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Treg paradigm in patients with type 2 diabetes mellitus: Relationship with diabetic nephropathy
Human Immunology 75 (2014) 289–296
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The alteration of Th1/Th2/Th17/Treg paradigm in patients with type 2 diabetes mellitus: Relationship with diabetic nephropathy Cuiping Zhang a,1, Chunchun Xiao b, Peng Wang c, Wenhua Xu d, Aimei Zhang a, Qing Li a, Xiucai Xu a,⇑,1 a
Central Laboratory of Medical Research Center, Affiliated Provincial Hospital of Anhui Medical University, Hefei, Anhui 230001, PR China Department of Endocrinology, Affiliated Provincial Hospital of Anhui Medical University, Hefei, Anhui 230001, PR China c Department of Nephrology, Affiliated Provincial Hospital of Anhui Medical University, Hefei, Anhui 230001, PR China d Department of Neurology, Affiliated Provincial Hospital of Anhui Medical University, Hefei, Anhui 230001, PR China b
a r t i c l e
i n f o
Article history: Received 14 August 2013 Accepted 4 February 2014 Available online 12 February 2014
a b s t r a c t T cells have been demonstrated to exert central roles in the development of type 2 DN (T2DN). To explore whether Th1/Th2/Th17/Treg paradigm plays an important role in the development of T2DN, we investigated the proportions of Th1/Th2/Th17/Treg cells and serum levels of relevant cytokines in T2DM patients with various degrees of nephropathy and controls. Moreover, we analyzed the relationships between the Th1/Th2/Th17/Treg paradigm or relevant cytokines with urine albumin:creatinine ratio (UACR). Our study demonstrated that the Th1/Th2/Th17/Treg paradigm skewed to Th1 and Th17 in T2DN patients. UACR was positively related to the proportions of Th1 and Th17 cells, as well as the ratio of Th17:Treg cells, and negatively related to the proportions of Treg cells. Furthermore, serum levels of IL-6, IL-17, IFN-c, TNF-a, IL-2 and IL-10 were increased in T2DN patients, and positively related to UACR. These data indicate that the alteration of Th1/Th2/Th17/Treg paradigm exists in T2DN patients, which may contribute to the enhanced immune activation and inflammation, and subsequent development and progression of T2DN. These findings may provide one new approach to the underlying mechanisms of the development of T2DN. Ó 2014 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
1. Introduction Diabetes mellitus (DM) is an increasingly prevalent metabolic disease characterized by absolute or relative insulin deficiency, resulting in hyperglycemia as well as an altered metabolism of glucose, fat, and protein. Diabetic nephropathy (DN), affecting over one-third of patients with type 1 DM and approximately 25% of all patients with type 2 DM, is an extremely common and progressive complication of DM, and deeply contributes to patient morbidity and mortality [1–4]. Basic pathological features of DN are hypertrophy of glomerular structures, accompanied by thickening of the basement membranes and accumulation of extracellular matrix components. The etiology and pathogenetic mechanisms of DN have not been clearly elucidated. Traditionally, metabolic and hemodynamic factors are the major causes of renal lesions in patients with DM, considered as one nonimmune disease [5–7]. However, serial studies have shown that inflammation plays a vital role in the process of DN [8–9]. T cells were proposed to participate in the development and progression of type 2 DN (T2DN) [10]. ⇑ Corresponding author. Fax: +86 551 62283574. 1
E-mail address: [email protected] (X. Xu). These authors contributed equally to this work.
CD4+ T helper (Th) cells are believed to play central roles in modulating immune responses. Based on the cytokine profiles and effector function, CD4+ T cells can be classified into T-helper 1 (Th1) cell, Th2 cell, Th17 cell, and CD4+CD25+ T regulatory (Treg) cell subsets [11–12]. Th1 cells produce mainly interferon (IFN)-c and promote the cell-mediated immunity [13], whereas Th2 cells suppress Th1cell responses, and contribute to humoral immunity [14]. A recent study has shown that not only Th1/Th2 imbalance but also Th17/ Treg imbalance contributed to the pathogenesis of some autoimmune/inflammatory diseases, such as rheumatoid arthritis [15], acute coronary syndrome [16], and type 1 and type 2 diabetes [17–18]. Th17 cells produce IL-17, TNF-a and IL-6, and induce inflammation in the pathogenesis of autoimmune diseases [19]. Immunosuppressive Treg cells exert important effects on the maintenance of immune homeostasis and immune tolerance by producing anti-inflammatory cytokines, such as IL-10 and transforming growth factor-b [20]. Recently, it was reported that the imbalance of Th17/Th1/Tregs may contribute to the development of T2DM and complications [18]. To explore whether Th1/Th2/Th17/Treg paradigm plays an important role in the development of T2DN, we investigated the Th1/Th2/Th17/Treg paradigm and serum levels of relevant cytokines in T2DM patients with various degrees of nephropathy and controls. Moreover, the relationships between
http://dx.doi.org/10.1016/j.humimm.2014.02.007 0198-8859/Ó 2014 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
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the Th1/Th2/Th17/Treg paradigm or relevant cytokines with urine albumin:creatinine ratio (UACR) were analyzed. Information obtained from this study may provide a better understanding of the pathogenesis of T2DN. 2. Materials and methods 2.1. Patients This case-control hospital-based study was conducted between April 2011 and March 2013 at our hospital. Ninety-three adult patients with T2DM were recruited from the diabetes mellitus and Endocrine center. T2DM was diagnosed according to the criteria of the American Diabetes Association [21]. Age-matched control subjects had normal fasting blood glucose and no clinical diagnosed diseases, and lacked family history of T2DM. This study was cross-sectional and blinded. All donors were nonsmokers. Albumin excretion in spot urine samples was evaluated on at least two separate occasions and the patients were divided further into three groups according to the definition of abnormalities of albumin excretion advocated by the American Diabetes Association [22]. A urine albumin:creatinine ratio (UACR) of less than 30 mg/g Cr was defined as normoalbuminuria, a UACR of 30–300 mg/g Cr as microalbuminuria, and a UACR of greater than 300 mg/g Cr as macroalbuminuria. The patients with DN enrolled in our study were characterized as a UACR above 30 mg/g Cr and were diagnosed by excluding other causes of glomerulonephritis and by the presence of characteristic pathologic findings of renal biopsy. No patient was on treatment with anti-inflammatory drugs and/or immunosuppressive agents known to interfere with the immune system prior to the examination. The patients with hypertension were receiving calcium channel blocker (CCB) inhibitors. In all groups, patients with cardiovascular disease had been excluded from our study. None had glomerulonephritis, chronic renal failure, congestive heart failure, hypo and hyperthyroidism, acute or chronic infectious disease, other inflammatory disease, or chronic-immunemediated disorders. The study was approved by the ethics committee of Anhui provincial hospital, and written informed consent was obtained from all participants before participation.
