Induction of type I IFNs by intracellular DNA-sensing pathways

A successful antimicrobial immune response involves the coordinate action of cells and soluble factors, with the cytokine family of type I interferons (IFNs) having a central role. Type I IFNs are not only crucial in conferring immediate antimicrobial, most importantly antiviral effects, but they also have an essential role in bridging the innate with the adaptive immune response. Therefore, production of these key cytokines must be tightly controlled. To this effect the host has evolved a set of pattern recognition receptors (PRRs) that reliably and specifically detect the presence of microbial pathogens before mounting an IFN response. Most PRR pathways that are known to induce type I IFNs are triggered upon recognition of nucleic acids. This mode of sensing is not straightforward, as large amounts of RNA and DNA are also present within the host. Nevertheless, in some cases distinct molecular features that are present within foreign nucleic acids but absent in endogenous nucleic acids, allow the host to reliably discriminate between 'self' and 'non-self'. At the same time, compartmentalization of PRRs within subcellular organelles that are usually devoid of host nucleic acids, but are sites of pathogen localization, is another principle that enables the host to distinguish self from non-self. The latter mode of sensing applies to the detection of microbial DNA within the cytoplasm, a compartment in which host DNAs are usually not present. Despite the past years' tremendous progress in the field of innate immunity, our understanding of cytoplasmic DNA sensing mechanisms is only beginning to form/take form. In this review, we outline the recent advancements in the elucidation of intracellular DNA-sensing pathways and discuss the future directions of this emerging field.     

Serum deprivation induced apoptosis in macrophage is mediated by autocrine secretion of type I IFNs

Apoptosis can be triggered by different forms of cellular stress. We here show that serum deprivation induces the expression and secretion of type I interferons and results in apoptosis in RAW 264.7 cell in a caspase dependent manner. Administration of either IFN-α or IFN-β antibody partially inhibits apoptosis while the two antibodies used together totally prevents RAW264.7 cell from apoptosis. GM-CSF, but not M-CSF and IL-3, protects serum deprivation induced apoptosis. Inhibition of JAKs also prevents macrophages from apoptosis. Activation of MAPKs is not required for serum deprivation induced apoptosis. Our results are the first to demonstrate that serum deprivation-induced apoptosis acts through autocrine secretion of type I interferons.     

Glucitol induction in Bacillus subtilis is mediated by a regulatory factor, GutR.

Expression of the glucitol dehydrogenase gene (gutB) is suggested to be regulated both positively and negatively in Bacillus subtilis. A mutation in the gutR locus results in the constitutive expression of gutB. The exact nature of this mutation and the function of gutR are still unknown. Cloning and characterization of gutR indicated that this gene is located immediately upstream of gutB and is transcribed in the opposite direction relative to gutB. GutR is suggested to be a 95-kDa protein with a putative helix-turn-helix motif and a nucleotide binding domain at the N-terminal region. At the C-terminal region, a short sequence of GutR shows homology with two proteins, Cyc8 (glucose repression mediator protein) and GsiA (glucose starvation-inducible protein), known to be directly or indirectly involved in catabolite repression. Part of the C-terminal conserved sequence from these proteins shows all the features observed in the tetratricopeptide motif found in many eucaryotic proteins. To study the functional role of gutR, chromosomal gutR was insertionally inactivated. A total loss of glucitol inducibility was observed. Reintroduction of a functional gutR to the GutR-deficient strain through integration at the amyE locus restores the inducibility. Therefore, GutR serves as a regulatory factor to modulate glucitol induction. The nature of the gutR1 mutation was also determined. A single amino acid change (serine-289 to arginine-289) near the putative nucleotide binding motif B in GutR is responsible for the observed phenotype. Possible models for the action of GutR are discussed.     

