Anti-Inflammatory Effect of Combined Sitagliptin and Vitamin D3 on Cytokines Profile in Patients with Type 2 Diabetes Mellitus

Abstract

Studies indicated that imbalance of proinflammatory and anti-inflammatory cytokines may contribute to development of type 2 diabetes mellitus (T2DM). We hypothesized that sitagliptin and VitD3 may exert more anti-inflammatory effects on the regulation of cytokine balance in T2DM. Nonnephropathic and nephropathic T2DM patients were divided into the subgroups, based on treatments. The effect of 8 months sitagliptin, alone or together with 2 months of VitD3, on serum IFN-γ, IL-4, IL-17, IL-6, IL-21, TGF-β, and IL-37 levels was determined using enzyme-linked immunosorbent assay. Increased levels of interferon (IFN)-γ and IL-17 in untreated (without sitagliptin and VitD3) nephropathic and nonnephropathic patients and decreased levels of IL-37 in untreated nephropathic patients were observed compared with healthy controls. Treatment with sitagliptin plus VitD3 reduced the levels of IFN-γ and IL-17 in both nonnephropathic and nephropathic patients compared with untreated patients. The level of IL-37 was enhanced in patients treated with sitagliptin or sitagliptin plus VitD3, compared with untreated patients. Sitagliptin plus VitD3 treatment increased the levels of IL-4 in nonnephropathic patients. These findings indicated that the sitagliptin plus VitD3 was more effective to reduce the increased proinflammatory IFN-γ and IL-17 cytokines in T2DM patients.

Introduction

It has been reported that the immune system triggers inflammation in type 2 diabetes mellitus (T2DM) and induces insulin resistance as well as diabetic complications, including nephropathy (Donath and Shoelson 2011). Approximately one-fourth of T2DM patients have nephropathy (Zhang and others 2014).

CD4⁺ T helper (Th) cells are basically classified into proinflammatory (Th1, Th2, Th17, and Tfh cells) and anti-inflammatory (Th2 and Treg cell) subsets, based on the function and cytokines profile (Zhang and others 2014; Alahgholi-Hajibehzad and others 2015a). Th1 and Th2 cells are identified by the production of interferon (IFN) -γ and interleukin (IL)-4, respectively. Th17 cells produce IL-17 and IL-6 and induce inflammatory conditions in the autoimmune/inflammatory diseases (Zhang and others 2014; Alahgholi-Hajibehzad and others 2015a). T follicular helper (Tfh) cells activate B cells by the production of IL-21 and promote immune response in the germinal centers (Alahgholi-Hajibehzad and others 2017). Regulatory T (Treg) cells are characterized by the secretion of regulatory IL-10, TGF-β, and IL-37 cytokines (Alahgholi-Hajibehzad and others 2015a; Xu and others 2015).

IL-37 has been identified as an anti-inflammatory cytokine that shifts the immune system away from inflammation (Dinarello and Bufler 2013) and reduces the insulin resistance in mice and humans (Ballak and others 2014). It has been speculated that IL-37 plays a regulatory role in immunosuppression of Tregs. IL-37 was expressed in CD4⁺CD25⁺ Tregs, and the levels of IL-37 were elevated with the enhanced activity of Tregs. It suppresses the secretion of proinflammatory cytokines, including IL-1, IL-6, and TNF-α (Shuai and others 2015). With evidences accumulated, IL-37 is recognized as a typical anti-inflammatory cytokine related to the autoimmune disease, liver inflammatory injury, obesity, and cancer. The serum levels of IL-37 were changed in autoimmune and inflammatory diseases (Boraschi and others 2011; Moschen and others 2011; Shuai and others 2015).

Active form of vitamin D, vitamin D3 (VitD3), has protective effects on immune system. It suppresses production of inflammatory Th cytokines such as IFN-γ and IL-17, and increases production of anti-inflammatory cytokines such as IL-10 and results in a shift from Th1 or Th17 cells to Th2/Treg cells in vitro (Jeffery and others 2009; Sheikh and others 2018a). VitD has also been shown to improve severity of existing autoimmune diseases (Alahgholi-Hajibehzad and others 2015b). Administration of supplemental VitD reduced the insulin sensitivity and improved the insulin secretion (von Hurst and others 2010).

Sitagliptin is currently being used in the treatment of T2DM. It has been shown that sitagliptin exerts a potent anti-inflammatory effect on immune function (Makdissi and others 2012). The suppressive effects of sitagliptin on Th cell functions are probably mediated by a complex alteration of cytokines production (Yazbeck and others 2009). In addition, sitagliptin treatment may change the proportion of Th subpopulations in patients with T2DM (Sromova and others 2016).

