The allogeneic umbilical cord mesenchymal stem cells regulate the function of T helper 17 cells from patients with rheumatoid arthritis in an in vitro co-culture system

Abstract

Background: Previous in vivo studies have shown that mesenchymal stem cell (MSC) transplantation significantly improves the condition of a number of autoimmune diseases including autoimmune cerebrospinal meningitis, multiple sclerosis, glomerulonephritis and systemic lupus erythematosus.

Methods: To investigate the immunoregulatory effect of stem cell transplantation, human umbilical cord MSCs were co-cultured with peripheral blood mononuclear cells (PBMCs) from patients with rheumatoid arthritis (RA). Orphan nuclear receptor gamma (ROR-γ) mRNA and protein expression was detected with real-time PCR and Western blotting. Interleukin (IL)-17, IL-6 and tumor necrosis factor (TNF-α) in the cell culture supernatant were measured using a flow cytometric bead capture method.

Results: After 72 hours of co-culture, the mRNA and protein expression levels of ROR-γ in co-cultured PBMCs were decreased compared with that in PBMC of RA patients cultured alone (p < 0.05). Moreover, the decrement was positively related to the disease activity of RA (p < 0.05). Decreased secretion of IL-17, TNF-α and IL-6 were also found in co-culture supernatants of PBMCs from patients with severe and moderate disease activity, but not in supernatant from PBMCs cultured alone. The decreased cytokine expression levels were positively correlated to the concentrations of MSCs. In contrast, PBMCs from healthy controls or patients with mild RA did not show significant differences in ROR-γ expression or cytokine secretion following co-culture with MSCs as compared with those cultured alone.

Conclusions: In vitro co-culture with MSCs down-regulated the inflammatory response of PBMCs from RA patients with severe disease activity, but had no significant effect on PBMCs from healthy controls or patients with mild disease activity, suggesting that the immunoregulatory role of MSCs may associate with the occurrence of inflammatory mediators.

Figure 1

Identification of the isolated human umbilical cord MSCs. MSCs (3rd passage) exhibited negative for CD45 and CD34, weak positive for CD71 and positive for CD105, CD44 and CD29 as analyzed by flow cytometry.

Figure 2

Expression of ROR-γ. a, mRNA expression of ROR-γ; b, Protein expression of ROR-γ in different groups after co-culture as measured by Western blotting. β-actin was used as control; c, ROR-γ expression levels in groups of MSCs: PBMC mixed ratio at 1:1 group (n = 6). *: P < 0.05, vs. 1:1 within the expression level.

Figure 3

Cytokine IL-17 in PBMC. Cytokine TNF-α, Cytokine IL-6 expression levels in different mixed cell culture (n = 6). a, Cytokine IL-17 in PBMC (pg/mL); b, Cytokine TNF-α (pg/mL); c, Cytokine IL-6 (pg/mL).