LF15-0195 generates tolerogenic dendritic cells by suppression of NF-κB signaling through inhibition of IKK activity

Abstract

LF15-0195 (LF) is a potent, less toxic analog of the immunosuppressant 15-deoxyspergualine, which we previously reported to prevent graft rejection and to induce permanent tolerance in a murine cardiac transplantation model. However, the underlying mechanism of action of LF required elucidation. In this study, dendritic cells (DC) treated with LF before activation with tumor necrosis factor alpha (TNF-alpha)/lipopolysaccharide (LPS) failed to express maturation markers (major histocompatibility complex II, CD40, CD86) and interleukin-12. LF prevented, in a concentration-dependent manner, the activation and nuclear translocation of nuclear factor-kappaB (NF-kappaB) in DC following addition of TNF-alpha/LPS. Yet-activated and active IkappaB kinases (IKKs) were inhibited in cells pretreated with LF, thereby preventing the phosphorylation of IkappaB and release of NF-kappaB, a key regulator of genes associated with the maturation of DC. LF-induced inhibition of IKK activity was reversed in a dose-dependent manner by the overexpression of IKK. The T helper cell type 2 (Th2) differentiation of naïve T cells promoted by LF-treated DC in vitro correlates with Th2 polarization observed in transplant recipients made tolerant by LF. These data demonstrated that LF-induced blockade of NF-kappaB signaling at the level of IKK promoted the generation of tolerogenic DC that inhibited Th1 polarization and increased Th2 polarization in vitro and in vivo.

Treatment of Autoimmune Arthritis Using RNA Interference-Modulated Dendritic Cells

“…We sought to expand on our previous studies in which chemical manipulation of DCs was able to generate a cellular population capable of suppressing initiation and onset of CIA when pulsed with CII (23). The previous studies used DCs treated with LF 15-0195, a small molecule inhibitor of IKK (13), which blocks DC maturation and upregulation of costimulatory molecules. In the current studies, we used siRNA to selectively inhibit costimulatory molecule expression on DCs prior to pulsing with Ags.…”

“…We and others have previously reported that the state of transplantation tolerance is associated with DCs expressing low levels of costimulatory molecules resembling an immature state (13,19). Although there are data supporting CD80 and CD86 in terms of the qualitative state of T cell activation, the role of CD40 is widely recognized as essential for stimulating T cell-mediated immunity.…”

“…One common feature of immune regulatory DCs appears to be reduced expression of costimulatory molecules. NF-kB-inhibited DCs, whether by proteasome inhibition (11), decoy oligonucleotides (12), or IKK suppression (13), all inhibit immune responses and are all associated with reduced costimulatory molecule expression. Another means of DC manipulation is treatment with 1a,25-dihydroxyvitamin D3 (calcitriol), which has been demonstrated to cause immune modulation by generating DC populations with an immature phenotype and ability to activate regulatory T cells (Tregs) (9,14).…”

“…Although there are data supporting CD80 and CD86 in terms of the qualitative state of T cell activation, the role of CD40 is widely recognized as essential for stimulating T cell-mediated immunity. The suppression of these molecules is believed to inhibit T cell activation and in many cases will instead stimulate Tregs to inhibit immune responses in an Ag-specific manner (13,19).…”

Immune Modulation and Tolerance Induction by RelB-Silenced Dendritic Cells through RNA Interference

“…Thus, NF-B is a key factor controlling DC maturation. In the context of transplantation, we and others have reported that inhibition of NF-B, using pharmacological inhibitors, endows DC with the ability to induce Treg formation and Th2 polarization (17,59).…”