Nephropathy is the commonest cause of end-stage renal disease in the patients. It is also known as an independent risk factor for cardiovascular disease (Wu and others 2011). The pathogenesis and inflammatory status of the T2DM disease may differ between nephropathic and nonnephropathic patients. More increased infiltration of activated proinflammatory T cells as well as inflammatory cytokines in the kidneys has been reported in patients with diabetic nephropathy (Navarro and Mora 2006). Some studies have been carried out revealing sitagliptin ability to not only control blood glucose but also decrease dysfunction in the periphery and kidney, due to anti-inflammatory properties. It is becoming clearer that T2DM management must envision not only the improvement in glycemic control but also the prevention of inflammatory condition and the nephropathy (Mega and others 2017).

Overall, previous studies have been indicated that Th cells are skewed toward proinflammatory subsets in the patients with T2DM. The imbalance of Th cell subsets may contribute to the development of T2DM (Zhang and others 2014; Francisco and others 2016). Combined treatment with sitagliptin and VitD has been shown to improve β-cell function and attenuate disease severity in patient with latent autoimmune diabetes (Rapti and others 2016). We hypothesized that sitagliptin plus VitD3 may exert more anti-inflammatory effects on the regulation of cytokine balance in the T2DM patients. To our knowledge, no data are available on the effect of combined administration of sitagliptin and VitD3 in the T2DM patients. We investigated the mono- or combined effects of sitagliptin and VitD3 on routine Th1/Th2/Th17/Tfh and Treg cell cytokines profile in nephropathic and nonnephropathic patients with T2DM.

Materials and Methods

Diabetic patients and healthy controls

In the current study, patients with type 2 diabetic mellitus (T2DM, 54 female and 25 male, mean age: 50.4 ± 8.14 years, range: 23–60 years) were recruited from the Internal Medicine Department of Shahid Beheshti Hospital, Hamadan University of Medical Science (UMSHA). The diagnosis of the patients was according to American Diabetes Association 2016. T2DM patients were identified on the basis of random blood glucose >200 mg/dL, fasting blood glucose >125 mg/dL, and HbA1c>6.5%. In this study, T2DM patients were classified into 6 subgroups based on the nephropathy condition and their treatments at the same time as follows:

DNN: Diabetic Nephropathy-Negative patients without sitagliptin and VitD3.

DNNS: Diabetic Nephropathy-Negative patients with sitagliptin.

DNNSD3: Diabetic Nephropathy-Negative patients with sitagliptin and VitD3.

DNP: Diabetic Nephropathy-Positive patients without sitagliptin and VitD3.

DNPS: Diabetic Nephropathy-Positive patients with sitagliptin.

DNPSD3: Diabetic Nephropathy-Positive patients with sitagliptin and VitD3.

The diagnosis of Total Diabetic Nephropathy-Positive patients (29 female and 12 male, mean age: 50 + 10.43 years, range: 23–60 years) was based on the presence of albuminuria (>30 mg/dL), serum creatinine >1.4 mg/dL, and/or decreased glomerular filtration rate (GFR <60 mL/min/1.73 m²). The diagnosis of Total Diabetic Nephropathy-Negative patients (25 female and 13 male, mean age: 50.92 + 6.79 years, range: 28–60 years) was based on the absence of albuminuria, serum creatinine <1.3 mg/dL, and/or decreased GFR (GFR >60 mL/min/1.73 m²).

All patients received metformin (1500 mg/dL) and gliclazide (120 mg/dL) for at least 1 year. The DNNS, DNNSD3, DNPS, and DNPSD3 groups received 100 mg/day of sitagliptin for last 8 months. The DNNSD3 and DNPSD3 groups received 1,000 IU/day of VitD3 at the last 2 months of sitagliptin therapy. The medications were prescribed by physician based on clinical and paraclinical data. The mean serum level of 25- VitD was slightly below the normal range (30 ng/mL) in total patients (25.26 ± 5.54 ng/mL) and HCs (28.38 ± 7.6 ng/mL), without significant differences between them. The mean serum level was measured in the DNNSD3 and DNPSD3 group, before (DNNSD3: 24.46 ± 3.91 ng/mL, DNPSD3: 24.71 ± 4.18 ng/mL) and after (DNNSD3: 35.23 ± 6.65 ng/mL, DNPSD3: 35.9 ± 6.65 ng/mL) VitD3 treatment. The details of the subjects are demonstrated in Table 1.

Table 1.

