Shrutie Sharma

The AIM2 inflammasome is essential for host defense against cytosolic bacteria and DNA viruses

Inflammasomes regulate the activity of caspase-1 and the maturation of interleukin 1β (IL-1β) and IL-18. AIM2 has been shown to bind DNA and engage the caspase-1-activating adaptor protein ASC to form a caspase-1-activating inflammasome. Using Aim2-deficient mice, we identify a central role for AIM2 in regulating caspase-1-dependent maturation of IL-1β and IL-18, as well as pyroptosis, in response to synthetic double-stranded DNA. AIM2 was essential for inflammasome activation in response to Francisella tularensis, vaccinia virus and mouse cytomegalovirus and had a partial role in the sensing of Listeria monocytogenes. Moreover, production of IL-18 and natural killer cell–dependent production of interferon-γ, events critical in the early control of virus replication, were dependent on AIM2 during mouse cytomegalovirus infection in vivo. Collectively, our observations demonstrate the importance of AIM2 in the sensing of both bacterial and viral pathogens and in triggering innate immunity.

Mouse, but not Human STING, Binds and Signals in Response to the Vascular Disrupting Agent 5,6-Dimethylxanthenone-4-Acetic Acid

Vascular disrupting agents such as 5,6-dimethylxanthenone-4-acetic acid (DMXAA) represent a novel approach for cancer treatment. DMXAA has potent antitumor activity in mice and, despite significant preclinical promise, failed human clinical trials. The antitumor activity of DMXAA has been linked to its ability to induce type I IFNs in macrophages, although the molecular mechanisms involved are poorly understood. In this study, we identify stimulator of IFN gene (STING) as a direct receptor for DMXAA leading to TANK-binding kinase 1 and IFN regulatory factor 3 signaling. Remarkably, the ability to sense DMXAA was restricted to murine STING. Human STING failed to bind to or signal in response to DMXAA. Human STING also failed to signal in response to cyclic dinucleotides, conserved bacterial second messengers known to bind and activate murine STING signaling. Collectively, these findings detail an unexpected species-specific role for STING as a receptor for an anticancer drug and uncover important insights that may explain the failure of DMXAA in clinical trials for human cancer.

Innate Immune Sensing of DNA

When a pathogen attacks, the immune system rapidly mobilizes host defenses in order to reduce the microbial burden and limit damage to the host [1]. Innate immunity is the first line of defense and relies on germ line–encoded pattern recognition receptors (PRRs) such as the Toll-like receptors (TLRs), which sense microbial products that are not normally found on or in mammalian cells. The considerable potency of nucleic acids as triggers of the innate immune response has gained appreciation over the last few years. In particular, nucleic acid sensing of viruses is central to anti-viral defenses through recognition of viral genomes or nucleic acids generated during viral replication. Distinct classes of nucleic acid sensing molecules have been uncovered that function in different cell types and subcellular compartments to coordinate innate defenses (reviewed in [2]). While recognition of RNA molecules is dependent on members of the TLR family and cytosolic RNA helicases, the mechanisms underlying the sensing of DNA have been less well defined. It has been known for over a decade that DNA, the most recognizable unit of life, is a potent trigger of inflammatory responses in cells. The discovery of TLR-9, a receptor for hypomethylated CpG-rich DNA, partially explained these findings [3]. TLR9 is localized to the endosomal compartment and in humans is expressed in B cells as well as in plasmacytoid dendritic cells (pDCs). However, it became clear that the immune stimulatory activity of microbial DNA was not compromised in many cells lacking TLR9 [4]. These observations prompted new efforts to understand how DNA triggers immune responses, an endeavor that has led to the discovery of several new DNA recognition receptors and fresh insights into infectious as well as autoimmune diseases.

