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.

PTH promotes bone catabolism by targeting bone marrow 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, RANKL/OPG production and osteoclastogenic activity. Therefore, T cells play an essential permissive role in hyperparathyroidism as they influence SC proliferation, lifespan and function through CD40L. T cell-SC cross-talk pathways may thus provide pharmacological targets for PTH induced bone disease.

Increased production of tumor necrosis factor ␣ (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 upregulation 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.CTLA-4Ig ͉ reactive oxygen species ͉ T cells ͉ osteoporosis ͉ tumor necrosis factor P ostmenopausal bone loss is induced by ovarian involution and the resulting increase in follicle-stimulating hormone production through multiple effects on bone marrow (BM) and bone cells (1-4), but the most relevant consequence of natural menopause and ovariectomy (ovx) is a cytokine-driven increase in osteoclast (OC) formation (2, 4). OC formation occurs when bone marrow macrophages (BMMs) are costimulated by the essential osteoclastogenic factors receptor activator of nuclear factor-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) (2), but after menopause TNF is produced in greater amounts in the BM and up-regulates OC formation (4, 5). TNF stimulates the production of RANKL and M-CSF by bone and BM cells (6, 7), augments the responsiveness of OC precursors to RANKL (8, 9), and induces additional osteoclastogenic cytokines such as IL-1, IL-6, and IL-7 (4). Studies with genetic and pharmacological models have demonstrated the pivotal role of TNF in ovx-induced bone loss in rodents and humans (10-13) and have provided insights on the estrogen (E)-regulated sources of TNF. Ovx upregulates the production of TNF by BM T lymphocytes (8, 12) by increasing thymic T cell output (14) and by stimulating the antigen (Ag)-dependent activation and expansion of T cells in the BM (15). Attesting to the relevance of T cell-produced TNF, T cell-deficient nude mice are protected against the loss of cortical and trabecular bone induced by ovx (8,12,16). Furthermore, the capacity of ovx to induce bone loss is restored by adoptive transfer into nude mice of T cells from wild-type (WT) but not of those from TNF Ϫ/Ϫ mice (12).T cell activation is caused by the presentation to T cells of Ag fragments bound to MHC molecules expressed on Ag-presenting cells (APCs) (17). Ovx up-regulates the capacity of BMMs to present Ag to CD4ϩ T cells (4, 15), but no information is available on how ovx activates CD8ϩ T cells and on whether ovx has additional stimulatory effects on BM DCs, t...

Hydrogen sulfide (H2S) is a gasotransmitter known to regulate bone formation and bone mass in unperturbed mice. However, it is presently unknown whether H2S plays a role in pathologic bone loss. Here we show that ovariectomy (ovx), a model of postmenopausal bone loss, decreases serum H2S levels and the bone marrow (BM) levels of two key H2S-generating enzymes, cystathione β-synthase (CBS) and cystathione γ-lyase (CSE). Treatment with the H2S-donor GYY4137 (GYY) normalizes serum H2S in ovx mice, increases bone formation, and completely prevents the loss of trabecular bone induced by ovx. Mechanistic studies revealed that GYY increases murine osteoblastogenesis by activating Wnt signaling through increased production of the Wnt ligands Wnt16, Wnt2b, Wnt6, and Wnt10b in the BM. Moreover, in vitro treatment with 17β-estradiol upregulates the expression of CBS and CSE in human BM stromal cells (hSCs), whereas an H2S-releasing drug induces osteogenic differentiation of hSCs. In summary, regulation of H2S levels is a novel mechanism by which estrogen stimulates osteoblastogenesis and bone formation in mice and human cells. Blunted production of H2S contributes to ovx-induced bone loss in mice by limiting the compensatory increase in bone formation elicited by ovx. Restoration of H2S levels is a potential novel therapeutic approach for postmenopausal osteoporosis.

Chemokines are involved in a number of inflammatory pathologies and some of them show a pivotal role in the modulation of osteoclast development. Therefore, we evaluated the role of CXCL12 chemokine on osteoclast differentiation and function and we analyzed its expression on synovial and bone tissue biopsies from rheumatoid arthritis (RA) patients. Osteoclasts were obtained by 7 days in vitro differentiation with RANKL and M-CSF of CD11b positive cells in the presence or absence of CXCL12. The total number of osteoclast was analyzed by Tartrate-resistant acid phosphatase (TRAP)-staining and bone-resorbing activity was assessed by pit assay. MMP-9 and TIMP-1 release was evaluated by ELISA assay. CXCL12 expression on biopsies from RA patients was analyzed by immunohistochemistry. Osteoclasts obtained in the presence of CXCL12 at 10 nM concentration displayed a highly significant increase in bone-resorbing activity as measured by pit resorption assay, while the total number of mature osteoclasts was not affected. The increased resorption is associated with overexpression of MMP-9. Immunostaining for CXCL12 on synovial and bone tissue biopsies from both rheumatoid arthritis (RA) and osteoarthritis (OA) samples revealed a strong increase in the expression levels under inflammatory conditions. CXCL12 chemokine showed a clear activating role on mature osteoclast by inducing bone-resorbing activity and specific MMP-9 enzymatic release. Moreover, since bone and synovial biopsies from RA patients showed an elevated CXCL12 expression, these findings may provide useful tools for achieving a full elucidation of the complex network that regulates osteoclast function in course of inflammatory diseases.