Study Of The Modulation Of Regulatory T Cell Stability By E3 Ubiquitin Ligase MDM2 And The Function Of Energy Sensor AMPK In Regulatory T Cells

Regulatory T cells?Treg cells?are crucial for maintaining immune tolerance and immune homeostasis.Forkhead box P3?FOXP3?is the key transcription factor of Treg cells and play an important role in Treg cell suppressive function.The transcription factor FOXP3 is regulated by many aspects,such as transcription regulation,protein complex regulation,and the regulation of post-translation modifications,which prefect the mechanisms of Treg cell development and function.Among them,the post-translation modifications of FOXP3 protein include acetylation,phosphorylation and ubiquitination,which affect the stability and function of FOXP3and Treg cells.In recent years,it has been found that several E3 ubiquitin ligases or deubiquitinases regulate the K48-linked polyubiquitination of FOXP3 protein,affecting the function of FOXP3 protein and Treg cells,however,other types of ubiquitination of FOXP3 protein are largely unclear.Due to the different sites and types,ubiquitination not only causes protein degradation,but also stabilizes the function of protein,therefore,there are other enzymes that regulate the ubiquitination of FOXP3 protein to be found.The E3 ubiquitin ligase MDM2?murine double minute2?mediates the ubiquitination and degradation of tumor suppressor p53,and a recent study has found that it can also negatively regulate T cell activation,however,it is unknown whether it modulates the function of Treg cells.Furthermore,phosphorylation occurring at different sites can upregulate or downregulate the function of FOXP3.Previous studies have shown that Treg cells exhibit higher level of AMPK?AMP-activated protein kinase?activation.After treatment with AMPK activator Metformin,the proportion and number of Treg cells in mice lymph nodes increased significantly.Although there is a correlation between the protein kinase AMPK and Treg cells,it remains unclear whether AMPK,an energy sensor,regulates the function and metabolism of Treg cells.In the first part,we found that MDM2 enhances the stability of FOXP3 protein through ubiquitination,therefore positively regulating Treg cell function.Knockdown of MDM2 in human primary Treg cells,led to the downregulation of CD25??chain of Interleukin-2?IL-2?receptor?and CTLA-4?cytotoxic T?lymphocyte antigen 4?but the upregulation of IL-2 and IFN-??interferon-??,indicating that the ability of FOXP3to regulate downstream genes was impaired,and Treg cells were transiting into effector T cells?Teff cells,especially type 1 T helper cells?.The capacity of Treg cells to inhibit the proliferation of Teff cells was also reduced.At the same time,the protein level of FOXP3 in Treg cells was significantly decreased after MDM2knockdown,suggesting that MDM2 knockdown resulted in the instability of FOXP3protein.Conversely,overexpression of MDM2 in Treg cells,led to the enhanced stability of FOXP3 protein,and the ability of FOXP3 to promote CD25 and CTLA-4expression and to inhibit the proliferation of Teff cells.Mechanistically,MDM2interacts with FOXP3,and mainly mediates multi-mono ubiquitination and K63-linked polyubiquitination of FOXP3,thus stabilizing the protein level of FOXP3.We also found the sites on FOXP3 protein that could be ubiquitinated by MDM2.In addition,TCR/CD28 signal could upregulate the expression level of MDM2 and its mediated FOXP3 ubiquitination in Treg cells.Our finding that MDM2 regulates the immunosuppressive activity of Treg cells,on the one hand,helps us understand the relationship among MDM2,Treg cells and tumors;on the other hand,it also provides a new target for the treatment of autoimmune diseases.In the second part,we found that the protein kinase AMPK interacts with FOXP3and mediates the phosphorylation of FOXP3,suggesting that AMPK may play a role in Treg cells.We bred mice in which AMPK is specifically depleted in Treg cells(Foxp3^CrePRKAA1^fl/flPRKAA2^fl/fl)and explored the function of AMPK in Treg cells.Under steady-state condition,AMPK deficiency in Treg cells did not affect the activation and cytokine production of Teff cells,as well as the proportion of Treg cells in immune organs.In the mouse fasting model,after two days'fasting,the level of IFN-?production from CD4⁺T and CD8⁺T cells was decreased in spleens of Foxp3^YFP-CreFP-Cre mice,while the level of IFN-?production from CD4⁺T and CD8⁺T cells in spleens of Foxp3^CrePRKAA1^fl/flPRKAA2^fl/fll/fl mice was unaffected,indicating that the defective mice were resistant to fasting-induced reduction of cytokines from T cells.After influenza A virus infection,Foxp3^CrePRKAA1^fl/flPRKAA2^fl/fll/fl mice did not show an obvious phenotype.In the mouse model of fasting and influenza A virus infection,after fasting,Foxp3^CrePRKAA1^fl/flPRKAA2^fl/fll/fl mice were more susceptible to infection,while Foxp3^YFP-CreFP-Cre mice were resistant to influenza A virus infection,and the levels of IFN-?and Granzyme B from CD8⁺T cells in lungs of the latter were significantly increased.Due to the unaffeted proportion of Treg cells,the suppressive activity of Treg cells could be weakened.The study of the function and mechanism of energy sensor and protein kinase AMPK in Treg cells is helpful for us to understand the relationship between Treg cell metabolism and function,and to provide new ideas for the treatment of diseases targeting Treg cells.