The Immunity Study Of The Hepatitis B Vaccine Combined With Human Beta-defensin-2in BALB/c Mice

Objective:To explore the function of human beta-defensin2on the dendritic cellmaturation and the immunological effective on hepatitis B vaccine in mice as juvant.Methods:1. With the recombinant mouse rmIL-4(10ng/mL) amd rmGM-CSF(20ng/mL),mouse bone marrow cells were cultured for6days and the cellular morphology wasobserved by microscope continuously. At the same time, the cell surface molecules(CD11c, MHCII, CD80, CD86) were detected by flow cytometry(FCM).2. The mouse bone marrow cells which cultured for6days under rmIL-4andrmGM-CSF were cultured continually for another24h with PRMI1640containing0ng/mL,12.5ng/mL,25ng/mL,50ng/mL and100ng/mL HBD2, respectively. ThemRNA expressions of IL-6and IL-12p40were measured by qRT-PCR.and themolecules of MHCII,CD11c,CD80and CD86on the surface of the cell were examedby FCM.3. By using PBS as Control,300ng recombinant HBD2was injected into theintothe hind leg of BALB/c mice by intromuscle injection.The serum and spleen cellswere collected at4h,8h and12h, respectively. IL-12p70and IFN-γ in the serum of themice were detected by ELISA and the gene expression of IL-6, IL-12p40and IFN-γin spleen cells were tested by qRT-PCR.4.40BALB/c mice,4-6week old, were randomly divided into4groups. Themice in the first three groups were inoculated in the inner thigh muscles with200ulPBS,3ug hepatitis B vaccine and300ng HBD2, respectively. The mice in the4thgroup(named the combined group in the folowing text) were injected with300ngHBD2as above firstly, and4hours later3ug hepatitis B vaccine was administered atthe same site as HBD2injection for each mouse.The procedure of immunization forthe mice of all4groups was3times vaccination with2weeks interval.5. On the11th,22th,33th and42th day when the first injection began, the OD450 vaule of IL-2, the concentration of HBsAb, IFN-γ and IL-12p70in the serum of theimmumized mice were tested by ELISA. When the mice were sacrificed on the42thday of immunization procedure, the spleen was removed aseptically from the bodyand weighed to calculate the the spleen index according to the formation, the spleenindex=spleen weight(mg)/body weight(g). The gene expression of IL-2, IFN-γ andIL-12p40in spleen cells was measured by qRT-PCR, and HBsAg specific IFN-γsecreted by spleen cell from immumized mice was tested by ELISA after they werestimulated with hepatitis B vaccine in vitro for18h. At last, the percentages ofCD3+CD4+T cells and CD3+CD8+T cells in the splenic cell suspension weremeasured by FCM.Results:1. Under the observation of the inverted microscope, the mouse bone marrowcells cultured with rmIL-4and rmGM-CSF were found keep round shape andadherent to the wall partly on the1thday, a large number of point-like colony on the2thday, apparent larger cell colony on the3thday, and some star sharacteristics of DCwith semi-suspension growth and larger diameter continunously on the5thday. On the6thday, more cells were separated from the colony and switched into suspension stategrowth. The volume of the cell became bigger by successive and CD11c of86.2%,CD80of75.1%, CD86of53.7%and MHCII of44.8%were found on the surface ofthe cell by FCM.2. The results of FCM test for the BMDCs cultured with50ng/mL HBD2for24hshowed that91.32%CD11c+,92.65%CD80+,73.75%MHCII+, and73.81%CD86+on the surface of the cell was confirmed. It was relatively higher than BMDCscultured without HBD2. The percentages of MHCII+and CD86+cells in HBD2cultured group were statisticully significant higher than the cells cultured withoutHBD2(P<0.05). With the concentration of HBD2increasing from12.5ng/mL,25ng/mL to50ng/mL, the relative expression levels of IL-12p40gene increased from1.31±0.02,1.54±0.01to2.99±0.18according to the results of qRT-PCR detection, butwhen the concentration of HBD2reached at100ng/mL, it decreased to1.41±0.09unexpectedly. When BMDCs were cultured with50ng/mL HBD2, IL-12p40gene expression level was statistically significant hgher than the level in cells culturedwithout HBD2(P<0.01). IL-6gene expression levels showed the obviousdose-response relationship, along with HBD2concentration raising from12.5ng/mL(1.25±0.07),25ng/mL (1.58±0.09),50ng/mL (1.92±0.04), to100ng/mL (3.16±0.1).There was statistical significance between100ng/mL HBD2group and0ng/mLgroup (P<0.01).3. The serum IFN-γ and IL-12p70had no significant change at all time points inPBS group, but serum IFN-γ contents in HBD2group raised obviously from25.21±1.87pg/mL of4h,35.40±1.77pg/mL of8h, to42.04±1.59pg/mL of12h. It reachedpeak level at12h, and was statisticaly higher than other time groups(P<0.05). Theserum IL-12p70content in HBD2group