Study On Exhausting Mechanism Of NKG2A Mediated Liver NK Cells In Alveolar Echinococcosis

Objective:Hepatic alveolar echinococcosis（AE）is a fatal zoonosis caused by Echinococcus multilocularis（E.m）and its invasive growth is similar to"liver cancer".At present,a large number of studies have shown that host immune status and prelesion immune microenvironment affect parasitism,lesion activity,progression and prognosis of E.multilocularis infection.The aim of this study was to investigate the functional mechanism of the inhibitory surface receptor NKG2A on the liver NK cells exhaustion during AE.Methods:Part one,1.Using flow cytometry technic to investigated the percentages and functional changes of liver NK cells and the maturation in mice infected with E.multilocularis;Using the RT-to detect of NK cell-related molecule expression in liver tissue of infected mice;2.By in vivo NK cell depletion experiment to observed the differences of size,number,total liver quality,hepatic pathological changes,prelesion fibrosis area between the experimental and control group;3.Through the flow cytometry technic to detect the changes of activation and inhibitory receptors on liver NK cell and the changes of NKG2A⁺NK cell secretion of cytokines at different time after inoculation in mice;Part two,1.Lymphocytes were isolated from fresh liver tissue specimens from 10hepatic AE patients,Collagenase digestion and lymphocyte isolation were performed on10 fresh liver tissue specimens from patients with hepatic AE,testing of NKG2A molecule expression on liver NK cell in lesion close liver tissues（CLT）and distal liver tissues（DLT）,analysis of the correlation between NKG2A⁺expression and lesion size,alkaline phosphatase（ALP）and other clinical data;investigate to the changes of cytokines and cytotoxic substances secretion by liver NK cells and NKG2A⁺NK cells in CLT and DLT;cytokines secretion were compared between NKG2A⁺NK and NKG2A^-NK cells in CLT;2.The expression of NKG2A molecules in CTL and DLT was detected by immunohistochemical technique in clinical liver tissue from 36 hepatic AE patients,in the same time,analysis of the correlation between the NKG2A expression and the lesion activity;Masson andα-SMA staining were used to analyze the correlation between pre-lesion liver fibrosis and activity of hepatic focus;3.Lymphocytes isolated from clinical liver tissue specimens of 10 AE patients and analyzed of CD56^brightNK and CD56^dimNK cells subsets and their NKG2A receptor expression;Part three,1.NK cells were purified from normal human peripheral blood and stimulated by parasite protein in vitro for 24 hours,analysis of expression NKG2A on NK cell and the secretion of cytokines by NKG2A⁺NK cells.Results:Part one,1.The percentage of liver NK cells in infected mice were significantly lower than control mice at 2,4 and 12 weeks after infection（P=0.0023,P=0.0430,P=0.0031）,but there was no significant difference between the two groups at 24 weeks after infection（P>0.05）;At 2,4 and 12 weeks after infection,the function of IFN-γsecretion from liver NK cells was significantly lower in infected group than control group（P=0.0001,P=0.0002,P=0.0009）,but at 24 weeks,there was no significant difference in the function of IFN-γsecretion between the two groups（P>0.05）;Besides,There was no significant difference in the function of granzyme B and TNF-αsecreted by liver NK cells between the two groups at all time points（P>0.05）;We also observed that the percentage of CD27⁺CD11b⁺NK（the most powerful cell subset during the maturation process of NK cells）in 2 and 4 weeks after infection was significantly lower in infection group than control group（P<0.0001,P<0.0001）,but the percentage of CD27+CD11b+NK gradually increased at the 12 weeks after infection,and that was significantly higher than control group（P=0.0031）;2.In vivo NK cell depletion,the number and volume of liver lesion,total liver weight in depletion mice were significantly higher than control（P=0.0054,P=0.0007,P=0.0205）;After NK cell depleted,the number of inflammatory foci and infectious foci with metacestode structures were higher than control mice（P=0.0047,P=0.0198）,but the repair granuloma Focus was no significant difference between the two groups（P>0.05）;3.The expression of NKG2A molecule m RNA was significantly higher than control group at 2 and 24 weeks after infection（P=0.0049,P<0.0001）,but there was no difference at 12 weeks（P>0.05）;NK