Dipeptidyl peptidase 4 (DPP4, also known as CD26) is a homodimer protein existing in the soluble form in the blood or in the membrane form on the surface of various cell types, including T cells and endothelial cells (Ohnuma
DPP4/CD26 has also been reported to play important roles in immune cells, including T helper 1 (Th1) cells (Ohnuma
Although diverse DPP4 inhibitors have been extensively studied as synthetic therapeutic drugs to suppress DPP4 activity in the treatment of type 2 diabetes, little is known about their effects on other DPP4-related diseases including cardiovascular disorders, and autoimmune diseases such as type 1 diabetes, rheumatoid arthritis, Grave’s disease, allograft rejection, inflammatory bowel disease, multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE) (Yazbeck
This study aimed to elucidate whether mDPP4 enzymatic activity is essential for T cell signaling pathways for cytokine expression and whether DPP4 inhibition by therapeutic drugs affects the biologic profiles of T cells. We investigated the effects of the antidiabetic drugs evogliptin and sitagliptin in activated CD4+CD26+ Th1 cells in terms of mDPP4 activity, cytokine expression, cell profiles, and cell surface marker profiles.
The human Th1 cell lines CD4+CD26–Jurkat E6 and CD4+CD26+H9 T cells were obtained from the Korean Type Culture Collection (KTCC, Seoul, Korea). The cells were cultured in RPMI 1640 medium (Cat. LM011-01; WELGENE Inc., Gyeongsan, Korea) supplemented with 10% fetal bovine serum (FBS; Ref. 26140-079, GibcoTM, PA, USA) and 1% penicillin–streptomycin (Ref. 15140-0122, GibcoTM) at 37°C under a 5% CO2 atmosphere.
The T lymphocytes were stimulated with various agents, including pokeweed mitogen (PWM; Cat. L9379, Sigma-Aldrich Co., MO, USA) as a primary activator, phorbol 12-myristate 13-acetate (PMA; Cat. P1585, Sigma-Aldrich Co.), and Dynabeads human T-activator CD3/CD28 (Cat. 111.31D, Invitrogen, MA, USA) used as the positive control to activate the T lymphocytes. Evogliptin (Suganon; Dong-A ST, Seoul, Korea) and sitagliptin (Januvia; Merck & Co., Inc., NJ, USA) were used as the synthetic therapeutic DPP-4 inhibitors. Diprotin A (Cat. 416200, Calbiochem, MO, USA) as a peptidyl peptide inhibitor and berberine (Cat. 141433-60-5, Sigma-Aldrich Co.), a natural compound extracted from
The cell pellets were washed and suspended in ice-cold DPBS (Cat. 14190-144, GibcoTM); subsequently, the cell count and viability were immediately measured. The cells were stained using an Accustain 4X kit (Cat. AD4K-200, NanoEnTek Inc., Seoul, Korea) in a 1:1 ratio for counting total cells and non-viable cells, and 20 µL of the sample mixture was loaded onto the Accuchip. The cell number and viability were measured using an Adam MC automatic cell counter (NanoEnTek Inc.).
