Innate Immune Response to Cytoplasmic DNA: Mechanisms and Diseases.

DNA has been known to be a potent immune stimulus for more than half a century. However, the underlying molecular mechanisms of DNA-triggered immune response have remained elusive until recent years. Cyclic GMP-AMP synthase (cGAS) is a major cytoplasmic DNA sensor in various types of cells that detect either invaded foreign DNA or aberrantly located self-DNA. Upon sensing of DNA, cGAS catalyzes the formation of cyclic GMP-AMP (cGAMP), which in turn activates the ER-localized adaptor protein MITA (also named STING) to elicit the innate immune response. The cGAS-MITA axis not only plays a central role in host defense against pathogen-derived DNA but also acts as a cellular stress response pathway by sensing aberrantly located self-DNA, which is linked to the pathogenesis of various human diseases. In this review, we summarize the spatial and temporal mechanisms of host defense to cytoplasmic DNA mediated by the cGAS-MITA axis and discuss the association of malfunctions of this axis with autoimmune and other diseases.

Intracellular Nucleic Acid Detection in Autoimmunity.

Protective immune responses to viral infection are initiated by innate immune sensors that survey extracellular and intracellular space for foreign nucleic acids. The existence of these sensors raises fundamental questions about self/nonself discrimination because of the abundance of self-DNA and self-RNA that occupy these same compartments. Recent advances have revealed that enzymes that metabolize or modify endogenous nucleic acids are essential for preventing inappropriate activation of the innate antiviral response. In this review, we discuss rare human diseases caused by dysregulated nucleic acid sensing, focusing primarily on intracellular sensors of nucleic acids. We summarize lessons learned from these disorders, we rationalize the existence of these diseases in the context of evolution, and we propose that this framework may also apply to a number of more common autoimmune diseases for which the underlying genetics and mechanisms are not yet fully understood.

Serotonin reduction in post-acute sequelae of viral infection.

Post-acute sequelae of COVID-19 (PASC, "Long COVID") pose a significant global health challenge. The pathophysiology is unknown, and no effective treatments have been found to date. Several hypotheses have been formulated to explain the etiology of PASC, including viral persistence, chronic inflammation, hypercoagulability, and autonomic dysfunction. Here, we propose a mechanism that links all four hypotheses in a single pathway and provides actionable insights for therapeutic interventions. We find that PASC are associated with serotonin reduction. Viral infection and type I interferon-driven inflammation reduce serotonin through three mechanisms: diminished intestinal absorption of the serotonin precursor tryptophan; platelet hyperactivation and thrombocytopenia, which impacts serotonin storage; and enhanced MAO-mediated serotonin turnover. Peripheral serotonin reduction, in turn, impedes the activity of the vagus nerve and thereby impairs hippocampal responses and memory. These findings provide a possible explanation for neurocognitive symptoms associated with viral persistence in Long COVID, which may extend to other post-viral syndromes.

Early cellular mechanisms of type I interferon-driven susceptibility to tuberculosis.

Mycobacterium tuberculosis (Mtb) causes 1.6 million deaths annually. Active tuberculosis correlates with a neutrophil-driven type I interferon (IFN) signature, but the cellular mechanisms underlying tuberculosis pathogenesis remain poorly understood. We found that interstitial macrophages (IMs) and plasmacytoid dendritic cells (pDCs) are dominant producers of type I IFN during Mtb infection in mice and non-human primates, and pDCs localize near human Mtb granulomas. Depletion of pDCs reduces Mtb burdens, implicating pDCs in tuberculosis pathogenesis. During IFN-driven disease, we observe abundant DNA-containing neutrophil extracellular traps (NETs) described to activate pDCs. Cell-type-specific disruption of the type I IFN receptor suggests that IFNs act on IMs to inhibit Mtb control. Single-cell RNA sequencing (scRNA-seq) indicates that type I IFN-responsive cells are defective in their response to IFNγ, a cytokine critical for Mtb control. We propose that pDC-derived type I IFNs act on IMs to permit bacterial replication, driving further neutrophil recruitment and active tuberculosis disease.

A tissue injury sensing and repair pathway distinct from host pathogen defense.

