A TLR9/TRIF pathway induces tolerance via TGF-beta and tryptophan catabolism in plasmacytoid dendritic cells

Cytosine-phosphate-guanosine (CpG)-rich oligodeoxynucleotides (ODNs) stimulate Toll-like receptor 9 (TLR9)-dependent signaling and activate the innate and acquired responses of the immune system. The immunostimulatory effects of CpG-ODN are being evaluated in clinical trials as a novel therapeutic approach for treatment of human diseases. Here we show that pre-treatment in vitro of plasmacytoid dendritic cells with a low-dose (1 µg/ml) of a CpG-ODN phosphorothioate (CpG 1826) induced, as expected, immunostimulatory effects in vivo. However, pre-treatment of the same cells with a higher concentration of the ODN (10 µg/ml) induced tolerogenic effects, which allowed us to unveil a previously undescribed role for the adaptor TRIF and TRAF6 proteins in TLR9 signaling. The low-dose CpG stimulation activated the TLR9/MyD88-dependent NF-κB pathway and the production of the pro-inflammatory cytokine IL-23, whereas the high-dose CpG stimulation triggered the TLR9/TRIF pathway, leading to activation of the noncanonical NF-κB pathway, production of the anti-inflammatory cytokine TGF-β, and subsequent induction of the immunosuppressive pathway of tryptophan catabolism. Thus, opposite in vivo outcomes of TLR9 stimulation by a CpG-ODN phosphorothioate (CpG 1826) can occur as a function of dosage. Our findings may be relevant not only for an increased understanding of the complexity of TLR signaling and for an optimal exploitation of CpG-ODNs as immune adjuvants, but also for the development of nonconventional forms of CpG-based immunotherapy in human allergy and autoimmunity