The presence of DNA in the cytoplasm of mammalian cells is a danger signal that triggers host immune responses such as the production of type I interferons. Cytosolic DNA induces interferons through the production of cyclic guanosine monophosphate–adenosine monophosphate (cyclic GMP-AMP, or cGAMP), which binds to and activates the adaptor protein STING. Through biochemical fractionation and quantitative mass spectrometry, we identified a cGAMP synthase (cGAS), which belongs to the nucleotidyltransferase family. Overexpression of cGAS activated the transcription factor IRF3 and induced interferon-β in a STING-dependent manner. Knockdown of cGAS inhibited IRF3 activation and interferon-β induction by DNA transfection or DNA virus infection. cGAS bound to DNA in the cytoplasm and catalyzed cGAMP synthesis. These results indicate that cGAS is a cytosolic DNA sensor that induces interferons by producing the second messenger cGAMP.

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Cyclic GMP-AMP Synthase is a Cytosolic DNA Sensor that Activates the Type-I Interferon Pathway

Over the past several decades, much has been revealed about the nature of the host innate immune response to microorganisms, with the identification of pattern recognition receptors (PRRs) and pathogen-associated molecular patterns, which are the conserved microbial motifs sensed by these receptors. It is now apparent that these same PRRs can also be activated by non-microbial signals, many of which are considered as damage-associated molecular patterns. The sterile inflammation that ensues either resolves the initial insult or leads to disease. Here, we review the triggers and receptor pathways that result in sterile inflammation and its impact on human health.

Sterile inflammation: sensing and reacting to damage

Inflammasomes are key signalling platforms that detect pathogenic microorganisms and sterile stressors, and that activate the highly pro-inflammatory cytokines interleukin-1β (IL-1β) and IL-18. In this Review, we discuss the complex regulatory mechanisms that facilitate a balanced but effective inflammasome-mediated immune response, and we highlight the similarities to another molecular signalling platform - the apoptosome - that monitors cellular health. Extracellular regulatory mechanisms are discussed, as well as the intracellular control of inflammasome assembly, for example, via ion fluxes, free radicals and autophagy.

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Activation and regulation of the inflammasomes

Autophagy, a cellular process for organelle and protein turnover, regulates innate immune responses. Here we demonstrate that depletion of the autophagic proteins LC3B and beclin 1 enhanced the activation of caspase-1 and secretion of interleukin 1β (IL-1β) and IL-18. Depletion of autophagic proteins promoted the accumulation of dysfunctional mitochondria and cytosolic translocation of mitochondrial DNA (mtDNA) in response to lipopolysaccharide (LPS) and ATP in macrophages. Release of mtDNA into the cytosol depended on the NALP3 inflammasome and mitochondrial reactive oxygen species (ROS). Cytosolic mtDNA contributed to the secretion of IL-1β and IL-18 in response to LPS and ATP. LC3B-deficient mice produced more caspase-1-dependent cytokines in two sepsis models and were susceptible to LPS-induced mortality. Our study suggests that autophagic proteins regulate NALP3-dependent inflammation by preserving mitochondrial integrity.

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Autophagy proteins regulate innate immune responses by inhibiting the release of mitochondrial DNA mediated by the NALP3 inflammasome

Inflammasomes are multiprotein signalling platforms that control the inflammatory response and coordinate antimicrobial host defences. They are assembled by pattern-recognition receptors following the detection of pathogenic microorganisms and danger signals in the cytosol of host cells, and they activate inflammatory caspases to produce cytokines and to induce pyroptotic cell death. The clinical importance of inflammasomes reaches beyond infectious disease, as dysregulated inflammasome activity is associated with numerous hereditary and acquired inflammatory disorders. In this Review, we discuss the recent developments in inflammasome research with a focus on the molecular mechanisms that govern inflammasome assembly, signalling and regulation.

Inflammasomes: mechanism of assembly, regulation and signalling

The detection of intracellular microbial DNA is critical to appropriate innate immune responses; however, knowledge of how such DNA is sensed is limited. Here we identify IFI16, a PYHIN protein, as an intracellular DNA sensor that mediates the induction of interferon-β (IFN-β). IFI16 directly associated with IFN-β-inducing viral DNA motifs. STING, a critical mediator of IFN-β responses to DNA, was recruited to IFI16 after DNA stimulation. Lowering the expression of IFI16 or its mouse ortholog p204 by RNA-mediated interference inhibited gene induction and activation of the transcription factors IRF3 and NF-κB induced by DNA and herpes simplex virus type 1 (HSV-1). IFI16 (p204) is the first PYHIN protein to our knowledge shown to be involved in IFN-β induction. Thus, the PYHIN proteins IFI16 and AIM2 form a new family of innate DNA sensors we call 'AIM2-like receptors' (ALRs).

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IFI16 is an innate immune sensor for intracellular DNA.

