Nucleic Acid Receptors Research Articles

Mechanisms and Effects of Activation of Innate Immunity by Mitochondrial Nucleic Acids.

In recent years, a growing number of roles have been identified for mitochondria in innate immunity. One principal mechanism is that translocation of mitochondrial nucleic acid species from the mitochondrial matrix to the cytosol and endolysosomal lumen in response to an array of microbial and non-microbial environmental stressors has been found to serve as a second messenger event in the cell signaling of the innate immune response. Thus, mitochondrial DNA and RNA have been shown to access the cytosol through several regulated mechanisms involving remodeling of the mitochondrial inner and outer membranes and to access lysosomes via vesicular transport, thereby activating cytosolic (e.g., cyclic GMP-AMP synthase [cGAS]; retinoic acid-inducible gene-I [RIG-I]-like receptors) and endolysosomal (Toll-like Receptor [TLR]7, -9) nucleic acid receptors that induce type I interferons and pro-inflammatory cytokines. In this mini-review, we discuss these molecular mechanisms of mitochondrial nucleic acid mislocalization and their roles in host defense, autoimmunity, and auto-inflammatory disorders. The emergent paradigm is one in which host-derived DNA interestingly serves as a signal amplifier in the innate immune response and also as an alarm signal for disturbances in organellar homeostasis. The apparent vast excess of mitochondria and mitochondrial DNA nucleoids per cell may thus serve to sensitize the cell response to stressors while ensuring an underlying reserve of intact mitochondria to sustain cellular metabolism. An improved understanding of these molecular mechanisms will hopefully afford future opportunities for therapeutic intervention in human disease.

Innate Immune Sensing of Adeno-Associated Virus Vectors.

Adeno-associated virus (AAV) based viral vectors are widely used in human gene therapy and form the basis of approved treatments for several genetic diseases. Immune responses to vector and transgene products, however, substantially complicate these applications in clinical practice. The role of innate immune recognition of AAV vectors was initially unclear, given that inflammatory responses early after vector administration were typically mild in animal models. However, more recent research continues to identify innate immune pathways that are triggered by AAV vectors and that serve to provide activation signals for antigen-presenting cells and initiation of adaptive immune responses. Sensing of the AAV genome by the endosomal DNA receptor toll-like receptor 9 (TLR9) promotes early inflammatory response and interferon expression. Thus, activation of the TLR9>MyD88 pathway in plasmacytoid dendritic cells (pDCs) leads to the conditioning of antigen cross-presenting DCs through type I interferon (IFN-I) and ultimately CD8+ T cell activation. Alternatively, pDCs may also promote CD8+ T cell responses in a TLR9-independent manner by the production of IL-1 cytokines, thereby activating the IL-1R1>MyD88 signaling pathway. AAV can induce cytokine expression in monocyte-derived DCs, which in turn increases antibody formation. Binding of AAV capsid to complement components likely further elevates B cell activation. At high systemic vector doses in humans and in non-human primates, AAV vectors can trigger complement activation, with contributions by classical and alternative pathways, leading to severe toxicities. Finally, evidence for activation of TLR2 by the capsid and of additional innate receptors for nucleic acids has been presented. These observations show that AAV vectors can initiate several and likely redundant innate immune pathways resulting in an exaggerated adaptive immune response.

Synthetic Genes For Dynamic Regulation Of DNA‐Based Receptors

AbstractWe present a strategy to control dynamically the loading and release of molecular ligands from synthetic nucleic acid receptors using in vitro transcription. We demonstrate this by engineering three model synthetic DNA‐based receptors: a triplex‐forming DNA complex, an ATP‐binding aptamer, and a hairpin strand, whose ability to bind their specific ligands can be cotranscriptionally regulated (activated or inhibited) through specific RNA molecules produced by rationally designed synthetic genes. The kinetics of our DNA sensors and their genetically generated inputs can be captured using differential equation models, corroborating the predictability of the approach used. This approach shows that highly programmable nucleic acid receptors can be controlled with molecular instructions provided by dynamic transcriptional systems, illustrating their promise in the context of coupling DNA nanotechnology with biological signaling.

Synthetic Genes For Dynamic Regulation Of DNA-Based Receptors.