Characteristics of the Diabetic Patients and Healthy Control Subjects

	DNN	DNNS	DNNSD3	DNP	DNPS	DNPSD3	HC
	n = 12	n = 14	n = 12	n = 14	n = 14	n = 13	n = 27
Age (years)	53 ± 6.78	50.18 ± 7.56	49.72 ± 5.60	52.5 ± 8.75	51.53 ± 9.7	45.83 ± 12.18	47.72 ± 6.56
Age (years)	55 (40–60)	51.5 (28–60)	50 (41–60)	55 (26–58)	54 (23–60)	51 (25–59)	49.5 (30–58)
Gender Female/Male	8/4	9/5	8/4	10/4	10/4	9/4	16/11
Duration of disease (years)	4.8 ± 2.39	4.63 ± 2.01	3.25 ± 1.58	4.58 ± 2.46	4.77 ± 2.58	5.08 ± 2.53	—
Duration of disease (years)	5 (1–8)	5 (2–8)	2.5 (2–6)	4.5 (1–8)	5 (2–10)	5 (2–10)
Weight (kg)	68.22 ± 11.61	69.5 ± 7.54	68.57 ± 9.91	72.91 ± 10.13	75 ± 10.46	71.41 ± 11.85	66.9 ± 10.23
Weight (kg)	68 (48–90)	70 (54–80)	67 (54–80)	72.5 (58–95)	80 (52–86)	74 (50–86)	66.5 (46–86)
Height (m)	1.47 ± 0.55	1.61 ± 0.12	1.60 ± 0.14	1.65 ± 0.06	1.67 ± 0.07	1.64 ± 0.08	1.65 ± 0.07
Height (m)	1.63 (1.55–1.77)	1.59 (1.45–1.88)	1.58 (1.45–1.88)	1.63 (1.57–1.79)	1.67 (1.56–1.78)	1.61 (1.54–1.76)	1.63 (1.58–1.81)
BMI (kg/m²)	25.83 ± 3.82	26.74 ± 4.02	27.02 ± 4.96	26.82 ± 4.04	26.35 ± 2.39	25.88 ± 2.7	24.20 ± 2.69
BMI (kg/m²)	24.59 (22.2–33.05)	24.78 (22.63–34.72)	24.02 (22.63–34.72)	26.4 (20.08–36.2)	26.93 (20.83–29.38)	26.73 (20.83–29.69)	24.59 (17.97–28.6)
HbA1c (%)	7.5 ± 0.93	7.41 ± 0.78	7.8 ± 0.81	7.34 ± 0.93	7.71 ± 0.77	7.62 ± 0.75	4.65 ± 0.56^a
HbA1c (%)	7.3 (6.5–8.8)	7.1 (6.6–8.7)	8 (6.5–8.7)	7 (6.3–8.8)	7.9 (6.5–8.5)	7.7 (6.5–8.5)	4.8 (4–5.9)
FBS (mg/dL)	180.67 ± 32.19	150.36 ± 42.16	159.29 ± 30.63	187.6 ± 56.89	185.78 ± 56.51	182.67 ± 43.30	86.64 ± 6.69^b
FBS (mg/dL)	162 (148–239)	138 (121–268)	142 (135–220)	186.5 (125–264)	160 (130–260)	180 (130–250)	86 (72–97)
Random blood glucose (mg/dL)	292.18 ± 72.96	239.25 ± 38.79	283 ± 97.89	281.1 ± 69.64	340.4 ± 139.81	336.71 ± 132.94	112.76 ± 8.69^c
Random blood glucose (mg/dL)	272 (209–465)	238.5 (202–278)	260 (202–410)	281 (199–376)	320 (204–560)	320 (204–560)	86 (72–97)
Serum Creatinine (mg/dL)	0.95 ± 0.14	0.89 ± 0.14	0.85 ± 0.11	1.57 ± 0.2^d	1.64 ± 0.15^e	1.65 ± 0.18^f	1.01 ± 0.16^g
Serum Creatinine (mg/dL)	0.92 (0.7–1.2)	0.87 (0.7–1.21)	0.91 (0.7–0.95)	1.6 (1.3–1.83)	1.6 (1.5–1.9)	1.6 (1.4–1.9)	1 (0.78–1.3)
GFR (mL/min/1.73 m²)	73.44 ± 18.63	78.2 ± 12.88	75.8 ± 11.77	47.33 ± 4.50^h	40.5 ± 8.08ⁱ	41.87 ± 8.45^j	74.28 ± 14.16^k
GFR (mL/min/1.73 m²)	68 (60–121)	75 (66–105)	69 (66–91)	49 (39–52)	41.5 (28–55)	41.5 (28–55)	73 (46–96)
Albuminuria (mg/day)	12.8 ± 8.49	16 ± 10.39	9.6 ± 8.02	451.22 ± 240.61^l	704.63 ± 261.65^m	697.55 ± 236.29ⁿ	12.21 ± 5.54^o
Albuminuria (mg/day)	11 (5–24)	19 (5–26)	5 (5–9)	438 (77–775)	561 (395–1122)	715 (410–1120)	12.5 (5–21)
Triglycerides (mg/dL)	157.62 ± 63.85	128.22 ± 58.39	137 ± 60.29	135.86 ± 49.73	147.33 ± 41.08	162.29 ± 45.45	123.47 ± 40.84
