Significance And Regulation Of TLR5 And Foxp3 In Allo-Transplantation Mice

ObjectivesThe significance of Toll-like receptor family(TLRs) on immune response has been highly concerned in recent years.TLRs can specifically identify the pathogen -associated molecular patterns(PAMPs),and play important roles not only in the activation of innate immunity,but also in the induction and regulation of adaptive immunity.So the TLRs can also be regard as an important bridge between innate immunity and adaptive immunity.TLRs can be activated by autologous antigens, allogenic antigens and heterologous antigens.The latest study in 2008 showed that existence of natural Tregs was necessary for the long-term survival of heart and skin allograft in mice,the CpG-specific activator of TLR9 can inhibit the function of Tregs and promote the differentiation of Th1 effective T cells to inhibit the survival of the allograft.Activation of TLR7 can promote the allograft rejection;activation of TLR4 can inhibit the long-term graft of heart survival,prevented the accumulation of the CD4~+/Foxp3~+ Treg within grafts.So we propose that the activation of TLRs may be critical in both the allograft tolerance and rejection. Recently it has been shown that CD4~+CD25~+ regulatory T cells(Treg) participate in transplantation tolerance which induced by a variety of ways,Treg is not only play a critical role in maintaining self-tolerance but also has great significance in preventing allograft rejection.So how to promote the Tregs's expansion effectively is a key factor for the induction of transplantation tolerance.It was found recently that TLR5 is the only negative regulatory TLRs which may be directly or indirectly involved in the process of autoimmune diseases.TLR5 can be activated by bacterial Flagellin,a specific ligand to TLR5,and mediate the immune response by stimulating the production of TNF-α,IL-1β,IL-8 and other pro-inflammatory cytokine through the activation of NF-κB(nuclear transcription factor-κB).Previous studies showed that Treg's functions were mainly regulated by cytokines or by the co-stimulatory molecules on APCs,but new evidences was shown that Tregs can respond to foreign antigens through TLRs directly.Recently TLR5 was found selectively high-expressed on Trges,and Flagellin can enhance the inhibition function of Treg's on effective T cells.A paper in 2008 reported that Flagellin can protect the body from damage of chemicals,bacteria,viruses and radiation through binding to TLR5,but how the TLR5 regulate the Treg's activity is still not be elucidated until now.Rapamycin(Rapa) and Cyclosporin A(CsA) are widely used as immunosuppressive drugs to avoid allograft rejection in clinic nowadays,and as a tool for inducing immune tolerance in the experimental model.Recently it was reported that Rapa in vitro can selectively amplify the natural CD4~+CD25~+ regulatory T cells in normal mice without blocking the CD4~+ T cell's expansion and activation-induced apoptosis,but the exact mechanism is still unclear.CsA can inhibit the TCR-mediated T cell activation and IL-2 production which indicated that CsA and Rapa have different effects on Tregs.The previous study in our laboratory found that the ratio of CD4~+CD25~+ Foxp3~+ was higher in allo-transplantation tolerance model induced by Rapa,but even using Rapa in vivo,the proportion of Treg was still at a relatively low level.How to regulate the activity of Tregs and promote its expansion in vivo are so urgent for inducing transplantation tolerance.We proposed that the use of Flagellin may activate TLR5 and enhance the expression of Foxp3 in vivo which may useful in induction of long term transplantation tolerance. This paper detected the expression of TLR5 and Foxp3 in different times post allotransplantation.A skin allo-transplantation model was established using mouse; Rapa,CsA were injected into mice for tolerance induction,then the effects of Rapa, CsA on expression of TLR5 and Foxp3 and the relationship between them in allo-transplantation were analyzed.In order to prove the effects of Flagellin on Foxp3,we purified Flagellin from Salmonella paratyphoid A,and then separated splenic T cells from naive and model mice of acute allograft rejection,and treated with Flagellin,Rapa,CsA for a certain time to study the effects on TLR5 and Foxp3 expression.The results were shown as followings:Methods1.The extraction and purification of FlagellinSlamonella Paratyphoid A were cultured with LB liquid nutrient culture medium, washed with PBS,then Crude flagella protein(Flagellin) was obtained through high speed centrifuge,different pH treatment,Sephacryl S-200 gel column separation and SDS-PAGE.The specificity of purified flagellin was proved by anti-flagellin antibody specifically binding in