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Cytosolic DNA sensors regulating type I interferon induction
Abstract
Type I interferon (IFN) induction is a crucial anti-pathogen response mediated by innate immune stimulation. Although it has been appreciated for some time that the presence of pathogen DNA within a cell leads to a type I IFN response, it is only in the past few years that some of the key signalling proteins and DNA sensors that regulate this response have been uncovered. Here, we review the nature of these DNA sensors, which include a new family of pattern recognition receptors termed the AIM2-like receptors, and consider the implications of their discovery for understanding emerging principles of innate immune DNA sensing. Furthermore, we discuss how their discovery provides a rationale as to why accumulation of self-DNA mediates IFN-dependent autoimmunity.
... To follow up on the finding of enhanced type I interferon and associated viral and viral defence signalling in Dex-iPS-Mg, we reasoned that one possible cause of this signal might be genomic self-DNA in the cytoplasm 58 (Keating et al. 2011). In this ectopic location, the self-DNA would be perceived as an invading virus and trigger an anti-viral defence and transcription of type I interferon 59 (Honda et al. 2006). ...
... To follow up on the finding of enhanced type I interferon and associated viral and viral defence signalling in Dex-iPS-Mg, we reasoned that one possible cause of this signal might be genomic self-DNA in the cytoplasm 58 (Keating et al. 2011). In this ectopic location, the self-DNA would be perceived as an invading virus and trigger an anti-viral defence and transcription of type I interferon 59 (Honda et al. 2006). ...
Early life stress, prenatal exposure to glucocorticoids (GCs), confers a higher risk of psychiatric and neurodevelopmental disorders in children. Increasingly, the importance of microglia in these disorders has been recognised. Studies on GCs exposure during microglial development have been limited, and there are few, if any, human studies. We established an in vitro model of ELS by continuous pre-expoure of human iPS-microglia to GCs during primitive haematopoiesis (the critical stage of iPS-microglial differentiation) and then examined how this exposure affected the microglial phenotype as they differentiated and matured to microglia. The iPS-microglia predominately expressed glucocorticoid receptors over mineralocorticoid receptors, and the GR-α splice variant. Chronic GCs exposure during primitive haematopoiesis was able to recapitulate in vivo ELS effects. Thus pre-exposure to prolonged GCs resulted in increased type I interferon signalling, the presence of Cyclic GMP-AMP synthase-positive (cGAS) micronuclei, and cellular senescence in the matured iPS-microglia. The findings from this in vitro ELS model have ramifications for the responses of microglia in the pathogenesis of GC-mediated ELS- associated disorders such as schizophrenia, attention-deficit hyperactivity disorder and autistic spectrum disorder.
Highlights
Human iPS-derived-microglia predominantly express glucocorticoid receptor NR3C1 compared with mineralocorticoid receptor NR3C2, and a predominant splice variant of the NR3C1 of GR-α.
GC expression shows a differentiation-linked increment from iPSC to iPS-microglia.
An early-life stress model was established by exposing iPSC to glucocorticoids during primitive haematopoiesis.
RNA-seq analysis revealed that this early glucocorticoid exposure led to enhanced type I interferon inducible gene expression in the subsequent iPS-microglia.
Furthermore, micronuclei formation and cellular senescence markers were upregulated in the iPSC-microglia, indicating genomic instability due to early chronic GC exposure.
These findings have ramifications for the microglial responses in ELS linked neurodevelopmental disorders such as schizophrenia, attention-deficit hyperactivity disorder and autistic spectrum disorder.
Graphical abstract
... However, using a range of knockout mice deficient in specific pathways of the innate system, we have previously shown that RNA-sensing pathways do not participate in the regulation of HBV replication in a mouse model; meanwhile, MyD88 is implicated in the HBV clearance . Since the cytosolic DNA sensing pathway was only first discovered recently, there are limited studies on the recognition of HBV DNA, particularly in the antigen-presenting cells (APCs) (Kato et al., 2011;Keating et al., 2011) Dendritic cells (DCs) and macrophages are among the APCs that process antigen material to activate the immune system's natural killer (NK) or T cells and B cells (Banchereau & Steinman, 1998). During viral or bacterial infection, identifying pathogen-associated molecular (PAMPs) by immature DCs causes their activation and subsequent maturation (Buchta & Bishop, 2014;Kumar et al., 2009). ...
... Unlike TLRs that have been well identified and characterized as pattern recognition receptors (PRRs) that respond to nucleic acid in the endosomes or extracellular, cytosolic PRRs which sense viral and host nucleic acids in the cytoplasm have only recently been discovered (Kato et al., 2011;Keating et al., 2011). Among recent works on the role of cytosol DNA sensing pathway during viral infection, Horan et al. (2013) reported that DNA viruses like the herpes simplex virus (HSV) capsid were ubiquitinated in the cytoplasm and destroyed by the proteasome, resulting in DNA release into the cytoplasm. ...
Hepatitis B virus (HBV) is the etiological agent that causes a self-limiting or chronic infection in the hepatocytes of about 250 million people worldwide. The role of adaptive immune system during HBV infection has been well studied. However, the innate immune system's responses against HBV during the early stage of infection largely remain unclear. In this study, we found that HBV genomic DNA or Salmon Sperm DNA (SSD) was able to induce the innate immune response in the macrophages cell line RAW264.7 but not the hepatocyte cell line, HepG2, indicating that hepatocytes may lack of a functional DNA-sensing pathway and hence are unable to respond to the presence of foreign DNA in the cytosol with type 1 IFN response. Thus, we hypothesized that non-parenchymal cells like the Antigen Presenting Cells (APC) might be crucial in triggering the initial immune response to suppress the virus replication and link the innate and adaptive responses. Using bone marrow-derived DCs (BMDC) as a model, this study demonstrated that HBV genomic DNA is able to induce cytokines like TNF-alpha, IL-6, and IL-12p40 secretion. We also examined the activation and maturation of BMDCs when exposed to the HBV genomic DNA intracellularly and extracellularly. A significant shift of CD86+ and CD40+ cell populations was observed during extracellular exposure of BMDC to Poly I:C and HBV genomic DNA, indicating that TLRs may be vital in the uptake of the extracellular viral DNA to activate the BMDCs. Moreover, transfection of intracellular nucleic acid stimuli, including HBV genomic DNA as well induced BMDCs maturation. Our findings highlight the critical function of DCs in antiviral response as a potential connection between the innate and adaptive immune systems during HBV pathogenesis. Nevertheless, further study is required to determine the role of cytosol DNA sensing pathway in DCs during HBV infection.
... A number of DNA and RNA sensors have been discovered in recent years [9,10]. They are widely expressed (i.e., also outside of the immune system) and they play key roles in the cell-intrinsic defense against viral infection, where they detect non-self (e.g., viral) DNA or RNA and relay this information to the innate and adaptive immune systems through the production of predominantly IFNβ (and less IFNα) and upregulating MHC and other immune recognition molecules. ...
... The L1 encoded proteins ORF1p/p40 and ORF2p/ p145 may play additional roles in SLE similar to the recently recognized role of L1 in cellular senescence and the associated production of IFNβ and age-related inflammation [23]. The mechanism for this involves the ORF2p protein, which is a reverse transcriptase that generates RNA:DNA hybrids and dsDNA that trigger the DNA sensor cGAS [10]. This same mechanism drives type I IFN production in certain forms of the interferonopathy known as Aicardi-Goutières syndrome [35], in which inhibitors of the reverse transcriptase reduced the interferon gene signature in a small clinical trial [36]. ...
Background
Most patients with systemic lupus erythematosus (SLE) have IgG autoantibodies against the RNA-binding p40 (ORF1p) protein encoded by the L1 retroelement. This study tested if these autoantibodies are also present in children with pediatric SLE (pSLE) and if the p40 protein itself could be detected in immune cells.
Methods
Autoantibodies in the plasma of pSLE patients ( n = 30), healthy children ( n = 37), and disease controls juvenile idiopathic arthritis (JIA) ( n = 32) and juvenile dermatomyositis (JDM) ( n = 60), were measured by ELISA. Expression of p40 in immune cells was assessed by flow cytometry. Markers of neutrophil activation and death were quantitated by ELISA.
Results
IgG and IgA autoantibodies reactive with p40 were detected in the pSLE patients, but were low in healthy controls and in JIA or JDM. pSLE patients with active disease (13 of them newly diagnosed) had higher titers than the same patients after effective therapy ( p = 0.0003). IgG titers correlated with SLEDAI ( r = 0.65, p = 0.0001), ESR ( r = 0.43, p = 0.02), and anti-dsDNA antibodies ( r = 0.49, p < 0.03), and inversely with complement C3 ( r = -0.55, p = 0.002) and C4 ( r = -0.51, p = 0.006). p40 protein was detected in a subpopulation of CD66b ⁺ granulocytes in pSLE, as well as in adult SLE patients. Myeloperoxidase and neutrophil elastase complexed with DNA and the neutrophil-derived S100A8/A9 were elevated in plasma from pSLE patients with active disease and correlated with anti-p40 autoantibodies and disease activity.
Conclusions
Children with active SLE have elevated IgG and IgA autoantibodies against L1 p40, and this protein can be detected in circulating granulocytes in both pediatric and adult SLE patients. P40 expression and autoantibody levels correlate with disease activity. Markers of neutrophil activation and death also correlate with these autoantibodies and with disease activity, suggesting that neutrophils express L1 and are a source of p40.
... RNA is the primary trigger of interferon production through binding to TLR7 and is of central importance in SLE. [17][18][19][20] Furthermore, a subset of female patients with SLE have been shown to have increased dosage of TLR7 through incomplete X chromosome inactivation. As such, these patients may be more sensitive to circulating extracellular RNA than women with a single dosage of TLR7, or male patients. ...
Background
Circulating, extracellular RNA is the primary trigger of type I interferon in systemic lupus erythematosus (SLE), and interferon is known to play a central pathogenic role in the disease. RSLV-132 is a catalytically active human RNase molecule fused to human IgG1 Fc designed to digest RNA and thereby decrease the chronic inflammation associated with SLE. The drug was evaluated in a cohort of patients with SLE with moderate-severe cutaneous disease activity and the presence of RNA immune complexes. The primary objective of the study was the assessment of the impact of 13 doses of 10 mg/kg RSLV-132 over 6 months on the mean Cutaneous Lupus Erythematosus Disease Area and Severity Index (CLASI) score.
Methods
Sixty-five patients meeting the entry criteria of a baseline CLASI score of 10 or greater and positivity of at least one of five autoantibodies to RNA-binding proteins (SM/RNP, SSA/Ro, SSB/La, Sm, RNP) were randomly assigned (2:1) to receive 13 doses of RSLV-132 10 mg/kg or placebo, respectively. Participants received study drug for 24 weeks on days 1, 8, 15, 29, 43, 57, 71, 85, 99, 113, 127, 141 and 155 with an end-of-treatment visit on day 169 and a follow-up visit at the end of the study on day 215. The primary objective was assessed on days 85 and 169. Secondary objectives included assessment of systemic disease activity using the Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2K), the British Isles Lupus Assessment Group 2004 Index and the Physician’s Global Assessment. Data from these instruments were used to calculate the SLE Responder Index 4 (SRI-4) and the British Isles Lupus Assessment Group-based Composite Lupus Assessment (BICLA) scores.
Results
The mean CLASI score change from baseline at day 169 was −5.7 (±7.0) in the placebo group and −6.2 (±8.5) in the RSLV-132 group. A subgroup of participants with moderate-severe systemic disease activity and high baseline SLEDAI scores (≥9) were analysed with respect to BICLA and SRI-4 responses. The RSLV-132 treated participants in the high SLEDAI subgroup had a greater percentage of BICLA responses (62% vs 44%) and SRI-4 responses (23% vs 11%) as compared with placebo. A second subgroup of participants with high baseline CLASI scores (≥21) were analysed with respect to BICLA and SRI-4 responses. The RSLV-132 treated participants in the high CLASI subgroup had a greater percentage of BICLA responses (28% vs 8%) and SRI-4 responses (39% vs 8%) as compared with placebo.
Conclusions
Six months of RSLV-132 therapy consisting of a weekly loading dose of RSLV-132 for 1 month, followed by 5 months of biweekly administrations did not significantly improve the mean CLASI score relative to placebo in this cohort of patients with SLE. The study entry criteria selected patients with moderate-severe cutaneous disease activity and no minimum SLEDAI score, which resulted in a wide range of systemic disease activity from inactive to severe as measured by SLEDAI. When the participants with higher SLEDAI and CLASI scores were analysed, a trend towards clinical improvement favouring RSLV-132 was observed. The results warrant further evaluation of RSLV-132 in SLE and suggest that patients with more active systemic disease are most likely to benefit from RNase therapy.
... Specifically, aside from cGAS, cytosolic DNA sensors such as absent in melanoma 2 (AIM2), DEAD box helicase 41 (DDX41), interferon gammainducible protein (IFI16), Z-DNA binding protein (ZBP1), RNA polymerase III, and LRR binding FLII interacting protein 1 (LRRFIP1) also elicit IFN-I response. 23 Moreover, DDX41, IFI16, and ZBP1 induce IFN-I responses in a cGAS-independent and STING-dependent manner. 24 To study the cGAS specificity of Svg3-induced IFN-I responses, first, by RNA sequencing (RNA-seq), we analyzed the transcriptomic changes in RAW 264.7 macrophages transfected with Svg3, with blank Lipofectamine 2000 as a control. ...
Activating cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) holds great potential for cancer immunotherapy by eliciting type-I interferon (IFN-I) responses. Yet, current approaches to cGAS-STING activation rely on STING agonists, which suffer from difficult formulation, poor pharmacokinetics, and marginal clinical therapeutic efficacy. Here, we report nature-inspired oligonucleotide, Svg3, as a cGAS agonist for cGAS-STING activation in tumor combination immunotherapy. The hairpin-shaped Svg3 strongly binds to cGAS and enhances phase separation to form Svg3-cGAS liquid-like droplets. This results in cGAS-specific immunoactivation and robust IFN-I responses. Remarkably, Svg3 outperforms several state-of-the-art STING agonists in murine and human cells/tissues. Nanoparticle-delivered Svg3 reduces tumor immunosuppression and potentiates immune checkpoint blockade therapeutic efficacy of multiple syngeneic tumor models in wild-type mice, but in neither cGas−/− nor Sting−/− mice. Overall, these results demonstrate the great potential of Svg3 as a cGAS agonistic oligonucleotide for cancer combination immunotherapy.
... We next studied the evolution of the innate immune proteins, beginning with cGAS and its broader family of CD-NTase enzymes. Following infections or cellular damage, cGAS binds cytosolic DNA and generates cyclic GMP-AMP (cGAMP) [33][34][35][36], which then activates downstream immune responses via STING [35,[37][38][39]. Another eukaryotic CD-NTase, 2 0 5 0 -Oligoadenylate Synthetase 1 (OAS1), synthesizes 2 0 ,5 0 -oligoadenylates that bind and activate Ribonuclease L (RNase L) [40]. ...
Animals use a variety of cell-autonomous innate immune proteins to detect viral infections and prevent replication. Recent studies have discovered that a subset of mammalian antiviral proteins have homology to antiphage defense proteins in bacteria, implying that there are aspects of innate immunity that are shared across the Tree of Life. While the majority of these studies have focused on characterizing the diversity and biochemical functions of the bacterial proteins, the evolutionary relationships between animal and bacterial proteins are less clear. This ambiguity is partly due to the long evolutionary distances separating animal and bacterial proteins, which obscures their relationships. Here, we tackle this problem for 3 innate immune families (CD-NTases [including cGAS], STINGs, and viperins) by deeply sampling protein diversity across eukaryotes. We find that viperins and OAS family CD-NTases are ancient immune proteins, likely inherited since the earliest eukaryotes first arose. In contrast, we find other immune proteins that were acquired via at least 4 independent events of horizontal gene transfer (HGT) from bacteria. Two of these events allowed algae to acquire new bacterial viperins, while 2 more HGT events gave rise to distinct superfamilies of eukaryotic CD-NTases: the cGLR superfamily (containing cGAS) that has diversified via a series of animal-specific duplications and a previously undefined eSMODS superfamily, which more closely resembles bacterial CD-NTases. Finally, we found that cGAS and STING proteins have substantially different histories, with STING protein domains undergoing convergent domain shuffling in bacteria and eukaryotes. Overall, our findings paint a picture of eukaryotic innate immunity as highly dynamic, where eukaryotes build upon their ancient antiviral repertoires through the reuse of protein domains and by repeatedly sampling a rich reservoir of bacterial antiphage genes.
... 4,5 The molecular mechanism of IFN-I induction is stimulated through several intracellular DNA, which is recognized by cytosolic DNA binding proteins such as cyclic GMP-AMP synthase (cGAS), DNA-dependent activator of IFN-regulatory factors (DAI), Interferon gamma inducible protein 16 (IFI16), Melanoma differentiation-associated gene 5 (MDA5), Retinoic acid-inducible gene I (RIG-I), and Toll-like receptor 9 (TLR9). [6][7][8] Upon activation, DNA sensors bind to their ligand leading to IFN-I expression. The secreted IFN-I signals through an IFN-I receptor (IFNAR) to stimulate the phosphorylation of JAK-STAT signaling and regulate IFN-γ induction and expression of many interferon-stimulated genes (ISGs), 9,10 such as MX Dynamin Like GTPase 1 (Mx1), C-X-C Motif Chemokine Ligand 10 (CXCL10) and Interferon-stimulated gene 15 (ISG15), which can fight against infection. ...
