Mouse superkiller-2-like helicase DDX60 is dispensable for type I IFN induction and immunity to multiple viruses.

IFN-α/ß allow cells to fight virus infection by inducing the expression of many genes that encode effectors of antiviral defense. One of these, the Ski2-like DExH-box helicase DDX60, was recently implicated in resistance of human cells to hepatitis C virus, as well as in induction of IFN-α/ß by retinoic acid inducible gene 1-like receptors (RLRs) that detect the presence of RNA viruses in a cell-intrinsic manner. Here, we sought to investigate the role of DDX60 in IFN-α/ß induction and in resistance to virus infection. Analysis of fibroblasts and myeloid cells from Ddx60-deficient mice revealed no impairment in IFN-α/ß production in response to RLR agonists, RNA viruses, or other stimuli. Moreover, overexpression of DDX60 did not potentiate IFN induction and DDX60 did not interact with RLRs or capture RLR agonists from virally infected cells. We also failed to identify any impairment in Ddx60-deficient murine cells or mice in resistance to infection with influenza A virus, encephalomyocarditis virus, Sindbis virus, vaccinia virus, or herpes simplex virus-1. These results put in question the reported role of DDX60 as a broad-acting positive regulator of RLR responses and hint at the possibility that it may function as a restriction factor highly specific for a particular virus or class of viruses.

GM-CSF Mouse Bone Marrow Cultures Comprise a Heterogeneous Population of CD11c(+)MHCII(+) Macrophages and Dendritic Cells.

Dendritic cells (DCs) are key players in the immune system. Much of their biology has been elucidated via culture systems in which hematopoietic precursors differentiate into DCs under the aegis of cytokines. A widely used protocol involves the culture of murine bone marrow (BM) cells with granulocyte-macrophage colony-stimulating factor (GM-CSF) to generate BM-derived DCs (BMDCs). BMDCs express CD11c and MHC class II (MHCII) molecules and share with DCs isolated from tissues the ability to present exogenous antigens to T cells and to respond to microbial stimuli by undergoing maturation. We demonstrate that CD11c(+)MHCII(+) BMDCs are in fact a heterogeneous group of cells that comprises conventional DCs and monocyte-derived macrophages. DCs and macrophages in GM-CSF cultures both undergo maturation upon stimulation with lipopolysaccharide but respond differentially to the stimulus and remain separable entities. These results have important implications for the interpretation of a vast array of data obtained with DC culture systems.

Intestinal intraepithelial lymphocyte activation promotes innate antiviral resistance.

Unrelenting environmental challenges to the gut epithelium place particular demands on the local immune system. In this context, intestinal intraepithelial lymphocytes (IEL) compose a large, highly conserved T cell compartment, hypothesized to provide a first line of defence via cytolysis of dysregulated intestinal epithelial cells (IEC) and cytokine-mediated re-growth of healthy IEC. Here we show that one of the most conspicuous impacts of activated IEL on IEC is the functional upregulation of antiviral interferon (IFN)-responsive genes, mediated by the collective actions of IFNs with other cytokines. Indeed, IEL activation in vivo rapidly provoked type I/III IFN receptor-dependent upregulation of IFN-responsive genes in the villus epithelium. Consistent with this, activated IEL mediators protected cells against virus infection in vitro, and pre-activation of IEL in vivo profoundly limited norovirus infection. Hence, intraepithelial T cell activation offers an overt means to promote the innate antiviral potential of the intestinal epithelium.

Antiviral immunity via RIG-I-mediated recognition of RNA bearing 5'-diphosphates.

Mammalian cells possess mechanisms to detect and defend themselves from invading viruses. In the cytosol, the RIG-I-like receptors (RLRs), RIG-I (retinoic acid-inducible gene I; encoded by DDX58) and MDA5 (melanoma differentiation-associated gene 5; encoded by IFIH1) sense atypical RNAs associated with virus infection. Detection triggers a signalling cascade via the adaptor MAVS that culminates in the production of type I interferons (IFN-α and ß; hereafter IFN), which are key antiviral cytokines. RIG-I and MDA5 are activated by distinct viral RNA structures and much evidence indicates that RIG-I responds to RNAs bearing a triphosphate (ppp) moiety in conjunction with a blunt-ended, base-paired region at the 5'-end (reviewed in refs 1, 2, 3). Here we show that RIG-I also mediates antiviral responses to RNAs bearing 5'-diphosphates (5'pp). Genomes from mammalian reoviruses with 5'pp termini, 5'pp-RNA isolated from yeast L-A virus, and base-paired 5'pp-RNAs made by in vitro transcription or chemical synthesis, all bind to RIG-I and serve as RIG-I agonists. Furthermore, a RIG-I-dependent response to 5'pp-RNA is essential for controlling reovirus infection in cultured cells and in mice. Thus, the minimal determinant for RIG-I recognition is a base-paired RNA with 5'pp. Such RNAs are found in some viruses but not in uninfected cells, indicating that recognition of 5'pp-RNA, like that of 5'ppp-RNA, acts as a powerful means of self/non-self discrimination by the innate immune system.

