Regulatory T Cells and Diabetes Mellitus

Abstract

Immune system dysfunction causes dysregulation of immune homeostasis, which in turn leads to autoimmune diseases. Regulatory T cells (Tregs) are a specialized T cell subpopulation that maintain peripheral tolerance and immune homeostasis. Diabetic patients are at an increased risk of developing cardiovascular diseases; thus, in terms of coronary risk, diabetes mellitus (DM) is considered coronary heart disease equivalent. Accumulating evidence indicates that Tregs play an important role in protecting against the development of various cardiovascular diseases. In this review, we provide an overview of the role of Tregs in the pathogenesis of DM, including type 1 DM, type 2 DM, latent autoimmune diabetes of adults, and gestational DM. In addition, we discuss the role of Tregs in diabetic complications, including cardiovascular diseases, nephropathy, neuropathy, and retinopathy. Tregs play a beneficial role in the pathogenesis of DM and diabetic complications, although the precise molecular mechanisms underlying the protective effect of Tregs against DM are still obscure. Collectively, modification of Tregs may provide a promising and novel future strategy for the prevention and therapy of DM and diabetic complications.

Introduction

Diabetes mellitus (DM) has become a major threat to global public health, with increased morbidity and mortality in the past several years. CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs), which constitute a specialized T cell subpopulation, maintain peripheral tolerance and immune homeostasis.¹ In recent years, the value of immunotherapy for treating autoimmune diseases has been increasingly recognized; additionally, the therapy has shown promise for treating DM as well. Thus, considering the important role of Tregs in immunity, we aimed to discuss the role of Tregs in DM.

Type 1 DM

Type 1 DM (T1DM) is a severe chronic autoimmune disease and its autoimmune nature has been widely established.² Innate and adaptive immunity are implicated in the onset and development of T1DM, with the infiltration of immune cells, including T lymphocytes, dendritic cells (DCs), macrophages, and B lymphocytes, which affect pancreatic β cells and participate in immune regulation of the disease.³ Most research has considered the destruction of insulin-secreting pancreatic β cells, caused by autoimmunity, as the main pathogenesis of T1DM.² Aberrant activation of effector T cells (Teffs) has been implicated in the pathogenesis of T1DM and considered the principal driver of disease initiation.

Chronic inflammation not only promotes the early induction and augmentation of immune assault against pancreatic β cells but also decreases their frequency and function.⁴ In prediabetic mice and subjects newly diagnosed with T1DM, islets are infiltrated by activated inflammatory cells, including macrophages and Teffs.⁵ Islet-associated macrophages produce proinflammatory cytokines that induce the activation of T cells and mediate β cell destruction and apoptosis, which results in autoimmune diabetes in the nonobese diabetic (NOD) mouse models, whereas the inactivation of macrophages prevents disease progression in NOD mice.^6,7 Inflammatory cytokine-mediated destruction of pancreatic β cells is considered a key pathological event in T1DM pathogenesis.^8,9

In the past decade, the association of Tregs with T1DM pathogenesis has been the focus in T1DM research. Treg treatment can effectively suppress the activation and infiltration of Teffs and macrophages and inhibit proinflammatory cytokine production and local inflammatory response.^10,11 A series of studies have documented that Tregs prolong the survival of pancreatic islets, and highlighted the importance of Tregs in limiting T1DM.¹² Deletion of Tregs in mice with anti-CD25 antibody (PC61) injection abrogates the protective role of vaccines and induces T1DM progression.¹³

Increasing evidence has revealed that aberrant levels and function of Tregs contribute to autoimmune destruction, which in turn leads to the pathogenesis of T1DM.¹⁴ T1DM progression is facilitated by reduction in the number and suppressive activity of Tregs.¹⁵ The frequency of Tregs in peripheral blood is decreased in T1DM patients compared with that in healthy controls.^16,17 Long-term T1DM patients reportedly showed a further decrease in Tregs as compared with newly onset T1DM patients.¹⁸ Moreover, number of Tregs was significantly reduced in T1DM children with uncontrolled (hemoglobin A1c ≥7%) versus controlled disease (hemoglobin A1c <7%).¹⁹ In addition, Treg frequency was lower in NOD than in nondiabetes mice.^20,21 The ratio of CD4⁺ Teffs and Tregs progressively increased in the inflamed islets of NOD mice, and this ratio imbalance contributed to the development of T1DM.²² However, Grant et al. described that normal or increased circulating Treg frequencies were observed relative to health in some autoimmune disease. One potential explanation is a lack of standardization between studies due to the absence of a uniformly recognized marker that was able to reliably define a homogeneous human Treg population. Moreover, it was also important to point out that Treg frequencies were influenced by disease stage and treatment regimen.²³

