The innate immune system detects infection by employing germline-encoded receptors specific for conserved microbial molecules. Recognition of microbial ligands leads to the production of cytokines, such as type I interferons (IFN), that are essential for successful pathogen elimination. Cytosolic detection of pathogen-derived DNA is one major mechanism of IFN induction1,2, and requires signaling via Tank Binding Kinase 1 (TBK1), and its downstream transcription factor, Interferon Regulatory Factor 3 (IRF3). In addition, a transmembrane protein called STING (STimulator of INterferon Genes; also called MITA, ERIS, MPYS, TMEM173) functions as an essential signaling adaptor linking cytosolic detection of DNA to the TBK1/IRF3 signaling axis3–7. Recently, unique nucleic acids called cyclic dinucleotides, which function as conserved signaling molecules in bacteria8, were also shown to induce a STING-dependent type I interferon response9–12. However, a mammalian sensor of cyclic dinucleotides has not been identified. Here we report evidence that STING itself is an innate immune sensor of cyclic dinucleotides. We demonstrate that STING binds directly to radiolabelled cyclic diguanylate monophosphate (c-di-GMP) and that this binding is competed by unlabelled cyclic dinucleotides but not by other nucleotides or nucleic acids. Furthermore, we identify mutations in STING that selectively affect the response to cyclic dinucleotides without affecting the response to DNA. Thus, STING appears to function as a direct sensor of cyclic dinucleotides, in addition to its established role as a signaling adaptor in the interferon response to cytosolic DNA. Cyclic dinucleotides have shown promise as novel vaccine adjuvants and immunotherapeutics9,13. Our results provide insight into the mechanism by which cyclic dinucleotides are sensed by the innate immune system.

The pathway causing CD4 T-cell death in HIV-infected hosts remains poorly understood. Apoptosis has been proposed as the key mechanism for CD4 T-cell loss. We now show that caspase-3-mediated apoptosis accounts for the death of only a small fraction of productively infected cells. The remaining >95% of quiescent lymphoid CD4 T-cells die by caspase-1-mediated pyroptosis triggered by abortive viral infection. Pyroptosis corresponds to an intensely inflammatory form of programmed cell death where cytoplasmic contents and pro-inflammatory cytokines including IL-1β, are released. This death pathway thus links the two signature events in HIV infection––CD4 T-cell depletion and chronic inflammation––and creates a vicious pathogenic cycle where dying CD4 T-cells release inflammatory signals that attract more cells to die. This cycle can be broken by caspase-1 inhibitors shown to be safe in humans, raising the possibility of a new class of “anti-AIDS” therapeutics targeting the host rather than the virus.

SUMMARY The presence of foreign DNA in the cytosol of mammalian cells elicits a potent antiviral interferon response. Recently, cytosolic DNA was proposed to induce the synthesis of cyclic-GMP-AMP (cGAMP) upon binding to an enzyme called cGAMP synthase (cGAS). cGAMP activates an interferon response by binding to a downstream receptor called STING. Here we identify natural variants of human STING that are poorly responsive to cGAMP, yet unexpectedly, are normally responsive to DNA and cGAS signaling. We explain this paradox by demonstrating that the cGAS product is actually a non-canonical cyclic-di-nucleotide, cyclic[G(2′ -5′)pA(3′ -5′)p], which contains a single 2′ -5′ phosphodiester bond. Cyclic[G(2′ -5′)pA(3′ -5′)p] potently activates diverse human STING receptors and may therefore be a useful adjuvant or immunotherapeutic. Our results indicate that human STING variants have evolved that can distinguish conventional (3′ -5′) cyclic-di-nucleotides, known only to be produced by bacteria, from the non-canonical cyclic-di-nucleotide produced by mammalian cGAS.

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The progressive depletion of quiescent “bystander” CD4 T-cells, which are non-permissive to HIV infection, is a principal driver of the acquired immunodeficiency syndrome (AIDS). These cells undergo abortive infection characterized by the cytosolic accumulation of incomplete HIV reverse transcripts. These viral DNAs are sensed by an unidentified host sensor that triggers an innate immune response, leading to caspase-1 activation and pyroptosis. Using unbiased proteomic and targeted biochemical approaches as well as two independent methods of lentiviral shRNA-mediated gene knockdown in primary CD4 T-cells, we identify Interferon gamma Inducible protein 16 (IFI16) as a host DNA sensor required for CD4 T-cell death due to abortive HIV infection. These findings provide insights into a key host pathway that plays a central role in CD4 T-cell depletion during disease progression to AIDS.

SUMMARY The nucleotide-binding domain and leucine-rich repeat containing (NLR) proteins regulate innate immunity. Although the positive regulatory impact of NLRs is clear, their inhibitory roles are not well defined. We showed Nlrx1−/− mice exhibited increased expression of antiviral signaling molecules IFN-β, STAT2, OAS1 and IL-6 after influenza virus infection. Consistent with increased inflammation, Nlrx1−/− mice exhibited marked morbidity and histopathology. Infection of these mice with an influenza strain that carries a mutated NS-1 protein, which normally prevents IFN induction by interaction with RNA and the intracellular RNA sensor RIG-I, further exacerbated IL-6 and type I IFN signaling. NLRX1 also weakened cytokine responses to the 2009 H1N1 pandemic influenza virus in human cells. Mechanistically, Nlrx1 deletion led to constitutive interaction of MAVS and RIG-I. Additionally, an inhibitory function is identified for NLRX1 during LPS-activation of macrophages where the MAVS-RIG-I pathway was not involved. NLRX1 interacts with TRAF6 and inhibits NF-κB activation. Thus, NLRX1 functions as a checkpoint of overzealous inflammation.

Highlights d Upon demyelination, Trem2 -/microglia fail to upregulate lipid metabolism genes d Trem2 -/microglia accumulate cholesteryl ester derived from myelin cholesterol d Accumulation is rescued by ACAT1 inhibitor and LXR agonist d Apoe -/glia defective in cholesterol transport also accumulate cholesteryl ester

The innate immune system responds to unique molecular signatures that are widely conserved among microbes but that are not normally present in host cells. Compounds that stimulate innate immune pathways may be valuable in the design of novel adjuvants, vaccines, and other immunotherapeutics. The cyclic dinucleotide cyclic-di–guanosine monophosphate (c-di-GMP) is a recently appreciated second messenger that plays critical regulatory roles in many species of bacteria but is not produced by eukaryotic cells. In vivo and in vitro studies have previously suggested that c-di-GMP is a potent immunostimulatory compound recognized by mouse and human cells. We provide evidence that c-di-GMP is sensed in the cytosol of mammalian cells via a novel immunosurveillance pathway. The potency of cytosolic signaling induced by c-di-GMP is comparable to that induced by cytosolic delivery of DNA, and both nucleic acids induce a similar transcriptional profile, including triggering of type I interferons and coregulated genes via induction of TBK1, IRF3, nuclear factor κB, and MAP kinases. However, the cytosolic pathway that senses c-di-GMP appears to be distinct from all known nucleic acid–sensing pathways. Our results suggest a novel mechanism by which host cells can induce an inflammatory response to a widely produced bacterial ligand.