IL-12p40 impairs mesenchymal stem cell-mediated bone regeneration via CD4+ T cells

Abstract

Severe or prolonged inflammatory response caused by infection or biomaterials leads to delayed healing or bone repair failure. This study investigated the important roles of the proinflammatory cytokines of the interleukin-12 (IL-12) family, namely, IL-12 and IL-23, in the inflammation-mediated inhibition of bone formation in vivo. IL-12p40^-/- mice lacking IL-12 and IL-23 exhibited enhanced bone formation. IL-12 and IL-23 indirectly inhibited bone marrow mesenchymal stem cell (BMMSC) differentiation by stimulating CD4⁺ T cells to increase interferon γ (IFN-γ) and IL-17 levels. Mechanistically, IL-17 synergistically enhanced IFN-γ-induced BMMSC apoptosis. Moreover, INF-γ and IL-17 exerted proapoptotic effects by upregulating the expression levels of Fas and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), as well as by activating the caspase cascade in BMMSCs. IL-12p40 depletion in mice could promote ectopic bone formation. Thus, IL-12p40 is an attractive therapeutic target to overcome the inflammation-mediated inhibition of bone formation in vivo.

Figure 1

IL-12p40^−/− mice increased bone formation and inhibited bone resorption. (a) Cytokine expression in different time periods after implantation in C57BL/6J mice. n=5 per group. (b) Increased cortical bone thickness and trabecular bone in micro-CT images of the distal femur of IL-12p40^−/− mice compared with WT mice. n=5 per group. (c–f) BMD (c), BV/TV (d), Tb.Th (e), and Tb.Sp (f) as measured by micro-CT. n=5 per group. (g) H&E staining of femur sections from 8-week-old WT and IL-12p40^−/− mice. n=5 per group. Scale bars, 200 μm. (h) ALP staining and quantitative analysis by ImageJ showed a more osteoblast surface in the IL-12p40^−/− mice compared with the WT mice. n=5 per group. Scale bars, 100 μm. (i) TRAP staining and quantitative analysis by ImageJ showed that osteoclast surface was reduced in the IL-12p40^−/− mice compared with the WT mice. n=5 per group. Scale bars, 100 μm. (j and k) ELISA of serum concentrations of OCN (j) and CTX-1 (k) in 8-week-old WT versus IL-12p40^−/− mice. n=5–8 per group. (l) Representative images (left) of new bone formation and quantification of the MAR (right) as assessed by double calcein labeling. n=5 per group. Scale bars, 25 μm. All values are given as the mean±S.D. of three independent experiments. *P<0.05, **P<0.01, ***P<0.001

Figure 2

IL-12 and IL-23 indirectly inhibited osteogenic differentiation of BMMSCs. (a) Schema of the experimental procedures. New bone formation in nude mice was detected with H&E staining after implantation of BMMSCs and β-TCP plus IL-12 and IL-23 or no cytokine (control). Con, control; B, bone; CT, connective tissue. n=5 per group. Scale bars, 50 μm. (b) The Col1 expression levels of implants plus IL-12 and IL-23 or no cytokine in nude mice were shown by immunohistochemistry. n=5 per group. Scale bars, 20 μm. (c and e) Alizarin Red S staining of cultured BMMSCs after treatment with osteogenic medium plus different concentrations of IL-12 (c) or IL-23 (e). CM, control medium; OM, osteogenic medium. (d and f) Relative expression levels of osteoblast markers in BMMSCs indicated in (c and e) were quantified by RT-PCR. (g) Alizarin Red S staining of cultured BMMSCs after treatment with osteogenic medium plus IL-12 (200 ng/ml), IL-23 (200 ng/ml), or both. (h) Relative expression levels of osteoblast markers in BMMSCs indicated in (g) were quantified by RT-PCR. (i) Alizarin Red S staining of cultured BMMSCs after treatment with osteogenic medium plus supernatants of CD4⁺ T cells that were stimulated with IL-12 and IL-23 or none (control). The relative levels of mRNA expression were normalized to β-actin. All values are given as the mean±S.D. of three independent experiments. *P<0.05, **P<0.01, ***P<0.001

Figure 3

IL-12 and IL-23 upregulated IFN-γ and IL-17 to suppress BMMSC differentiation. (a) Expression of IFN-γ or IL-17 in CD4⁺ T cells stimulated with IL-12 (200 ng/ml) or IL-23 (200 ng/ml). (b) Concentrations of serum IFN-γ and IL-17 after implantation in WT and IL-12p40^−/− mice. (c, e, g, and h) Alizarin Red S staining of cultured BMMSCs after treatment with osteogenic medium plus different concentrations of IL-17 (c), IFN-γ (e), or both (g and h). (d and f) Relative expression levels of osteoblast markers indicated in (c and e) were quantified by RT-PCR. (i) Western blot analysis of the groups indicated in (h). (j) Relative expression levels of osteoblast markers in BMMSCs indicated in (h) were quantified by RT-PCR. The relative levels of mRNA expression were normalized to β-actin. All values are given as the mean±S.D. of three independent experiments. *P<0.05, **P<0.01, ***P<0.001

