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

Abstract

T cell-produced cytokines play a pivotal role in the bone loss caused by inflammation, infection, and estrogen deficiency. IFN-gamma is a major product of activated T helper cells that can function as a pro- or antiresorptive cytokine, but the reason why IFN-gamma has variable effects in bone is unknown. Here we show that IFN-gamma 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-alpha. Analysis of the in vivo effects of IFN-gamma in 3 mouse models of bone loss - ovariectomy, LPS injection, and inflammation via silencing of TGF-beta signaling in T cells - reveals that the net effect of IFN-gamma in these conditions is that of stimulating bone resorption and bone loss. In summary, IFN-gamma 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-gamma signaling may thus represent a novel strategy to simultaneously reduce inflammation and bone loss in common forms of osteoporosis.

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-γR^–/– 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 μg/ml ). *P < 0.05 compared with vehicle-treated controls. The proliferation (F) and rate of apoptosis (G) of maturing osteoclasts were determined by [³H]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.

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 × 10⁶ IU/kg body weight) was injected subcutaneously twice a week into 16-week-old WT mice, T cell–deficient 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.

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 μCT 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.

Figure 4. Effects of LPS treatment for 3 weeks in WT and IFN-γ^–/– mice.

LPS (25 mg/kg body weight) was injected once a week in 16-week-old mice. All mice were sacrificed at the end of the LPS treatment and bone, BM, and serum samples analyzed. (A) In vivo BMD measurements by DXA were obtained at baseline and weekly thereafter. (B) Analysis of BV/TV by μCT. (C) Serum CTX. (D) Serum osteocalcin. (E) Antigen presentation assay. (F) Measurement of CD4 and CD69 expression by flow cytometric analysis. 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. ^#P < 0.05 compared with LPS-treated WT mice.

Figure 5. Silencing of IFN-γ production reduces the increase in bone loss and T cell activation induced by the disruption of TGF-β signaling in T cells.

In vivo BMD measurements by DXA (A), μCT analysis of trabecular bone (B), serum CTX (C) and T cell activation assays (D), and serum osteocalcin measurements (E) were carried out in 16-week-old intact WT, CD4dnTGFβIIR, CD4dnTGFβIIR/IFN-γ^–/–, and IFN-γ^–/– mice. The percentages in A represent the change compared with age-matched WT controls. All data are expressed as mean ± SD. *P < 0.05 compared with age-matched WT control mice. ^#P < 0.05 compared with CD4dnTGFβIIR mice.

Figure 6. Silencing IFN-γ production reduces the increase in antigen presentation and T cell cytokine production induced by the disruption of TGF-β signaling in bone T cells.

BMMs and spleen T cells were purified from spleens of 16-week-old WT, CD4dnTGFβIIR, CD4dnTGFβIIR/IFN-γ^–/–, and IFN-γ^–/– mice and pooled by group. Antigen presentation (A) and CIITA expression (B) were assayed in BMMs. Cytokine levels (C and D) were measured by ELISA in CM of T cells cultured for 3 days in the presence of 100 ng/ml PMA and 100 ng/ml ionomycin. All data are expressed as mean ± SD. *P < 0.01 compared with WT mice; ^#P < 0.05 compared with CD4dnTGFβIIR mice.

Figure 7. Silencing IFN-γ production reduces the increase in bone resorption induced by the disruption of TGF-β signaling in T cells.

T cells from 16-week-old WT (WT-T), CD4dnTGFβIIR (CD4dnTGFβIIR-T), and CD4dnTGFβIIR/IFN-γ^–/– (CD4dnTGFβIIR/IFN-γ^–/––T) mice were transferred into 16-week-old T cell–deficient nude mice. Spine BMD measurement (A), μCT analysis (B), serum CTX measurement (C), and serum osteocalcin measurement (D) were performed 4 weeks after transplantation. The percentages in A represent the change compared with baseline. All data are expressed as mean ± SD. *P < 0.05 compared with nude mice transplanted with T cells from WT mice; ^#P < 0.05 compared with nude mice transferred with T cells from CD4dnTGFβIIR mice.