Triglycerides (mg/dL)	160 (80–249)	112 (64–215)	128 (64–215)	126 (81–217)	149 (103–190)	165 (92–230)	123 (59–200)
Total cholesterol (mg/dL)	188.5 ± 45.16	152.33 ± 34.66	158.8 ± 43.99	128.61 ± 53.30	166.67 ± 42.94	172.67 ± 34.33	168.73 ± 38.54
Total cholesterol (mg/dL)	178.5 (131–249)	147 (111–212)	153 (111–212)	129 (22.3–200)	173.5 (116–218)	180.5 (116–212)	168 (115–234)
LDL (mg/dL)	94.74 ± 35.51	80.19 ± 21.60	76 ± 29.04	90.25 ± 26.90	122.83 ± 48.9	109.86 ± 31.41	106.27 ± 42.98
LDL (mg/dL)	107 (23–131)	77.5 (55–127)	66 (55–127)	96.8 (46–120)	132.5 (62–195)	96 (73–150)	110 (32–189)
HDL (mg/dL)	59.85 ± 20.35	49.1 ± 24.46	52.25 ± 25.99	52.42 ± 21.21	63.83 ± 26.37	73.14 ± 25.47	50.33 ± 11.83
HDL (mg/dL)	48 (44–99)	39 (31–99)	44.5 (31–110)	42 (35–92)	61.5 (28–110)	90 (28–97)	49 (36–80)
Hct (%)	68.71 ± 29.81	52.96 ± 24.16	40.32 ± 2.24	69.95 ± 33.56	50.71 ± 28.80	67.2 ± 33.76	41.06 ± 4.79
Hct (%)	69.35 (38.6–99.9)	42.6 (37.6–96.9)	39.4 (37.6–42.7)	72.15 (36.6–98.9)	39.75 (12.9–96)	91 (12.9–96)	41.1 (29.9–49)
Hb (gr/dL)	14.3 ± 2.82	15.37 ± 3.15	15.66 ± 3.16	14.88 ± 2.30	13 ± 1.47	13.73 ± 3.65	13.65 ± 1.63
Hb (gr/dL)	13.6 (10.8–19.8)	13.6 (12.5–19.7)	14.9 (12.6–19.7)	15.6 (12.2–17.8)	12.9 (10.6–15.7)	12.9 (8–19)	13.75 (9.7–16.6)
PLT ( × 10³)	254.71 ± 60.19	249.78 ± 621.61	250 ± 511.02	219.67 ± 72.02	239.25 ± 50.71	223.67 ± 517.01	209.53 ± 419.67
PLT ( × 10³)	236 (169–339)	226 (187–372)	242 (187–320)	207.5 (142–339)	256.50 (155–295)	226 (155–295)	209 (150–310)
RBC ( × 10⁶)	4.66 ± 0.46	4.73 ± 0.45	4.60 ± 0.53	4.18 ± 1.44	5.33 ± 0.73	4.63 ± 0.73	4.64 ± 0.74
RBC ( × 10⁶)	4.79 (3.81–5.12)	4.72 (4–5.42)	4.6 (4–5.42)	4.76 (2.53–5.25)	4.95 (4.56–6.51)	4.66 (3.87–5.9)	4.65 (3.31–5.97)
WBC ( × 10³)	7.13 ± 19.23	6.74 ± 1.78	6.58 ± 2.14	6.53 ± 1.2	7.72 ± 2.84	7.62 ± 3.68	62.60 ± 20.90
WBC ( × 10³)	6 (4.9–9.8)	7.3 (4.3–9.9)	6 (4.3–9.9)	6.38 (5.30–8.60)	8.2 (1.03–9.9)	8.4 (10.30–11.60)	60 (37–98)
Lymphocytes (%)	38.28 ± 9.58	40.08 ± 6.82	42.8 ± 7.59	36.25 ± 10.99	37.5 ± 6.82	35.5 ± 2.58	38.43 ± 7.57
Lymphocytes (%)	33 (30–53)	38.35 (31–50)	45 (31–50)	41 (20–43)	36.5 (29–50)	36 (31–39)	39.5 (25–50)
Neutrophils (%)	50.85 ± 6.20	54.36 ± 6.36	49 ± 7.2	58.25 ± 8.77	54 ± 7.07	56.33 ± 4.41	54.37 ± 5.43
Neutrophils (%)	48 (45–60)	53 (47–67)	48 (40–60)	55.5 (51–71)	55.5 (42–62)	56.5 (51–62)	54.5 (43–62)
Eosinophils (%)	5.24 ± 3.63	2.73 ± 1.65	4 ± 2.7	4 ± 1	3.25 ± 2.60	3.5 ± 2.7	2.92 ± 1.97
Eosinophils (%)	6 (1–10)	2.7 (1–5)	4 (1–8)	4 (3–5)	2 (1–7)	2 (1–7)	2.5 (1–7)
Basophils (%)	1.4 ± 0.54	2 ± 1.41	1.75 ± 0.5	1.66 ± 0.57	1.66 ± 0.57	1.5 ± 0.57	1.57 ± 0.53
Basophils (%)	1 (1–2)	1.5 (1–4)	2 (1–2)	2 (1–2)	2 (1–2)	1.5 (1–2)	2 (1–2)
Monocytes (%)	2.64 ± 2.47	2.37 ± 0.91	2.8 ± 1.09	3.75 ± 2.21	4.75 ± 3.10	3.66 ± 2.73	3.66 ± 2.16
Monocytes (%)	2 (1–8)	2 (1–4)	2 (2–4)	4 (1–6)	4.5 (1–9)	2.5 (1–8)	3 (1–7)