We present a strategy to control dynamically the loading and release of molecular ligands from synthetic nucleic acid receptors using in vitro transcription. We demonstrate this by engineering three model synthetic DNA-based receptors: a triplex-forming DNA complex, an ATP-binding aptamer, and a hairpin strand, whose ability to bind their specific ligands can be cotranscriptionally regulated (activated or inhibited) through specific RNA molecules produced by rationally designed synthetic genes. The kinetics of our DNA sensors and their genetically generated inputs can be captured using differential equation models, corroborating the predictability of the approach used. This approach shows that highly programmable nucleic acid receptors can be controlled with molecular instructions provided by dynamic transcriptional systems, illustrating their promise in the context of coupling DNA nanotechnology with biological signaling.

Glabridin improves autoimmune disease in Trex1-deficient mice by reducing type I interferon production

BackgroundThe cGAS-STING signaling pathway is an essential section of the natural immune system. In recent years, an increasing number of studies have shown a strong link between abnormal activation of the cGAS-STING signaling pathway, a natural immune pathway mediated by the nucleic acid receptor cGAS, and the development and progression of autoimmune diseases. Therefore, it is important to identify an effective compound to specifically downregulate this pathway for disease.MethodsThe effect of Glabridin (Glab) was investigated in BMDMs and Peripheral blood mononuclear cell (PBMC) by establishing an in vitro model of cGAS-STING signaling pathway activation. An activation model stimulated by DMXAA was also established in mice to study the effect of Glab. On the other hand, we investigated the possible mechanism of action of Glab and the effect of Glab on Trex1-deficient mice.ResultsIn this research, we report that Glab, a major component of licorice, specifically inhibits the cGAS-STING signaling pathway by inhibiting the level of type I interferon and inflammatory cytokines (IL-6 and TNF-α). In addition, Glab has a therapeutic effect on innate immune diseases caused by abnormal cytoplasmic DNA in Trex1-deficient mice. Mechanistically, Glab can specifically inhibit the interaction of STING with IRF3.ConclusionGlab is a specific inhibitor of the cGAS-STING signaling pathway and may be used in the clinical therapy of cGAS-STING pathway-mediated autoimmune diseases.

Circulating NETs Promote Platelet-TLR9 Dependent Acute Chest Syndrome in Sickle Cell Disease

Acute chest syndrome (ACS), a type of acute lung injury is one of the primary reasons for mortality among Sickle Cell Disease (SCD) patients. Although new evidence suggests that circulating neutrophil extracellular traps (cNETs) promote lung injury in SCD by triggering occlusion of pulmonary arterioles by neutrophil-platelet-erythrocyte thrombo-inflammatory aggregates, the molecular mechanism underlying cNETs-dependent lung vaso-occlusion remains unknown. Surprisingly, platelets are one of the few cell types that express Toll-like receptor 9 (TLR9), a receptor for double-stranded DNA on the surface (rather than in endosomes), however, the role of platelet-TLR9 in promoting pulmonary thrombo-inflammation in SCD has never been studied. Using intravital (in vivo) lung microscopy in live SCD mice and imaging flow cytometry (in vitro) of SCD mice plasma, we show for the first time that lung vaso-occlusion and elevated levels of cNETs in knock-in humanized SCD mice challenged with 10 μmol/kg oxy-hemoglobin (oxy-Hb) is associated with significant upregulation of TLR9 surface expression in platelets. Importantly, TLR9 inhibition using a function blocking Ab led to significant reduction by ~6 and ~4 fold in the frequency and size of lung vaso-occlusions in SCD mice challenged with IV oxy-Hb. Identical to SCD mice, TLR9 surface expression was also significantly higher in SCD than control human platelets, and the expression was further upregulated following treatment of SCD patient platelet-rich-plasma (PRP) with oxy-Hb. Remarkably, elevated TLR9 expression in SCD patient platelets was concomitant to increased phosphorylation of both downstream TLR9 pathway effector tank-binding-kinase-1 (TBK-1) and the TBK-1 substrate interferon regulatory factor-3 (IRF3), which is the major transcription factor driving IFN-1 response. Taken together, our current findings suggest for the first time that activation of nucleic acid receptor TLR9 on the surface of platelets by cNETs contributes to lung vaso-occlusion and acute chest syndrome in SCD.

