Tuftsin prevents the negative immunoregulation of neuropilin-1highCD4+CD25+Regulatory T cells and improves survival rate in septic mice

Abstract

Our previous research showed that neuropilin (Nrp) -1highCD4+CD25+Regulatory T cells (Tregs) exhibited primary negative immunoregulation in sepsis induced immune dysfunction. Tuftsin is the typical ligand of Nrp-1. Herein, we investigated the potential therapeutic value and mechanisms of tuftsin in sepsis. Sepsis per se markedly decreased the serum concentration of tuftsin, administration of tuftsin improved the survival rate of septic mice with cecal ligation and puncture (CLP). In vitro study, tuftsin prevented the negative immunoregulation of Nrp-1highCD4+CD25+Tregs, including weakening the expression of forkhead/winged helix transcription factor (Foxp)- 3/cytotoxic T lymphocyte associated antigen (CTLA)-4, inhibiting the secretion of transforming growth factor (TGF)-β, and weakening the immunosuppressive function of Nrp-1highCD4+CD25+Tregs to conventional CD4+CD25-T cells. Tuftsin markedly inhibited the demethylation of Foxp3-Tregs specific demethylated region (TSDR) of Nrp-1highCD4+CD25+Tregs. Tuftsin could represent a new potential therapeutic agentia to improve the outcome of septic mice, and associate with preventing the negative immunoregulation of Tregs via Nrp-1.

Keywords: negative immunoregulation; neuropilin-1; regulatory T cells; sepsis; tuftsin.

Figure 1. The grade- and time- dependent responses between sepsis and the serum concentration of tuftsin

The results showed the effects of various severity of sepsis on the serum concentration of tuftsin after CLP A. The time-dependent response between various severity of sepsis and the serum concentration of tuftsin B. Data were represented as mean ± standard deviation (SD), and analyzed by software of SPSS 17.0 with a one-way ANOVA, n=4 per group, *P<0.05, **P<0.01.

Figure 2. The grade- and time- dependent responses between sepsis and the expression of Nrp-1 on splenic CD4⁺CD25⁺Tregs

The results showed the effects of various severity of sepsis on the average fluorescence level of Nrp-1 on splenic CD4⁺CD25⁺Tregs after CLP A. The time-dependent response between various severity of sepsis and the average fluorescence level of Nrp-1 B. Immunofluorescence confocal microscopy using antibodies to Nrp-1 (C, red). Data were represented as mean ± standard deviation (SD), and analyzed by software of SPSS 17.0 with a one-way ANOVA, n=4 per group, *P<0.05, **P<0.01.

Figure 3. The dose-dependent response between tuftsin and the 48- hrs survival rate

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 4. The impact of tuftsin on the expressions of Foxp-3

Tuftsin weakened the expression of Foxp-3 of Nrp-1^highCD4⁺CD25⁺Tregs from 12 to 24 hrs A. Tuftsin had no effect on the expression of Foxp-3 of Nrp-1^lowCD4⁺CD25⁺Tregs B. The expression of Foxp-3 of Nrp-1^highCD4⁺CD25⁺Tregs were subjected to flow cytometric analysis by flow cytometer at 12 hrs C. Data were represented as mean ± standard deviation (SD), and analyzed by software of SPSS 17.0 with a one-way ANOVA, n=4 per group, *P<0.05, **P<0.01.

Figure 5. The impact of tuftsin on the expressions of CTLA-4

Tuftsin weakened the expression of CTLA-4 of Nrp-1^highCD4⁺CD25⁺Tregs from 12 to 24 hrs A. Tuftsin had no effect on the expression of CTLA-4 of Nrp-1^lowCD4⁺CD25⁺Tregs B. The expression of CTLA-4 of Nrp-1^highCD4⁺CD25⁺Tregs were subjected to flow cytometric analysis by flow cytometer at 24 hrs C. Data were represented as mean ± standard deviation (SD), and analyzed by software of SPSS 17.0 with a one-way ANOVA, n=4 per group, *P<0.05, **P<0.01.

Figure 6. The impact of tuftsin on the secretion of TGF-β

Tuftsin inhibited the secretion of TGF-β from Nrp-1^highCD4⁺CD25⁺Tregs from 12 to 24 hrs A. Tuftsin had no effect on the secretion of TGF-β from Nrp-1^lowCD4⁺CD25⁺Tregs B. Data were represented as mean ± standard deviation (SD), and analyzed by software of SPSS 17.0 with a one-way ANOVA, n=4 per group, *P<0.05, **P<0.01.

Figure 7. The regulating ability of tuftsin on Nrp-1^highCD4⁺CD25⁺Tregs to the proliferative activity of conventional CD4⁺CD25⁻ T cells

Tuftsin weakened the suppressive function of Nrp-1^highCD4⁺CD25⁺Tregs to the proliferative activity of conventional CD4⁺CD25⁻ T cells. Data were represented as mean ± standard deviation (SD), and analyzed by software of SPSS 17.0 with a one-way ANOVA, n=4 per group, *P<0.05, **P<0.01.

Figure 8. The regulating ability of tuftsin on Nrp-1^highCD4⁺CD25⁺Tregs to the apoptosis of conventional CD4⁺CD25⁻ T cells

Tuftsin weakened the suppressive function of Nrp-1^highCD4⁺CD25⁺Tregs to the apoptosis of conventional CD4⁺CD25⁻ T cells A. The apoptotic rate of CD4⁺CD25⁻ T cells was analyzed with annexin-V-FITC/PI flow cytometry at 24 hours after co-cultured with Nrp-1^highCD4⁺CD25⁺Tregs B. Data were represented as mean ± standard deviation (SD), and analyzed by software of SPSS 17.0 with a one-way ANOVA, n=4 per group, *P<0.05, **P<0.01.

Figure 9. The regulating ability of tuftsin on Nrp-1^highCD4⁺CD25⁺Tregs to the secretive ability of conventional CD4⁺CD25⁻ T cells

Tuftsin weakened the suppressive function of Nrp-1^highCD4⁺CD25⁺Tregs to the secretive ability of conventional CD4⁺CD25⁻ T cells. Data were represented as mean ± standard deviation (SD), and analyzed by software of SPSS 17.0 with a one-way ANOVA, n=4 per group, *P<0.05, **P<0.01.

Figure 10. The impact of tuftsin on the methylation of Foxp 3-TSDR of Nrp-1^highCD4⁺CD25⁺Tregs

Tuftsin markedly promoted the methylation of Foxp3-TSDR in the stimulated of LPS at 24 hours in a dose-dependent manner A. Tuftsin markedly promoted the methylation of Foxp3-TSDR in splenic Nrp-1^highCD4⁺CD25⁺ Tregs of septic mice at 24 hours in a dose-dependent manner B. Data were represented as mean ± standard deviation (SD), and analyzed by software of SPSS 17.0 with a one-way ANOVA, n=4 per group, *P<0.05, **P<0.01.