The E3 Ubiquitin Ligase HERC5 Promotes Antimycobacterial Immunity In Macrophages Through Regulating ISGylation And Degradation Of PTEN

HERC5(homologous HERC6 in mice)is an E3 enzyme which mediates interferon(IFN)-stimulated gene 15(ISG15)-induced ISGylation of protein targets to inhibit protein function in general,thereby plays a vital role in kinds of physiological functions.However,the direct role of HERC5 in macrophages after M.tuberculosis infection has not been studied.In the present study,we showed that HERC5 plays a vital role in promoting inflammatory immune response.HERC5 deficiency in macrophages enhanced the bacterial growth both in vitro and in vivo.We demonstrated that HERC5 increased pro-inflammatory cytokines production of macrophages via positively regulating AKT-mTOR signaling pathway.Finally,we found that HERC5 regulated PTEN ISGylation and degradation,thereby promoted activation of AKT/mTOR signal pathway.Our findings uncover an essential role of HERC5 in ISGylation of PTEN and contribute to the understanding of its protective effects on immunity during M.tuberculosis infection.HERC5 may serve as a potential target for therapeutic intervention against tuberculosis.Methods1.The mRNA and protein expression levels of HERC5(mHERC6)in mouse bone marrow derived macrophage(mBMDM),human monocyte-derived macrophage(hMDM)and human monocytic leukemia(THP-1)cells with MTB infection were detected by real-time quantitative PCR(qRT-PCR)and western blot(WB);2.After C57BL/6 wild-type and Herc6fl/fl-Lyz2-Cre mice were infected with H37Rv,colony-forming unit(CFU)experiment was determined by plating serial dilutions of spleens and lungs homogenates at 1 and 4 weeks after infection.At the same time,take the eyeball blood,and the expression of TNF-α,IL-6,IL-1β and IFN-βin serum,spleens and lungs homogenates were detected by Enzyme-linked immunosorbent assay(ELISA);3.HERC5-overexpressing cell line THP-1-HERC5 cells,Herc6fl/fl-Lyz2-Cre mBMDM and their control groups were infected H37Rv respectively,the survival of macrophage intracellular bacteria were detected by CFU,the expression levels of inflammation cytokines were detected by qRT-PCR and ELISA;4.The phosphorylation changes of signaling pathway molecules such as TBK1,JNK,ERK,AKT,NF-κB,p38,mTOR and the expression changes of PTEN and LC3 were also detected by WB;5,Herc6fl/fl,Herc6fl/fl-Lyz2-Cre mBMDM and THP-1,THP-1-HERC5 cells were infected by H37Rv respectively,the interaction between PTEN,HERC5(mHERC6),ISG15 were detected by COIP;6.UBE1L,UBCH8,PTEN-HA,ISG15-Myc and HERC5-Flag or HERC5-Flag(C994A)were co-transfected into 293T cells,and the interaction between PTEN,HERC5 and ISG15 were detected by COIP;7.Knocking down Pten in Herc6fl/fl Herc6fl/fl-Lyz2-Cre mBMDM,intracellular bacterias were detected by CFU and the expression levels of inflammation cytokines were detected by qRT-PCR after H37Rv infection;8.Empty virus,mHERC6,mHERC6(C970A)lentiviral vectors were transfcted into/Herc6fl/fl-Lyz2-Cre mBMDM respectively,the mRNA expression levels of TNF-α,IL-6,IL-1β and IFN-β were detected by qRT-PCR after H37Rv infection,and bacterial burden was detected by CFU after infection.Results1.H37Rv infection induced HERC5(mHERC6)expression of macrophages;2.HERC5(mHERC6)positively regulated bacterial clearance and the production of TNF-α,IL-6 and IL-1β both in vivo and vitro,3.HERC5(mHERC6)positively regulated PI3K/AKT/mTOR signaling;4.HERC5(mHERC6)promoted the ISGylation of PTEN through Cys994.ConclusionIn this study,we demonstrated that HERC5 interacted with PTEN and then catalyzed ISGylation of PTEN through its ubiquitin activity on Cys994 after infection with MTB,which activated the PI3K/AKT/mTOR signaling,facilitated the expression of pro-inflammatory cytokines TNF-α,IL-6 and IL-1β for the activation of an innate antibacterial immune response.