Self-nucleic Acids Research Articles

Neurophysiological phenotypes are uncoupled from toll-like receptor (TLR)-mediated systemic disease in a model of Systemic Lupus Erythematosus (SLE)

Abstract SLE is a multisystemic autoimmune disease with diverse clinical presentations, including neuropsychiatric symptoms (NPSLE). Prior work implicates uncontrolled MyD88-dependent TLR activation, increased interferon regulatory factor 5 (IRF5) activity and elevated type I interferon (IFN) levels in disease. Immune complexes containing self-nucleic acids can activate endosomal TLRs 7/8/9, which require signaling adaptor MyD88 to modulate downstream gene expression through transcription factor 1) IRF7 to upregulate IFN, which can subsequently act on the IFN-α/β receptor (IFNAR) or 2) IRF5 to upregulate proinflammatory genes. SLE- and NPSLE-prone mice with conditional deletion of signaling mediators MyD88, IRF5 and IFNAR in dendritic cells and tissue resident-macrophages were generated. MyD88 and IRF5 deletion ameliorated systemic disease phenotypes including splenomegaly, lymphadenopathy and glomerulonephritis, but NPSLE-associated phenotypes in the brain, including expansion of total and disease-associated microglia, macrophages and extravascular T cells, persisted. In contrast, IFNAR deletion worsened systemic disease, yet reversed several NPSLE-associated phenotypes. These discoveries suggest an uncoupling of systemic and neurophysiological phenotypes in SLE. Namely, neurophysiological phenotypes manifest independently from the TLR-dependent pathway that drives systemic disease and are instead in part linked to aberrant signaling through IFNAR via mechanisms yet unexplored.

Type I Interferon Signalling and Ischemic Stroke: Mechanisms and Therapeutic Potentials.

Type I interferon (IFN-I) signalling is intricately involved in the pathogenesis of multiple infectious diseases, autoimmune diseases, and neurological diseases. Acute ischemic stroke provokes overactivation of IFN-I signalling within the injured brain, particularly in microglia. Following cerebral ischemia, damage-associated molecular patterns (DAMPs) released from injured neural cells elicit marked proinflammatory episodes within minutes. Among these, self-nucleic acids, including nuclear DNA and mitochondrial DNA (mtDNA), have been recognized as a critical alarm signal to fan the flames of neuroinflammation, predominantly via inducing IFN-I signalling activation in microglia. The concept of interferon-responsive microglia (IRM), marked by upregulation of a plethora of IFN-stimulated genes, has been emergingly elucidated in ischemic mouse brains, particularly in aged ones. Among the pattern recognition receptors responsible for IFN-I induction, cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) plays integral roles in potentiating microglia-driven neuroinflammation and secondary brain injury after cerebral ischemia. Here, we aim to provide an up-to-date review on the multifaceted roles of IFN-I signalling, the detailed molecular and cellular mechanisms leading to and resulting from aberrant IFN-I signalling activation after cerebral ischemia, and the therapeutic potentials. A thorough exploration of these above points will inform our quest for IFN-based therapies as effective immunomodulatory therapeutics to complement the limited repertoire of thrombolytic agents, thereby facilitating the translation from bench to bedside.

UNC93B1 variants underlie TLR7-dependent autoimmunity.

UNC93B1 is critical for trafficking and function of nucleic acid-sensing Toll-like receptors (TLRs) TLR3, TLR7, TLR8, and TLR9, which are essential for antiviral immunity. Overactive TLR7 signaling induced by recognition of self-nucleic acids has been implicated in systemic lupus erythematosus (SLE). Here, we report UNC93B1 variants (E92G and R336L) in four patients with early-onset SLE. Patient cells or mouse macrophages carrying the UNC93B1 variants produced high amounts of TNF-α and IL-6 and upon stimulation with TLR7/TLR8 agonist, but not with TLR3 or TLR9 agonists. E92G causes UNC93B1 protein instability and reduced interaction with TLR7, leading to selective TLR7 hyperactivation with constitutive type I IFN signaling. Thus, UNC93B1 regulates TLR subtype-specific mechanisms of ligand recognition. Our findings establish a pivotal role for UNC93B1 in TLR7-dependent autoimmunity and highlight the therapeutic potential of targeting TLR7 in SLE.