2.4. Flow cytometric staining and analysis For Treg analysis, cell surface staining was performed with fluorescein isothiocyanate (FITC)-conjugated anti-human CD4 (13B8.2 clone; Beckman Coulter-Immunotech, Marseille, France), phycoerythrin (PE)-conjugated anti-human CD25 (B1.49.9 clone, Beckman Coulter-Immunotech) and their appropriate isotype controls for 20 min at 4 °C in the dark. After being washed with ice-cold phosphate buffered solution (PBS), cells were fixed and permeabilized with the Fix/Perm reagent (eBioscience, San Diego, CA, USA), washed twice with ice-cold permeabilization buffer (eBioscience), and incubated with allophycocyanin (APC)-conjugated anti-human Foxp3 (PCH101 clone, eBioscience) or its isotype control antibody. Cells were then washed again with ice-cold permeabilization buffer and analyzed by FCM. For intracellular cytokine analysis, cells were stained with FITC-conjugated anti-human CD3 (UCHT clone, Beckman Coulter-Immunotech) and Percp-cy5.5-conjugated anti-human CD8 (RPA-T8 clone, eBioscience) for 20 min at room temperature, fixed and permeabilized with Fix/Perm solution (Beckman Coulter-Immunotech), and stained with PE-conjugated anti-human IFN-c (4S.B3 clone, eBioscience), APC-conjugated anti-human IL-4 (8D4–8 Clone, eBioscience) or PE-conjugated anti-human IL-17A (eBio64CAP17 clone, eBioscience). Stained cells were assayed using a FACSCanto™II flow cytometry (BD Biosciences, San Jose, CA, USA) and then analyzed with FACSDiva 6.0 software. The frequency of Th1 (CD3+ CD8 IFN-c+), Th2 (CD3+ CD8 IL-4+) and Th17 (CD3+ CD8 IL-17+) was expressed as a percentage of CD3+ CD8 T cells by sequential gating on lymphocytes and CD3+ CD8 T cells, while the frequency of Treg (CD4+ CD25+ Foxp3+) expressed as a percentage of CD4+ T cells by sequential gating on lymphocytes and CD4+ T cells. 2.5. Cytokine measurement Cytokine profiles in serum were detected using human Th1/ Th2/Th17 cytometric bead array (CBA) kit, according to the manufacturer’s instructions (BD Biosciences). The minimum detectable concentrations of IL-2, IL-4, IL-6, IL-10, TNF-a, IFN-c and IL-17A were 2.6, 4.9, 2.4, 4.5, 3.8, 3.7, and 18.9 pg/ml, respectively. Intraand inter-assay coefficients of variation for all these detections were <5%. All samples were examined in duplicate. 2.6. Statistical analysis
2.2. Blood samples Blood samples (5–10 ml) were collected from all the participants in a fasting state in the morning. Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized venous blood by Ficoll density gradient for analysis of flow cytometry (FCM). Serum was separated from the blood samples and stored at 80 °C until analyzed. 2.3. Cell preparation and Th cells analysis For the analysis of Th1, Th2 and Th17 cells, PBMCs (2 106 cells/ ml) were suspended in complete culture medium (400 ll RPMI 1640 supplemented with 100 lg/ml streptomycin, 100 U/mL penicillin and 10% heat-inactivated fetal calf serum; Gibco BRL, USA). The cells were stimulated with 100 ng/mL of phorbol myristate acetate – PMA (Sigma–Aldrich, USA) plus 1 lg/mL of ionomycin (Enzo Life Sciences, Inc., USA) for 4 h, in the presence of 10 lg/mL of brefeldin A (ENZO, USA). After 4 h of culture in 37 °C under a 5% CO2 environment, the contents of the wells were transferred to 5 ml sterile tubes and centrifuged at 1200 rpm for 5 min. For Treg analysis, 100 ll of PBMCs (1 106) was added to sterile tubes for flow cytometric staining.
Values were mainly expressed as the mean ± standard deviation (SD). Data were analyzed using statistical software (SPSS 16.0; LEAD Technologies, Inc., Chicago, IL, USA). For a comparison of the different subgroups of the T2DM patients and the control subjects, the data were first analyzed by One-way analysis of variance (ANOVA). Comparison between the two groups was carried out with LSD test. Correlations between two variables were assessed with Spearman’s rank correlation coefficient test and regression tests. A p value <0.05 was considered to be statistically significant. 3. Results 3.1. Patient characteristics As shown in Table 1, there were no significant differences in age, gender, or high-density lipoprotein-cholesterol (HDL-C) concentrations between the diabetic patients and control subjects. Furthermore, no significant differences in fasting plasma glucose (FPG), hemoglobin A1c (HbA1c) and body mass index (BMI) were observed among patients with and without albuminuria. Systolic and diastolic blood pressures of our patients were within the range of the reference values due to the medical treatment.
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C. Zhang et al. / Human Immunology 75 (2014) 289–296 Table 1 Clinical characteristics of all type 2 diabetes mellitus patients. Item
Control (n = 30)
Normoalbuminuria (n = 32)
Microalbuminuria (n = 30)
Macroalbuminuria (n = 31)
Age Gender (male/female) Patients with hypertension Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) TC (mmol/L) TG (mmol/L) HDL-C (mmol/L) LDL-C (mmol/L) VLDL-C (mmol/L) FPG (mmol/L) HbA1c (%) Duration (years) BMI (kg/m2) Serum Cr (lmol/L) UACR (mg/g)
56.9 ± 6.93 20/10 0 120 ± 2.64 76 ± 1.23 4.77 ± 0.46 1.42 ± 0.28 1.35 ± 0.19 3.48 ± 0.47 0.69 ± 0.15 5.06 ± 0.63 5.05 ± 0.62 – 22.12 ± 1.41 83.78 ± 18.29 –
58.47 ± 9.76 22/10 20 125 ± 1.41 80 ± 1.58 4.83 ± 0.93 2.39 ± 0.49* 1.26 ± 0.3 3.6 ± 0.71 0.65 ± 0.12 8.88 ± 2.34* 8.05 ± 1.05* 5.06 ± 2.64 25.18 ± 1.71* 86.96 ± 15.59 19.13 ± 5.17
60.13 ± 11.07 22/8 23 130 ± 2.77 80 ± 1.77 5.06 ± 0.48 2.24 ± 0.61* 1.18 ± 0.13 3.54 ± 0.96 0.65 ± 0.11 8.62 ± 1.78* 8.01 ± 1.24* 11.3 ± 4.37** 25.76 ± 2.28* 96.38 ± 16.52* 86.85 ± 54.52**
56.74 ± 10.13 16/15 25 135 ± 2.43 84 ± 1.43 6.71 ± 0.49**** 3.08 ± 0.54**** 1.22 ± 0.33 4.41 ± 1.41**** 0.82 ± 0.19**** 8.79 ± 1.76* 8.02 ± 1.92* 10 ± 4.44** 24.92 ± 2.53* 150.2 ± 25.06**** 1006.59 ± 563.36***
Values are expressed as mean ± SD. TC: total cholesterol; TG: total triglyceride; HDL-C: high-density lipoprotein-cholesterol; LDL-C: low-density lipoprotein-cholesterol; VLDL-C: very low-density lipoprotein-cholesterol; FPG: fasting plasma glucose; HbA1c: hemoglobin A1c; Duration: diabetes duration; BMI: body mass index; Cr: creatinine; UACR: urine albumin: creatinine ratio. * P < 0.05 vs. control. ** P < 0.05 vs. normoalbuminuria groups. *** P < 0.05 vs. normoalbuminuria and microalbuminuria groups. **** P < 0.05 vs. control, normoalbuminuria and microalbuminuria groups.
3.2. Th1/Th2/Th17/Treg paradigm skewed to Th1 and Th17 in patients with T2DN
3.4. Correlation between the Th1/Th2/Th17/Treg paradigm with the duration of the diabetic disease
Flow cytometry was used to determine the Th1/Th2/Th17/Treg paradigm in T2DM patients with various degrees of nephropathy. Compared to control subjects, the proportions of Th1 (CD3+ CD8 IFN-c+) and Th17 (CD3+ CD8 IL-17A+) cells increased in diabetic patients with or without albuminuria, while the proportion of Treg (CD4+ CD25+ Foxp3+) cells, but not Th2 (CD3+ CD8 IL-4+) cells, was markedly decreased (Figs. 1 and 2A and B), resulting in elevated ratios of Th1/Th2 cells and Th17/Treg cells (Figs. 1 and 2C). Moreover, the alteration of Th1/Th2/Th17/Treg paradigm was more evident in diabetic patients with the development of urinary albumin excretion (P < 0.05), although there were no significant differences in the proportions of Th1 and the ratio of Th1/Th2 cells between patients with normoalbuminuria and microalbuminuria. In contrast, there was no significant difference in the proportion of Th2 cells among patients with and without albuminuria (Figs. 1 and 2A and B).
The proportion of Th17 cells, as well as the ratio of Th17/Treg cells, were positively related to diabetes duration (Fig. 4D and F; P < 0.05), whereas the proportion of Treg cells was negatively related to diabetes duration (Fig. 4E; P < 0.05). Moreover, the data showed that the correlation between the ratio of Th17/Treg cells with diabetes duration was more significant than the correlations between the individual proportions of Th17 and Treg with diabetes duration (0.32 vs. 0.245 and 0.293). However, we found no significant relationships between diabetes duration with Th1 cells or Th2 cells (Fig. 4A and B; P > 0.05).