At the end of each incubation, the cell supernatant was harvested and stored at –20°C until cytokine quantification. The cell supernatant was analyzed for 18 cytokines, including IL-1β (Cat. 171B5001M), IL-2 (Cat. 171B5003M), IL-4 (Cat. 171B5004M), IL-5 (Cat. 171B5005M), IL-6 (Cat. 171B5006M), IL-7 (Cat. 171B5007M), IL-8 (Cat. 171B5008M), IL-10 (Cat. 171B5010M), IL-12p70 (Cat. 171B5011M), IL-13 (Cat. 171B5012M), IL-15 (Cat. 171B5013M), and IL-17 (Cat. 171B5014M); granulocyte-macrophage colony-stimulating factor (GM-CSF; Cat. 171B5018M); interferon-gamma (IFN-γ; Cat. 171B5019M); tumor necrosis factor-alpha (TNF-α; Cat. 171B5026M); basic fibroblast growth factor (bFGF; Cat. 171B5016M); vascular endothelial growth factor (VEGF; Cat. 171B5027M); and platelet-derived growth factor (PDGF-BB; Cat. 171B5024M) using a Bio-Plex Pro assay (BIO-RAD Laboratories, Inc., CA, USA), and for 6 matrix metalloproteinases (MMPs), including MMP-1 (Cat. LMP901B), MMP-2 (Cat. LMP902C), MMP-3 (Cat. LMP513B), MMP-8 (Cat. LMP908B), MMP-9 (Cat. LMP911B), and MMP-12 (Cat. LMP919B) using a Luminex® performance assay (R&D Systems, Inc., MN, USA). The Luminex® performance assay is a multiplex immunoassay based on xMAP technology developed by LuminexTM that uses analyte-specific capture antibodies coupled to different magnetic microspheres with a distinct color code. After a series of reactions, the microspheres react with the biotinylated detection antibody and streptavidin–phycoerythrin as a fluorescent reporter. The microspheres were read using the flow-based dual laser system (635 and 532 nm) of the Bio-Plex 200 system (BIO-RAD Laboratories, Inc.) to determine the fluorescence intensity (FI) of each cytokine. Standard curves were fitted to determine the concentration of each cytokine in every batch. The measurement was performed in three different batches to confirm that the results were reproducible and the coefficient of variation (CV) was under 25%.
After the cell pellets were washed with DPBS, the DPP4 enzymatic activity of the cells was measured kinetically using substrate buffers containing 100 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid,
The cell surface markers were analyzed by harvesting a million cultured cells and washing with phosphate-buffered saline (PBS). The washed cells were resuspended with 100 µL of DPBS and stained with 5 µL of fluorescently labeled monoclonal antibodies in the dark for 20 min at room temperature. The antibodies used were mouse anti-human CD3-eFluor 450 (Cat. 48-0037-41, clone OKT3, eBioscienceTM, CA, USA), mouse anti-human CD4-PerCP-Cyanine5.5 (Cat. 45-0049-41, clone RPA-T4, eBioscienceTM), mouse anti-human CD8-FITC 5 (Cat. 11-0087-41, clone SK1, eBioscienceTM), mouse anti-human CD26-PE (Cat. 12-0269-41, clone 2A6, eBioscienceTM), and mouse anti-human CD28-APC (Cat. 17-0289-41, clone CD28.2, eBioscienceTM). The labeled cells were washed twice with DPBS and suspended in 200 µL of DPBS. CD3 was analyzed at Ex 405 nm/Em 445 nm; CD4, Ex 488 nm/Em 695 nm; CD8a, Ex 488 nm/Em 520 nm; CD26, Ex 488 nm/Em 578 nm; and CD28, Ex 633 nm/Em 660 nm using the BD FACSCantoTM system (BD Biosciences, CA, USA) and FACSDiva software (BD Biosciences).
Cell cycle progression, including DNA synthesis, was detected by harvesting the cultured cells and washing them twice with PBS. The cells were fixed with ice-cold 70% ethanol at –20°C for 2 h. After the cells were washed with PBS, they were stained with a DNA-staining solution containing propidium iodide (PI; Cat. P4170, Sigma-Aldrich Co.), RNase (Cat. R6513, Sigma-Aldrich Co.), and triton X-100 (Cat. 108603, Merck & Co., Inc.) in the dark for 20 min at room temperature. Cell cycle progression was analyzed at an Ex 488 nm/Em 585 nm using the BD FACSCaliburTM system (BD Biosciences) and Cell Quest Pro program; next, DNA synthesis (S phase) was determined using the ModFit LT 3.0 DNA analysis program (Verity Software House, ME, USA). For the apoptosis analysis, a commercially available Annexin V Apoptosis Detection Kit FITC (Cat. 88-8005, eBioscienceTM) was used, which contains recombinant annexin V conjugated to fluorescein (FITC annexin V) and a solution of the red-fluorescent PI, a nucleic acid-binding dye. The cultured cells were washed once with cold PBS and then with annexin-binding buffer. The cells were resuspended in 100 µL of annexin-binding buffer, following which 5 µL of annexin V-FITC was added; then, the mixture was incubated for 10 min in the dark at room temperature. After incubation, the cells were washed with annexin-binding buffer, and 200 µL of the same buffer and 5 µL of PI staining solution were added. Apoptosis was measured at Ex 530 nm/Em 695 nm using the BD FACSCantoTM system (BD Biosciences) and FACSDiva software (BD Biosciences). All analyses were performed in triplicate.