Background
Azithromycin (AZM) is a macrolide antibiotic that exhibits anti-inflammatory and anti-viral infection properties by enhancing type-I interferon (IFN-I) responses. The stimulator of interferon genes (STING) can directly induce IFN-I production. However, elevated IFN-I induces auto-immune phenotypes such as systemic lupus erythematosus (SLE). The effects of AZM and STING on the production of IFN-I are unclear.
Objective
Therefore, this study aims to evaluate the role of AZM and STING on IFN-I responses in macrophages.
Methods
RAW 264.7 macrophages were treated with AZM with and without a STING-agonist (DMXAA), and the maturation of macrophages was determined using flow cytometry. Gene expression and pro-inflammatory cytokines were analyzed using qPCR and ELISA, respectively. Moreover, protein expression was investigated using Western blot assays and immunofluorescence.
Results
Our results show that AZM significantly induced M1 phenotypes, promoting surface molecule expansion of CD80 and MHC-II and production of IL-6 and TNF-α cytokines on DMXAA-stimulated macrophages. Furthermore, we found that AZM-increased mRNA levels of interferon-stimulated genes (ISGs) could be due to the high expression of STNG-TBK1 signaling in the presence of DMXAA.
Conclusion
Our data suggest that AZM enhancement of IFN-I responses was STING dependent in DMXAA-stimulated macrophages. These data underline a novel approach to AZM action-mediated STING-TBK1 signaling for regulating IFN-I responses and may further augment the scientific basis and potential use of AZM in clinical applications.
... The result confirmed its increase in all groups receiving pIL-12 in combination with one of the plasmids or their combination. This was an expected result, as IFN-γ can be induced by transfected IL-12, as well as type I interferons [43], which are induced by the introduction of plasmid DNA that is recognized by endosomal or cytosolic DNA sensors [44,45]. Additionally, once antigenspecific immunity develops, IFN-γ is produced by antigen-stimulated lymphocytes [42]. ...
DNA vaccination is one of the emerging approaches for a wide range of applications, including prophylactic vaccination against infectious diseases and therapeutic vaccination against cancer. The aim of this study was to evaluate the feasibility of our previously optimized protocols for gene electrotransfer (GET)-mediated delivery of plasmid DNA into skin and muscle tissues on a model of COVID-19 vaccine. Plasmids encoding the SARS-CoV-2 proteins spike (S) and nucleocapsid (N) were used as the antigen source, and a plasmid encoding interleukin 12 (IL-12) was used as an adjuvant. Vaccination was performed in the skin or muscle tissue of C57BL/6J mice on days 0 and 14 (boost). Two weeks after the boost, blood, spleen, and transfected tissues were collected to determine the expression of S, N, IL-12, serum interferon-γ, the induction of antigen-specific IgG antibodies, and cytotoxic T-cells. In accordance with prior in vitro experiments that indicated problems with proper expression of the S protein, vaccination with S did not induce S-specific antibodies, whereas significant induction of N-specific antibodies was detected after vaccination with N. Intramuscular vaccination outperformed skin vaccination and resulted in significant induction of humoral and cell-mediated immunity. Moreover, both boost and adjuvant were found to be redundant for the induction of an immune response. Overall, the study confirmed the feasibility of the GET for DNA vaccination and provided valuable insights into this approach.
... A variety of cytosolic nucleic acid sensors can be activated to elicit IFN-I responses. Specifically, aside from cGAS, cytosolic DNA sensors such as absent in melanoma 2 (AIM2), DEAD box helicase 41 (DDX41), interferon gamma inducible protein (IFI16), Z-DNA binding protein (ZBP1), RNA polymerase III, and LRR binding FLII interacting protein 1 (LRRFIP1) also elicit IFN-I response [23] . Moreover, DDX41, IFI16, and ZBP1 induce IFN-I responses in a cGAS-independent and STING-dependent manner [24] . ...
Current cancer immunotherapy (e.g., immune checkpoint blockade (ICB)) has only benefited a small subset of patients. Cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) activation holds the potential to improve cancer immunotherapy by eliciting type-I interferon (IFN-I) responses in cancer cells and myeloid cells. Yet, current approaches to this end, mostly by targeting STING, have marginal clinical therapeutic efficacy. Here, we report a cGAS-specific agonistic oligonucleotide, Svg3, as a novel approach to cGAS-STING activation for versatile cancer immunotherapy. Featured with a hairpin structure with consecutive guanosines flanking the stem, Svg3 binds to cGAS and enhances cGAS-Svg3 phase separation to form liquid-like droplets. This results in cGAS activation by Svg3 for robust and dose-dependent IFN-I responses, which outperforms several state-of-the-art STING agonists in murine and human immune cells, and human tumor tissues. Nanocarriers efficiently delivers Svg3 to tissues, cells, and cytosol where cGAS is located. Svg3 reduces tumor immunosuppression and potentiates ICB therapeutic efficacy of multiple syngeneic tumors, in wildtype but neither cGas-/- nor goldenticket Sting-/- mice. Further, as an immunostimulant adjuvant, Svg3 enhances the immunogenicity of peptide antigens to elicit potent T cell responses for robust ICB combination immunotherapy of tumors. Overall, cGAS-agonistic Svg3 is promising for versatile cancer combination immunotherapy.
... Following infections or cellular damage, cGAS binds cytosolic DNA and generates cyclic GMP-AMP (cGAMP) [26][27][28][29], which then activates downstream immune responses via STING [28,[30][31][32]. OAS1 synthesizes 2',5'-oligoadenylates which bind and activate Ribonuclease L (RNase L) [33]. ...
Animals use a variety of cell-autonomous innate immune proteins to detect viral infections and prevent replication. Recent studies have discovered that a subset of mammalian antiviral proteins have homology to anti-phage defense proteins in bacteria, implying that there are aspects of innate immunity that are shared across the Tree of Life. While the majority of these studies have focused on characterizing the diversity and biochemical functions of the bacterial proteins, the evolutionary relationships between animal and bacterial proteins are less clear. This ambiguity is partly due to the long evolutionary distances separating animal and bacterial proteins, which obscures their relationships. Here, we tackle this problem for three innate immune families (CD-NTases [including cGAS], STINGs, and Viperins) by deeply sampling protein diversity across eukaryotes. We find that Viperins and OAS family CD-NTases are truly ancient immune proteins, likely inherited since the last eukaryotic common ancestor and possibly longer. In contrast, we find other immune proteins that arose via at least four independent events of horizontal gene transfer (HGT) from bacteria. Two of these events allowed algae to acquire new bacterial viperins, while two more HGT events gave rise to distinct superfamilies of eukaryotic CD-NTases: the Mab21 superfamily (containing cGAS) which has diversified via a series of animal-specific duplications, and a previously undefined eSMODS superfamily, which more closely resembles bacterial CD-NTases. Finally, we found that cGAS and STING proteins have substantially different histories, with STINGs arising via convergent domain shuffling in bacteria and eukaryotes. Overall, our findings paint a picture of eukaryotic innate immunity as highly dynamic, where eukaryotes build upon their ancient antiviral repertoires through the reuse of protein domains and by repeatedly sampling a rich reservoir of bacterial anti-phage genes.
... A key event in the innate immune response to viral infection is the recognition of cytoplasmic DNA by DNA sensors, which can induce the production of type I IFN, cytokines, and chemokines. The production of these factors is mediated through cascaded amplification reactions, thereby exerting antiviral innate immune functionality (30). DNAdependent activators of IFN-regulatory factors are the first DNA sensors thought to be required to respond to viral DNA (31,32). ...
Pseudorabies virus (PRV) is a double-stranded DNA virus that causes Aujeszky's disease and is responsible for economic loss worldwide. Transmembrane protein 41B (TMEM41B) is a novel endoplasmic reticulum (ER)-localized regulator of autophagosome biogenesis and lipid mobilization; however, the role of TMEM41B in regulating PRV replication remains undocumented. In this study, PRV infection was found to upregulate TMEM41B mRNA and protein levels both in vitro and in vivo. For the first time, we found that TMEM41B could be induced by interferon (IFN), suggesting that TMEM41B is an IFN-stimulated gene (ISG). While TMEM41B knockdown suppressed PRV proliferation, TMEM41B overexpression promoted PRV proliferation. We next studied the specific stages of the virus life cycle and found that TMEM41B knockdown affected PRV entry. Mechanistically, we demonstrated that the knockdown of TMEM41B blocked PRV-stimulated expression of the key enzymes involved in lipid synthesis. Additionally, TMEM41B knockdown played a role in the dynamics of lipid-regulated PRV entry-dependent clathrin-coated pits (CCPs). Lipid replenishment restored the CCP dynamic and PRV entry in TMEM41B knockdown cells. Together, our results indicate that TMEM41B plays a role in PRV infection via regulating lipid homeostasis. IMPORTANCE PRV belongs to the alphaherpesvirus subfamily and can establish and maintain a lifelong latent infection in pigs. As such, an intermittent active cycle presents great challenges to the prevention and control of PRV disease and is responsible for serious economic losses to the pig breeding industry. Studies have shown that lipids play a crucial role in PRV proliferation. Thus, the manipulation of lipid metabolism may represent a new perspective for the prevention and treatment of PRV. In this study, we report that the ER transmembrane protein TMEM41B is a novel ISG involved in PRV infection by regulating lipid synthesis. Therefore, our findings indicate that targeting TMEM41B may be a promising approach for the development of PRV vaccines and therapeutics.
... Zbp1 has been shown to play a role in innate immune response, DNA damage response, and apoptosis (Zheng and Kanneganti 2020;Nakad and Schumacher 2016). In particular, Zbp1 has been identified as a sensor of cytosolic DNA, and it can activate the STING-TBK1-IRF3 signaling pathway to induce the production of type I interferons (IFNs) and other cytokines (Keating et al. 2011;Kondo et al. 2013). Zbp1 has also been implicated in the regulation of cellular senescence, inflammation, and mitochondrial function (Barrera et al. 2021). ...
The Zbp1 gene has recently emerged as a potential therapeutic target for age-related diseases. Multiple studies have reported that Zbp1 plays a key role in regulating several aging hallmarks, including cellular senescence, chronic inflammation, DNA damage response, and mitochondrial dysfunction. Regarding cellular senescence, Zbp1 appears to regulate the onset and progression of senescence by controlling the expression of key markers such as p16INK4a and p21CIP1/WAF1. Similarly, evidence suggests that Zbp1 plays a role in regulating inflammation by promoting the production of pro-inflammatory cytokines, such as IL-6 and IL-1β, through activation of the NLRP3 inflammasome. Furthermore, Zbp1 seems to be involved in the DNA damage response, coordinating the cellular response to DNA damage by regulating the expression of genes such as p53 and ATM. Additionally, Zbp1 appears to regulate mitochondrial function, which is crucial for energy production and cellular homeostasis. Given the involvement of Zbp1 in multiple aging hallmarks, targeting this gene represents a potential strategy to prevent or treat age-related diseases. For example, inhibiting Zbp1 activity could be a promising approach to reduce cellular senescence and chronic inflammation, two critical hallmarks of aging associated with various age-related diseases. Similarly, modulating Zbp1 expression or activity could also improve DNA damage response and mitochondrial function, thus delaying or preventing the development of age-related diseases. Overall, the Zbp1 gene appears to be a promising therapeutic target for age-related diseases. In the current review, we have discussed the molecular mechanisms underlying the involvement of Zbp1 in aging hallmarks and proposed to develop effective strategies to target this gene for therapeutic purposes.
... Nevertheless, it seems that cell-intrinsic type I IFN likely contributed to the induction of IFN-response genes and that this cell-intrinsic IFN was dependent on ongoing reverse transcription. L1 ORF2p is also the reverse transcriptase responsible for the aberrant DNA [15,59] that drives type I IFN production and disease in patients with the interferonopathy Aicardi-Goutières syndrome [60]. In a small clinical trial in these patients, reverse transcriptase inhibition flat-lined the IFN-inducible gene signature for the duration of drug dosing [61]. ...
Abstract Background Patients with systemic lupus erythematosus (SLE) have autoantibodies against the L1-encoded open-reading frame 1 protein (ORF1p). Here, we report (i) which immune cells ORF1p emanates from, (ii) which L1 loci are transcriptionally active, (iii) whether the cells express L1-dependent interferon and interferon-stimulated genes, and (iv) the effect of inhibition of L1 ORF2p by reverse transcriptase inhibitors. Results L1 ORF1p was detected by flow cytometry primarily in SLE CD66b+CD15+ regular and low-density granulocytes, but much less in other immune cell lineages. The amount of ORF1p was higher in neutrophils from patients with SLE disease activity index (SLEDAI) > 6 (p = 0.011) compared to patients with inactive disease, SLEDAI
... Our study has several important limitations. While the use of HEK293T cells for characterization of FLAG-IFNLR1 isoforms was informed by prior published work [22,24], wild type HEK293T cells are largely unresponsive to IFNLs and are deficient in certain aspects of IFN signaling [49][50][51]. In addition, our cell model relies on protein overexpression, which results in supra-physiologic levels of receptor. ...
Interferon lambdas (IFNLs) are innate immune cytokines that induce antiviral cellular responses by signaling through a heterodimer composed of IL10RB and the interferon lambda receptor 1 (IFNLR1). Multiple IFNLR1 transcriptional variants are expressed in vivo and are predicted to encode distinct protein isoforms whose function is not fully established. IFNLR1 isoform 1 has the highest relative transcriptional expression and encodes the full-length functional form that supports canonical IFNL signaling. IFNLR1 isoforms 2 and 3 have lower relative expression and are predicted to encode signaling-defective proteins. To gain insight into IFNLR1 function and regulation, we explored how altering relative expression of IFNLR1 isoforms influenced the cellular response to IFNLs. To achieve this, we generated and functionally characterized stable HEK293T clones expressing doxycycline-inducible FLAG-tagged IFNLR1 isoforms. Minimal FLAG-IFNLR1 isoform 1 overexpression markedly increased IFNL3-dependent expression of antiviral and pro-inflammatory genes, a phenotype that could not be further augmented by expressing higher levels of FLAG-IFNLR1 isoform 1. Expression of low levels of FLAG-IFNLR1 isoform 2 led to partial induction of antiviral genes, but not pro-inflammatory genes, after IFNL3 treatment, a phenotype that was largely abrogated at higher FLAG-IFNLR1 isoform 2 expression levels. Expression of FLAG-IFNLR1 isoform 3 partially augmented antiviral gene expression after IFNL3 treatment. In addition, FLAG-IFNLR1 isoform 1 significantly reduced cellular sensitivity to the type-I IFN IFNA2 when overexpressed. These results identify a unique influence of canonical and non-canonical IFNLR1 isoforms on mediating the cellular response to interferons and provide insight into possible pathway regulation in vivo.
... RNA Pol III has been shown to recognize DNA from the Epstein-Barr virus (EBV) and herpes simplex virus-1 (HSV-1) (Fig. 1). 71,72 RNA Pol III converts dsDNA into 5′ppp RNA, which is detected by RIG-I, leading to the activation of inflammatory and IFN signaling pathways (Fig. 1). 72 DAI/ZBP1, on the other hand, is a dsDNA sensor that activates NF-κB and IRF via TBK1 and RIP1/3, respectively (Fig. 1). ...
Several studies over the last decade have identified intimate links between cellular metabolism and macrophage function. Metabolism has been shown to both drive and regulate macrophage function by producing bioenergetic and biosynthetic precursors as well as metabolites (and other bioactive molecules) that regulate gene expression and signal transduction. Many studies have focused on lipopolysaccharide-induced reprogramming, assuming that it is representative of most inflammatory responses. However, emerging evidence suggests that diverse pathogen-associated molecular patterns (PAMPs) are associated with unique metabolic profiles, which may drive pathogen specific immune responses. Further, these metabolic pathways and processes may act as a rheostat to regulate the magnitude of an inflammatory response based on the biochemical features of the local microenvironment. In this review, we will discuss recent work examining the relationship between cellular metabolism and macrophage responses to viral PAMPs and describe how these processes differ from lipopolysaccharide-associated responses. We will also discuss how an improved understanding of the specificity of these processes may offer new insights to fine-tune macrophage function during viral infections or when using viral PAMPs as therapeutics.
... To follow up on the finding of enhanced type I interferon and associated viral and viral defense signaling in Dex-iPS-Mg, we reasoned that one possible cause of this signal might be genomic self-DNA in the cytoplasm (Keating et al., 2011). In this ectopic location, the self-DNA would be perceived as an invading virus and trigger an anti-viral defense and transcription of type I interferon (Honda et al., 2006). ...
