Human Adipose Tissue-Derived Mesenchymal Stem Cells in Parkinson's Disease: Inhibition of T Helper 17 Cell Differentiation and Regulation of Immune Balance Towards a Regulatory T Cell Phenotype

Abstract

Background: Parkinson's disease (PD) is a neurodegenerative disorder displaying a typical neuroinflammation pathology that may result from an imbalance between regulatory T cells (Treg) and T helper 17 (Th17) cells. Human adipose tissue-derived mesenchymal stem cells (Ad-MSCs) exert immunomodulatory effects by inhibiting effector T cell responses and have been used to treat diverse immune disorders. We aimed to investigate the modulating effect of human Ad-MSCs on peripheral blood mononuclear cells (PBMCs) of patients with PD, focusing on differentiation into Th17 and Treg cells.

Methods: We isolated human peripheral blood CD4⁺T cells and co-cultured them with Ad-MSCs at a ratio of 4:1 under either Th17 or Treg cell polarizing conditions for 4 days to detect the proportions of IL-17-producing CD4⁺T (Th17) and CD4⁺CD25⁺Foxp3⁺regulatory T (Treg) cells by flow cytometry. We also determined the mRNA expression levels of the retinoid-related orphan nuclear receptor (RORγt) transcription factor and those of interleukin-6 receptor (IL-6R), interleukin-23 receptor (IL-23R), leukemia inhibitory factor (LIF), and LIF receptor (LIFR) by quantitative reverse transcription PCR. We detected levels of cytokines in the supernatant (including LIF, IL-6, IL-23, IL-10, and TGF-β) using ELISA.

Results: Our results showed that Ad-MSCs specifically inhibited the differentiation of PBMCs of patients with PD into IL-17-producing CD4⁺T cells by decreasing expressions of IL-6R, IL-23R, and RORγt (the key transcription factor for Th17 cells). Moreover, Ad-MSCs induced a functional CD4⁺CD25⁺Foxp3⁺T regulatory cell phenotype as evidenced by the secretion of IL-10. The levels of IL-6, IL-23, and TGF-β remained constant after co-culture under either the Th17 or the Treg cell polarizing condition. In addition, levels of LIF protein and its receptor mRNA were significantly increased under both polarizing conditions.

Conclusion: The present in vitro study found that Ad-MSCs from healthy participants were able to correct the imbalance between Th17 and Treg found in PBMCs of PD patients, which were correlated with an increase in LIF secretion and a decrease in expression of IL-6R, IL-23R, and RORγt. These findings should be confirmed by in vivo experiments.

Keywords: CD4+T cell; Parkinson’s disease; T helper 17 cell; T regulatory cell; adipose-derived mesenchymal stem cells; leukemia inhibitory factor; peripheral blood mononuclear cells.

Figure 1

Ad-MSCs inhibited the differentiation of CD4⁺T cells into Th17 cells. Percentage of Th17 (IL-17A⁺CD4⁺) cells analyzed by flow cytometry (A, B). After 4-day- co-culture with Ad-MSCs, the level of RORγt mRNA was analyzed using real-time PCR (C). *p<0.05.

Figure 2

Ad-MSCs suppressed the expression of cytokines and their receptors. Low levels of expression of IL-6R and IL-23R mRNAs in co-cultures under the Th17 polarized condition (A, B), and low levels of IL-6R mRNA under the Treg polarized condition (C, n=3). Levels of IL-6, IL-23, and TGF-β proteins showed similar results in CD4⁺T cell co-cultures under either of the polarized conditions (D–F). *p<0.05.

Figure 3

hAd-MSCs induced CD4+T cell differentiation into functional Treg cells. The proportion of Treg (CD4⁺CD25⁺ Foxp3⁺T) cells was analyzed by flow cytometry (A; B, n=4). Increased levels of IL-10 in the supernatants of co-cultured systems as tested by ELISA (C). *p<0.05.

Figure 4

LIF is the key inhibitory factor. The levels of LIF protein were significantly upregulated in the supernatant of the co-cultured systems under either of the polarizing conditions tested by ELISA (A, n=5; B, n=4), real-time PCR results showed increased expression of LIF mRNA (C, n=4; D, n=4). We found increased expressions of LIF-R mRNA by real-time PCR in co-cultures under both polarizing conditions (E; F, n=4). *p<0.05.