3.3. Correlations between the Th1/Th2/Th17/Treg paradigm with UACR or blood HbA1c levels UACR is the most common marker used in the clinic to measure diabetic nephropathy severity. We found the proportions of Th1 and Th17 cells were positively related to UACR (Fig. 3A and D; P < 0.05), while the proportion of Treg cells was negatively related to UACR (Fig. 3E; P < 0.05). Additionally, the ratio of Th17/Treg cells was positively related to UACR (Fig. 3F; P < 0.05). Moreover, the data showed that the correlation between UACR and the ratio of Th17/Treg cells was more significant than the correlations between UACR and the individual proportions of Th17 and Treg (0.753 vs. 0.571 and 0.706). However, there were no significant relationships between UACR and the proportion of Th2 cells or the ratio of Th1/Th2 cells (Fig. 3B, C; P > 0.05). HbA1c is the most common marker used in the clinic to measure the control of hyperglycemia. We evaluated the correlation of the Th1/Th2/Th17/Treg paradigm with the levels of blood HbA1c.
3.5. Expression profiles of serum cytokines and their correlations with UACR T2DN is associated with increased pro-inflammatory cytokine release [23]. The concentrations of serum cytokines including IL-2, IL-4, IL-6, IL-10, IL-17, IFN-c and TNF-a in 93 T2DM patients and 30 control subjects were detected using CBA. As seen in Fig 5, serum levels of IL-6, IL-17, IFN-c, TNF-a, IL-2 and IL-10 were increased in diabetic patients with the development of urinary albumin excretion, although no significant differences in IL-2 and IL-10 levels were observed between patients with normoalbuminuria and microalbuminuria. In contrast, there was no significant difference in serum IL-4 levels among patients with and without albuminuria. Furthermore, there were significant and positive correlations between serum concentrations of IL-6, IL-17, IFN-c, TNF-a, IL-2 and IL-10 with UACR (Fig. 6; P < 0.05). However, no such relationship was observed between serum IL-4 levels and UACR.
4. Discussion Beyond traditional metabolic and hemodynamic risk factors, recent studies have suggested that DN is now increasingly considered as an inflammatory process, and immune cells are involved in its development and progression [9,24]. T cells have been demonstrated to exert central roles in the development of T2DN.
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Fig. 1. Th1/Th2 paradigm in type 2 diabetic patients and normal control subjects. (A) Representative flow cytometry results of CD3+ CD8 IFN-c+, CD3+ CD8 IL-4+ cells from a single patient in each group. (B) The proportions of Th1 cells (CD3+ CD8- IFN-c+ cells) and Th2 cells (CD3+ CD8 IL-4+ cells) in each group. (C) Ratio of Th1/Th2 cells in each group. ⁄P < 0.05 vs. control, 4P < 0.05 vs. normoalbuminuria groups, #P < 0.05 vs. microalbuminuria groups.
Th1 cells, which produce large quantities of IFN-c, activate macrophages and promote the cell-mediated immunity. Th2 cells, which produce mainly IL-4 cytokines, suppress Th1 cell activation and contribute to humoral immunity [11]. Th1 and Th2-mediated immunity are reciprocally regulated and maintain a balance in immune-mediated disease [11]. It has been reported that Th1 cellular immunity, but not Th2 humoral immunity, in conjunction with Th1 and proinflammatory cytokines, predominantly mediate tissue injury in patients with DN [10]. Due to the discovery of Th17 and Treg cells, the Th1/Th2 paradigm has been expanded into the Th1/Th2/Th17/ Treg paradigm. Recently, Th17 and Treg cells, two novel T-cell subsets, have been demonstrated to originate from the same developmental lineage [25]. Th17/Treg intermediate cells will differentiate from the progenitor cells to Treg cells in the presence of transforming growth factor, to Th17 cells in the presence of IL-6 [26]. Th17 cells, which produce the hallmark cytokine IL-17A, were found playing vital roles in the pathogenesis of inflammatory and autoimmune diseases [27]. IL-17 has proinflammatory properties and plays a key role in neutrophil recruitment, activation and migration [28].
Increasing evidence has revealed the present of Th17 cells in type 1 DM in murine model and human type 1 DM [29–31]. Recently, T cells in T2DM patients have been shown to be skewed toward a proinflammatory phenotype that requires monocytes for maintenance and promotes chronic inflammation through increased IFN-c and IL17 production [32]. However, since serum levels of IL-17A are also elevated in patients without nephropathy, it is unlikely that IL17A is associated with nephropathic complications of T2DM [33]. The role of Th17 cells in T2DN still requires further evaluation. In contrast to Th17 cells, Treg cells exert important effects on the maintenance of immune tolerance, protecting the inflamed tissue against deleterious immune responses by down-regulating the function of innate and adaptive immune effector cells [34–36]. Dysfunction of CD4+CD25+ Tregs contributes to the development of autoimmune diseases in animals and human beings. A previous study has shown that CD4+CD25+Foxp3+ Treg cells may contribute to the development and progression of T2DN. However, the relationship between CD4+CD25+Foxp3+ Treg cells and pathogenesis of T2DN still requires further investigation [37].
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Fig. 2. Th17/Treg paradigm in type 2 diabetic patients and normal control subjects. (A) Representative flow cytometry results of CD3+ CD8 IL-17+, CD4+ CD25+ Foxp3+ cells from a single patient in each group. (B) The proportions of Th17 cells (CD3+ CD8 IL-17+ cells) and Treg cells (CD4+ CD25+ Foxp3+ cells) in each group. (C) Ratio of Th17/Treg cells in each group. ⁄P < 0.05 vs. control, 4P < 0.05 vs. normoalbuminuria groups, #P < 0.05 vs. microalbuminuria groups.
To the best of our knowledge, this is the first study which was performed to determine the Th1/Th2/Th17/Treg paradigm in T2DN. The present study showed that the level of CD4+CD25+ Foxp3+ Treg cells, but not Th2 cells, markedly decreased in the periphery of patents with T2DN, whereas Th1 and Th17 cells increased, indicating that the alteration of Th1/Th2/Th17/Treg paradigm exists in T2DN patients, which may contribute to immune activation and inflammation. Moreover, our data showed that UACR was positively related to the proportions of Th1 and Th17 cells, as well as the ratio of Th17/Treg cells, and negatively related to the proportions of Treg cells. The correlation between UACR and the ratio of Th17/Treg cells was more significant than the correlations between UACR and the individual proportions of Th17 and Treg. The results demonstrated that the relative ratio of Th17/Treg cells has more important roles in modulating the immunologic
microenvironment of diabetic nephropathy than the absolute proportions of Th17 and Treg cells. On the other hand, we found the proportions of Th1, Th2, Th17 cells, and Treg cells as well as the ratios of Th1/Th2 cells and Th17/Treg cells were not correlated with the blood HbA1c in T2DM patients with or without nephropathy (data not shown). Similar results were found in patents with T2DM [18]. Thus, hyperglycemia itself may not directly affect the Th1/Th2/Th17/Treg paradigm in patients with T2DN. Our study showed diabetes duration was positively related to the proportion of Th17 cells, and negatively related to the proportion of Treg cells. However, we found no significant relationships between diabetes duration with Th1 cells or Th2 cells. Longer disease is related to longer chronic inflammation, and so we speculate that it may have impact on the Th17/Treg paradigm rather than Th1/Th2 paradigm.
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Fig. 3. Correlations between the Th1/Th2/Th17/Treg paradigm and urine albumin:creatinine ratio (UACR) in all type 2 diabetes mellitus patients. Correlations were analyzed by Spearman’s rank correlation and regression tests. (A) Relationship between the proportion of Th1 cells with UACR. (B) Relationship between the proportion of Th2 cells with UACR. (C) Relationship between the ratio of Th1/Th2 cells with UACR. (D) Relationship between the proportion of Th17 cells with UACR. (E) Relationship between the proportion of Treg cells with UACR. (F) Relationship between the ratio of Th17/Treg cells with UACR. The p value and r value were indicated in the graphs.
Fig. 4. Correlations between the Th1/Th2/Th17/Treg paradigm and diabetes duration in type 2 diabetes mellitus patients. Correlations were analyzed by Spearman’s rank correlation and regression tests. (A) Relationship between the proportion of Th1 cells with diabetes duration. (B) Relationship between the proportion of Th2 cells with diabetes duration. (C) Relationship between the ratio of Th1/Th2 cells with diabetes duration. (D) Relationship between the proportion of Th17 cells with diabetes duration. (E) Relationship between the proportion of Treg cells with diabetes duration. (F) Relationship between the ratio of Th17/Treg cells with diabetes duration. The p value and r value were indicated in the graphs.
