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Synthetic oligonucleotides (ODNs) containing CpG motifs stimulate human plasmacytoid dendritic cells (pDCs) to produce type-1 interferons (IFNs) and proinflammatory cytokines. Previous studies demonstrated that interferon regulatory factors (IRFs) play a central role in mediating CpG-induced pDC activation. This work explores the inverse effects of IRF5 and IRF8 (also known as IFN consensus sequence-binding protein) on CpG-dependent gene expression in the human CAL-1 pDC cell line. This cell line shares many of the phenotypic and functional properties of freshly isolated human pDCs. Results from RNA interference and microarray studies indicate that IRF5 upregulates TLR9-driven gene expression whereas IRF8 downregulates the same genes. Several findings support the conclusion that IRF8 inhibits TLR9-dependent gene expression by directly blocking the activity of IRF5. First, the inhibitory activity of IRF8 is only observed when IRF5 is present. Second, proximity ligation analysis shows that IRF8 and IRF5 colocalize within the cytoplasm of resting human pDCs and cotranslocate to the nucleus after CpG stimulation. Taken together, these findings suggest that IRF5 and IRF8, two transcription factors with opposing functions, control TLR9 signaling in human pDCs., (Published 2015. This article is a U.S. Government work and is in the public domain in the USA.)

Alarmins are a group of structurally diverse host defense antimicrobial peptides that are important immune activators. In this article, we present a novel role for two potent alarmins, human β-defensin 2 and 3 (HBD2 and 3), in promoting IFN-α production by human plasmacytoid dendritic cells. We demonstrate that HBD2 and 3 activate pDCs by enhancing the intracellular uptake of CpG and self DNA and promote DNA-induced IFN-α production in a TLR9-dependent manner. Both CpG and host DNA form aggregates that resemble DNA nets when combined with HBD2 and 3. Isothermal titration calorimetry studies to elucidate the nature of HBD3/CpG complexes demonstrate involvement of enthalpy-driven interactions, in addition to hydrophobic interactions, with the formation of complexes at a molar ratio of 2:1 defensin/CpG. The i.v. administration of HBD3/CpG complexes induced proinflammatory cytokines like IL-12, IFN-γ, IL-6, IFN-α, and IL-10 in serum, associated with an increased recruitment of APCs in the spleen. Subcutaneous injections of these complexes showed enhanced infiltration of inflammatory cells at the injection site, indicating a potential pathophysiological role for alarmin/DNA complexes in contributing to inflammation. Intraperitoneal immunization of HBD3/CpG complexes with OVA enhanced both cellular and humoral responses to OVA, compared with OVA/HBD3 or OVA/CPG alone, indicative of a much more potent adjuvant effect of the HBD3/CpG complexes. Thus, the ability of defensins to enhance cellular uptake of nucleic acids can lead to improved vaccine formulations by promoting their uptake by various cells, resulting in an enhanced immune response.

Synthetic oligonucleotides (ODN) expressing CpG motifs mimic the ability of bacterial DNA to trigger the innate immune system via TLR9. Plasmacytoid dendritic cells (pDCs) make a critical contribution to the ensuing immune response. This work examines the induction of antiviral (IFN-β) and pro-inflammatory (IL-6) cytokines by CpG-stimulated human pDCs and the human CAL-1 pDC cell line. Results show that interferon regulatory factor-5 (IRF-5) and NF-κB p50 are key co-regulators of IFN-β and IL-6 expression following TLR9-mediated activation of human pDCs. The nuclear accumulation of IRF-1 was also observed, but this was a late event that was dependant on type 1 IFN and unrelated to the initiation of gene expression. IRF-8 was identified as a novel negative regulator of gene activation in CpG-stimulated pDCs. As variants of IRF-5 and IRF-8 were recently found to correlate with susceptibility to certain autoimmune diseases, 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., (Published 2013. This is a US Government work and is in the public domain in the USA. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Structurally distinct classes of synthetic CpG oligonucleotides (ODN) differentially activate human immune cells. K-type ODN trigger plasmacytoid dendritic cells (pDCs) to differentiate and produce TNF α . In contrast, D-type ODN stimulate large amounts of IFN α secretion from pDCs. The cell-surface receptor CXCL16 was previously shown to influence the nature and specificity of CpG ODN-induced immune activation. Here, we evaluated the expression and function of CXCL16 on pDC from healthy volunteers. We report that increased CXCL16 expression correlated with enhanced in vitro response exclusively to D-type CpG ODN. Conversely, enzymatic digestion of the receptor resulted in a decrease in IFN α production. Moreover, ox-LDL presence significantly inhibited D-ODN mediated IFN α production by pDCs. Coculture of enriched pDCs with the CXCR6 expressing Jurkat T cells decreased the activation threshold of these cells responding to D-ODN, suggesting that CXCL16/CXCR6 interaction may play an important role in modifying the response of pDCs to environmental danger signals.

