Study On The Molecular Mechanism Of Functional Depletion Of NK And CD8~+ T Cells Mediated By Magt1 In Chronic HBV Infection

BackgroundAs one of the most common infectious pathogens,hepatitis B virus?HBV?is a serious threat to human health.The data from world health organization?WHO?showed that the population of patients with chronic HBV infection in the world was up to 200 million,and that viral hepatitis continued to rank seventh in the world's cause of death.The population of patients infected with HBV virus has been up to nearly 100 million,ranking the first in the world.It is a therapeutic target to effectively remove HBV-DNA in hepatocytes during the hepatitis B treatment by antiviral therapy or hepatitis B virus-specific clearance.After the HBV virus infection,the patients'condition developed rapidly,and gradually developed into chronic hepatitis B.Then with the development of the disease,chronic hepatitis B could gradually progress for end-stage liver diseases,such as liver cirrhosis?LC?,hepatocellular carcinoma,liver failure?ALF?and so on.The prognosis of the diseases was poor,and the diseases were easy to relapse,which were serious threats to the safety of patients.Therefore,it has become an urgent and severe public health issue to explore the host antiviral immune mechanism and developing new therapeutic drugs,which need give priority to research.The activation of the host immune system,such as immune cells CD4⁺T cells,CD8⁺T cells and NK cells,played an important role in the fight against HBV infection and its immune injury in the liver.The activation of T cells and NK cells was dependent on the surface activation-related receptors,among which the programmed cell death-1?PD-1?,one of inhibitors,and NK cell receptor 2D?Natural killer cell group 2D,NKG2D?,the potent activated receptor,had important effects on regulation of the immune function of T cells and NK cells.The researches found that the co-stimulatory signaling pathway of PD-1 and its ligand PD-L had played an important negative regulation of many processes,for example,T cells'activation,proliferation and cytokine secretion.Magnesium ions(Mg²⁺),the divalent cations,were necessary for normal cellular metabolism and played an important role in the immunomodulation processes as a second messenger.As a highly selective transporter of Mg²⁺,MagT1 could exert the role of regulation of immune response by regulating concentration of intracellular free Mg²⁺.When MagT1 protein was lacked in the human immune cells,the free Mg²⁺concentration in the immune cells decreased significantly and the expression of NKG2D receptor in NK cells reduced,so that cytotoxic effects on EBV mediated by NK cells were significantly weakened,leading the body prone to EBV infection and tend to develop lymphoma.When the patients were orally treated with L-magnesium threonate(Neural Magtein^TM),the concentration of Mg²⁺in vivo was restored and the expression of NKG2D was up-regulated.So the antiviral activities of NK cells and CTL were enhancd,so that the replication of EBV virus was inhibited.Therefore,the results indicated that application of Mg²⁺for the clinical treatment of chronic hepatitis B could improve the immune activity of patients,which had a certain objective basis.Main contents and objectiveBased on the above background,the clinical patients with HBV were researched in our study.On the one hand,the blood of patients was collected to explore the reason of CD8⁺T and NK cells function depletion in the peripheral blood of patients with HBV,and try to provide a new target for treatment of HBV.On the other hand,in order to observe the effects of magnesium on the clinical efficacy,HBV patients were treatment with Neural Magtein^TM and entecavir.At the same time,to explore and verify whether the addition of the right amount of magnesium could restore the cytotoxicity of"immune depletion"of NK and CTL cells in the process of the treatment of HBV,and which could clean up HBV at the maximum.Main research resultsAiming at the mechanism research of CD8⁺T and NK cells function depletion in peripheral blood of patients with HBV,the results are as follows:?1?With the time of HBV infection,the concentration of Mg²⁺/Ca²⁺in peripheral blood of chronic HBV patients was not changed.?2?The concentration of Mg²⁺in CD8⁺T cells decreased with the prolongation of HBV infection time,which might be due to the disorder of Mg²⁺influx.However,the changes of Mg²⁺and Ca²⁺in NK cells were not correlated with HBV infection time.?3?With the prolongation of HBV infection time,the expression of MagT1 protein in PBMC cells and CD8⁺T cells were decreased gradually,but it did not change significantly in NK cells.?4?The expression of MagT1 protein in PBMC cells and CD8⁺T cells after HBV infection was caused by post-transcriptional regulation.The regulatory miRNAs included mi R-199a-5p,miR-199b-5p,miR-374c-5p,miR-655-3R,miR-9-5p,miR-124-3p and so on,especially mi R-374c-5p,mi R-655-3p and miR-124-3p.?5?In the late stage of HBV infection,with the up-regulation expression of PD-1 and the down-regulation expression of NKG2D,the activities of CD8⁺T and NK were been inhibited,and leaded to CD8⁺T and NK cell function depletion.Aiming at the efficacy and safety of Neural Magtein^TMM combined with entecavir in the treatment of HbeAg-positive chronic hepatitis,the results are as follows:?1?Neural Magtein^TM combined with entecavir in the treatment of hepatitis B is effective,Neural Magtein^TM can inhibit the replication and expression of HBV DNA,accelerate HBV-DNA to became negative,which has anti-HBV effect.?2?Neural Magtein^TM combined with entecavir could effectively reduced the level of HbeAg in the blood of patients with hepatitis B,but the serum levels of HbeAg in the treatment group and observation groups were not became negative after 12 months of continuous treatment,and we suspected that the reason was related to a shorter treatment of drugs.?3?Neural Magtein^TM could effectively and rapidly supplemented the level of serum Mg²⁺in patients with hepatitis B,which might inhibit the level of serum ALT in patients.However,the concentration of AST in blood,platelet activation,the number of white blood cells and the concentration of Ca²⁺and K⁺were no significantly changed.?4?Neural Magtein^TM could increased the expression level of MAGT1 protein in PBMC cells of patients with hepatitis B,and the combination of drugs could significantly increased the expression of protein NKG2D in PBMC and significantly decreased the expression of PD-1.?5?Neural Magtein^TM could significantly promoted the expression of NKG2D and MAGT1 in CD8⁺T cells of patients with hepatitis B and could down-regulated the expression of PD-1 protein.Conlusion:In summary,this study revealed the mechanism of MAGT1-mediated disturbance of Mg²⁺homeostasis lead to exhausted of HBV-infected NK and CD8⁺T cells.With the prolongation of HBV infection time,the infection could lead to down-regulation of the expression of transmembrane protein MagT1 and cause Mg²⁺influx disorder,and then cell surface inhibition receptor PD-1 expression was upregulated and activated receptor NKG2D expression was down-regulation,resulting in function depletion of CD8⁺T cells and NK cells in peripheral blood in patients with hepatitis B virus infection.In addition,supplement of the corresponding Mg²⁺agent(Neural Magtein^TM)could restore the immune status of NK and CD8⁺T cells in HBV infection in the clinical study.