Roberto Pacifici

Estrogen deficiency and bone loss: an inflammatory tale

Estrogen plays a fundamental role in skeletal growth and bone homeostasis in both men and women. Although remarkable progress has been made in our understanding of how estrogen deficiency causes bone loss, the mechanisms involved have proven to be complex and multifaceted. Although estrogen is established to have direct effects on bone cells, recent animal studies have identified additional unexpected regulatory effects of estrogen centered at the level of the adaptive immune response. Furthermore, a potential role for reactive oxygen species has now been identified in both humans and animals. One major challenge is the integration of a multitude of redundant pathways and cytokines, each apparently capable of playing a relevant role, into a comprehensive model of postmenopausal osteoporosis. This Review presents our current understanding of the process of estrogen deficiency-mediated bone destruction and explores some recent findings and hypotheses to explain estrogen action in bone. Due to the inherent difficulties associated with human investigation, many of the lessons learned have been in animal models. Consequently, many of these principles await further validation in humans.

Interleukin-1 receptor antagonist and tumor necrosis factor binding protein decrease osteoclast formation and bone resorption in ovariectomized mice.

To investigate the contribution of IL-1, IL-6, and TNF to the increased osteoclastogenesis induced by estrogen deficiency, ovariectomized (ovx) mice were treated with either IL-1 receptor antagonist (IL-1ra), a competitive inhibitor of IL-1, TNF binding protein (TNFbp), an inhibitor of TNF, or the anti-IL-6 antibody (Ab) 20F3 for the first 2 wk after surgery. ovx increased the bone marrow cells secretion of IL-1 and TNF, but not IL-6, and the formation of TRAP-positive osteoclast-like multinucleated cells (MNCs) in bone marrow cultures treated with 1,25(OH)2D3. The increase in MNC formation induced by ovx was prevented by in vivo treatment with either 17 beta estradiol, IL-1ra, TNFbp, or anti-IL-6 Ab. However, the percent change in MNC formation induced by the anti-IL-6 Ab was similar in ovx and sham-operated animals, whereas IL-1ra and TNFbp were effective only in ovx mice. MNC formation was also decreased by in vitro treatment of bone marrow cultures with IL-1ra and TNFbp, but not with anti-IL-6 Ab. Ovx also increased bone resorption in vivo and in vitro, as assessed by the urinary excretion of pyridinoline cross links and the formation of resorption pits, respectively. IL-1ra, TNFbp and estrogen decreased bone resorption in vivo and in vitro whereas the anti-IL-6 Ab inhibited bone resorption in vitro but not in vivo. In conclusion, these data indicate that IL-1 and TNF play a direct role in mediating the effects of ovx on osteoclastogenesis and bone resorption. The data also suggest that IL-6 is not essential for increasing bone resorption in the early postovariectomy period.

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

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 formation 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 summary, 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 simultaneously reduce inflammation and bone loss in common forms of osteoporosis.

IL-7 induces bone loss in vivo by induction of receptor activator of nuclear factor κB ligand and tumor necrosis factor α from T cells

IL-7, a powerful lymphopoietic cytokine, is elevated in rheumatoid arthritis (RA) and known to induce bone loss when administered in vivo. IL-7 has been suggested to induce bone loss, in part, by stimulating the proliferation of B220+ cells, a population capable of acting as early osteoclast (OC) precursors. However, the mechanism by which IL-7 leads to differentiation of precursors into mature OCs remains unknown. We previously reported that, in vitro, IL-7 up-regulated T cell cytokines including receptor activator of nuclear factor κB ligand (RANKL). To demonstrate the importance of T cells to the bone-wasting effect of IL-7 in vivo, we have now examined IL-7-induced bone loss in T cell-deficient nude mice. We show that T cell-replete mice undergo significant osteoclastic bone loss after IL-7 administration, concurrent with induction of RANKL and tumor necrosis factor α (TNF-α) secretion by splenic T cells. In contrast, nude mice were resistant to IL-7-induced bone loss and showed no detectable increase in either RANKL or TNF-α, despite an up-regulation of B220+ cells. Importantly, T cell adoptive transfer into nude mice restored IL-7-induced bone loss, and RANKL and TNF-α secretion, demonstrating that T cells are essential mediators of IL-7-induced bone loss in vivo.

Estrogen deficiency induces bone loss by increasing T cell proliferation and lifespan through IFN-γ-induced class II transactivator

Expansion of the pool of tumor necrosis factor (TNF)-α-producing T cells is instrumental for the bone loss induced by estrogen deficiency, but the responsible mechanism is unknown. Here we show that ovariectomy up-regulates IFN-γ-induced class II transactivator, a multitarget immune modulator, resulting in increased antigen presentation by macrophages, enhanced T cell activation, and prolonged lifespan of active T cells. Up-regulation of class II transactivator derives from increased production of IFN-γ by T helper 1 cells, resulting from enhanced secretion of IL-12 and IL-18 by macrophages. The resulting T cell expansion and bone loss are prevented in vivo by both blockade of antigen presenting cell-induced T cell activation, and silencing of IFN-γ receptor signaling. Thus, increased IFN-γ-induced class II transactivator expression and the resulting enhanced T cell proliferation and lifespan are critical to the bone wasting effect of estrogen deficiency.