Statistical analyses were performed using IBM SPSS version 21 (NY, USA) and Prism 5 (GraphPad Software, CA, USA). A paired sample
The expression of CD26 on the cell surface of Th1 cells, as shown in previous studies (Dong
Next, we tested the inhibitory effects of the antidiabetic drugs evogliptin and sitagliptin on the mDPP4 enzymatic activity of CD26 in the PWM-activated Th1 cells. Evogliptin and sitagliptin are antihyperglycemic agents that bind directly at a DPP4 catalytic region and are used to treat type 2 diabetes (Fig. 2A; White
In addition to these antidiabetic DPP4 inhibitors, we also tested other known DPP4 inhibitors with antihyperglycemic effects, such as diprotin A and berberine (Fig. 3A). Diprotin A (Ile-Pro-Ile) is a peptidyl DPP IV inhibitor that binds to the DPP4 enzyme as a substrate. Berberine is a natural compound that inhibits the DPP4 enzyme, albeit its mechanism of action is still unclear (Lee
To further determine the time-course effects of the antidiabetic drugs, we treated the PWM-stimulated H9 cells with evogliptin and sitagliptin for 3, 6, 12, and 24 h at concentrations of 2 ng/mL and 2 µg/mL, respectively, and analyzed the mDPP4 activity and Th1 cell-specific cytokines. Although mDPP4 enzymatic activity was the highest at 12 h after PWM treatment, the increase in mDPP4 activity induced by PWM was independent of co-treatment with the antidiabetic drugs (Fig. 4A). Evogliptin and sitagliptin efficiently inhibited mDPP4 activity in the PWM-treated and untreated H9 Th1 cells at all time points. The expression of Th1 cell-specific cytokines, including IL-2, IL-10, IL-13, GM-CSF, IFN-γ, and TNF-α, gradually increased over time in the PWM-activated cells, whereas VEGF exceptionally decreased at 24 h after treatment with PWM (Fig. 4B). However, the expression levels of the cytokines were not affected by co-treatment with the antidiabetic drugs in H9 Th1 cells. When we further investigated the cell profiles, including cell count, cell viability, DNA synthesis, and apoptosis, in cultured cells prepared from the same batches, the results of which are shown in Fig. 4, evogliptin and sitagliptin did not change any of the cell profiles (Supplementary Fig. 4). In summary, our results indicate that evogliptin and sitagliptin are specific inhibitors that control the mDPP4 enzymatic activity of CD26 on the surface of Th1 cells but do not influence the expression of Th1 cell-specific cytokines and cell profiles, including cell count, viability, DNA synthesis, and apoptosis, in the PWM-activated H9 Th1 cells.
Since diprotin A and berberine did not inhibit the mDPP4 activity of H9 Th1 cells after 12-h treatment, we analyzed their effects at various time points at a high concentration of 20 µg/mL. Despite the change in the incubation time from 3 to 24 h, the high dose of diprotin A and berberine did not reduce the mDPP4 activity in H9 Th1 cells (Fig. 5A). In addition, diprotin A did not influence cytokine expression at any time point (Fig. 5B). In contrast, from 6 to 24 h, berberine treatment significantly inhibited the expression of Th1 cell-specific cytokines except VEGF in the PWM-activated H9 Th1 cells. Diprotin A did not affect the overall cell profiles of the PWM-activated H9 cells, but berberine decreased the cell count, viability, and DNA synthesis at 24 h (Supplementary Fig. 5). These results suggest that diprotin A inhibits neither mDPP4 enzymatic activity nor Th1 cell-specific cytokine expression despite the different incubation time and that berberine may exhibit cytotoxic effects even without mDPP4 inhibitory activity, leading to decreased cytokine expression and cellular growth in H9 Th1 cells.
