The type-I interferon (IFN-alpha/beta) response is critical to immunity against viruses and can be triggered in many cell types by cytosolic detection of viral infection, or in differentiated plasmacytoid dendritic cells by the Toll-like receptor 9 (TLR9) subfamily, which generates signals via the adaptor MyD88 to elicit robust IFN induction. Using mice deficient in the Irf7 gene (Irf7-/- mice), we show that the transcription factor IRF-7 is essential for the induction of IFN-alpha/beta genes via the virus-activated, MyD88-independent pathway and the TLR-activated, MyD88-dependent pathway. Viral induction of MyD88-independent IFN-alpha/beta genes is severely impaired in Irf7-/- fibroblasts. Consistently, Irf7-/- mice are more vulnerable than Myd88-/- mice to viral infection, and this correlates with a marked decrease in serum IFN levels, indicating the importance of the IRF-7-dependent induction of systemic IFN responses for innate antiviral immunity. Furthermore, robust induction of IFN production by activation of the TLR9 subfamily in plasmacytoid dendritic cells is entirely dependent on IRF-7, and this MyD88-IRF-7 pathway governs the induction of CD8+ T-cell responses. Thus, all elements of IFN responses, whether the systemic production of IFN in innate immunity or the local action of IFN from plasmacytoid dendritic cells in adaptive immunity, are under the control of IRF-7.

Central to innate immunity is the sensing of pathogen-associated molecular patterns by cytosolic and membrane-associated receptors. In particular, DNA is a potent activator of immune responses during infection or tissue damage, and evidence indicates that, in addition to the membrane-associated Toll-like receptor 9, an unidentified cytosolic DNA sensor(s) can activate type I interferon (IFN) and other immune responses. Here we report on a candidate DNA sensor, previously named DLM-1 (also called Z-DNA binding protein 1 (ZBP1)), for which biological function had remained unknown; we now propose the alternative name DAI (DNA-dependent activator of IFN-regulatory factors). The artificial expression of otherwise IFN-inducible DAI (DLM-1/ZBP1) in mouse fibroblasts selectively enhances the DNA-mediated induction of type I IFN and other genes involved in innate immunity. On the other hand, RNA interference of messenger RNA for DAI (DLM-1/ZBP1) in cells inhibits this gene induction programme upon stimulation by DNA from various sources. Moreover, DAI (DLM-1/ZBP1) binds to double-stranded DNA and, by doing so, enhances its association with the IRF3 transcription factor and the TBK1 serine/threonine kinase. These observations underscore an integral role of DAI (DLM-1/ZBP1) in the DNA-mediated activation of innate immune responses, and may offer new insight into the signalling mechanisms underlying DNA-associated antimicrobial immunity and autoimmune disorders.

The activation of Toll-like receptors (TLRs) is central to innate and adaptive immunity. All TLRs use the adaptor MyD88 for signalling, but the mechanisms underlying the MyD88-mediated gene induction programme are as yet not fully understood. Here, we demonstrate that the transcription factor IRF-5 is generally involved downstream of the TLR-MyD88 signalling pathway for gene induction of proinflammatory cytokines, such as interleukin-6 (IL-6), IL-12 and tumour-necrosis factor-alpha. In haematopoietic cells from mice deficient in the Irf5 gene (Irf5-/- mice), the induction of these cytokines by various TLR ligands is severely impaired, whereas interferon-alpha induction is normal. We also provide evidence that IRF-5 interacts with and is activated by MyD88 and TRAF6, and that TLR activation results in the nuclear translocation of IRF-5 to activate cytokine gene transcription. Consistently, Irf5-/- mice show resistance to lethal shock induced by either unmethylated DNA or lipopolysaccharide, which correlates with a marked decrease in the serum levels of proinflammatory cytokines. Thus, our study identifies IRF-5 as a new, principal downstream regulator of the TLR-MyD88 signalling pathway and a potential target of therapeutic intervention to control harmful immune responses.

Robust type-I interferon (IFN-alpha/beta) induction in plasmacytoid dendritic cells, through the activation of Toll-like receptor 9 (TLR9), constitutes a critical aspect of immunity. It is absolutely dependent on the transcription factor IRF-7, which interacts with and is activated by the adaptor MyD88. How plasmacytoid dendritic cells, but not other cell types (such as conventional dendritic cells), are able to activate the MyD88-IRF-7-dependent IFN induction pathway remains unknown. Here we show that the spatiotemporal regulation of MyD88-IRF-7 signalling is critical for a high-level IFN induction in response to TLR9 activation. The IFN-inducing TLR9 ligand, A/D-type CpG oligodeoxynucleotide (CpG-A), is retained for long periods in the endosomal vesicles of plasmacytoid dendritic cells, together with the MyD88-IRF-7 complex. However, in conventional dendritic cells, CpG-A is rapidly transferred to lysosomal vesicles. We further show that conventional dendritic cells can also mount a robust IFN induction if CpG-A is manipulated for endosomal retention using a cationic lipid. This strategy also allows us to demonstrate endosomal activation of the IFN pathway by the otherwise inactive TLR9 ligand B/K-type oligodeoxynucleotide (CpG-B). Thus, our study offers insights into the regulation of TLR9 signalling in space, potentially suggesting a new avenue for therapeutic intervention.

