HIV/Tat CAN Affect Mouse Neuronal Skeleton Rearrangement And Anti-apoptotic Protein Expression By Activating MDSCs

Background and ObjectiveInjury to the central nervous system?CNS?in HIV-infected people leads to HIV-related neurocognitive disorders?HAND?.The pathogenesis of HAND is still unclear.After HIV infection,it does not directly cause neuronal damage,but enters the central nervous system by infecting monocytes or the like through the blood-brain barrier?BBB?.The study found that the protein released by HIV virus is one of the main factors that promote the development of HAND.The HIV regulatory protein Tat is a key viral trans-neuronal injury factor and is a transcriptional transactivator,which is important for transcriptional regulation and viral replication.Tat can cause neuronal damage by producing oxidative stress,causing cognitive dysfunction.Recent studies have found that the proportion of myeloid-derived suppressor cells?MDSCs?in peripheral blood of HIV-infected patients after antiretroviral therapy?ART?is higher than that of healthy people.Studies have found that healthy human MDSCs can be directly infected by HIV,and can promote the replication and spread of the virus,HIV infection or HIV Tat protein can stimulate the expansion of human MDSCs.Although the immunosuppressive function of MDSCs has been demonstrated in many tumors,there have been few studies on their role in infectious diseases,particularly retroviral infections that cause immunodeficiency.The cytoskeleton maintains the basic physiological functions of the cells,and skeletal system abnormalities often occur under pathological conditions.Apoptosis is a physiological process in which the body clears harmful cells to maintain normal cell proliferation.The expression of anti-apoptotic proteins is involved in maintaining normal apoptosis regulation.There is currently no study on the effect of MD Ts-infected MDSCs on neuronal cells.The aim of this study was to investigate the influence on different subtypes and function of HIV/Tat1-86 protein on MDSCs.By establishing the non-contact co-culture model of MDSCs and neurons,explore the effect of neuronal cytoskeleton and inhibitor of apoptosis protein expression of MDSCs treated with HIV/Tat_1-86,then to study the mechanism of HIV/Tat_1-86 on neuronal damage.Methods1.Cell culture:The bone marrow cells of the thigh bone of adult Balb/c mice were taken out,and the Gr-1+CD11b+MDSCs cultured in the bone marrow cells of Balb/c mice were sorted by flow cytometry?FACS?.Group:treated with different concentrations of HIV/Tat_1-86?0,12.5,25,50,100 nM?for 12 h;the MDSCs subpopulation cells were sorted using the MESC subgroup sorting kit,and the subpopulations were divided into subgroups.Group 5,treated with different concentrations of HIV/Tat_1-86?0,12.5,25,50,100 nM?for 12h;the primary neurons of cerebral cortex were cultured in Balb/c fetal rat cerebral cortex and divided into 4groups,namely:neuronal group;neuron+MDSCs group;neuron+HIV/Tat_1-86-MDSCs group;neuron+HIV/Tat1_-86-MDSCs+inhibitor group.2.Flow cytometry was used to detect the changes of the proportion of cells in each subtype of MDSCs:after treatment of MDSCs at different concentrations of HIV/Tat_1-86,anti-mouse CD11b-labeled MDSCs,G-MDSCs and M-MDSCs subtypes were labeled with Ly6G and Ly6C,respectively.3.ELISA method for detection of MDSC cytokines:HIV/Tat_1-86 at different concentrations?0,12.5,25,50,100 nM?after treatment of MDSC,cell culture supernatants were used to ELISA detect 5 groups of MDSC cytokines reactive oxygen species?ROS?,Arginase 1?Arg1?and inducible nitric oxide synthase?iNOS?secretion.4.Treatment of neuronal cells:HIV/Tat_1-86?100 nM?treated with MDSCs for 12hours(represented by HIV/Tat_1-86-MDSCs)and untreated MDSCs were non-contacted with mouse neurons via Transwell chamber Co-culture models are processed differently by grouping.5.Fluorescence microscopy was used to observe the rearrangement of neuronal skeleton:FI-Phalloidin was used to stain the microfilaments?F-actin?of mouse neuronal cells,and the changes in F-actin rearrangement were calculated by fluorescence microscopy and photographing.6.Western-blot was used to detect the expression of anti-apoptotic protein Survivin protein in neurons.7.Statistical analysis of data:All experiments were repeated at least three times,and the data were processed with SPSS 21.0 statistical software.P<0.05 was considered statistically significant.Results1.Effect of HIV/Tat1-86 on MDSCs:Different concentrations of HIV/Tat_1-86?0,12.5,25,50,100 nM?in G-MDSCs and M-MDSCs,the ratio of the two subtypes was concentration dependent The expression was more pronounced at 100 nM?P<0.01?,with the proliferation of M-MDSCs being more pronounced.2.Effect of HIV/Tat1-86 on cell function of MDSCs:Statistical analysis of data:The levels of cytokine Arg1,ROS and iNOS released by MDSCs treated with HIV/Tat_1-86 at different concentrations of 0,12.5,25,50 and 100 nM increased significantly,and the increase of 100 nM was statistically significant?P<0.05?.3.Effect of HIV/Tat1-86MDSCs on neurons:HIV/Tat1-86?100 nM?treatment for12 hours After non-contact co-culture of MDSCs with neurons for 12 hours,compared with the control group the expression of phalloidin in neurons was significantly reduced?P<0.01?,Survivin expression decreased?P<0.01?,but after blocking the effect of the effector molecules Arg1,ROS,iNOS,the expression of phalloidin in neurons increased significantly?P<0.01?,and the expression of Survivin was significantly increased?P<0.01?.ConclusionsHIV/Tat_1-86-86 can promote the proliferation and activation of MDSCs,and the proliferation of M-MDSCs is more obvious.The increased secretion of cytokines Arg1,ROS and iNOS in activated MDSCs can cause neuronal skeleton rearrangement,and the expression of anti-apoptotic molecule Survivin is decreased,resulting in increased neuronal apoptosis.