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

Abstract

Increased production of tumor necrosis factor alpha (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.

Fig. 1.

Effects (mean ± SD) of ovx on DC expression of MHCII and CD80, Ag presentation, and T cell activation. (A) Ovx up-regulates the percentage of CD80+ but not that of MHCII+ BM DCs. FACS analysis was carried out by gating CD11c+ cells and analyzing for MHCII and CD80 expression. Dot plot shows one of three independent experiments. (B) Ovx increases the number of BM DCs, MHCII+ DCs, and CD80+ DCs. (C) Ovx increases Ag presentation by BMMs and BM DCs. (D) Ovx increases Ag presentation by BM but not spleen (SPL) and lymph node (LN) DCs. (E) Ovx decreases CIITA mRNA expression in BM DCs. (F and G) Ovx increases the expression of the activation marker CD69 in CD4+ and CD8+ BM T cells. (H) TNF levels in conditioned medium from coculture of BM DCs, OT-II T cells, and ovalbumin; n = 4 mice per group in each one of triplicate experiments. *, P < 0.05; **, P < 0.001 compared with sham mice.

Fig. 2.

Ovx induces oxidative stress and T cell activation in the BM. (A and B) Densitometric quantification of Western blots. (A) Effect of ovx on intracellular levels of APE-1/Ref-1. (B) Effect of ovx on BM Prx-1 levels. (C) Effect of ovx on GSH levels in the BM. (D) Ovx increases protein carbonylation in BM lysates. (A–D) All data are the mean ± SD of three independent experiments (n = 3 mice per group; *, P < 0.05, compared with the corresponding sham group). (E) H₂O₂ causes a dose-dependent increase in the relative number of mature BM DC (CD80+ MHCII^hi CD11c+ cells). Whole BM was cultured in α-MEM with 10% FBS for 24 h and treated with the indicated concentrations of H₂O₂. Cell viability was confirmed by trypan blue exclusion. FACS analysis was carried out by gating MHCII^hi cells and analyzing for CD11c and CD80 expression. Dot plot shows one of three independent experiments. (F) H₂O₂ increases CD69 expression in CD4+ and CD8+ BM T cells. (E and F) Data are the mean ± SD of three independent experiments (n = 4 mice per group; *, P < 0.05; **, P < 0.01 compared with unstimulated BM).

Fig. 3.

Effects (mean ± SD) of in vivo treatment of ovx mice with 100 mg/kg per day i.p. NAC for 4 weeks. (A) Ag presentation by BM and spleen DCs. (B) Percentage CD80+ BM DCs. (C) Percentage CD4+CD69+ BM T cells. (D) Percentage CD8+CD69+ BM T cells. (E) Levels of TNF in 24-h BM cultures stimulated with phorbol 12-myristate 13-acetate and ionomycin. (F) Femur BMD as measured in vivo by DXA at 2 and 4 weeks. (G) μCT measurements of percentage total volume occupied by trabecular bone volume (BV/TV) in vertebras harvested 4 weeks after surgery (n = 7 mice per group; *, P < 0.05 compared with sham mice; **, P < 0.05 compared with baseline).

Fig. 4.

CTLA4-Ig prevents T cell activation, T cell TNF production, OC formation, and the bone loss induced by ovx. All data are shown as means ± SD. Ovx- and sham-operated mice were treated with CTLA4-Ig (500 μg/mouse) or isotype-matched Irr-Ig (500 μg/mouse) three times per week for 4 weeks. (A) Percentage CD4+CD69+ BM T cells. (B) Percentage CD8+CD69+ BM T cells. (C) TNF levels in 24-h BM T cell culture medium. (D) Serum CTX levels. (E) Serum osteocalcin levels. (F) No. of OCs in 8-day BM cultures stimulated with subsaturating doses of RANKL and M-CSF. (G) In vivo measurements of femoral BMD by DXA. (H) Epiphyseal trabecular volume over total volume and trabecular thickness (Tb.Th) as measured by μCT in femurs at 4 weeks from surgery (n = 7–8 mice per group; *, P < 0.05 compared with Irr-Ig-treated sham controls; **, P < 0.05 compared with baseline.