Human and mouse CD4+CD25+ Tregs have ability to induce M2 macrophage differentiation from monocytes [38–39]. Macrophage activation and accumulation may be the main causes for the development of diabetic complications [40–42]. Therefore, it is speculated that the decreased CD4+CD25+ Tregs and elevated ratio of Th17/Treg cells may exert less immunosuppressive effects on mono-
cytes or macrophages, and likely prompt an inflammatory state, which may contribute to the occurrence of diabetic complication. This hypothesis is supported by our present observation of elevated IL-6, IL-17, IFN-c and TNF-a levels in blood of patients with T2DN. The present study showed that serum levels of IL-6, IL-17, IFN-c, TNF-a, IL-2 and IL-10, but not IL-4, were increased in
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Fig. 5. Serum concentrations of cytokines in type 2 diabetic patients and normal control subjects. (A) The concentration of serum IFN-c. (B) The concentration of serum TNFa. (C) The concentration of serum IL-2. (D) The concentration of serum IL-6. (E) The concentration of serum IL-4. (F) The concentration of serum IL-10. (G) The concentration of serum IL-17. ⁄P < 0.05 vs. control, 4P < 0.05 vs. normoalbuminuria groups, #P < 0.05 vs. microalbuminuria groups.
Fig. 6. Correlations between serum cytokine concentrations with urine albumin:creatinine ratio (UACR) in type 2 diabetes mellitus patients. Correlations were analyzed by Spearman’s rank correlation and regression tests. (A) Relationship between the concentrations of IFN-c with UACR. (B) Relationship between the concentrations of TNF-a with UACR. (C) Relationship between the concentrations of IL-2 with UACR. (D) Relationship between the concentrations of IL-6 with UACR. (E) Relationship between the concentrations of IL-4 with UACR. (F) Relationship between the concentrations of IL-10 with UACR. (G) Relationship between the concentrations of IL-17 with UACR. The p value and r value were indicated in the graphs.
T2DN patients, and positively related to UACR. However, no significant difference in IL-2 and IL-10 levels was observed between patients with microalbuminuria and normoalbuminuria. Previous studies have shown that concentrations of pro-inflammatory cytokines were elevated in T2DN patients [23,43]. Some studies have also revealed elevated IL-10 levels in DN patients may indirectly promote the progression of DN [44,23,45]. All the above results indicate that inflammation participates in the pathogenesis of T2DN. Although multiple risk factors, such as genetic, metabolic, and inflammatory, contribute to the initiation and progression of both DM and DN, the pathogenesis of T2DN has been not fully under-
stood. Whether the alteration of Th1/Th2/Th17/Treg paradigm is just a consequence of T2DN or also a contributor to T2DN development needs to be identified. Based on the recent studies [10,46], there may be a bidirectional regulation loop between T2DN and CD4+ T cells during the development and progression of T2DN, possibly via inflammatory and anti-inflammatory cytokines. In conclusion, the present study demonstrated for the first time that the Th1/Th2/Th17/Treg paradigm skewed to Th1 and Th17 in T2DN patients, which may contribute to the enhanced immune activation and inflammation, and subsequent development and progression of T2DN. These findings may provide a better understanding of the pathogenesis of T2DN.
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Disclosure The authors declare no financial or commercial conflict of interest. Acknowledgments This work was supported by National Natural Science Foundation of China (No. 81171605). We are grateful to the Departments of Endocrinology and Nephrology for the peripheral blood samples, as well as to all the healthy donors. References [1] Caramori ML, Mauer M. Diabetes and nephropathy. Curr Opin Nephrol Hypertens 2003;12:273–82. [2] Remuzzi G, Schieppati A, Ruggenenti P. Clinical practice. Nephropathy in patients with type 2 diabetes. N Engl J Med 2002;346:1145–51. [3] Ritz E, Rychlik I, Locatelli F, Halimi S. End-stage renal failure in type 2 diabetes: a medical catastrophe of worldwide dimensions. Am J Kidney Dis 1999;34:795–808. [4] Wolf G, Ritz E. Diabetic nephropathy in type 2 diabetes prevention and patient management. J Am Soc Nephrol 2003;14:1396–405. [5] Cooper ME. Interaction of metabolic and haemodynamic factors in mediating experimental diabetic nephropathy. Diabetologia 2001;44:1957–72. [6] Gross JL, de Azevedo MJ, Silveiro SP, Canani LH, Caramori ML, Zelmanovitz T. Diabetic nephropathy: diagnosis, prevention, and treatment. Diabetes Care 2005;28:164–76. [7] Wolf G. New insights into the pathophysiology of diabetic nephropathy: from haemodynamics to molecular pathology. Eur J Clin Invest 2004;34:785–96. [8] Navarro-Gonzalez JF, Mora-Fernandez C. The role of inflammatory cytokines in diabetic nephropathy. J Am Soc Nephrol 2008;19:433–42. [9] Rivero A, Mora C, Muros M, Garcia J, Herrera H, Navarro-Gonzalez JF. Pathogenic perspectives for the role of inflammation in diabetic nephropathy. Clin Sci (Lond) 2009;116:479–92. [10] Wu CC, Chen JS, Lu KC, Chen CC, Lin SH, Chu P, et al. Aberrant cytokines/ chemokines production correlate with proteinuria in patients with overt diabetic nephropathy. Clin Chim Acta 2010;411:700–4. [11] Abbas AK, Murphy KM, Sher A. Functional diversity of helper T lymphocytes. Nature 1996;383:787–93. [12] Mosmann TR, Sad S. The expanding universe of T-cell subsets: Th1, Th2 and more. Immunol Today 1996;17:138–46. [13] Mosmann TR, Cherwinski H, Bond MW, Giedlin MA, Coffman RL. Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins. J Immunol 1986;136:2348–57. [14] Adkins B, Leclerc C, Marshall-Clarke S. Neonatal adaptive immunity comes of age. Nat Rev Immunol 2004;4:553–64. [15] Boissier MC, Assier E, Falgarone G, Bessis N. Shifting the imbalance from Th1/ Th2 to Th17/treg: the changing rheumatoid arthritis paradigm. Joint Bone Spine 2008;75:373–5. [16] Cheng X, Yu X, Ding YJ, Fu QQ, Xie JJ, Tang TT, et al. The Th17/Treg imbalance in patients with acute coronary syndrome. Clin Immunol 2008;127:89–97. [17] Ryba-Stanislawowska M, Skrzypkowska M, Mysliwiec M, Mysliwska J. Loss of the balance between CD4(+)Foxp3(+) regulatory T cells and CD4(+)IL17A(+) Th17 cells in patients with type 1 diabetes. Hum Immunol 2013;74:701–7. [18] Zeng C, Shi X, Zhang B, Liu H, Zhang L, Ding W, et al. The imbalance of Th17/ Th1/Tregs in patients with type 2 diabetes: relationship with metabolic factors and complications. J Mol Med (Berl) 2012;90:175–86. [19] Bettelli E, Oukka M, Kuchroo VK. T(H)-17 cells in the circle of immunity and autoimmunity. Nat Immunol 2007;8:345–50. [20] Sakaguchi S, Ono M, Setoguchi R, Yagi H, Hori S, Fehervari Z, et al. Foxp3+ CD25+ CD4+ natural regulatory T cells in dominant self-tolerance and autoimmune disease. Immunol Rev 2006;212:8–27. [21] Report of the Expert Committee on the Diagnosis and Classification of Diabetes Mellitus. Diabetes Care 1997;20:1183–97. [22] Bloomgarden ZT. American Diabetes Association annual meeting, 1997, and the Teczem Consultant Meeting. Diabetic nephropathy. Diabetes Care 1998;21:315–9.