One form of early life stress, prenatal exposure to glucocorticoids (GCs), confers a higher risk of psychiatric and neurodevelopmental disorders in later life. Increasingly, the importance of microglia in these disorders is recognized. Studies on GCs exposure during microglial development have been limited, and there are few, if any, human studies. We established an in vitro model of ELS by continuous pre-exposure of human iPS-microglia to GCs during primitive hematopoiesis (the critical stage of iPS-microglial differentiation) and then examined how this exposure affected the microglial phenotype as they differentiated and matured to microglia, using RNA-seq analyses and functional assays. The iPS-microglia predominantly expressed glucocorticoid receptors over mineralocorticoid receptors, and in particular, the GR-α splice variant. Chronic GCs exposure during primitive hematopoiesis was able to recapitulate in vivo ELS effects. Thus, pre-exposure to prolonged GCs resulted in increased type I interferon signaling, the presence of Cyclic GMP-AMP synthase-positive (cGAS) micronuclei, cellular senescence and reduced proliferation in the matured iPS-microglia. The findings from this in vitro ELS model have ramifications for the responses of microglia in the pathogenesis of GC- mediated ELS-associated disorders such as schizophrenia, attention-deficit hyperactivity disorder and autism spectrum disorder.
... As expected, the introducing of Alu RNAs into the cytoplasm of ARPE-19 cells triggered the robust expression of IFN (Fig. 1a). The intracellular nucleic acidstimulated expression of IFN is dependent on the transcriptional factor, interferon regulatory factor 3 (IRF3) and the phosphorylation level of IRF3 can be used to reflect its activation [22][23][24]. We then detected the phosphorylation of IRF3 using immunoblotting with the specific antibodies against the phosphorylated IRF3. ...
Background
The degeneration of retinal pigmented epithelium (RPE) cells results in severe diseases, such as age-related macular degeneration (AMD) that causes blindness in millions of individuals.
Results
We report that targeting GMP-AMP (cGAMP) synthase (cGAS) alleviates Alu RNA-induced immune responses and cytotoxicity in RPE. We find that the deletion of cGAS in RPE inhibits the Alu RNA-stimulated interferon production. cGAS deficiency also protects RPE from cell death triggered by Alu RNA. Importantly, two natural chemicals, epigallocatechin gallate (EGCG) and resveratrol (RSVL), are effective in suppressing the immunogenic and cytotoxic effect of Alu RNA in RPE.
Conclusions
Our findings further demonstrate the crucial role of cGAS in the Alu RNA-induced RPE damage and present EGCG and RSVL as potential therapies for AMD and other RPE degeneration-related conditions.
... This dimerizes STING, which then recruits and activates TBK1 to phosphorylate IRF3, promoting IRF3 dimerization and nuclear translocation, thus inducing IRF3-mediated IFN transcription (31). In addition, AIM2 recognizes specific DNA sequences in a cell typedependent manner (32,33), while cGAS senses cytosolic DNA in a DNA sequence-independent but DNA length-dependent manner in most cell types. Regardless of different types of cytosolic DNA sensors, hyperactivation of cytosolic DNA sensing and signaling results in autoimmune disease (34), while suppression of cytosolic DNA sensing contributes to evasion of immune destruction during tumorigenesis as well as resistance to cancer immunotherapies (35). ...
The innate immune response is the first-line host defense against pathogens. Cytosolic nucleic acids, including both DNA and RNA, represent a special type of danger signal to initiate an innate immune response. Activation of cytosolic nucleic acid sensors is tightly controlled in order to achieve the high sensitivity needed to combat infection while simultaneously preventing false activation that leads to pathologic inflammatory diseases. In this review, we focus on post-translational modifications of key cytosolic nucleic acid sensors that can reversibly or irreversibly control these sensor functions. We will describe phosphorylation, ubiquitination, SUMOylation, neddylation, acetylation, methylation, succinylation, glutamylation, amidation, palmitoylation, and oxidation modifications events (including modified residues, modifying enzymes, and modification function). Together, these post-translational regulatory modifications on key cytosolic DNA/RNA sensing pathway members reveal a complicated yet elegantly controlled multilayer regulator network to govern innate immune activation.
... The fold expression of Ifn-β1 after pDNA electrotransfer to skin increased more than 2000-fold, implicating the activation of several DNA sensors [25,33,[63][64][65]. The mRNAs of the five DNA sensors upregulated in keratinocytes or fibroblasts, Ddx60, Zbp1, Aim2, Ifi202, and Ifi204, were upregulated in the skin. ...
Skin, the largest organ in the body, provides a passive physical barrier against infection and contains elements of the innate and adaptive immune systems. Skin consists of various cells, including keratinocytes, fibroblasts, endothelial cells and immune cells. This diversity of cell types could be important to gene therapies because DNA transfection could elicit different responses in different cell types. Previously, we observed the upregulation and activation of cytosolic DNA sensing pathways in several non-tumor and tumor cell types as well in tumors after the electroporation (electrotransfer) of plasmid DNA (pDNA). Based on this research and the innate immunogenicity of skin, we correlated the effects of pDNA electrotransfer to fibroblasts and keratinocytes to mouse skin using reverse transcription real-time PCR (RT-qPCR) and several types of protein quantification. After pDNA electrotransfer, the mRNAs of the putative DNA sensors DEAD (AspGlu-Ala-Asp) box polypeptide 60 (Ddx60), absent in melanoma 2 (Aim2), Z-DNA binding protein 1 (Zbp1), interferon activated gene 202 (Ifi202), and interferon-inducible protein 204 (Ifi204) were upregulated in keratinocytes, while Ddx60, Zbp1 and Ifi204 were upregulated in fibroblasts. Increased levels of the mRNAs and proteins of several cytokines and chemokines were detected and varied based on cell type. Mouse skin experiments in vivo confirmed our in vitro results with increased expression of putative DNA sensor mRNAs and of the mRNAs and proteins of several cytokines and chemokines. Finally, with immunofluorescent staining, we demonstrated that skin keratinocytes, fibroblasts and macrophages contribute to the immune response observed after pDNA electrotransfer.
... At present, four distinct PRR families are known, including NOD-like receptors (NLRs), C-type lectin receptors (CLRs), transmembrane proteins like Toll-like receptors (TLRs), and cytoplasmic proteins like RIG-I-like receptors (RLRs). 94,95 As the new cytoplasmic PRRs and cytosol DNA sensors, cGAS and AIM2 have an inter-regulatory relationship (Fig. 1) and have been extensively investigating recently as the important part of the inflammatory and immune systems. 18,96 It has previously been reported in many studies that the inflammasome activation and type-I IFN signal transduction balance each other during the inflammatory response. ...
Non-targeted effect is an important complement to the classical target theory of radiation biology which takes nuclear genomic DNA as the core target. The principle of radiation target theory is to assume that an organism or cell has one or more sensitive points or targets, hit and inactivation of which directly by radiation leads to considerable damage and the death event. Recent findings indicate that not only cell nucleus but also other cellular parts can be considered as possible targets. Mitochondrion is considered as a critical organelle where the non-targeted effect is initiated. A series of recent studies have provided substantial evidence and solid data which profoundly facilitate the understanding of radiation-induced non-targeted effects emitted from mitochondrion in the irradiated cells, such the major apparent performances, signaling pathways and biological significance. Mitochondrial genome is more sensitive to genotoxic than nuclear genome. Ionizing radiation can induce mtDNAs double-strand breaks directly or indirectly via increased mitochondrial ROS. Under stress conditions, mitochondrial DNA (mtDNA) fragments are released into the cytoplasm. The cytosol mtDNAs are sensed by cGAS and AIM2 proteins and they activate the corresponding signaling pathways, generating relevant inflammatory and immune responses. These newly developed mitochondrial DNA-initiating pathways may boost the development of targeted therapies for preventing normal tissue toxicity as well as radio-immunotherapy, an emerging trend for cancer therapies. Here we focus and discuss the mechanisms and biological significance of mtDNA-triggering cGAS/AIM2 signaling pathways of immune response from the aspect of non-targeted effect of radiation.
... Therefore, the role of PYHIN proteins in IRF7 regulation is likely to be complex and there may be fundamental differences in this regard in mouse versus human. In contrast to the five human PYHINs, the mouse locus encodes 13 PYHINs, with no direct MNDA ortholog (even though one of the mouse genes is named Mnda) 52,53 . ...
Type I interferons (IFNs) are critical for anti-viral responses, and also drive autoimmunity when dysregulated. Upon viral sensing, monocytes elicit a sequential cascade of IFNβ and IFNα production involving feedback amplification, but how exactly this cascade is regulated in human cells is incompletely understood. Here we show that the PYHIN protein myeloid cell nuclear differentiation antigen (MNDA) is required for IFNα induction in monocytes. Unlike other PYHINs, this is not due to a pathogen sensing role, but rather MNDA regulated expression of IRF7, a transcription factor essential for IFNα induction. Mechanistically, MNDA is required for recruitment of STAT2 and RNA polymerase II to the IRF7 gene promoter, and in fact MNDA is itself recruited to the IRF7 promoter after type I IFN stimulation. These data implicate MNDA as a critical regulator of the type I IFN cascade in human myeloid cells and reveal a new role for human PYHINs in innate immune gene induction.
... Parmi les mécanismes similaires, c'est-à-dire détectant l'ADN bactérien au niveau cytosolique et déclenchant la production des interférons de type I par l'activation de STING, on peut également citer le DAI (DNA-dependent activator of IFNregulatory factors), également connu sous le nom de ZBP-1 (Z-DNA binding protein) (Keating, et al. 2011). De même, la protéine IFI16 (interferon-inducible 16), dont l'équivalent est IFI204 chez la souris, est capable d'activer STING en réponse à certaines bactéries intracellulaires (Hansen, et al. 2014) et, de façon plus récemment démontrée, à des germes extracellulaires tels que Staphylococcus aureus (Chen, et al. 2019). ...
Le sepsis résulte d’une dysfonction de la réponse immunitaire de l’hôte en réponse à une infection, menant à une dysfonction endothéliale, qui peut aboutir à une coagulation intravasculaire disséminée (CIVD). Les mécanismes demeurant mal compris, nous avons étudié le rôle des interférons (IFNs) de type I et de leur voie de signalisation (STAT1) sur la dysfonction endothéliale au cours du sepsis. Nous avons montré que les souris présentant un déficit global en récepteur de l’IFN-α (Ifnar1-/-), ou en Stat1 ciblé sur l’endothélium, étaient protégées face au choc septique induit par ligature et perforation cӕcale (CLP), en lien avec une réduction des marqueurs d’inflammation, de dysfonction endothéliale, et de coagulopathie. Nous avons conforté ces résultats, chez l’Homme, en montrant une corrélation entre le taux plasmatique d’IFN-α d’une part, et la présence d’une CIVD et le niveau de microparticules endothéliales circulantes CD105+ d’autre part. Ainsi, notre travail confirme que l’inhibition de STAT1, ciblée sur l’endothélium, réduit la dysfonction endothéliale au cours du sepsis, ouvrant des perspectives thérapeutiques centrées sur l’endothélium.
... This, together with the high contagiousness and the short incubation times of FMD, makes the development of new antiviral strategies to induce fast protection crucial, especially during outbreak situations. The group of Dr. Grubman has demonstrated that the delivery of IFN genes by replication-defective adenovirus Type 5 (Ad5) induces fast protection against foot-and-mouth disease virus (FMDV) in pigs [13][14][15][16][17]. Certain DNA or RNA molecules are also being studied with this aim, because they can trigger the expression of IFNs in vivo after impacting on cytosolic nucleic acid sensors or endosomal toll-like receptors [3,[18][19][20][21]. However, the impact on several of these pattern recognition receptors depends, among other aspects, on the particular sequence [22], and the entry and intracellular trafficking of the molecules [19,[23][24][25]. ...
The huge variety of viruses affecting swine represents a global threat. Since vaccines against highly contagious viruses last several days to induce protective immune responses, antiviral strategies for rapid control of outbreak situations are needed. The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), an insect virus, has been demonstrated to be an effective vaccine vector for mammals. Besides the ability to display or transduce heterologous antigens, it also induces strong innate immune responses and provides IFN-mediated protection against lethal challenges with viruses like foot-and-mouth disease virus (FMDV) in mice. Thus, the aim of this study was to evaluate the ability of AcMNPV to induce IFN production and elicit antiviral activity in porcine peripheral blood mononuclear cells (PBMCs). Our results demonstrated that AcMNPV induced an IFN-α-mediated antiviral activity in PBMCs in vitro. Moreover, the inoculation of AcMNPV in piglets led to the production of type I and II IFNs in sera from inoculated animals and antiviral activities against vesicular stomatitis virus (VSV) and FMDV measured by in vitro assays. Finally, it was demonstrated that the pseudotyping of AcMNPV with VSV-G protein, but not the enrichment of the AcMNPV genome with specific immunostimulatory CpG motifs for the porcine TLR9, improved the ability to induce IFN-α production in PBMCs in vitro. Together, these results suggest that AcMNPV is a promising tool for the induction of IFNs in antiviral strategies, with the potential to be biotechnologically improved.
... nucleic acids: double-stranded (ds) or single-stranded (ss) DNA and RNA is essential to initiate innate immunity. PRR families include the retinoic acid-inducible gene I (RIG-I)-like receptors, toll-like receptors (TLRs), and a diverse member of cytosolic DNA sensors (Bowie and Haga, 2005;Kaisho and Akira, 2006;Yoneyama and Fujita, 2008;Beutler, 2009;Kawai and Akira, 2009;Yoneyama and Fujita, 2009;Barber, 2011;Keating et al., 2011;Thompson et al., 2011;Paludan and Bowie, 2013;Dempsey and Bowie, 2015). Once PAMPs are sensed by PRRs, the recognition subsequently mediates intracellular signaling pathways and activates transcription factors, interferon (IFN) regulatory factors (IRFs) or nuclear factor kB (NF-kB), which in turn leads to the increased production of antiviral interferons and proinflammatory cytokines (Lee and Kim, 2007;Mogensen, 2009). ...
Human Ku70 is a well-known endogenous nuclear protein involved in the non-homologous end joining pathway to repair double-stranded breaks in DNA. However, Ku70 has been studied in multiple contexts and grown into a multifunctional protein. In addition to the extensive functional study of Ku70 in DNA repair process, many studies have emphasized the role of Ku70 in various other cellular processes, including apoptosis, aging, and HIV replication. In this review, we focus on discussing the role of Ku70 in inducing interferons and proinflammatory cytokines as a cytosolic DNA sensor. We explored the unique structure of Ku70 binding with DNA; illustrated, with evidence, how Ku70, as a nuclear protein, responds to extracellular DNA stimulation; and summarized the mechanisms of the Ku70-involved innate immune response pathway. Finally, we discussed several new strategies to modulate Ku70-mediated innate immune response and highlighted some potential physiological insights based on the role of Ku70 in innate immunity.
... Innate immune systems respond by recognizing pathogens or dangers from invading host cells through pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) (Zhou et al., 2014). Once PAMPs and DAMPs are detected by several kinds of host pattern recognition receptors (PRRs), the immune system undergoes successive activation and response stages and ultimately returns to homeostasis (Keating et al., 2011). ...
Chicken has an impaired innate immune system compared with mammals. Some key innate immune genes, such as Retinoic acid-inducible gene I (RIG-I), Toll like receptor 8 (TLR8), Absent in melanoma 2 (AIM2) and IFN regulatory factor 3 (IRF3), are inactivated or missing due to DNA Insertion, gene partial deletion, or gene total deletion. A predicted N-terminal deleted chicken Cyclic GMP-AMP synthase (chcGAS) gene, which is proven as the most essential cytosolic DNA sensor in other species, be obtained from the GenBank database. The large fragment deletion makes the sequence accuracy and functional integrity of the predicted chcGAS open to dispute. Here, the exact chcGAS gene was first experimentally determined by 5' and 3' rapid amplification of cDNA ends (RACE) PCR, which specifically lacked 83 amino acids in the DNA binding domain. In addition, the conservation and feasibility of cGAS-STING signaling among different species were conducted by bioinformatics to explore the possibility of the existence of the conserved pathway in chickens. The basic characteristics of the chcGAS, such as macroscopic and microscopic distribution patterns of chcGAS have been studied. In order to better reseach the function of chGAS, a chcGAS knockout chicken cell line has been generated by CRISPR/CAS9. Together, chicken owns an N-terminal deleted cGAS gene, and more experimental evidences are urgently needed to verify the functional integrity of chcGAS.
... Whether an "antitumor" IFN response may be aimed toward eliminating (pre)malignant cells and the development of cutaneous squamous cell carcinoma or other cancers associated with HS is not fully understood (91). On the other hand, changes in the IFN gene signature may be secondary to dysregulated immune responses from ongoing cytotoxicity and the sensing of nucleic acid material from dying cells (92). pDCs are one of the most potent sources of type I IFNs and respond potently to alarmins, including danger-associated molecular patterns and pathogen-associated molecular patterns (80). ...
