Figure 7. The anabolic effect of low-dose RANKL treatment in ovariectomized mice is mediated through CD8 T-cells.

To confirm that decrease in bone erosion and effector T-cell levels is mediated via Tc_REG, β2M^−/− mice lacking CD8 T-cells, were sham-operated (S; n=6), ovariectomized (OVX; n=6) and ovariectomized then treated with low-dose RANKL (ldRL = 0.125 mg/kg; n=6). In some experiments mice are also treated with high-dose RANKL (hdRL = 1 mg/kg). A. Low-dose RANKL treatment of ovariectomized mice lacking CD8 T-cells does not decrease T_EFF: Ovariectomy of β2M^−/− mice increased effector T-cells (CD3⁺ and CD44⁺) relative to sham-operated (indicated by S). However, unlike treatment of CD8 T-cell-replete mice, no decrease in levels of effector T-cells was observed due to low-dose RANKL (ldRL) treatment in β2M^−/− mice. B. Low-dose RANKL treatment of ovariectomized mice lacking CD8 T-cells does not decrease bone resorption: Bone resorption levels, as assayed by serum CTX, increase in CD8 T-cell (β2M^−/−) deficient ovariectomized mice relative to sham-operated mice. A modest increase in serum CTX is observed in ovariectomized β2M^−/− mice treated with low-dose RANKL. C. Low-dose RANKL treatment of ovariectomized mice lacking CD8 T-cells does not increase bone volume or bone density: Ovariectomy leads to significant levels of bone loss relative in β2M^−/− mice. Low-dose RANKL leads to further decrease in bone volume (BV/TV) and bone mineral density (BMD) in β2M^−/− mice, indicating that CD8 T-cells in WT mice protect against bone loss. D. Representative μCT renderings for panels C and E. E. Low-dose RANKL treatment of ovariectomized mice is bone anabolic but not in mice lacking CD8 T-cells: In WT mice low-dose RANKL is anti-resorptive in ovariectomized mice but promotes bone loss in estrogen-replete mice. Treatment with high-dose RANKL (1mg/kg; n= 6 mice per group) leads to bone resorption in both estrogen-deficient and replete mice. In CD8 T-cell-deficient mice low dose RANKL leads to bone loss in both estrogen-replete (Sham) and ovariectomized (OVX) mice. Percentage of bone volume lost (or gained; BV/TV) relative to reference state (untreated sham surgery or ovariectomy - indicated by horizontal dashed line) is also given. The results represent mean ± standard deviation (error bars). P values were calculated using Mann-Whitney test; *** = P < 0.001 and * = P < 0.05. F. A model of the negative feedback loop between osteoclast-induced Tc_REG: In ovariectomized mice increased levels of proinflammatory cytokines TNFα and IL-17 suppress DLL4 expression needed for Tc_REG induction (left panel). In the absence of Tc_REG and in the presence of the proinflammatory cytokine the activated osteoclasts have increased bone resorption leading to osteoporosis. Treatment with low-dose RANKL (right panel) reverses the repression of DLL4 in ovariectomized mice leading to induction of Tc_REG by osteoclasts to engage the negative feedback loop. Induction of Tc_REG not only limits osteoclastogenesis but also reduced the proinflammatory effector T-cells. The reduction in T_EFF leads to increased bone formation rate.