This led to the upregulation of IFN-stimulated genes known to enhance host resistance to virus infection [8-12]. “K” ODN also upregulate the expression of IL-6, which contributes to the activation of multiple pro-inflammatory genes Y-27632 cell line and the subsequent shift from

innate to adaptive immunity [8-12]. The current study was designed to identify the key signaling pathway(s) responsible for the upregulation of IFN-β and IL-6, as these would provide important insights into the pattern of “K” ODN mediated activation of human pDCs. Previous efforts to examine the signaling cascade(s) triggered by the interaction of TLR9 with CpG DNA focused primarily on murine myeloid DCs (mDCs), monocytes, and macrophages [13]. Studies examining the regulation of IL-6 by “K” ODN in mice documented a role for interferon regulatory factor-5 (IRF-5) and the binding of the NF-κB transcription factors p50/p65/c-Rel to the IL-6 promoter [14, 15], while IRF-1 was identified as a key mediator of IFN-β induction by “K” ODN [16]. Yet, there is reason to question whether those findings are applicable to human pDC, as there are fundamental differences in the signaling cascades utilized by mDCs versus pDCs and mice versus humans [2, 13, 17-20]. The rarity of pDCs in human peripheral blood (they constitute only 0.2–0.5% of the PBMC pool) and ease with which they become activated during the purification process

complicates their use [6, 7]. Thus, studies of human pDCs were supplemented by analyses of the human CAL-1 pDC-like cell line to provide novel insights into the regulation of TLR9-mediated activation of human pDCs. CAL-1 cells express TLR9 and mirror the response of primary human Anti-infection Compound Library pDCs to CpG ODN, as reflected by similar patterns of cytokine induction [12, 21, 22]. siRNA knockdown studies identified the transcription factors IRF-5 and NF-κB p50 as key early regulators of both IL-6 and IFN-β gene expression in CAL-1 cells. Proximity ligation assays (PLAs) demonstrated PtdIns(3,4)P2 that IRF-5 and NF-κB p50 but not p65 significantly co-localized within the nucleus of these cells within 30

min of stimulation, consistent with these factors cooperatively mediating gene expression. In contrast to data derived from murine studies, IRF-8 was identified as a negative regulator of IFN-β and IL-6 expression, indicating that IRF-5 and IRF-8 compete to control gene expression following “K” ODN stimulation in human pDCs. This work also demonstrates that endogenous IRF-5 and IRF-7 are associated with MyD88 in resting CAL-1 cells but stimulation with “K” ODN leads to the activation only of IRF-5. As IRF-5 and IRF-8 variants are associated with autoimmune diseases such as lupus [23-28], these findings are relevant to our understanding of the pharmacologic effects of “K” ODN and the role of TLR9 ligation under physiologic, pathologic, and therapeutic conditions. CAL-1 cells share many phenotypic and functional properties of human pDCs [12, 21, 22].