Pathogen infection and tissue injury are universal insults that disrupt homeostasis. Innate immunity senses microbial infections and induces cytokines/chemokines to activate resistance mechanisms. Here, we show that, in contrast to most pathogen-induced cytokines, interleukin-24 (IL-24) is predominately induced by barrier epithelial progenitors after tissue injury and is independent of microbiome or adaptive immunity. Moreover, Il24 ablation in mice impedes not only epidermal proliferation and re-epithelialization but also capillary and fibroblast regeneration within the dermal wound bed. Conversely, ectopic IL-24 induction in the homeostatic epidermis triggers global epithelial-mesenchymal tissue repair responses. Mechanistically, Il24 expression depends upon both epithelial IL24-receptor/STAT3 signaling and hypoxia-stabilized HIF1α, which converge following injury to trigger autocrine and paracrine signaling involving IL-24-mediated receptor signaling and metabolic regulation. Thus, parallel to innate immune sensing of pathogens to resolve infections, epithelial stem cells sense injury signals to orchestrate IL-24-mediated tissue repair.

Innate immune recognition of cancer.

The observation that a subset of cancer patients show evidence for spontaneous CD8+ T cell priming against tumor-associated antigens has generated renewed interest in the innate immune pathways that might serve as a bridge to an adaptive immune response to tumors. Manipulation of this endogenous T cell response with therapeutic intent-for example, using blocking antibodies inhibiting PD-1/PD-L1 (programmed death-1/programmed death ligand 1) interactions-is showing impressive clinical results. As such, understanding the innate immune mechanisms that enable this T cell response has important clinical relevance. Defined innate immune interactions in the cancer context include recognition by innate cell populations (NK cells, NKT cells, and γδ T cells) and also by dendritic cells and macrophages in response to damage-associated molecular patterns (DAMPs). Recent evidence has indicated that the major DAMP driving host antitumor immune responses is tumor-derived DNA, sensed by the stimulator of interferon gene (STING) pathway and driving type I IFN production. A deeper knowledge of the clinically relevant innate immune pathways involved in the recognition of tumors is leading toward new therapeutic strategies for cancer treatment.

A family of conserved bacterial virulence factors dampens interferon responses by blocking calcium signaling.

Interferons (IFNs) induce an antimicrobial state, protecting tissues from infection. Many viruses inhibit IFN signaling, but whether bacterial pathogens evade IFN responses remains unclear. Here, we demonstrate that the Shigella OspC family of type-III-secreted effectors blocks IFN signaling independently of its cell death inhibitory activity. Rather, IFN inhibition was mediated by the binding of OspC1 and OspC3 to the Ca2+ sensor calmodulin (CaM), blocking CaM kinase II and downstream JAK/STAT signaling. The growth of Shigella lacking OspC1 and OspC3 was attenuated in epithelial cells and in a murine model of infection. This phenotype was rescued in both models by the depletion of IFN receptors. OspC homologs conserved in additional pathogens not only bound CaM but also inhibited IFN, suggesting a widespread virulence strategy. These findings reveal a conserved but previously undescribed molecular mechanism of IFN inhibition and demonstrate the critical role of Ca2+ and IFN targeting in bacterial pathogenesis.

Microbiota-Induced Type I Interferons Instruct a Poised Basal State of Dendritic Cells.

Environmental signals shape host physiology and fitness. Microbiota-derived cues are required to program conventional dendritic cells (cDCs) during the steady state so that they can promptly respond and initiate adaptive immune responses when encountering pathogens. However, the molecular underpinnings of microbiota-guided instructive programs are not well understood. Here, we report that the indigenous microbiota controls constitutive production of type I interferons (IFN-I) by plasmacytoid DCs. Using genome-wide analysis of transcriptional and epigenetic regulomes of cDCs from germ-free and IFN-I receptor (IFNAR)-deficient mice, we found that tonic IFNAR signaling instructs a specific epigenomic and metabolic basal state that poises cDCs for future pathogen combat. However, such beneficial biological function comes with a trade-off. Instructed cDCs can prime T cell responses against harmless peripheral antigens when removing roadblocks of peripheral tolerance. Our data provide fresh insights into the evolutionary trade-offs that come with successful adaptation of vertebrates to their microbial environment.

New frontiers in the cGAS-STING intracellular DNA-sensing pathway.

