of Type I IFN in Dendritic Cells TLR-Dependent and -Independent Induction CpG-B Oligodeoxynucleotides Inhibit

CpG oligodeoxynucleotides (ODNs) signal through TLR9 to induce type I IFN (IFN- ab ) in dendritic cells (DCs). CpG-A ODNs are more efﬁcacious than CpG-B ODNs for induction of IFN- ab . Because IFN- ab may contribute to autoimmunity, it is important to identify mechanisms to inhibit induction of IFN- ab . In our studies, CpG-B ODN inhibited induction of IFN- ab by CpG-A ODN, whereas induction of TNF- a and IL-12p40 by CpG-A ODN was not affected. CpG-B inhibition of IFN- ab was observed in FLT3 ligand-induced murine DCs, puriﬁed murine myeloid DCs, plasmacytoid DCs, and human PBMCs. CpG-B ODN inhibited induction of IFN- ab by agonists of multiple receptors, including MyD88-dependent TLRs (CpG-AODN signaling via TLR9, or R837 or Sendai virus signaling via TLR7) and MyD88-independent receptors (polyinosinic:polycytidylic acid signaling via TLR3 or ds break-DNA signalingviaacytosolicpathway). CpG-BODNdidnotinhibittheIFN- ab positivefeedbackloopsecond-waveIFN- ab ,becauseIFN-ab –induced expression of IFN- ab was unaffected, and CpG-B inhibition of IFN- ab was manifested in IFN- ab R 2 / 2 DCs, which lack the positive feedback mechanism. Rather, CpG-B ODN inhibited early TLR-induced ﬁrst wave IFN- a 4 and IFN- b . Chromatin immunoprecipitation revealed that association of IFN regulatory factor 1 with the IFN- a 4 and IFN- b promoters was induced by CpG-A ODN but not CpG-B ODN. Moreover, CpG-A–induced association of IFN regulatory factor 1 with these promoters was inhibited by CpG-B ODN. Our studies demonstrate a novel mechanism of transcriptional regulation of ﬁrst-wave IFN- ab that selectively inhibits induction of IFN- ab downstream of multiple receptors and may provide targets for future therapeutic inhibition of IFN- ab expression in vivo. The Journal of Immunology , 2010, 184: 3367–3376. ODNs induce IFN- ab and to develop agents to inhibit induction of IFN- ab by self- and nonself-DNA. Our studies describe selective inhibition of IFN- ab induction by CpG-B ODNs. High concentrations of CpG-B ODNs speciﬁcally inhibited the induction of IFN- ab by agonists of TLR9 and other receptors without inhibiting other responses (e.g., induction of TNF- a and IL-12p40). This inhibition was evident as early as 3 h and for a period of at least 48 h in all cell types tested: FLT 3 ligand (FLT3L)-induced murine bone marrow-derived DCs, puriﬁed murine mDCs, puriﬁed murine pDCs, and humanPBMCs. This mechanism inhibited induction of IFN- ab by agonists of a range of receptors, including MyD88-dependentTLRs(TLR9,TLR7),aMyD88-independentTLR(TLR3),andnon-TLRsignals(e.g.,acytosolicpathwayforsignalingbydsB-DNAandperhapsRIG-I–likeproteins)(19),suggestingthat the mechanism affected downstream signaling at a point where sig-nalingfromthesereceptorsconvergesfortheinductionofIFN- ab . On the other hand, inhibition was upstream of mechanisms involved in IFN- ab positive feedback signaling (second-wave IFN- ab ). Fur-thermore, chromatin immunoprecipitation (ChIP) demonstrated that CpG-B ODN inhibited association of IRF1 with IFN- b and IFN- a 4 promoters. Our results indicate a novel pathway for transcriptional regulation that results in inhibition of ﬁrst-wave IFN- ab . duplicate assays). Statistical analysis was done to compare the mean response for the ﬁve different donors under each condition, and signiﬁcance was assessed with one-way ANOVA and Student-Newman-Keuls method. Results reveal statistically signiﬁcant ( p , 0.05) induction of IFN- a by CpG-AODN (comparison of PBMCs with CpG ODN-A2336 to PBMCs with medium alone), higher induction of IFN- a by CpG ODN-A2336 than CpG ODN-B2006( p , 0.05),andstatisticallysigniﬁcant( p , 0.05)inhibitionofCpG-A– inducedIFN- a productionby CpG-BODN(comparisonofPBMCswithCpG ODN-A2336 to A3/B3).