cell The expression of inhibitory receptor（NKG2A）on liver NK cells were detected by flow cytometry,the expression of NKG2A on liver NK cells was significantly increased in infection group compared with the control group all time points（P=0.0058,P=0.0326,P=0.0156,P=0.0156）;The IFN-γfrom NKG2A⁺NK cell was significantly lower after infection than control group at 2,4 and 12 weeks after infected with E.multilocularis（P=0.0099,P=0.0050,P=0.0100）,but there was no difference at 24 weeks（P>0.05）,however,the granzyme B and TNF-αsecretion were no difference all time points（P>0.05）.Part two,1)The percentage of NKG2A expression on total liver NK cells in CLT was significantly higher than in DLT（P=0.0137）;Meanwhile,the expression of NKG2A on liver CD56 ^dimNK cells was significantly higher in CLT than in DLT（P=0.0480）;The expression of NKG2A on total liver NK cells was negatively correlated with ALP（P=0.0234,R=-0.7212）,but there was no correlation between with ALT and AST;There was no correlation between the maximum diameter of the lesion with the percentage of NKG2A expression in total NK cells,this result need to prove by increased sample size;The ability of IFN-γproduction of total liver NK cells was significantly down regulated in CLT than in DLT（P=0.0488）,but the production of granzyme B,perforin and TNF-αhad no difference（P>0.05）;The expression of NKG2A on total liver NK cells in CLT was negatively correlated with the secretion of IFN-γ（P=0.0438,R=-0.6606）;The production ability of IFN-γand granzyme B of NKG2A⁺NK cells were significantly decreased in CLT than in DLT（P=0.0408,P=0.0371）,but there was no difference in production of TNF-αand perforin（P>0.05）;2)Using the immunohistochemical method,analysis of the expression of NKG2A in CLT was significantly higher than DLT（P=0.0001）;There was a positive correlation between the expression area of NKG2A in CLT/DLT and PET-CT（P=0.0065,R=0.735）;Masson staining was used to detect the area of fibrosis around high-activity and low-activity lesions.The area of fibrosis around high-activity lesions was significantly lower than low-activity lesions（P=0.0398）;The positive expression area of NKG2A in CLT was negatively correlated with the fibrosis area around the lesion（P=0.0229,R=-0.6742）,but there was no correlation with the positive area of hepatic stellate cells by alpha-SMA staining（P=0.2149,R=0.4064）;3)We also observed that the percentage of total NK cells,CD56^brightNK cells and CD56^dimNK cells,percentage of total liver NK cells and CD56^brightNK cells in CLT and DLT tissues showed no significant difference（P>0.05）,but the percentage of CD56^dimNK in total NK cells significantly decreased in CLT（P=0.0059）.Part three,1.NK cells were purified from peripheral blood of healthy volunteers,the expression of the inhibitory receptor NKG2A was significantly increased after stimulation with E.multilocularis protein（Emp）.Besides,the secretion of IFN-γby NK cells and NKG2A⁺NK cells were significantly decreased（P=0.0094,P=0.0355,P=0.0460）;2.The expression of NKG2A increased and the secretion of IFN-γby NK cells and NKG2A⁺NK significantly decreased after stimulation by TGF-β1（P=0.0266,P<0.0001,P=0.0049）.Moreover,the function of IFN-γsecretion by NK cells decreased significantly in TGF-β1 stimulated group than Emp.Conclusion:1.The up-regulation of NKG2A expression on liver NK cells might mediate the exhaustion of hepatic NK cells function after infected with E.multilocularis,this functional exhaustion might be mainly represent by down-regulation of IFN-γsecretion.This lead to reduction of fibrous area of around the lesion and reduce the limitation from fibrous area for lesion,finally causes increased growth in liver lesion;2.Increased expression of NKG2A on total liver NK cells and CD56^dimNK cells in CLT,this may lead to decreased secretion of IFN-γ,reduced fibrosis around the lesion and faster growth of the lesion in hepatic AE patients;3.Emp stimulates may mediate the exhaustion of IFN-γsecretion by NK cells through the up-regulation of NKG2A expression,which may be one of the reasons that E.multilocularis successfully escaped the immune surveillance by NK cells.This study provide a theoretical basis for blocking NK cell surface receptor NKG2A might be reversing immune escape of E.multilocularis.It have important scientific significance for deepening the study of echinococcosis and exploring new immunotherapy of E.multilocularis.