1,720,985 research outputs found
Discovery of a Novel cGAMP Exporter that Regulates Innate Antiviral Immunity
No full textThesis (Ph.D.)--University of Washington, 2022The DNA sensor cyclic GMP-AMP synthase (cGAS) is important for antiviral and anti-tumor immunity. cGAS generates cyclic GMP-AMP (cGAMP), a diffusible cyclic dinucleotide that activates the antiviral response through the adapter protein Stimulator of Interferon Genes (STING). cGAMP cannot passively cross cell membranes, but recent advances have established a role for extracellular cGAMP as an “immunotransmitter” that can be imported into cells. The mechanism by which cGAMP exits cells remains unknown. Here, we identify ABCC1/MRP1 as an ATP-dependent cGAMP exporter. We show that ABCC1 overexpression enhances cGAMP export and limits STING signaling, and that loss of ABCC1 reduces cGAMP export and potentiates STING signaling. We demonstrate that ABCC1 deficiency exacerbates cGAS-dependent autoimmunity in the Trex1-/- mouse model of Aicardi-Goutières syndrome. These studies identify ABCC1-mediated cGAMP export as a key regulatory mechanism that limits cell intrinsic activation of STING and ameliorates STING-dependent autoimmune disease
Human DNA-PK activates a STING-independent DNA sensing pathway
No full textThesis (Ph.D.)--University of Washington, 2019Recognition of foreign nucleic acids is critical for antiviral defense. Detection of DNA is mediated by the cGAS-STING pathway, which activates a potent type I interferon response. This pathway is broadly required for antiviral defense across cell types and species, and its relevance in context of infection, cancer, and autoimmunity has been thoroughly established. However, we have discovered an additional STING-independent DNA sensing pathway (SIDSP) in human cells. Using STING KO human cell lines, we find that the induction of interferon is indeed abrogated at early timepoints, but surprisingly, at later timepoints, we observe a robust DNA-induced interferon response. Here we identify DNA-PK as the sensor for the SIDSP and demonstrate that its kinase activity is required for the antiviral response. We show that a heat shock protein HSPA8/HSC70 is phosphorylated after DNA stimulation and acts as a marker for the SIDSP. Finally, we explore how DNA viruses antagonize both the cGAS-STING pathway and the SIDSP. Our work highlights the importance of nucleic acid sensing for both host and virus and has implications for modulating DNA sensing in order to improve therapies for cancer or autoimmunity
Regulation and Endogenous Activators of the Cell-Intrinsic Antiviral Response
Get PDFThesis (Ph.D.)--University of Washington, 2014The primary objective of immune sensors is to differentiate self from non-self. Detection of viral genomes leads to activation of the antiviral response, accompanied by the production of type I interferons. However, these viral nucleic acids are very similar to host produced RNA and DNA. We investigate how human variants in SKIV2L, an RNA metabolism enzyme, can lead to erroneous interferon production by allowing an accumulation of self-RNA. While studying sources of the endogenous ligands that can accumulate, we considered the role of RNase L in the antiviral response. While the OAS/2-5A system and RNase L have been studied for many years, our findings suggest a new model for their function. We reveal that RNase L acts as a negative regulator of the antiviral response by eliciting global translational inhibition through cleavage of ribosomal RNA. We go on to show that the different 2-5A species synthesized by OAS proteins are endogenous immunostimulatory ligands, and that the OAS/2-5A system generates ligands for crosstalk between the RNA and DNA sensing and signaling networks within every cell. These studies have far-reaching implications for the detection of endogenous RNA ligands, their metabolism, and their association with autoimmune disorders. Furthermore, the reappraisal of RNase L and the new role for 2-5A species as endogenous activators of the antiviral response come with their own clinical implications, and allow for a more complete understanding of the early cellular response to viral pathogens
The role of ADAR1 in innate immune regulation and cell biology
No full textThesis (Ph.D.)--University of Washington, 2016-12University of Washington Abstract The Role of ADAR1 in Innate Immune Regulation and Cell Biology Kathleen M. Pestal Chair of Supervisory Committee: Associate Professor Daniel B. Stetson Department of Immunology Mutations in ADAR, which encodes the ADAR1 RNA editing enzyme, cause Aicardi–Goutières syndrome (AGS), a severe inflammatory disease associated with an aberrant type I interferon response. Aicardi–Goutières syndrome has previously been shown to result from ineffectual negative regulation of the cytosolic DNA sensing pathway. Here, we demonstrate that ADAR1 is a specific and essential negative regulator of the MDA5-MAVS RNA sensing pathway. Moreover, we uncovered an MDA5-MAVS-independent function for ADAR1 in the development of multiple organs, including the kidney and intestines. We also discovered a cell-intrinsic role for ADAR1 in B cell development. We showed that the p150 isoform of ADAR1 exclusively regulated the MDA5 pathway, whereas both the p150 and p110 isoforms contributed to development. Abrupt deletion of ADAR1 in adult mice revealed that both of these functions were required throughout life. Our findings define genetically distinct roles for both ADAR1 isoforms in vivo, with implications for the human diseases caused by ADAR mutations
Nucleic Acid Detection in Anti-Viral Responses and Autoimmunity