Figure 4

IL-17 synergistically promoted IFN-γ-induced BMMSC apoptosis in vitro and in vivo. (a) Western blot analysis of BMMSCs treated with IL-17 (200 ng/ml), IFN-γ (200 ng/ml), or both. (b) Percentage of surviving BMMSCs after treatment with different concentrations of IL-17, IFN-γ, or both. **P<0.01, ***P<0.001 (versus IFN-γ/IL-17). ^##P<0.01, ^###P<0.001 (versus IFN-γ/IFN-γ+IL-17). (c) Ratio of surviving BMMSCs after treatment with various concentrations of IFN-γ (10–200 ng/ml) alone or in combination with IL-17 (200 ng/ml). (d) Ratio of surviving BMMSCs after treatment with various concentrations of IL-17 (10–200 ng/ml) alone or in combination with IFN-γ (200 ng/ml). (e and f) Relative expression levels of Fas (e) or TRAIL (f) in BMMSCs treated with IL-17 (200 ng/ml), IFN-γ (200 ng/ml), or both were quantified by RT-PCR. *P<0.05, **P<0.01, ***P<0.001 (versus IFN-γ/IFN-γ+IL-17). (g) Western blot analysis of cleaved caspase expression in BMMSCs after treatment with IL-17 (200 ng/ml), IFN-γ (200 ng/ml), or both. (h) Apoptosis of BMMSCs after treatment with IL-17 (200 ng/ml), IFN-γ (200 ng/ml), or both was detected by TUNEL staining. (i) Quantitative analysis of the apoptosis cells indicated in (h). (j) Cell apoptosis of BMMSCs after treatment with IL-17 (200 ng/ml), IFN-γ (200 ng/ml), or both, as demonstrated by toluidine blue staining. (k and l) The cleaved caspase 3 (k) and TUNEL (l) expression levels of implants in WT and IL-12p40^−/− mice were shown by immunohistochemistry. n=4 per group. Scale bars, 20 μm. The relative levels of mRNA expression were normalized to β-actin. All values are given as the mean±S.D. of three independent experiments. *P<0.05, **P<0.01, ***P<0.001

Figure 5

Inhibition of apoptosis could partially rescue osteogenic differentiation inhibited by IFN-γ and IL-17. (a and b) Knockdown of Fas (a) and TRAIL (b) expression by siRNA. NC, negative control. (c) Western blot analysis of cleaved caspase expression in BMMSCs after siRNA-mediated inhibition of Fas and TRAIL and treatment with IL-17 (200 ng/ml), IFN-γ (200 ng/ml), or both. (d) Ratio of surviving BMMSCs after siRNA-mediated inhibition of Fas and TRAIL and treatment with IL-17 (200 ng/ml) combined with IFN-γ (200 ng/ml). (e) Alizarin Red S staining of cultured BMMSCs after siRNA-mediated inhibition of Fas and TRAIL, and treatment with osteogenic medium alone or plus IL-17 (200 ng/ml) and IFN-γ (200 ng/ml). (f) Ratio of surviving BMMSCs after treatment with either a caspase 3 inhibitor (Z-DEVD-FMK) or a pan-caspase inhibitor (Z-VAD-FMK) alone or in combination with IL-17 (200 ng/ml) and IFN-γ (200 ng/ml). (g) Alizarin Red S staining of cultured BMMSCs after treatment with osteogenic medium and Z-DEVD-FMK or Z-VAD-FMK alone or in combination with IL-17 (200 ng/ml) and IFN-γ (200 ng/ml). The relative levels of mRNA expression were normalized to β-actin. All values are given as the mean±S.D. of three independent experiments. *P<0.05, **P<0.01, ***P<0.001

Figure 6

Depletion of IL-12p40 promoted BMMSC-mediated bone formation. (a) Schema of the experimental procedures. BMMSCs mixed with β-TCP were subcutaneously implanted into the dorsal surface of WT or IL-12p40^−/− mice. After 8 weeks, the implants were harvested. (b) New bone formation after implantation of BMMSCs and β-TCP in WT or IL-12p40^−/− mice was detected with H&E staining. B, bone; CT, connective tissue. n=5 per group. Scale bars, 50 μm. (c) Quantitative analysis of new bone volume by ImageJ showed significantly increased new bone volume in IL-12p40^−/− mice. n=5 per group. (d) Col1 expression levels of implants in WT or IL-12p40^−/− mice were shown by immunohistochemistry. n=5 per group. Scale bars, 20 μm. (e) Quantification of Col1 expression in implants. n=5 per group. (f) Proposed role of IL-12 and IL-23 in fracture healing. With severe inflammation in the body, immune cells accumulate to the damaged tissue. These cells release large amounts of IL-12 and IL-23, which further promote the immune cells to produce other inflammatory factors (e.g., IFN-γ and IL-17). Locally produced IFN-γ and IL-17 can affect the function of BMMSCs and lead to BMMSC apoptosis. All values are given as the mean±S.D. of three independent experiments. *P<0.05, **P<0.01