Hidradenitis suppurativa (HS) is a chronic inflammatory skin disease characterized by recurrent abscesses, nodules, and sinus tracts in areas of high hair follicle and sweat gland density. These sinus tracts can present with purulent drainage and scar formation. Dysregulation of multiple immune pathways drives the complexity of HS pathogenesis and may account for the heterogeneity of treatment response in HS patients. Using transcriptomic approaches, including single-cell sequencing and protein analysis, we here characterize the innate inflammatory landscape of HS lesions. We identified a shared upregulation of genes involved in interferon (IFN) and antimicrobial defense signaling through transcriptomic overlap analysis of differentially expressed genes (DEGs) in datasets from HS skin, diabetic foot ulcers (DFUs), and the inflammatory stage of normal healing wounds. Overlap analysis between HS- and DFU-specific DEGs revealed an enrichment of gene signatures associated with monocyte/macrophage functions. Single-cell RNA sequencing further revealed monocytes/macrophages with polarization toward a pro-inflammatory M1-like phenotype and increased effector function, including antiviral immunity, phagocytosis, respiratory burst, and antibody-dependent cellular cytotoxicity. Specifically, we identified the STAT1/IFN-signaling axis and the associated IFN-stimulated genes as central players in monocyte/macrophage dysregulation. Our data indicate that monocytes/macrophages are a potential pivotal player in HS pathogenesis and their pathways may serve as therapeutic targets and biomarkers in HS treatment.
... Like the escalated apoptosis of beta cells as well as aberrant apoptotic cell clearance causing a leakage of cellular content as well as exposure of autoantigens , cause acceleration of insulitis as well as autoimmunity.Further DNA collection from cellular apoptosis can result in exaggerated type 1 IFNs generation.Another e.g. of Pro-Inflammatory substances generated via leukocytes ,like IL-1β, TNF-α,IFNƴ, as well as other soluble mediators ,can stimulate beta cells apoptosis [20,33]. All the cytokines can activate cytosolic transduction pathways which control the pathways that control the apoptosis of influenced beta cells .Like IL-1β, as well as TNF-α work by activation of the NFκB(Nuclear factor -kappa B), as well as MAPK (Mitogen activated protein pathways) as well as IFNƴ, basically act through the JAK(Janus kinase)STAT(signal transducers as well as activators of transcription)pathway . ...
Type 1 Diabetes Mellitus(T1DM),an autoimmune disease has the property of selective damage of insulin-generating pancreatic beta cells-via autoreactive Tcells , total insulin deficiency followed by ultimate hyperglycemia.Genetic as well as environmental factors have significance in the pathogenesis of T1DM.Earlier we had reviewed the etiopathogenesis of T1DM by genetic along with environmental factors like viral factors, celiac disease , role of gut microbiome in genetically predisposed individuals.Although greater than fifty regions carrying genetic chances add to the etiology in addition to pathogenesis of Type 1 Diabetes Mellitus along with by utilization of GWAS(genome wide association studies), the exact underlying molecular modes are still not clear.Significantly, greater than 60% of the isolated candidate genes or genes linked to a specific chromosomal region with association of T1DM with expression inpancreatic islets as well as βcells,that Implies the possibility of such genes to be acting at the pancreatic βcells, hence having a crucial part in the pathogenesis of T1DM.Thus we conducted a Systematic review utilizing Pubmed Search Engine along with Google Scholar, Web Of Science,Cochrane Library , utilizing the MeSH terms ;T1DM;Candidate Genes;Role of interferon(IFN) signaling pathway ;mechanism of action of all genes;GWAS from 1990 till date in aug 2020. We got a total of 130 articles out of which we selected 85 articles for this review.No meta-analysis was conducted.Thus we have reviewed by concentrating on candidate genes, that get expressed as well as work at the βcells region by controlling the reactions of the innate immune system along with action against viruses , influencing the proneness to proapoptotic stimuli as well as affecting the pancreatic beta cell phenotype.
... The hSTING role as an adaptor protein is to recruit and activate TANK binding kinase (TBK1) (Ishikawa et al., 2009) and inhibitory kappa B kinases (IKK). Activation of hSTING triggers type I interferon response protecting against bacterial or viral infections and cancer (Cui et al., 2019;Feng et al., 2020;Fuertes et al., 2013;Keating et al., 2011;Zhong et al., 2008). On the other hand, hSTING over-activation can be associated with auto-immune and inflammatory disorders (Haag et al., 2018;Kumar, 2019). ...
Human stimulator of interferon genes (hSTING) is a signaling adaptor protein that triggers innate immune system by response to cytosolic DNA and second messenger cyclic dinucleotides (CDNs). Natural CDNs contain purine nucleobase with different phosphodiester linkage types (3′-3′, 2′-2′ or mixed 2′-3′-linkages) and exhibit different binding affinity towards hSTING, ranging from micromolar to nanomolar. High-affinity CDNs are considered as suitable candidates for treatment of chronic hepatitis B and cancer. We have used molecular dynamics simulations to investigate dynamical aspects of binding of natural CDNs (specifically, 2'-2'-cGAMP, 2'-3'-cGAMP, 3'-3'-cGAMP, 3'-3'-c-di-AMP, and 3'-3'-c-di-GMP) with hSTINGwt protein. Our results revealed that CDN/hSTINGwt interactions are controlled by the balance between fluctuations (conformational changes) in the CDN ligand and the protein dynamics. Binding of different CDNs induces different degrees of conformational/dynamics changes in hSTINGwt ligand binding cavity, especially in α1-helices, the so-called lid region and α2-tails. The ligand residence time in hSTINGwt protein pocket depends on different contribution of R232 and R238 residues interacting with oxygen atoms of phosphodiester groups in ligand, water distribution around interacting charged centers (in protein residues and ligand) and structural stability of closed conformation state of hSTINGwt protein. These findings may perhaps guide design of new compounds modulating hSTING activity.
... T he innate immune system depends on germline-encoded pattern recognition receptors to sense microbe-associated molecular patterns and initiate immune responses (1). Fish share most of the key antiviral pathways with humans and mice, which rely on a number of pattern recognition receptors, such as TLRs (2), retinoic acidinducible gene I (RIG-I)like receptors (RLRs) (3), and cytosolic DNA sensors (CDSs) (4). RLRs consist of RIG-I, melanoma differentiationassociated protein 5 (MDA5), and laboratory of genetics and physiology 2 (LGP2), which recognize viral dsRNA and activate IFN-b promoter stimulator-1 (IPS-1, also known as mitochondrial antiviral signaling protein [MAVS]) for downstream signal transmission (3). ...
IFN-β promoter stimulator-1 (IPS-1)- and stimulator of IFN genes (STING)-mediated type I IFNs play a critical role in antiviral responses. Myxovirus resistance (Mx) proteins are pivotal components of the antiviral effectors induced by IFNs in many species. An unprecedented expansion of Mx genes has occurred in fish. However, the functions and mechanisms of Mx family members remain largely unknown in fish. In this study, we found that grass carp (Ctenopharyngodon idella) MxG, a teleost-specific Mx protein, is induced by IFNs and viruses, and it negatively regulates both IPS-1- and STING-mediated antiviral responses to facilitate grass carp reovirus, spring viremia of carp virus, and cyprinid herpesvirus-2 replication. MxG binds and degrades IPS-1 via the proteasomal pathway and STING through the lysosomal pathway, thereby negatively regulating IFN1 antiviral responses and NF-κB proinflammatory cytokines. MxG also suppresses the phosphorylation of STING IFN regulatory factor 3/7, and it subsequently downregulates IFN1 and NF-κB1 at the promoter, transcription, and protein levels. GTPase and GTPase effector domains of MxG contribute to the negative regulatory function. On the contrary, MxG knockdown weakens virus replication and cytopathic effect. Therefore, MxG can be an ISG molecule induced by IFNs and viruses, and degrade IPS-1 and STING proteins in a negative feedback manner to maintain homeostasis and avoid excessive immune responses after virus infection. To our knowledge, this is the first identification of a negative regulator in the Mx family, and our findings clarify a novel mechanism by which the IFN response is regulated.
... Notably, distinct from other DNA sensors that stimulate interferon production, AIM2 activation in macrophages triggers the formation of a multiprotein complex named inflammasome, leading to activation of the protease procaspase 1 that cleaves pro-IL-1β and pro-IL-18 in triggering proptosis, 23 a process antagonized by p202. 24 These PRRs are celltype or DNA-sequence specific, 25,26 thus excluding their function as a universal cytosolic DNA sensor. In 2013, the cyclic GMP-AMP (cGAMP) synthase (cGAS) was identified as one of the most important cytosolic DNA sensors, 27 given that cGAS recognizes and responds to cytosolic DNA in a DNA-sequence-independent but DNA length-dependent manner in various cell types. ...
Sensing invasive cytosolic DNA is an integral component of innate immunity. cGAS was identified in 2013 as the major cytosolic DNA sensor that binds dsDNA to catalyze the synthesis of a special asymmetric cyclic-dinucleotide, 2′3′-cGAMP, as the secondary messenger to bind and activate STING for subsequent production of type I interferons and other immune-modulatory genes. Hyperactivation of cGAS signaling contributes to autoimmune diseases but serves as an adjuvant for anticancer immune therapy. On the other hand, inactivation of cGAS signaling causes deficiency to sense and clear the viral and bacterial infection and creates a tumor-prone immune microenvironment to facilitate tumor evasion of immune surveillance. Thus, cGAS activation is tightly controlled. In this review, we summarize up-to-date multilayers of regulatory mechanisms governing cGAS activation, including cGAS pre- and post-translational regulations, cGAS-binding proteins, and additional cGAS regulators such as ions and small molecules. We will also reveal the pathophysiological function of cGAS and its product cGAMP in human diseases. We hope to provide an up-to-date review for recent research advances of cGAS biology and cGAS-targeted therapies for human diseases.
... 20 Once inside the cell, exDNA is detected by TLR-9 (Toll-like receptor 9) located in the endosome membrane and is activated by unmethylated sequences. [49][50][51][52][53][54][55] On the other hand, the DNA bound to extracellular vesicles (EVs) is biologically functional and serves like an intercellular messenger, and can regulate the biological functions of the target cells by increasing DNA-coding mRNA and protein levels stimulating their proliferation and induce phenotype changes. 16,38 The EVs contain DNA fragments randomly selected from the entire genome spanning the 5ʹ promoter region, gene coding region, and 3ʹ untranslated region of chromatin-associated double-stranded DNA. ...
Nowadays, extracellular DNA or circulating cell-free DNA is considered to be a molecule with clinical applications (diagnosis, prognosis, monitoring of treatment responses, or patient follow-up) in diverse pathologies, especially in cancer. Nevertheless, because of its molecular characteristics, it can have many other functions. This review focuses on the participation of extracellular DNA (exDNA) in fundamental processes such as cell signaling, coagulation, immunity, evolution through horizontal transfer of genetic information, and adaptive response to inflammatory processes. A deeper understanding of its role in each of these processes will allow development of better tools to monitor and control pathologies, as well as helping to generate new therapeutic options, beyond the applicability of DNA in liquid biopsy.
... The results showed increased expression of Ifn-β1 and Tnf-α after GET of both therapeutic and empty control plasmid, which indicates on the activation of the DNA sensing pathways. This effect is mediated by the presence of plasmid DNA in the cytosol and the accumulation of DNA in cytosol after IR, which activates several DNA sensing pathways in immune, tumor and non-tumor cells [56][57][58][71][72][73]. Even though IR alone can trigger DNA sensing pathways by DNA accumulation [57], the increased expression of cytokines after IR was not observed in our current study, probably due to the too low dose of IR (i.e., 5 Gy). ...
Targeting tumor vasculature through specific endothelial cell markers represents a promising approach for cancer treatment. Here our aim was to construct an antibiotic resistance gene-free plasmid encoding shRNAs to simultaneously target two endothelial cell markers, CD105 and CD146, and to test its functionality and therapeutic potential in vitro when delivered by gene electrotransfer (GET) and combined with irradiation (IR). Functionality of the plasmid was evaluated by determining the silencing of the targeted genes using qRT-PCR. Antiproliferative and antiangiogenic effects were determined by the cytotoxicity assay tube formation assay and wound healing assay in murine endothelial cells 2H-11. The functionality of the plasmid construct was also evaluated in malignant melanoma tumor cell line B16F10. Additionally, potential activation of immune response was measured by induction of DNA sensor STING and proinflammatory cytokines by qRT-PCR in endothelial cells 2H-11. We demonstrated that the plasmid construction was successful and can efficiently silence the expression of the two targeted genes. As a consequence of silencing, reduced migration rate and angiogenic potential was confirmed in 2H-11 endothelial cells. Furthermore, induction of DNA sensor STING and proinflammatory cytokines were determined, which could add to the therapeutic effectiveness when used in vivo. To conclude, we successfully constructed a novel plasmid DNA with two shRNAs, which holds a great promise for further in vivo testing.
... V iruses infect all groups of living things and produce a variety of diseases. Host cell detection of viral infection depends on the recognition of pathogen-associated molecular patterns (PAMPs) from viral proteins or nucleic acids by the pattern recognition receptors (PRRs) such as Tolllike receptors (TLRs), RIG-I-like receptors (RLRs), NOD-like receptors (NLRs), and cytoplasmic DNA or RNA sensors [1][2][3][4][5][6] . The PRRs, once stimulated by their appropriate ligands, trigger distinct intracellular signaling pathways that converge on the activation of IFN regulatory factor 3 (IRF3) and/or IFN regulatory factor 7 (IRF7), which then translocate into the nucleus and activate the production of interferons (IFNs) that play important roles in inhibiting virus replication 7,8 . ...
Bone morphogenetic protein (BMP) is a kind of classical multi-functional growth factor that plays a vital role in the formation and maintenance of bone, cartilage, muscle, blood vessels, and the regulation of adipogenesis and thermogenesis. However, understanding of the role of BMPs in antiviral immunity is still limited. Here we demonstrate that Bmp8a is a newly-identified positive regulator for antiviral immune responses. The bmp8a−/− zebrafish, when infected with viruses, show reduced antiviral immunity and increased viral load and mortality. We also show for the first time that Bmp8a interacts with Alk6a, which promotes the phosphorylation of Tbk1 and Irf3 through p38 MAPK pathway, and induces the production of type I interferons (IFNs) in response to viral infection. Our study uncovers a previously unrecognized role of Bmp8a in regulation of antiviral immune responses and provides a target for controlling viral infection. Zhang, Liu and colleagues identify the role of Bmp8a in antiviral immunity in zebrafish and provide mechanistic insight into its function. Bmp8a could serve as a future target for investigative studies of antiviral immune responses.
... To counteract these ancient foes, evolution has produced several cellular mechanisms for the detection of nonself RNA and DNA (11). Five principal pathways operate in the cytosol and on the cytosolic surface of intracellular organelles: the DNA-sensor "cyclic GMP-AMP synthase" (cGAS) (101), the RNA sensors "retinoic acid-inducible gene I" (RIG-I) (102) and "melanoma differentiation-associated gene 5" (MDA5) (102)(103)(104), and the two kinases "protein kinase RNA-activated" (PKR) (105, 106) and DNA-activated protein kinase (DNA-PK). A sixth pathway responds to extracellular DNA or RNA brought into the cell by receptor-mediated endocytosis and is initiated by Toll-like receptors (TLRs) 3, 7, 8, and 9 in the endosome. ...
More than 200 human disorders include various manifestations of autoimmunity. The molecular events that lead to these diseases are still incompletely understood and their causes remain largely unknown. Numerous potential triggers of autoimmunity have been proposed over the years, but very few of them have been conclusively confirmed or firmly refuted. Viruses have topped the lists of suspects for decades, and it seems that many viruses, including those of the Herpesviridae family, indeed can influence disease initiation and/or promote exacerbations by a number of mechanisms that include prolonged anti-viral immunity, immune subverting factors, and mechanisms, and perhaps “molecular mimicry”. However, no specific virus has yet been established as being truly causative. Here, we discuss a different, but perhaps mechanistically related possibility, namely that retrotransposons or retroviruses that infected us in the past and left a lasting copy of themselves in our genome still can provoke an escalating immune response that leads to autoimmune disease. Many of these loci still encode for retroviral proteins that have retained some, or all, of their original functions. Importantly, these endogenous proviruses cannot be eliminated by the immune system the way it can eliminate exogenous viruses. Hence, if not properly controlled, they may drive a frustrated and escalating chronic, or episodic, immune response to the point of a frank autoimmune disorder. Here, we discuss the evidence and the proposed mechanisms, and assess the therapeutic options that emerge from the current understanding of this field.