Identification of an LGP2-associated MDA5 agonist in picornavirus-infected cells.

The RIG-I-like receptors RIG-I, LGP2, and MDA5 initiate an antiviral response that includes production of type I interferons (IFNs). The nature of the RNAs that trigger MDA5 activation in infected cells remains unclear. Here, we purify and characterise LGP2/RNA complexes from cells infected with encephalomyocarditis virus (EMCV), a picornavirus detected by MDA5 and LGP2 but not RIG-I. We show that those complexes contain RNA that is highly enriched for MDA5-stimulatory activity and for a specific sequence corresponding to the L region of the EMCV antisense RNA. Synthesis of this sequence by in vitro transcription is sufficient to generate an MDA5 stimulatory RNA. Conversely, genomic deletion of the L region in EMCV generates viruses that are less potent at stimulating MDA5-dependent IFN production. Thus, the L region antisense RNA of EMCV is a key determinant of innate immunity to the virus and represents an RNA that activates MDA5 in virally-infected cells. DOI: http://dx.doi.org/10.7554/eLife.01535.001.

Cytosolic sensing of viruses.

Cells are equipped with mechanisms that allow them to rapidly detect and respond to viruses. These defense mechanisms rely partly on receptors that monitor the cytosol for the presence of atypical nucleic acids associated with virus infection. RIG-I-like receptors detect RNA molecules that are absent from the uninfected host. DNA receptors alert the cell to the abnormal presence of that nucleic acid in the cytosol. Signaling by RNA and DNA receptors results in the induction of restriction factors that prevent virus replication and establish cell-intrinsic antiviral immunity. In light of these formidable obstacles, viruses have evolved mechanisms of evasion, masking nucleic acid structures recognized by the host, sequestering themselves away from the cytosol or targeting host sensors, and signaling adaptors for deactivation or degradation. Here, we detail recent advances in the molecular understanding of cytosolic nucleic acid detection and its evasion by viruses.

RIG-I detects viral genomic RNA during negative-strand RNA virus infection.

RIG-I is a key mediator of antiviral immunity, able to couple detection of infection by RNA viruses to the induction of interferons. Natural RIG-I stimulatory RNAs have variously been proposed to correspond to virus genomes, virus replication intermediates, viral transcripts, or self-RNA cleaved by RNase L. However, the relative contribution of each of these RNA species to RIG-I activation and interferon induction in virus-infected cells is not known. Here, we use three approaches to identify physiological RIG-I agonists in cells infected with influenza A virus or Sendai virus. We show that RIG-I agonists are exclusively generated by the process of virus replication and correspond to full-length virus genomes. Therefore, nongenomic viral transcripts, short replication intermediates, and cleaved self-RNA do not contribute substantially to interferon induction in cells infected with these negative strand RNA viruses. Rather, single-stranded RNA viral genomes bearing 5'-triphosphates constitute the natural RIG-I agonists that trigger cell-intrinsic innate immune responses during infection.

Transcriptional re-programming of primary macrophages reveals distinct apoptotic and anti-tumoral functions of IRF-3 and IRF-7.

The immunoregulatory transcriptional modulators - IFN-regulatory factor (IRF)-3 and IRF-7 - possess similar structural features but distinct gene-regulatory potentials. For example, adenovirus-mediated transduction of the constitutively active form of IRF-3 triggered cell death in primary human MPhi, whereas expression of active IRF-7 induced a strong anti-tumoral activity in vitro. To further characterize target genes involved in these distinct cellular responses, transcriptional profiles of active IRF-3- or IRF-7-transduced primary human MPhi were compared and used to direct further mechanistic studies. The pro-apoptotic BH3-only protein Noxa was identified as a primary IRF-3 target gene and an essential regulator of IRF-3, dsRNA and vesicular stomatitis virus-induced cell death. The critical role of IRF-7 and type I IFN production in increasing the immunostimulatory capacity of MPhi was also evaluated; IRF-7 increased the expression of a broad range of IFN-stimulated genes including immunomodulatory cytokines and genes involved in antigen processing and presentation. Furthermore, active IRF-7 augmented the cross-presentation capacity and tumoricidal activity of MPhi and led to an anti-tumor response against the B16 melanoma model in vivo. Altogether, these data further highlight the respective functions of IRF-3 and IRF-7 to program apoptotic, immune and anti-tumor responses.

Involvement of TBK1 and IKKepsilon in lipopolysaccharide-induced activation of the interferon response in primary human macrophages.