Leukemic Cell-Intrinsic Innate Nucleic Acid Receptor Signaling Governs Efficacy of Hypomethylating Agents and Immunosurveillance in AML

Introduction: Hypomethylating agents (HMA), such as Azaciditine (AZA), are a long-established treatment option for acute myeloid leukemia (AML). Besides epigenetic reprogramming of malignant cell clones, HMAs are recognized to facilitate antineoplastic immunity. In vitro assessments in solid tumor models suggest that a major mechanism underlying AZA-triggered immune responses may be the activation of innate nucleic acid sensing pathways. As such, the RNA- and DNA-sensing receptor systems, RIG-I/MDA-5 and cGAS/STING - and subsequent type I interferon (IFN-I) signaling seem to be triggered by the transcriptional upregulation of otherwise DNA-hypermethylated and thus silenced endogenous retroviruses (ERVs). In AML, it is unknown whether similar processes are involved and/or by which molecular mechanisms, HMAs modulate anti-leukemic immunity against AML blasts, and how these processes are affected during acquired HMA resistance. Methods: Murine (c1498 and Wehi-3B) and human (MOLM-13) AML cell lines were exposed to AZA in vitro and analyzed for expression of ERVs. Nucleic acid receptor sensing pathways and IFN-I genes are not usually DNA methylated by AZA at promoter regions. However, the re-expression of endogenous nucleic acids or ERVs could trigger their activation. Protein and transcriptional expression of major components of nucleic acid receptor sensing pathways and IFN-I genes were, therefore, analysed. To understand the importance of AML cell-intrinsic nucleic acid receptor signaling in HMA-induced immunosurveillance, syngeneic c1498 and Wehi-3B AML models were employed. Mice received 2 cycles of 2.5mg/kg AZA (6 days on, 5 days off, 6 days on) intraperitoneally to mimic the principal treatment course used in patients. To address the contribution of the nucleic acid-sensing pathways to AZA treatment efficacy in vitroand in vivo, AML cells with acquired AZA resistance (AZA-R) or gene engineered AML cells deficient in RNA receptors (RIG-I/MDA-5), or the RNA- and DNA-sensing adapter proteins (MAVS and STING) were employed. Results: A targeted ERV screen revealed upregulation of various ERV transcripts in c1498 and Wehi-3B cells and was similarly observed in MOLM-13 AML cells following AZA exposure in vitro. Increased RNA transcript and protein expression of RIG-I/MAVS and (to a lesser degree) STING was observed, resulting in activation of downstream constituents including higher phosphorylation of the transcription factors IRF3/7 and NF-kB, IFN-I, and other ISGs. This was abrogated in RIG-I and MAVS, but not STING, deficient murine and human AML cells. Interestingly, expression of RIG-I/MDA-5, STING and all other downstream constituents, but not MAVS, were diminished in AZA-R AML cells. In mice harboring MAVS deficient AML (c1498), HMA treatment failed to show anti-leukemic activity resulting in rapid disease progress and death of recipient animals. Loss of STING in leukemic cells also impacted AZA treatment efficacy, although to a much less pronounced degree. These findings were similarly reproduced with Wehi-3B AML-engrafted mice. Following antibody-mediated depletion of T cells but not NK cells, mice rapidly succumbed to AML disease and demonstrated no anti-leukemic effect of AZA treatment. CyTOF and flow cytometric analysis also revealed increased bone marrow and spleen infiltration as well as activation of CD4 +/CD8 + T cells, NK cells, and MHC-II + APCs/Myeloid cells in AZA treated mice bearing wildtype AML, compared to untreated. This was not observed in AZA-treated mice bearing MAVS-deficient or AZA-R AML. Conclusion: Our findings indicate that nucleic acid receptor signaling (specifically RIG-I/MAVS) within leukemic cells is imperative for AZA efficacy and immunosurveillance in AML. Whether these RNA-sensing receptors are triggered by ERVs or alternate (endogenous) RNAs remains to be determined. Elucidating the molecular mechanisms of how nucleic acid sensors within leukemic blasts shape treatment efficacy of AZA may guide development of systematic immunotherapeutic strategies for AZA-R AML by targeted combinatorial approaches.

