Pulsed low-dose RANKL as a potential therapeutic for postmenopausal osteoporosis

Abstract

A number of studies in model animal systems and in the clinic have established that RANKL promotes bone resorption. Paradoxically, we found that pulsing ovariectomized mice with low-dose RANKL suppressed bone resorption, decreased the levels of proinflammatory effector T cells and led to increased bone mass. This effect of RANKL is mediated through the induction of FoxP3⁺CD25⁺ regulatory CD8⁺ T cells (Tc_REG) by osteoclasts. Here, we show that pulses of low-dose RANKL are needed to induce Tc_REG, as continuous infusion of identical doses of RANKL by pump did not induce Tc_REG. We also show that low-dose RANKL can induce Tc_REG at 2, 3, 6, and 10 weeks after ovariectomy. Our results show that low-dose RANKL treatment in ovariectomized mice is optimal at once-per-month doses to maintain the bone mass. Finally, we found that treatment of ovariectomized mice with the Cathepsin K inhibitor odanacatib also blocked Tc_REG induction by low-dose RANKL. We interpret this result to indicate that antigens presented to CD8⁺ T cells by osteoclasts are derived from the bone protein matrix because Cathepsin K degrades collagen in the bone. Taken together, our studies provide a basis for using low-dose RANKL as a potential therapeutic for postmenopausal osteoporosis.

Figure 1. Pulsed RANKL is required for the bone anabolic effect of low-dose RANKL.

Twelve-week-old C57BL/6J mice were either sham-operated or OVX using pumps or pulsed with low-dose RANKL. Two weeks after surgery, ALZET pumps were implanted in the i.p. cavity that contained either PBS or RANKL. All groups were pulsed with PBS or with an equivalent dose of RANKL (0.125 mg/kg, pulsed twice 24 hours apart) as indicated in the plots. (A) Flow cytometry shows that pulsed but not continuous exposure to RANKL induced Tc_REG. (B) Quantification of FACS data of Tc_REG levels across all groups (6–8 mice/group). (C) Consistent with Tc_REG induction, decrease in T_EFF cells (gated on CD45⁺CD3⁺ cells, and then on CD44⁺) was only observed in mice pulsed with RANKL but not treated with RANKL delivered by pumps. (D) Decreased levels of bone resorption were observed in mice pulsed with low-dose RANKL but not in mice with continuous exposure to RANKL. Modestly higher (but statistically significant) levels of bone resorption were observed in mice treated with continuous RANKL. (E) Bone volume (BV/TV) decreased in mice where RANKL was delivered by pump but was restored to levels observed in sham-operated mice when pulsed with RANKL. (F) Consistent with serum CTX and BV/TV results, an increase in bone mineral density (BMD) was observed in mice treated with pulsed RANKL but decreased in mice where RANKL was delivered by pump. (G and H) Pulsed low-dose RANKL decreased osteoclast numbers. Quantitation of osteoclast numbers/mm bone (N.OC/BS) and osteoclast surface/bone surface (OC.S/BS) is shown in G, and representative images are shown in H from sham and ovariectomized mice treated with infused and pulsed RANKL. Arrowheads indicate Tartrate-resistant acid phosphatase–positive (TRAP⁺) cells. Data is from 6–8 mice per group. Group statistical significance was calculated using Mann-Whitney U test. ***P < 1 × 10⁻³; **P < 0.01; *P < 0.05.

Figure 2. The bone anabolic effect of low-dose RANKL is independent of time after ovariectomy.

Sham surgery or ovariectomy (OVX) was performed on mice at 12 weeks of age. At times (in weeks) indicated after OVX, 2 doses of RANKL (0.125 mg/kg) were administered 24 hours apart. Ten days after first treatment, mice were sacrificed. Low-dose RANKL (A) induced Tc_REG and (B) decreased proinflammatory effector cells in the femora, measured using flow-cytometry. (C) Treatment decreased serum CTX levels. Measurements of proximal tibia by μCT show increased bone mass (BV/TV) (D) and bone mineral density (BMD) (E) across time course. Data is from 6–12 mice per group. Group statistical significance was calculated using Mann-Whitney U test. ****P < 1 × 10⁻⁴; ***P < 1 × 10⁻³; **P < 0.01.

Figure 3. Pulsing RANKL once per month is optimal to limit bone resorption and inflammation.

(A) To assess the kinetics of a single treatment, C57BL/6J ovariectomized mice were treated with low-dose RANKL (administered twice 24 hours apart, 2 weeks after OVX). (B)Although serum CTX levels start to rise at day 35, bone mass as assessed by μCT (BV/TV) was relatively stable at days 25 and 35. (C–E) To assess the effect of repeated dosing, 12-week-old OVX mice then treated every 30 days over a span of 90 days. (C) Serum CTX levels for each of the 4 groups. (D) Representative animals were sacrificed (5–7 mice/group), and BV/TV of the proximal tibias is shown. (E) Cells that secrete TNFα and IL-17A could be detected in peripheral blood 120 days after OVX (6 mice/group). Multiplexed ELISA used media collected after 30 hours of culturing to quantify cytokines. Groups were ovariectomized and treated with low-dose RANKL or zoledronic acid. Group statistical significance was calculated using Mann-Whitney U test. ****P < 1 × 10⁻⁴; ***P < 1 × 10⁻³; **P ≤ 0.01, *P < 0.05.

Figure 4. Active antigen processing by osteoclasts is required for Tc_REG induction.

(A) FoxP3^eGFP reporter mice were treated with odanacatib (Odn), and then low-dose RANKL was administered. In the presence of Odn, Tc_REG (GFP⁺CD8⁺ cells) induction was not observed (right panel). (B) The results are quantified from 6 animals/group. (C) In culture, BM cell–derived osteoclasts are able to induce FoxP3 in OVA-specific OT-I CD8⁺ T cells when pulsed with peptide antigen (SIINFEKL).