Swift elimination of undesirable cells is an important feature in tumour suppression and immunity. The tumour suppressor p53 and interferon-a and -b (IFN-a/b) are essential for the induction of apoptosis in cancerous cells and in antiviral immune responses, respectively, but little is known about their interrelationship. Here we show that transcription of the p53 gene is induced by IFN-a/b, accompanied by an increase in p53 protein level. IFN-a/b signalling itself does not activate p53; rather, it contributes to boosting p53 responses to stress signals. We show examples in which p53 gene induction by IFN-a/b contributes to tumour suppression. Furthermore, we show that p53 is activated in virally infected cells to evoke an apoptotic response and that p53 is critical for antiviral defence of the host. Our study reveals a hitherto unrecognized link between p53 and IFN-a/b in tumour suppression and antiviral immunity, which may have therapeutic implications.The tumour suppressor p53, activated in response to DNA damage, induces cell cycle arrest or apoptosis through transcriptional activation of its target genes, hence having a central role in tumour suppression [1][2][3][4][5] . So far, it is not known whether p53 contributes to the immune responses that lead to the eradication of pathogens such as viruses. On the other hand, IFN-a/b, both of which are essential cytokines for antiviral immunity, are sometimes referred to as 'negative growth factors' , and manifest anti-oncogenic activities [6][7][8][9][10] . In fact, IFN-a/b are used for the treatment of some forms of human cancer but the molecular basis for the treatment is poorly understood [11][12][13] . Until know, little if anything has been known about the link between the p53 and IFN-a/b system. Induction of p53 protein by IFN-a/bWhen wild-type mouse embryonic fibroblasts (MEFs) were stimulated with IFN-b, a notable increase in the level of p53 was observed (Fig. 1a). A similar observation was made in cells stimulated with IFN-a (data not shown) but not with IFN-g ( Supplementary Fig. 1). The increase in p53 level with IFN-b treatment was dose-dependent, with about fourfold induction achieved at a high concentration of IFN-b (Fig. 1b). The p53 induction by IFN-b was also observed in two human hepatic cancer cell lines, HepG2 and HLE, p53 function being abrogated in the latter cells by a mutation in the DNA-binding domain 14 (Fig. 1c). We performed a pulse-chase experiment to examine whether the observed p53 protein induction is secondary to suppression of the p53 degradation pathway by IFN-b, typically the MDM2-mediated pathway [15][16][17] . However, no difference was observed in the half-life of p53 (40-45 min) between the IFN-treated and untreated MEFs (Fig. 1d), suggesting that p53 protein synthesis is induced by IFN-b stimulation. Induction of the p53 gene by IFN-a/bInformation is limited about the induction of the p53 gene 18 , and the above results prompted us to examine whether IFN-a/b induces p53 gene transcription. Inspection of mouse and human p53...

The activation of innate immune responses by nucleic acids is crucial to protective and pathological immunities and is mediated by the transmembrane Toll-like receptors (TLRs) and cytosolic receptors. However, it remains unknown whether a mechanism exists that integrates these nucleic-acid-sensing systems. Here we show that high-mobility group box (HMGB) proteins 1, 2 and 3 function as universal sentinels for nucleic acids. HMGBs bind to all immunogenic nucleic acids examined with a correlation between affinity and immunogenic potential. Hmgb1(-/-) and Hmgb2(-/-) mouse cells are defective in type-I interferon and inflammatory cytokine induction by DNA or RNA targeted to activate the cytosolic nucleic-acid-sensing receptors; cells in which the expression of all three HMGBs is suppressed show a more profound defect, accompanied by impaired activation of the transcription factors interferon regulatory factor 3 (IRF3) and nuclear factor (NF)-kappaB. The absence of HMGBs also severely impairs the activation of TLR3, TLR7 and TLR9 by their cognate nucleic acids. Our results therefore indicate a hierarchy in the nucleic-acid-mediated activation of immune responses, wherein the selective activation of nucleic-acid-sensing receptors is contingent on the more promiscuous sensing of nucleic acids by HMGBs. These findings may have implications for understanding the evolution of the innate immune system and for the treatment of immunological disorders.

Toll-like receptor (TLR) activation is central to immunity, wherein the activation of the TLR9 subfamily members TLR9 and TLR7 results in the robust induction of type I IFNs (IFN-␣͞␤) by means of the MyD88 adaptor protein. However, it remains unknown how the TLR signal ''input'' can be processed through MyD88 to ''output'' the induction of the IFN genes. Here, we demonstrate that the transcription factor IRF-7 interacts with MyD88 to form a complex in the cytoplasm. We provide evidence that this complex also involves IRAK4 and TRAF6 and provides the foundation for the TLR9-dependent activation of the IFN genes. The complex defined in this study represents an example of how the coupling of the signaling adaptor and effector kinase molecules together with the transcription factor regulate the processing of an extracellular signal to evoke its versatile downstream transcriptional events in a cell. Thus, we propose that this molecular complex may function as a cytoplasmic transductional-transcriptional processor.

IFN regulatory factor ͉ macrophage ͉ MyD88