The cGAS-STING intracellular DNA-sensing pathway has emerged as a key element of innate antiviral immunity and a promising therapeutic target. The existence of an innate immune sensor that can be activated by any double-stranded DNA (dsDNA) of any origin raises fundamental questions about how cGAS is regulated and how it responds to "foreign" DNA while maintaining tolerance to ubiquitous self-DNA. In this review, we summarize recent evidence implicating important roles for cGAS in the detection of foreign and self-DNA. We describe two recent and surprising insights into cGAS-STING biology: that cGAS is tightly tethered to the nucleosome and that the cGAMP product of cGAS is an immunotransmitter acting at a distance to control innate immunity. We consider how these advances influence our understanding of the emerging roles of cGAS in the DNA damage response (DDR), senescence, aging, and cancer biology. Finally, we describe emerging approaches to harness cGAS-STING biology for therapeutic benefit.

Self-Recognition of an Inducible Host lncRNA by RIG-I Feedback Restricts Innate Immune Response.

The innate RNA sensor RIG-I is critical in the initiation of antiviral type I interferons (IFNs) production upon recognition of "non-self" viral RNAs. Here, we identify a host-derived, IFN-inducible long noncoding RNA, lnc-Lsm3b, that can compete with viral RNAs in the binding of RIG-I monomers and feedback inactivate the RIG-I innate function at late stage of innate response. Mechanistically, binding of lnc-Lsm3b restricts RIG-I protein's conformational shift and prevents downstream signaling, thereby terminating type I IFNs production. Multivalent structural motifs and long-stem structure are critical features of lnc-Lsm3b for RIG-I binding and inhibition. These data reveal a non-canonical self-recognition mode in the regulation of immune response and demonstrate an important role of an inducible "self" lncRNA acting as a potent molecular decoy actively saturating RIG-I binding sites to restrict the duration of "non-self" RNA-induced innate immune response and maintaining immune homeostasis, with potential utility in inflammatory disease management.

ABCC1 transporter exports the immunostimulatory cyclic dinucleotide cGAMP.

The DNA sensor cyclic GMP-AMP synthase (cGAS) is important for antiviral and anti-tumor immunity. cGAS generates cyclic GMP-AMP (cGAMP), a diffusible cyclic dinucleotide that activates the antiviral response through the adaptor protein stimulator of interferon genes (STING). cGAMP cannot passively cross cell membranes, but recent advances have established a role for extracellular cGAMP as an "immunotransmitter" that can be imported into cells. However, the mechanism by which cGAMP exits cells remains unknown. Here, we identifed ABCC1 as a direct, ATP-dependent cGAMP exporter in mouse and human cells. We show that ABCC1 overexpression enhanced cGAMP export and limited STING signaling and that loss of ABCC1 reduced cGAMP export and potentiated STING signaling. We demonstrate that ABCC1 deficiency exacerbated cGAS-dependent autoimmunity in the Trex1-/- mouse model of Aicardi-Goutières syndrome. Thus, ABCC1-mediated cGAMP export is a key regulatory mechanism that limits cell-intrinsic activation of STING and ameliorates STING-dependent autoimmune disease.

The gut microbiota prime systemic antiviral immunity via the cGAS-STING-IFN-I axis.

The microbiota are vital for immune homeostasis and provide a competitive barrier to bacterial and fungal pathogens. Here, we investigated how gut commensals modulate systemic immunity and response to viral infection. Antibiotic suppression of the gut microbiota reduced systemic tonic type I interferon (IFN-I) and antiviral priming. The microbiota-driven tonic IFN-I-response was dependent on cGAS-STING but not on TLR signaling or direct host-bacteria interactions. Instead, membrane vesicles (MVs) from extracellular bacteria activated the cGAS-STING-IFN-I axis by delivering bacterial DNA into distal host cells. DNA-containing MVs from the gut microbiota were found in circulation and promoted the clearance of both DNA (herpes simplex virus type 1) and RNA (vesicular stomatitis virus) viruses in a cGAS-dependent manner. In summary, this study establishes an important role for the microbiota in peripheral cGAS-STING activation, which promotes host resistance to systemic viral infections. Moreover, it uncovers an underappreciated risk of antibiotic use during viral infections.

IRGB10 Liberates Bacterial Ligands for Sensing by the AIM2 and Caspase-11-NLRP3 Inflammasomes.