The Sixth Sense: Self-nucleic acid sensing in the brain.

Our innate immune system uses pattern recognition receptors (PRRs) as a first line of defense to detect microbial ligands and initiate an immune response. Viral nucleic acids are key ligands for the activation of many PRRs and the induction of downstream inflammatory and antiviral effects. Initially it was thought that endogenous (self) nucleic acids rarely activated these PRRs, however emerging evidence indicates that endogenous nucleic acids are able to activate host PRRs in homeostasis and disease. In fact, many regulatory mechanisms are in place to finely control and regulate sensing of self-nucleic acids by PRRs. Sensing of self-nucleic acids is particularly important in the brain, as perturbations to nucleic acid sensing commonly leads to neuropathology. This review will highlight the role of nucleic acid sensors in the brain, both in disease and homeostasis. We also indicate the source of endogenous stimulatory nucleic acids where known and summarize future directions for the study of this growing field.

Aicardi-Goutières syndrome: A monogenic type I interferonopathy.

Aicardi-Goutières syndrome (AGS) is a rare monogenic autoimmune disease that primarily affects the brains of children patients. Its main clinical features include encephalatrophy, basal ganglia calcification, leukoencephalopathy, lymphocytosis and increased interferon-α (IFN-α) levels in the patient's cerebrospinal fluid (CSF) and serum. AGS may be caused by mutations in any one of nine genes (TREX1, RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1, ADAR1, IFIH1, LSM11 and RNU7-1) that result in accumulation of self-nucleic acids in the cytoplasm or aberrant sensing of self-nucleic acids. This triggers overproduction of type I interferons (IFNs) and subsequently causes AGS, the prototype of type I interferonopathies. This review describes the discovery history of AGS with various genotypes and provides the latest knowledge of clinical manifestations and causative genes of AGS. The relationship between AGS and type I interferonopathy and potential therapeutic methods for AGS are also discussed in this review.

Stray self-nucleic acids: the emerging drivers of neurodegeneration.

Emergent evidence has shown that abnormal buildup of stray self-nucleic acids is a pathological feature observed across many neurodegenerative conditions. Here, we discuss how these self-nucleic acids act as a driver of disease by triggering harmful inflammatory responses. Understanding these pathways and targeting them has the potential to prevent neuronal death at the early stages of disease.

Rosmarinic acid ameliorates autoimmune responses through suppression of intracellular nucleic acid-mediated type I interferon expression

Recognition of intracellular nucleic acids is a vital step for host to mount prompt immune responses against microbial pathogens. However, inappropriate response to self-nucleic acids leads to sustained type I interferon (IFN) production, which is implicated in the development of several autoimmune diseases, such as Aicardi–Goutières syndrome (AGS). Therefore, effective confinement of intracellular nucleic acid-induced IFN expression is a potential strategy for the treatment of such autoimmune diseases. In this study, we found that rosmarinic acid (RA), a natural compound isolated from rosemary, inhibits intracellular nucleic acid-stimulated IFN expression. Mechanistic investigation revealed that RA binds to both G3BP1 and cGAS, and impairs cGAS activation through disrupting the binding of DNA with cGAS. More importantly, we showed that RA could effectively attenuate the expression of IFN-stimulated genes (ISGs) in the well-established cell models for AGS. Thus, our study provides a promising compound for the treatment of autoimmune responses induced by aberrant nucleic acid-sensing.

Aberrant B Cell Signaling in Autoimmune Diseases.