Toll-like receptors (TLRs) may need to cooperate with each other to be effective in detecting imminent infection and trigger immune responses. Understanding is still limited about the intracellular mechanism of this cooperation. We found that when certain TLRs are involved, dendritic cells (DCs) establish unidirectional intracellular cross-talk, in which the MyD88-independent TRIF-dependent pathway amplifies the MyD88-dependent DC function through a JNK-dependent mechanism. The amplified MyD88-dependent DC function determines the induction of the T cell response to a given vaccine in vivo. Therefore, our study revealed an underlying TLR mechanism governing the functional, nonrandom interplay among TLRs for recognition of combinatorial ligands that may be dangerous to the host, providing important guidance for design of novel synergistic molecular vaccine adjuvants.

Treatment with synthetic oligodeoxynucleotides containing CpG motifs (CpG ODNs) is remarkably protective against otherwise lethal infection. Here, we describe an essential role for the transcription factor T-bet in mediating the protective function of CpG ODNs. Loss of T-bet in conventional CD11c(hi) dendritic cells (DCs) and in plasmacytoid DCs impaired production of IFNs. Strikingly, in contrast to Rag2-/- mice, Rag2-/- mice that also lacked T-bet (DKO) could not be rescued from lethal Listeria monocytogenes infection by prior treatment with CpG ODN. Rescue was achieved by adoptive transfer of CD11c(hi) DCs from WT, but not T-bet-/-, CpG ODN-treated donor mice. We conclude that T-bet in DCs is required for the adjuvant activity of CpG ODN in infection, revealing its vital role in innate immunity.

Synthetic oligodeoxynucleotides containing CpG motifs (CpG ODN) activate the innate immune system by interacting with Toll-like receptor 9. The resultant immune response increases host resistance to infection by a variety of pathogenic microorganisms, including Listeria monocytogenes. There is a considerable interest in harnessing the immunoprotective properties of CpG ODN, yet little is known of the cell phenotype(s) responsible for mediating this protection. This work demonstrates that treatment of mice with CpG ODN increases the number of Thy1.2+, CD11c+ dendritic cells (Thy1.2+ DC) in the spleen, which are both necessary and sufficient for transferring resistance to infection from CpG-treated donors to naive recipients. These CpG-activated Thy1.2+ DC are distinct from conventional (CD11c(hi), Thy1.2-) or plasmacytoid DC (mPDCA+), and secrete IFN-gamma that contributes to protection. These findings suggest that a novel Thy1.2+ DC subset plays a critical role in mediating the immunoprotective activity of CpG DNA.

Unmethylated CpG DNA binds to the Toll-like receptor 9 (TLR9) and activates NF-kappaB to induce cytokine genes in dendritic cells (DCs). IFN regulatory factor (IRF)-8/IFN consensus sequence binding protein is a transcription factor important for development and activation of DCs. We found that DCs from IRF-8(-/-) mice were unresponsive to CpG and failed to induce TNF-alpha and IL-6, targets of NF-kappaB. Revealing a signaling defect selective for CpG, these cytokines were robustly induced in IRF-8(-/-) DCs in response to LPS that signals through TLR4. IRF-8(-/-) DCs expressed TLR9, adaptor myeloid differentiation factor 88, and other signaling molecules, but CpG failed to activate NF-kappaB in -/- cells. This was due to the selective inability of -/- DCs to activate I-kappaB kinase alphabeta, the kinases required for NF-kappaB in response to CpG. IRF-8 reintroduction fully restored CpG activation of NF-kappaB and cytokine induction in -/- DCs. Together, TLR signals that activate NF-kappaB are diverse among different TLRs, and TLR9 signaling uniquely depends on IRF-8 in DCs.

Plasmacytoid dendritic cells (DC) are professional APC and a major source of type I IFN following viral infection. We previously showed that histamine alters the cytokine profiles of maturing monocyte-derived DC resulting in a change from Th1 to Th2 in their T cell polarizing function. In this study, we show that human plasmacytoid DC, activated by either CpG oligodeoxynucleotides or viral infection, also respond to histamine through H2 receptors, leading to a marked down-regulation of IFN-alpha and TNF-alpha and a moderate switch in their capacity to polarize naive T cells. Our findings provide an explanation for low levels of type I IFN frequently observed in atopic individuals.

Dendritic cells (DC) excel at presenting antigen to T cells and thus make a key contribution to the induction of primary and secondary immune responses. DC matured in vitro and pulsed with antigen show promise for the immunotherapy of cancer and infectious diseases. Synthetic oligonucleotides (ODN) expressing immunomodulatory "CpG motifs" were found to boost APC function in mice. Current results demonstrate that the recently identified "D" type of CpG ODN stimulate human peripheral blood monocytes to mature into functionally active DC over 2-4 days. The transition from monocyte to DC is characterized by the up-regulation of CD83, CD86, CD80, CD40 and the down-regulation of CD14. These DC support antigen-specific humoral and cellular responses in vitro and in vivo. The differentiation of these monocytes is mediated by plasmacytoid DC, which respond to D type ODN by secreting IFN-alpha. Since D type CpG motifs are present in bacterial and viral DNA, the maturation of monocytes into functional DC may reflect a physiologic response that can be harnessed therapeutically through the use of CpG ODN.