Western Blot analysis.2.Experiments in vivoThe model of skin allo-transplantation was established.The recipient BALB/c mice were injected with CsA or Rapa after transplantation and then grafts survival situation was observed.Five time points(1,7,10,14,21 days) post transplantation were selected,spleen cells of recipients were purified on each point to detect the expression of TLR5 and Foxp3 mRNA with RT-PCR.3.Experiments in vitroT cells were purified on the 10 days post transplantation from recipient's mice,and treated with different concentrations of flagellin for different time in vitro,also treated with Rapa,CsA and/or definite concentration of Flagellin in vitro.The expression of Foxp3 and TLR5 mRNA was measured by RT-PCR.Same treatment to the T cells from naive BALB/c mice was done as control.Results1.The result of SDS-PAGE revealed a single protein band staining in molecule weight of 52kD.The results of Western Blot which use anti-Flagellin antibodies showed a positive stain,indicated that the extraction of Flagellin was successful.2.Compared with the control group(12.8±1.6 d),the treatment with CsA (22.0±1.9d),Rapa(23.8±2.1d) in vivo can prolong the survival of allograft significantly(p＜0.05);Grafts survival of Rapa group was similar as CSA group, but the general condition was better in Rapa group.3.The expression of TLR5 and Foxp3 in control group after allograft transplantation increased,the most obviously on 10 days post transplantation,and then the expression of Foxp3 declined rapidly and the expression of TLR5 stayed at high level until the 14 day post transplantation.The expression of TLR5 and Foxp3 mRNA in 1,7,10,14,21 days post transplantation was significantly positive correlated.4.With the treatment with CsA,The expression of TLR5 and Foxp3 were significantly increased on the 7th day post transplantation,and reached a peak at 10th day.The expression of Foxp3 returned to the beginning level at 14th day and the expression of TLR5 returned to the beginning level at the 21 st day.Compared with the control group,the expression of TLR5 mRNA in model mice treated with Rapa was significantly higher at 1,7,10,14,21 days,much higher than the CsA and the control group until 21st day.The expression of Foxp3 in group treated with Rapa significantly increased at 7th day and reached the peak at 10th day,then returned to the beginning level at 14th day.5.Experiments in vitro showed that:the expression of TLR5 mRNA was the highest when treated with Flagellin of 100ng/ml for 6 hours in T cells from acute rejection period of model mice,the expression of Foxp3 mRNA at the highest level with Flagellin of 1000ng/ml for 6h.Flagellin can promote the expression of Foxp3 in all concentration assayed.Flagellin,Flagellin+Rapa,Flagellin+CsA could promote the expression of TLR5 mRNA,and the effect of Flagellin+Rapa was more obviously.Treated with CsA or Rapa alone have little effect,CsA,Rapa,and Flagellin+Rapa treatment can promote the expression of Foxp3 mRNA,in which Flagellin+Rapa combined treatment showed effect significantly.Besides,Flagellin, Rapa,Flagellin+Rapa,and Flagellin+CsA can promote naive BABL/c mouse T cells to express TLR5 mRNA significantly(p＜0.05),in which Flagellin+Rapa combined treatment group increased more significantly,while treated with the CsA alone has little effect.The Foxp3 mRNA were expressed significantly in Flagellin treatment group,Rapa group,Flagellin+Rapa combined treatment group,Flagellin+CsA combined treatment group(p＜0.05),and Flagellin+Rapa combined treatment group increased more obviously.Conclusions1.Rapa or CsA treatment in vivo can significantly prolong skin allograft survival; Rapa has been shown a better effect than CsA in inhibiting graft rejection and improves the state of the recipients.2.The expression of TLR5 and Foxp3 was higher in the acute rejection of allotransplantation.Rapa can significantly enhance the expression of TLR5 and Foxp3 in allograft rejection,particularly enhance the expression of Foxp3 for long time.CsA also promote the expression of these two genes,but the time was much shorter than Rapa.3.Successfully extracted and purified Flagellin.4.Rapa in vivo can promote the expression of TLR5 and Foxp3 of T cells,and Flagellin can collaborate to strengthen the role of Rapa.CsA can enhance the expression of Foxp3 in T cells in the peak of allo-rejection,but has no significant effect on the expression of TLR5,with no synergy to Flagellin.Flagellin can promote the expression of TLR5 and Foxp3 in T cells in allo-rejection in vitro,and can promote the effect of Rapa. 5.Both Rapa and Flagellin can promote the expression of TLR5 and Foxp3 in normal naive T cells in vitro and promote each other.CsA has little effect on the expression of TLR5 and Foxp3 in normal T cells.