Updated roles of cGAS-STING signaling in autoimmune diseases.

Natural immunity, the first line for the body to defense against the invasion of pathogen, serves as the body's perception of the presence of pathogens depends on nucleic acid recognition mechanisms. The cyclic GMP-AMP synthase-stimulator of the interferon gene (cGAS-STING) signaling pathway is considered an essential pattern recognition and effector pathway in the natural immune system and is mainly responsible for recognizing DNA molecules present in the cytoplasm and activating downstream signaling pathways to generate type I interferons and some other inflammatory factors. STING, a crucial junction protein in the innate immune system, exerts an essential role in host resistance to external pathogen invasion. Also, STING, with the same character of inflammatory molecules, is inseparable from the body's inflammatory response. In particular, when the expression of STING is upregulated or its related signaling pathways are overactivated, the body may develop serious infectious disorders due to the generation of excessive inflammatory responses, non-infectious diseases, and autoimmune diseases. In recent years, accumulating studies indicated that the abnormal activation of the natural immune cGAS-STING signaling pathway modulated by the nucleic acid receptor cGAS closely associated with the development and occurrence of autoimmune diseases (AID). Thereof, to explore an in-depth role of STING and its related signaling pathways in the diseases associated with inflammation may be helpful to provide new avenues for the treatment of these diseases in the clinic. This article reviews the activation process of the cGAS-STING signaling pathways and its related important roles, and therapeutic drugs in AID, aiming to improve our understanding of AID and achieve better diagnosis and treatment of AID.

Potential of Nucleic Acid Receptor Ligands to Improve Vaccination Efficacy against the Filarial Nematode Litomosoides sigmodontis.

More than two-hundred-million people are infected with filariae worldwide. However, there is no vaccine available that confers long-lasting protection against filarial infections. Previous studies indicated that vaccination with irradiated infective L3 larvae reduces the worm load. This present study investigated whether the additional activation of cytosolic nucleic acid receptors as an adjuvant improves the efficacy of vaccination with irradiated L3 larvae of the rodent filaria Litomosoides sigmodontis with the aim of identifying novel vaccination strategies for filarial infections. Subcutaneous injection of irradiated L3 larvae in combination with poly(I:C) or 3pRNA resulted in neutrophil recruitment to the skin, accompanied by higher IP-10/CXCL10 and IFN-β RNA levels. To investigate the impact on parasite clearance, BALB/c mice received three subcutaneous injections in 2-week intervals with irradiated L3 larvae in combination with poly(I:C) or 3pRNA prior to the challenge infection. Vaccination with irradiated L3 larvae in combination with poly(I:C) or 3pRNA led to a markedly greater reduction in adult-worm counts by 73% and 57%, respectively, compared to the immunization with irradiated L3 larvae alone (45%). In conclusion, activation of nucleic acid-sensing immune receptors boosts the protective immune response against L. sigmodontis and nucleic acid-receptor agonists as vaccine adjuvants represent a promising novel strategy to improve the efficacy of vaccines against filariae and potentially other helminths.

Increased IKKϵ protein stability ensures efficient type I interferon responses in conditions of TBK1 deficiency.

TBK1 and IKKϵ are related, crucial kinases in antiviral immune signaling pathways downstream of cytosolic nucleic acid receptors such as cGAS and RIG-I-like receptors. Upon activation, they phosphorylate the transcription factors IRF3 and IRF7 and thereby initiate the expression of type I interferons and antiviral effectors. While point mutation-induced loss of TBK1 kinase activity results in clinical hyper-susceptibility to viral infections, a complete lack of TBK1 expression in humans is unexpectedly not associated with diminished antiviral responses. Here, we provide a mechanistic explanation for these so-far unexplained observations by showing that TBK1 controls the protein expression of its related kinase IKKϵ in human myeloid cells. Mechanistically, TBK1 constitutively diminishes the protein stability of IKKϵ independent of TBK1 kinase activity but dependent on its interaction with the scaffold protein TANK. In consequence, depletion of TBK1 protein but not mutation-induced kinase deficiency induces the upregulation of IKKϵ. Due to the functional redundancy of the kinases in cGAS-STING and RIG-I-like receptor signaling in human myeloid cells, enhanced IKKϵ expression can compensate for the loss of TBK1. We show that IKKϵ upregulation is crucial to ensure unmitigated type I interferon production in conditions of TBK1 deficiency: While the type I interferon response to Listeria monocytogenes infection is maintained upon TBK1 loss, it is strongly diminished in cells harboring a kinase-deficient TBK1 variant, in which IKKϵ is not upregulated. Many pathogens induce TBK1 degradation, suggesting that loss of TBK1-mediated destabilization of IKKϵ is a critical backup mechanism to prevent diminished interferon responses upon TBK1 depletion.

