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The cGAS/STING sensor system drives innate immune responses to intracellular microbial double-stranded DNA (dsDNA) and bacterial cyclic nucleotide second messengers (e.g., c-di-AMP). STING-dependent cell-intrinsic responses can increase resistance to microbial infection and speed pathogen clearance. Correspondingly, STING activation and signaling are known to be targeted for suppression by effectors from several bacterial pathogens. Whether STING responses are also positively regulated through sensing of specific bacterial effectors is less clear. We find that STING activation through dsDNA, by its canonical ligand 2'-3' cGAMP, or the small molecule DMXAA is potentiated following intracellular delivery of the AB 5 toxin family member pertussis toxin from Bordetella pertussis or the B subunit of cholera toxin from Vibrio cholerae . Entry of pertussis toxin or cholera toxin B into mouse macrophages triggers markers of endoplasmic reticulum (ER) stress and enhances ligand-dependent STING responses at the level of STING receptor activation in a manner that is independent of toxin enzymatic activity. Our results provide an example in which STING responses integrate information about the presence of relevant ER-transiting bacterial toxins into the innate inflammatory response and may help to explain the in vivo adjuvant effects of catalytically inactive toxins., Competing Interests: The authors declare no conflict of interest.

The unique cell biology of Toll-like receptor 4 (TLR4) allows it to initiate two signal-transduction cascades: a signal dependent on the adaptors TIRAP (Mal) and MyD88 that begins at the cell surface and regulates proinflammatory cytokines, and a signal dependent on the adaptors TRAM and TRIF that begins in the endosomes and drives the production of type I interferons. Negative feedback circuits to limit TLR4 signals from both locations are necessary to balance the inflammatory response. We describe a negative feedback loop driven by autocrine-paracrine prostaglandin E 2 (PGE 2 ) and the PGE 2 receptor EP4 that restricted TRIF-dependent signals and the induction of interferon-β through the regulation of TLR4 trafficking. Inhibition of PGE 2 production or antagonism of EP4 increased the rate at which TLR4 translocated to endosomes and amplified TRIF-dependent activation of the transcription factor IRF3 and caspase-8. This PGE 2 -driven mechanism restricted TLR4-TRIF signaling in vitro after infection of macrophages by the Gram-negative pathogens Escherichia coli or Citrobacter rodentium and protected mice against mortality induced by Salmonella enteritidis serovar Typhimurium. Thus, PGE 2 restricted TLR4-TRIF signaling specifically in response to lipopolysaccharide.

The host protein Stimulator of Interferon Genes (STING) has been shown to be essential for recognition of both viral and intracellular bacterial pathogens, but its regulation remains unclear. Previously, we reported that mitochondrial membrane potential regulates STING-dependent IFN-β induction independently of ATP synthesis. Because mitochondrial membrane potential controls calcium homeostasis, and AMP-activated protein kinase (AMPK) is regulated, in part, by intracellular calcium, we postulated that AMPK participates in STING activation; however, its role has yet to be been defined. Addition of an intracellular calcium chelator or an AMPK inhibitor to either mouse macrophages or mouse embryonic fibroblasts (MEFs) suppressed IFN-β and TNF-α induction following stimulation with the STING-dependent ligand 5,6-dimethyl xanthnone-4-acetic acid (DMXAA). These pharmacological findings were corroborated by showing that MEFs lacking AMPK activity also failed to up-regulate IFN-β and TNF-α after treatment with DMXAA or the natural STING ligand cyclic GMP-AMP (cGAMP). As a result, AMPK-deficient MEFs exhibit impaired control of vesicular stomatitis virus (VSV), a virus sensed by STING that can cause an influenza-like illness in humans. This impairment could be overcome by pretreatment of AMPK-deficient MEFs with type I IFN, illustrating that de novo production of IFN-β in response to VSV plays a key role in antiviral defense during infection. Loss of AMPK also led to dephosphorylation at Ser-555 of the known STING regulator, UNC-51-like kinase 1 (ULK1). However, ULK1-deficient cells responded normally to DMXAA, indicating that AMPK promotes STING-dependent signaling independent of ULK1 in mouse cells., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Toll-like receptors (TLRs) are major receptors of the host innate immune system that recognize conserved pathogen-associated molecular patterns (PAMPs) of invading microbes. Activation of TLR signaling culminates in the expression of multiple genes in a coordinate and kinetically defined manner. In this review, we summarize the current studies describing the chromatin landscape of TLR-responsive inflammatory genes and how changes to this chromatin landscape govern cell type-specific and temporal gene expression. We further elaborate classical endotoxin tolerance and epigenetic mechanisms controlling tolerance and interferon priming effects on inflammatory promoters.

