USP4 Interacts And Positively Regulates IRF8 Function Via K48-linked Deubiquitination In Regulatory T Cells

Objective To explore whether USP4 could impact regulatory T cells function through deubiquitination of IRF8,which might offer mechanism and new target for treatment of immunity disease and tumor related to regulatory T cells.Methods and Materials We resorted to co-immunoprecipitation in HEK293 T cells to study whether USP4 could interact with IRF8.To further confirm the binding between those two proteins,we performed the co-immunoprecipitation in human regulatory T cells.Moreover,we performed His-pull down assay to detect the ubiquitination of IRF8 via USP4 in HEK293 T cells.Then,We checked the ubiquitination of IRF8 by USP4 via knocking down USP4 in regulatory T cells.At the same time,we also analyzed the expression of a variety of cytokine upon knocking down USP4 in regulatory T cells.We further performed suppressive assay to figure out whether USP4 is crucial for function of regulatory T cells.We also consult the GSE datato compare USP4 and IRF8 of tumor infiltrated regulatory T cells in colon cancer and para-carcinoma tissue on colon cancer mouse model.Furthermore,we analyzed the immunohistochemical staining of human colon cancer and para-carcinoma tissue to compare the expression of USP4 and IRF8.Results We found that USP4 could interact with IRF8 and stabilize it’s protein level in HEK293 T cell,which also existed in human regulatory T cells.The His-pull down assay in HEK293 T cells showed that USP4 mediated the polyubiquitination of IRF8 via K48.Depletion of USP4 promoted the polyubiquitination of IRF8 and the upregulation of type 2 inflammatory cytokine gene expression in regulatory T cells.Consistently,treated regulatory T cells with USP4 inhibitor facilitated the polyubiquitination of IRF8.In addition,deficiency of USP4 alleviated regulatory T cells suppressive function.GSE data showed that the expression of USP4 and IRF8 in mouse tumor filtrated regulatory T cells from colon para-carcinoma tissue was higher than cancer tissue.Consistently,human immunohistochemical staining indicated higher expression of USP4 and IRF8 in colon para-carcinoma tissue than cancer tissue.Conclusions USP4 interacts with and stabilizes IRF8 via K48-linked deubiquitination to promote the suppressive function of regulatory T cells.Compared to colon cancer tissue,human colon para-carcinoma tissue have higher expression of USP4 and IRF8.