Study On The Role Of Th17Cells In HBV Infected Murine Model And HBV Antigen Regulate Th17Cells Differentiation

BackgroundThe hepatitis B virus （HBV）, a small, enveloped virus with a circular, partiallydouble-stranded DNA genome, is a major cause of infectious liver disease throughoutthe world. Although HBV has been described as a typical non-cytopathic virus, it isable to induce tissue damage with variable severity by stimulating immune responses.The function of these responses is to eliminate the intracellular viruses via destructingthe virus-infected cells, but the consequence of these responses can be complicated:the immune responses could be protective can be protective and beneficial to live inone hand, and could be damaging and detrimental. Worldwide400million peoplesuffer from a chronic HBV infection and approximately1million people die annuallyfrom HBV-related disease, such as liver failure, liver cirrhosis, and hepatocarcinoma.Due to its species-specificity, the studies on HBV is always lack of an ideal animalmodel, so that the immune pathological mechanism of HBV infection is not clear yet.Th17Cells is a recent discovered CD⁴⁺T helper subset, which are characterizedby the ability to secrete IL-17A, IL-17F, IL-21, IL-22, and IL-6. RORγt is a criticaltranscription factor for the differentiation of Th17cells. It has been demonstrated thatTh17cells is critical for some immune diseases. Recently, it was found that Th17cells and cytokines secreted by these cells were increased in HBV-related disease.This discovery prompted several immunological questions:1) whether Th17cellsplay an important role in HBV-related liver injury; and2) how does HBV antigenaffect the differentiation of Th17cells. Therefore, we established the murine model ofHBV acute infection by i.v. injection of Ad-HBV which resulted in expressed HBVantigen （HBsAg and HBcAg） in the liver. Utilizing this model, we examined whetheradoptive transfer of HBV antigen-specific CD⁴⁺T cells, which were induced to differentiate into Th17cells from naive CD⁴⁺T cells by HBV antigen, to Ad-HBVinfected mice could induce liver inflammation. In addition, we tested whether HBVantigen affected the differentiation of Th17cells and investigated the mechanism ofHBV antigen induced differentiation of Th17cells. Part1: Construction of recombinant adenoviruses with1.3copiesHBV genomeAimConstruction of recombinant adenoviruses with1.3copies HBV genome.Methods1.3-copy overlength genome of HBV carried by pcDNA3.1plasmid-was cloned intothe adenovirus shuttle vector pAdTrack. After confirmation of the constructed HBVgenome by sequencing, the linearized resultant plasmid was transformed into E. coliBJ5183which contained pAdEasy-1for homologous recombination. Afterconfirmation of the constructed HBV genome by sequencing, Plasmids pAd-GFP/HBV1.3were linearized and transfected into the packaging cells （HEK293AD cells）using Lipofectamine2000. After transfection, cells were harvested and subjected tothree freeze/thaw cycles, and Ad-HBV was purified by Cesium chloride gradientcentrifugation. Through the intensity of green fluorescent protein expression,;Ad-HBV infected the HBV DNA within the adnovirus geome was measured byFQ-PCR.ResultsConfirmed by restriction endonuclease, plasmids with pAdTrack-HBV andpAd-GFP/HBV1.3containing HBV1.3fragment were successfully constructed.Quantified by the intensity of green fluorescent protein （GFP） in HEK293AD cells,recombinated adenovirus Ad-GFP/HBV1.3carrying HBV1.3fragment wassuccessfully obtained. The titer of purified Ad-GFP/HBV1.3after Cesium chloridegradient centrifugation is around7.89×1012copies/ml.ConclusionsThe adenoviruses containing HBV1.3fragment were constructed successfully. Part2: Establishment of a murine model with infection of HBV andinvestigation of the early stage immune response in C57BL/6micechallenged by Ad-HBVAim1．Establishment of a murine model with acute infection containing HBV1.3fragment（Ad-HBV）.2．