Circulating cytokine profile and modulation of regulatory T cells in chronic hepatitis B patients with type 2 diabetes mellitus

Abstract

The risk of hepatitis B virus (HBV) infection is higher in patients with diabetes mellitus, and diabetes mellitus is one of the metabolic complications of HBV infection. However, the cytokine profile of chronic hepatitis B (CHB) patients with type 2 diabetes mellitus (T2DM) is not fully understood. The aim of this study was to investigate the cytokine expression profile in CHB patients with T2DM, and to assess the regulatory function of cytokines to regulatory T cells (Tregs). Forty-four T2DM patients, 39 CHB patients, 17 patients with CHB and T2DM, and 21 control subjects were enrolled. Cytokine levels in the plasma were measured by Luminex multiplex assay. CD4+CD25+CD127dim/- Tregs were detected by flow cytometry. Tregs were purified and stimulated with recombinant human interleukin-15 (IL-15). The regulation of IL-15 on Tregs function was investigated by measuring cell number, IL-10/IL-35 secretion, and mRNA expression of immune checkpoint molecules in a Tregs+PBMC co-culture system. We found that levels of IL-1α, IL-6, and IL-33 were upregulated, while IFN-α, IL-2, IL-7, and IL-15 were downregulated in T2DM and CHB patients. CHB patients with T2DM had even lower plasma IL-7 and IL-15 levels. Tregs percentage was elevated in T2DM and CHB patients. CHB patients with T2DM had increased levels of Tregs, which correlated negatively with IL-15. Tregs showed stronger inhibitory activity in CHB patients with T2DM than in controls, T2DM, and CHB patients, which presented as reduction in cellular proliferation and induction of IL-10/IL-35 secretion. IL-15 suppressed Tregs function and inhibited the expression of immune checkpoint molecules in Tregs. The current data suggest that insufficient IL-15 levels and decreased responsiveness of Tregs to IL-15 signaling might contribute to strong immune dysfunction in CHB patients with T2DM.

Figure 1.

CD4⁺CD25⁺CD127^dim/− Tregs analysis in control (n ═ 21), T2DM (n ═ 44), CHB (n ═ 39), and CHB+T2DM group (n ═ 17). (A) CD4⁺CD25⁺CD127^dim/− Tregs were analyzed by flow cytometry. PBMCs were stained with anti-CD3-APC, anti-CD4-PerCP, anti-CD25-FITC, and anti-CD127-PE. The flow dots for CD25⁺CD127^dim/− cells within CD3⁺CD4⁺ cells in the control, T2DM, CHB, and CHB+T2DM group were shown. (B) CD4⁺CD25⁺CD127^dim/− Tregs proportion within CD3⁺CD4⁺ cells was compared between the control, T2DM, CHB, and CHB+T2DM groups. Statistical analysis was performed using one-way ANOVA and SNK-q test. (C) Correlation between Tregs proportion and plasma IL-7 levels was analyzed in the CHB+T2DM group. (D) Correlation between Tregs proportion and plasma IL-15 levels was analyzed in the CHB+T2DM group. Pearson correlation analysis was performed for correlation analysis. T2DM: Type 2 diabetes mellitus; CHB: Chronic hepatitis B; Tregs: Regulatory T cells; IL: Interleukin; PBMCs: Peripheral blood mononuclear cells.

Figure 2.

Inhibitory activity analysis of CD4⁺CD25⁺CD127^dim/− Tregs in the control (n ═ 7), T2DM (n ═ 9), CHB (n ═ 11), and CHB+T2DM group (n ═ 9). 5 × 10⁴ of purified CD4⁺CD25⁺CD127^dim/− Tregs were co-cultured with 2 × 10⁵ of autologous PBMCs for 72 hours. (A) Cell number was determined by CCK-8 method and was compared between the control, T2DM, CHB, and CHB+T2DM groups. (B) IL-10 levels in the cultured supernatants were measured by ELISA and were compared between the control, T2DM, CHB, and CHB+T2DM groups. (C) IL-35 levels in the cultured supernatants were measured by ELISA and were compared between the control, T2DM, CHB, and CHB+T2DM groups. Statistical analysis was performed using one-way ANOVA and SNK-q test. T2DM: Type 2 diabetes mellitus; CHB: Chronic hepatitis B; Tregs: Regulatory T cells; IL: Interleukin; PBMCs: Peripheral blood mononuclear cells.

Figure 3.

Inhibitory activity analysis of CD4⁺CD25⁺CD127^dim/− Tregs in response to IL-15 stimulation in the control (n ═ 7), T2DM (n ═ 9), CHB (n ═ 11), and CHB+T2DM group (n ═ 9). Purified CD4⁺CD25⁺CD127^dim/− Tregs were stimulated with either 10 ng/mL or 100 ng/mL recombinant human IL-15 for 24 hours. 5×  10⁴ of stimulated Tregs were co-cultured with 2 × 10⁵ of autologous PBMCs for 72 hours. (A) Cell number was determined by CCK-8 method and was compared between no stimulation, 10 ng/mL of IL-15 stimulation, and 100 ng/mL of IL-15 stimulation in each group. (B) IL-10 levels in the cultured supernatants were measured by ELISA and were compared between no stimulation, 10 ng/mL of IL-15 stimulation, and 100 ng/mL of IL-15 stimulation in each group. (C) IL-35 levels in the cultured supernatants were measured by ELISA and were compared between no stimulation, 10 ng/mL of IL-15 stimulation, and 100 ng/mL of IL-15 stimulation in each group. Statistical analysis was performed using one-way ANOVA and SNK-q test. T2DM: Type 2 diabetes mellitus; CHB: Chronic hepatitis B; Tregs: Regulatory T cells; IL: Interleukin.

Figure 4.

The influence of IL-15 stimulation to IL-15Rα and immune checkpoint molecules expression in CD4⁺CD25⁺CD127^dim/− Tregs in the control (n ═ 6), T2DM (n ═ 10), CHB (n ═ 12), and CHB+T2DM group (n ═ 8). Purified CD4⁺CD25⁺CD127^dim/− Tregs were stimulated with either 10 ng/mL or 100 ng/mL recombinant human IL-15 for 24 hours. mRNA relative levels corresponding to IL-15Rα, CTLA-4, LAG-3, PD-1, and TIM-3 were semi-quantified by real-time PCR. (A) IL-15Rα mRNA relative levels were compared between no stimulation, 10 ng/mL of IL-15 stimulation, and 100 ng/mL of IL-15 stimulation in each group. (B) CTLA-4 mRNA relative levels were compared between no stimulation, 10 ng/mL of IL-15 stimulation, and 100 ng/mL of IL-15 stimulation in each group. (C) LAG-3 mRNA relative levels were compared between no stimulation, 10 ng/mL of IL-15 stimulation, and 100 ng/mL of IL-15 stimulation in each group. (D) PD-1 mRNA relative levels were compared between no stimulation, 10 ng/mL of IL-15 stimulation, and 100 ng/mL of IL-15 stimulation in each group. (E) TIM-3 mRNA relative levels were compared between no stimulation, 10 ng/mL of IL-15 stimulation, and 100 ng/mL of IL-15 stimulation in each group. Statistical analysis was performed using one-way ANOVA and SNK-q test. T2DM: Type 2 diabetes mellitus; CHB: Chronic hepatitis B; Tregs: Regulatory T cells; IL: Interleukin.