The inflammasome is an intracellular signaling complex, which on recognition of pathogens and physiological aberration, drives activation of caspase-1, pyroptosis, and the release of the pro-inflammatory cytokines IL-1ß and IL-18. Bacterial ligands must secure entry into the cytoplasm to activate inflammasomes; however, the mechanisms by which concealed ligands are liberated in the cytoplasm have remained unclear. Here, we showed that the interferon-inducible protein IRGB10 is essential for activation of the DNA-sensing AIM2 inflammasome by Francisella novicida and contributed to the activation of the LPS-sensing caspase-11 and NLRP3 inflammasome by Gram-negative bacteria. IRGB10 directly targeted cytoplasmic bacteria through a mechanism requiring guanylate-binding proteins. Localization of IRGB10 to the bacterial cell membrane compromised bacterial structural integrity and mediated cytosolic release of ligands for recognition by inflammasome sensors. Overall, our results reveal IRGB10 as part of a conserved signaling hub at the interface between cell-autonomous immunity and innate immune sensing pathways.

Vaginal Exposure to Zika Virus during Pregnancy Leads to Fetal Brain Infection.

Zika virus (ZIKV) can be transmitted sexually between humans. However, it is unknown whether ZIKV replicates in the vagina and impacts the unborn fetus. Here, we establish a mouse model of vaginal ZIKV infection and demonstrate that, unlike other routes, ZIKV replicates within the genital mucosa even in wild-type (WT) mice. Mice lacking RNA sensors or transcription factors IRF3 and IRF7 resulted in higher levels of local viral replication. Furthermore, mice lacking the type I interferon (IFN) receptor (IFNAR) became viremic and died of infection after a high-dose vaginal ZIKV challenge. Notably, vaginal infection of pregnant dams during early pregnancy led to fetal growth restriction and infection of the fetal brain in WT mice. This was exacerbated in mice deficient in IFN pathways, leading to abortion. Our study highlights the vaginal tract as a highly susceptible site of ZIKV replication and illustrates the dire disease consequences during pregnancy.

Protein kinase R and the integrated stress response drive immunopathology caused by mutations in the RNA deaminase ADAR1.

The RNA deaminase ADAR1 is an essential negative regulator of the RNA sensor MDA5, and loss of ADAR1 function triggers inappropriate activation of MDA5 by self-RNAs. Mutations in ADAR, the gene that encodes ADAR1, cause human immune diseases, including Aicardi-Goutières syndrome (AGS). However, the mechanisms of MDA5-dependent disease pathogenesis in vivo remain unknown. Here we generated mice with a single amino acid change in ADAR1 that models the most common human ADAR AGS mutation. These Adar mutant mice developed lethal disease that required MDA5, the RIG-I-like receptor LGP2, type I interferons, and the eIF2α kinase PKR. A small-molecule inhibitor of the integrated stress response (ISR) that acts downstream of eIF2α phosphorylation prevented immunopathology and rescued the mice from mortality. These findings place PKR and the ISR as central components of immunopathology in vivo and identify therapeutic targets for treatment of human diseases associated with the ADAR1-MDA5 axis.

A PGE2-MEF2A axis enables context-dependent control of inflammatory gene expression.

Tight control of inflammatory gene expression by antagonistic environmental cues is key to ensure immune protection while preventing tissue damage. Prostaglandin E2 (PGE2) modulates macrophage activation during homeostasis and disease, but the underlying mechanisms remain incompletely characterized. Here we dissected the genomic properties of lipopolysaccharide (LPS)-induced genes whose expression is antagonized by PGE2. The latter molecule targeted a set of inflammatory gene enhancers that, already in unstimulated macrophages, displayed poorly permissive chromatin organization and were marked by the transcription factor myocyte enhancer factor 2A (MEF2A). Deletion of MEF2A phenocopied PGE2 treatment and abolished type I interferon (IFN I) induction upon exposure to innate immune stimuli. Mechanistically, PGE2 interfered with LPS-mediated activation of ERK5, a known transcriptional partner of MEF2. This study highlights principles of plasticity and adaptation in cells exposed to a complex environment and uncovers a transcriptional circuit for IFN I induction with relevance for infectious diseases or cancer.

Decreased Expression of the Host Long-Noncoding RNA-GM Facilitates Viral Escape by Inhibiting the Kinase activity TBK1 via S-glutathionylation.