Values expressed as means (±SD) and medians (interquartile range). P < 0.05 was considered significant. Significance was tested using ANOVA.

a–c

P < 0.001; compared with each patient subgroups.

d,h,l

P < 0.001; compared with DNN group.

e,i,m

P < 0.001; compared with DNNS group.

f,j,n

P < 0.001; compared with DNNSD3 group.

g,k,o

P < 0.001; compared with each patient subgroups.

DNN, Diabetic Nephropathy-Negative patients without sitagliptin and VitD3; DNNS, Diabetic Nephropathy-Negative patients with sitagliptin; DNNSD3, Diabetic Nephropathy-Negative patients with sitagliptin and VitD3; DNP, Diabetic Nephropathy-Positive patients without sitagliptin and VitD3; DNPS, Diabetic Nephropathy-Positive patients with sitagliptin; DNPSD3, Diabetic Nephropathy-Positive patients with sitagliptin and VitD3; BMI, body mass index; FPG, fasting plasma glucose; HbA1C, hemoglobin A1C; Cr, creatinine; GFR, glomerular filtration rate; LDL, low-density lipoprotein; HDL, high-density lipoprotein; Hb, hemoglobin; Hct, hematocrit; RBC, red blood cell; WBC, white blood cell; HC, healthy control.

The exclusion criteria were HbA1c>9%, autoimmune/chronic inflammatory diseases, neoplastic/coronary disease, liver dysfunction, and current treatment with insulin, glucocortids, or any immunosupprsssive medications. Twenty seven individuals (16 female and 11 male, mean age: 47.72 + 6.56 years, range: 30–58 years) without T2DM or autoimmune disease were selected as healthy control subjects (HCs). The HCs were matched for sex and age as much as possible. Informed consent was obtained from all participants and the study was approved by the Ethics Committee of UMSHA (no. IR.UMSHA.REC.1395.374).

Cytokines assay

The blood samples (10 mL) were centrifuged at 3,000 rpm for 15 min and the serum samples were stored at −80°C until cytokine assay. The levels of IFN-γ (BioLegend), IL-4 (Invitrogen), IL-17 (BioLegend), IL-6 (BioLegend), IL-21 (Invitrogen), TGF-β (Invitrogen), and IL-37 (Invitrogen) were determined using enzyme-linked immunosorbent assay kits (ELISA) according to the manufacturer's instructions. Before analysis, all thawed samples were centrifuged to remove debris.

Statistical analysis

The Statistical Package for Social Sciences (SPSS, Version 21) was used for the analysis. Graphs were drawn by GraphPad Prism software version 6.07. Data were checked for normality by the Shapiro–Wilk test. ANOVA was used to evaluate differences among groups. Data are shown as mean ± standard error of the mean. P-value <0.05 was considered significant.

