Prominent Bone Loss Mediated by RANKL and IL-17 Produced by CD4+ T Cells in TallyHo/JngJ Mice

Increasing evidence that decreased bone density and increased rates of bone fracture are associated with abnormal metabolic states such as hyperglycemia and insulin resistance indicates that diabetes is a risk factor for osteoporosis. In this study, we observed that TallyHo/JngJ (TH) mice, a polygenic model of type II diabetes, spontaneously developed bone deformities with osteoporotic features. Female and male TH mice significantly gained more body weight than control C57BL/6 mice upon aging. Interestingly, bone density was considerably decreased in male TH mice, which displayed hyperglycemia. The osteoblast-specific bone forming markers osteocalcin and osteoprotegerin were decreased in TH mice, whereas osteoclast-driven bone resorption markers such as IL-6 and RANKL were significantly elevated in the bone marrow and blood of TH mice. In addition, RANKL expression was prominently increased in CD4+ T cells of TH mice upon T cell receptor stimulation, which was in accordance with enhanced IL-17 production. IL-17 production in CD4+ T cells was directly promoted by treatment with leptin while IFN-γ production was not. Moreover, blockade of IFN-γ further increased RANKL expression and IL-17 production in TH-CD4+ T cells. In addition, the osteoporotic phenotype of TH mice was improved by treatment with alendronate. These results strongly indicate that increased leptin in TH mice may act in conjunction with IL-6 to preferentially stimulate IL-17 production in CD4+ T cells and induce RANKL-mediated osteoclastogenesis. Accordingly, we propose that TH mice could constitute a beneficial model for osteoporosis

Altered CD4+ T cell functions in TH mice.

<p>All B6 and TH mice were analyzed at 8 weeks of age (n = 8). (A) Bone marrow cells were stained with fluorescence-conjugated anti-Ly6C Ab (BD Pharmingen) and analyzed using FACS Calibur and CellQuest program (BD Biosciences). (B) Single cell suspensions of thymus and lymph node were harvested and stained with Abs against CD4 and CD8, followed by flow cytometry analysis. (C) CD4+ T cells were isolated from the lymph node and stimulated with anti-CD3 and anti-CD28 for 24 h. Cells were then collected for real time-PCR to determine the relative expression levels of IL-4 and IFN-γ. (D-F) CD4+ T cells were stimulated for 48 h. IFN-γ production was analyzed in the cell supernatant by ELISA (D) and intracellular cytokine staining (E). (F) T cells were stained with anti-IFN-γ and anti-IL-4 Abs (BD PHarmingen), followed by flow cytometry. (G) T cells were stimulated and subsequently treated with TGF-β and IL-6 for 48 h. IL-17 was measured in the cell supernatant by ELISA. (H) T cells were stimulated with anti-CD3/28 for 48 h, followed by incubation with anti-RANKL Ab and flow cytometry analysis.</p

Increased serum levels of leptin and inflammatory cytokines in TH mice.

<p>Whole blood was collected from mice (males, n = 6) at 8 weeks and used to measure the serum levels of leptin (A), OCN (B), OPG (C), IL-6 (D), IFN-γ (E), and TGF-β (F). All data are expressed as the mean ± SEM. P value was calculated by student t-test.</p

Restoration of BMD in TH mice by testosterone and alendronate.

<p>B6 and TH mice (n = 6 each group) were orchiectomized at 8 weeks of age and subsequently injected with vehicle (sesame oil) or testosterone (50 mg/kg) for 6 weeks. (A) Micro-CT images of trabecular bone were reconstructed, and one representative image is presented in each group. (B) BMD was calculated using a micro-CT scanner and micoView. All results are expressed as the mean ± SEM for 6 mice. (C) Serum testosterone levels of 8 week-old WT and TH mice (n =  5) were determined using a high-sensitivity ELISA kit. The testosterone/estradiol ratio was determined after determination of serum estradiol level. (D) Relative expression level of aromatase was quantitated in testis by real time PCR and normalization to the level of β-actin (n = 4). (E) TH mice at 4 weeks of age (n = 7) were orally administered alendronate (ALD, 5 mg/kg/day) for 4 weeks. BMD and BMC were determined in B6 and TH mice at 8 weeks of age using a micro-CT scanner.</p

Leptin-mediated IL-17 production in CD4+ T cells.

<p>CD4+ T cells were isolated from B6 mice (8 weeks, n = 3) and stimulated with anti-CD3/28 Ab for 24 h. (A-C) Leptin was added to the cells for an additional 24 h. Supernatant was used in ELISA for measurement of IFN-γ (A) and IL-17 (B). Cells were used to determine the relative expression level of IL-17 by real time-PCR (C). (D-F) Stimulated CD4+ T cells were further treated with either control (-αIFN-γ) or anti-IFN-γ Ab (+αIFN-γ) for 24 h and then harvested to determine RANKL expression by real time-PCR (D). Culture supernatants were used to measure the expression of IFN-γ (E) and IL-17 (F) by ELISA.</p