Increased production of IL-7 uncouples bone formation from bone resorption during estrogen deficiency

Postmenopausal bone loss stems from the inability of osteoblastic activity to match the increase in osteoclastic bone resorption induced by estrogen deficiency. However, the mechanism that uncouples osteoblast from osteoclast activities remains unexplained. We show that ovariectomy enhances the production of the osteoclastogenic cytokine IL-7, and that its neutralization in vivo prevents ovariectomy-induced bone loss. Surprisingly, serum osteocalcin levels, a biochemical marker of bone formation, suggested that the bone-sparing effects of IL-7 neutralization were due not only to inhibition of bone resorption, but also to stimulation of bone formation. Consistent with these data, addition of IL-7 to neonatal calvarial organ cultures blocked new bone formation, and injection of IL-7 into mice in vivo inhibited bone formation as measured by calcein incorporation into long bones. The antianabolic effects of IL-7 were consistent with an observed downregulation of the osteoblast-specific transcription factor core-binding factor alpha1/Runx2. Thus, because it targets both the osteoclast and the osteoblast pathways, IL-7 is central to the altered bone turnover characteristic of estrogen deficiency.

The role of T lymphocytes in bone metabolism

Summary:  Recent findings from animal models suggest that the bone loss induced by estrogen deficiency may stem in large measure from a pathological upregulation of the adaptive immune response. While the role of activated T cells in the osteoporosis driven by inflammatory conditions and infection has been well documented, only recently has the role of T cells in the bone destruction associated with estrogen deficiency begun to be appreciated. In vivo and in vitro models of postmenopausal osteoporosis demonstrate that estrogen deficiency leads to an increase in the adaptive immune function that culminates in an increased production of tumor necrosis factor α (TNF) by activated T cells. TNF increases osteoclast (OC) formation and bone resorption both directly and by augmenting the sensitivity of maturing OCs to the essential osteoclastogenic factor receptor activator of nuclear factor κB ligand. The activation and expansion of TNF‐producing T cells are key steps in estrogen deficiency‐driven bone loss and are regulated by multiple interacting cytokines including transforming growth factor‐β, interleukin‐7, and interferon‐γ, as well as by the process of antigen presentation. Herein, we review the experimental evidence that suggests estrogen prevents bone loss by regulating T‐cell function and immune cell bone interactions.

T lymphocytes amplify the anabolic activity of parathyroid hormone through Wnt10b signaling.

Intermittent administration of parathyroid hormone (iPTH) is used to treat osteoporosis because it improves bone architecture and strength, but the underlying cellular and molecular mechanisms are unclear. Here, we show that iPTH increases the production of Wnt10b by bone marrow CD8+ T cells and induces these lymphocytes to activate canonical Wnt signaling in preosteoblasts. Accordingly, in responses to iPTH, T cell null mice display diminished Wnt signaling in preosteoblasts and blunted osteoblastic commitment, proliferation, differentiation, and life span, which result in decreased trabecular bone anabolism and no increase in strength. Demonstrating the specific role of lymphocytic Wnt10b, iPTH has no anabolic activity in mice lacking T-cell-produced Wnt10b. Therefore, T-cell-mediated activation of Wnt signaling in osteoblastic cells plays a key permissive role in the mechanism by which iPTH increases bone strength, suggesting that T cell osteoblast crosstalk pathways may provide pharmacological targets for bone anabolism.

Oxidative stress causes bone loss in estrogen-deficient mice through enhanced bone marrow dendritic cell activation

Increased production of tumor necrosis factor α (TNF) in the bone marrow (BM) in response to both oxidative stress and T cell activation contributes to the bone loss induced by estrogen deficiency, but it is presently unknown whether oxidative stress causes bone loss through T cells. Here we show that ovariectomy causes an accumulation in the BM of reactive oxygen species, which leads to increased production of TNF by activated T cells through up-regulation of the costimulatory molecule CD80 on dendritic cells. Accordingly, bone loss is prevented by treatment of ovariectomized mice with either antioxidants or CTLA4-Ig, an inhibitor of the CD80/CD28 pathway. In summary, reactive oxygen species accumulation in the BM is an upstream consequence of ovariectomy that leads to bone loss by activating T cells through enhanced activity of BM dendritic cells, and these findings suggest that the CD80/CD28 pathway may represent a therapeutic target for postmenopausal bone loss.

T Cells Potentiate PTH-Induced Cortical Bone Loss through CD40L Signaling

Parathyroid hormone (PTH) promotes bone catabolism by targeting bone marrow (BM) stromal cells (SCs) and their osteoblastic progeny. Here we show that a continuous infusion of PTH that mimics hyperparathyroidism fails to induce osteoclast formation, bone resorption, and cortical bone loss in mice lacking T cells. T cells provide proliferative and survival cues to SCs and sensitize SCs to PTH through CD40 ligand (CD40L), a surface molecule of activated T cells that induces CD40 signaling in SCs. As a result, deletion of T cells or T cell-expressed CD40L blunts the bone catabolic activity of PTH by decreasing bone marrow SC number, the receptor activator of nuclear factor-kappaB ligand (RANKL)/OSTEOPROTEGERN (OPG) ratio, and osteoclastogenic activity. Therefore, T cells play an essential permissive role in hyperparathyroidism as they influence SC proliferation, life span, and function through CD40L. T cell-SC crosstalk pathways may thus provide pharmacological targets for PTH-induced bone disease.