX ( B ) and serum osteocalcin levels ( C ); and T cell production of TNF ( D ), RANKL ( E ), and IFN- γ  ( F ) were measured 3 weeks after initial IFN- γ  injection. The percentages in A  represent the change compared with baseline. All data are expressed as mean ± SD. * P  < 0.05 compared with vehicle-treated controls. ND, not detectable.  research article 126  The Journal of Clinical Investigation   http://www.jci.org Volume 117 Number 1 January 2007 of IFN- γ , RANKL, and TNF- α  (37). IFN- γ  further stimulates T cell activation and cytokine secretion by promoting antigen presenta-tion. As a result, CD4dnTGF  β  IIR   mice have a lower bone density and increased bone turnover compared with WT controls (37). In order to assess the role of IFN- γ  in the impaired bone modeling induced by the silencing of TGF- β  signaling in T cells, CD4dnTGF  β  IIR   mice were crossed with  IFN- γ –/–  mice to generate mice ( CD4dnTGF  β  IIR/  IFN- γ –/– ) with T cells lacking both IFN- γ  production and TGF- β  signaling. Spine BMD was measured in WT, CD4dnTGF  β  IIR  , CD4dnTGF  β  IIR/IFN- γ –/– , and  IFN- γ –/–  mice at 8, 12, and 16 weeks of age (Figure 5A). At the latter time point, WT mice achieve their peak BMD, and bone remodeling ensues (38–41). All groups of genetic mutants were found to have progressively lower BMD values relative to WT mice, consistent with a modeling defect. However, silencing of IFN- γ  production decreased significant-ly the modeling defect caused by the lack of TGF- β  signaling in T cells, as shown by the finding of higher BMD values in CD4dnTGF  β  IIR/  IFN- γ –/–  mice as compared with CD4dnTGF  β  IIR   mice.These findings were confirmed by μ CT analysis of trabecular bone in the epiphyses of distal femurs harvested at 16 weeks of age. Specifically, CD4dnTGF  β  IIR/IFN- γ –/–  mice had BV/TV val-ues lower than those in WT controls but higher than those in CD4dnTGF  β  IIR   mice. Thus, the decrease in BV/TV induced by the lack of TGF- β  signaling in T cells was partly rescued by the silencing of IFN- γ  production (Figure 5B). Similarly, the increas-es in CTX levels (Figure 5C) and in the number of CD69 +  T cells (Figure 5D) characteristic of CD4dnTGF  β  IIR   mice were less severe in CD4dnTGF  β  IIR/IFN- γ –/–  mice, while serum osteocalcin was not affected by the silencing of TGF- β  signaling and/or IFN- γ  produc-tion (Figure 5E). Antigen presentation by macrophages (Figure 6A) and the macrophage mRNA levels of class II transactivator ( CIITA ) (Figure 6B), a key inducer of MHC class II gene expression, were higher in CD4dnTGF  β  IIR   mice than in WT and CD4dnTGF  β  IIR/  IFN- γ –/–  mice. This is consistent with a high production of IFN- γ  by activated T cells in CD4dnTGF  β  IIR   mice. As activated T cells induce bone destruction through enhanced production of osteoclastogenic cytokines by T cells, we measured TNF- α  and RANKL production in T cell CM from all groups of mice following a 72-hour culture in the presence of PMA and ionomycin. Compared with that in T cells from WT mice, TNF- α  and RANKL production was 4- to 6-fold higher in CD4dnTGF  β  IIR   T cells and 2- to 4-fold higher in CD4dnTGF  β  IIR/IFN- γ –/–  T cells (Figure 6, C and D). Together, these findings confirm that silenc-ing of TGF- β  signaling in T cells causes bone loss in part by direct-ly promoting the T cell production of TNF- α  and RANKL and in part by promoting the secretion of IFN- γ , which further stimulates T cell activation through increased antigen presentation.To quantify the relevance of IFN- γ  in the bone loss induced by silencing of TGF- β  signaling in T cells, we adoptively transferred T cells from WT, CD4dnTGF  β  IIR  , and CD4dnTGF  β  IIR/IFN- γ –/–  mice into T cell–deficient nude mice. The transfer of T cells from CD4dnTGF  β  IIR   mice caused an approximately 2-fold larger decrease in spine BMD (Figure 7A) and femur BV/TV (Figure 7B) and a great-er increase in CTX levels (Figure 7C) than the transfer of both WT T cells and CD4dnTGF  β  IIR/IFN- γ –/–  T cells. In contrast, transfer of T cells from CD4dnTGF  β  IIR   or CD4dnTGF  β  IIR/IFN- γ –/–  mice to nude mice failed to change serum levels of osteocalcin when compared with the transfer of T cells from WT mice (Figure 7D). Thus, the IFN- γ –mediated pathway accounted for approximately 50% of the bone loss induced by the silencing of TGF- β  signaling in T cells. Figure 3 Effects of ovx in WT and IFN- γ  –/–   mice. Sixteen-week-old WT or IFN- γ  –/–   mice were subjected to sham operation or ovx, and BMD ( A ); BV/TV (by μ CT analysis) ( B ); serum CTX ( C ) and serum osteocalcin levels ( D ); and T cell activation (by cytometric detection) ( E ) were examined 4 weeks after surgery. The percentages in A  represent the change compared with baseline. All data are expressed as mean ± SD. * P  < 0.05 compared with WT sham controls.
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