Cytoplasmic DNA Research Articles

Systems pharmacology dissection of pharmacological mechanisms of Xiaochaihu decoction against human coronavirus

BackgroundAlthough coronavirus disease 2019 (COVID-19) pandemic is still rage worldwide, there are still very limited treatments for human coronaviruses (HCoVs) infections. Xiaochahu decoction (XCHD), which is one of the traditional Chinese medicine (TCM) prescriptions in Qingfeipaidu decoction (QFPDD), is widely used for COVID-19 treatment in China and able to relieve the symptoms of fever, fatigue, anorexia, and sore throat. To explore the role and mechanisms of XCHD against HCoVs, we presented an integrated systems pharmacology framework in this study.MethodsWe constructed a global herb-compound-target (H-C-T) network of XCHD against HCoVs. Multi-level systems pharmacology analyses were conducted to highlight the key XCHD-regulated proteins, and reveal multiple HCoVs relevant biological functions affected by XCHD. We further utilized network-based prediction, drug-likeness analysis, combining with literature investigations to uncover the key ani-HCoV constituents in XCHD, whose effects on anit-HCoV-229E virus were validated using cytopathic effect (CPE) assay. Finally, we proposed potential molecular mechanisms of these compounds against HCoVs via subnetwork analysis.ResultsBased on the systems pharmacology framework, we identified 161 XCHD-derived compounds interacting with 37 HCoV-associated proteins. An integrated pathway analysis revealed that the mechanism of XCHD against HCoVs is related to TLR signaling pathway, RIG-I-like receptor signaling pathway, cytoplasmic DNA sensing pathway, and IL-6/STAT3 pro-inflammatory signaling pathway. Five compounds from XCHD, including betulinic acid, chrysin, isoliquiritigenin, schisandrin B, and (20R)-Ginsenoside Rh1 exerted inhibitory activity against HCoV-229E virus in Huh7 cells using in vitro CPE assay.ConclusionOur work presented a comprehensive systems pharmacology approach to identify the effective molecules and explore the molecular mechanism of XCHD against HCoVs.

Type IV pili facilitated natural competence in Fusobacterium nucleatum

ObjectivesMany bacterial species naturally take up DNA from their surroundings and recombine it into their chromosome through homologous gene transfer (HGT) to aid in survival and gain advantageous functions. Herein we present the first characterization of Type IV pili facilitated natural competence in Fusobacterium nucleatum, which is a Gram-negative, anaerobic bacterium that participates in a range of infections and diseases including periodontitis, preterm birth, and cancer. MethodsHere we used bioinformatics on multiple Fusobacterium species, as well as molecular genetics to characterize natural competence in strain F. nucleatum subsp. nucleatum ATCC 23726. ResultsWe bioinformatically identified components of the Type IV conjugal pilus machinery and show this is a conserved system within the Fusobacterium genus. We next validate Type IV pili in natural competence in F. nucleatum ATCC 23726 and show that gene deletions in key components of pilus deployment (pilQ) and cytoplasmic DNA import (comEC) abolish DNA uptake and chromosomal incorporation. We next show that natural competence may require native F. nucleatum DNA methylation to bypass restriction modification systems and allow subsequent genomic homologous recombination. ConclusionsIn summary, this proof of principle study provides the first characterization of natural competence in Fusobacterium nucleatum and highlights the potential to exploit this DNA import mechanism as a genetic tool to characterize virulence mechanisms of an opportunistic oral pathogen.

STING signaling in inflammaging: a new target against musculoskeletal diseases.