[23] Wong CK, Ho AW, Tong PC, Yeung CY, Kong AP, Lun SW, et al. Aberrant activation profile of cytokines and mitogen-activated protein kinases in type 2 diabetic patients with nephropathy. Clin Exp Immunol 2007;149:123–31. [24] Navarro-Gonzalez JF, Mora-Fernandez C, Muros de Fuentes M, Garcia-Perez J. Inflammatory molecules and pathways in the pathogenesis of diabetic nephropathy. Nat Rev Nephrol 2011;7:327–40. [25] Harrington LE, Hatton RD, Mangan PR, Turner H, Murphy TL, Murphy KM, et al. Interleukin 17-producing CD4+ effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages. Nat Immunol 2005;6:1123–32. [26] Zheng SG, Wang J, Horwitz DA. Cutting edge: Foxp3+CD4+CD25+ regulatory T cells induced by IL-2 and TGF-beta are resistant to Th17 conversion by IL-6. J Immunol 2008;180:7112–6. [27] Crome SQ, Wang AY, Levings MK. Translational mini-review series on Th17 cells: function and regulation of human T helper 17 cells in health and disease. Clin Exp Immunol 2010;159:109–19. [28] Korn T, Bettelli E, Oukka M, Kuchroo VK. IL-17 and Th17 Cells. Annu Rev Immunol 2009;27:485–517. [29] Bending D, De la Pena H, Veldhoen M, Phillips JM, Uyttenhove C, Stockinger B, et al. Highly purified Th17 cells from BDC2.5NOD mice convert into Th1-like cells in NOD/SCID recipient mice. J Clin Invest 2009;119:565–72. [30] Bradshaw EM, Raddassi K, Elyaman W, Orban T, Gottlieb PA, Kent SC, et al. Monocytes from patients with type 1 diabetes spontaneously secrete proinflammatory cytokines inducing Th17 cells. J Immunol 2009;183:4432–9. [31] Honkanen J, Nieminen JK, Gao R, Luopajarvi K, Salo HM, Ilonen J, et al. IL-17 immunity in human type 1 diabetes. J Immunol 2010;185:1959–67. [32] Jagannathan-Bogdan M, McDonnell ME, Shin H, Rehman Q, Hasturk H, Apovian CM, et al. Elevated proinflammatory cytokine production by a skewed T cell compartment requires monocytes and promotes inflammation in type 2 diabetes. J Immunol 2011;186:1162–72. [33] Arababadi MK, Nosratabadi R, Hassanshahi G, Yaghini N, Pooladvand V, Shamsizadeh A, et al. Nephropathic complication of type-2 diabetes is following pattern of autoimmune diseases? Diabetes Res Clin Pract 2010;87:33–7. [34] Roncarolo MG, Battaglia M. Regulatory T-cell immunotherapy for tolerance to self antigens and alloantigens in humans. Nat Rev Immunol 2007;7:585–98. [35] Costantino CM, Baecher-Allan CM, Hafler DA. Human regulatory T cells and autoimmunity. Eur J Immunol 2008;38:921–4. [36] Bluestone JA, Tang Q. How do CD4+CD25+ regulatory T cells control autoimmunity? Curr Opin Immunol 2005;17:638–42. [37] Xu J, Su HL, Wang JH, Zhang CH. Role of CD4+CD25+Foxp3+ regulatory T cells in type 2 diabetic nephropathy. Nan Fang Yi Ke Da Xue Xue Bao 2009;29:137–9. [38] Tiemessen MM, Jagger AL, Evans HG, van Herwijnen MJ, John S, Taams LS. CD4+CD25+Foxp3+ regulatory T cells induce alternative activation of human monocytes/macrophages. Proc Natl Acad Sci USA 2007;104:19446–51. [39] Liu G, Ma H, Qiu L, Li L, Cao Y, Ma J, et al. Phenotypic and functional switch of macrophages induced by regulatory CD4+CD25+ T cells in mice. Immunol Cell Biol 2011;89:130–42. [40] Lopes-Virella MF, Virella G. The role of immune and inflammatory processes in the development of macrovascular disease in diabetes. Front Biosci 2003;8:s750–68. [41] Westcott DJ, Delproposto JB, Geletka LM, Wang T, Singer K, Saltiel AR, et al. MGL1 promotes adipose tissue inflammation and insulin resistance by regulating 7/4hi monocytes in obesity. J Exp Med 2009;206:3143–56. [42] Wueest S, Rapold RA, Schumann DM, Rytka JM, Schildknecht A, Nov O, et al. Deletion of Fas in adipocytes relieves adipose tissue inflammation and hepatic manifestations of obesity in mice. J Clin Invest 2010;120:191–202. [43] Moriwaki Y, Yamamoto T, Shibutani Y, Aoki E, Tsutsumi Z, Takahashi S, et al. Elevated levels of interleukin-18 and tumor necrosis factor-alpha in serum of patients with type 2 diabetes mellitus: relationship with diabetic nephropathy. Metabolism 2003;52:605–8. [44] Mysliwska J, Zorena K, Semetkowska-Jurkiewicz E, Rachon D, Suchanek H, Mysliwski A. High levels of circulating interleukin-10 in diabetic nephropathy patients. Eur Cytokine Netw 2005;16:117–22. [45] Zamauskaite A, Yaqoob MM, Madrigal JA, Cohen SB. The frequency of Th2 type cells increases with time on peritoneal dialysis in patients with diabetic nephropathy. Eur Cytokine Netw 1999;10:219–26. [46] Jefferson JA, Shankland SJ, Pichler RH. Proteinuria in diabetic kidney disease: a mechanistic viewpoint. Kidney Int 2008;74:22–36.
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The alteration of Th1/Th2/Th17/Treg paradigm in patients with type 2 diabetes mellitus: Relationship with diabetic nephropathy Cuiping Zhang a,1, Chunchun Xiao b, Peng Wang c, Wenhua Xu d, Aimei Zhang a, Qing Li a, Xiucai Xu a,⇑,1 a
Central Laboratory of Medical Research Center, Affiliated Provincial Hospital of Anhui Medical University, Hefei, Anhui 230001, PR China Department of Endocrinology, Affiliated Provincial Hospital of Anhui Medical University, Hefei, Anhui 230001, PR China c Department of Nephrology, Affiliated Provincial Hospital of Anhui Medical University, Hefei, Anhui 230001, PR China d Department of Neurology, Affiliated Provincial Hospital of Anhui Medical University, Hefei, Anhui 230001, PR China b
a r t i c l e
i n f o
Article history: Received 14 August 2013 Accepted 4 February 2014 Available online 12 February 2014
a b s t r a c t T cells have been demonstrated to exert central roles in the development of type 2 DN (T2DN). To explore whether Th1/Th2/Th17/Treg paradigm plays an important role in the development of T2DN, we investigated the proportions of Th1/Th2/Th17/Treg cells and serum levels of relevant cytokines in T2DM patients with various degrees of nephropathy and controls. Moreover, we analyzed the relationships between the Th1/Th2/Th17/Treg paradigm or relevant cytokines with urine albumin:creatinine ratio (UACR). Our study demonstrated that the Th1/Th2/Th17/Treg paradigm skewed to Th1 and Th17 in T2DN patients. UACR was positively related to the proportions of Th1 and Th17 cells, as well as the ratio of Th17:Treg cells, and negatively related to the proportions of Treg cells. Furthermore, serum levels of IL-6, IL-17, IFN-c, TNF-a, IL-2 and IL-10 were increased in T2DN patients, and positively related to UACR. These data indicate that the alteration of Th1/Th2/Th17/Treg paradigm exists in T2DN patients, which may contribute to the enhanced immune activation and inflammation, and subsequent development and progression of T2DN. These findings may provide one new approach to the underlying mechanisms of the development of T2DN. Ó 2014 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
1. Introduction Diabetes mellitus (DM) is an increasingly prevalent metabolic disease characterized by absolute or relative insulin deficiency, resulting in hyperglycemia as well as an altered metabolism of glucose, fat, and protein. Diabetic nephropathy (DN), affecting over one-third of patients with type 1 DM and approximately 25% of all patients with type 2 DM, is an extremely common and progressive complication of DM, and deeply contributes to patient morbidity and mortality [1–4]. Basic pathological features of DN are hypertrophy of glomerular structures, accompanied by thickening of the basement membranes and accumulation of extracellular matrix components. The etiology and pathogenetic mechanisms of DN have not been clearly elucidated. Traditionally, metabolic and hemodynamic factors are the major causes of renal lesions in patients with DM, considered as one nonimmune disease [5–7]. However, serial studies have shown that inflammation plays a vital role in the process of DN [8–9]. T cells were proposed to participate in the development and progression of type 2 DN (T2DN) [10]. ⇑ Corresponding author. Fax: +86 551 62283574. 1
E-mail address: [email protected] (X. Xu). These authors contributed equally to this work.
