Analysis Of Changes In The γδT Cells In A Mouse Model Of Acute Hepatitis B Virus Infection By Hydrodynamics-based In Vivo Transfection With HBV Plasmid

Objective: γδT cells are one important type of innate immune cell. Ourprevious studies have shown that γδT cells had some abnormal phenotypeor function in the patients with chronic Hepatitis B. In this study, a mousemodel of acute hepatitis B virus infection was constructed for detecting thechange in the proportion, the activation phenotype, or the cytokineexpression of liver γδT cells, and exploring the role of γδT cells in theclearance mechanism of acute HBV infection. This study might providesome data to reveal the effect of γδT cells in the early stage of HBVinfection or persistent HBV infection.Methods:1. A mouse model of acute hepatitis B virus infection was constructedby hydrodynamics-based injection of plasmid containing full-lengthHBV genome (pcDNA3.1-HBV1.3).2. The expression of HBsAg or HBcAg in liver of model mice was detected with immunohistochemistry method, and the level of serumHBsAg or HBeAg was determined by electrochemiluminescenceimmunoassay (ECLIA).3. The proportion of total γδT cells in liver, spleen or peripheral blood ofpcDNA3.1-HBV1.3plasmid (pHBV group) or pcDNA3.1controlplasmid (pcDNA group) was detected by Flow Cytometry (FCM) at thedifferent time points after plasmid injection.4. The proportion of CD25+or CD69+γδT cells in the liver of mouse inpHBV group was detected by FCM.5. The proportion of IFN-γ+, or TNF-α+γδT cells in the liver of mouse inpHBV group was detected by FCM.6. The data was statistically analyzed by SPSS software to find whetherthese differences were significant.Results：1. Construction of mouse model of acute hepatitis B virus infection1.1HBsAg was positive in the serum of pHBV group on the first dayafter plasmid injection (33.64±14.88COI), and HBsAg reached itspeak level at5day (146.92±24.23COI) followed by gradual decline.1.2For HBeAg in pHBV group, it was positive and got the maximumlevel at the first day (2.26±2.34COI), then its level decreased.1.3In pHBV group, the rate of HBsAg-or HBcAg-positive hepatocytes got the highest rate at day1(10～20%), and then declined rapidly (＜5%).1.4HBsAg or HBcAg expression in liver, or HBsAg or HBeAg levels inserum at each time point were detected negative in pcDNA group.2. The changes in the proportion of γδT cells in liver, spleen, orperipheral blood of the mouse model of acute hepatitis B virusinfection2.1For the normal control mice (NC), the proportion of γδT cells in liver(4.5±0.6％) was significantly higher than that in spleen (2.1±0.3％) orin peripheral blood (1.2±0.6％)(p<0.01).2.2In pHBV group, the proportion of liver γδT cells increased to8.3±4.3%at the first day, then decreased following a quick rise to8.0±1.2%at the10th day. The difference in the proportion betweenfirst day and NC, the5th or15th day was significant (p<0.05).2.3There were no significant differences in the proportion of spleen orblood γδT cells among all groups (p>0.05).2.4When compared the pcDNA groups and NC, the statistics resultsshowed no significant differences in the proportion of liver, spleen orblood γδT cells (p>0.05).3. The changes in the surface CD25or CD69expression on liver γδT cells of pHBV mice3.1In the liver of NC mice, the proportion of CD25+or CD69+γδT was3.1±0.5％or9.9±2.7％.3.2In the pHBV group, the proportion of liver CD25+γδT cells got thepeak of8.4±3.7%, but no significant differences were found amongthese groups (p>0.05).3.3The percentage of liver CD69+γδT cells decreased gradually, and thepercentage at the10th day was the lowest which was significantdifference from that at other days or NC (p<0.05).4. The changes in the proportion of liver IFN-γ+γδT or TNF-α+γδT cellsof pHBV mice4.1The proportion of liver IFN-γ+or TNF-α+γδT cells in NC mice was5.1±0.4％or4.7±0.6％。4.2The proportion of liver IFN-γ＋γδT in pHBV mice increased to8.6±3.2%at first day and then declined, which has no significantdifference among these groups (p>0.05).4.3There showed a upward trend in the proportion of liver TNF-α+γδTcells of pHBV mice, but no significant differences were found amongthese groups (p>0.05). Conclusion：1. A mouse model of acute hepatitis B virus infection was successfullyconstructed.2. There were some changes in the proportion, activation phenotype, orthe cytokine producing of liver γδT of this model.3. All these results showed some role of γδT cells in the process of acuteHBV infection and clearance.