Neuropilin-1highCD4⁺CD25⁺ Regulatory T Cells Exhibit Primary Negative Immunoregulation in Sepsis

Abstract

Regulatory T cells (Tregs) appear to be involved in sepsis-induced immune dysfunction; neuropilin-1 (Nrp-1) was identified as a surface marker for CD4(+)CD25(+)Tregs. In the current study, we investigated the negative immunoregulation of Nrp-1(high)CD4(+)CD25(+)Tregs and the potential therapeutic value of Nrp-1 in sepsis. Splenic CD4(+)CD25(+)Tregs from cecal ligation and puncture (CLP) mouse models were further segregated into Nrp-1(high)Tregs and Nrp-1(low)Tregs; they were cocultured with CD4(+)CD25(-) T cells. The expression of forkhead/winged helix transcription factor-3 (Foxp-3), cytotoxic T-lymphocyte associated antigen-4 (CTLA-4), membrane associated transforming growth factor-β (TGF-β(m+)), apoptotic rate, and secretive ability [including TGF-β and interleukin-10 (IL-10)] for various types of Tregs, as well as the immunosuppressive ability of Tregs on CD4(+)CD25(-) T cells, were determined. Meanwhile, the impact of recombinant Nrp-1 polyclonal antibody on the demethylation of Foxp-3-TSDR (Treg-specific demethylated region) was measured in in vitro study. Sepsis per se markedly promoted the expression of Nrp-1 of CD4(+)CD25(+)Tregs. Foxp-3/CTLA-4/TGF-β(m+) of Nrp-1(high)Tregs were upregulated by septic challenge. Nrp-1(high)Tregs showed strong resilience to apoptosis and secretive ability and the strongest immunosuppressive ability on CD4(+)CD25(-) T cells. In the presence of lipopolysaccharide (LPS), the recombinant Nrp-1 polyclonal antibody reduced the demethylation of Foxp-3-TSDR. Nrp-1(high)Tregs might reveal primary negative immunoregulation in sepsis; Nrp-1 could represent a new potential therapeutic target for the study of immune regulation in sepsis.

Figure 1

The grade- and time-dependent responses between sepsis and the expression of Nrp-1 in splenic CD4⁺CD25⁺Tregs. The results showed the effects of various severities of sepsis on the expression of Nrp-1 in splenic CD4⁺CD25⁺Tregs at 24 hours after CLP ((a), (e)). The grade-dependent impact between sepsis and the 24-hour survival rate (b). The effects of midgrade sepsis on the expression of Nrp-1 of splenic CD4⁺CD25⁺Tregs at 12, 24, 48, and 72 hours after CLP ((c), (f)). The time-dependent response between sepsis and the 72-hour survival rate (d). Data was represented as mean ± standard deviation (SD) and analyzed by SPSS 17.0 software with a one-way ANOVA, n = 4 per group (P < 0.01). The survival rate was analyzed by Kaplan-Meier via the log-rank test, n = 30 per group (P < 0.05 or P < 0.01).

Figure 2

Sepsis markedly upregulated the expression of Foxp-3/CTLA-4/TGF-β ^m+ on Nrp-1^highCD4⁺CD25⁺Tregs. Midgrade septic model at 24 hours after CLP was used to investigate the effect of Nrp-1 on the negative immunoregulation of Tregs in sepsis. The expressions of Nrp-1 and Foxp-3 ((a), (c)) and CTLA-4 and TGF-β ^m+ ((b), (c)) were subjected to flow cytometric analysis by flow cytometer. Data was represented as mean ± standard deviation (SD) and analyzed by SPSS 17.0 software with a one-way ANOVA, n = 4 per group (P < 0.01).

Figure 3

Nrp-1^highCD4⁺CD25⁺Tregs showed the strongest resilience to apoptosis in sepsis. The apoptotic rate of Tregs was analyzed with annexin-V-FITC/PI flow cytometry at 24 hours after midgrade sepsis. Data was represented as mean ± standard deviation (SD) and analyzed by SPSS 17.0 software with a one-way ANOVA, n = 4 per group (P < 0.05 or P < 0.01).

Figure 4

Nrp-1^highCD4⁺CD25⁺Tregs showed the strong ability to secrete cytokines. Sepsis could markedly enhance the release of IL-10 and TGF-β. Data was represented as mean ± standard deviation (SD) and analyzed by SPSS 17.0 software with a one-way ANOVA, n = 4 per group (P < 0.01).

Figure 5

Nrp-1^highCD4⁺CD25⁺Tregs showed the strong ability to inhibit the proliferation and increase the apoptosis of CD4⁺CD25⁻ T cells. Three different subtype Tregs groups (CD4⁺CD25⁺Tregs, Nrp-1^highCD4⁺CD25⁺Tregs, and Nrp-1^lowCD4⁺CD25⁺Tregs) were cocultured with CD4⁺CD25⁻ T cells for 24 hours in a ratio of 1 : 1; the proliferative ability of CD4⁺CD25⁻ T cells (a) and apoptotic rate of CD4⁺CD25⁻ T cells ((b), (c)) were determined. Data was represented as mean ± standard deviation (SD) and analyzed by SPSS 17.0 software with a one-way ANOVA, n = 4 per group (P < 0.05 or P < 0.01).

Figure 6

Nrp-1^highCD4⁺CD25⁺Tregs showed the strong ability to suppress cytokine release of CD4⁺CD25⁻ T cells. Three different subtype Tregs (CD4⁺CD25⁺Tregs, Nrp-1^highCD4⁺CD25⁺Tregs, and Nrp-1^lowCD4⁺CD25⁺Tregs) were cocultured with CD4⁺CD25⁻ T cells for 24 hours, and the secretion of IFN-γ and IL-4 of CD4⁺CD25⁻ T cells was measured. Data was represented as mean ± standard deviation (SD) and analyzed by SPSS 17.0 software with a one-way ANOVA, n = 4 per group (P < 0.05 or P < 0.01).

Figure 7

Recombinant Nrp-1 polyclonal antibody markedly downregulated the demethylation of Foxp-3-TSDR in the stimulated LPS at 24 hours in a dose-dependent manner. Data was represented as mean ± standard deviation (SD) and analyzed by SPSS 17.0 software with a one-way ANOVA, n = 4 per group (P < 0.05 or P < 0.01).