IL-17 down-regulates the immunosuppressive capacity of olfactory ecto-mesenchymal stem cells in murine collagen-induced arthritis

Abstract

Olfactory ecto-mesenchymal stem cells (OE-MSCs) are a population of cells which has been recognized as a new resident stem cell type in the olfactory lamina propria. OE-MSCs have been shown to exert their immunosuppressive capacity by modulating T cell responses, including up-regulation of regulatory T cells (Tregs) and down-regulation of Th1/Th17 cells. As an inflammatory cytokine, IL-17 plays a critical role in orchestrating the inflammatory response during the development of collagen-induced arthritis (CIA). However, it is unclear whether the increased level of IL-17 may affect the immunosuppressive function of OE-MSCs under inflammatory condition. In this study, we found that IL-17 could significantly reduce the suppressive capacity of OE-MSCs on CD4+ T cells and down-regulate the suppressive factors produced by OE-MSCs. Notably, IL-17 treatment abolished the capacity of OE-MSCs in inducing Treg expansion. In addition, knockdown of IL-17R in OE-MSCs significantly enhanced their therapeutic effect in ameliorating CIA upon adoptive transfer. Moreover, IL-17R knockdown-OE-MSCs could efficiently induce Tregs expansion and reduce Th1 and Th17 responses. Taken together, all these data suggest that IL-17R knockdown in OE-MSCs may provide a novel strategy in maintaining their immunosuppressive properties for the treatment of autoimmune diseases.

Keywords: IL-17; Immune response; Immunity; Immunology and Microbiology Section; Treg; collagen-induced arthritis; olfactory ecto-mesenchymal stem cells; suppressive capacity.

Figure 1. IL-17 reduces the suppressive capacity of OE-MSCs

A. OE-MSCs were stained for IL-17R with anti-IL-17R antibody (thick line histogram) or rat IgG2a (solid gray histogram) and then analyzed using flow cytometry. B. RNA isolated from OE-MSCs was subjected to RT-PCR to measure IL-17R mRNA expression. C., D. OE-MSCs were stimulated with or without IL-17 (0, 10, 20, 50 ng/ml) for 48 h, and then cells were harvested to co-cultured with CD4⁺ T cells in the presence of anti-CD3 mAb and anti-CD28 mAb for 72 h. Suppression of T-cell proliferation was measured by [H]-thymidine incorporation (C), and the status of cells in the co-culture system was also observed under the microscope (100×, D). Data are presented as mean ± SD pooled from three independent experiments. ***p < 0.001, **p < 0.01.

Figure 2. IL-17 down-regulates the suppressive factors of OE-MSCs

A., B. OE-MSCs were stimulated with or without IL-17 (20 ng/ml) for 48 h, and then cells were harvested to co-culture with CD4⁺ T cells in the presence of anti-CD3 mAb and anti-CD28 mAb for 72 h, (A) and then OE-MSCs were collected to extract total RNA, and qRT-PCR was used to analyze the mRNA expression of PD-L1, iNOS, IL-10 and TGF-β, (B) and the supernatant was harvested to detect NO, IL-10 and TGF-β. Data are presented as mean ± SD pooled from three independent experiments. **p < 0.01, *p < 0.05.

Figure 3. IL-17 reduces the capacity of OE-MSCs in inducing the expansion of Tregs

OE-MSCs were treated with or without IL-17 (20 ng/ml) for 48 h, and then cells were harvested to co-culture with CD4⁺ T cells in the presence of anti-CD3 mAb and anti-CD28 mAb for 72 h, and then the proportion of CD4⁺CD25⁺Foxp3⁺ Treg cells in the co-culture system was analyzed by flow cytometry. Data are presented as mean ± SD pooled from three independent experiments. **p < 0.01, N.S represents no significance.

Figure 4. IL-17R knockdown OE-MSCs efficiently ameliorates the development of CIA

A. Graphic scheme of CIA induction and OE-MSCs administration. DBA/1J mice were immunized with CII/CFA on day 0 and boosted with CII/IFA on day 21. Treatment groups were intravenously injected with OE-MSCs (1×10⁶) transfected with IL-17R siRNA or negative control on day 27 after CII/CFA immunization. Mice were then sacrificed on day 42. B. IL-17 levels on day 0, day 14, day 27 and day 42 during the CIA development were detected by ELISA. C., D. Clinical score (C) and incidence of arthritis development (D) in immunized mice treated with OE-MSCs transfected with IL-17R siRNA or negative control were monitored every 3 days (n=6 per group). E. Serum levels of CII-specific autoantibodies from OE-MSCs transfected with IL-17R siRNA or negative control were detected by ELISA. F. Photos of hind paws from CIA mice treated with OE-MSCs transfected with IL-17R siRNA or negative control. G. Representative section of hind paws stained with hematoxylin and eosin, then assessed for histopathological scores of joint tissue from three groups. Results are expressed as mean ± SD. **p < 0.01,*p < 0.05, N.S represents no significance.

Figure 5. IL-17R knockdown OE-MSCs enhances Treg cells and reduces Th1 and Th17 cell responses in collagen-induced arthritis

A., B., C. CD4⁺CD25⁺Foxp3⁺ Treg cells (A), CD4⁺IFN-γ⁺ Th1 (B) and CD4⁺ IL-17A⁺ Th17 (C) in the draining lymph nodes of CIA mice treated with OE-MSCs transfected with IL-17R siRNA or negative controls (n = 3/group). D., E. Serum levels of IFN-γ (D) and IL-17 (E) in CIA mice in these three groups were measured by ELISA. Results are expressed as mean ± SD. **p < 0.01,*p < 0.05. N.S represents no significance.