No full textThesis (Ph.D.)--University of Washington, 2012Viruses infect all living organisms. Therefore, hosts evolved extensive means to detect and destroy viral pathogens. Pattern recognition receptors that detect viral RNA or DNA genome are important components of the innate immune response against viruses. In vertebrates, viral nucleic acid is detected by two mechanisms that both result in the production of antiviral type I IFN. The first mechanism involves toll-like receptor detection of non-cell autonomous viral nucleic acid and allows uninfected, specialized innate immune cells to initiate an adaptive immune response to eliminate infection. Second, cell intrinsic receptors allow all infected cells to detect viral infection and induce a type I IFN response. The interferon stimulatory DNA (ISD) pathway specifically detects of viral DNA genomes. Chronic activation of the ISD pathway is the cause of lethal autoimmune disease in Trex1 deficient mice and humans thereby coupling innate immune detection to initiation of autoimmunity. The ISD pathway induces type I IFN by STING-dependent activation of TBK1 and IRF3. However, little is known about the DNA sensors proteins of the ISD pathway. Here we use the novel model of Trex1 autoimmunity to identify Trex1 interacting proteins that may be involved in ISD sensing. We discovered that Trex1 is localized to the inner nuclear membrane in addition to the endoplasmic reticulum and interacts with nuclear partners Brd7, Btf3l4, Chaf1a, Pias1, Rfc2 and Sae2. This data implies that negative regulation of the ISD pathway may occur in the nucleus and not the cytoplasm as previously thought. In addition, we identified new members and characterized the 13 members of the AIM2-like receptor (ALR) family of ISD sensors. Here we show the ALR family of nuclear proteins is highly divergent across mammals reflecting each species' unique struggle with specific viral pathogens. We also discovered the ALR proteins fit into two functional categories based on their ability to interact with STING and induce type I IFN or interact with ASC to activate the IL-1β inflammasome. Together our data imply nuclear detection of DNA occurs utilizing highly species-specific sensors and provides a functional framework for further study of the ISD sensors
ADAR1 Regulation of Innate RNA Sensing in Immune Disease
No full textThesis (Ph.D.)--University of Washington, 2021Detection of nucleic acids and production of type I interferons (IFNs) are principal elements of antiviral defense, but can cause autoimmune disease if dysregulated. Loss of function mutations in the human ADAR gene cause Aicardi-Goutières Syndrome (AGS), a rare and severe autoimmune disease that resembles congenitally acquired viral infection. Our lab and others defined ADAR1 as an essential negative regulator of an RNA-sensing pathway. Specifically, accumulation of endogenous ADAR1 RNA substrates within cells triggers type I IFN production through the anti-viral MDA5/MAVS pathway, highlighting the connection between innate antiviral responses and autoimmunity, with important implications for the treatment of AGS and related diseases. However, the mechanisms of MDA5-dependent disease pathogenesis in vivo remain unknown. Here, we introduce a knockin mouse that models the most common ADAR AGS mutation in humans. In defining this model we confirm that the unique z-alpha domain of ADAR1 is required, along with the deaminase domain, for MDA5 regulation. We establish that it is haploinsufficiency paired with an otherwise non-deleterious allele that drives disease, and may explain the dominance of this allele amongst the broader population. We used this new Adar-mutant mouse model to confirm several putative effectors of disease that results from ADAR1 loss of function. These mice develop lethal disease that requires MDA5, the RIG-I-like receptor LGP2, type I interferons, and the eIF2α kinase PKR. We additionally show that a small molecule inhibitor of the integrated stress response (ISR) that acts downstream of eIF2α phosphorylation prevents immunopathology, and rescues the mice from mortality. We also determined that haploinsufficiency is essential to the progression of disease, and demonstrate increased MDA5 signaling in p150 and Adar1 heterozygous mice. We describe a new set of mice that will allow in vitro and in vivo interrogation of the RNAs that activate MDA5 in the absence of sufficient ADAR1 editing. Our findings place PKR and the ISR as central components of immunopathology in vivo and identify new therapeutic targets for treatment of human diseases associated with the ADAR1-MDA5 axis, and shed light on how decreased ADAR1 activity may be leveraged for cancer treatments
DNA tumor virus oncogenes antagonize the cGAS-STING DNA sensing pathway
No full textThesis (Ph.D.)--University of Washington, 2015-12A key aspect of antiviral immunity is the induction of type I interferons (IFN) to mediate the effective clearance of a viral infection. Cyclic GMP-AMP synthase (cGAS) detects intracellular DNA and signals through the adapter protein STING to initiate a type I IFN-mediated antiviral response to DNA viruses. These viruses, some of which have evolved with their hosts for millions of years, have likely developed means to prevent activation of the cGAS-STING pathway, but such virus-encoded antagonists remain largely unknown. Here, we identify the viral oncogenes of the DNA tumor viruses, including E7 from human papillomavirus (HPV) and E1A from adenovirus, as potent and specific inhibitors of the cGAS-STING pathway. We show that the LXCXE motif of these oncoproteins, which is essential for blockade of the Retinoblastoma tumor suppressor, is also important for cGAS-STING pathway antagonism. We find that E1A and E7 bind to STING, and that silencing of these oncogenes in human tumor cells restores cGAS-STING pathway signaling. Our findings reveal a host-virus conflict that may have shaped the evolution of viral oncogenes, with implications for the origins of the DNA viruses that cause cancer in humans
Engagement of programmed cell death by nucleic acid sensing
Get PDFThesis (Ph.D.)--University of Washington, 2017During infection, the sensing of foreign nucleic acids inside an infected cell is often the first line of immunological defense. When DNA or RNA is sensed, one outcome is the production of anti-viral signaling molecules such as type I interferons. Additionally, cells can undergo programmed cell death. Here we describe the mechanism of induction of the programmed cell death pathway, necroptosis, following detection of cytosolic DNA or RNA. Necroptosis is a lytic and proinflammatory form of cell death, that can eliminate infected cells and alert and instruct the immune system. We show that following sensing of foreign nucleic acids, induction of necroptosis relies on the production of both anti-viral interferon, as well pro-inflammatory molecule, tumor necrosis factor
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
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