Results

Cytokine changes in nonnephropathic and nephropathic T2DM patients based on the treatments

To investigate the effects of sitagliptin with or without VitD3 on cytokine levels, the total T2DM patients were divided into the subgroups based on the nephropathy condition and the treatments as follows: Diabetic nephropathy-negative patients without sitagliptin and VitD3 (DNN group), diabetic nephropathy-negative patients with sitagliptin (DNNS group), diabetic nephropathy-negative patients with sitagliptin and VitD3 (DNNSD3 group), diabetic nephropathy-positive patients without sitagliptin and VitD3 (DNP group), diabetic nephropathy-positive patients with sitagliptin (DNPS group), and diabetic nephropathy-positive patients with sitagliptin and VitD3 (DNPSD3 group). Serum cytokine levels of IFN-γ, IL-4, IL-17, IL-6, IL-21, TGF–β, and IL-37 were assessed using ELISA.

We observed that the level of IFN-γ was significantly enhanced in both DNN and DNNS subgroups compared with HC subjects (P ≤ 0.001, Fig. 1A). The IFN-γ levels were significantly reduced in DNNSD3 subgroup compared with DNN and DNNS subgroups (P ≤ 0.001, Fig. 1A). Its levels were slightly enhanced in DNP and DNPS groups in comparison with HC group, but not quite statistically significant. The level of this cytokine was diminished in DNPSD3 subgroup compared with DNP, DNPS, and HC groups (P ≤ 0.001, Fig. 1A).

FIG. 1.

Cytokine levels in patients with T2DM with or without nephropathy receiving sitagliptin and VitD3. Cytokine levels of (A) IFN-γ, (B) IL-4, (C) IL-17, (D) IL-6) (E) IL-21, (F) TGF-β, and (G) IL-37 were evaluated in serum of DNN (n = 12), DNNS (n = 14), DNNSD3 (n = 12), DNP (n = 14), DNPS (n = 14), DNPSD3 (n = 13), and HC (n = 27) group. Data expressed as means ± SEM. P < 0.05 was considered significant. ANOVA test was used to calculate the differences between groups. *P ≤ 0.001, **P = 0.04. DNN, Diabetic Nephropathy-Negative patients without sitagliptin and VitD3; DNNS, Diabetic Nephropathy-Negative patients with sitagliptin; DNNSD3, Diabetic Nephropathy-Negative patients with sitagliptin and VitD3; DNP, Diabetic Nephropathy-Positive patients without sitagliptin and VitD3; DNPS, Diabetic Nephropathy-Positive patients with sitagliptin; DNPSD3, Diabetic Nephropathy-Positive patients with sitagliptin and VitD3; HC, healthy control.

The level of IL-4 did not change among the DNN subgroup and HC subjects (Fig. 1B). Its levels were obviously increased in DNNSD3 group compared with DNN and HC groups (P ≤ 0.001, Fig. 1B). The level of this cytokine was slightly increased in DNPSD3 group in comparison to DNP, DNPS, and HC groups, but not quite statistically significant. Consumption of sitagliptin did not change the level of IL-4 in the patients.

An increase in IL-17 levels was observed in the DNN, DNNS, DNP, and DNPS subgroups in comparison to HC group (P ≤ 0.001, Fig. 1C). The level of this cytokine was reduced in the DNNSD3 group compared with DNN group (P ≤ 0.001, Fig. 1C). The IL17 levels were significantly decreased in the DNPSD3 subgroup in comparison with DNP and DNPS subgroups (P ≤ 0.001, Fig. 1C). There were no significant differences between the DNN and DNNS subgroups and also between the DNP and DNPS subgroups.

No significant changes were found in IL-6, IL-21, and TGF-β cytokine levels between the patients and HC subjects (Fig. 1D–F). There were no significant changes among patient subgroups.

The IL-37 levels were enhanced in the DNNS and DNNSD3 groups compared with DNN group (P = 0.04, Fig. 1G). IL-37 levels were obviously diminished in DNP subgroup in comparison to HC group (P ≤ 0.001, Fig. 1G), while its levels were increased in the DNPS and DNPSD3 groups compared with DNP group (P ≤ 0.001, Fig. 1G).

Discussion

Recent studies have suggested that inflammatory process and immune system are involved in development and progression of T2DM (Donath and Shoelson 2011). Sitagliptin and VitD3 have been demonstrated to exert regulatory roles in the pathogenesis of T2DM (von Hurst and others 2010; Makdissi and others 2012). In this study, we investigate the mono- or combined effects of sitagliptin and VitD3 therapy on routine Th1/Th2/Th17/Tfh and Treg cell cytokines profile, including IFN-γ, IL-4, IL-17, IL-6, IL-21, TGF-β, and IL-37 in nephropathic and nonnephropathic T2DM patients in vivo.