Suppression of the effector phase of inflammatory arthritis by double-stranded RNA is mediated by type I IFNs

Innate immune receptors that recognize nucleic acids, such as TLRs and RNA helicases, are potent activators of innate immunity that have been implicated in the induction and exacerbation of autoimmunity and inflammatory arthritis. Polyriboinosine-polyribocytidylic acid sodium salt (poly(IC)) is a mimic of dsRNA and viral infection that activates TLR3 and the RNA helicases retinoic acid-induced gene-1 and melanoma differentiation-associated gene-5, and strongly induces type I IFN production. We analyzed the effects of systemic delivery of poly(IC) on the inflammatory effector phase of arthritis using the collagen Ab-induced and KRN TCR-transgenic mouse serum-induced models of immune complex-mediated experimental arthritis. Surprisingly, poly(IC) suppressed arthritis, and suppression was dependent on type I IFNs that inhibited synovial cell proliferation and inflammatory cytokine production. Administration of exogenous type I IFNs was sufficient to suppress arthritis. These results suggest a regulatory role for innate immune receptors for dsRNA in modulating inflammatory arthritis and provide additional support for an anti-inflammatory function of type I IFNs in arthritis that directly contrasts with a pathogenic role in promoting autoimmunity in systemic lupus.     

Guanylate binding protein 4 negatively regulates virus-induced type I IFN and antiviral response by targeting IFN regulatory factor 7

IRF7 is known as the master regulator in virus-triggered induction of type I IFNs (IFN-I). In this study, we identify GBP4 virus-induced protein interacting with IRF7 as a negative regulator for IFN-I response. Overexpression of GBP4 inhibits virus-triggered activation of IRF7-dependent signaling, but has no effect on NF-κB signaling, whereas the knockdown of GBP4 has opposite effects. Furthermore, the supernatant from Sendai virus-infected cells in which GBP4 have been silenced inhibits the replication of vesicular stomatitis virus more efficiently. Competitive coimmunoprecipitation experiments indicate that overexpression of GBP4 disrupts the interactions between TRAF6 and IRF7, resulting in impaired TRAF6-mediated IRF7 ubiquitination. Our results suggest that GBP4 is a negative regulator of virus-triggered IFN-I production, and it is identified as a novel protein targeting IRF7 and inhibiting its function.     

Cutting edge: TLR4 activation mediates liver ischemia/reperfusion inflammatory response via IFN regulatory factor 3-dependent MyD88-independent pathway

The triggering molecular mechanism of ischemia-reperfusion injury (IRI), which in clinical settings results in excessive and detrimental inflammatory responses, remains unclear. This study analyzes the role of the TLR system in an established murine model of liver warm ischemia followed by reperfusion. By contrasting in parallel TLR knockout mice with their wild-type counterparts, we found that TLR4, but not TLR2, was specifically required in initiating the IRI cascade, as manifested by liver function (serum alanine aminotransferase levels), pathology, and local induction of proinflammatory cytokines/chemokines (TNF-alpha, IL-6, IFN-inducible protein 10). We then investigated the downstream signaling pathway of TLR4 activation. Our results show that IFN regulatory factor 3, but not MyD88, mediated IRI-induced TLR4 activation leading to liver inflammation and hepatocellular damage. This study documents the selective usage of TLR in a clinically relevant noninfectious disease model, and identifies a triggering molecular mechanism in the pathophysiology of liver IRI.     

Identification of a second group of type I IFNs in fish sheds light on IFN evolution in vertebrates

In this report, three type I IFN genes were identified in rainbow trout (rt) Oncorhynchus mykiss and are classified into two groups based on their primary protein sequences: group I containing two cysteine residues; and group II containing four cysteines residues. The group I rtIFNs were induced in fibroblasts (RTG-2 cells), macrophages (RTS-11 cells), and head kidney leukocytes when stimulated with polyinosinic:polycytidylic acid, whereas group II IFN was up-regulated in head kidney leukocytes but not in RTG-2 and RTS-11 cells. Recombinant group I rtIFNs were potent at inducing Mx expression and eliciting antiviral responses, whereas recombinant group II rtIFN was poor in these activities. That two subgroups of type I IFN exist in trout prompted a survey of the genomes of several fish species, including zebrafish, medaka, threespine stickleback and fugu, the amphibian Xenopus tropicalis, the monotreme platypus and the marsupial opossum, to gain further insight into possible IFN evolution. Analysis of the sequences confirmed that the new IFN subgroup found in trout (group II IFN) exists in other fish species but was not universally present in fish. The IFN genes in amphibians were shown for the first time to contain introns and to conserve the four cysteine structure found in all type I IFNs except IFN-betaepsilon and fish group I IFN. The data overall support the concept that different vertebrate groups have independently expanded their IFN types, with deletion of different pairs of cysteines apparent in fish group I IFN and IFN-betaepsilon of mammals.     

