Knockout Of MiR-146a In Regulatory T Cells Suppressed Heart Transplantation Rejection In Mice

Background and purpose:Rejection is one of the main factors affecting the survival rate and quality of life of transplant patients.Regulatory T cells(Tregs),characterized by the expression of the transcription factor forkhead cassette 3(Foxp3),are essential for maintaining the immune tolerance of the recipients.However,strategies to improve Treg function are needed.However,further studies are needed to rationally induce Tregs and maintain their functional properties in organ transplantation.Notably,microRNA(miRNA)plays an important regulatory role in the function and metabolism of Tregs in both normal and immune environments.Mir-146a,one of the widely expressed miRNAs in Tregs,responds to immune signaling by negatively regulating TRAF6,IRAK1 and STAT1.Therefore,the purpose of this study is to enhance the function of Tregs by regulating miR-146a,and then suppress the immune response.Methods:This study is mainly divided into three parts.In the first part,miR-146a conditional gene knockout mice(miR-146a CKO)were constructed by Cre-Lox P system,and identified and screened by PCR.In the second part,CKO Tregs were co-cultured with monocytes and CD4~+T cells in vitro,and the effects of miR-146a knockout on Treg function were evaluated from the changes of macrophage polarization and biological behaviors(proliferation, apoptosis,migration and differentiation)of CD4~+T cells.The mechanism of miR-146a regulating Treg function was analyzed by Western blot and RT-PCR.In the third part,a mouse heart transplantation model was established by microsurgical technique with CKO as recipients and BALB/c mice as donors to analyze the effect of knockout of miR-146a in Tregs on rejection.Western blot was used to analyze the mechanism of miR-146a regulating Treg function during transplant immunity.Results:In the first part,after CKO mice were constructed by the Cre-Lox P system.The loxP sequence of 350 bp and the Cre sequence of 346 bp could be amplified by PCR.Moreover,miR-146a was low expressed in Tregs in peripheral blood,spleen and thymus of CKO mice,indicating that miR-146a was successfully constructed and could be used in subsequent experiments.In the second part,knockout of miR-146a promoted Treg expansion,enhanced the inhibitory effect of Tregs on macrophages and CD4~+T cells,as shown by inhibiting M1 macrophage polarization and promoting M2 macrophage polarization;inhibiting CD4~+T proliferation and migration and promoting CD4~+T cell apoptosis,but impairing the ability of Tregs to inhibit Th1 responses.Further mechanistic exploration revealed that knockout of miR-146a regulated Treg function mainly through the IFN-γ/STAT1 pathway.In the third part,knockout of miR-146a transiently prolonged allograft survival;reduced CD4~+and CD8~+T cell infiltration into allografts while increasing Treg infiltration;and reduced T lymphocyte ratio and M1 macrophage polarization while promoting M2 macrophage polarization and Treg expansion.The mechanism of miR-146a regulation of Tregs in the immune environment were similar to the results in the second part of the normal environment,that is,knockout of miR-146a in the immune environment still regulated Treg function through IFN-γ/STAT1.When knockout of miR-146a combined with IFN-γ/STAT1 blockade significantly prolonged allograft survival and enhanced Treg suppression of Th1 responses.Conclusions:The results showed that miR-146a knockout promoted the expansion of Tregs in vivo,enhanced the inhibition of Tregs in vitro,but impaired the ability of Tregs to inhibit the Th1 response.Mechanically,miR-146a regulates Tregs mainly viaIFN-γ/STAT1.Knockout of miR-146a exerted a certain therapeutic effect in a mouse heart transplantation models and slightly prolonged allograft survival.SynergisticIFN-γ/STAT1 blockade significantly improved allograft survival and reversedimpaired function of Tregs.Our data will provide new strategies for improving Treg function to suppress clinical rejection.