Myd88 knockdown with RNA interference induces in vitro immune hyporesponsiveness in dendritic cells from rhesus monkeys.

Abstract

Immature dendritic cells (imDCs) are activated and mature to initiate an adaptive immune response, resulting in allograft rejection and transplantation failure. Myeloid differentiation factor 88 (Myd88) is a key factor in the Toll-like receptor (TLR) signaling pathway. Here, we investigated the effect of Myd88 silencing on DC function and immune response. CD34 + cells were isolated from the bone marrow of rhesus monkeys by the immunomagnetic bead method and then infected with an adenovirus expressing Myd88-specific short hairpin RNA (sh-Myd88). sh-NC (nontargeting negative control)- or sh-Myd88-infected DCs were treated with lipopolysaccharide (LPS) for another 48h to induce DCS maturation. The maturation of DCs was identified by immunofluorescence staining for MHCII, CD80, and CD86. DC apoptosis was examined using Annexin V/PI staining. DC-related cytokine levels (IFN-γ and IL-12) were assessed by ELISA. A mixed lymphocyte reaction (MLR) was performed to test the effect of Myd88-silenced DCs on T lymphocytes in vitro. The results showed that compared with control or sh-NC-infected DCs, Myd88-silenced DCs had lower MHCII, CD80, CD86, and DC-related cytokine (IFN-γ and IL-12) levels. Myd88 did not affect the apoptosis of DCs. MLR demonstrated that Myd88 silencing could effectively block LPS-activated T cell proliferation in vitro. These data were consistent with the characteristics of tolerogenic DCs. In conclusion, our data indicated that Myd88 silencing could inhibit the maturation of imDCs and alleviate immune rejection, which provides a reference for immune tolerance in clinical liver transplantation.