Following entry into a host cell, the obligate intracellular pathogen, Chlamydia trachomatis , establishes an intracellular niche within a membrane derived vacuole called the chlamydial inclusion. The resulting inclusion membrane is modified by the pathogen and is a hybrid host-chlamydial structure. From within this intracellular niche, C. trachomatis must orchestrate numerous host-pathogen interactions to surreptitiously acquire nutrients from its host and to limit detection by the host innate immune system. C. trachomatis mediates many of these interactions with the host, in part, by using a family of type III secreted membrane proteins, termed inclusion membrane proteins (Incs). Incs are embedded within the inclusion membrane, and some function to recruit host proteins to the inclusion. Two such recruited host proteins are leucine r ich r epeat F lightless-1 interacting p rotein 1 (LRRF1/LRRFIP1) and its binding partner Flightless 1 (FLI1/FLII). LRRF1 interacts with Inc protein Ct226. However, interactions of FLI1 with candidate Incs or with LRRF1 during infection have not been defined. We hypothesized that FLI1 recruitment to the inclusion would be dependent on LRRF1 localization. To test this hypothesis, we used siRNA targeting lrrf1 or fli1, revealing that FLI1 can localize to the inclusion independently of LRRF1. Therefore, to further characterize FLI1 localization, we developed and characterized a series of CRISPRi knockdown and complementation strains in C. trachomatis serovar L2 that target ct226 and co-transcribed candidate Incs, ct225 and ct224 , to understand the mechanisms of FLI1 and LRRF1 localization to the inclusion. Our results indicate that FLI1 is recruited to the inclusion by multiple mechanisms.
IMPORTANCE
Chlamydia trachomatis is a leading cause of both preventable infectious blindness and bacterial sexually transmitted infections worldwide. Since C. trachomatis must grow and replicate within human host cells, it has evolved several ways of manipulating the host to establish a successful infection. As such, it is important to describe the interactions between host proteins and chlamydial proteins to understand which strategies C. trachomatis uses to shape its intracellular environment. This study looks in detail at such interactions of two host proteins, FLI1 and LRRF1, during chlamydial infection. Importantly, the series of knockdown and complement strains developed in this study suggest these proteins have both independent and overlapping mechanisms for localization, which ultimately will dictate how these proteins function during chlamydial infection.
PYHIN proteins AIM2 and IFI204 sense pathogen DNA, while other PYHINs have been shown to regulate host gene expression through as-yet unclear mechanisms. We characterize mouse PYHIN IFI207, which we find is not involved in DNA sensing but rather is required for cytokine promoter induction in macrophages. IFI207 co-localizes with both active RNA polymerase II (RNA Pol II) and IRF7 in the nucleus and enhances IRF7-dependent gene promoter induction. Generation of Ifi207-/- mice shows no role for IFI207 in autoimmunity. Rather, IFI207 is required for the establishment of a Klebsiella pneumoniae lung infection and for Klebsiella macrophage phagocytosis. These insights into IFI207 function illustrate that PYHINs can have distinct roles in innate immunity independent of DNA sensing and highlight the need to better characterize the whole mouse locus, one gene at a time.
Lupus nephritis is a severe organ manifestation of systemic lupus erythematosus, and its pathogenesis involves complex etiology and mechanisms. Despite significant knowledge gains and extensive efforts put into understanding the development and relapsing disease activity, lupus nephritis remains a substantial cause of morbidity and mortality in lupus patients. Current therapies retain a significant unmet medical need regarding rates of complete response, preventing relapse of lupus nephritis, progression of chronic kidney disease to kidney failure, drug toxicity, and pill burden-related drug non-adherence. Connected to progression of chronic kidney disease are the associated risks for disabling or even lethal cardiovascular events, as well as chronic kidney disease-related secondary immunodeficiency and serious infections. In this regard, biomarkers are needed that can predict treatment response to specific drugs to enable personalized precision medicine. A series of clinical trials with innovative immunomodulatory drugs are ongoing and raise expectations for improvements in the management of lupus nephritis. Here, we review how new developments in pathogenesis connect with current and future perspectives for the management of lupus nephritis.
Cyclic dinucleotides (CDNs) are second messengers that activate stimulator of interferon genes (STING). The cGAS-STING pathway plays a promising role in cancer immunotherapy. Here, we describe the synthesis of CDNs containing 7-substituted 7-deazapurine moiety. We used mouse cyclic GMP-AMP synthase and bacterial dinucleotide synthases for the enzymatic synthesis of CDNs. Alternatively, 7-(het)aryl 7-deazapurine CDNs were prepared by Suzuki-Miyaura cross-couplings. New CDNs were tested in biochemical and cell-based assays for their affinity to human STING. Eight CDNs showed better activity than 2'3'-cGAMP, the natural ligand of STING. The effect on cytokine and chemokine induction was also evaluated. The best activities were observed for CDNs bearing large aromatic substituents that point above the CDN molecule. We solved four X-ray structures of complexes of new CDNs with human STING. We observed π-π stacking interactions between the aromatic substituents and Tyr240 that are involved in the stabilization of CDN-STING complexes.
Cyclic GMP-AMP synthase (cGAS) is a predominant and ubiquitously expressed cytosolic onfirmedDNA sensor that activates innate immune responses by producing a second messenger, cyclic GMP-AMP (cGAMP), and the stimulator of interferon genes (STING). cGAS contains a highly disordered N-terminus, which can sense genomic/chromatin DNA, while the C terminal of cGAS binds dsDNA liberated from various sources, including mitochondria, pathogens, and dead cells. Furthermore, cGAS cellular localization dictates its response to foreign versus self-DNA. Recent evidence has also highlighted the importance of dsDNA-induced post-translational modifications of cGAS in modulating inflammatory responses. This review summarizes and analyzes cGAS activity regulation based on structure, sub-cellular localization, post-translational mechanisms, and Ca2+ signaling. We also discussed the role of cGAS activation in different diseases and clinical outcomes.
Inflammation plays a role in the etiopathogenesis of age-related macular degeneration (AMD). A retrospective case-control study was conducted to assess the significance of the neutrophil-lymphocyte ratio (NLR) as a systemic inflammatory indicator in dry AMD. Clinical diagnosis and complete blood count (CBC) results were extracted from medical records for patients with dry AMD and age/sex-matched controls. This study included 90 patients diagnosed with dry AMD and 270 controls without AMD. There were no significant differences in the CBC results between the cases and controls. Patients with dry AMD had a slightly higher mean NLR than controls; however, this increase was not significant (P = .13). In the NLR model, age and sex were significant factors affecting the NLR values in the dry AMD group (P = .03 and 0.01, respectively). The NLR alone cannot predict dry AMD. Therefore, exploring other routine laboratory measurements may shed light on early disease prediction and prevention.
Mitochondrial DNA (mtDNA) is a potent agonist of the cyclic GMP-AMP (cGAMP) synthase (cGAS) – Stimulator of Interferon Genes (STING) – type I interferon (IFN-I) pathway. However, the kinetics of mtDNA detection by cGAS or other nucleic acid sensors and the exact immunostimulatory features of mtDNA remain poorly defined. Here, we show that robust and sustained IFN-I responses downstream of mtDNA stress require the nucleic acid sensor Z-DNA binding protein 1 (ZBP1) in a cell death independent manner. Cells experiencing persistent mtDNA release display robust ZBP1 expression, as well as a marked increase in the cytoplasmic pool of cGAS. Biochemical and microscopy analyses reveal that the receptor-interacting protein homotypic interaction motifs (RHIMs) of ZBP1 bind the N-terminus of cGAS, leading to its entrapment in the cytoplasm. Moreover, we show that genetic and pharmacologic induction of mtDNA stress leads to the mitochondrial and cytoplasmic accumulation of Z-form DNA. Nuclease treatment or deletion of Z-DNA binding domains of ZBP1 reduce its interaction with cGAS and impair cGAMP production downstream of mtDNA stress. Finally, we uncover that ZBP1 is a novel regulator of IFN-I-mediated disease pathology, working in tandem with the cGAS-STING pathway to sense mtDNA instability and sustain IFN-I signaling that contributes to cardiac remodeling and heart failure. These results provide new insight into the molecular mechanisms of mtDNA sensing by the innate immune system and reveal that ZBP1 is a cooperative partner for cGAS. Moreover, our findings highlight ZBP1 as a potential therapeutic target in heart failure and other disorders where mtDNA instability drives disease-promoting IFN-I and inflammatory responses.
Graphical abstract
The cell-autonomous innate immune system enables animal cells to resist viral infection. This system comprises an array of sensors that, after detecting viral molecules, activate the expression of antiviral proteins and the interferon response. The repertoire of immune sensors and antiviral proteins has long been considered to be derived from extensive evolutionary innovation in vertebrates, but new data challenge this dogma. Recent studies show that central components of the cell-autonomous innate immune system have ancient evolutionary roots in prokaryotic genes that protect bacteria from phages. These include the cyclic GMP–AMP synthase (cGAS)–stimulator of interferon genes (STING) pathway, Toll/IL-1 receptor (TIR) domain-containing pathogen receptors, the viperin family of antiviral proteins, SAMHD1-like nucleotide-depletion enzymes, gasdermin proteins and key components of the RNA interference pathway. This Perspective details current knowledge of the elements of antiviral immunity that are conserved from bacteria to humans, and presents possible evolutionary scenarios to explain the observed conservation. The cell-autonomous innate immune system has long been considered an evolutionary innovation of metazoans; however, recent evidence challenges this dogma. This Perspective describes the components of antiviral immunity that are conserved from bacteria to humans, and presents potential evolutionary scenarios to explain the observed conservation.
Purpose
To assess the significance of neutrophil-lymphocyte ratio (NLR) as an indicator of inflammation in Saudi patients with dry age-related macular degeneration (AMD).
Study design
A retrospective case-control study.
Methods
Clinical diagnosis along with complete blood count (CBC) results were extracted from hospital and laboratory information systems for patients with dry AMD and age- gender- matched controls attending the ophthalmology clinic at King Abdulaziz medical city, Jeddah, between January 2018 and December 2020. NLR was calculated by dividing the neutrophil by the lymphocyte count.
Results
This study captured 90 patients diagnosed with dry AMD and 270 control subjects without AMD. The mean of ages 70 and 71 years old for cases and controls, respectively. In univariate analysis, there were no significant differences in CBC results between cases and control. In NLR, dry AMD patients have a slightly higher mean than the control group; however, this increase was not statistically significant (P-value 0.8). In the NLR model, age and gender were statistically significant factors affecting the NLR values in dry AMD (P-value 0.03, 0.01 respectively).
Conclusion
as a systemic inflammatory biomarker, NLR alone could not predict dry AMD. However, the slight increase in the NLR values may be helpful if augmented with other laboratory measurements to aid in early disease prediction.
Purpose: To assess the significance of neutrophil-lymphocyte ratio (NLR) as an inflammatory indicator in patients with dry age-related macular degeneration (AMD).
Study design: A retrospective case-control study.
Methods: Clinical diagnosis along with complete blood count (CBC) results were extracted from hospital and laboratory information systems for patients with dry-AMD and age/gender-matched controls attending the ophthalmology clinic at King Abdulaziz medical city, Jeddah, Saudi Arabia, between 2018-2020. NLR was calculated by dividing the neutrophil by the lymphocyte count.
Results: This study captured 90 patients diagnosed with dry-AMD and 270 control subjects without AMD. The mean of ages 70 and 71 years old for cases and controls, respectively. In univariate analysis, there were no significant differences in CBC results between cases and control. In NLR, dry-AMD patients have a slightly higher mean than the control group; however, this increase was not statistically significant (P-value 0.8). In the NLR model, age and gender were statistically significant factors affecting the NLR values in dry-AMD (P-value 0.03, 0.01 respectively).
Conclusion: as a systemic inflammatory biomarker, NLR alone could not predict dry-AMD. However, the slight increase in the NLR values may be helpful if augmented with other laboratory measurements to aid in early disease prediction.
The induction of type I interferons (IFN) is critical for antiviral innate immune response. The rapid activation of antiviral innate immune responses is the key to successful clearance of evading pathogens. To achieve this, a series of proteins, including the pathogen recognition receptors (PRRs), the adaptor proteins, the accessory proteins, kinases, and the transcription factors, are all involved and finely orchestrated. The magnitude and latitude of type I IFN induction however are distinctly regulated in different tissues. A set of interferon simulated genes (ISGs) are then expressed in response to type I IFN signaling to set the cells in the antiviral state. In this review, how type I IFN is induced by viral infections by intracellular PRRs and how type I IFN triggers the expression of downstream effectors will be discussed.
Cytokines induced by Zika virus (ZIKV) infection are crucial to control the replication and dissemination of the virus. Despite their role in mediating the events that protect the host, cytokines can also be associated with symptoms and with ZIKV immunopathology. In recent outbreaks, ZIKV infection caused neurological complications such as Guillain-Barré syndrome and the congenital Zika syndrome (CZS), including microcephaly and system nervous malformation. This chapter describes the cytokines involved in the acute phase of ZIKV infection, including those induced in women who were infected during pregnancy and their possible association with CZS. We also describe the cytokines related to nonneurological complications such as ocular damage, testis infection, and hearing loss problems following ZIKV infection. Understanding the role of cytokines and the underlying mechanisms in the ZIKV infection will open possibility for therapeutic and prophylactical interventions.
STING protein (stimulator of interferon genes) plays an important role in the innate immune system. A number of potent compounds regulating its activity have been reported, mostly derivatives of cyclic dinucleotides (CDNs), natural STING agonists. Here, we aim to provide complementary information to large-scale "ligand-profiling" studies by probing the importance of STING-CDN protein-ligand interactions on the protein side. We examined in detail six typical CDNs each in complex with 13 rationally devised mutations in STING: S162A, S162T, Y167F, G230A, R232K, R232H, A233L, A233I, R238K, T263A, T263S, R293Q, and G230A/R293Q. The mutations switch on and off various types of protein-ligand interactions: π-π stacking, hydrogen bonding, ionic pairing, and nonpolar contacts. We correlated experimental data obtained by differential scanning fluorimetry, X-ray crystallography, and isothermal titration calorimetry with theoretical calculations. This enabled us to provide a mechanistic interpretation of the differences in the binding of representative CDNs to STING. We observed that the G230A mutation increased the thermal stability of the protein-ligand complex, indicating an increased level of ligand binding, whereas R238K and Y167F led to a complete loss of stabilization (ligand binding). The effects of the other mutations depended on the type of ligand (CDN) and varied, to some extent. A very good correlation (R2 = 0.6) between the experimental binding affinities and interaction energies computed by quantum chemical methods enabled us to explain the effect of the studied mutations in detail and evaluate specific interactions quantitatively. Our work may inspire development of high-affinity ligands against the common STING haplotypes by targeting the key (sometimes non-intuitive) protein-ligand interactions.
Signaling through stimulator of interferon genes (STING) leads to the production of type I interferons (IFN-Is) and inflammatory cytokines. A gain-of-function mutation in STING was identified in an autoinflammatory disease (STING-associated vasculopathy with onset in infancy; SAVI). The expression of cyclic GMP-AMP, DNA-activated cGAS-STING pathway, increased in a proportion of patients with SLE. The STING signaling pathway may be a candidate for targeted therapy in SLE. Here, we demonstrated that disruption of STING signaling ameliorated lupus development in Fcgr2b-deficient mice. Activation of STING promoted maturation of conventional dendritic cells and differentiation of plasmacytoid dendritic cells via LYN interaction and phosphorylation. The inhibition of LYN decreased the differentiation of STING-activated dendritic cells. Adoptive transfer of STING-activated bone marrow-derived dendritic cells into the FCGR2B and STING double-deficiency mice restored lupus phenotypes. These findings provide evidence that the inhibition of STING signaling may be a candidate targeted treatment for a subset of patients with SLE.
Plasmacytoid dendritic cells (pDCs) are important mediators of antiviral immunity through their ability to produce large amounts of type I interferons (IFNs) on viral infection. This function of pDCs is linked to their expression of Toll-like receptor 7 (TLR7) and TLR9, which sense viral nucleic acids within the early endosomes. Exclusion of self nucleic acids from TLR-containing early endosomes normally prevents pDC responses to them. However, in some autoimmune diseases, self nucleic acids can be modified by host factors and gain entrance to pDC endosomes, where they activate TLR signalling. Several pDC receptors negatively regulate type I IFN responses by pDCs during viral infection and for normal homeostasis.
Recent studies in cell lines and genetically engineered mice have demonstrated that cytosolic dsDNA could activate dendritic cells (DCs) to become effector APCs. Recognition of DNA might be a major factor in antimicrobial immune responses against cytosolic pathogens and also in human autoimmune diseases such as systemic lupus erythematosus. However, the role of cytosolic dsDNA in human DC activation and its effects on effector T and B cells are still elusive. In this study, we demonstrate that intracellular dsDNA is a potent activator of human monocyte-derived DCs as well as primary DCs. Activation by dsDNA depends on NF-κB activation, partially on the adaptor molecule IFN-promoter stimulator-1 and the novel cytosolic dsDNA receptor IFI16, but not on the previously recognized dsDNA sentinels absent in melanoma 2, DNA-dependent activator of IFN regulatory factor 3, RNA polymerase III, or high-mobility group boxes. More importantly, we report for the first time, to our knowledge, that human dsDNA-activated DCs, rather than LPS- or inflammatory cytokine mixture-activated DCs, represent the most potent inducers of naive CD4(+) T cells to promote Th1-type cytokine production and generate CD4(+) and CD8(+) cytotoxic T cells. dsDNA-DCs, but not LPS- or mixture-activated DCs, induce B cells to produce complement-fixing IgG1 and IgG3 Abs. We propose that cytosolic dsDNA represents a novel, more effective approach to generate DCs to enhance vaccine effectiveness in reprogramming the adaptive immune system to eradicate infectious agents, autoimmunity, allergy, and cancer.