Stimulator of Interferon Gene (STING) is a critical signaling linker protein that plays a crucial role in the intrinsic immune response, particularly in the cytoplasmic DNA-mediated immune response in both pathogens and hosts. It is also involved in various signaling processes in vivo. The musculoskeletal system provides humans with morphology, support, stability, and movement. However, its aging can result in various diseases and negatively impact people's lives. While many studies have reported that cellular aging is a leading cause of musculoskeletal disorders, it also offers insight into potential treatments. Under pathological conditions, senescent osteoblasts, chondrocytes, myeloid cells, and muscle fibers exhibit persistent senescence-associated secretory phenotype (SASP), metabolic disturbances, and cell cycle arrest, which are closely linked to abnormal STING activation. The accumulation of cytoplasmic DNA due to chromatin escape from the nucleus following DNA damage or telomere shortening activates the cGAS-STING signaling pathway. Moreover, STING activation is also linked to mitochondrial dysfunction, epigenetic modifications, and impaired cytoplasmic DNA degradation. STING activation upregulates SASP and autophagy directly and indirectly promotes cell cycle arrest. Thus, STING may be involved in the onset and development of various age-related musculoskeletal disorders and represents a potential therapeutic target. In recent years, many STING modulators have been developed and used in the study of musculoskeletal disorders. Therefore, this paper summarizes the effects of STING signaling on the musculoskeletal system at the molecular level and current understanding of the mechanisms of endogenous active ligand production and accumulation. We also discuss the relationship between some age-related musculoskeletal disorders and STING, as well as the current status of STING modulator development.

Cytoplasmic DNA sensing boosts CD4+ T cell metabolism for inflammatory induction

Abstract DNA accumulation is associated with the development of autoimmune inflammatory diseases. However, the pathological role and underlying mechanism of cytoplasmic DNA accumulation in CD4+ T cells have not been well established. Here, we show that Trex1 deficiency-induced endogenous DNA accumulation in CD4+ T cells greatly promoted their induction of autoimmune inflammation in a lupus-like mouse model. Mechanistically, the accumulated DNA in CD4+ T cells was sensed by the KU complex, then triggered the activation of DNA-PKcs and ZAK and further facilitated the activation of AKT, which exacerbated glycolysis, thereby promoting the inflammatory responses. Accordingly, blocking the DNA sensing pathway in CD4+ T cells by genetic knockout of Zak or using our newly developed ZAK inhibitor iZAK2 attenuated all pathogenic characteristics in a lupus-like inflammation mouse model induced with Trex1-deficient CD4+ T cells. Overall, our study demonstrated a causal link between DNA-sensing and metabolic reprogramming in CD4+ T cells for inflammatory induction and suggested inhibition of the DNA sensing pathway may be a potential therapy for the treatment of inflammatory diseases.

Vaccinia E5 is a major inhibitor of the DNA sensor cGAS

The DNA sensor cyclic GMP-AMP synthase (cGAS) is critical in host antiviral immunity. Vaccinia virus (VACV) is a large cytoplasmic DNA virus that belongs to the poxvirus family. How vaccinia virus antagonizes the cGAS-mediated cytosolic DNA-sensing pathway is not well understood. In this study, we screened 80 vaccinia genes to identify potential viral inhibitors of the cGAS/Stimulator of interferon gene (STING) pathway. We discovered that vaccinia E5 is a virulence factor and a major inhibitor of cGAS. E5 is responsible for abolishing cGAMP production during vaccinia virus (Western Reserve strain) infection of dendritic cells. E5 localizes to the cytoplasm and nucleus of infected cells. Cytosolic E5 triggers ubiquitination of cGAS and proteasome-dependent degradation via interacting with cGAS. Deleting the E5R gene from the Modified vaccinia virus Ankara (MVA) genome strongly induces type I IFN production by dendritic cells (DCs) and promotes DC maturation, and thereby improves antigen-specific T cell responses.