(Invited) Designed DNA Nano-Switches As Sensitive Electrochemical Biosensors

Functional nucleic acid receptors (aptamers) have emerged as effective and robust recognition elements for state-of-the-art biosensors. Analytical readouts from aptamer-based biosensors (whether optical, electrochemical, or otherwise) derive primarily from global-scale conformational changes induced in the aptamer domain by analyte binding. Herein, we describe a unique biosensor design principle that represents a distinct alternative to this paradigm; particularly we demonstrate the ready applicability of this design principle in the de novo creation of electrochemical sensors for a clinical analyte of current interest. The function of the class of biosensors we describe, termed “DNA nano-switches”, is designed to depend on the integrity of duplex DNA-mediated charge transfer between an electrode and a redox label.[1] Thomas, J. M.; Chakraborty, B.; Sen, D.; Yu, H.-Z. J. Am. Chem. Soc. 2012, 134, 13823–13833.[2] Tang, Y.; Ge, B.; Sen, D.; Yu, H.-Z. Chem. Soc. Rev. 2014, 43, 518–529[3] Ma, F.; Qi, L.; Einarson, O.; Sen, D.; Yu, H.-Z. Anal. Chem. 2019, 91, 8244−8251. Figure 1

Z-DNA binding protein 1 promotes heatstroke-induced cell death.

Heatstroke is a heat stress-induced, life-threatening condition associated with circulatory failure and multiple organ dysfunctions. If global warming continues, heatstroke might become a more prominent cause of mortality worldwide, but its pathogenic mechanism is not well understood. We found that Z-DNA binding protein 1 (ZBP1), a Z-nucleic acid receptor, mediated heatstroke by triggering receptor-interacting protein kinase 3 (RIPK3)-dependent cell death. Heat stress increased the expression of ZBP1 through heat shock transcription factor 1 (HSF1) and activated ZBP1 through a mechanism independent of the nucleic acid sensing action. Deletion of ZBP1, RIPK3, or both mixed lineage kinase domain-like (MLKL) and caspase-8 decreased heat stress-induced circulatory failure, organ injury, and lethality. Thus, ZBP1 appears to have a second function that orchestrates host responses to heat stress.

Abstract LBA010: Therapeutic stimulation of nucleic acid receptor RIG-I enhances efficacy of kinase inhibitor treatment in oncogene-driven tumors

