Oxidative Stress Induced by High Glucose Aggravates Immunosuppression in Diabetes Mellitus by Promoting Effector T Cell Apoptosis and Regulatory T Cell Differentiation

Objective  To explore the regulatory mechanisms underlying the increased proportion of CD4⁺ Foxp3⁺ regulatory T (Treg) cells in late-stage diabetes mellitus (DM) with poorly-controlled blood glucose, and to identify new approaches and therapeutic targets for the prevention and treatment of secondary infections in the late stage of DM.

Methods  Wild-type C57BL/6 mice aged 6 to 8 weeks were randomly assigned to two groups, the experimental and the control groups (n = 5 per group). Mice in the experimental group were injected with streptozotocin (STZ) to induce the mouse model of type 1 diabetes mellitus (T1D), while those in the control group received injection of an an equal volume of 0.1 mol/L citrate buffer. In addition, wild-type C57BL/6 mice aged 6 to 8 weeks were fed with high-fat diet for 2 months and subsequently randomly assigned to two groups, the experimental and the control groups (n = 3 per group). Mice in the experimental group were injected with low-dose STZ for multiple times to induce the mouse model of type 2 diabetes mellitus (T2D), while those in the control group received an equal volume of 0.1 mol/L citrate buffer. The spleen and peripheral lymph nodes of the mice were collected 2 weeks after the stable onset of diabetes, and T cell immune responses were examined by flow cytometry. Naive T cells isolated by immunomagnetic beads were cultured to investigate the mechanisms by which high glucose regulates T cell differentiation and function. The frequency of Treg cells and effector T (Teff) cells, the expression levels of Ki67, a cell proliferation marker, cell apoptosis rate, and intracellular reactive oxygen species (ROS) levels in the mouse tissue single cell suspension and T cell culture samples were assessed by multicolor flow cytometry.

Results  Late-stage T1D and T2D mice with poorly-managed blood glucose exhibited increased peripheral CD4⁺ Foxp3⁺ Treg frequencies (P < 0.05). In these diabetic mice with poorly-managed blood glucose, the expression of Ki67 in Treg cells was significantly upregulated (P < 0.05), while the apoptosis of non-Treg cells (Foxp3^－ non-Treg cells) increased markedly (P < 0.05). Under high-glucose treatment conditions, the ROS levels in Teff cells increased significantly, and the cell apoptosis also increased significantly. High-glucose treatment induced the activation of transforming growth factor-β (TGF-β) and promoted the differentiation of Treg cells, whereas blocking the TGF-β signaling pathway or neutralizing ROS completely inhibited high glucose-induced Treg differentiation (P < 0.01).

Conclusion  Sustained hyperglycemic internal environment in poorly-controlled diabetic mice causes high level of ROS production in Teff cells by inducing oxidative stress, which leads to increased apoptosis of Teff cells, promotes the differentiation of Treg cells by activating TGF-β, and ultimately leads to exacerbated immunosuppressive environment in the late stages of DM. Inhibiting the high level of ROS in late-stage diabetic patients may be conducive to mitigating Teff apoptosis and increasing the frequencies of Treg cells, and may offer new perspectives for improving hyperglycemia-induced immunosuppression and secondary infections in the late stage of DM.