Investigation of the early stage immune response in C57BL/6mice challenged byAd-HBVMethodsAd-HBV was delivered into C57BL/6mice using the tail vein injection method. OnDay1,2,3,5,7after injection the serum, spleen and liver were collected. The levelsof ALT were measured with improved LaiShi method. The levels of HBsAg, HBeAg,anti-HBs, anti-HBe and anti-HBc in blood serum were determined with time-resolvedimmunofluoro metric assay （IFMA）, and the titers of HBV DNA in blood serumwere analyzed by fluorogenic quantitative polymerase chain reaction （FQ-PCR）. Thelevels of Cytokine INF-γ, IL-4and IL-17produced by liver cells were detected withELISA, which were induced by HBV antigens （HBsAg and HBcAg）. HBV cccDNAwere analyzed in the liver by Real-Time polymerase chain reaction （Real-Time-PCR）.Viral specific proteins （HBsAg and HBcAg） in the liver tissue were assayed byimmunohistochemi cal staining. The green fluorescent protein （GFP） werequantified in the liver tissue. In addi tion, the inflammatory area were assayed in theliver tissue by HE staining.ResultsResults showed that the levels of serum ALT were elevated mildly and inflammationappeared in liver tissue after C67BL/6mice were injected with either Ad-HBV or Ad,which were no significant difference between the two groups. The levels of serum ALT and liver histological changes begun to returned back to normal at day3. HBsAgand HBV DNA in mice serum could be detected1day after Ad-HBV injection.HBsAg, HBcAg and HBV cccDNA were detected in mice liver, whose changes weremost obvious3days after injection. Anti-HBs and anti-HBc could be determined inmice serum respectively5days and7days after infection, and anti-HBe could not befound in mice serum. The levels of INF-γ, IL-4and IL-17did not changed in livercells treated with HBsAg and HBcAg.Conclusions1． a murine model with acute infection of HBV by i.v. injection of Ad-HBV wasestablished successfully.2．Ad-HBV did not cause liver injury in the mice in early period of HBV infection.3． the specific immune response of C57BL/6mice induced by Ad-HBV in earlyperiod was not detected. Part3: Studies on the role of HBV antigen specific Th17cells in theliver of mice infected by Ad-HBVAim1．Induction of HBV antigen-specific Th17cells.2．The role of HBV antigen specific Th17cells in the liver of Ad-HBV infected miceMethods1．Induction of HBV antigen-specific CD⁴⁺T cells in C57BL/6mice were executed byintranasal inoculation of5×106Ad-HBV （in200ul PBS）. After4week, mice wereimmunized with Ad-HBV again. The immunized mice were sacrificed after1week,spleen cells were stimulated with20μg/ml HBsAg and20μg/ml HBcAg in thepresence/absence of1ng/mL TGF-β,10ng/mL IL-6,10ng/mL IL-23,5μg/mLanti-IFN-γ mAb, and5μg/mL anti-IL-4mAb. After72hours, CD⁴⁺T cells in thisculture were separated using MACS, and these cells was restimulated under theconditions described above with mitomycin C-treated C57BL/6spleen cells（5:1）.Then, CD⁴⁺T cells were expanded in the presence of high concentrations of ConA.The levels of Cytokines including IL-17, IL-22, INF-γ, and IL-4produced by thesecells were detected by ELISA. IL-17+CD⁴⁺T cells、 INF-γ+CD⁴⁺T cells, andL-⁴⁺CD⁴⁺T cells in the these cells were analyzed by FACS. The mRNA levels ofIL-17, IL-22, and RORγt were quantitatied by Real-Time-PCR.2． Establishment of a liver injury model induced by HBV antigen-specific CD⁴⁺Tcells. C57BL/6mice were treated first with i.v. injection of Ad-HBV （in200μl saline）by tail and3days later with cells transfer. Cultured HBV antigen-specific CD⁴⁺Tcells were washed and resuspended in saline, and2×107cells （in200μl saline） wereinjected i.v. by the mice tail. The mice were sacrificed24hours later, and the serumwere collected to determine alanine aminotransferase（ALT）. The liver tissue sampleswere fixed in10%formaldehyde and embedded in paraffin, and sections were stainedwith HE. Results1．The proportion of IL-17+CD⁴⁺T cells in HBV antigen combined cytokines groupswas significantly higher than that in antigen treated group and PBS group, as well asthe protein and mRNA levels of IL-17and IL-22（p<0.01）. Meanwhile, the mRNAlevels of RORγt were increased significantly. These results indicated that na ve CD⁴⁺T cell can be induced to differentiate into Th17cells in mice immunized with HBV byHBV antigen combined cytokines. The percentage of INF-γ+CD⁴⁺T cells and therelease of INF-γin antigen treated group were significantly higher than in the othertwo groups. The percentage of IL-17+CD⁴⁺T cells and the releasing of IL-17andIL-22in antigen treated group were higher than in the PBS treated group. Accordingto the percentage of IL-⁴⁺CD⁴⁺T cells and the release of IL-4, there was nosignificant difference in different groups.2．Higher serum ALT, inflammatory response, swollen cells, necrosis and structuraldisorder in liver could be found in mice infected by Ad-HBv which is adaptivelytransferred to cells with both HBV antigen combined cytokines and HBV antigen.Conclusions1．HBV antigen-specific Th17cells were induced successfully in vitro.2． HBV antigen specific Th17cells cause the liver injury in mice infected withAd-HBV. Part4: HBV antigen regulates Th17cells differentiationAims1．