The mucosal epithelium is the initial target for respiratory pathogens of all types. While type I interferon (IFN) signaling is traditionally associated with antiviral immunity, we demonstrate that the extracellular bacterial pathogen Streptococcus pneumoniae activates the type I IFN cascade in airway epithelial and dendritic cells. This response is dependent upon the pore-forming toxin pneumolysin. Pneumococcal DNA activates IFN-beta expression through a DAI/STING/TBK1/IRF3 cascade. Tlr4(-/-), Myd88(-/-), Trif(-/-), and Nod2(-/-) mutant mice had no impairment of type I IFN signaling. Induction of type I IFN signaling contributes to the eradication of pneumococcal carriage, as IFN-alpha/beta receptor null mice had significantly increased nasal colonization with S. pneumoniae compared with that of wild-type mice. These studies suggest that the type I IFN cascade is a central component of the mucosal response to airway bacterial pathogens and is responsive to bacterial pathogen-associated molecular patterns that are capable of accessing intracellular receptors.
IMPORTANCE: The bacterium Streptococcus pneumoniae is a leading cause of bacterial pneumonia, leading to upwards of one million deaths a year worldwide and significant economic burden. Although it is known that antibody is critical for efficient phagocytosis, it is not known how this pathogen is sensed by the mucosal epithelium. We demonstrate that this extracellular pathogen activates mucosal signaling typically activated by viral pathogens via the pneumolysin pore to activate intracellular receptors and the type I interferon (IFN) cascade. Mice lacking the receptor to type I IFNs have a reduced ability to clear S. pneumoniae, suggesting that the type I IFN cascade is central to the mucosal clearance of this important pathogen.
The IFN-inducible IFI16 and AIM2 proteins act as innate immune sensors for cytosolic double-stranded DNA (dsDNA). On sensing dsDNA, the IFI16 protein induces the expression of IFN-β whereas the AIM2 protein forms an inflammasome, which promotes the secretion of IL-1β. Given that the knockdown of IFI16 expression in human diploid fibroblasts (HDF) delays the onset of cellular senescence, we investigated the potential roles for the IFI16 and AIM2 proteins in cellular senescence. We found that increased IFI16 protein levels in old (vs. young) HDFs were associated with the induction of IFN-β. In contrast, increased levels of the AIM2 protein in the senescent (vs. old) HDFs were associated with increased production of IL-1β. The knockdown of type I IFN-α receptor subunit, which reduced the basal levels of the IFI16 but not of the AIM2, protein delayed the onset of cellular senescence. Accordingly, increased constitutive levels of IFI16 and AIM2 proteins in ataxia telangiectasia mutated (ATM) HDFs were associated with the activation of the IFN signaling and increased levels of IL-1β. The IFN-β treatment of the young HDFs, which induced the expression of IFI16 and AIM2 proteins, activated a DNA damage response and also increased basal levels of IL-1β. Interestingly, the knockdown of AIM2 expression in HDFs increased the basal levels of IFI16 protein and activated the IFN signaling. In contrast, the knockdown of the IFI16 expression in HDFs decreased the basal and dsDNA-induced activation of the IFN signaling. Collectively, our observations show differential roles for the IFI16 and AIM2 proteins in cellular senescence and associated secretory phenotype.
Cytosolic foreign DNA is detected by pattern recognition receptors and mainly induces type I IFN production. We found that transfection of different types of DNA into various untreated cells induces type III IFN (IFN-λ1) rather than type I IFN, indicating the presence of uncharacterized DNA sensor(s). A pull-down assay using cytosolic proteins identified that Ku70 and Ku80 are the DNA-binding proteins. The knockdown studies and the reporter assay revealed that Ku70 is a novel DNA sensor inducing the IFN-lambda1 activation. The functional analysis of IFNL1 promoter revealed that positive-regulatory domain I and IFN-stimulated response element sites are predominantly involved in the DNA-mediated IFNL1 activation. A pull-down assay using nuclear proteins demonstrated that the IFN-λ1 induction is associated with the activation of IFN regulatory factor-1 and -7. Thus, to our knowledge, we show for the first time that Ku70 mediates type III IFN induction by DNA.
MPYS, also known as STING and MITA, is an interferon (IFN)β stimulator essential for host defense against RNA, DNA viruses and intracellular bacteria. MPYS also facilitates the adjuvant activity of DNA vaccines. Here, we report identification of a distinct human MPYS haplotype that contains three non-synonymous single nucleotide polymorphisms (SNPs), R71H-G230A-R293Q (thus, named the HAQ haplotype). We estimate, in two cohorts (1,074 individuals), that ∼3% of Americans are homozygous for this HAQ haplotype. HAQ MPYS exhibits a > 90% loss in the ability to stimulate IFNβ production. Furthermore, fibroblasts and macrophage cells expressing HAQ are defective in Listeria monocytogenes infection-induced IFNβ production. Lastly, we find that the loss of IFNβ activity is due primarily to the R71H and R293Q SNPs in HAQ. We hypothesize that individuals carrying HAQ may exhibit heightened susceptibility to viral infection and respond poorly to DNA vaccines.
Type I interferons (IFNs) are central regulators of the innate and adaptive immune responses to viral and bacterial infections.
Type I IFNs are induced upon cytosolic detection of microbial nucleic acids, including DNA, RNA, and the bacterial second
messenger cyclic-di-GMP (c-di-GMP). In addition, a recent study demonstrated that the intracellular bacterial pathogen Listeria monocytogenes stimulates a type I IFN response due to cytosolic detection of bacterially secreted c-di-AMP. The transmembrane signaling
adaptor Sting (Tmem173, Mita, Mpys, Eris) has recently been implicated in the induction of type I IFNs in response to cytosolic
DNA and/or RNA. However, the role of Sting in response to purified cyclic dinucleotides or during in vivo L. monocytogenes infection has not been addressed. In order to identify genes important in the innate immune response, we have been conducting
a forward genetic mutagenesis screen in C57BL/6 mice using the mutagen N-ethyl-N-nitrosourea (ENU). Here we describe a novel mutant mouse strain, Goldenticket (Gt), that fails to produce type I IFNs upon L. monocytogenes infection. By genetic mapping and complementation experiments, we found that Gt mice harbor a single nucleotide variant (T596A) of Sting that functions as a null allele and fails to produce detectable protein. Analysis of macrophages isolated from Gt mice revealed that Sting is absolutely required for the type I interferon response to both c-di-GMP and c-di-AMP. Additionally, Sting is required for the response to c-di-GMP and L. monocytogenes in vivo. Our results provide new functions for Sting in the innate interferon response to pathogens.
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).
Innate recognition of viruses is mediated by pattern recognition receptors (PRRs) triggering expression of antiviral interferons (IFNs) and proinflammatory cytokines. In mice, Toll-like receptor 2 (TLR2) and TLR9 as well as intracellular nucleotide-sensing pathways have been shown to recognize herpes simplex virus (HSV). Here, we describe how human primary macrophages recognize early HSV infection via intracellular pathways. A number of inflammatory cytokines, IFNs, and IFN-stimulated genes were upregulated after HSV infection. We show that early recognition of HSV and induction of IFNs and inflammatory cytokines are independent of TLR2 and TLR9, since inhibition of TLR2 using TLR2 neutralizing antibodies did not affect virus-induced responses and the macrophages were unresponsive to TLR9 stimulation. Instead, HSV recognition involves intracellular recognition systems, since induction of tumor necrosis factor alpha (TNF-α) and IFNs was dependent on virus entry and replication. Importantly, expression of IFNs was strongly inhibited by small interfering RNA (siRNA) knockdown of MAVS, but this MAVS-dependent IFN induction occurred independently of the recently discovered polymerase III (Pol III)/RIG-I DNA sensing system. In contrast, induction of TNF-α was largely independent of MAVS, suggesting that induction of inflammatory cytokines during HSV infection proceeds via a novel pathway. Transfection with ODN2006, a broad inhibitor of intracellular nucleotide recognition, revealed that nucleotide-sensing systems are employed to induce both IFNs and TNF-α. Finally, using siRNA knockdown, we found that MDA5, but not RIG-I, was the primary mediator of HSV recognition. Thus, innate recognition of HSV by human primary macrophages occurs via two distinct intracellular nucleotide-sensing pathways responsible for induction of IFNs and inflammatory cytokine expression, respectively.
Toll-like receptor 9 (TLR9) senses microbial DNA and triggers type I IFN responses in plasmacytoid dendritic cells (pDCs). Previous studies suggest the presence of myeloid differentiation primary response gene 88 (MyD88)-dependent DNA sensors other than TLR9 in pDCs. Using MS, we investigated C-phosphate-G (CpG)-binding proteins from human pDCs, pDC-cell lines, and interferon regulatory factor 7 (IRF7)-expressing B-cell lines. CpG-A selectively bound the aspartate-glutamate-any amino acid-aspartate/histidine (DExD/H)-box helicase 36 (DHX36), whereas CpG-B selectively bound DExD/H-box helicase 9 (DHX9). Although the aspartate-glutamate-alanine-histidine box motif (DEAH) domain of DHX36 was essential for CpG-A binding, the domain of unknown function 1605 (DUF1605 domain) of DHX9 was required for CpG-B binding. DHX36 is associated with IFN-alpha production and IRF7 nuclear translocation in response to CpG-A, but DHX9 is important for TNF-alpha and IL-6 production and NF-kappaB activation in response to CpG-B. Knocking down DHX9 or DHX36 significantly reduced the cytokine responses of pDCs to a DNA virus but had no effect on the cytokine responses to an RNA virus. We further showed that both DHX9 and DHX36 are localized within the cytosol and are directly bound to the Toll-interleukin receptor domain of MyD88 via their helicase-associated domain 2 and DUF domains. This study demonstrates that DHX9/DHX36 represent the MyD88-dependent DNA sensors in the cytosol of pDCs and suggests a much broader role for DHX helicases in viral sensing.
Signal transducer and activator of transcription 4 (STAT4) is a transcription factor mainly activated by interleukin 12, which promotes the secretion of type 2 interferon (IFN) by T-helper 1 cells. We assessed the association of STAT4 gene polymorphism and primary Sjögren's syndrome (pSS) and its functional relevance. We analyzed STAT4 rs7582694 polymorphism in an exploratory cohort of 186 pSS patients and 152 controls, and in a replication cohort of 192 pSS patients and 483 controls, all Caucasian. mRNA levels of STAT4α, STAT4β, STAT1, and the type 1 IFN-induced genes PKR, MX1 and IFITM1 were assessed in peripheral blood mononuclear cells (PBMCs) from 30 pSS patients. STAT4 rs7582694 C allele was associated with pSS in both cohorts (odds ratio (OR) 1.57, 95% confidence interval (CI) 1.27–1.93, P=2.3 × 10−5). The association was increased for homozygous subjects, which suggests a recessive effect of the STAT4 at-risk allele. STAT4α, STAT4β and STAT1 mRNA levels in PBMCs were not significantly associated with rs7582694 genotypes, however the mRNA levels of STAT4α and type 1 IFN-induced genes were strongly correlated: PKR (P=4 × 10−3, r=0.51), MX1 (P=2 × 10−4, r=0.63) and IFITM1 (P=8 × 10−3, r=0.47), suggesting that STAT4 might be involved in not only type 2 IFN production but also in type 1 IFN-mediated effects.
The human cytomegalovirus (HCMV) virion protein pUL83 (also termed pp65) inhibits the expression of interferon-inducible cellular genes. In this work we demonstrate that pUL83 is also important for efficient induction of transcription from the viral major immediate-early promoter. Infection with a mutant virus containing a premature translation termination codon in the UL83 open reading frame (ORF) (UL83Stop) resulted in decreased transcription from the major immediate-early promoter in a time- and multiplicity-dependent manner. Expression of pUL83 alone is capable of transactivating the promoter in a reporter assay, and pUL83 associates with the promoter in infected cells. To investigate the mechanism by which the protein regulates the major immediate-early promoter, we utilized a mutant virus expressing an epitope-tagged pUL83 from its own promoter to identify protein binding partners for pUL83 during infection. We identified and confirmed the interaction of pUL83 with cellular IFI16 family members throughout the course of HCMV infection. pUL83 recruits IFI16 to the major immediate-early promoter, and IFI16 binding at the promoter is dependent upon the presence of pUL83. Consistent with the results obtained with the UL83Stop virus, infection of IFI16 knockdown cells with wild-type virus resulted in decreased levels of immediate-early transcripts compared to those of control cells. These data identify a previously unknown role for pUL83 in the initiation of the human cytomegalovirus gene expression cascade.
Macrophages respond to cytosolic nucleic acids by activating cysteine protease caspase-1 within a complex called the inflammasome. Subsequent cleavage and secretion of proinflammatory cytokines IL-1beta and IL-18 are critical for innate immunity. Here, we show that macrophages from mice lacking absent in melanoma 2 (AIM2) cannot sense cytosolic double-stranded DNA and fail to trigger inflammasome assembly. Caspase-1 activation in response to intracellular pathogen Francisella tularensis also required AIM2. Immunofluorescence microscopy of macrophages infected with F. tularensis revealed striking colocalization of bacterial DNA with endogenous AIM2 and inflammasome adaptor ASC. By contrast, type I IFN (IFN-alpha and -beta) secretion in response to F. tularensis did not require AIM2. IFN-I did, however, boost AIM2-dependent caspase-1 activation by increasing AIM2 protein levels. Thus, inflammasome activation was reduced in infected macrophages lacking either the IFN-I receptor or stimulator of interferon genes (STING). Finally, AIM2-deficient mice displayed increased susceptibility to F. tularensis infection compared with wild-type mice. Their increased bacterial burden in vivo confirmed that AIM2 is essential for an effective innate immune response.
Francisella tularensis, the causative agent of tularemia, infects host macrophages, which triggers production of the proinflammatory cytokines interleukin 1beta (IL-1beta) and IL-18. We elucidate here how host macrophages recognize F. tularensis and elicit this proinflammatory response. Using mice deficient in the DNA-sensing inflammasome component AIM2, we demonstrate here that AIM2 is required for sensing F. tularensis. AIM2-deficient mice were extremely susceptible to F. tularensis infection, with greater mortality and bacterial burden than that of wild-type mice. Caspase-1 activation, IL-1beta secretion and cell death were absent in Aim2(-/-) macrophages in response to F. tularensis infection or the presence of cytoplasmic DNA. Our study identifies AIM2 as a crucial sensor of F. tularensis infection and provides genetic proof of its critical role in host innate immunity to intracellular pathogens.
Inflammasomes regulate the activity of caspase-1 and the maturation of interleukin 1beta (IL-1beta) 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-1beta 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-gamma, 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.
Legionella pneumophila is a gram-negative bacterial pathogen that replicates in host macrophages and causes a severe pneumonia called Legionnaires' Disease. The innate immune response to L. pneumophila remains poorly understood. Here we focused on identifying host and bacterial factors involved in the production of type I interferons (IFN) in response to L. pneumophila. It was previously suggested that the delivery of L. pneumophila DNA to the host cell cytosol is the primary signal that induces the type I IFN response. However, our data are not easily reconciled with this model. We provide genetic evidence that two RNA-sensing proteins, RIG-I and MDA5, participate in the IFN response to L. pneumophila. Importantly, these sensors do not seem to be required for the IFN response to L. pneumophila DNA, whereas we found that RIG-I was required for the response to L. pneumophila RNA. Thus, we hypothesize that bacterial RNA, or perhaps an induced host RNA, is the primary stimulus inducing the IFN response to L. pneumophila. Our study also identified a secreted effector protein, SdhA, as a key suppressor of the IFN response to L. pneumophila. Although viral suppressors of cytosolic RNA-sensing pathways have been previously identified, analogous bacterial factors have not been described. Thus, our results provide new insights into the molecular mechanisms by which an intracellular bacterial pathogen activates and also represses innate immune responses.
Human cytomegalovirus (HCMV) is a member of the betaherpesvirus family that, unlike other herpesviruses, triggers a strong innate immune response in infected cells that includes transcription of the beta interferon gene via activation of interferon regulatory factor 3 (IRF3). IRF3 activation requires signaling from pattern recognition receptors that is initiated by their interaction with specific pathogen-associated molecules. However, while IRF3-activating pathways are increasingly well characterized, the cellular molecules involved in HCMV-mediated IRF3-dependent beta interferon transcription are virtually unknown. We undertook a systematic examination of new and established IRF3-terminal pathway components to identify those that are essential to HCMV-triggered IRF3 activation. We show here that IRF3 activation induced by HCMV infection involves the newly identified protein STING but, in contrast to infections with other herpesviruses, occurs independently of the adaptor molecule IPS-1. We also show that the protein DDX3 contributes to HCMV-triggered expression of beta interferon. Moreover, we identify Z-DNA binding protein 1 (ZBP1) as being essential for IRF3 activation and interferon beta expression triggered by HCMV, as well as being sufficient to enhance HCMV-stimulated beta interferon transcription and secretion. ZBP1 transcription was also found to be induced following exposure to HCMV in a JAK/STAT-dependent manner, thus perhaps also contributing to a positive feedback signal. Finally, we show that constitutive overexpression of ZBP1 inhibits HCMV replication. ZBP1 was recently identified as a cytosolic pattern recognition receptor of double-stranded DNA, and thus, we propose a model for HCMV-mediated IRF3 activation that involves HCMV-associated DNA as the principal innate immune-activating pathogen-associated molecular pattern.