were42.72±0.91at4h,39.82±0.45at8h,and32.08±1.68at12h. It reached the peak level at4h and was statisticaly higher thanother time groups (P<0.05); The values of serum IFN-γ and IL-12p70in all HBD2groups were also significantly higher than PBS group by ELISA. The levels of IL-6,IFN-γ and IL-12p40mRNA expression in the spleen cells from HBD2groups testedby qRT-PCR and the peak level of their gene expression was at4h. They were6.43±0.27,9.66±0.8and13.37±0.53, respectively, and were also statistically higherthan the level at other time point (P<0.05).4. There were no statistical difference for the spleen index of the mice in allgroups (P>0.05). Serum HBsAb levels were measured dynamically by ELISA on the11th,22th,33thand42thday, respectively, after the first immunization. The resultsshowed that S/C.O values of HBsAb in Hepatitis B vaccine group and the combinedimmunization group became higher and higher with the increasing of immunizationfrequency (P<0.05), and the S/C.O values in the combined group were statisticallyhigher than those of Hepatitis B vaccine group at each same time point of the test.They were0.42±0.03vs0.12±0.008on11thday,1.55±0.12vs1.17±0.072on22thday,2.03±0.14vs1.76±0.07on33thday, and3.31±0.176vs2.88±0.18on42thday,respectively. The OD450values of IL-2, and the concentrations of IFN-γ and IL-12p70in serum were also test by ELISA on2weeks after the3thimmunization. The resultsshowed that all3items from the combined immunization group were consistently thehighest one among total4groups. IL-2in combined immunization group was0.181± 0.02, and it was statistically higher than HBD2group and Hepatitis B vaccinegroup(P<0.05). But there unexpectively was no statistical difference when it wascompared with PBS group(0.12±0.012)(P>0.05). The levels of serum IFN-γ andIL-12p70in the combined immunization group were33.56±1.61pg/mL and40.18±3.02pg/mL, and they were all significantly higher than other groups(P<0.05).The mRNA expression levels of IL-2, IL-12p40and IFN-γ in splenic tissue fromthe immunized mice were tested by qRT-PCR. The results showed that the mRNAlevels of IL-2, IL-12p40from the combined immunization group were2.50±0.41and2.47±0.17, and they were significantly higher than the values from PBSgroup(1.02±0.06and1.03±0.08), HBD2group(1.76±0.3and1.26±0.105), andhepatitis B vaccine group(1.009±0.4and0.94±0.14), respectively(P<0.05). Theexpression level of IFN-γ mRNA in the combined immunization group (1.97±0.10)was also statistically higher than PBS group (1.02±0.19) and hepatitis B vaccinegroup(1.18±0.17)(P<0.05).The positive percentages of CD4+T cells tested by FCM were31.52±2.58%inHBD2group,27.36±1.59%in hepatitis B vaccine group and32.9±1.88%in thecombined immunization group, respectively, and they were statistically higher thanthe value of PBS group (14.85±0.59%)(P<0.05). Although the positive percentage ofCD4+T cells in the combined immunization group was highest one, there was nostatistical difference when it was compared with observations from the hepatitis Bvaccine group and HBD2group(P>0.05). The highest positive percentage of CD8+Tcell appeared in HBD2group, it was24.263±2.62%and there were significantdifference when it was compared with the other groups(P<0.05). However the ratio ofCD4+/CD8+T cell in the combined immunization group was1.75±0.07and it wasalso significantly higher than other group (P<0.05).At last, the HBsAg-specific IFN-γ content in the culture supernatant of thesplentic cells from each immunized mouse was measured by ELISA, in vitro. Theconcentrations of HBsAg-specific IFN-γ were17.57±0.93pg/mL in PBS group，21.92±2.97pg/mL in HBD2group,30.24±4.03pg/mL in hepatitis B vaccine groupand57.74±3.86pg/mL in combined immunization group, respectively. These resultsexpressed that the combined group owned the highest level of the HBsAg-specific IFN-γ and it was statistically higher than any other groups(P<0.05). The value ofHBsAg-specific IFN-γ from hepatitis B vaccine group were significantly higher thanthe value of PBS group(P<0.05), but there were consistently no significant differencebetween hepatitis B vaccine group and HBD2group(P>0.05), and between HBD2group and PBS group(P>0.05). It was oppsite to the statistical result which narratedabove that the serum values of IFN-γ in HBD2immunized mice was significantlyhigher than the one from hepatitis B vaccine group(P<0.05).Conclusion:HBD2could enhance the specific humoral immunity of the hepatitis B vaccineand promote its Th1type cellular immune function in mice, and perhaps itsmechanism is the function of enhancing the maturation of dendritic cells. HBD2maybe able to play a more important role in the prevention of HBV infection and theremoval of the invaded intracellular virus.