The Profile of CD4⁺CD25⁺CD127^low/- Regulator T Cell and the Associated Expression of Cytokines and Transcription Factors in Immune Thrombocytopenia Murine Model

Background

Regulator T cell (Treg cell) may be associated with immune thrombocytopenia (ITP). Our previous study has showed that the profile of CD4⁺CD25⁺CD127^low/-Treg cells presented significantly decreased in ITP patients and weakened immunosuppressive activity. This cellular defect about immune modulation may play important roles in the pathogenesis of ITP. The precise reason for these abnormalities remains being elucidated. To further investigate the role of Treg cells in ITP，we detected the profiles of Treg cells and the associated mRNA expression of cytokines and Transcription factors in CD4+CD25+ Treg cells in ITP murine model .

Methods

1. The profiles of Treg cell in peripheral blood in ITP patients were examined with flow cytometry through intracellular cytokines analysis. Treg cells were identified as those that were CD4⁺CD25⁺CD127^low/-.

2. ITP was induced by daily intraperitoneal injection of anti-platelet membrane CD41 antibody（MWReg30）into BALB/c murine and the controls were daily intraperitoneal injection of IgG antibodies.

3. The proportion of Treg cells in peripheral blood and spleen mononuclear cells were measured by FCM analysis. Treg cells were identified as those that were CD4⁺CD25⁺CD127^low/-

4. The mRNA expression of Treg cells associated transcription factors (Foxp3, Smad7, STAT5 and Akt-1) and cytokines (IL-10, TGF-β) in CD4⁺CD25⁺ T cells which were enriched from spleen mononuclear cells were measured by real-time PCR.

5. Statistical analysis. All values were analyzed using SPSS version 18.0 software. We used the Kolmogorov-Smirnov goodness-of-fit model to assess the normality of the data. Because of the data did not show a normal distribution, the values were presented as median (range) and statistical significance was evaluated using a Wilcoxon rank-sum test. Spearman’s test was used for correlation analysis. Differences were considered significant at P <0.05.

Results

1. We detected the expression of intracellular cytokines. In peripheral blood of ITP patients, the median of Treg cells was 3.9 % (3.1–6.3 %), respectively, significantly lower than the controls of which that was 6.0 % (5.3–7.85 %, p <0.05).

2. The percentage of CD4⁺CD25⁺CD127^low/-Treg cells was significantly lower in both splenocyte and peripheral blood of ITP murine as compared with that in normal controls (p<0.05).

3. ITP murine had lower mRNA expression of IL-10, TGF-β and Foxp3 in splenocyte CD4⁺CD25⁺T cells (p<0.05). The expression of Smad7 mRNA was significantly higher than the controls. No significantly difference of the STAT5 and Akt-1 mRNA expression were observed between ITP murine and controls both in splenocyte and peripheral blood.

Conclusions

Our study data supported a low Treg polarization of the immune response in ITP murine model. The lower mRNA expressions of IL-10 and TGF-β indicated that the indirect immunosuppressive effect of Treg cells had impaired. The lower mRNA expressions of TGF-β and Foxp3 accelerated effects on the proliferation and differentiation of Treg cells, and the higher mRNA expression of Smad 7 inhibited the proliferation and differentiation of Treg cells. Results indicated that disorder proliferation and differentiation of Treg cells involved in the pathogenesis of ITP.