Aberrant B cell signaling plays a critical in role in various systemic and organ-specific autoimmune diseases. This is supported by genetic evidence by many functional studies in B cells from patients or specific animal models and by the observed efficacy of small-molecule inhibitors. In this review, we first discuss key signal transduction pathways downstream of the B cell receptor (BCR) that ensure that autoreactive B cells are removed from the repertoire or functionally silenced. We provide an overview of aberrant BCR signaling that is associated with inappropriate B cell repertoire selection and activation or survival of peripheral B cell populations and plasma cells, finally leading to autoantibody formation. Next to BCR signaling, abnormalities in other signal transduction pathways have been implicated in autoimmune disease. These include reduced activity of several phosphates that are downstream of co-inhibitory receptors on B cells and increased levels of BAFF and APRIL, which support survival of B cells and plasma cells. Importantly, pathogenic synergy of the BCR and Toll-like receptors (TLR), which can be activated by endogenous ligands, such as self-nucleic acids, has been shown to enhance autoimmunity. Finally, we will briefly discuss therapeutic strategies for autoimmune disease based on interfering with signal transduction in B cells.

The nucleic acid sensor RIG-I as an inducer of anti-tumor response

Abstract The immune microenvironment plays a critical role in tumor progression. Recently, approaches to target Pattern Recognition Receptors (PRRs) have emerged as a promising strategy to enhance immune recognition of tumors. Among the PRRs, those which recognize DNA and RNA in the cells are unique in their ability to respond to both pathogenic and self-nucleic acids that are generated as a result of aberrant cell division/transcription. We previously showed that targeting the DNA sensor three prime exonuclease 1 (TREX1) enhanced immune responses in mouse tumor models. Here we show that the RNA sensor Retinoic acid Induced Gene 1 (RIG-I) acts as a robust immune activator across cancer types. According cBioportal data, expression of RIG-I seems to be a prognostic factor for progression free survival. To functional evaluate RIG-I response in tumor models, we used a hairpin RNA derived from H1N1 sequence. Interestingly, activation of RIG-I increased expression of TREX1 revealing a potential crosstalk between the two pathways. Both breast and colorectal tumor cell lines produce IFN-I after RIG-I activation. Consistently, RNAseq showed upregulation of IFN-I response genes and proinflammatory cytokines across different cell lines. Activation of RIG-I in the tumor cells decreased tumor burden with a concomitant broad cellular and molecular anti-tumor response. Thus, results showed an increase in NK and DC population, while the transcription profile was upregulation of innate immune response genes. Interestingly, tumor growth and NK results were consistent when tumor was implanted in immunocompromised mouse model. Taken together, our findings illustrate how tumor cell RIG-I is a potent inducer of cellular and molecular immune responses.

The Signal Peptide and Chaperone UNC93B1 Both Influence TLR8 Ectodomain Intracellular Endosomal Localization.

Toll-like receptor 8 (TLR8) is a single-stranded RNA sensing receptor and is localized in the cellular compartments, where it encounters foreign or self-nucleic acids and activates innate and adaptive immune responses. However, the mechanism controlling intracellular localization TLR8 is not completely resolved. We previously revealed the intracellular localization of TLR8 ectodomain (ECD), and in this study, we investigated the mechanism of the intracellular localization. Here we found that TLR8 ECDs from different species as well as ECDs from different TLRs are all intracellularly localized, similarly to the full-length porcine TLR8. Furthermore, porcine, bovine, and human TLR8 ECDs are all localized in cell endosomes, reflecting the cellular localization of TLR8. Intriguingly, none of post-translational modifications at single sites, including glycosylation, phosphorylation, ubiquitination, acetylation, and palmitoylation alter porcine TLR8-ECD endosomal localization. Nevertheless, the signal peptide of porcine TLR8-ECD determines its endosomal localization. On the other hand, signaling regulator UNC93B1 also decides the endosomal localization of porcine, bovine, and human TLR8 ECDs. The results from this study shed light on the mechanisms of not only TLR8 intracellular localization but also the TLR immune signaling.