Mitotic clustering of pulverized chromosomes from micronuclei

Complex genome rearrangements can be generated by the catastrophic pulverization of missegregated chromosomes trapped within micronuclei through a process known as chromothripsis1–5. As each chromosome contains a single centromere, it remains unclear how acentric fragments derived from shattered chromosomes are inherited between daughter cells during mitosis6. Here we tracked micronucleated chromosomes with live-cell imaging and show that acentric fragments cluster in close spatial proximity throughout mitosis for asymmetric inheritance by a single daughter cell. Mechanistically, the CIP2A–TOPBP1 complex prematurely associates with DNA lesions within ruptured micronuclei during interphase, which poises pulverized chromosomes for clustering upon mitotic entry. Inactivation of CIP2A–TOPBP1 caused acentric fragments to disperse throughout the mitotic cytoplasm, stochastically partition into the nucleus of both daughter cells and aberrantly misaccumulate as cytoplasmic DNA. Mitotic clustering facilitates the reassembly of acentric fragments into rearranged chromosomes lacking the extensive DNA copy-number losses that are characteristic of canonical chromothripsis. Comprehensive analysis of pan-cancer genomes revealed clusters of DNA copy-number-neutral rearrangements—termed balanced chromothripsis—across diverse tumour types resulting in the acquisition of known cancer driver events. Thus, distinct patterns of chromothripsis can be explained by the spatial clustering of pulverized chromosomes from micronuclei.

Immediate effects of acute Mars mission equivalent doses of SEP and GCR radiation on the murine gastrointestinal system-protective effects of curcumin-loaded nanolipoprotein particles (cNLPs).

Missions beyond low Earth orbit (LEO) will expose astronauts to ionizing radiation (IR) in the form of solar energetic particles (SEP) and galactic cosmic rays (GCR) including high atomic number and energy (HZE) nuclei. The gastrointestinal (GI) system is documented to be highly radiosensitive with even relatively low dose IR exposures capable of inducing mucosal lesions and disrupting epithelial barrier function. IR is also an established risk factor for colorectal cancer (CRC) with several studies examining long-term GI effects of SEP/GCR exposure using tumor-prone APC mouse models. Studies of acute short-term effects of modeled space radiation exposures in wildtype mouse models are more limited and necessary to better define charged particle-induced GI pathologies and test novel medical countermeasures (MCMs) to promote astronaut safety. In this study, we performed ground-based studies where male and female C57BL/6J mice were exposed to γ-rays, 50 MeV protons, or 1 GeV/n Fe-56 ions at the NASA Space Radiation Laboratory (NSRL) with histology and immunohistochemistry endpoints measured in the first 24 h post-irradiation to define immediate SEP/GCR-induced GI alterations. Our data show that unlike matched γ-ray controls, acute exposures to protons and iron ions disrupts intestinal function and induces mucosal lesions, vascular congestion, epithelial barrier breakdown, and marked enlargement of mucosa-associated lymphoid tissue. We also measured kinetics of DNA double-strand break (DSB) repair using gamma-H2AX- specific antibodies and apoptosis via TUNEL labeling, noting the induction and disappearance of extranuclear cytoplasmic DNA marked by gamma-H2AX only in the charged particle-irradiated samples. We show that 18 h pre-treatment with curcumin-loaded nanolipoprotein particles (cNLPs) delivered via IV injection reduces DSB-associated foci levels and apoptosis and restore crypt villi lengths. These data improve our understanding of physiological alterations in the GI tract immediately following exposures to modeled space radiations and demonstrates effectiveness of a promising space radiation MCM.

ISG15: A link between innate immune signaling, DNA replication, and genome stability.

Interferon stimulated gene 15 (ISG15) encodes a ubiquitin-like protein that is highly induced upon activation of interferon signaling and cytoplasmic DNA sensing pathways. As part of the innate immune system ISG15 acts to inhibit viral replication and particle release via the covalent conjugation to both viral and host proteins. Unlike ubiquitin, unconjugated ISG15 also functions as an intracellular and extra-cellular signaling molecule to modulate the immune response. Several recent studies have shown ISG15 to also function in a diverse array of cellular processes and pathways outside of the innate immune response. This review explores the role of ISG15 in maintaining genome stability, particularly during DNA replication, and how this relates to cancer biology. It puts forth the hypothesis that ISG15, along with DNA sensors, function within a DNA replication fork surveillance pathway to help maintain genome stability.

