IFN-γ regulates human dental pulp stem cells behavior via NF-κB and MAPK signaling

Abstract

During caries, dental pulp expresses a range of pro-inflammatory cytokines in response to the infectious challenge. Interferon gamma (IFN-γ) is a dimerized soluble cytokine, which is critical for immune responses. Previous study has demonstrated that IFN-γ at relative high concentration (100 ng/mL) treatment improved the impaired dentinogenic and immunosuppressive regulatory functions of disease-derived dental pulp stem cells (DPSCs). However, little is known about the regulatory effects of IFN-γ at relative low concentration on healthy DPSC behavior (including proliferation, migration, and multiple-potential differentiation). Here we demonstrate that IFN-γ at relatively low concentrations (0.5 ng/mL) promoted the proliferation and migration of DPSCs, but abrogated odonto/osteogenic differentiation. Additionally, we identified that NF-κB and MAPK signaling pathways are both involved in the process of IFN-γ-regulated odonto/osteogenic differentiation of DPSCs. DPSCs treated with IFN-γ and supplemented with pyrrolidine dithiocarbamate (PDTC, an NF-κB inhibitor) or SB203580 (a MAPK inhibitor) showed significantly improved potential for odonto/osteogenic differentiation of DPSCs both in vivo and in vitro. These data provide important insight into the regulatory effects of IFN-γ on the biological behavior of DPSCs and indicate a promising therapeutic strategy for dentin/pulp tissue engineering in future endodontic treatment.

Figure 1. Characterization of human dental pulp stem cells (DPSCs).

The fibroblast-like monoclonal cell isolated from primary cells (a) monoclonal cells at day 14 (d). This clone was able to differentiate into the mesenchymal lineages: osteogenic (alizarin red staining) (b) adipogenic (oil-red o staining) (c) when cultured in appropriate differentiation conditions in vitro compared to control groups (e,f), respectively. Analysis of molecular surface antigen markers in DPSCs by flow cytometry indicated that cells were negative for CD34 and CD45, whereas they were positive for CD146, CD90, CD105, CD29; PE-conjugated non-specific mouse IgG1 served as negative controls (g). Bar 100 μm.

Figure 2. The effects of IFN-γ on the proliferative capacity of DPSCs.

DPSCs were treated with IFN-γ at different concentrations for the indicated time. Cell viability was evaluated using the MTT (a) and EdU incorporation assays (b,c). Scale bars = 50 μm. The mRNA expression of cyclin B1, PCNA, cyclin D1 and P21 was analysed by Q-PCR (d). Statistical analysis was performed using one-way ANOVA. Data are shown as the mean ± SD. *P < 0.05 when compared with the control group.

Figure 3. The effects of IFN-γ on the migration of DPSCs.

Cell migration was evaluated with a two-chamber Transwell system. The migratory cells were stained with crystal violet (a). For the wound healing assay, cells were incubated with IFN-γ at different concentrations for 24 h. Photomicrographs of the scratch were taken at 0 and 24 h post wounding (b). Statistical analysis was performed using one-way ANOVA. Data are shown as the mean ± SD. *P < 0.05 when compared with the control group. ^#P < 0.05 when compared with the IFN-γ 0.5 ng/mL group. Scale bars = 100 μm.

Figure 4. The effects of IFN-γ on the odonto/osteogenic differentiation of DPSCs.

DPSCs were cultured in odonto/osteogenic medium containing IFN-γ at different concentrations for 2 weeks with or without the MAPK inhibitor (SB203580: 20 μΜ, U0126: 25 μΜ, SP600125: 25 μΜ), or NF-κB inhibitor (PDTC: 20 μΜ). Alizarin red staining and alizarin red quantification were used to evaluate the formation of calcium nodules (a–d). The protein expression of ALP, DSPP, OCN and β-actin was analysed by Western blotting (e, Supplementary Fig. 1a). The relative band intensities were determined by densitometry (f). Statistical analysis was performed using one-way ANOVA. Data are shown as the mean ± SD. *P < 0.05 when compared with the control. ^#P < 0.05 when compared with the IFN-γ group. Scale bars = 100 μm.

Figure 5. Involvement of NF-κB and MAPK signaling pathways in the IFN-γ-induced inhibition odonto/osteogenic differentiation of DPSCs.

DPSCs were cultured in odonto/osteogenic medium containing 0.5 ng/ml IFN-γ with or without MAPK inhibitor (SB203580: 20 μΜ, U0126: 25 μΜ, SP600125: 25 μΜ), or NF-κB inhibitor (PDTC: 20 μΜ). The protein expression of P65, p-P65, P38, p-P38, ERK, p-ERK, JNK, p-JNK was analysed by Western blotting (a, Supplementary Fig. 1b). The relative band intensities were determined by densitometry (b–e). Statistical analysis was performed by using one-way ANOVA. Data are shown as means ± SD. *P < 0.05 when compared with the control group. ^#P < 0.05 when compared with the IFN-γ 60 min group.

Figure 6. Micro-computed tomographic (CT) analyses of bone structural and mineral density changes.

Three dimensional reconstruction images from micro-CT analysis (a). IFN-γ decreased the BMD, BV/TV, Tb. Th and the Tb. N parameters, but increased the Tb. Sp parameter (b–f). TNF-κB inhibitor (PDTC) and MAPK inhibitor (SB203580) reversed the effects induced by IFN-γ in DPSCs in vivo. (BMD: bone mineral density; BV/TV: bone volume/total volume; Tb. Th: trabecular thickness; Tb. N: trabecular number; Tb. Sp: trabecular separation). *P < 0.05 when compared with the control group. ^#P < 0.05 when compared with the IFN-γ group. (n = 4 for each group).

Figure 7. Histological analyses of dental pulp stem cells (DPSCs) seeded on NF-gelatin scaffolds after 4 weeks of ectopic in vivo implantation in immunodeficient mice.

Histology of control cell-scaffold constructs (a,e,i), IFN-γ treated cell-scaffold constructs (b,f,j), cell-scaffold constructs co-stimulated with IFN-γ and PDTC (c,g,k) and cell-scaffold constructs co-stimulated with IFN-γ and SB203580 (d,h,l). H&E staining (a–d), Masson’s trichrome staining (e–h), Van Gieson staining (i–l). Scale bars = 100 μm. Black arrows: the newly formed ECM-like architecture. Red arrows: mineralized tissue consisting of newly formed dentin-like matrix. Quantitative analyses of the newly formed dentin-like matrix areas quantified using Image-Pro Plus 6.0 software (m). *P < 0.05 when compared with the control group. ^#P < 0.05 when compared with the IFN-γ group. (n = 4 for each group).