RNA is sensed by Toll-like receptor 7 (TLR7) and TLR8 or by the RNA helicases LGP2, Mda5 and RIG-I to trigger antiviral responses. Much less is known about sensors for DNA. Here we identify a novel DNA-sensing pathway involving RNA polymerase III and RIG-I. In this pathway, AT-rich double-stranded DNA (dsDNA) served as a template for RNA polymerase III and was transcribed into double-stranded RNA (dsRNA) containing a 5'-triphosphate moiety. Activation of RIG-I by this dsRNA induced production of type I interferon and activation of the transcription factor NF-kappaB. This pathway was important in the sensing of Epstein-Barr virus-encoded small RNAs, which were transcribed by RNA polymerase III and then triggered RIG-I activation. Thus, RNA polymerase III and RIG-I are pivotal in sensing viral DNA.
Detection of viral nucleic acids is central to antiviral immunity. Recently, DAI/ZBP1 (DNA-dependent activator of IRFs/Z-DNA binding protein 1) was identified as a cytoplasmic DNA sensor and shown to activate the interferon regulatory factor (IRF) and nuclear factor-kappa B (NF-kappaB) transcription factors, leading to type-I interferon production. DAI-induced IRF activation depends on TANK-binding kinase 1 (TBK1), whereas signalling pathways and molecular components involved in NF-kappaB activation remain elusive. Here, we report the identification of two receptor-interacting protein (RIP) homotypic interaction motifs (RHIMs) in the DAI protein sequence, and show that these domains relay DAI-induced NF-kappaB signals through the recruitment of the RHIM-containing kinases RIP1 and RIP3. We show that knockdown of not only RIP1, but also RIP3 affects DAI-induced NF-kappaB activation. Importantly, RIP recruitment to DAI is inhibited by the RHIM-containing murine cytomegalovirus (MCMV) protein M45. These findings delineate the DAI signalling pathway to NF-kappaB and suggest a possible new immune modulation strategy of the MCMV.
We report here the identification and characterization of a protein, ERIS, an endoplasmic reticulum (ER) IFN stimulator, which is a strong type I IFN stimulator and plays a pivotal role in response to both non-self-cytosolic RNA and dsDNA. ERIS (also known as STING or MITA) resided exclusively on ER membrane. The ER retention/retrieval sequence RIR was found to be critical to retain the protein on ER membrane and to maintain its integrity. ERIS was dimerized on innate immune challenges. Coumermycin-induced ERIS dimerization led to strong and fast IFN induction, suggesting that dimerization of ERIS was critical for self-activation and subsequent downstream signaling.
TANK-binding kinase-1 (TBK1) and the inducible IkappaB kinase (IKK-i) have recently been shown to activate type I IFN responses elicited by intracellular detection of RNA or DNA from infecting viruses. Detection of viral RNA is mediated by retinoic acid inducible gene-I or melanoma differentiation-associated gene-5 pathways in which TBK1 and IKK-i have been demonstrated to play redundant roles in IFN activation. In this study, we have examined whether such redundancy occurs in the type I IFN response to DNA viral challenges by examining induction of IFNs and IFN-mediated signaling and gene programs in TBK1(-/-) macrophages. In contrast to the normal IFN responses in TBK1(-/-) macrophages infected with an RNA virus, IFN responses were severely abrogated during DNA virus infections in TBK1(-/-) macrophages. Because both TBK1 and IKK-i are expressed in macrophages, our studies suggest that TBK1 and IKK-i differ functionally in DNA virus-mediated IFN responses; however, they are redundant in RNA virus-mediated IFN responses. Confirmatively, reconstitution of TBK1(-/-)IKK-i(-/-) fibroblasts revealed that TBK1 rescued IFN responses to transfected B-DNA to a much stronger degree than IKK-i. Finally, we demonstrate the requirement for the TBK1-IFN regulatory factor-3 pathway in host defense against a DNA virus infection in vivo.
The innate immune system senses nucleic acids by germline-encoded pattern recognition receptors. RNA is sensed by Toll-like receptor members TLR3, TLR7 and TLR8, or by the RNA helicases RIG-I (also known as DDX58) and MDA-5 (IFIH1). Little is known about sensors for cytoplasmic DNA that trigger antiviral and/or inflammatory responses. The best characterized of these responses involves activation of the TANK-binding kinase (TBK1)-interferon regulatory factor 3 (IRF3) signalling axis to trigger transcriptional induction of type I interferon genes. A second, less well-defined pathway leads to the activation of an 'inflammasome' that, via caspase-1, controls the catalytic cleavage of the pro-forms of the cytokines IL1beta and IL18 (refs 6, 7). Using mouse and human cells, here we identify the PYHIN (pyrin and HIN domain-containing protein) family member absent in melanoma 2 (AIM2) as a receptor for cytosolic DNA, which regulates caspase-1. The HIN200 domain of AIM2 binds to DNA, whereas the pyrin domain (but not that of the other PYHIN family members) associates with the adaptor molecule ASC (apoptosis-associated speck-like protein containing a caspase activation and recruitment domain) to activate both NF-kappaB and caspase-1. Knockdown of Aim2 abrogates caspase-1 activation in response to cytoplasmic double-stranded DNA and the double-stranded DNA vaccinia virus. Collectively, these observations identify AIM2 as a new receptor for cytoplasmic DNA, which forms an inflammasome with the ligand and ASC to activate caspase-1.
Primary Sjögren's syndrome (SS) shares many features with systemic lupus erythematosus (SLE). Here we investigated the association of the three major polymorphisms in IRF5 and STAT4 found to be associated with SLE, in patients from Sweden and Norway with primary SS. These polymorphisms are a 5-bp CGGGG indel in the promoter of IRF5, the single nucleotide polymorphism (SNP) rs10488631 downstream of IRF5 and the STAT4 SNP rs7582694, which tags the major risk haplotype of STAT4. We observed strong signals for association between all three polymorphisms and primary SS, with odds ratios (ORs) >1.4 and P-values <0.01. We also found a strong additive effect of the three risk alleles of IRF5 and STAT4 with an overall significance between the number of risk alleles and primary SS of P=2.5 x 10(-9). The OR for primary SS increased in an additive manner, with an average increase in OR of 1.78. For carriers of two risk alleles, the OR for primary SS is 1.43, whereas carriers of five risk alleles have an OR of 6.78. IRF5 and STAT4 are components of the type I IFN system, and our findings emphasize the importance of this system in the etiopathogenesis of primary SS.
The expression of pattern-recognition receptors (PRRs) by immune and tissue cells provides the host with the ability to detect and respond to infection by viruses and other microorganisms. Significant progress has been made from studying this area, including the identification of PRRs, such as Toll-like receptors and RIG-I-like receptors, and the description of the molecular basis of their signalling pathways, which lead to the production of interferons and other cytokines. In parallel, common mechanisms used by viruses to evade PRR-mediated responses or to actively subvert these pathways for their own benefit are emerging. Accumulating evidence on how viral infection and PRR signalling pathways intersect is providing further insights into the function of the pathways involved, their constituent proteins and ways in which they could be manipulated therapeutically.
DNA-dependent activator of IFN regulatory factors (IRF; DAI, also known as ZBP1 or DLM-1) is a cytosolic DNA sensor that initiates IRF3 and NF-kappaB pathways leading to activation of type I IFNs (IFNalpha, IFNbeta) and other cytokines. In this study, induction of NF-kappaB is shown to depend on the adaptor receptor-interacting protein kinase (RIP)1, acting via a RIP homotypic interaction motif (RHIM)-dependent interaction with DAI. DAI binds to and colocalizes with endogenous RIP1 at characteristic cytoplasmic granules. Suppression of RIP1 expression by RNAi abrogates NF-kappaB activation as well as IFNbeta induction by immunostimulatory DNA. DAI also interacts with RIP3 and this interaction potentiates DAI-mediated activation of NF-kappaB, implicating RIP3 in regulating this RHIM-dependent pathway. The role of DAI in activation of NF-kappaB in response to immunostimulatory DNA appears to be analogous to sensing of dsRNA by TLR3 in that both pathways involve RHIM-dependent signaling that is mediated via RIP1, reinforcing a central role for this adaptor in innate sensing of intracellular microbes.
Interferons have been shown to exert antiviral, cell growth regulatory, and immunomodulatory effects on target cells (16, 37). Both type I (a and b) and type II (g) interferons regulate cellular activities by specifically inducing the expression or activation of endogenous proteins that perform distinct biolog- ical functions. The HIN-200 (hematopoietic interferon-induc- ible nuclear proteins with a 200-amino-acid repeat) gene fam- ily found on human and mouse chromosome 1 is positively regulated by type I and II interferons (15, 27, 30). The HIN- 200 family of proteins consists of a number of highly homolo- gous human and murine proteins with similar primary amino acid sequences and biological characteristics. The human HIN- 200 family members include IFI 16 (39), the myeloid nuclear differentiation antigen (MNDA) (2, 18), and AIM-2 (absent in melanoma) (17), while the mouse HIN-200 family members include p202 (3), p203 (20), p204 (3), and D3 (38). There are a number of reviews that elegantly outline the biochemical characteristics of these proteins and their patterns of expres- sion (15, 27, 30). Recently, there have been a number of re- ports examining possible cellular functions of HIN-200 pro- teins. It is now clear that these proteins play a role in modulating cell growth and perhaps in vivo differentiation, and the newest member of the HIN-200 family, AIM-2, has been identified as a possible tumor suppressor protein. Some family members are transcriptional regulators, acting either directly by binding DNA at a promoter or indirectly by modulating the function of other cellular transcription factors. Clearly, there is much still to learn about the biological functions of these proteins and how significant a role they play during an inter- feron response. While it is clear that the human and mouse family members share many biochemical and structural char- acteristics, it is unclear which, if any, of the currently identified murine HIN-200 proteins are functional homologs of the dif- ferent human HIN-200 family members. This review will briefly cover the structural and biochemical properties of HIN- 200 proteins but will concentrate on the molecular and biolog- ical functions of these molecules.
Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease that affects over one million people in the United States. SLE is characterized by the presence of anti-nuclear antibodies (ANA) directed against naked DNA and entire nucleosomes. It is thought that the resulting immune complexes accumulate in vessel walls, glomeruli and joints and cause a hypersensitivity reaction type III, which manifests as glomerulonephritis, arthritis and general vasculitis. The aetiology of SLE is unknown, but several studies suggest that increased liberation or disturbed clearance of nuclear DNA-protein complexes after cell death may initiate and propagate the disease. Consequently, Dnase1, which is the major nuclease present in serum, urine and secreta, may be responsible for the removal of DNA from nuclear antigens at sites of high cell turnover and thus for the prevention of SLE (refs 7-11). To test this hypothesis, we have generated Dnase1-deficient mice by gene targeting. We report here that these animals show the classical symptoms of SLE, namely the presence of ANA, the deposition of immune complexes in glomeruli and full-blown glomerulonephritis in a Dnase1-dose-dependent manner. Moreover, in agreement with earlier reports, we found Dnase1 activities in serum to be lower in SLE patients than in normal subjects. Our findings suggest that lack or reduction of Dnase1 is a critical factor in the initiation of human SLE.
Systemic lupus erythematosus (SLE) is a highly prevalent human autoimmune diseases that causes progressive glomerulonephritis, arthritis and an erythematoid rash. Mice deficient in deoxyribonuclease I (Dnase1) develop an SLE-like syndrome. Here we describe two patients with a heterozygous nonsense mutation in exon 2 of DNASE1, decreased DNASE1 activity and an extremely high immunoglobulin G titer against nucleosomal antigens. These data are consistent with the hypothesis that a direct connection exists between low activity of DNASE1 and progression of human SLE.
Apoptosis is often accompanied by the degradation of chromosomal DNA. Caspase-activated DNase (CAD) is an endonuclease that is activated in dying cells, whereas DNase II is present in the lysosomes of macrophages. Here, we show that CAD(-/-) thymocytes did not undergo apoptotic DNA degradation. But, when apoptotic cells were phagocytosed by macrophages, their DNA was degraded by DNase II. The thymus of DNase II(-/-)CAD(-/-) embryos contained many foci carrying undigested DNA and the cellularity was severely reduced due to a block in T cell development. The interferon-beta gene was strongly up-regulated in the thymus of DNase II(-/-)CAD(-/-) embryos, suggesting that when the DNA of apoptotic cells is left undigested, it can activate innate immunity leading to defects in thymic development.
Indirect evidence suggests that type-I interferons (IFN-alpha/beta) play a significant role in the pathogenesis of lupus. To directly examine the contribution of these pleiotropic molecules, we created congenic NZB mice lacking the alpha-chain of IFN-alpha/betaR, the common receptor for the multiple IFN-alpha/beta species. Compared with littermate controls, homozygous IFN-alpha/betaR-deleted NZB mice had significantly reduced anti-erythrocyte autoantibodies, erythroblastosis, hemolytic anemia, anti-DNA autoantibodies, kidney disease, and mortality. These reductions were intermediate in the heterozygous-deleted mice. The disease-ameliorating effects were accompanied by reductions in splenomegaly and in several immune cell subsets, including B-1 cells, the major producers of anti-erythrocyte autoantibodies. Decreases of B and T cell proliferation in vitro and in vivo, and of dendritic cell maturation and T cell stimulatory activity in vitro were also detected. Absence of signaling through the IFN-alpha/betaR, however, did not affect increased basal levels of the IFN-responsive p202 phosphoprotein, encoded by a polymorphic variant of the Ifi202 gene associated with the Nba2 predisposing locus in NZB mice. The data indicate that type-I IFNs are important mediators in the pathogenesis of murine lupus, and that reducing their activity in the human counterpart may be beneficial.
Dendritic cell (DC) activation by nucleic acid-containing immunoglobulin (Ig)G complexes has been implicated in systemic lupus erythematosus (SLE) pathogenesis. However, the mechanisms responsible for activation and subsequent disease induction are not completely understood. Here we show that murine DCs are much more effectively activated by immune complexes that contain IgG bound to chromatin than by immune complexes that contain foreign protein. Activation by these chromatin immune complexes occurs by two distinct pathways. One pathway involves dual engagement of the Fc receptor FcgammaRIII and Toll-like receptor (TLR)9, whereas the other is TLR9 independent. Furthermore, there is a characteristic cytokine profile elicited by the chromatin immune complexes that distinguishes this response from that of conventional TLR ligands, notably the induction of BAFF and the lack of induction of interleukin 12. The data establish a critical role for self-antigen in DC activation and explain how the innate immune system might drive the adaptive immune response in SLE.
Inflammasomes are cytoplasmic sensors of foreign molecules, including pathogens, and function to induce caspase-1 activation and IL-1β cytokine maturation. Whether such a mechanism exists in the nucleus and is effective against nuclear replicating pathogens is unknown. Nuclear replicating herpesvirus KSHV is associated with Kaposi Sarcoma, an angioproliferative tumor characterized by an inflammatory microenvironment including IL-1β. We demonstrate that during KSHV infection of endothelial cells, interferon gamma-inducible protein 16 (IFI16) interacts with the adaptor molecule ASC and procaspase-1 to form a functional inflammasome. This complex was initially detected in the nucleus and subsequently in the perinuclear area. KSHV gene expression and/or latent KSHV genome is required for inflammasome activation and IFI16 colocalizes with the KSHV genome in the infected cell nucleus. Caspase-1 activation by KSHV was reduced by IFI16 and ASC silencing. Our studies reveal IFI16 as a nuclear pathogen sensor and demonstrate that the inflammasome also functions in the nucleus.
Viral infection triggers innate immune sensors to produce type I interferon. However, infection of T cells and macrophages with human immunodeficiency virus (HIV) does not trip those alarms. How HIV avoids activating nucleic acid sensors is unknown. Here we found that the cytosolic exonuclease TREX1 suppressed interferon triggered by HIV. In Trex1(-/-) mouse cells and human CD4(+) T cells and macrophages in which TREX1 was inhibited by RNA-mediated interference, cytosolic HIV DNA accumulated and HIV infection induced type I interferon that inhibited HIV replication and spreading. TREX1 bound to cytosolic HIV DNA and digested excess HIV DNA that would otherwise activate interferon expression via a pathway dependent on the kinase TBK1, the adaptor STING and the transcription factor IRF3. HIV-stimulated interferon production in cells deficient in TREX1 did not involve known nucleic acid sensors.