Sensing of cytoplasmic chromatin by cGAS activates innate immune response in SARS-CoV-2 infection

The global coronavirus disease 2019 (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a positive-sense RNA virus. How the host immune system senses and responds to SARS-CoV-2 infection remain largely unresolved. Here, we report that SARS-CoV-2 infection activates the innate immune response through the cytosolic DNA sensing cGAS-STING pathway. SARS-CoV-2 infection induces the cellular level of 2′3′-cGAMP associated with STING activation. cGAS recognizes chromatin DNA shuttled from the nucleus as a result of cell-to-cell fusion upon SARS-CoV-2 infection. We further demonstrate that the expression of spike protein from SARS-CoV-2 and ACE2 from host cells is sufficient to trigger cytoplasmic chromatin upon cell fusion. Furthermore, cytoplasmic chromatin-cGAS-STING pathway, but not MAVS-mediated viral RNA sensing pathway, contributes to interferon and pro-inflammatory gene expression upon cell fusion. Finally, we show that cGAS is required for host antiviral responses against SARS-CoV-2, and a STING-activating compound potently inhibits viral replication. Together, our study reported a previously unappreciated mechanism by which the host innate immune system responds to SARS-CoV-2 infection, mediated by cytoplasmic chromatin from the infected cells. Targeting the cytoplasmic chromatin-cGAS-STING pathway may offer novel therapeutic opportunities in treating COVID-19. In addition, these findings extend our knowledge in host defense against viral infection by showing that host cells’ self-nucleic acids can be employed as a “danger signal” to alarm the immune system.

Role of nucleic acid sensing in the pathogenesis of type 1 diabetes.

During infections, nucleic acids of pathogens are also engaged in recognition via several exogenous and cytosolic pattern recognition receptors, such as the toll-like receptors, retinoic acid inducible gene-I-like receptors, and nucleotide-binding and oligomerization domain-like receptors. The binding of the pathogen-derived nucleic acids to their corresponding sensors initiates certain downstream signaling cascades culminating in the release of type-I interferons (IFNs), especially IFN-α and other cytokines to induce proinflammatory responses towards invading pathogens leading to their clearance from the host. Although these sensors are hardwired to recognize pathogen associated molecular patterns, like viral and bacterial nucleic acids, under unusual physiological conditions, such as excessive cellular stress and increased apoptosis, endogenous self-nucleic acids like DNA, RNA, and mitochondrial DNA are also released. The presence of these self-nucleic acids in extranuclear compartments or extracellular spaces or their association with certain proteins sometimes leads to the failure of discriminating mechanisms of nucleic acid sensors leading to proinflammatory responses as seen in autoimmune disorders, like systemic lupus erythematosus, psoriasis and to some extent in type 1 diabetes (T1D). This review discusses the involvement of various nucleic acid sensors in autoimmunity and discusses how aberrant recognition of self-nucleic acids by their sensors activates the innate immune responses during the pathogenesis of T1D.

Regulation of the nucleic acid-sensing Toll-like receptors.

Many of the ligands for Toll-like receptors (TLRs) are unique to microorganisms, such that receptor activation unequivocally indicates the presence of something foreign. However, a subset of TLRs recognizes nucleic acids, which are present in both the host and foreign microorganisms. This specificity enables broad recognition by virtue of the ubiquity of nucleic acids but also introduces the possibility of self-recognition and autoinflammatory or autoimmune disease. Defining the regulatory mechanisms required to ensure proper discrimination between foreign and self-nucleic acids by TLRs is an area of intense research. Progress over the past decade has revealed a complex array of regulatory mechanisms that ensure maintenance of this delicate balance. These regulatory mechanisms can be divided into a conceptual framework with four categories: compartmentalization, ligand availability, receptor expression and signal transduction. In this Review, we discuss our current understanding of each of these layers of regulation.

Selectins impair regulatory T cell function and contribute to systemic lupus erythematosus pathogenesis.