Viruses have evolved multiple strategies to evade elimination by the immune system. Here we examined the contribution of host long noncoding RNAs (lncRNAs) in viral immune evasion. By functional screening of lncRNAs whose expression decreased upon viral infection of macrophages, we identified a lncRNA (lncRNA-GM, Gene Symbol: AK189470.1) that promoted type I interferon (IFN-I) production and inhibited viral replication. Deficiency of lncRNA-GM in mice increased susceptibility to viral infection and impaired IFN-I production. Mechanistically, lncRNA-GM bound to glutathione S-transferase M1 (GSTM1) and blocked GSTM1 interaction with the kinase TBK1, reducing GSTM1-mediated S-glutathionylation of TBK1. Decreased S-glutathionylation enhanced TBK1 activity and downstream production of antiviral mediators. Viral infection reprogrammed intracellular glutathione metabolism and furthermore, an oxidized glutathione mimetic could inhibit TBK1 activity and promote viral replication. Our findings reveal regulation of TBK1 by S-glutathionylation and provide insight into the viral mediated metabolic changes that impact innate immunity and viral evasion.

microRNA-induced translational control of antiviral immunity by the cap-binding protein 4EHP.

Type I interferons (IFNs) are critical cytokines in the host defense against invading pathogens. Sustained production of IFNs, however, is detrimental to the host, as it provokes autoimmune diseases. Thus, the expression of IFNs is tightly controlled. We report that the mRNA 5' cap-binding protein 4EHP plays a key role in regulating type I IFN concomitant with controlling virus replication, both in vitro and in vivo. Mechanistically, 4EHP suppresses IFN-ß production by effecting the miR-34a-induced translational silencing of Ifnb1 mRNA. miR-34a is upregulated by both RNA virus infection and IFN-ß induction, prompting a negative feedback regulatory mechanism that represses IFN-ß expression via 4EHP. These findings demonstrate the direct involvement of 4EHP in virus-induced host response, underscoring a critical translational silencing mechanism mediated by 4EHP and miR-34a to impede sustained IFN production. This study highlights an intrinsic regulatory function for miRNA and the translation machinery in maintaining host homeostasis.

Dynamic control of gene expression by ISGF3 and IRF1 during IFNß and IFNγ signaling.

Type I interferons (IFN-I, including IFNß) and IFNγ produce overlapping, yet clearly distinct immunological activities. Recent data show that the distinctness of global transcriptional responses to the two IFN types is not apparent when comparing their immediate effects. By analyzing nascent transcripts induced by IFN-I or IFNγ over a period of 48 h, we now show that the distinctiveness of the transcriptomes emerges over time and is based on differential employment of the ISGF3 complex as well as of the second-tier transcription factor IRF1. The distinct transcriptional properties of ISGF3 and IRF1 correspond with a largely diverse nuclear protein interactome. Mechanistically, we describe the specific input of ISGF3 and IRF1 into enhancer activation and the regulation of chromatin accessibility at interferon-stimulated genes (ISG). We further report differences between the IFN types in altering RNA polymerase II pausing at ISG 5' ends. Our data provide insight how transcriptional regulators create immunological identities of IFN-I and IFNγ.

Differential Activation of the Transcription Factor IRF1 Underlies the Distinct Immune Responses Elicited by Type I and Type III Interferons.

Type I and III interferons (IFNs) activate similar downstream signaling cascades, but unlike type I IFNs, type III IFNs (IFNλ) do not elicit strong inflammatory responses in vivo. Here, we examined the molecular mechanisms underlying this disparity. Type I and III IFNs displayed kinetic differences in expression of IFN-stimulated genes and proinflammatory responses, with type I IFNs preferentially stimulating expression of the transcription factor IRF1. Type III IFNs failed to induce IRF1 expression because of low IFNλ receptor abundance and insufficient STAT1 activation on epithelial cells and thus did not activate the IRF1 proinflammatory gene program. Rather, IFNλ stimulation preferentially induced factors implicated in tissue repair. Our findings suggest that IFN receptor compartmentalization and abundance confer a spatiotemporal division of labor where type III IFNs control viral spread at the site of the infection while restricting tissue damage; the transient induction of inflammatory responses by type I IFNs recruits immune effectors to promote protective immunity.