Our results showing the lack of inhibitory effects of diprotin A and berberine on the mDPP4 activity of H9 Th1 cells are contradictory to those of previous studies showing their activities as DPP4 inhibitors, which could be attributed to the different assay systems used: previous DPP4 enzymatic assays were performed using the recombinant DPP4 protein (rDPP4; (Barzegar
The highly transient inhibition of DPP4 enzymatic activity by diprotin A could be the reason we did not observe any inhibition of mDPP4 activity in the PWM-activated H9 Th1 cells co-treated with diprotin A. Next, we evaluated the DPP4 inhibitory effects of these inhibitors in H9 Th1 cells for less than 3 h (Fig. 6E-6H). Consistent with the results obtained using rDPP4, evogliptin and sitagliptin were highly potent in inhibiting the mDPP4 activity of H9 Th1 cells even after 30 min incubation (Fig. 6E, 6F). Diprotin A also exhibited dose-dependent inhibition of mDPP4 activity in H9 Th1 cells after 30 min and 1 h incubation, but not after 3 h incubation, whereas berberine had no effect (Fig. 6G, 6H).
Thus, we observed that evogliptin and sitagliptin were potent inhibitors of both the rDPP4 protein and mDPP4 in CD4+CD26+ H9 Th1 cells after incubation for as little as 30 min until 24 h. Conversely, diprotin A transiently inhibited both the rDPP4 protein and mDPP4 in H9 Th1 cells, but this was not noted for more than 1 h. Berberine did not actively inhibit DPP4 activity regardless of the assay systems used. Therefore, these results suggest that evogliptin and sitagliptin are more effective mDPP4 inhibitors without any adverse effects on Th1 cells than the peptidyl DPP4 inhibitor, diprotin A.
In this study, we investigated the effects of evogliptin and sitagliptin, which are antidiabetic drugs and DPP4 inhibitors, on the mDPP4 enzymatic activity of CD26 and Th1 cell-specific cytokine expression in PWM-activated CD4+CD26+ H9 Th1 cells. PWM treatment stimulated the substantial expression of Th1 cell-specific cytokines such as IL-2, IL-10, TNF-α, IFN-γ, IL-13, and GM-CSF in the H9 Th1 cells (Fig. 1C). These results were consistent with those of previous studies showing that CD26 expression was closely related to T cell signaling pathways for Th1 cell-specific cytokine expression in PWM-activated Th1 cells (Yan
Evogliptin and sitagliptin efficiently inhibited mDPP4 activity in a dose-dependent manner but did not alter cytokine expression in PWM-treated H9 Th1 cells. According to the IC50 values of the drugs, evogliptin was more potent than sitagliptin in inhibiting mDPP4 enzymatic activity in the PWM-activated H9 cells as well as rDPP4 activity
While the non-enzymatic activities of CD26 are essential for T cell signaling pathways, whether mDPP4 enzymatic activity is also critical for the immune responses of T cells is not yet known. Sitagliptin treatment of patients with type 2 diabetes did not affect CD4+ T cell activation (White
Our results showed that the treatment of PWM-activated H9 Th1 cells with DPP4 inhibitors did not change the cell profiles such as cell viability and CD3+CD4+ or CD26+CD28+ subset populations (Supplementary Fig. 2, 3). In addition, potent DPP4 inhibitors had no or little effect on Th1 cell-specific cytokine expression in the PWM-activated Th1 cells, although they directly inhibited DPP4 enzymatic activity in these cells. Despite our limitation using a Th1 cell line rather than primary cells, these results suggest that mDPP4 enzymatic activity may not be required for Th1 cell-specific cytokine expression. Thus, patients with type 2 diabetes treated with evogliptin or sitagliptin may not exhibit adverse effects on T cell immune responses, though the effects of long-term treatment with these drugs should be evaluated
Our study investigated Th1 cell immune responses in PWM-activated H9 cells, after treatment with clinical DPP4 inhibitors such as evogliptin and sitagliptin. Our results suggest that the non-enzymatic activity of CD26 is more important than its enzymatic activity for the regulation of Th1 cell immune responses for the treatment of patients with type 2 diabetes.
This work was supported in part by the National Research Foundation of Korea (NRF) grants funded by the Korea government (MSIT) (No. 2020R1A2C2013827) and by the grants from Korea University and the Biomedical Research Institute, Seoul National University Hospital. Evogliptin and sitagliptin were provided by the Department of Clinical Pharmacology and Therapeutics, Seoul National University Hospital.
All authors declare no conflict of interest.