Innate immune inflammatory responses are subject to complex layers of negative regulation at intestinal mucosal surfaces. Although the type I IFN system is critical for amplifying antiviral immunity, it has been shown to play a homeostatic role in some models of autoimmune inflammation. Type I IFN is triggered in the gut by select bacterial pathogens, but whether and how the type I IFN might regulate innate immunity in the intestinal environment have not been investigated in the context of Salmonella enterica serovar Typhimurium (ST). ST infection of human or murine macrophages reveals that IFN-β selectively restricts the transcriptional responses mediated by both the TLRs and the NOD-like receptors. Specifically, IFN-β potently represses ST-dependent innate induction of IL-1 family cytokines and neutrophil chemokines. This IFN-β-mediated transcriptional repression was independent of the effects of IFN-β on ST-induced macrophage cell death, but significantly dependent on IL-10 regulation. We further evaluated ST pathogenesis in vivo following oral inoculation of mice lacking IFN-β. We show that IFN-β(-/-) mice exhibit greater resistance to oral ST infection and a slower spread of ST to distal sterile sites. This work provides mechanistic insight into the relationship between ST and type I IFN, and demonstrates an additional mechanism by which IFN-β may promote spread of enteric pathogens., (Copyright © 2015 by The American Association of Immunologists, Inc.)

The Toll like receptors (TLRs) and the type I interferons have critical roles to play in innate immunity. In this review we will discuss new developments relating to the important area of TLR/IFN cross regulation., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The cell surface/endosomal Toll-like Receptors (TLRs) are instrumental in initiating immune responses to both bacteria and viruses. With the exception of TLR2, all TLRs and cytosolic RIG-I-like receptors (RLRs) with known virus-derived ligands induce type I interferons (IFNs) in macrophages or dendritic cells. Herein, we report that prior ligation of TLR2, an event previously shown to induce "homo" or "hetero" tolerance, strongly "primes" macrophages for increased Type I IFN production in response to subsequent TLR/RLR signaling. This occurs by increasing activation of the transcription factor, IFN Regulatory Factor-3 (IRF-3) that, in turn, leads to enhanced induction of IFN-β, while expression of other pro-inflammatory genes are suppressed (tolerized). In vitro or in vivo "priming" of murine macrophages with TLR2 ligands increase virus-mediated IFN induction and resistance to infection. This priming effect of TLR2 is mediated by the selective upregulation of the K63 ubiquitin ligase, TRAF3. Thus, we provide a mechanistic explanation for the observed antiviral actions of MyD88-dependent TLR2 and further define the role of TRAF3 in viral innate immunity.

The chemotherapeutic agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA) is a potent inducer of type I IFNs and other cytokines. This ability is essential for its chemotherapeutic benefit in a mouse cancer model and suggests that it might also be useful as an antiviral agent. However, the mechanism underlying DMXAA-induced type I IFNs, including the host proteins involved, remains unclear. Recently, it was reported that the antioxidant N-acetylcysteine (NAC) decreased DMXAA-induced TNF-α and IL-6, suggesting that oxidative stress may play a role. The goal of this study was to identify host proteins involved in DMXAA-dependent signaling and determine how antioxidants modulate this response. We found that expression of IFN-β in response to DMXAA in mouse macrophages requires the mitochondrial and endoplasmic reticulum resident protein STING. Addition of the antioxidant diphenylene iodonium (DPI) diminished DMXAA-induced IFN-β, but this decrease was independent of both the NADPH oxidase, Nox2, and de novo generation of reactive oxygen species. Additionally, IFN-β up-regulation by DMXAA was inhibited by agents that target the mitochondrial electron transport chain and, conversely, loss of mitochondrial membrane potential correlated with diminished innate immune signaling in response to DMXAA. Up-regulation of Ifnb1 gene expression mediated by cyclic dinucleotides was also impaired by DPI, whereas up-regulation of Ifnb1 mRNA due to cytosolic double-stranded DNA was not. Although both stimuli signal through STING, cyclic dinucleotides interact directly with STING, suggesting that recognition of DMXAA by STING may also be mediated by direct interaction.