Viruses are detected by different classes of pattern recognition receptors (PRRs), such as Toll‐like receptors and RIG‐like helicases. Engagement of PRRs leads to activation of interferon (IFN)‐regulatory factor 3 (IRF3) and IRF7 through IKKe and TBK1 and consequently IFN‐β induction. Vaccinia virus (VACV) encodes proteins that manipulate host signalling, sometimes by targeting uncharacterised proteins. Here, we describe a novel VACV protein, K7, which can inhibit PRR‐induced IFN‐β induction by preventing TBK1/IKKe‐mediated IRF activation. We identified DEAD box protein 3 (DDX3) as a host target of K7. Expression of DDX3 enhanced Ifnb promoter induction by TBK1/IKKe, whereas knockdown of DDX3 inhibited this, and virus‐ or dsRNA‐induced IRF3 activation. Further, dominant‐negative DDX3 inhibited virus‐, dsRNA‐ and cytosolic DNA‐stimulated Ccl5 promoter induction, which is also TBK1/IKKe dependent. Both K7 binding and enhancement of Ifnb induction mapped to the N‐terminus of DDX3. Furthermore, virus infection induced an association between DDX3 and IKKe. Therefore, this study shows for the first time the involvement of a DEAD box helicase in TBK1/IKKe‐mediated IRF activation and Ifnb promoter induction.

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Viral targeting of DEAD box protein 3 reveals its role in TBK1/IKKe-mediated IRF activation

Cytosolic DNA sensors regulating type I interferon induction

Recognition of viruses by pattern recognition receptors (PRRs) causes interferon-β (IFN-β) induction, a key event in the anti-viral innate immune response, and also a target of viral immune evasion. Here the vaccinia virus (VACV) protein C6 is identified as an inhibitor of PRR-induced IFN-β expression by a functional screen of select VACV open reading frames expressed individually in mammalian cells. C6 is a member of a family of Bcl-2-like poxvirus proteins, many of which have been shown to inhibit innate immune signalling pathways. PRRs activate both NF-κB and IFN regulatory factors (IRFs) to activate the IFN-β promoter induction. Data presented here show that C6 inhibits IRF3 activation and translocation into the nucleus, but does not inhibit NF-κB activation. C6 inhibits IRF3 and IRF7 activation downstream of the kinases TANK binding kinase 1 (TBK1) and IκB kinase-e (IKKe), which phosphorylate and activate these IRFs. However, C6 does not inhibit TBK1- and IKKe-independent IRF7 activation or the induction of promoters by constitutively active forms of IRF3 or IRF7, indicating that C6 acts at the level of the TBK1/IKKe complex. Consistent with this notion, C6 immunoprecipitated with the TBK1 complex scaffold proteins TANK, SINTBAD and NAP1. C6 is expressed early during infection and is present in both nucleus and cytoplasm. Mutant viruses in which the C6L gene is deleted, or mutated so that the C6 protein is not expressed, replicated normally in cell culture but were attenuated in two in vivo models of infection compared to wild type and revertant controls. Thus C6 contributes to VACV virulence and might do so via the inhibition of PRR-induced activation of IRF3 and IRF7.

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Vaccinia virus protein C6 is a virulence factor that binds TBK-1 adaptor proteins and inhibits activation of IRF3 and IRF7.

The detection of nucleic acids by the innate immune system is an essential host response during viral infection. In recent years, a number of immune sensors capable of recognizing cytosolic DNA have been identified and include the PYHIN family members AIM2, IFI16, and p204 as well as the enzyme, cGAS. Activation of these receptors leads to the induction of antiviral genes including Type‐1 interferons and chemokines such as CCL5. We have carried out extensive expression profiling of these DNA sensors and other members of the PYHIN family in highly purified primary astrocytes and microglia and have demonstrated that both cell types express the majority of these proteins at the mRNA level. In microglia, several family members are highly upregulated in response to IFN‐β treatment while both cell types induce robust proinflammatory and antiviral cytokine production (e.g., IL‐6, CCL5, IFN‐β) in the presence of immune stimulatory DNA and RNA. The production of IL‐6 is partially dependent on the interferon receptor as is IFN‐β itself. Furthermore, we have found that p204 and AIM2 are upregulated in a Type I IFN dependent fashion in vivo, in a murine model of chronic neurodegeneration. Given the propensity of inflammatory responses to cause neuronal damage, increased expression and activation of these receptors, not only during viral infection but also during sterile inflammatory responses, has the potential to exacerbate existing neuroinflammation leading to further damage and impaired neurogenesis. GLIA 2015;63:812–825

https://onlinelibrary.wiley.com/doi/pdf/10.1002/glia.22786

Marcin Baran

Papers

IFI16 is an innate immune sensor for intracellular DNA.

Viral targeting of DEAD box protein 3 reveals its role in TBK1/IKKe-mediated IRF activation

Cytosolic DNA sensors regulating type I interferon induction

Vaccinia virus protein C6 is a virulence factor that binds TBK-1 adaptor proteins and inhibits activation of IRF3 and IRF7.

DNA sensors are expressed in astrocytes and microglia in vitro and are upregulated during gliosis in neurodegenerative disease.