Effect of HBV antigen on the differentiation of Th17cells in mice2．The mechanism of HBV antigen regulation on Th17cells differentiation.Methods1． The mice were immunized with recombinant HBsAg, HBcAg, and HBeAgrespectively in the presence of adjuvants. Spleen cells from the immunized mice werestimulated with20μg/ml HBsAg,2.5μg/ml HBcAg, and2.5μg/ml HBeAgcorrespondingly for24hours. The population of IL-17+CD⁴⁺T cells was analyzed byFACS. CD⁴⁺T cells separated from the spleen cells by MACS were stimulated withMPMs under HBsAg, HBcAg, and HBeAg respectively. After72h, the levels ofCytokine IL-17and IL-22produced by these cells, and the mRNA levels of IL-17,IL-22, and RORγt in these CD⁴⁺T cells were detected.2．To study the mechanism of HBV antigen regulation on Th17cells differentiation,the culture media and cells described above also were harvested at24hour, for IL-6and IL-23determination by ELISA, TLRs, IL-6and IL-23mRNA levels detection byReal-Time PCR. In addition, mice peritoneal macrophages （MPMs） were incubatedwith virous concentration of recombinant HBsAg, recombinant HBcAg andrecombinant HBeAg. After24h, the mRNA levels of TLR7, IL-6and IL-23wereanalyzed by real time-PCR, the concentration of IL-6and IL-23in medium wereassayed by ELISA, and the protein levels of TLR7were detected via western blot.After MPMs were transfected with TLR7specific siRNA, the silence efficieny wasdetermined via analyzing the protein level of TLR7, the levels of Cytokine IL-17andIL-22produced by these cells, and the levels mRNA of IL-17, IL-22, and RORγt inthe CD⁴⁺T cells treated above were detected, and the mRNA and protein levels ofIL-6and IL23in MPMs treated above were detected by real time-PCR and ELISArespectively. Results1．The proportion of IL-17+CD⁴⁺T cells in spleen cells from the mice immunized byHBsAg, HBcAg and HBeAg were higher than in adjuvant control group. The mRNAlevels of IL-17, IL-22and RORγt in CD⁴⁺T cells from the mice immunized byHBsAg, HBcAg and HBeAg were higher than in adjuvant control group, as well asthe secretion of IL-17and IL-22（P <0.01）. The mRNA level and secretion of IL-17were higher in HBcAg and HBeAg groups than in HBsAg group, with the highestIL-17levels in HBcAg group. The mRNA level and secretion of IL-22were higher inHBsAg group than in HBcAg and HBeAg groups, while there was no differencebetween HBcAg and HBeAg. The mRNA level of RORγt was higher in HBcAg groupthan in the other two groups.2．The mRNA levels of TLR2,3,4,7, and9in spleen cells from mice immunized byHBsAg, HBcAg and HBeAg were analyzed by quantitative PCR. The mRNA levelsof TLR17were higher in spleen cells treated by each HBV antigen than in theadjuvant group. The up-regulation of TLR4mRNA was detected only in spleen cellstreated with HBcAg The upregulation of TLR2mRNA was found only in spleen cellstreated with HBeAg. The mRNA levels of TLR9were down-regulated in spleen cellstreated with each HBV antigen. While, the mRNA levels of TLR3had no changes inspleen cells treated by any HBV antigen. The mRNA level and secretion of IL-6isincreased in spleen cells treated with HBsAg, HBcAg and HBeAg, while the mRNAlevels and secretion of IL-23is increased only in spleen cells treated with HBcAg andHBeAg.3． The mRNA and protein levels of TLR7, as well as IL-6and IL-23, wereup-regulated in MPMs treated by HBcAg or HBeAg in a dose-dependent manner,The up-regulation of TLR7and IL-6could be detected in MPMs treated with HBsAg.The protein expression of TLR7in MPMs was silenced effectively by specific siRNA.The percentage of IL-17+CD⁴⁺T cells, the levels of Cytokine IL-17and IL-22produced by these cells, and the mRNA levels of IL-17, IL-22, and RORγt in theCD⁴⁺T cells treated above were abolished by specific TLR7-siRNA; The up-regulation of IL-6in MPMs mediated via HBcAg, HBcAg and HBeAg wereabolished by specific TLR7-siRNA. The upregulation of IL-23in MPMs mediated byHBcAg and HBeAg was abrogated by specific TLR7-siRNA.Conclusions1． HBsAg, HBcAg, and HBeAg may regulate Th17cells differentiation, whicheffect are strong to weak, respectively via HBcAg, HBeAg and HBsAg2．Th17cells challenged by HBcAg and HBeAg secreted IL-17mainly, while Th17cells induced by HBsAg secreted IL-22mainly.3． HBcAg and HBeAg regulate Th17cells differentiation by TLR7with increasedsecretion of IL-6and IL-23in MPMs. and HBsAg regulate Th17cells differentiationby TLR7with increased secretion of IL-6in MPMs.