Mitigating alloimmune rejection using Circular RNA AEBP2-silenced DC in heart transplantation: A new target for tolerogenic DCs
Qingfeng Zhou1, Amal Abuomar1, Raj Thapa1, Serina Chahal2, Tan Ze Wang3, Adam Greasley3, Xiufen Zheng1,2,3,4.
1Department of Surgery, Western University, London, ON, Canada; 2Department of Microbiology &Immunology, Western University, London, ON, Canada; 3Department of Pathology &Laboratory Medicine, Western University, London, ON, Canada; 4Department of Surgery, Lawson Health Research Institute, London, ON, Canada
Introduction: Circular RNA (circRNA), a new type of single -stranded RNAs generated by back-splicing, plays vital roles in cellular activities. However, the impact of circRNAs on dendritic cell (DCs)-mediated immune modulation and allo immune rejection is not well investigated. Our microarray assays show that circRNA is differentially expresed in DCs. One of circRNA known as circAENP which is backspliced from the AEBP2 gene is highly expressed in mature immune reactive DCs. We hypothesize that silencing of circAEBP2 in DC will induce immunosuppressive DCs which can prevent allograft rejection after heart transplantation
Methods: Bone marrow- derived DCs were cultured in vitro. Day 5 of cultured DCs were transfected with siRNA specifically targeting circAEBP2 for 48 h. DC phenotyping was conducted by measuring the expression of CD11c, MHCII, and costimulatory molecules (CD40 and CD80) by flow cytometry. MLRs were conducted to measure DCs’ function to activate allogeneic naïve T cells and to generate regulatory T cells (Treg) in vitro. CircAEBP2 silenced DCs were administrated to recipient mice seven days prior to MHC-full mismated mouse heart transplantation. Allograft survival was observed to assess the impact of circAEBP2 silenced immunosuppressive DCs on alloimmune rejection in organ transplantation. Histopathological changes of transplanted hearts were assessed
Results: Knockdown of circAEBP2 reduced the expression of costimulatory molecules (CD 40 and CD80) and proinflammatory cytokines IL12 and IFNr in DCs, reduced DCs to activate naïve allogeneic both CD4+ and CD8+T cells, while promoted Treg generation. Silencing of circAEBP2 also reduced the expression of phosphorylated p65 and its nuclear translocation, suggesting that silencing of AEBP2 attenuates the activation of the NFKb pathway. Pre-treatment with circAEBP2-silenced DCs prolonged allograft survival after heart transplantation, reduced fibrosis of transplanted hearts, and attenuated donor-antigen specific T response.
Conclusion: Silencing of circASEBP2 can induce immunosuppressive DCs, which in turn, can mitigate alloimmune rejection after heart transplantation. This study highlights that circAENP2 is a novel target for the induction of tolerogenic DCs which can induce donor antigen specific immune suppression.
The study was supported by grants funded by the Canadian Institute of Health Research and the Natural Sciences and Engineering Research Council of Canada.
[1] Immune rejection, dendritic cell, circular RNA, circAEBP2, heart transplantation
