CXCL4 promoted the production of CD4⁺CD25⁺FOXP3⁺treg cells in mouse sepsis model through regulating STAT5/FOXP3 pathway

Abstract

Background: CXCL4 plays an essential role in the regulation of multiple immune diseases. However, the underlying role of CXCL4 is still not clear in sepsis. Aim: In the present study, we aimed to investigate the function of CXCL4 in sepsis.

Methods: Sepsis model was constructed on mouse. Flow cytometry was used to determine the ratio of CD4⁺CD25⁺FOXP3⁺Treg cells. ELISA assays were used to determine the levels of CXCL4, IL-6, IL-10, and TNF-α respectively. Western blot was used to examine protein contents.

Results: Our results suggested that the serum level of CXCL4 was upregulated in patients with sepsis and positively associated with the ratio of human CD4⁺CD25⁺FOXP3⁺Treg cells. To further examine the role of CXCL4 in sepsis, we constructed the mouse sepsis model. Our results indicated that the mouse antibody of CXCL4 treatment reduced the expression of urine creatinine and urea nitrogen in sepsis model. Moreover, the frequency of CD25⁺FOXP3⁺ mouse regulatory T cells (Tregs) cells was decreased in mouse CD4⁺ T cells in the presence of mouse CXCL4 antibody. Further, the mouse recombinant protein CXCL4 was used to culture normal mouse CD4⁺ T cells in vitro. Our finding indicated that the recombinant protein CXCL4 promoted the percentage of mouse CD25⁺FOXP3⁺Treg cells and enhanced the phosphorylation of STAT5 in mouse CD4⁺ T cells in a dose-dependent manner. However, these effects were significantly reversed by the STAT5 inhibitor (p < .001). Conclusion: our findings not only indicated the function and signalling pathway of CXCL4 in CD4⁺ T cells but also provided novel insight and target in sepsis treatment.

Keywords:

• Sepsis
• Treg cells
• CXCL4
• STAT5
• FOXP3

Acknowledgments

Tao Yang and Jinjun Bian designed this project and wrote the manuscript; Tao Xu, Jie Zhao and Xiaolin Wang, performed the experiments; Yan Meng, Zhenzhen Zhao and Rui Bao analysed the data and edited diagrams. Rui Bao provided the technical assistance. All authors have contributed to read and agreed the final content of manuscript for submission.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The authors confirm that the data supporting the findings of this study are available within the article.

Additional information

Funding

This research was supported by the National Natural Science Foundation of China [NO.81501646, 81471895, and 81671939] and "234 Discipline Climbing Project" of the First Affiliated Hospital of the Naval Medical University (NO. 2019YXK016).