Dysfunctional Tregs have been considered a central factor in the progressive breakdown of self-tolerance and the T1DM process.²² The suppressive activity of Tregs on Teffs is attenuated in T1DM patients as compared with that in healthy controls, with the insufficient control of autoreactive T cells by Tregs contributing to the pathogenesis of T1DM.²⁴ Meanwhile, in the mouse models of T1DM, the suppressor capacity of Tregs is reduced²⁵ and the progression of autoimmune diabetes is associated with impaired suppressive function of Tregs.²⁶ These findings suggest that the deficiency or dysfunction of Tregs is associated with the development of T1DM in both T1DM patients and mouse models.

Restoring Tregs to Treat T1DM

Considering Treg insufficiency in T1DM and the role of Teffs in destroying insulin-producing β cells, any efforts to restore and induce functional Tregs, suppress the diabetogenic T cell response, and reset the balance between Teffs and Tregs may protect the remaining β cells and prevent disease progression. Several lines of evidence have indicated the potential therapeutic use of Tregs in T1DM, and increasing number and function of Tregs has been the goal of various strategies used in T1DM therapy (Fig. 1).

Figure 1.

Underlying mechanisms of Tregs in T1DM. Tregs suppress the activation and infiltration of Teffs and macrophages and prevent proinflammatory cytokine production through direct cell-to-cell contact and production of immunosuppression cytokines such as TGF-β, IL-10, and IL-35. In addition, Tregs inhibit B cells and reduce inflammatory DC subset number and their cytokine levels. DC, dendritic cell; IL-10, interleukin-10; IL-35, interleukin-35; T1DM, type 1 diabetes mellitus; TGF-β, transforming growth factor-β; Teffs, effector T cells; Tregs, regulatory T cells. Color images are available online.

Exogenous Treg infusion is a direct method to selectively increase number of Tregs and proportion in vivo. Previous animal studies have shown that Tregs injected into host mice could suppress Teff activation and protect against the development of diabetes.²⁷ Treg therapy to restore immune balance in the clinical application of T1DM has also been reported. In a human experiment, the administration of Tregs preserved β cell function in T1DM children within 2 months after diagnosis, which suggested that Treg infusion prolonged remission in recent-onset T1DM.¹⁵ In another study, 12 T1DM children received an infusion of autologous expanded ex vivo Tregs, which prolonged the survival of pancreatic islet β cells and resulted in a lower requirement for exogenous insulin in the majority of patients at 1 year, with two children having absolutely no insulin requirement.¹² Encouraging evidence documented that isolation, expansion, and reinfusion of autologous Tregs in T1DM patients conferred a long-term survival in vivo (>1 year after transfer).²⁸ Importantly, Treg administration was safe with no infusion reactions or other cell therapy-related high-grade adverse events occurring in the donors.²⁸ Therefore, these findings supported the therapeutic effect of Tregs in T1DM and showed that expanding number of Tregs by direct infusion is feasible and promising.

Some strategies focused on efficient Treg induction through multiple pharmacological methods to treat diabetes. Interleukin (IL)-2 is essential for Treg generation and maintaining Treg suppressive function.²⁹ T1DM is associated with reduced IL-2 signaling, and altered IL-2 signaling is a typical feature in long-term T1DM patients.¹⁸ T1DM patients present an increased apoptosis of peripheral Tregs, partially mediated by IL-2 deprivation.³⁰ Low-dose IL-2 administration reportedly increased number of Tregs and improved Treg function to limit the activation of Teffs, and led to diabetes prevention and reversal in mouse models.^31,32 Clinical outcome for T1DM patients using IL-2 therapy has also been observed in prior studies. In a single-center clinical study to establish an effective dose of IL-2 for application in T1DM, 24 adult patients (18–55 years) with established T1DM were divided into control and IL-2 groups who received different doses of IL-2.³³ IL-2 treatment increased Treg proportion dose dependently, and IL-2-treated patients did not exhibit any severe adverse effects or detrimental changes in glucose metabolism variables, which suggested the efficiency and safety of using these levels of IL-2 in T1DM patients.³³

In addition, high-dose vitamin D administration for 3 months enhanced the number of Tregs in male T1DM patients, which was associated with improved HbA1C value and decreased insulin demands.³⁴ Cholecalciferol supplementation significantly increased the suppressive activity of Tregs in young patients with T1DM and might serve as a potential candidate for developing immunomodulatory combination therapy for T1DM.³⁵ Tregs expansion by the glucocorticoid-induced TNFR-related (Gitr) costimulatory receptor could delay/prevent DM development in NOD mice.³⁶ Therefore, providing a long-term homeostatic immune regulation by increasing the Treg frequency may be an effective treatment for T1DM.