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by a loss of tolerance toward self-nucleic acids, autoantibody production, interferon expression and signaling, and a defect in the regulatory T (Treg) cell compartment. In this work, we identified that platelets from patients with active SLE preferentially interacted with Treg cells via the P-selectin/P-selectin glycoprotein ligand-1 (PSGL-1) axis. Selectin interaction with PSGL-1 blocked the regulatory and suppressive properties of Treg cells and particularly follicular Treg cells by triggering Syk phosphorylation and an increase in intracytosolic calcium. Mechanistically, P-selectin engagement on Treg cells induced a down-regulation of the transforming growth factor-β axis, altering the phenotype of Treg cells and limiting their immunosuppressive responses. In patients with SLE, we found an up-regulation of P- and E-selectin both on microparticles and in their soluble forms that correlated with disease activity. Last, blocking P-selectin in a mouse model of SLE improved cardinal features of the disease, such as anti-dsDNA antibody concentrations and kidney pathology. Overall, our results identify a P-selectin-dependent pathway that is active in patients with SLE and validate it as a potential therapeutic avenue.

Priming of the cGAS-STING-TBK1 Pathway Enhances LPS-Induced Release of Type I Interferons.

Cytoplasmic nucleic acids sensing through cGAS-STING-TBK1 pathway is crucial for the production of antiviral interferons (IFNs). IFN production can also be induced by lipopolysaccharide (LPS) stimulation through Toll-like receptor 4 (TLR4) in appropriate conditions. Of note, both IFN production and dysregulated LPS-response could play a role in the pathogenesis of Systemic Lupus Erythematosus (SLE). Indeed, LPS can trigger SLE in lupus-prone mice and bacterial infections can induce disease flares in human SLE. However, the interactions between cGAS and TLR4 pathways to IFNs have been poorly investigated. To address this issue, we studied LPS-stimulation in cellular models with a primed cGAS-STING-TBK1 pathway. cGAS-stimulation was naturally sustained by undigested self-nucleic acids in fibroblasts from DNase2-deficiency interferonopathy, whilst it was pharmacologically obtained by cGAMP-stimulation in THP1 cells and murine bone marrow-derived dendritic cells. We showed that cells with a primed cGAS-STING-TBK1 pathway displayed enhanced IFNs production after TLR4-challenge. STING-inhibition did not affect IFN production after LPS alone, but prevented the amplified IFN production in cGAMP-primed cells, suggesting that functional STING is required for priming-dependent enhancement. Furthermore, we speculated that an increased PIK3AP1 expression in DNase2-deficient fibroblasts may link cGAMP-priming with increased LPS-induced IFN production. We showed that both the hyper-expression of PIK3API and the enhanced LPS-induced IFN production can be contrasted by STING inhibitors. Our results may explain how bacterial LPS can synergize with cGAS-pathway in promoting the development of SLE-like autoimmunity.

The Role of Nucleases and Nucleic Acid Editing Enzymes in the Regulation of Self-Nucleic Acid Sensing.

Detection of microbial nucleic acids by the innate immune system is mediated by numerous intracellular nucleic acids sensors. Upon the detection of nucleic acids these sensors induce the production of inflammatory cytokines, and thus play a crucial role in the activation of anti-microbial immunity. In addition to microbial genetic material, nucleic acid sensors can also recognize self-nucleic acids exposed extracellularly during turn-over of cells, inefficient efferocytosis, or intracellularly upon mislocalization. Safeguard mechanisms have evolved to dispose of such self-nucleic acids to impede the development of autoinflammatory and autoimmune responses. These safeguard mechanisms involve nucleases that are either specific to DNA (DNases) or RNA (RNases) as well as nucleic acid editing enzymes, whose biochemical properties, expression profiles, functions and mechanisms of action will be detailed in this review. Fully elucidating the role of these enzymes in degrading and/or processing of self-nucleic acids to thwart their immunostimulatory potential is of utmost importance to develop novel therapeutic strategies for patients affected by inflammatory and autoimmune diseases.