CD4+ T helper (Th) cells are believed to play central roles in modulating immune responses. Based on the cytokine profiles and effector function, CD4+ T cells can be classified into T-helper 1 (Th1) cell, Th2 cell, Th17 cell, and CD4+CD25+ T regulatory (Treg) cell subsets [11–12]. Th1 cells produce mainly interferon (IFN)-c and promote the cell-mediated immunity [13], whereas Th2 cells suppress Th1cell responses, and contribute to humoral immunity [14]. A recent study has shown that not only Th1/Th2 imbalance but also Th17/ Treg imbalance contributed to the pathogenesis of some autoimmune/inflammatory diseases, such as rheumatoid arthritis [15], acute coronary syndrome [16], and type 1 and type 2 diabetes [17–18]. Th17 cells produce IL-17, TNF-a and IL-6, and induce inflammation in the pathogenesis of autoimmune diseases [19]. Immunosuppressive Treg cells exert important effects on the maintenance of immune homeostasis and immune tolerance by producing anti-inflammatory cytokines, such as IL-10 and transforming growth factor-b [20]. Recently, it was reported that the imbalance of Th17/Th1/Tregs may contribute to the development of T2DM and complications [18]. To explore whether Th1/Th2/Th17/Treg paradigm plays an important role in the development of T2DN, we investigated the Th1/Th2/Th17/Treg paradigm and serum levels of relevant cytokines in T2DM patients with various degrees of nephropathy and controls. Moreover, the relationships between
http://dx.doi.org/10.1016/j.humimm.2014.02.007 0198-8859/Ó 2014 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
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the Th1/Th2/Th17/Treg paradigm or relevant cytokines with urine albumin:creatinine ratio (UACR) were analyzed. Information obtained from this study may provide a better understanding of the pathogenesis of T2DN. 2. Materials and methods 2.1. Patients This case-control hospital-based study was conducted between April 2011 and March 2013 at our hospital. Ninety-three adult patients with T2DM were recruited from the diabetes mellitus and Endocrine center. T2DM was diagnosed according to the criteria of the American Diabetes Association [21]. Age-matched control subjects had normal fasting blood glucose and no clinical diagnosed diseases, and lacked family history of T2DM. This study was cross-sectional and blinded. All donors were nonsmokers. Albumin excretion in spot urine samples was evaluated on at least two separate occasions and the patients were divided further into three groups according to the definition of abnormalities of albumin excretion advocated by the American Diabetes Association [22]. A urine albumin:creatinine ratio (UACR) of less than 30 mg/g Cr was defined as normoalbuminuria, a UACR of 30–300 mg/g Cr as microalbuminuria, and a UACR of greater than 300 mg/g Cr as macroalbuminuria. The patients with DN enrolled in our study were characterized as a UACR above 30 mg/g Cr and were diagnosed by excluding other causes of glomerulonephritis and by the presence of characteristic pathologic findings of renal biopsy. No patient was on treatment with anti-inflammatory drugs and/or immunosuppressive agents known to interfere with the immune system prior to the examination. The patients with hypertension were receiving calcium channel blocker (CCB) inhibitors. In all groups, patients with cardiovascular disease had been excluded from our study. None had glomerulonephritis, chronic renal failure, congestive heart failure, hypo and hyperthyroidism, acute or chronic infectious disease, other inflammatory disease, or chronic-immunemediated disorders. The study was approved by the ethics committee of Anhui provincial hospital, and written informed consent was obtained from all participants before participation.
2.4. Flow cytometric staining and analysis For Treg analysis, cell surface staining was performed with fluorescein isothiocyanate (FITC)-conjugated anti-human CD4 (13B8.2 clone; Beckman Coulter-Immunotech, Marseille, France), phycoerythrin (PE)-conjugated anti-human CD25 (B1.49.9 clone, Beckman Coulter-Immunotech) and their appropriate isotype controls for 20 min at 4 °C in the dark. After being washed with ice-cold phosphate buffered solution (PBS), cells were fixed and permeabilized with the Fix/Perm reagent (eBioscience, San Diego, CA, USA), washed twice with ice-cold permeabilization buffer (eBioscience), and incubated with allophycocyanin (APC)-conjugated anti-human Foxp3 (PCH101 clone, eBioscience) or its isotype control antibody. Cells were then washed again with ice-cold permeabilization buffer and analyzed by FCM. For intracellular cytokine analysis, cells were stained with FITC-conjugated anti-human CD3 (UCHT clone, Beckman Coulter-Immunotech) and Percp-cy5.5-conjugated anti-human CD8 (RPA-T8 clone, eBioscience) for 20 min at room temperature, fixed and permeabilized with Fix/Perm solution (Beckman Coulter-Immunotech), and stained with PE-conjugated anti-human IFN-c (4S.B3 clone, eBioscience), APC-conjugated anti-human IL-4 (8D4–8 Clone, eBioscience) or PE-conjugated anti-human IL-17A (eBio64CAP17 clone, eBioscience). Stained cells were assayed using a FACSCanto™II flow cytometry (BD Biosciences, San Jose, CA, USA) and then analyzed with FACSDiva 6.0 software. The frequency of Th1 (CD3+ CD8 IFN-c+), Th2 (CD3+ CD8 IL-4+) and Th17 (CD3+ CD8 IL-17+) was expressed as a percentage of CD3+ CD8 T cells by sequential gating on lymphocytes and CD3+ CD8 T cells, while the frequency of Treg (CD4+ CD25+ Foxp3+) expressed as a percentage of CD4+ T cells by sequential gating on lymphocytes and CD4+ T cells. 2.5. Cytokine measurement Cytokine profiles in serum were detected using human Th1/ Th2/Th17 cytometric bead array (CBA) kit, according to the manufacturer’s instructions (BD Biosciences). The minimum detectable concentrations of IL-2, IL-4, IL-6, IL-10, TNF-a, IFN-c and IL-17A were 2.6, 4.9, 2.4, 4.5, 3.8, 3.7, and 18.9 pg/ml, respectively. Intraand inter-assay coefficients of variation for all these detections were <5%. All samples were examined in duplicate. 2.6. Statistical analysis
2.2. Blood samples Blood samples (5–10 ml) were collected from all the participants in a fasting state in the morning. Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized venous blood by Ficoll density gradient for analysis of flow cytometry (FCM). Serum was separated from the blood samples and stored at 80 °C until analyzed. 2.3. Cell preparation and Th cells analysis For the analysis of Th1, Th2 and Th17 cells, PBMCs (2 106 cells/ ml) were suspended in complete culture medium (400 ll RPMI 1640 supplemented with 100 lg/ml streptomycin, 100 U/mL penicillin and 10% heat-inactivated fetal calf serum; Gibco BRL, USA). The cells were stimulated with 100 ng/mL of phorbol myristate acetate – PMA (Sigma–Aldrich, USA) plus 1 lg/mL of ionomycin (Enzo Life Sciences, Inc., USA) for 4 h, in the presence of 10 lg/mL of brefeldin A (ENZO, USA). After 4 h of culture in 37 °C under a 5% CO2 environment, the contents of the wells were transferred to 5 ml sterile tubes and centrifuged at 1200 rpm for 5 min. For Treg analysis, 100 ll of PBMCs (1 106) was added to sterile tubes for flow cytometric staining.
Values were mainly expressed as the mean ± standard deviation (SD). Data were analyzed using statistical software (SPSS 16.0; LEAD Technologies, Inc., Chicago, IL, USA). For a comparison of the different subgroups of the T2DM patients and the control subjects, the data were first analyzed by One-way analysis of variance (ANOVA). Comparison between the two groups was carried out with LSD test. Correlations between two variables were assessed with Spearman’s rank correlation coefficient test and regression tests. A p value <0.05 was considered to be statistically significant. 3. Results 3.1. Patient characteristics As shown in Table 1, there were no significant differences in age, gender, or high-density lipoprotein-cholesterol (HDL-C) concentrations between the diabetic patients and control subjects. Furthermore, no significant differences in fasting plasma glucose (FPG), hemoglobin A1c (HbA1c) and body mass index (BMI) were observed among patients with and without albuminuria. Systolic and diastolic blood pressures of our patients were within the range of the reference values due to the medical treatment.