Type 2 DM

Although autoimmunity is a well-known pathogenic component in T1DM, the hypothesis that the progression of type 2 DM (T2DM) also features autoimmunity is increasingly being accepted. T2DM affects different components of the immune system, and immune activation may precede the manifestation of T2DM. The conclusion of immune disorders being associated with T2DM progress has been accepted and the role of Tregs in T2DM has been subject to intensive research.

Insulin resistance is a core element of T2DM.³⁷ It is believed that the deficiency and dysfunction of Tregs are correlated with insulin resistance. In mouse models, decreased number of Tregs culminated in insulin resistance.³⁸ Deleting Tregs by using anti-CD25 antibody in a db/db mouse model exaggerated insulin resistance, as evidenced by enhanced fasting blood glucose and impaired insulin sensitivity, whereas adoptive transfer of Tregs improved insulin sensitivity.³⁹ Ample evidence has revealed that Tregs primarily protect against insulin resistance,⁴⁰ and that they might participate in the pathogenesis of T2DM.³⁸

T2DM is a chronic inflammatory disease. T2DM patients show increased levels of circulating proinflammatory cytokines (IFN-γ and IL-17), which are associated with the inflammatory milieu of T2DM.⁴¹ Increased proinflammatory cytokine secretion both precedes and maintains insulin resistance, whereas suppressed proinflammatory cytokine production protects rodents against insulin resistance.⁴¹ Tregs inhibit the recruitment and activation of Teffs and macrophages and decrease proinflammatory cytokine production, thus counteracting the local inflammatory response.⁴² Feuerer et al. reported that Tregs influenced the inflammatory state of adipose tissues and improved metabolic parameters, thus regulating and prohibiting insulin resistance.⁴³ Moreover, Tregs accumulation in visceral adipose tissue through Ag/MHCII and IL-33 may play a critical role in lipid metabolism and prevention of metabolic related disorders.⁴⁴ Impaired Treg proliferation and function enhanced the adipose macrophage inflammation and insulin resistance, whereas adoptive transfer of Tregs reversed adipose inflammation and restored insulin sensitivity in B7 knockout mice.⁴⁵ It has been argued that Tregs improve insulin resistance by limiting the proinflammatory milieu.³⁹

Human studies have provided evidence to support that Tregs are aberrant in T2DM. Number of Tregs in the peripheral blood of T2DM patients was significantly decreased compared with those in the healthy population.^46,47 Moreover, the remaining circulating T cells in T2DM patients exhibited an attenuated differentiation toward a regulatory phenotype.⁴⁸ In addition, lower incidence of DM is associated with increased serum levels of IL-10, a cytokine produced by activated Tregs.⁴⁹ Treg-associated anti-inflammatory cytokine (transforming growth factor [TGF-β] and IL-10) levels were lower in patients with newly diagnosed T2DM than in the control individuals,⁵⁰ suggesting that the ability of Tregs to limit the inflammatory response might be impaired in T2DM patients. Increased serum levels of proinflammatory cytokines (IL-6 and TNF-α), as well as decreased anti-inflammatory IL-10 levels in T2DM patients, may suppress Tregs and the ratio of Tregs to Th1 and Th17 cells.⁵¹

Restoring Tregs to Treat T2DM

Targeting Treg response presents a promising therapy for insulin resistance and T2DM.⁵² In a mouse model of T2DM, induction of Tregs by anti-CD3 antibody plus β-glucosylceramide suppressed adipose inflammation and ameliorated insulin resistance and pathological abnormalities of the pancreas, suggesting that inducing Tregs might be an effective immunological approach for treating T2DM.⁵³ Metformin, as a biguanide antidiabetic drug, has been successfully used for treating T2DM. Metformin treatment significantly increased Tregs and decreased Th17 cells in the spleens of obese mice, which might be an antidiabetic mechanism of this drug.⁵⁴

Traditional Chinese Medicine has also been used for T2DM therapy. The Dangguiliuhuang decoction (DGLHD) promotes glucose uptake, normalizes glucose levels, and improves insulin resistance.⁵⁵ In both in vivo and in vitro experiments, DGLHD increased Treg differentiation and generation and suppressed T lymphocyte proliferation and DC activation, suggesting that the DGLHD therapy is effective against insulin resistance by modulating abnormal immune and metabolic homeostasis.⁵⁵