Murine dendritic cell type I IFN production induced by human IgG-RNA immune complexes is IFN regulatory factor (IRF)5 and IRF7 dependent and is required for IL-6 production

Dendritic cell (DC) activation by nucleic acid-containing IgG complexes is implicated in systemic lupus erythematosus (SLE) pathogenesis. However, it has been difficult to definitively examine the receptors and signaling pathways by which this activation is mediated. Because mouse FcgammaRs recognize human IgG, we hypothesized that IgG from lupus patients might stimulate mouse DCs, thereby facilitating this analysis. In this study, we show that sera and purified IgG from lupus patients activate mouse DCs to produce IFN-alpha, IFN-beta, and IL-6 and up-regulate costimulatory molecules in a FcgammaR-dependent manner. This activation is only seen in sera with reactivity against ribonucleoproteins and is completely dependent on TLR7 and the presence of RNA. As anticipated, IFN regulatory factor (IRF)7 is required for IFN-alpha and IFN-beta production. Unexpectedly, however, IRF5 plays a critical role in IFN-alpha and IFN-beta production induced not only by RNA-containing immune complexes but also by conventional TLR7 and TLR9 ligands. Moreover, DC production of IL-6 induced by these stimuli is dependent on a functional type I IFNR, indicating the need for a type I IFN-dependent feedback loop in the production of inflammatory cytokines. This system may also prove useful for the study of receptors and signaling pathways used by immune complexes in other human diseases.     

Cutting edge: a murine,IL-12-independent pathway of IFN-gamma induction by gram-negative bacteria based on STAT4 activation by Type I IFN and IL-18 signaling

IFN-alphabeta is a potent immunoregulatory cytokine involved in the defense against viral and bacterial infections. In this study, we describe an as yet undefined IFN-alphabeta-dependent pathway of IFN-gamma induction in mice. This pathway is based on a synergism of IFN-alphabeta and IL-18, and is independent of IL-12 signaling yet dependent on STAT4. In contradiction to current dogma, we show further that IFN-alphabeta alone induces tyrosine phosphorylation of STAT4 in murine splenocytes of different mouse strains. This pathway participates in the induction of IFN-gamma by Gram-negative bacteria and is therefore expected to play a role whenever IFN-alpha or IFN-beta and IL-18 are produced concomitantly during bacterial, viral, or other infections.     

Stat1-independent induction of SOCS-3 by interferon-gamma is mediated by sustained activation of Stat3 in mouse embryonic fibroblasts

Using microarray technology, we previously demonstrated that IFN-gamma induces suppressor of cytokine signaling-3 (SOCS-3) in Stat1-/- mouse embryonic fibroblasts and bone marrow-derived macrophages. In this study, we have investigated the mechanism by which SOCS-3 is induced by Stat1-independent signal transduction pathway. Tyrosine kinases Jak1 and Jak2 are required for SOCS-3 induction by IFN-gamma in mouse embryonic fibroblasts. IFN-gamma stimulated strong and sustained activation of Stat1 whereas Stat3 activation was weak and transient in wild-type fibroblasts. In contrast, Stat3 is activated strongly and in a sustained manner in Stat1-/- fibroblasts. The Src kinase inhibitor SU6656 suppressed IFN-gamma activation of Stat3 in both wild-type and Stat1-/- fibroblasts. However, SU6656 inhibited IFN-gamma induction of SOCS-3 completely in Stat1-/- but not in wild-type fibroblasts. Knock down of Stat3 by short interfering RNA abrogated Stat3 activation and SOCS-3 induction by IFN-gamma in Stat1-/- fibroblasts. In human fibrosarcoma cell line 2fTGH, IFN-gamma activated Stat1 but not Stat3. SOCS-3 induction by IFN-gamma is strictly Stat1-dependent. The Stat1 docking site is required for SOCS-3 induction by IFN-gamma in human lung adenocarcinoma cells. We propose a model in which sustained activation of Stat1 or Stat3 mediates SOCS-3 induction by IFN-gamma in wild-type and Stat1-/- mouse embryonic fibroblasts, respectively.