Sitagliptin Induces Tolerogenic Human Dendritic Cells

Abstract

Sitagliptin, an anti-diabetic drug, is a dipeptidyl peptidase (DPP)-4/CD26 inhibitor with additional anti-inflammatory and immunomodulatory properties. In this study, we investigated for the first time the effect of sitagliptin on the differentiation and functions of human dendritic cells generated from monocytes (MoDCs) for 4 days using the standard GM-CSF/IL-4 procedure. LPS/IFN-γ treatment for an additional 24 h was used for maturation induction of MoDCs. Sitagliptin was added at the highest non-cytotoxic concentration (500 µg/mL) either at the beginning (sita 0d protocol) or after MoDC differentiation (sita 4d protocol). Sitagliptin impaired differentiation and maturation of MoDCs as judged with the lower expression of CD40, CD83, CD86, NLRP3, and HLA-DR, retention of CD14 expression, and inhibited production of IL-β, IL-12p70, IL-23, and IL-27. In contrast, the expression of CD26, tolerogenic DC markers (ILT4 and IDO1), and production of immunoregulatory cytokines (IL-10 and TGF-β) were increased. Generally, the sita 0d protocol was more efficient. Sitagliptin-treated MoDCs were poorer allostimulators of T-cells in MoDC/T-cell co-culture and inhibited Th1 and Th17 but augmented Th2 and Treg responses. Tolerogenic properties of sitagliptin-treated MoDCs were additionally confirmed by an increased frequency of CD4+CD25+CD127- FoxP3+ Tregs and Tr1 cells (CD4+IL-10+FoxP3-) in MoDC/T-cell co-culture. The differentiation of IL-10+ and TGF-β+ Tregs depended on the sitagliptin protocol used. A Western blot analysis showed that sitagliptin inhibited p65 expression of NF-kB and p38MAPK during the maturation of MoDCs. In conclusion, sitagliptin induces differentiation of tolerogenic DCs, and the effect is important when considering sitagliptin for treating autoimmune diseases and allotransplant rejection.

Keywords: CD26 expression; dendritic cells; dipeptidyl peptidase 4 inhibitors; regulatory T cells; tolerance.

Figure 1

The effect of different concentrations of sitagliptin on the metabolic activity (A) and apoptosis/necrosis (B,C) in immature MoDC cultures. Immature (Im) MoDCs were generated by incubating human monocytes for 5 days in culture medium supplemented with Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF/IL-4), as described in the Materials and Methods. ImMoDCs were treated with double-increasing concentrations of sitagliptin starting from 31.25 to 1000 µg/mL for 24 h. Cytotoxicity was evaluated using MTT and apoptosis/necrosis assays. Values are given as mean ± SD (n = 3). * p < 0.05, ** p < 0.01 compared with corresponding controls (non-treated imMoDCs). (B) Plots of apoptosis/necrosis of one representative experiment.

Figure 2

The effect of sitagliptin (500 µg/mL) on the expression of CD1a and CD14 on imMoDCs. Sitagliptin was applied at the beginning of Mo differentiation (sita 0d) or after imMoDC differentiation (sita 4d). CD1a/CD14 expression was analyzed on day 5 of cell cultures. (A) Plots of CD1a/CD14 expression of one representative experiment. (B) Results are presented as mean ± SD (n = 3). *** p < 0.005 compared with corresponding controls (non-treated imMoDCs).

Figure 3

The effect of sitagliptin on the expression of different markers by im- and mMoDCs. Sitagliptin was applied at the beginning of Mo differentiation (sita 0d) or after imMoDC differentiation (sita 4d) at the concentration of 500 µg/mL. The plots are for one representative experiment.

Figure 4

The effect of sitagliptin on the expression of different markers by im- and mMoDCs. Sitagliptin was applied at the beginning of Mo differentiation (sita 0d) or after imMoDC differentiation (sita 4d) at the concentration of 500 µg/mL. Results are presented as mean ± SD (n = 3). * p < 0.05; ** p < 0.01; *** p < 0.005 compared with corresponding controls (non-treated MoDCs).

Figure 5

The effect of sitagliptin on the expression of CD26 by MoDCs. Sitagliptin (500 µg/mL) was applied at the beginning of Mo differentiation (sita 0d) or after imMoDC differentiation (sita 4d). CD26/HLA-DR expression was analyzed on day 5 of cell cultures. (A) The plots of marker expression by im- and mMoDCs for one representative experiment are shown. (B) The results are presented as mean ± SD (n = 3). * p < 0.05; ** p < 0.01; compared with corresponding controls (non-treated imMoDCs).

