The regulation of the Treg/Th17 balance by mesenchymal stem cells in human systemic lupus erythematosus

Abstract

Background and objective: Umbilical cord (UC)-derived mesenchymal stem cells (MSCs) have shown immunoregulation of various immune cells. The aim of this study was to investigate the mechanism of UC MSCs in the regulation of peripheral regulatory T cells (Treg) and T helper 17 (Th17) cells in patients with systemic lupus erythematosus (SLE).

Methods: Thirty patients with active SLE, refractory to conventional therapies, were given UC MSCs infusions. The percentages of peripheral blood CD4+CD25+Foxp3+ regulatory T cells (Treg) and CD3+CD8-IL17A+ Th17 cells and the mean fluorescence intensities (MFI) of Foxp3 and IL-17 were measured at 1 week, 1 month, 3 months, 6 months, and 12 months after MSCs transplantation (MSCT). Serum cytokines, including transforming growth factor beta (TGF-β), tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), and IL-17A were detected using ELISA. Peripheral blood mononuclear cells from patients were collected and co-cultured with UC MSCs at ratios of 1:1, 10:1, and 50:1, respectively, for 72 h to detect the proportions of Treg and Th17 cells and the MFIs of Foxp3 and IL-17 were determined by flow cytometry. The cytokines in the supernatant solution were detected using ELISA. Inhibitors targeting TGF-β, IL-6, indoleamine 2,3-dioxygenase (IDO), and prostaglandin E2 were added to the co-culture system, and the percentages of Treg and Th17 cells were observed.

Results: The percentage of peripheral Treg and Foxp3 MFI increased 1 week, 1 month, and 3 months after UC MSCs transplantation, while the Th17 proportion and MFI of IL-17 decreased 3 months, 6 months, and 12 months after the treatment, along with an increase in serum TGF-β at 1 week, 3 months, and 12 months and a decrease in serum TNF-α beginning at 1 week. There were no alterations in serums IL-6 and IL-17A before or after MSCT. In vitro studies showed that the UC MSCs dose-dependently up-regulated peripheral Treg proportion in SLE patients, which was not depended on cell-cell contact. However, the down-regulation of Th17 cells was not dose-dependently and also not depended on cell-cell contact. Supernatant TGF-β and IL-6 levels significantly increased, TNF-α significantly decreased, but IL-17A had no change after the co-culture. The addition of anti-TGF-β antibody significantly abrogated the up-regulation of Treg, and the addition of PGE2 inhibitor significantly abrogated the down-regulation of Th17 cells. Both anti-IL-6 antibody and IDO inhibitor had no effects on Treg and Th17 cells.

Conclusions: UC MSCs up-regulate Treg and down-regulate Th17 cells through the regulation of TGF-β and PGE2 in lupus patients.

Figures

Figure 1

UC MSCs up-regulate Treg and down-regulate Th17 cell in lupus patients in vivo. Thirty refractory lupus patients were given UC MSCs transplantation. The peripheral CD4+CD25+Foxp3+ Treg percentage (a), the mean fluorescence of Foxp3 (b), the CD3+CD8-IL-17A+ Th17 cell percentage (c), and the mean fluorescence of IL-17 (d) were assessed by flow cytometry at different visit times. The serum levels of TGF-β (e), TNF-α (f), IL-6 (g), and IL-17A (h) were determined using ELISA. cDNA was synthesized from the PBMC of patients pre- and post-MSCT, and the relative mRNA expression of RORC, IL-6, TGF-β, Foxp3, and IL-23R was assessed by real-time PCR. PBMCs from healthy individuals were used as the control (I). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2

UC MSCs up-regulate Treg and down-regulate Th17 cell in lupus patients in vitro. PBMCs from lupus patients were co-cultured with UC MSCs at different ratios (50:1, 10:1, 1:1); 72 h later, the percentages of CD4+CD25+Foxp3+ Treg (a) and CD4+IL17A+ Th17 (b) were assessed by flow cytometry. The culture supernatant levels of TGF-β (c), IL-6 (d), TNF-α (e), and IL-17A (f) were determined using ELISA. In the co-cultures of lupus PBMC and UC MSCs (10:1), transwells were used, and 72 h later, the percentages of CD4+CD25+Foxp3+ Treg (g) and CD4+IL17A+ Th17 (h) were assessed by flow cytometry. *P < 0.05, **P < 0.01, ***P < 0.001, n.s., no significance.

Figure 3

SLE patients' PBMC and sera enhance UC MSCs to produce functional factors. PBMC from active lupus patients (sPBMC) and healthy individuals (hPBMC) were co-cultured with UC MSCs (10:1); 72 h later, the culture supernatants were collected and the levels of TGF-β (a), IL-6 (b), and PGE2 (c) were assessed by ELISA. The culture supernatant kynurenine levels were assessed by HPLC (d). Sera from healthy controls and active SLE patients were collected and used to stimulate UC MSCs in vitro at a concentration of 10%. FBS was used as control. After 48 h, the mRNA levels of TGF-β (e), COX-2 (f), IL-6 (g), and IDO (h) were analyzed by real-time PCR. *P < 0.05, **P < 0.01.

Figure 4

TGF-β mediates the up-regulation on Treg cells in lupus patients. In the co-culture of lupus patients PBMC and UC MSCs (10:1), an anti-TGF-β antibody and an anti-IL6 antibody, alone or in combination, was added; 72 h later, the percentages of CD4+CD25+Foxp3+ Treg (a), CD4+IL17A+ Th17 (c), and the MFI of Foxp3 (b) were analyzed by flow cytometry. TGF-β siRNA-UC MSCs were used to down-regulate TGF-β expression on UC MSCs (d), and the percentages of CD4+CD25+Foxp3+ Treg (e) and CD4+IL17A+ Th17 (f) were assessed by flow cytometry. *P < 0.05, **P < 0.01.

Figure 5

PGE2 mediates the down-regulation on Th17 cell in lupus patients. In the co-culture of lupus patients PBMC and UC MSCs (10:1), a PGE2 inhibitor (CAY10404) and an IDO inhibitor (1-MT), alone or in combination, was added; 72 h later, the percentage of CD4+IL17A+Th17 (a), MFI of IL17 (b) and the percentage of CD4+CD25+Foxp3+ Treg (c) were analyzed by flow cytometry. *P < 0.05, **P < 0.01.