Type I interferons (IFNs) are secreted cytokines that orchestrate diverse immune responses to infection. Although typically considered to be most important in the response to viruses, type I IFNs are also induced by most, if not all, bacterial pathogens. Although diverse mechanisms have been described, bacterial induction of type I IFNs occurs upon stimulation of two main pathways: (i) Toll-like receptor (TLR) recognition of bacterial molecules such as lipopolysaccharide (LPS); (ii) TLR-independent recognition of molecules delivered to the host cell cytosol. Cytosolic responses can be activated by two general mechanisms. First, viable bacteria can secrete stimulatory ligands into the cytosol via specialized bacterial secretion systems. Second, ligands can be released from bacteria that lyse or are degraded. The bacterial ligands that induce the cytosolic pathways remain uncertain in many cases, but appear to include various nucleic acids. In this review, we discuss recent advances in our understanding of how bacteria induce type I interferons and the roles type I IFNs play in host immunity.
Intracellular nucleic acid sensors detect microbial RNA and DNA and trigger the production of type I interferon. However, the cytosolic nucleic acid-sensing system remains to be fully identified. Here we show that the cytosolic nucleic acid-binding protein LRRFIP1 contributed to the production of interferon-beta (IFN-beta) induced by vesicular stomatitis virus (VSV) and Listeria monocytogenes in macrophages. LRRFIP1 bound exogenous nucleic acids and increased the expression of IFN-beta induced by both double-stranded RNA and double-stranded DNA. LRRFIP1 interacted with beta-catenin and promoted the activation of beta-catenin, which increased IFN-beta expression by binding to the C-terminal domain of the transcription factor IRF3 and recruiting the acetyltransferase p300 to the IFN-beta enhanceosome via IRF3. Therefore, LRRFIP1 and its downstream partner beta-catenin constitute another coactivator pathway for IRF3-mediated production of type I interferon.
Virus infection in mammals elicits a variety of defense responses that are initiated by signals from virus-sensing receptors expressed by the host. These receptors include the ubiquitously expressed RIG-I-like receptor (RLR) family of RNA helicases. RLRs are cytoplasmic proteins that act in cell-intrinsic antiviral defense by recognizing RNAs indicative of virus presence. Here, we highlight recent progress in understanding how RLRs discriminate between the RNA content of healthy versus virus-infected cells, functioning as accurate sensors of virus invasion.
Aicardi-Goutières syndrome (AGS) is a genetically determined encephalopathy demonstrating phenotypic overlap both with the sequelae of congenital infection and with systemic lupus erythematosus (SLE). Recent molecular advances have revealed that AGS can be caused by mutations in any one of five genes, most commonly on a recessive basis but occasionally as a dominant trait. Like AGS, SLE is associated with a perturbation of type I interferon metabolism. Interestingly then, heterozygous mutations in the AGS1 gene TREX1 underlie a cutaneous subtype of SLE-called familial chilblain lupus, and mutations in TREX1 represent the single most common cause of monogenic SLE identified to date. Evidence is emerging to show that the nucleases defective in AGS are involved in removing endogenously produced nucleic acid (NA) species, and that a failure of this removal results in activation of the immune system. This hypothesis explains the phenotypic overlap of AGS with congenital infection and some aspects of SLE, where an equivalent type I interferon-mediated innate immune response is triggered by viral and self NAs, respectively. The combined efforts of clinicians, geneticists, immunologists and cell biologists are producing rapid progress in the understanding of AGS and overlapping autoimmune disorders. These studies provide important insights into the pathogenesis of SLE and beg urgent questions about the development and use of immunosuppressive therapies in AGS and related phenotypes.
The innate immune system is critical for the early detection of invading pathogens and for initiating cellular host defence countermeasures, which include the production of type I interferon (IFN). However, little is known about how the innate immune system is galvanized to respond to DNA-based microbes. Here we show that STING (stimulator of interferon genes) is critical for the induction of IFN by non-CpG intracellular DNA species produced by various DNA pathogens after infection. Murine embryonic fibroblasts, as well as antigen presenting cells such as macrophages and dendritic cells (exposed to intracellular B-form DNA, the DNA virus herpes simplex virus 1 (HSV-1) or bacteria Listeria monocytogenes), were found to require STING to initiate effective IFN production. Accordingly, Sting-knockout mice were susceptible to lethal infection after exposure to HSV-1. The importance of STING in facilitating DNA-mediated innate immune responses was further evident because cytotoxic T-cell responses induced by plasmid DNA vaccination were reduced in Sting-deficient animals. In the presence of intracellular DNA, STING relocalized with TANK-binding kinase 1 (TBK1) from the endoplasmic reticulum to perinuclear vesicles containing the exocyst component Sec5 (also known as EXOC2). Collectively, our studies indicate that STING is essential for host defence against DNA pathogens such as HSV-1 and facilitates the adjuvant activity of DNA-based vaccines.
Type I interferons (IFNs) are important for antiviral and autoimmune responses. Retinoic acid-induced gene I (RIG-I) and mitochondrial antiviral signaling (MAVS) proteins mediate IFN production in response to cytosolic double-stranded RNA or single-stranded RNA containing 5'-triphosphate (5'-ppp). Cytosolic B form double-stranded DNA, such as poly(dA-dT)*poly(dA-dT) [poly(dA-dT)], can also induce IFN-beta, but the underlying mechanism is unknown. Here, we show that the cytosolic poly(dA-dT) DNA is converted into 5'-ppp RNA to induce IFN-beta through the RIG-I pathway. Biochemical purification led to the identification of DNA-dependent RNA polymerase III (Pol-III) as the enzyme responsible for synthesizing 5'-ppp RNA from the poly(dA-dT) template. Inhibition of RNA Pol-III prevents IFN-beta induction by transfection of DNA or infection with DNA viruses. Furthermore, Pol-III inhibition abrogates IFN-beta induction by the intracellular bacterium Legionella pneumophila and promotes the bacterial growth. These results suggest that RNA Pol-III is a cytosolic DNA sensor involved in innate immune responses.
The cellular innate immune system is essential for recognizing pathogen
infection and for establishing effective host defence. But critical
molecular determinants responsible for facilitating an appropriate
immune response-following infection with DNA and RNA viruses, for
example-remain to be identified. Here we report the identification,
following expression cloning, of a molecule (STING; stimulator of
interferon genes) that appears essential for effective innate immune
signalling processes. It comprises five putative transmembrane regions,
predominantly resides in the endoplasmic reticulum and is able to
activate both NF-κB and IRF3 transcription pathways to induce
expression of type I interferon (IFN-α and IFN-β ) and exert
a potent anti-viral state following expression. In contrast, loss of
STING rendered murine embryonic fibroblasts extremely susceptible to
negative-stranded virus infection, including vesicular stomatitis virus.
Further, STING ablation abrogated the ability of intracellular B-form
DNA, as well as members of the herpesvirus family, to induce IFN-β,
but did not significantly affect the Toll-like receptor (TLR) pathway.
Yeast two-hybrid and co-immunoprecipitation studies indicated that STING
interacts with RIG-I and with SSR2 (also known as TRAPβ), which is
a member of the translocon-associated protein (TRAP) complex required
for protein translocation across the endoplasmic reticulum membrane
following translation. Ablation by RNA interference of both TRAPβ
and translocon adaptor SEC61β was subsequently found to inhibit
STING's ability to stimulate expression of IFN-β. Thus, as well as
identifying a regulator of innate immune signalling, our results imply a
potential role for the translocon in innate signalling pathways
activated by select viruses as well as intracellular DNA.
Viral infection triggers activation of transcription factors such as NF-kappaB and IRF3, which collaborate to induce type I interferons (IFNs) and elicit innate antiviral response. Here, we identified MITA as a critical mediator of virus-triggered type I IFN signaling by expression cloning. Overexpression of MITA activated IRF3, whereas knockdown of MITA inhibited virus-triggered activation of IRF3, expression of type I IFNs, and cellular antiviral response. MITA was found to localize to the outer membrane of mitochondria and to be associated with VISA, a mitochondrial protein that acts as an adaptor in virus-triggered signaling. MITA also interacted with IRF3 and recruited the kinase TBK1 to the VISA-associated complex. MITA was phosphorylated by TBK1, which is required for MITA-mediated activation of IRF3. Our results suggest that MITA is a critical mediator of virus-triggered IRF3 activation and IFN expression and further demonstrate the importance of certain mitochondrial proteins in innate antiviral immunity.
Detection of nucleic acids and induction of type I interferons (IFNs) are principal elements of antiviral defense but can cause autoimmunity if misregulated. Cytosolic DNA detection activates a potent, cell-intrinsic antiviral response through a poorly defined pathway. In a screen for proteins relevant to this IFN-stimulatory DNA (ISD) response, we identify 3' repair exonuclease 1 (Trex1). Mutations in the human trex1 gene cause Aicardi-Goutieres syndrome (AGS) and chilblain lupus, but the molecular basis of these diseases is unknown. We define Trex1 as an essential negative regulator of the ISD response and delineate the genetic pathway linking Trex1 deficiency to lethal autoimmunity. We show that single-stranded DNA derived from endogenous retroelements accumulates in Trex1-deficient cells, and that Trex1 can metabolize reverse-transcribed DNA. These findings reveal a cell-intrinsic mechanism for initiation of autoimmunity, implicate the ISD pathway as the cause of AGS, and suggest an unanticipated contribution of endogenous retroelements to autoimmunity.
DNA from bacteria has stimulatory effects on mammalian immune cells, which depend on the presence of unmethylated CpG dinucleotides in the bacterial DNA. In contrast, mammalian DNA has a low frequency of CpG dinucleotides, and these are mostly methylated; therefore, mammalian DNA does not have immuno-stimulatory activity. CpG DNA induces a strong T-helper-1-like inflammatory response. Accumulating evidence has revealed the therapeutic potential of CpG DNA as adjuvants for vaccination strategies for cancer, allergy and infectious diseases. Despite its promising clinical use, the molecular mechanism by which CpG DNA activates immune cells remains unclear. Here we show that cellular response to CpG DNA is mediated by a Toll-like receptor, TLR9. TLR9-deficient (TLR9-/-) mice did not show any response to CpG DNA, including proliferation of splenocytes, inflammatory cytokine production from macrophages and maturation of dendritic cells. TLR9-/- mice showed resistance to the lethal effect of CpG DNA without any elevation of serum pro-inflammatory cytokine levels. The in vivo CpG-DNA-mediated T-helper type-1 response was also abolished in TLR9-/- mice. Thus, vertebrate immune systems appear to have evolved a specific Toll-like receptor that distinguishes bacterial DNA from self-DNA.
Plasmacytoid dendritic cell (P-DC) precursors in peripheral blood produce large amounts of interferon (IFN)-alpha/beta when triggered by viruses. However, when incubated with interleukin-3 and CD40 ligand, the same precursors differentiate into mature DCs that stimulate naïve CD4(+) T cells to produce Th2 cytokines. We recently reported that P-DCs accumulate in nasal mucosa of experimentally induced allergic rhinitis, supporting a role for this DC subset in Th2-dominated inflammation. Here we examined whether P-DCs accumulate in cutaneous lesions of lupus erythematosus (LE), a disorder associated with increased IFN-alpha/beta production. Our results showed that P-DCs were present in 14 out of 15 tissue specimens of cutaneous LE lesions, but not in normal skin. Importantly, the density of P-DCs in affected skin correlated well (r(s) = 0.79,P < 0.0005) with the high number of cells expressing the IFN-alpha/beta-inducible protein MxA, suggesting that P-DCs produce IFN-alpha/beta locally. Accumulation of P-DCs coincided also with the expression of L-selectin ligand peripheral lymph node addressin on dermal vascular endothelium, adding further support to the notion that these adhesion molecules are important in P-DC extravasation to peripheral tissue sites. Together, our findings suggested that P-DCs are an important source of IFN-alpha/beta in cutaneous LE lesions and may therefore be of pathogenic importance.
Systemic lupus erythematosus (SLE) patients have increased levels of interferon-alfa (IFN-a) in the circulation but a reduced number of functionally intact natural IFN-a producing cells (IPC) in peripheral blood. In search for tissue localisation of activated IPC, we investigated skin biopsies from SLE patients for the occurrence of such cells.
Eleven SLE patients with inflammatory skin lesions and six healthy controls were biopsied. An immunohistochemical technique (IH) and in situ hybridisation (ISH) were used to detect intracellular IFN-a protein and IFN-a mRNA, respectively.
In all 11 biopsies from SLE lesions, a high number of IPC were detected by IH. In the nonlesional SLE biopsies we could also demonstrate IPC in 10=11 patients. In 6=11 SLE patients, IFN- a mRNA containing cells could be detected in the specimens. A low number of IPC were detected in 1=6 healthy controls by IH, but no ISH positive cells were seen.
Our results demonstrate that SLE patients have active IPC in both dermal lesions and in noninflammatory skin. A recruitment of IPC from blood to peripheral tissues may explain the low number of circulating natural IPC in SLE patients. Because the type I IFN system is involved in the SLE disease process, these results are of interest for the understanding of the pathogenesis in SLE.
Dendritic cells (DCs) are important in regulating both immunity and tolerance. Hence, we hypothesized that systemic lupus erythematosus (SLE), an autoimmune disease characterized by autoreactive B and T cells, may be caused by alterations in the functions of DCs. Consistent with this, monocytes from SLE patients' blood were found to function as antigen-presenting cells, in vitro. Furthermore, serum from SLE patients induced normal monocytes to differentiate into DCs. These DCs could capture antigens from dying cells and present them to CD4-positive T cells. The capacity of SLE patients' serum to induce DC differentiation correlated with disease activity and depended on the actions of interferon-alpha (IFN-alpha). Thus, unabated induction of DCs by IFN-alpha may drive the autoimmune response in SLE.
Type I Interferons (IFN-I) are immunoregulatory cytokines that enhance activation and survival of many cellular components of the immune system. In the present work, we evaluated the effect of IFN-I on the development of the lymphoproliferative disorder in Fas-defective lpr mice. We report that sustained injection of polyinosinic:polycytidylic acid, a potent inducer of IFN-I, in B6 lpr mice resulted in a dramatic aggravation of the renal disease, higher titers of autoantibodies, a 10-fold increase in serum Ig and accumulation of activated lymphocytes. Moreover, introducing a null mutation for the IFN-I-Receptor gene into the lpr background resulted in dramatic decrease of immune complexes deposition in the kidney and reduced lymphadenopathy. While several recent reports correlated serum levels of IFN-alpha with disease activity in systemic Lupus erythematosus patients, our findings establish a causal link from IFN-I production to the onset and severity of another related autoimmune syndrome.
The interferon-inducible p200 (IFI-200) family of proteins is among the numerous gene products induced by interferons (IFNs), which are important regulators of cell growth, immunomodulation and host resistance to tumors and viral infections. The members of this family of proteins are highly homologous to one another and consist of five murine proteins including p202, p203, p204 and p205 as well as three human homologues; IFI-16, myeloid cell nuclear differentiation antigen (MNDA) and absent in melanoma (AIM) 2. They possess at least one copy of a conserved 200 amino-acid motif which exists in two types; the a and b domains. Most of the IFI-200 proteins also possess a domain in apoptosis and interferon response (DAPIN)/PYRIN domain, which is a conserved motif associated with protein-protein interactions in the regulation of apoptotic and inflammatory signaling pathways. The p200 proteins have been implicated in cell cycle regulation and differentiation based on their ability to interact with and modulate the activities of multiple transcriptional factors such as Rb and p53, and there are significant findings that link mutations in their genetic loci to the incidence of cancer. Here, we describe the structure and biological activities of these proteins, and discuss recent studies that describe their relevant roles in processes regulating cell proliferation and differentiation.
Previously, we showed that bacterial DNA and vertebrate DNA/cationic liposome complexes stimulate potent inflammatory responses in cultured mouse macrophages. In the present study, we examined whether endocytosis and subsequent acidification are associated with these responses. The endocytosis inhibitor, cytochalasin B, reduced tumor necrosis factor alpha (TNF-alpha) production by a plasmid DNA (pDNA)/cationic liposome complex. The endosomal acidification inhibitor, monensin, inhibited cytokine production by pDNA or a calf thymus DNA/liposome complex. These results suggest, similarly to CpG motif-dependent responses, that endocytosis and subsequent endosomal acidification are also required for these inflammatory responses. It is intriguing that another inhibitor of endosomal acidification, bafilomycin A, stimulated the production of TNF-alpha mRNA and its protein after removal of the pDNA/liposome complex and inhibitors, although it inhibited the release of interleukin-6. Similar phenomena were observed in the activation of macrophages by CpG oligodeoxynucleotide, calf thymus DNA, and Escherichia coli DNA complexed with liposomes. Moreover, bafilomycin A also induced a high degree of TNF-alpha release after stimulation with naked pDNA. These results suggest that bafilomycin A increases TNF-alpha production induced by DNA at the transcriptional level via an as-yet unknown mechanism. Furthermore, we investigated the contribution of Toll-like receptor 9 (TLR9), the receptor of CpG motifs, to the cell activation by the DNA/cationic liposome complex using the macrophages from TLR9-/- mice. We observed a reduced inflammatory cytokine release from macrophages of TLR9-/- mice compared with wild-type mice. However, the cytokine production was not completely abolished, suggesting that the DNA/cationic liposome complex can induce macrophage activation via TLR9-dependent and -independent pathways.