The Parenteral Vitamin C Improves Sepsis and Sepsis-Induced Multiple Organ Dysfunction Syndrome via Preventing Cellular Immunosuppression

Abstract

Cellular immunosuppression appears to be involved in sepsis and sepsis-induced multiple organ dysfunction syndrome (MODS). Recent evidence showed that parenteral vitamin C (Vit C) had the ability to attenuate sepsis and sepsis-induced MODS. Herein, we investigated the impact of parenteral Vit C on cellular immunosuppression and the therapeutic value in sepsis. Using cecal ligation and puncture (CLP), sepsis was induced in WT and Gulo^-/- mice followed with 200 mg/Kg parenteral Vit C administration. The immunologic functions of CD4⁺CD25⁺ regulatory T cells (Tregs) and CD4⁺CD25^- T cells, as well as the organ functions, were determined. Administration of parenteral Vit C per se markedly improved the outcome of sepsis and sepsis-induced MODS of WT and Gulo^-/- mice. The negative immunoregulation of Tregs was inhibited, mainly including inhibiting the expression of forkhead helix transcription factor- (Foxp-) 3, cytotoxic T lymphocyte associated antigen- (CTLA-) 4, membrane associated transforming growth factor-β (TGF-β^m+), and the secretion of inhibitory cytokines [including TGF-β and interleukin- (IL-) 10], as well as CD4⁺ T cells-mediated cellular immunosuppression which was improved by parenteral Vit C in WT and Gulo^-/- septic mice. These results suggested that parenteral Vit C has the ability to improve the outcome of sepsis and sepsis-induced MODS and is associated with improvement in cellular immunosuppression.

Figure 1

The impact of parenteral Vit C treatment on the 72-hour survival rate of septic mice and sepsis-induced MODS. The 72-hour survival rate of WT mice and Gulo^−/− mice was improved on administration of 200 mg/Kg parenteral Vit C twice after CLP (a). Parenteral Vit C improved the biomarkers of liver (b and c), renal (d), respiratory (e and f), and circulatory (g) function in CLP-exposed WT mice and Vit C deficient Gulo^−/− mice. The survival rate was analyzed by Kaplan-Meier via the log-rank test, n = 50 per group. WT (CLP) versus WT (CLP + Vit C), p < 0.01. Gulo^−/− (CLP) versus Gulo^−/− (CLP + Vit C), p < 0.01. WT (CLP) versus Gulo^−/− (CLP), p < 0.05. Data were represented as mean ± standard deviation (SD) and analyzed by software SPSS 17.0 with one-way ANOVA, n = 4 per group, ^∗p < 0.05 or ^∗∗p < 0.01.

Figure 2

Parenteral Vit C markedly weakened the stability of CD4⁺CD25⁺ Tregs in sepsis. The expressions of Foxp-3, CTLA-4, and TGF-β^m+, as well as the apoptotic ability of splenic CD4⁺CD25⁺ Tregs, were subjected to flow cytometric analysis by flow cytometer (g). Parenteral Vit C downregulated the expression of Foxp-3 (a), CTLA-4 (b), and TGF-β^m+ (c) of CD4⁺CD25⁺ Tregs in sepsis. Parenteral Vit C did not alter the apoptotic ability of splenic CD4⁺CD25⁺ Tregs in sepsis (d). Parenteral Vit C inhibited the secretion of TGF-β (e) and IL-10 (f) from CD4⁺CD25⁺ Tregs in sepsis. Data were represented as mean ± standard deviation (SD) and analyzed by software SPSS 17.0 with one-way ANOVA, n = 4 per group, ^∗p < 0.05 or ^∗∗p < 0.01.

Figure 3

The impact of parenteral Vit C on the immunosuppressive function of CD4⁺CD25⁺ Tregs. The parenteral Vit C inhibited the immunosuppressive function of CD4⁺CD25⁺ Tregs which upregulated the proliferative response (a) and release of IFN-γ (c) but significantly downregulated the apoptotic rate (b) and release of IL-4 (d) of splenic conventional CD4⁺CD25⁻ T cells. Data were represented as mean ± standard deviation (SD) and analyzed by software SPSS 17.0 with one-way ANOVA, n = 4 per group, ^∗p < 0.05 or ^∗∗p < 0.01.

Figure 4

The impact of parenteral Vit C on the mRNA expression of Foxp-3, Helios, CTLA-4, TGF-β, IL-10, and the methylation level of Foxp3-TSDR mRNA in CD4⁺CD25⁺ Tregs; CD4⁺CD25⁺ Tregs were harvested for SYBR Green and Methylation-Sensitive RT-PCRT from every group after CLP. The parenteral Vit C inhibited the expression of Foxp-3 (a), Helios (c), CTLA-4 (d), TGF-β  (e), and IL-10 (f) but increased the methylation level of Foxp3-TSDR (b) in CD4⁺CD25⁺ Tregs. Data were represented as mean ± standard deviation (SD) and analyzed by software SPSS 17.0 with one-way ANOVA, n = 4 per group, ^∗p < 0.05 or ^∗∗p < 0.01.

Figure 5

The impact of parenteral Vit C on CD4⁺ T cells-mediated cellular immunosuppression in sepsis. Treatment with 200 mg/Kg parenteral Vit C upregulated the proliferative response of CD4⁺CD25⁻ T cells of WT mice and Gulo^−/− mice in sepsis (a). The apoptotic rate of splenic CD4⁺CD25⁻ T cells was analyzed with Annexin-V-FITC/PI by flow cytometry at 24 hours after CLP-induced midgrade sepsis (e). The apoptotic level of splenic CD4⁺CD25⁻ T cells was decreased when 200 mg/Kg parenteral Vit C was administered after CLP to WT mice and Gulo^−/− mice in sepsis (b). Parenteral Vit C increased the serum level of IFN-γ (c) as well as decreasing the serum levels of IL-4 (d) of WT mice and Gulo^−/− mice in sepsis. Data were represented as mean ± standard deviation (SD) and analyzed by software SPSS 17.0 with one-way ANOVA, n = 4 per group, ^∗p < 0.05 or ^∗∗p < 0.01.