Figure 6

The effect of sitagliptin (500 µg/mL) on cytokine production by MoDCs and their allostimulatory capacity. Day-4 imMoDCs were induced to mature for the next 24 h, as described in the Materials and Methods. The levels of cytokine in supernatants, standardized to an equal number of MoDCs (5 × 10⁵) of both im- and mMoDCs (A) were analyzed on day 5 of cell cultivation. Day-5 im- and mMoDCs were co-cultivated with purified CellTrace Far Red-stained T cells for 4 days, and after that, the proliferation was analyzed using flow cytometry (B,C). 7-Aminoactinomycin D (7-AAD) was used to detect viable cells. Results are presented as mean ± SD (n = 3). * p < 0.05; ** p < 0.01; compared with corresponding controls (non-treated MoDCs). (B) Plots of allogeneic T-cell proliferation of one representative experiment.

Figure 7

The effect of MoDCs treated with sitagliptin (500 µg/mL) on their Th polarization capability. ImMoDCs and mMoDCs were co-cultivated with purified allogeneic T cells for 4 days. (A) The levels of Th cytokines were determined in culture supernatants (standardized to an equal number of cells (1 × 10⁵ T cells) and intracellularly (B,C), as described in the Materials and Methods. Results are presented as mean ± SD (n = 3). * p < 0.05; ** p < 0.01; compared with corresponding controls (non-treated MoDCs). (B) shows the plots of Th cytokine expression of one representative experiment.

Figure 8

The expression of tolerogenic markers by control and sitagliptin-treated MoDCs. Sitagliptin was used at a concentration of 500 µg/mL. The expression of ILT3, ILT4, and IDO-1 was analyzed on day 5 MoDCs. Results are presented as mean ± SD (n = 3). * p < 0.05; ** p < 0.01; compared with corresponding controls (non-treated MoDCs).

Figure 9

Tolerogenic properties of MoDCs treated with sitagliptin (500 µg/mL). ImMoDCs and mMoDCs were co-cultivated with purified allogeneic T cells for 4 days. Tregs were identified as FoxP3+ cells in CD4+CD25+CD127- cells. The numbers of cells marked in red on flow cytometric plots (middle vertical rows) represent the percentage of CD25+CD127-FoxP3+ cells, whereas the numbers marked in black on the same histograms represent the percentages of Tregs within total CD4+ T cells. TGF-β+ Tregs were identified within CD4+CD25+CD127-FoxP3+ cells and expressed as %. The plots and histograms are from one of three different experiments.

Figure 10

The subsets of Tregs and levels of tolerogenic cytokines (TGF-β and IL-10) in co-culture supernatants. Control MoDCs and MoDCs treated with sitagliptin (500 µg/mL) were co-cultured with purified allogeneic T cells, as described in the Materials and Methods. (A) Tregs were identified as FoxP3+ cells in CD4+CD25+CD127- cells and presented in %. The percentages of TGF-β+ Tregs were expressed as relative values (%) of the cells calculated on the basis of the number of total Tregs used as 100%. (B) The levels of IL-10 and TGF-β were determined in MoDC/T-cell co-culture supernatants and standardized to an equal number of cells (1 × 10⁵ T cells). Results are presented as mean ± SD (n = 3). * p < 0.05; ** p < 0.01; compared with corresponding controls (non-treated MoDCs).

Figure 11

The effect of MoDCs treated with sitagliptin (500 µg/mL) on the induction of Tr1 cells. Tr1 cells were identified as CD4+Foxp3-IL-10+ T cells within the IFN-γ-IL-4-CD4+ T-cell population. (A) Plots of one experiment are shown. (B) The results are presented as mean ± SD (n = 3). * p < 0.05 compared with corresponding controls (non-treated MoDCs).

Figure 12

The effect of sitagliptin (500 µg/mL) on the activation of p65 NF-kB and p38 MAPK in MoDCs. The activation of p65 NF-kB and p38 MAPK was analyzed using Western blot by the phosphorylated (p) and total forms of these molecules, as described in the Materials and Methods. Values are from two different experiments (A); supported by the original blots (B). * p < 0.05; ** p < 0.01 compared with corresponding controls as indicated by bars. GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) was used as a loading control.