Cell Surface Expression of Endosomal Toll-Like Receptors-A Necessity or a Superfluous Duplication?

Timely and precise delivery of the endosomal Toll-like receptors (TLRs) to the ligand recognition site is a critical event in mounting an effective antimicrobial immune response, however, the same TLRs should maintain the delicate balance of avoiding recognition of self-nucleic acids. Such sensing is widely known to start from endosomal compartments, but recently enough evidence has accumulated supporting the idea that TLR-mediated signaling pathways originating in the cell membrane may be engaged in various cells due to differential expression and distribution of the endosomal TLRs. Therefore, the presence of endosomal TLRs on the cell surface could benefit the host responses in certain cell types and/or organs. Although not fully understood why, TLR3, TLR7, and TLR9 may occur both in the cell membrane and intracellularly, and it seems that activation of the immune response can be initiated concurrently from these two sites in the cell. Furthermore, various forms of endosomal TLRs may be transported to the cell membrane, indicating that this may be a normal process orchestrated by cysteine proteases—cathepsins. Among the endosomal TLRs, TLR3 belongs to the evolutionary distinct group and engages a different protein adapter in the signaling cascade. The differently glycosylated forms of TLR3 are transported by UNC93B1 to the cell membrane, unlike TLR7, TLR8, and TLR9. The aim of this review is to reconcile various views on the cell surface positioning of endosomal TLRs and add perspective to the implication of such receptor localization on their function, with special attention to TLR3. Cell membrane-localized TLR3, TLR7, and TLR9 may contribute to endosomal TLR-mediated inflammatory signaling pathways. Dissecting this signaling axis may serve to better understand mechanisms influencing endosomal TLR-mediated inflammation, thus determine whether it is a necessity for immune response or simply a circumstantial superfluous duplication, with other consequences on immune response.

Research in practice: Disturbance in intracellular nucleic acid metabolism promotes lupus erythematosus

Lupus erythematosus (LE) is an autoimmune disease with a broad spectrum of cutaneous manifestations. It is characterized by a loss of tolerance to nuclear antigens, including endogenous nucleic acids, autoantibody formation and upregulation of the typeI interferon pathway. This can be induced by an immune response to unrestricted extracellular self-antigens. In addition, the molecular characterization of rare monogenic subtypes of LE has revealed insights into the role of cell-intrinsic sensing of endogenous self-nucleic acids in inducing a typeI interferon response. This pathway can also initiate and sustain LE. The enhanced understanding of innate and adaptive immune responses in LE is a prerequisite for the development of more targeted therapies.

Signaling Through Nucleic Acid Sensors and Their Roles in Inflammatory Diseases.

Recognition of pathogen-derived nucleic acids by pattern-recognition receptors (PRRs) is essential for eliciting antiviral immune responses by inducing the production of type I interferons (IFNs) and proinflammatory cytokines. Such responses are a prerequisite for mounting innate and pathogen-specific adaptive immune responses. However, host cells also use nucleic acids as carriers of genetic information, and the aberrant recognition of self-nucleic acids by PRRs is associated with the onset of autoimmune or autoinflammatory diseases. In this review, we describe the mechanisms of nucleic acid sensing by PRRs, including Toll-like receptors, RIG-I-like receptors, and DNA sensor molecules, and their signaling pathways as well as the disorders caused by uncontrolled or unnecessary activation of these PRRs.

Loosening the grip on nuclear cGAS.

Maintaining ignorance to self-nucleic acids can prevent inflammatory diseases such as Aicardi–Goutieres syndrome (AGS). A new study finds that mutations in LSM11 and RNU7-1, which encode components of the histone messenger RNA–preprocessing complex, cause AGS by loosening the binding of cyclic GMP–AMP synthase (cGAS) to nucleosomes, thus enabling cGAS activation and induction of type I interferons.