Interferon (IFN) is an important effector of the innate immune response, induced by different viral or bacterial components through Toll-like receptor-dependent and -independent mechanisms. In human macrophages and macrophage-activated killer cells, we demonstrate that (i) the type I IFN response to lipopolysaccharide (LPS) is weak compared to the host response to virus infection; (ii) there is a temporal difference in the induction of tank-binding kinase-1 (TBK1) and IkappaB kinase (IKK)-related kinase epsilon (IKKepsilon) kinase activities in response to LPS, with TBK1 activated early and IKKepsilon induced in the late phase of IFN induction; and (iii) interferon regulatory factor (IRF)-7 is induced following LPS treatment, but there is no evidence that IRF-7 becomes activated by phosphorylation in vivo. Specifically, TBK1 kinase activity is rapidly increased after LPS stimulation (15 min) whereas IKKepsilon activation occurs at 8 h. RNA interference-mediated inhibition of TBK1 and IKKepsilon expression in macrophages interfere with IFNB and IRF7 gene expression following LPS activation. Macrophage priming with rIFN-alpha increased IRF-7 expression, led to a sharp up-regulation of the IFNB gene and to a rapid induction of IFNA2 upon LPS stimulation. These data support a differential role of TBK1 and IKKepsilon in the downstream response mediated by IRF-3 and IRF-7 to LPS in primary human macrophages.

Distinct roles for IFN regulatory factor (IRF)-3 and IRF-7 in the activation of antitumor properties of human macrophages.

When properly activated, macrophages can be tumoricidal, thus making them attractive additions to standard cancer therapies. To this end, tolerance and activity of human autologous IFN-gamma-activated macrophages, produced in large scale for clinical use (MAK cells), have been assessed in pilot trials in cancer patients. In the present study, we tested the hypothesis that activation of IFN regulatory factor (IRF)-3 and IRF-7, with subsequent type I IFN production, may be involved in the acquisition of new antitumor functions by macrophages. Adenoviral vectors were generated for the delivery of constitutively active forms of IRF-3 (Ad-IRF-3) or IRF-7 (Ad-IRF-7) into primary human macrophages. Cell death was observed in Ad-IRF-3-transduced macrophages, whereas Ad-IRF-7-transduced macrophages produced type I IFNs and displayed increased expression of genes encoding tumor necrosis factor (TNF)-related apoptosis-inducing ligand, interleukin (IL)-12, IL-15, and CD80, persisting for at least 96 hours. Expression of iNOS, TNF-alpha, FasL, IL-1, and IL-6 genes was unaltered by Ad-IRF-7 transduction. Interestingly, Ad-IRF-3 or Ad-IRF-7 transduction negatively regulated the transcription of protumorigenic genes encoding vascular endothelial growth factor and matrix metalloproteinase-2. Furthermore, Ad-IRF-7-transduced macrophages exerted a cytostatic activity on different cancer cell lines, including SK-BR-3, MCF-7, and COLO-205; the latter cells were shown previously to be insensitive to MAK cells. In conclusion, transduction of active forms of IRF-3 or IRF-7 differentially modulate the apoptotic and antitumor properties of primary macrophages, with active IRF-7 leading to the acquisition of novel antitumor effector functions.

Distinct functions of IRF-3 and IRF-7 in IFN-alpha gene regulation and control of anti-tumor activity in primary macrophages.

Type I IFN (IFN-alpha/beta) have important biological functions ranging from immune cell development and activation, to tumor cell killing and most importantly inhibition of virus replication. Following viral infection or activation of Toll-like receptors (TLRs) via distinct ligands, IFN-alpha/beta are produced. Two members of the interferon regulatory factor (IRF) family - IRF-3 and IRF-7 - are the major modulators of IFN gene expression. Activation of IRF-3 and IRF-7 by TBK1/IKKvarepsilon mediated phosphorylation promotes IFN gene expression and potentiates the production of IFN responsive genes important to the development of an effective antiviral immune response. IFN treatment can augment anti-tumor properties and they are potentially key players in cancer therapy. For example, adoptive transfer of IFN-gamma-activated macrophages can mediate tumor cell killing via direct cell-cell contact, as well as release of soluble cytotoxic pro-inflammatory molecules. A recent study investigated whether IRF-3 and IRF-7 could mediate the acquisition of new anti-tumor effector functions in macrophages. Adenovirus mediated transduction of the active form of IRF-7 into primary macrophages resulted in the production of type I IFN, upregulation of target genes including TRAIL and increased tumoricidal activity of macrophages; in contrast, the active form of IRF-3 led to induction of cell death. These studies indicate that IRF-7 transduced macrophages may be an attractive candidate for in vivo adoptive therapy of cancer.