Abstract Introduction: A major challenge in the treatment of kinase driven tumors is the inevitable emergence of resistance and the limited efficacy of subsequent treatments including immunotherapy. Methods: To gain deeper insights into the adaptive processes of cells surviving kinase inhibition we studied the mechanisms enabling cell persistence despite targeted treatment across various in vitro and in vivo tumor models. This included transcriptomic profiles of EGFR-, BRAF-, HER2- or ALK-driven colon, melanoma and lung cancer cells and of melanoma patients during kinase inhibition. To investigate underlying mechanisms we employed CRISPR/Cas9 and overexpression experiments and stimulation of nucleic acid receptors (NARs) TLR3, cGAS and RIG-I with synthetic agonists to assess combination therapies. Finally, we used humanized tumor xenografts and a syngeneic EGFR-mutant lung cancer model to validate the inflammatory effects of kinase inhibition and the efficacy of adding RIG-I agonist IVT4 to kinase inhibition in vivo. Results: Analysis of dynamic transcriptomic changes during kinase inhibition by time-series RNA-seq of oncogene-dependent cells displayed rapid induction of an interferon gene signature and suppression of MYC- and E2F-target genes. This was accompanied by induction of p27 protein levels, cell cycle arrest and an increase of b-galactosidase positive cells, indicating a senescence-associated inflammatory response that occurred in a cell autonomous manner in vitro and in vivo. In vivo, EGFR inhibition also increased T-cells, but anti-PD-1 therapy following EGFR inhibitor pre-treatment did not prevent tumor recurrence. Interestingly, the kinase inhibitor induced interferon signature was independent of caspase activation and of the nucleic acid sensing machinery. Instead, functional experiments indicated that induction of interferon signaling was dependent on inhibition of MAPK signaling and subsequent IRF1 activation, providing a potential mechanism of action. Numerous innate immune pathway members including NARs MDA-5, TLR3 and RIG-I were upregulated, which we aimed to exploit therapeutically. Evaluation of NAR agonists showed that only stimulation of RIG-I with its agonist IVT4 evoked cytokine secretion and synergistically enhanced cell death in vitro. In humanized xenografts and syngenic immunocompetent mouse models adding IVT4 led to additional tumor shrinkage after EGFR-inhibition and decreased T cell exhaustion. Effects of IVT4 on tumor volumes were lower in immunodeficient xenografts and after NK- or CD8-cell depletion in the syngeneic model indicating that immunological influences in addition to tumor cell intrinsic effects contribute to the efficacy in vivo. Conclusion: Taken together, our findings show that kinase inhibition may promote tumor cell-autonomous, senescence-associated inflammatory signaling and may sensitize oncogene-driven tumors to therapeutic intervention using RIG-I agonist IVT4. Our results thus provide a novel treatment strategy and a basis to explore combining immunotherapy with kinase inhibition in the future. Citation Format: Johannes Brägelmann, Carina Lorenz, Sven Borchmann, Kazuya Nishii, Julia Wegner, Jenny Ostendorp, David Ast, Alena Heimsoeth, Philipp Lohneis, Thomas Zillinger, Roland Ullrich, Kadoaki Ohashi, Martin Schlee, Martin Sos. Therapeutic stimulation of nucleic acid receptor RIG-I enhances efficacy of kinase inhibitor treatment in oncogene-driven tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr LBA010.

CGAS-like receptors: ancient catchers of viral nucleic acids

Living organisms have evolved conserved 'catchers' for detecting and handling molecular patterns from invading viruses. Slavik et al. and Holleufer et al. recently identified cyclic GMP-AMP (cGAMP) synthase (cGAS)-like receptors that detect double-stranded viral RNA in Drosophila. cGAS-like receptors constitute a new expanding family of pattern recognition receptors (PRRs) for viral nucleic acids.

Bacterial death and TRADD-N domains help define novel apoptosis and immunity mechanisms shared by prokaryotes and metazoans.

Several homologous domains are shared by eukaryotic immunity and programmed cell-death systems and poorly understood bacterial proteins. Recent studies show these to be components of a network of highly regulated systems connecting apoptotic processes to counter-invader immunity, in prokaryotes with a multicellular habit. However, the provenance of key adaptor domains, namely those of the Death-like and TRADD-N superfamilies, a quintessential feature of metazoan apoptotic systems, remained murky. Here, we use sensitive sequence analysis and comparative genomics methods to identify unambiguous bacterial homologs of the Death-like and TRADD-N superfamilies. We show the former to have arisen as part of a radiation of effector-associated α-helical adaptor domains that likely mediate homotypic interactions bringing together diverse effector and signaling domains in predicted bacterial apoptosis- and counter-invader systems. Similarly, we show that the TRADD-N domain defines a key, widespread signaling bridge that links effector deployment to invader-sensing in multicellular bacterial and metazoan counter-invader systems. TRADD-N domains are expanded in aggregating marine invertebrates and point to distinctive diversifying immune strategies probably directed both at RNA and retroviruses and cellular pathogens that might infect such communities. These TRADD-N and Death-like domains helped identify several new bacterial and metazoan counter-invader systems featuring underappreciated, common functional principles: the use of intracellular invader-sensing lectin-like (NPCBM and FGS), transcription elongation GreA/B-C, glycosyltransferase-4 family, inactive NTPase (serving as nucleic acid receptors), and invader-sensing GTPase switch domains. Finally, these findings point to the possibility of multicellular bacteria-stem metazoan symbiosis in the emergence of the immune/apoptotic systems of the latter.

