*Purpose: Induction of immune tolerance is a major challenge in heart transplants. There is no robust treatment with fewer side effects for preventing chronic rejection. Recent studies have shown that circular RNA (circRNA), a new class of endogenously expressed non-coding RNAs, functions as a master gene. It plays an important role in the physiological and pathological development of cells. However, the role of circRNAs in dendritic cells (DCs) remains unknown. The objectives of this study are to investigate the role of circular RNA FSCN1 (circFSCN1) in DC development and immune function and to investigate whether circFSCN1 silenced tolerogenic DCs can prevent immune rejection and induce immune tolerance in heart transplant.

*Methods: Murine bone marrow-derived DCs were cultured in vitro. CircRNA expression was detected by circRNA microarrays and qRT-PCR. DCs were transfected with siRNA targeting circRNA FSCN1 in vitro. DC maturation was determined by measuring the expression of MHC II and co-stimulatory molecules (CD40, CD80, and CD83) by flow cytometry. DC’s function was assessed by the capacity of DC to activate T cells in a Mixed Lymphocytes Reaction (MLR). Treg generation in vitro was measured by staining with CD4, CD25, and Foxp3. The effect of circFSCN1 silenced DCs on preventing allograft rejection was investigated in a murine heterotopic cardiac transplant where recipients were treated with circFSCN1 silenced DCs. Graft rejection was determined by the cessation of the heartbeat.

*Results: Our microarray results showed that the expression profiles of circRNAs in mature DCs were significantly different compared with immature immunosuppressive DCs. circFSCN1 was significantly up-regulated in mature DCs compared to immature DCs. Treatment with immunosuppressive molecules such as TGF-β, Rapamycin, and Dexamethasone reduced circFSCN1 expression in DCs. Silencing of circFSCN1 using siRNA reduced the expression of CD40, CD80, and CD83, but not MHCII, indicating that circFSCN1 is critical for DC development. Knockdown of circFSCN1 in DCs significantly impaired DCs to activate naïve allogeneic T cells as well as promoting Treg generation in vitro, leading to inducing more tolerogenic DCs. More than 60% of heart grafts from mice treated with circFSCN1-silenced tolerogenic DCs survived over 100 days post-transplant, while grafts from control mice treated with control DCs were rejected around 40 days, indicating tolerogenic DCs induced by circFSCN1 siRNA significantly prolonged the allograft survival and induced immune tolerance in heart transplant.

*Conclusions: CircFSCN1 is a novel regulator for the induction of tolerogenic DCs. Administration of circFSCN1 silenced tolerogenic DCs can successfully prevent allograft rejection and induce immune tolerance in organ transplants. This study not only provides insights into the role of circRNA in DC development and function but also identifies a new potential target for preventing transplant rejection.

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