Human Gingiva-Derived Mesenchymal Stem Cells Ameliorate Streptozoticin-induced T1DM in mice via Suppression of T effector cells and Up-regulating Treg Subsets

Abstract

There is yet no cure for type 1 diabetes (T1DM) so far. A significant body of evidence has demonstrated that bone marrow-derived mesenchymal stem cells (BMSCs) showed great potential in controlling T1DM. But there exists much difficulty in using BMSCs as a clinical therapy. We here test whether a new population of mesenchymal stem cells from human gingiva (GMSCs), which has many advantages over BMSCs, can delay or prevent progress of T1DM. GMSCs were adoptively transferred to multiple low-dose streptozotocin (STZ)-induced T1DM. Blood glucose levels and disease severities were analyzed. T cells subsets in blood, spleen and lymph nodes were detected dynamically by flow cytometry. GMSC distribution was dynamically analyzed. We found that infusion of GMSCs but not fibroblast cells significantly controlled blood glucose levels, delayed diabetes onset, ameliorated pathology scores in pancreas, and down-regulated production of IL-17 and IFN-γ in CD4⁺ and CD8⁺ T cells in spleens, pancreatic lymph nodes (pLN) and other lymph nodes. GMSCs also up-regulated the levels of CD4⁺ Treg induced in the periphery. Mechanismly, GMSCs could migrate to pancreas and local lymph node and function through CD39/CD73 pathway to regulate effector T cells. Thus, GMSCs show a potential promise in treating T1DM in the clinic.

Figure 1

Phenotypic and functional characteristics of GMSCs. GMSCs and fibroblast cells were stained with a series of sufure makers and used for in vitro suppressive assay. (a) Representative flow data showed related phenotypes of GMSC and fibroblast cells. (b) Statistics analysis of each marker expression in two cell types (GMSCs were collected from ten donors and fibroblast cells from commercial resource). (c and d), Representative flow data showed that only GMSCs have suppressive function on mouse T cells even in the ratio of 1:25 (GMSCs or fibroblast cells: CD25⁻ T cells) (c) and statistic analysis (d) Data are presented as the mean ± SEM from 4 independent experiments. ***P < 0.001, **P < 0. 01.

Figure 2

GMSCs ameliorated the development and severity of T1DM in mice. T1DM was induced in male C57BL/6-FoxP3^gfp mice using multiple low doses of STZ injection, 1 × 10⁶ GMSCs or human skin fibroblast cells were injected into mice via intraperitoneal route on days 0, 7, 14, 21, 28. (a) Incidence of diabetes. Blood glucose concentration exceeding 300 mg/dL in two consecutive daily measurements was considered diabetes. (b) Non-fasting blood glucose of mice in three groups. (c) Body weight in different groups. Data are presented as the mean ± SEM from two independent experiments (n = 5). ***P < 0.001, versus the fibroblast or model groups.

Figure 3

GMSCs decreased the severity of insulitis in mice model of T1DM. Pancreas were harvested on day 10, fixed in 10% formalin, embedded in paraffin. Six sections per pancreas, at least three individual pancreases from each group, were evenly sectioned and separated by 200 μm, stained with H&E, anti-insulin and anti-glucogon antibodies. (a) Hematoxylin and eosin staining and immunohistochemistry examination of pancreas from three groups. (b) Insulitis scores of pancreas on day 10. (c,d) Insulin-positive beta cells and glucogon-positive alpha cells in islets area were revealed by immunohistochemistry using the anti-insulin and anti-glucogon antibody on day 10. Data are presented as the mean ± SEM from two independent experiments using at least three individual pancreases from each group. *P < 0.05, **P < 0.01, versus the fibroblast or model groups.

Figure 4

GMSCs down-regulated IL-17 and IFN-γ expression on CD4⁺ and CD8⁺ T cells in STZ-induced T1DM model. T1DM was induced using multiple low dose STZ injection and 1 × 10⁶ GMSCs or fibroblast cells were injected into mice via intraperitoneal route on days 0, 7, 14, 21, 28. pLN was harvested on day 10. Lymphocytes were isolated and then stimulated in vitro with PMA (50 ng/ml) and ionomycin (500 ng/ml) for 5 hours, with brefeldin A (10 μg/ml) added in the last 4 hours, and intracellular expression of IFN-γ and IL-17 on CD4⁺ and CD8⁺ T cells was analyzed by flow cytometry. (a,b) Representative flow data of IFN-γ and IL-17 expression gated on CD4⁺ T cells and CD8⁺ T cells in draining LN (pLN). (c,d) Expression of cytokines, including IFN-γ, IL-17 on CD4⁺ T and CD8⁺ T cells in the draining LNs of T1DM mice. Data are presented as the mean ± SEM from two independent experiments (n = 5). *P < 0.05, **P < 0.01, versus the fibroblast or model groups.

Figure 5

GMSCs up-regulated Treg production in STZ-induced T1DM mice model. Foxp3^gfp reporter C57BL/6 mice were injected with GMSCs (1 × 10⁶). CD4⁺ Foxp3⁺ (GFP⁺) Treg frequency was counted in the spleens and LN on day 30 after GMSC injection. (a.b) Representative flow data of CD4⁺ Foxp3⁺ frequency in day 30 spleen and LN in each groups. (c) statistics analysis of CD4⁺ Foxp3⁺ (GFP⁺) Treg frequency in slpeens and LN on day 30. Data are representative of two separate experiments and mean ± SEM of each group was shown (n = 3). *P < 0.05, ***P < 0.001 versus the control groups. (d) GMSCs increased the frequency of Helios^- Foxp3⁺ and Nrp-1⁻ Foxp3⁺ iTregs in T1DM mice model. Representative flow data of the Helios and Nrp-1 expression in LN. Cells were gated on CD4⁺ GFP⁺ cells.

Figure 6

Intraperitoneal injected GMSCs migrated to pancreas and local lymph nodes. GMSCs were labeled with CFSE before i.p. injection and GMSCs organ distribution and engraftment was then examined at different time points post-injection by flow cytometry. (a) Representative flow data (%) showed that i.p. injected GMSCs could home to mesenteric lymph nodes (MLN) and pancreas lymph node (pLN) in a large percentage and the target organ—pancreas, regardless of a lower percentage at different time points. (b) Statistic chart for (a). Each time point had two mice and mean ± SEM of each time point in different tissues was shown. Experments were repeated twice with the similar results.

Figure 7

GMSCs inhibited the differentiation of mouse CD4⁺ T effector cells through CD39/CD73 signals. (a) Naïve CD4⁺ T cells were cultured without (Ctrl) or with GMSCs (GMSCs:naïve CD4⁺ T cells = 1:10 or 1:20) for 3 days under Th17 or Th1 cell-polarization conditions. Expression of intracellular cytokines (IL-17 and IFN-γ) in each T helper cell subset was analyzed by flow cytometry (representative data shown); (b) statistics analysis for (a); (c and d) GMSCs were pre-treated with solube factors (APCP, POM-1, 1-MT; DMSO or isotype were used as controls) for 24 hours, then GMSCs were washed completely with PBS in well, and suppression of CD4⁺ T cell differentiation was determined (GMSCs:naïve CD4⁺ T cells = 1:20). Bars showed the mean ± SEM of 3 separate experiments. *P < 0.05, **P < 0.01, ***P < 0.001.