| Objective: The main aim of this research is to investigate the effect of TIM-4 blockade on Th17 immunoresponses,and the potential mechanism of miR-21 and related lnc RNA;We also aim to establish a mice allogeneic skin transplantation model with the treatment of anti-TIM-4 +/-rapamycin and observe their effects on skin graft survival and Th17 responses,which provides a new basis for further seeking potent immunosuppressive agents and suitable immunosuppressant combinations.Methods:(1)According to our previous results of gene array and the predictive analysis by Freiburg RNA Tools software,we found that miR-21 may bind with lnc RNA Malat1,Kcnq1ot1,Gm12054,Gm25925,and interfere with these potential lnc RNAs.These lnc RNAs may act as a sponge of miR-21 and inhibit the effect of miR-21.To screen out the most effective lnc RNA(s)that interfere with miR-21,C57BL/6 mouse splenocytes were transfected with miR-21 inhibitor and corresponding NC,the interference efficiency of miR-21 inhibitor and the expression of related lnc RNA(s)were tested by Real-time fluorescence quantitative PCR(qRT-PCR).(2)To verify the interaction between miR-21 and screened lnc RNA(Malat1),si-Malat1 and si-NC were transfected into C57BL/6 mouse splenocytes,the interference efficiency of si-Malat1 and the relative expression of miR-21 were detected by qRT-PCR.(3)Our previous results uncovered miR-21 silencing inhibits Th17 cells.To verify that Malat1 may act as a sponge of miR-21 and has the ability to offset the effect of miR-21 on Th17.While stimulated with CD3/CD28 mAb and IL-2,MACS-purified splenic CD4~+T cells were transfected with si-Malat1 and si-NC respectively,the relative expression of IL-17 a was detected by qRT-PCR.(4)To prove the effect of TIM-4 blockade on Th17 cells and explore the potential regulatory mechanism of miR-21 and Malat1,C57BL/6 were immunized with alloantigen by i.p injection with splenocytes of Balb/c,the control group and experiments group were respectively treated with isotype and anti-TIM-4 mAb.The mouse splenocytes were collected on the day 10 after immunization.The percentages of Th17 cells from the control and experimental groups were analyzed by flow cytometry(FCM).The level of IL-17 a m RNA from MACS-purified splenic CD4~+T cells and the relative expression of miR-21 and Malat1 were measured by qRT-PCR.(5)To explore whether anti-TIM-4+/-rapamycin synergistically promote the survival of skin grafts and observe the changes of Th17 cells.Balb/c skins were engrafted onto C57BL/6 mice,the mice allogeneic skin transplantation model was set as isotype,anti-TIM-4,rapamycin,rapamycin+isotype,rapamycin+anti-TIM-4 groups respectively,the survival time of skin allografts was assessed.Histological staining of the skin allografts by H-E was made on the day 8 after transplantation.The frequencies of Th17 cells were analyzed by FCM.Results:(1)The mouse splenocytes were transfected with miR-21 inhibitor,the interference efficiency was up to ?80%.The expression of Malat1 in the miR-21 inhibitor treated group was significantly higher than that in NC group(0.79±0.04 vs 0.25±0.11,p<0.01).After the splenocytes were transfected with si-Malat1,the interference efficiency was up to 45%.The relative expression of miR-21 in si-Malat1 group was significantly higher than that in si-NC group(0.0000413 ± 0.000005.3 vs 0.0000056±0.00000065,p<0.01).While stimulated with CD3/CD28 mAb,MACS-purified splenic CD4~+T cells were transfected with si-Malat1.The relative expression of IL-17 a in si-Malat1 group was significantly higher than that in si-NC group(0.0039±0.00024 vs 0.0021± 0.00023,p<0.01).(2)In the allo-immunization model,in which treated with isotype(control group)or anti-TIM-4(experimental group),the percentages of Th17 cells were significantly decreased in anti-TIM-4 group(2.82±0.17 vs 1.83±0.07,p<0.01).The relative expression of IL-17 a in CD4~+T cells from experimental group was significantly decreased vs.NC(0.00026±0.000009 vs 0.00079±0.000009,p<0.01).The relative expression of miR-21 in splenocytes and splenic CD4~+T cells was significantly decreased vs.NC(splenocytes,0.4±0.12 vs 1.0±0,p<0.01;CD4~+T cells,0.02 ± 0.007 vs 1.0±0,p<0.0001).The relative expression of Malat1 increased significantly(0.3±0.059 vs 0.12±0.012,p<0.05).(3)In mice allogeneic skin transplantation model,the survival of skin allografts was not significantly prolonged between anti-TIM-4 and isotype treated groups.But the H-E staining results showed that immune cells infiltrated the skin allografts in the anti-TIM-4-treated recipients were slightly decreased vs.isotyped-treated group.Compared with isotype,anti-TIM-4,rapamycin and rapamycin+isotype groups,the treatment of anti-TIM-4 combined with rapamycin markedly prolonged skin allograft survival(MST;anti-TIM-4+rapamycin 16.5 day vs isotype 7 day;vs anti-TIM-4 8.5 day;vs rapamycin 10 day;vs rapamycin+isotype 8 day).H-E staining showed a significant decrease in immune cells infiltrated the skin allografts in the rapamycin combined with anti-TIM-4 group.(4)In mice allogeneic skin transplantation model,compared with isotype group,the percentages of Th17 cells were significantly decreased in anti-TIM-4 group(1.21±0.15 vs 2.81±0.31,p<0.05).The percentages of Th17 cells were significantly decreased in rapamycin+anti-TIM-4 treated mice,compared with rapamycin and rapamycin+isotype groups respectively(anti-TIM-4+rapamycin,1.0±0.015 vs rapamycin,1.3±0.09,p<0.05 vs rapamycin+isotype,2.03±0.21,p<0.05).Interestingly,we observed that the percentages of Th17 cells were significantly increased in rapamycin+isotype vs.rapamycin only treated mice(rapamycin+isotype,2.03±0.21 vs rapamycin,1.3±0.09.p<0.05).This surprising result may be due to the fact that the isotype antibody is Rat Ig G2 a,a partially heteroantigenic protein that elicits a background immune response.This result also confirmed that the Fc domain of isotype control antibody could non-specifically bind to the FC receptors expressed in the mouse cells(B cells,macrophages,etc.,)and non-specifically promote proinflammatory response[1].Conclusions:1.Lnc RNA Malat1 may act as a sponge of miR-21 and has the ability to offset the effect of miR-21 on Th17.2.In vivo blockade of TIM-4 significantly inhibits Th17 cells,and this change is closely associated with a decrease in miR-21 and an increase in Malat1.3.The combination of anti-TIM-4 and rapamycin treatments acted synergistically to promote the survival of skin grafts. |
PDF Full Text Request