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C. Zhang et al. / Human Immunology 75 (2014) 289–296 Table 1 Clinical characteristics of all type 2 diabetes mellitus patients. Item
Control (n = 30)
Normoalbuminuria (n = 32)
Microalbuminuria (n = 30)
Macroalbuminuria (n = 31)
Age Gender (male/female) Patients with hypertension Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) TC (mmol/L) TG (mmol/L) HDL-C (mmol/L) LDL-C (mmol/L) VLDL-C (mmol/L) FPG (mmol/L) HbA1c (%) Duration (years) BMI (kg/m2) Serum Cr (lmol/L) UACR (mg/g)
56.9 ± 6.93 20/10 0 120 ± 2.64 76 ± 1.23 4.77 ± 0.46 1.42 ± 0.28 1.35 ± 0.19 3.48 ± 0.47 0.69 ± 0.15 5.06 ± 0.63 5.05 ± 0.62 – 22.12 ± 1.41 83.78 ± 18.29 –
58.47 ± 9.76 22/10 20 125 ± 1.41 80 ± 1.58 4.83 ± 0.93 2.39 ± 0.49* 1.26 ± 0.3 3.6 ± 0.71 0.65 ± 0.12 8.88 ± 2.34* 8.05 ± 1.05* 5.06 ± 2.64 25.18 ± 1.71* 86.96 ± 15.59 19.13 ± 5.17
60.13 ± 11.07 22/8 23 130 ± 2.77 80 ± 1.77 5.06 ± 0.48 2.24 ± 0.61* 1.18 ± 0.13 3.54 ± 0.96 0.65 ± 0.11 8.62 ± 1.78* 8.01 ± 1.24* 11.3 ± 4.37** 25.76 ± 2.28* 96.38 ± 16.52* 86.85 ± 54.52**
56.74 ± 10.13 16/15 25 135 ± 2.43 84 ± 1.43 6.71 ± 0.49**** 3.08 ± 0.54**** 1.22 ± 0.33 4.41 ± 1.41**** 0.82 ± 0.19**** 8.79 ± 1.76* 8.02 ± 1.92* 10 ± 4.44** 24.92 ± 2.53* 150.2 ± 25.06**** 1006.59 ± 563.36***
Values are expressed as mean ± SD. TC: total cholesterol; TG: total triglyceride; HDL-C: high-density lipoprotein-cholesterol; LDL-C: low-density lipoprotein-cholesterol; VLDL-C: very low-density lipoprotein-cholesterol; FPG: fasting plasma glucose; HbA1c: hemoglobin A1c; Duration: diabetes duration; BMI: body mass index; Cr: creatinine; UACR: urine albumin: creatinine ratio. * P < 0.05 vs. control. ** P < 0.05 vs. normoalbuminuria groups. *** P < 0.05 vs. normoalbuminuria and microalbuminuria groups. **** P < 0.05 vs. control, normoalbuminuria and microalbuminuria groups.
3.2. Th1/Th2/Th17/Treg paradigm skewed to Th1 and Th17 in patients with T2DN
3.4. Correlation between the Th1/Th2/Th17/Treg paradigm with the duration of the diabetic disease
Flow cytometry was used to determine the Th1/Th2/Th17/Treg paradigm in T2DM patients with various degrees of nephropathy. Compared to control subjects, the proportions of Th1 (CD3+ CD8 IFN-c+) and Th17 (CD3+ CD8 IL-17A+) cells increased in diabetic patients with or without albuminuria, while the proportion of Treg (CD4+ CD25+ Foxp3+) cells, but not Th2 (CD3+ CD8 IL-4+) cells, was markedly decreased (Figs. 1 and 2A and B), resulting in elevated ratios of Th1/Th2 cells and Th17/Treg cells (Figs. 1 and 2C). Moreover, the alteration of Th1/Th2/Th17/Treg paradigm was more evident in diabetic patients with the development of urinary albumin excretion (P < 0.05), although there were no significant differences in the proportions of Th1 and the ratio of Th1/Th2 cells between patients with normoalbuminuria and microalbuminuria. In contrast, there was no significant difference in the proportion of Th2 cells among patients with and without albuminuria (Figs. 1 and 2A and B).
The proportion of Th17 cells, as well as the ratio of Th17/Treg cells, were positively related to diabetes duration (Fig. 4D and F; P < 0.05), whereas the proportion of Treg cells was negatively related to diabetes duration (Fig. 4E; P < 0.05). Moreover, the data showed that the correlation between the ratio of Th17/Treg cells with diabetes duration was more significant than the correlations between the individual proportions of Th17 and Treg with diabetes duration (0.32 vs. 0.245 and 0.293). However, we found no significant relationships between diabetes duration with Th1 cells or Th2 cells (Fig. 4A and B; P > 0.05).
3.3. Correlations between the Th1/Th2/Th17/Treg paradigm with UACR or blood HbA1c levels UACR is the most common marker used in the clinic to measure diabetic nephropathy severity. We found the proportions of Th1 and Th17 cells were positively related to UACR (Fig. 3A and D; P < 0.05), while the proportion of Treg cells was negatively related to UACR (Fig. 3E; P < 0.05). Additionally, the ratio of Th17/Treg cells was positively related to UACR (Fig. 3F; P < 0.05). Moreover, the data showed that the correlation between UACR and the ratio of Th17/Treg cells was more significant than the correlations between UACR and the individual proportions of Th17 and Treg (0.753 vs. 0.571 and 0.706). However, there were no significant relationships between UACR and the proportion of Th2 cells or the ratio of Th1/Th2 cells (Fig. 3B, C; P > 0.05). HbA1c is the most common marker used in the clinic to measure the control of hyperglycemia. We evaluated the correlation of the Th1/Th2/Th17/Treg paradigm with the levels of blood HbA1c.
3.5. Expression profiles of serum cytokines and their correlations with UACR T2DN is associated with increased pro-inflammatory cytokine release [23]. The concentrations of serum cytokines including IL-2, IL-4, IL-6, IL-10, IL-17, IFN-c and TNF-a in 93 T2DM patients and 30 control subjects were detected using CBA. As seen in Fig 5, serum levels of IL-6, IL-17, IFN-c, TNF-a, IL-2 and IL-10 were increased in diabetic patients with the development of urinary albumin excretion, although no significant differences in IL-2 and IL-10 levels were observed between patients with normoalbuminuria and microalbuminuria. In contrast, there was no significant difference in serum IL-4 levels among patients with and without albuminuria. Furthermore, there were significant and positive correlations between serum concentrations of IL-6, IL-17, IFN-c, TNF-a, IL-2 and IL-10 with UACR (Fig. 6; P < 0.05). However, no such relationship was observed between serum IL-4 levels and UACR.
4. Discussion Beyond traditional metabolic and hemodynamic risk factors, recent studies have suggested that DN is now increasingly considered as an inflammatory process, and immune cells are involved in its development and progression [9,24]. T cells have been demonstrated to exert central roles in the development of T2DN.
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Fig. 1. Th1/Th2 paradigm in type 2 diabetic patients and normal control subjects. (A) Representative flow cytometry results of CD3+ CD8 IFN-c+, CD3+ CD8 IL-4+ cells from a single patient in each group. (B) The proportions of Th1 cells (CD3+ CD8- IFN-c+ cells) and Th2 cells (CD3+ CD8 IL-4+ cells) in each group. (C) Ratio of Th1/Th2 cells in each group. ⁄P < 0.05 vs. control, 4P < 0.05 vs. normoalbuminuria groups, #P < 0.05 vs. microalbuminuria groups.
Th1 cells, which produce large quantities of IFN-c, activate macrophages and promote the cell-mediated immunity. Th2 cells, which produce mainly IL-4 cytokines, suppress Th1 cell activation and contribute to humoral immunity [11]. Th1 and Th2-mediated immunity are reciprocally regulated and maintain a balance in immune-mediated disease [11]. It has been reported that Th1 cellular immunity, but not Th2 humoral immunity, in conjunction with Th1 and proinflammatory cytokines, predominantly mediate tissue injury in patients with DN [10]. Due to the discovery of Th17 and Treg cells, the Th1/Th2 paradigm has been expanded into the Th1/Th2/Th17/ Treg paradigm. Recently, Th17 and Treg cells, two novel T-cell subsets, have been demonstrated to originate from the same developmental lineage [25]. Th17/Treg intermediate cells will differentiate from the progenitor cells to Treg cells in the presence of transforming growth factor, to Th17 cells in the presence of IL-6 [26]. Th17 cells, which produce the hallmark cytokine IL-17A, were found playing vital roles in the pathogenesis of inflammatory and autoimmune diseases [27]. IL-17 has proinflammatory properties and plays a key role in neutrophil recruitment, activation and migration [28].
