The Preliminary Study Of TGF-β Inhibiting The Natural Killer Cells Functions In The Immune Escape Of Echinococcus Granulosus

ObjectiveThrough the establishment of animal models of Echinococcus granulosus (E. granulosus) infected mice,we explored whether E. granulosus promote TGF-β1secretion by spleen cells, and then induceCD4+CD25+T cell differentiation, and thus reduce the expression of activity receptor NKG2D of NK cells,resulting in the final the reduced efficiency of NK cells killing of E. granulosus, which have evolvedmechanisms of evading the host immune response.Methods1. We established a mouse model via protoscolices infection, and BALB/c mice were infected with PSCin experimental groups, and mice were injected with the same volume of PBS in control groups; the micewere euthanised at1,3,5,7,9and12days post-infection, and the CD4+/CD8+T cell ratio, the numberof CD4+CD25+T cells, and expression of activity receptor NKG2D of NK cells of in spleen cells weremeasured by flow cytometry; the relative mRNA levels of Foxp3and TGF-β1were quantified byreal-time quantitative PCR; the Yac-1target cells were co-cultured with spleen cells of mice, the lysisrate of Yac-1cells by NK cells were detected by LDH assay, and cytotoxic activity of spleen cells wereobserved.2. We established a mouse model via E. granulosus infection, TGF-β1receptor was blocked with specificinhibitors, the mice were euthanised at9days post-infection, and the CD4+/CD8+T cell ratio, the numberof CD4+CD25+T cells, and expression of activity receptor NKG2D of NK cell of in spleen cells weremeasured by flow cytometry; the cytotoxic activity of spleen cells were detected by LDH assay; theeffect of E. granulosus on TGF-β/Smad pathway was determined by western bolting.Results1. During the early stages of E. granulosus infection in BALB/c mice, the CD4+CD25+Foxp3+T cell ratiowas increased, there was a decreasing trend for the CD4+/CD8+T cell ratio compared with that in controlgroup; the number of CD4+CD25+T cells with prolonged E. granulosus infection tended to increase;there was increasing trend in relative expression of the Foxp3gene, and these differences werestatistically significant, except on the first day (P<0.05).2. During the early stages of E. granulosus infection in BALB/c mice, TGF-β1production was increased inthe peripheral blood: the result of enzyme-linked immunosorbent assay showed that the cytokinesTGF-β1levels from peripheral blood were in the infected mice significantly higher than those of thecontrol groups (P<0.05), which showed an increasing trend with increased infection time; Similarly, thelevels of TGF-β1mRNA in the E. granulosus-infected groups were also significantly higher than thoseof the control groups (P<0.05),which showed an increasing trend with time depandent. 3. During the early stages of E. granulosus infection in BALB/c mice, the number and cytotoxic activity ofNK cells decreased in mice: the lysis rate of Yac-1cells by NK cells was statistically significantcompared to the control groups (P<0.05); there was decreasing trend in the expression of activityreceptor NKG2D of NK cells with increased infection time, and the differences were statisticallysignificant compared to the control groups at1,3,9and12days post-infection (P<0.05), there wasdecreasing trend in the number of NK cells with increased infection time, and the differences werestatistically significant compared to pre-infection groups at1,3,9and12days post-infection (P<0.05),there was a positive correlation between the expression of activity receptor NKG2D and killing activityof NK cells, R2=0.679.4. During the early stages of E. granulosus infection in BALB/c mice, all these results were reversed afterTGF-β1receptors were inhibited by SB-525334: its downstream molecule phosphorylation of Smad2/3were decreased, the expression of activity receptor NKG2D of NK cells increased, the lysis rate of Yac-1cells by NK cells was restored, there was a positive correlation between the expression of activityreceptor NKG2D and killing activity of NK cells, the CD4+/CD8+T cell ratio was increased, the numberof CD4+CD25+T cells were decreased.Conclusion1. Through the establishment of animal models of E. granulosus infected mice，we observed: E. granulosuspromote TGF-β1secretion of spleen cells, and then induce CD4+CD25+T cell differentiation, and thusreduces expression of activity receptor NKG2D of NK cells, resulting in the final the reduced efficiencyof NK cells killing of E. granulosus, which have evolved mechanisms of evading the host immuneresponse.2. Inhibition of TGF-β signaling by the inhibitor of TGF-β1receptor SB-525334may have practicalsignificance for prevention of clinical relapse recurrence of hydatid disease.