704/DNA vaccines leverage cytoplasmic DNA stimulation to promote anti-HIV neutralizing antibody production in mice and strong immune response against alpha-fetoprotein in non-human primates

Genetic immunization is an attractive approach for prophylactic and therapeutic vaccination using synthetic vectors to deliver antigen-encoding nucleic acids. Recently, DNA delivered by a physical means or RNA by liposomes consisting of four different lipids demonstrated good protection in human phase III clinical trials and received Drugs Controller General of India and US FDA approval to protect against COVID-19, respectively. However, the development of a system allowing for efficient and simple delivery of nucleic acids while improving immune response priming has the potential to unleash the full therapeutic potential of genetic immunization. DNA-based gene therapies and vaccines have the potential for rapid development, as exemplified by the recent approval of Collategene, a gene therapy to treat human critical limb ischemia, and ZyCoV, a DNA vaccine delivered by spring-powered jet injector to protect against SARS-CoV2 infection. Recently, we reported amphiphilic block copolymer 704 as a promising synthetic vector for DNA vaccination in various models of human diseases. This vector allows dose sparing of antigen-encoding plasmid DNA. Here, we report the capacity of 704-mediated HIV and anti-hepatocellular carcinoma DNA vaccines to induce the production of specific antibodies against gp120 HIV envelope proteins in mice and against alpha-fetoprotein antigen in non-human primates, respectively. An investigation of the underlying mechanisms showed that 704-mediated vaccination did trigger a strong immune response by (1) allowing a direct DNA delivery into the cytosol, (2) promoting an intracytoplasmic DNA sensing leading to both interferon and NF-κB cascade stimulation, and (3) inducing antigen expression by muscle cells and presentation by antigen-presenting cells, leading to the induction of a robust adaptive response. Overall, our findings suggest that the 704-mediated DNA vaccination platform is an attractive method to develop both prophylactic and therapeutic vaccines.

High glucose-induced STING activation inhibits diabetic wound healing through promoting M1 polarization of macrophages

Diabetic wound (DW) is characterized by elevated pro-inflammatory cytokines and cellular dysfunction consistent with elevated reactive oxygen species (ROS) levels. Recent advances in immunology have dissected molecular pathways involved in the innate immune system where cytoplasmic DNA can trigger STING-dependent inflammatory responses and play an important role in metabolic-related diseases. We investigated whether STING regulates inflammation and cellular dysfunction in DW healing. We found that STING and M1 macrophages were increased in wound tissues from DW in patients and mice and delayed the wound closure. We also noticed that the massively released ROS in the High glucose (HG) environment activated STING signaling by inducing the escape of mtDNA to the cytoplasm, inducing macrophage polarization into a pro-inflammatory phenotype, releasing pro-inflammatory cytokines, and exacerbating endothelial cell dysfunction. In Conclusion, mtDNA-cGAS-STING pathway activation under diabetic metabolic stress is an important mechanism of DW refractory healing. While using STING gene-edited macrophages for wound treatment by cell therapy can induce the polarization of wound macrophages from pro-inflammatory M1 to anti-inflammatory M2, promote angiogenesis, and collagen deposition to accelerate DW healing. STING may be a promising therapeutic target for DW.

HP1γ Prevents Activation of the cGAS/STING Pathway by Preserving Nuclear Envelope and Genomic Integrity in Colon Adenocarcinoma Cells.

Chronic inflammatory processes in the intestine result in serious conditions such as inflammatory bowel disease (IBD) and cancer. An increased detection of cytoplasmic DNA sensors has been reported in the IBD colon mucosa, suggesting their contribution in mucosal inflammation. Yet, the mechanisms altering DNA homeostasis and triggering the activation of DNA sensors remain poorly understood. In this study, we show that the epigenetic regulator HP1γ plays a role in preserving nuclear envelope and genomic integrity in enterocytic cells, thereby protecting against the presence of cytoplasmic DNA. Accordingly, HP1 loss of function led to the increased detection of cGAS/STING, a cytoplasmic DNA sensor that triggers inflammation. Thus, in addition to its role as a transcriptional silencer, HP1γ may also exert anti-inflammatory properties by preventing the activation of the endogenous cytoplasmic DNA response in the gut epithelium.