Making Sense of Intracellular Nucleic Acid Sensing in Type I Interferon Activation in Sjögren's Syndrome.

Primary Sjögren’s syndrome (pSS) is a systemic autoimmune rheumatic disease characterized by dryness of the eyes and mucous membranes, which can be accompanied by various extraglandular autoimmune manifestations. The majority of patients exhibit persistent systemic activation of the type I interferon (IFN) system, a feature that is shared with other systemic autoimmune diseases. Type I IFNs are integral to anti-viral immunity and are produced in response to stimulation of pattern recognition receptors, among which nucleic acid (NA) receptors. Dysregulated detection of endogenous NAs has been widely implicated in the pathogenesis of systemic autoimmune diseases. Stimulation of endosomal Toll-like receptors by NA-containing immune complexes are considered to contribute to the systemic type I IFN activation. Accumulating evidence suggest additional roles for cytosolic NA-sensing pathways in the pathogenesis of systemic autoimmune rheumatic diseases. In this review, we will provide an overview of the functions and signaling of intracellular RNA- and DNA-sensing receptors and summarize the evidence for a potential role of these receptors in the pathogenesis of pSS and the sustained systemic type I IFN activation.

IRGM1 links mitochondrial quality control to autoimmunity.

Mitochondrial abnormalities have been noted in lupus, but the causes and consequences remain obscure. Autophagy-related genes ATG5, ATG7, and IRGM have been previously implicated in autoimmune disease. We reasoned that failure to clear defective mitochondria via mitophagy might be a foundational driver in autoimmunity by licensing mitochondrial (mt)DNA-dependent induction of type I interferon (IFN-I). Here, we show that mice lacking the GTPase IRGM1 (IRGM homologue) exhibited a type I interferonopathy with autoimmune features. Irgm1 deletion impaired execution of mitophagy with cell-specific consequences. In fibroblasts, mtDNA soiling of the cytosol induced cyclic GMP-AMP synthase (cGAS)–Stimulator of Interferon Genes (STING)-dependent IFN-I, whereas in macrophages, lysosomal TLR7 was activated. In vivo, Irgm1−/− tissues exhibited mosaic dependency upon nucleic acid receptors. Whereas salivary and lacrimal gland autoimmune pathology were abolished and lung pathology was attenuated by cGAS and STING deletion, pancreatic pathology remained unchanged. These findings reveal fundamental connections between mitochondrial quality control and tissue-selective autoimmune disease.

Human Cancer Cells Sense Cytosolic Nucleic Acids Through the RIG-I-MAVS Pathway and cGAS-STING Pathway.

Pattern recognition receptors (PRRs) are germline-encoded host sensors of the innate immune system. Some human cancer cells have been reported to express PRRs. However, nucleic acid sensors in human cancers have not been studied in detail. Therefore, we systematically analyzed the expression, molecular cascade, and functions of TLR3, RIG-I, MDA5, LGP2, cGAS, and STING in human cancer cells. TLR3, TRIF, RIG-I, MDA5, LGP2, and MAVS were expressed in 22 cell lines. The majority of cell lines responded to only RIG-I ligands 5′-ppp-dsRNA, Poly(I:C)-HMW, Poly(I:C)-LMW, and/or Poly(dA:dT), as revealed by IRF3 phosphorylation and IFN-β secretion. IFN-β secretion was inhibited by RIG-I and MAVS knockdown. cGAS and STING were co-expressed in 10 of 22 cell lines, but IFN-β secretion was not induced by STING ligands ISD, HSV60, VACV70, Poly(dG:dC), and 3′3′-cGAMP in cGAS and STING intact cell lines. Further experiments revealed that the cGAS–STING pathway was activated, as revealed by TBK1 and IRF3 phosphorylation and IFN-β and ISG mRNA expression. These results suggest that human epithelial cancer cells respond to cytosolic RNA through the RIG-I–MAVS pathway but only sense cytosolic DNA through the cGAS–STING pathway. These findings are relevant for cancer immunotherapy approaches based on targeting nucleic acid receptors.