This study has several main findings as follows: we found increased basic levels of IFN-γ and IL-17 in untreated (without sitagliptin and VitD3) nonnephropathic and nephropathic T2DM patients and decreased level of IL-37 in untreated nephropathic T2DM patients compared with HC subjects. Combined treatment with sitagliptin and VitD3 decreased the levels of IFN-γ and IL-17 in both nonnephropathic and nephropathic patients compared with untreated groups. In contrast, sitagliptin plus VitD3 treatment increased the levels of IL-4 in nonnephropathic patients. The level of IL-37 was enhanced in patients who received mono-sitagliptin or combined sitagliptin and VitD3 compared with untreated patients.

Th1 and Th17 cells, which produce IFN-γ and IL-17, activate the cell-mediated immunity (Alahgholi-Hajibehzad and others 2015a). Th1 and Th17 subsets contributed to the pathogenesis of autoimmune or inflammatory diseases (Boissier and others 2008; Cheng and others 2008). Previous studies proposed that the increased levels of IFN-γ and IL-17 are associated with inflammation and insulin resistance in diabetic patients (Jagannathan-Bogdan and others 2011; Sumarac-Dumanovic and others 2013). In this study, we confirmed that the serum concentrations of proinflammatory IFN-γ and IL-17 cytokines were elevated in patients with/without nephropathy.

The anti-inflammatory actions of VitD3 on T cell function and cytokine production have been shown in autoimmune diseases (Alahgholi-Hajibehzad and others 2015b). Previous studies showed that VitD3 can downregulate proinflammatory cytokines and enhance anti-inflammatory cytokines in vitro (Jeffery and others 2009; Sheikh and others 2018a). On the contrary, treatment with sitagliptin decreased the number of CD4⁺ T cells and the percentage of proinflammatory Th cell subsets in the patient with T2DM (Aso and others 2015). It has been demonstrated that 6 months of sitagliptin treatment can reduce plasma glucose and HbA1c in the diabetic patients (Katsuyama and others 2015). In this study, we observed that the levels of both IFN-γ and IL-17 were diminished in diabetic patients who were treated by combined sitagliptin and VitD3. With regard to medicine, sitagliptin and VitD3 were more effective anti-inflammatory actions in the reduction of proinflammatory cytokines in the T2DM patients.

The level of IFN-γ was lower than HC upon combined treatment in nephropathic patients, which could be as a result of fixed-dose combination of sitagliptin and VitD3. It is possible that the various doses of sitagliptin and VitD3 treatments may have a different effect on cytokine levels. Further complementary studies are required to confirm these possibilities. No significant difference was observed between the nephropathic and nonnephropathic patients in the parameters examined. We did not observe any significant changes in the serum level of IL-6, another cytokine for Th17 cells, in T2DM patients.

Th2 cells produce mainly IL-4 and inhibit Th1 cell activation and contribute to humoral immunity (O'Shea and others 2011). In the current study, the serum level of IL-4 was unchanged between the untreated patients and HC subjects. Previous study reported that VitD3 can enhance the production of intracellular IL-4 in activated CD4⁺ T cells (Sheikh and others 2018a). On the contrary, it has also been shown that sitagliptin enhanced the frequency of Th2 cells (by phenotype: CD4⁺IL-4⁺) in the PBMCs cultures in vitro (Pinheiro and others 2017). We confirmed that combined sitagliptin and VitD3 treatment increased the serum level of IL-4 in the T2DM patients in vivo, particularly in the nonnephropathic patients. On the contrary, the combined treatment decreased the levels of IFN-γ in the patients. Basically, the IL-4 and IFN-γ play major roles in the regulation of immune responses. These cytokines mutually have antagonistic functions. IL-4 promotes Th2 differentiation and inhibits Th1 cells and production of IFN-γ and vice versa. Because IL-4 and IFN-γ are opposing each other, it is possible that the increased IL-4 in the patients with combined treatments reduced the level of IFN-γ or vice versa.