Suppression of systemic autoimmunity by the innate immune adaptor STING

Significance Systemic lupus erythematosus (SLE) is a chronic systemic autoimmune disease that presents with a diverse array of clinical symptoms and afflicts over 1.5 million Americans. Current treatments involve immunosuppressive regimens associated with debilitating and adverse effects. With the description of a role for innate signaling in SLE, safe and efficient therapies that block Toll-like receptors also have been stymied by the relative short in vivo half lives of known inhibitors and the dangerous outcome of complete MyD88 blockade. Key natural regulators of the disease process are not well described but are more likely to provide disease-specific therapeutics with fewer adverse effects. In this study, we have identified a novel function for Stimulator of interferon genes as a suppressor of disease and a target for future SLE therapeutics. Cytosolic DNA-sensing pathways that signal via Stimulator of interferon genes (STING) mediate immunity to pathogens and also promote autoimmune pathology in DNaseII- and DNaseIII-deficient mice. In contrast, we report here that STING potently suppresses inflammation in a model of systemic lupus erythematosus (SLE). Lymphoid hypertrophy, autoantibody production, serum cytokine levels, and other indicators of immune activation were markedly increased in STING-deficient autoimmune-prone mice compared with STING-sufficient littermates. As a result, STING-deficient autoimmune-prone mice had significantly shorter lifespans than controls. Importantly, Toll-like receptor (TLR)-dependent systemic inflammation during 2,6,10,14-tetramethylpentadecane (TMPD)-mediated peritonitis was similarly aggravated in STING-deficient mice. Mechanistically, STING-deficient macrophages failed to express negative regulators of immune activation and thus were hyperresponsive to TLR ligands, producing abnormally high levels of proinflammatory cytokines. This hyperreactivity corresponds to dramatically elevated numbers of inflammatory macrophages and granulocytes in vivo. Collectively these findings reveal an unexpected negative regulatory role for STING, having important implications for STING-directed therapies.

Interferon γ-inducible Protein (IFI) 16 Transcriptionally Regulates Type I Interferons and Other Interferon-stimulated Genes and Controls the Interferon Response to both DNA and RNA Viruses

Background: IFI16 binds dsDNA and elicits a type I interferon response. Results: IFI16 knockdown cells show a decrease in interferon production and ISG expression in response to DNA and RNA ligands and cyclic dinucleotides. Conclusion: IFI16 transcriptionally regulates ISGs to enhance IFN responses to multiple IFN-inducing ligands. Significance: IFI16 has a broader role in the regulation of ISG expression. The interferon γ-inducible protein 16 (IFI16) has recently been linked to the detection of nuclear and cytosolic DNA during infection with herpes simplex virus-1 and HIV. IFI16 binds dsDNA via HIN200 domains and activates stimulator of interferon genes (STING), leading to TANK (TRAF family member-associated NF-κB activator)-binding kinase-1 (TBK1)-dependent phosphorylation of interferon regulatory factor (IRF) 3 and transcription of type I interferons (IFNs) and related genes. To better understand the role of IFI16 in coordinating type I IFN gene regulation, we generated cell lines with stable knockdown of IFI16 and examined responses to DNA and RNA viruses as well as cyclic dinucleotides. As expected, stable knockdown of IFI16 led to a severely attenuated type I IFN response to DNA ligands and viruses. In contrast, expression of the NF-κB-regulated cytokines IL-6 and IL-1β was unaffected in IFI16 knockdown cells, suggesting that the role of IFI16 in sensing these triggers was unique to the type I IFN pathway. Surprisingly, we also found that knockdown of IFI16 led to a severe attenuation of IFN-α and the IFN-stimulated gene retinoic acid-inducible gene I (RIG-I) in response to cyclic GMP-AMP, a second messenger produced by cyclic GMP-AMP synthase (cGAS) as well as RNA ligands and viruses. Analysis of IFI16 knockdown cells revealed compromised occupancy of RNA polymerase II on the IFN-α promoter in these cells, suggesting that transcription of IFN-stimulated genes is dependent on IFI16. These results indicate a broader role for IFI16 in the regulation of the type I IFN response to RNA and DNA viruses in antiviral immunity.