Certain foods appear to benefit T2DM patients. Black tea originates from Camellia sinensis and contains a large amount of the flavonoid catechin, which has anti-inflammatory and antioxidative properties. Its intake significantly reduces HbA1c levels, along with enhanced number of Tregs, in the peripheral blood of T2DM patients.⁵⁶ In a study, T2DM patients received Diabetea tea™ (DT; its main constituent is black tea supplemented with 12 other medicinal herbs) per day for 12 weeks. Besides decreased HbA1c levels, DT ingestion significantly increased IL-10 expression and Treg/Th17 ratio in the peripheral blood.⁵⁷

Latent Autoimmune Diabetes Of Adults

Latent autoimmune diabetes of adults (LADA) develops in adults and shares many features with both T1DM and T2DM. Tregs limit autoimmunity, and peripheral Tregs are important for understanding the immunological pathogenesis of diabetes in LADA patients.⁵⁸ In a study by Yang et al., CD4⁺CD25⁺ T cell proportion was high and Foxp3 mRNA expression in CD4⁺ T cells was significantly decreased in LADA patients compared with those in healthy individuals, which suggested an immune disorder and decreased number of Tregs in LADA patients.⁵⁹ It was assumed that the adoptive transfer of Tregs might be a potential therapy in LADA patients.⁵⁹ Li et al. also found, in a study including 15 LADA patients and 11 healthy controls, that Foxp3 mRNA expression was decreased in LADA patients compared with that in healthy individuals.⁶⁰

Gestational Diabetes Mellitus

Tregs may have a role in the development of gestational diabetes mellitus (GDM).⁶¹ As compared with non-GDM pregnant women, GDM patients present decreased circulating Tregs and Treg-associated cytokine levels (TGF-β and IL-10) as well as increased serum levels of proinflammatory cytokines (IL-6 and TNF-α).⁶² In addition, Treg suppressive activity is impaired in GDM versus healthy pregnancies, which indicates a functional defect in Tregs and a less effective maternal immune suppression in GDM women.⁶² GDM patients have a higher ratio of Th1/Tregs and Th17/Tregs in pregnant women than do control women without GDM.⁶³ Evidence supports that Tregs protect pregnant women against GDM development and thus, Treg dysregulation early in pregnancy increases GDM risk.⁶²

Diabetic Complications

Diabetes can cause various systemic complications and damage to the body, including cardiovascular disease, nephropathy, neuropathy, and retinopathy. Tregs may be able to abate end-organ damage in DM patients.⁶⁴ Evidence has shown that Tregs protect against various cardiovascular diseases, including atherosclerosis and myocardial infarction.⁶⁵ In type 2 diabetic nephropathy patients, Treg percentage and Tregs/Th17 ratio were lower than those in T2DM patients without nephropathy and in healthy controls.⁶⁶ In a db/db mouse model, deletion of Tregs by using an anti-CD25 antibody increased the symptoms of diabetic nephropathy, including albuminuria and impaired glomerular hyperfiltration, but adoptive transfer of Tregs improved diabetic nephropathy.³⁹ Evidence shows that Tregs might protect against diabetic nephropathy by limiting the proinflammatory milieu.⁶⁶ In addition, Treg percentages and TGF-β levels in peripheral blood were reported to be significantly decreased in patients with T2DM retinopathy compared with those in patients with nondiabetic retinopathy.⁴⁷ Adoptive transfer of purified Tregs could recruit Tregs to the retina and significantly reduce retinal neovascularization.⁶⁷

Conclusions

Accumulating evidence indicates that reduced number and impaired function of Tregs might be associated with both types of DM, T1DM and T2DM. Modification of Tregs may be an effective strategy to prevent DM and diabetic complications. To better understand the immunomodulatory mechanisms and interventions of DM, we have provided an overview of the roles of Tregs in the pathogenesis of DM, including T1DM, T2DM, LADA, and GDM. We have also discussed the roles of Tregs in the secondary conditions to diabetes, including cardiovascular diseases, nephropathy, neuropathy, and retinopathy. Further researches will be done to focus on the use of Tregs in random controlled trials and underlying mechanisms.

Footnotes

Authors' Contributions

S.L., A.G., Z.K., and Z.C. researched the data in the literature; D.M. drew the figure; and Q.W. and M.X. wrote and edited the article. M.X. is the guarantor of this study, has full access to all the data in the study, and takes responsibility for the integrity of data and the accuracy of data analysis.

Data Availability Statement

The data are available from the corresponding author upon reasonable request.

Author Disclosure

No competing financial interests exist.

Funding Information

No funding was received for this article.

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