Treg cells are critical for the normal function of the immune system that provides a mechanism to the prevention of autoimmune responses (Alahgholi-Hajibehzad and others 2015a). Treg cell populations produce low amounts of IL-10, TGF-β, and IL-37, and their suppressive activity is mainly mediated by the immunosuppressive cytokines (Alahgholi-Hajibehzad and others 2015a; Xu and others 2015). Some studies have been reported that the serum level of IL-10 was reduced in T2DM patients (Qiao and others 2016) and the sitagliptin increased the level of IL-10 (Satoh-Asahara and others 2013). In this study, we selected/investigated the level of IL-37 as a novel cytokine and TGF-β as a routine cytokine for Tregs in T2DM. IL-37, known as IL-1F7, is a novel anti-inflammatory/immunoregulatory cytokine that contributed to reduce the inflammation and insulin resistance in mice and humans (Ballak and others 2014). In addition, our previous study showed that CD4⁺CD25⁺CD45RA⁺ Tregs have a functional defect with normal frequencies in T2DM patients (Sheikh and others 2018b). In this study, no significant changes were observed in TGF-β cytokine levels between the patients and HC subjects. No significant effects of sitagliptin and VitD3 treatment were observed on serum TGF-β levels. We found decreased level of IL-37 in untreated nephropathic T2DM patients compared with HC subjects. We observed that 8 months of the sitagliptin/sitagliptin plus VitD3 treatments increased the level of IL-37 in serum of nonnephropathic and nephropathic T2DM patients. Cytokines are produced by a broad range of cells, and the source of the TGF-β and IL-37 may be different from each other. TGF-β, is expressed not only by FOXP3⁺ Treg but also is expressed by Th3 (rare subpopulation of Treg cells) (Alahgholi-Hajibehzad and others 2015a). IL-37 is mainly expressed in FOXP3⁺ Tregs, and the levels of IL-37 were elevated with the enhanced activity of CD4⁺CD25⁺ Tregs (Shuai and others 2015). It has been reported that the other cells (except lymphocytes) produce TGF-β or IL-37 in immune system (Alahgholi-Hajibehzad and others 2015a; Shuai and others 2015). In this study, we assessed the TGF-β and IL-37 in serum samples (consist of T cells, B cells, NK cells, and other immune cells) not in isolated Tregs. So, it is possible that TGF-β and IL-37 may have a different direction in the study. But this probability should be investigated/confirmed on isolated Tregs in the future studies.

Tfh were described in humans as CD4⁺ T cells that produce IL-21 (Alahgholi-Hajibehzad and others 2017). Tfh cells are proposed in pathogenesis of autoimmune diseases (Spolski and Leonard 2008). In this study, we did not observe any significant changes in level of IL-21 between the total patients and HC subjects.

This study offers in vivo immunomodulatory capability of sitagliptin or combined sitagliptin and VitD3 treatments in T2DM patients by inhibition of the proinflammatory cytokines and the induction of regulatory cytokines. The limitation of this study was the small sample size in evaluation of cytokines production at serum environment. Future studies confirmed this observation with more sample size on isolated CD4⁺ T cell or Treg cells. This study investigated the cytokines in serum, yet, flow cytometric analysis should be included in future studies. In addition, the anti-inflammatory effect of sitagliptin and VitD3 on cytokine signaling pathways should be investigated. Further studies are needed to further define the cytokine changes in T2DM subgroups, with correlations to the clinical parameters/complications such as cardiovascular/peripheral vascular disease, cerebrovascular events, neuropathy, nephropathy, retinopathy, and metabolic complications.

Conclusion

This study confirmed that the T2DM pathogenesis may relate to the increased IFN-γ and IL-17 proinflammatory cytokines and reduced IL-37 anti-inflammatory cytokine. Eight months of sitagliptin plus 2 months of VitD3 treatments significantly decreased the level of IFN-γ and IL-17 in nephropathic and nonnephropathic T2DM patients in vivo. In addition, IL-37 levels were enhanced in patients who received sitagliptin or combined sitagliptin and VitD3, compared with untreated patients. With these findings, combined therapy with sitagliptin and VitD3 was more effective to reduce IFN-γ and IL-17 cytokines in the T2DM patients compared to monotherapy with sitagliptin. Further studies are needed to confirm these observations in the isolated cell culture environments on intracellular cytokines production.

Footnotes

Acknowledgments

We are grateful to patients and healthy control participants. The study was funded by Vice-chancellor for Research and Technology, Hamadan University of Medical Sciences, Hamadan, Iran (No. 9510146104). Sh.B. and M.A.H. performed the experiments and analyzed the data. V.Sh., M.G., A.M.M., and M.A.A. took care of the donors. Z.T. and Z.Z. produced the figures and contributed to the ELISA experiments. I.S., A.Z. and M.A.H. wrote the article. All authors have approved the final article.

Author Disclosure Statement

No competing financial interests exist.

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