RNA-Editing-Initiated MAVS Signaling is a Key Epitranscriptomic Alteration in Human B Cell Lymphoma

Diffuse large B cell lymphoma (DLBCL) is one of the most common types of aggressive lymphoid malignancy. Genetically, it is a highly heterogeneous disease. Here, we explore the contribution of an epitranscriptomic modification (RNA editing) to DLBCL pathogenesis. We demonstrate that DNA mutations and RNA editing events are often mutually exclusive, suggesting that tumours can modulate pathway outcomes by altering sequence at either the genomic or the transcriptomic level. We further show that in DLBCL, RNA editing targets transcripts within known disease-driving pathways such as apoptosis, p53 and NF-κB signaling, as well as novel gene targets like MAVS, the essential downstream adaptor of RIG-I-like receptors and a central molecule in the innate response to RNA viruses. We next illustrate that ADAR1-mediated editing in the MAVS transcript correlates with increased MAVS mRNA and protein expression levels. Increased MAVS expression in DLBCL furthermore associates with increased interferon/NF-κB signaling and increased T cell exhaustion. We validate these findings in independent cohorts of DLBCL samples as well as another type of germinal center related B cell lymphoma, follicular lymphoma. Finally, using targeted RNA base editing tools to restore editing specifically within MAVS 3'UTR in ADAR1-deficient cells, we demonstrate that editing is causal to an increase in downstream signaling, in the absence of activation by canonical nucleic acid receptor sensing. Overall, ADAR1-mediated RNA editing represents a new mechanism underlying human B cell lymphomagenesis.

Endometrial expression of various genes (ISGs, PPARs, RXRs and MUC1) on day 16 post-ovulation in repeat breeder cows, with or without subclinical endometritis

Our objective was to elucidate differences in endometrial mRNA expressions of interferon-stimulated genes (ISG15, CTSL1, RSAD2, SLC2A1, CXCL10, and SLC27A6), peroxisome proliferator activated receptors (PPARA, PPARD, and PPARG), retinoic acid receptors (RXRA, RXRB, and RXRG), and mucin 1 (MUC1) in repeat breeder cows, with or without subclinical endometritis (RB + SE and RB, respectively) and normal cows on day 16 post-ovulation (n = 4 cows per group). The CXCL10 and SLC27A6 mRNA abundances were greater for normal cows compared to RB and RB + SE cows (P < 0.05 and P < 0.01 respectively) whereas ISG15 and SLC2A1 mRNA abundances were greater for normal cows compared to RB + SE (P < 0.05). The SLC27A6 mRNA abundances were greater for RB versus RB + SE (P < 0.01). Similarly, PPARD, PPARG, RXRA and RXRG mRNA abundances were greater for normal cows compared to RB and RB + SE (P < 0.01 and P < 0.05, respectively). Abundances of PPARD, PPARG, RXRA and RXRG mRNA were greater for RB versus RB + SE (P < 0.05) and MUC1 was lower in abundance in normal cows compared to RB or RB + SE (P < 0.05). Key predicted molecular functions were binding, signal transducer and transporter; key biological processes were cellular, localization and metabolic; key cellular components were cell part, membrane and organelle components; and key protein classes were nucleic acid binding, receptor, and transcription factors. Gene networking analysis highlighted interactions and pathways involving PAPRs, RXRs, and MUC1, notably among PPARD, PPARG, and MUC1. In conclusion, endometrial mRNA expressions of ISGs (CXCL10 and SLC27A6), PPAR isomers (PPARD and PPARG), and RXRs (RXRA and RXRG) were in lower abundances, whereas MUC1 expression was more abundant in RB or RB + SE compared to normal cows on day 16. In addition, ISG15 and SLC2A1 genes were less abundant in RB + SE versus RB or normal cows. Altered expression of these uterine genes and associated potential impairment in embryo elongation and implantation may promote embryonic loss in repeat breeder cows. Furthermore, interactions among PPARD, PPARG and MUC1 may be therapeutically exploitable.