Shear Stress Enhances the Paracrine-Mediated Immunoregulatory Function of Human Periodontal Ligament Stem Cells via the ERK Signalling Pathway

Abstract

Relevant immunomodulatory effects have been proposed following allogeneic cell-based therapy with human periodontal ligament stem cells (hPDLSCs). This study aimed to examine the influence of shear stress on the immunosuppressive capacity of hPDLSCs. Cells were subjected to shear stress at different magnitudes (0.5, 5 and 10 dyn/cm2). The expression of immunosuppressive markers was evaluated in shear stress-induced hPDLSCs using qRT-PCR, western blot, enzyme activity and enzyme-linked immunosorbent assays. The effects of a shear stress-derived condition medium (SS-CM) on T cell proliferation were examined using a resazurin assay. Treg differentiation was investigated using qRT-PCR and flow cytometry analysis. Our results revealed that shear stress increased mRNA expression of IDO and COX2 but not TGF-β1 and IFN-γ. IDO activity, kynurenine and active TGF-β1 increased in SS-CM when compared to the non-shear stress-derived conditioned medium (CTL-CM). The amount of kynurenine in SS-CM was reduced in the presence of cycloheximide and ERK inhibitor. Subsequently, T cell proliferation decreased in SS-CM compared to CTL-CM. Treg differentiation was promoted in SS-CM, indicated by FOXP3, IL-10 expression and CD4+CD25hiCD127lo/- subpopulation. In conclusion, shear stress promotes kynurenine production through ERK signalling in hPDLSC, leading to the inhibition of T cell proliferation and the promotion of Treg cell differentiation.

Keywords: IDO; T cells; TGF-β1; immunosuppression; mechanotransduction; periodontal ligament stem cells; shear stress.

Figure 1

The mRNA expression of immunosuppressive regulators in shear stress-induced hPDLSCs. After shear stress stimulation, the relative mRNA expression of IDO (A), TGF-β1 (B), IFN-γ (C) and COX2 (D) was detected using qRT-PCR. * p < 0.05 vs. control group.

Figure 2

Protein expression of immunosuppressive regulators in shear stress-induced hPDLSCs. Shear stress promoted IDO activity (A) and the kynurenine product (B) in hPDLSCs. Shear stress increased the secretion of active TGF-$\beta$1 in PDLSC-derived conditioned medium (C). TGF-$\beta$1 protein expression in hPDLSCs was determined by Western blot analysis (D). The quantitative analysis of Western blot band intensity of latent TGF-$\beta$1(E) and active TGF-$\beta$1 (F). The concentration of IFN-$\gamma$ in cell lysate (G) and conditioned medium (H) and the amount of COX2-independent PGE2 were determined by ELISA assay (I). * p < 0.05 vs. control group.

Figure 3

Shear stress enhanced IDO expression and IDO-catabolised kynurenine product in hPDLSCs via ERK1/2 signalling pathway. After shear stress stimulation (at 5 dyn/cm${}^2$), hPDLSCs treated with CHX were examined for IDO mRNA expression and kynurenine product (A,B). The activity of ERK1/2 was examined by Western blot analysis (C). The quantitative analysis of Western blot band intensity showed that ERK inhibitor attenuated P-ERK1/2 (D), but not ERK1/2 expression in shear stress-induced hPDLSCs (E). The addition of an ERK inhibitor inhibits the effect of shear stress-induced kynurenine secretion (F). However, shear stress did not affect TGF-$\beta$1 secretion (G). * p < 0.05 vs. non-shear stress without ERK inhibitor group. # p < 0.05 vs. control.

Figure 4

Shear stress attenuated CD4${}^{+}$ T cell proliferation and promoted Treg differentiation. All hPDLSC-derived conditioned media were indirectly co-cultured with CD4${}^{+}$ T cells. The proliferation of CD4${}^{+}$ T cells was assessed using a resazurin assay. SS-CM decreased the percentage of T cell proliferation (A). The mRNA expressions of FOXP3 (B) and IL-10 (C) were upregulated in SS-CM-treated CD4${}^{+}$ T cells. The flow cytometry analysis showed an increase in the percentage of CD4${}^{+}$CD25${}^{hi}$CD127${}^{lo/-}$ Treg population in SS-CM compared to CTL-CM and activated T cells (D,E). $ p < 0.05 vs. T cell alone group, * p < 0.05 vs. activated T cells group, # p < 0.05 vs. CTL-CM group.

Figure 5

Schematic diagram of shear stress activates an immunosuppressive ability of hPDLSCs via ERK-induced IDO and total and active TGF-$\beta$1. Shear stress enhances IDO-dependent kynurenine in hPDLSCs via ERK1/2 activation and increases total and active TGF-$\beta$1 in the extracellular matrix or conditioned medium. Increased kynurenine and TGF-$\beta$1 secretion inhibit the CD4${}^{+}$ T cell proliferation and promote Treg cell differentiation. Created with Biorender.com.