Increasing evidence has revealed the present of Th17 cells in type 1 DM in murine model and human type 1 DM [29–31]. Recently, T cells in T2DM patients have been shown to be skewed toward a proinflammatory phenotype that requires monocytes for maintenance and promotes chronic inflammation through increased IFN-c and IL17 production [32]. However, since serum levels of IL-17A are also elevated in patients without nephropathy, it is unlikely that IL17A is associated with nephropathic complications of T2DM [33]. The role of Th17 cells in T2DN still requires further evaluation. In contrast to Th17 cells, Treg cells exert important effects on the maintenance of immune tolerance, protecting the inflamed tissue against deleterious immune responses by down-regulating the function of innate and adaptive immune effector cells [34–36]. Dysfunction of CD4+CD25+ Tregs contributes to the development of autoimmune diseases in animals and human beings. A previous study has shown that CD4+CD25+Foxp3+ Treg cells may contribute to the development and progression of T2DN. However, the relationship between CD4+CD25+Foxp3+ Treg cells and pathogenesis of T2DN still requires further investigation [37].
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Fig. 2. Th17/Treg paradigm in type 2 diabetic patients and normal control subjects. (A) Representative flow cytometry results of CD3+ CD8 IL-17+, CD4+ CD25+ Foxp3+ cells from a single patient in each group. (B) The proportions of Th17 cells (CD3+ CD8 IL-17+ cells) and Treg cells (CD4+ CD25+ Foxp3+ cells) in each group. (C) Ratio of Th17/Treg cells in each group. ⁄P < 0.05 vs. control, 4P < 0.05 vs. normoalbuminuria groups, #P < 0.05 vs. microalbuminuria groups.
To the best of our knowledge, this is the first study which was performed to determine the Th1/Th2/Th17/Treg paradigm in T2DN. The present study showed that the level of CD4+CD25+ Foxp3+ Treg cells, but not Th2 cells, markedly decreased in the periphery of patents with T2DN, whereas Th1 and Th17 cells increased, indicating that the alteration of Th1/Th2/Th17/Treg paradigm exists in T2DN patients, which may contribute to immune activation and inflammation. Moreover, our data showed that UACR was positively related to the proportions of Th1 and Th17 cells, as well as the ratio of Th17/Treg cells, and negatively related to the proportions of Treg cells. The correlation between UACR and the ratio of Th17/Treg cells was more significant than the correlations between UACR and the individual proportions of Th17 and Treg. The results demonstrated that the relative ratio of Th17/Treg cells has more important roles in modulating the immunologic
microenvironment of diabetic nephropathy than the absolute proportions of Th17 and Treg cells. On the other hand, we found the proportions of Th1, Th2, Th17 cells, and Treg cells as well as the ratios of Th1/Th2 cells and Th17/Treg cells were not correlated with the blood HbA1c in T2DM patients with or without nephropathy (data not shown). Similar results were found in patents with T2DM [18]. Thus, hyperglycemia itself may not directly affect the Th1/Th2/Th17/Treg paradigm in patients with T2DN. Our study showed diabetes duration was positively related to the proportion of Th17 cells, and negatively related to the proportion of Treg cells. However, we found no significant relationships between diabetes duration with Th1 cells or Th2 cells. Longer disease is related to longer chronic inflammation, and so we speculate that it may have impact on the Th17/Treg paradigm rather than Th1/Th2 paradigm.
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Fig. 3. Correlations between the Th1/Th2/Th17/Treg paradigm and urine albumin:creatinine ratio (UACR) in all type 2 diabetes mellitus patients. Correlations were analyzed by Spearman’s rank correlation and regression tests. (A) Relationship between the proportion of Th1 cells with UACR. (B) Relationship between the proportion of Th2 cells with UACR. (C) Relationship between the ratio of Th1/Th2 cells with UACR. (D) Relationship between the proportion of Th17 cells with UACR. (E) Relationship between the proportion of Treg cells with UACR. (F) Relationship between the ratio of Th17/Treg cells with UACR. The p value and r value were indicated in the graphs.
Fig. 4. Correlations between the Th1/Th2/Th17/Treg paradigm and diabetes duration in type 2 diabetes mellitus patients. Correlations were analyzed by Spearman’s rank correlation and regression tests. (A) Relationship between the proportion of Th1 cells with diabetes duration. (B) Relationship between the proportion of Th2 cells with diabetes duration. (C) Relationship between the ratio of Th1/Th2 cells with diabetes duration. (D) Relationship between the proportion of Th17 cells with diabetes duration. (E) Relationship between the proportion of Treg cells with diabetes duration. (F) Relationship between the ratio of Th17/Treg cells with diabetes duration. The p value and r value were indicated in the graphs.
Human and mouse CD4+CD25+ Tregs have ability to induce M2 macrophage differentiation from monocytes [38–39]. Macrophage activation and accumulation may be the main causes for the development of diabetic complications [40–42]. Therefore, it is speculated that the decreased CD4+CD25+ Tregs and elevated ratio of Th17/Treg cells may exert less immunosuppressive effects on mono-
cytes or macrophages, and likely prompt an inflammatory state, which may contribute to the occurrence of diabetic complication. This hypothesis is supported by our present observation of elevated IL-6, IL-17, IFN-c and TNF-a levels in blood of patients with T2DN. The present study showed that serum levels of IL-6, IL-17, IFN-c, TNF-a, IL-2 and IL-10, but not IL-4, were increased in
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Fig. 5. Serum concentrations of cytokines in type 2 diabetic patients and normal control subjects. (A) The concentration of serum IFN-c. (B) The concentration of serum TNFa. (C) The concentration of serum IL-2. (D) The concentration of serum IL-6. (E) The concentration of serum IL-4. (F) The concentration of serum IL-10. (G) The concentration of serum IL-17. ⁄P < 0.05 vs. control, 4P < 0.05 vs. normoalbuminuria groups, #P < 0.05 vs. microalbuminuria groups.
Fig. 6. Correlations between serum cytokine concentrations with urine albumin:creatinine ratio (UACR) in type 2 diabetes mellitus patients. Correlations were analyzed by Spearman’s rank correlation and regression tests. (A) Relationship between the concentrations of IFN-c with UACR. (B) Relationship between the concentrations of TNF-a with UACR. (C) Relationship between the concentrations of IL-2 with UACR. (D) Relationship between the concentrations of IL-6 with UACR. (E) Relationship between the concentrations of IL-4 with UACR. (F) Relationship between the concentrations of IL-10 with UACR. (G) Relationship between the concentrations of IL-17 with UACR. The p value and r value were indicated in the graphs.
T2DN patients, and positively related to UACR. However, no significant difference in IL-2 and IL-10 levels was observed between patients with microalbuminuria and normoalbuminuria. Previous studies have shown that concentrations of pro-inflammatory cytokines were elevated in T2DN patients [23,43]. Some studies have also revealed elevated IL-10 levels in DN patients may indirectly promote the progression of DN [44,23,45]. All the above results indicate that inflammation participates in the pathogenesis of T2DN. Although multiple risk factors, such as genetic, metabolic, and inflammatory, contribute to the initiation and progression of both DM and DN, the pathogenesis of T2DN has been not fully under-
stood. Whether the alteration of Th1/Th2/Th17/Treg paradigm is just a consequence of T2DN or also a contributor to T2DN development needs to be identified. Based on the recent studies [10,46], there may be a bidirectional regulation loop between T2DN and CD4+ T cells during the development and progression of T2DN, possibly via inflammatory and anti-inflammatory cytokines. In conclusion, the present study demonstrated for the first time that the Th1/Th2/Th17/Treg paradigm skewed to Th1 and Th17 in T2DN patients, which may contribute to the enhanced immune activation and inflammation, and subsequent development and progression of T2DN. These findings may provide a better understanding of the pathogenesis of T2DN.
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