Cutting Edge: AIM2 and Endosomal TLRs Differentially Regulate Arthritis and Autoantibody Production in DNase II–Deficient Mice

Innate immune pattern recognition receptors sense nucleic acids from microbes and orchestrate cytokine production to resolve infection. Inappropriate recognition of host nucleic acids also results in autoimmune disease. In this study, we use a model of inflammation resulting from accrual of self DNA (DNase II−/− type I IFN receptor [Ifnar]−/−) to understand the role of pattern recognition receptor–sensing pathways in arthritis and autoantibody production. Using triple knockout (TKO) mice deficient in DNase II/IFNaR together with deficiency in either stimulator of IFN genes (STING) or absent in melanoma 2 (AIM2), we reveal central roles for the STING and AIM2 pathways in arthritis. AIM2 TKO mice show limited inflammasome activation and, similar to STING TKO mice, have reduced inflammation in joints. Surprisingly, autoantibody production is maintained in AIM2 and STING TKO mice, whereas DNase II−/− Ifnar−/− mice also deficient in Unc93b, a chaperone required for TLR7/9 endosomal localization, fail to produce autoantibodies to nucleic acids. Collectively, these data support distinct roles for cytosolic and endosomal nucleic acid–sensing pathways in disease manifestations.

RNA and β-Hemolysin of Group B Streptococcus Induce Interleukin-1β (IL-1β) by Activating NLRP3 Inflammasomes in Mouse Macrophages

Background: Group B Streptococcus (GBS) activates the NLRP3 inflammasome. Results: GBS RNA escapes the phagolysosome and activates the NLRP3 inflammasome in a β-hemolysin-dependent fashion. RNA-NLRP3 interaction and activation are enhanced by lysosomal leakage. Conclusion: RNA activates the NLRP3 inflammasome in synergy with phagolysosomal proteins. Significance: The NLRP3 inflammasome responds to bacterial invasion via RNA recognition subsequent to phagolysosomal degradation. The inflammatory cytokine IL-1β is critical for host responses against many human pathogens. Here, we define Group B Streptococcus (GBS)-mediated activation of the Nod-like receptor-P3 (NLRP3) inflammasome in macrophages. NLRP3 activation requires GBS expression of the cytolytic toxin, β-hemolysin, lysosomal acidification, and leakage. These processes allow the interaction of GBS RNA with cytosolic NLRP3. The present study supports a model in which GBS RNA, along with lysosomal components including cathepsins, leaks out of lysosomes and interacts with NLRP3 to induce IL-1β production.

Viral Defense: It Takes Two MAVS to Tango

To defend cells against viruses, the MAVS (mitochondrial antiviral signaling) adaptor protein initiates an antiviral signaling cascade from mitochondrial membranes. In this issue, Dixit et al. (2010) show that MAVS also localizes to the membranes of peroxisomes, where it rapidly induces expression of a subset of antiviral genes that curb viral replication until mitochondrial MAVS can induce a sustained antiviral response.

Cutting Edge: Novel Tmem173 Allele Reveals Importance of STING N Terminus in Trafficking and Type I IFN Production.

With the stimulator of IFN genes (STING) C terminus being extensively studied, the role of the N-terminal domain (NTD) of STING remains an important subject of investigation. In this article, we identify novel mutations in NTD of Sting of the MOLF strain in response to HSV and Listeria monocytogenes both in vitro and in vivo. These mutations are responsible for low levels of IFN-β caused by failure of MOLF STING to translocate from the endoplasmic reticulum. These data provide evidence that the NTD of STING affects DNA responses via control of trafficking. They also show that the genetic diversity of wild-derived mice resembles the diversity observed in humans. Several human alleles of STING confer attenuated IFN-I production similar to what we observe with the MOLF Sting allele, a crucial functional difference not apparent in classical inbred mice. Thus, understanding the functional significance of polymorphisms in MOLF STING can provide basic mechanistic insights relevant to humans.

Catenin' on to nucleic acid sensing

Many pathogens induce a type I interferon response via a pathway dependent on the kinase TBK1 and transcription factor IRF3. However, LRRFIP1, a cytosolic sensor of DNA and RNA, triggers interferon production by a β-catenin-dependent signal.