STING recruits WIPI2 for autophagosome formation

ABSTRACT Induction of autophagy is a primordial function of the cGAS-STING pathway. However, the molecular mechanisms regulating autophagosome formation during STING-induced autophagy remain largely unknown. Recently, we reported that STING directly interacts with WIPI2 to recruit WIPI2 onto STING-positive vesicles for LC3 lipidation and autophagosome formation. We found that STING and PtdIns3P competitively bind to the FRRG motif of WIPI2, resulting in a mutual inhibition between STING-induced and PtdIns3P-dependent autophagy. We also showed that STING-WIPI2 interaction is necessary for cells to clear cytoplasmic DNA and attenuate activated cGAS-STING signaling. In summary, by identifying the interaction between STING and WIPI2, our study revealed a mechanism that allows STING to bypass the canonical upstream machinery to induce autophagosome formation. Abbreviations: ATG: autophagy-related; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; cGAMP: cyclic GMP-AMP; cGAS: cyclic GMP-AMP synthase; ER: endoplasmic reticulum; ERGIC: ER-Golgi intermediate compartment; IRF3: interferon regulatory factor 3; PtdIns3P: phosphatidylinositol-3-phosphate; SQSTM1: sequestosome 1; STING: stimulator of interferon genes; TBK1: TANK-binding kinase 1; ULK1: unc-51 like autophagy activating kinase 1; WIPI2: WD repeat domain, phosphoinositide interacting 2

Cytoplasmic DNAs: Sources, sensing, and roles in the development of lung inflammatory diseases and cancer.

Cytoplasmic DNA is emerging as a pivotal contributor to the pathogenesis of inflammatory diseases and cancer, such as COVID-19 and lung carcinoma. However, the complexity of various cytoplasmic DNA-related pathways and their crosstalk remains challenging to distinguish their specific roles in many distinct inflammatory diseases, especially for the underlying mechanisms. Here, we reviewed the latest findings on cytoplasmic DNA and its signaling pathways in inflammatory lung conditions and lung cancer progression. We found that sustained activation of cytoplasmic DNA sensing pathways contributes to the development of common lung diseases, which may result from external factors or mutations of key genes in the organism. We further discussed the interplays between cytoplasmic DNA and anti-inflammatory or anti-tumor effects for potential immunotherapy. In sum, this review aids in understanding the roles of cytoplasmic DNAs and exploring more therapeutic strategies.

Abstract 1852: Systemic delivery of ST317, a cell penetrating STING pathway sensitizer, results in strong anti-tumor activity

Abstract Pharmacological activation of the STING pathway is being assessed as a strategy to stimulate both the innate and adaptive arms of the immune system to trigger an anti-tumor immune response in so-called "cold" tumors. Although the first generation of STING agonists dosed intratumorally showed a strong anti-tumor response in preclinical models, positive clinical data has so far been limited, potentially due to reduced efficacy in non-injected tumors. Newer classes of STING agonists that are delivered systemically are expected to deliver more consistent and predictable levels of STING activation, but may less tolerated due to untargeted activation of the immune system resulting in immune related adverse events. We have previously demonstrated that IFI16 promotes activation of the cGAMP-activated STING pathway by a process relying on IFI16 PYRIN domain-dependent protein-protein interactions1. Based on these findings, an IFI16 Pyrin domain-derived peptide incorporating a cell penetrating peptide that recapitulates the agonist-dependent activation of the STING pathway by IFI16, was developed. This peptide, ST317, acts as a STING sensitizer for effective and safe induction of anti-tumour immune responses only at sites where STING agonists are present. In vitro, ST317 sensitizes STING to pathway activators (Cytoplasmic DNA and cGAMP) and pharmacological STING agonists including ADU-S100 and diABZI compound 3, reducing the EC50 values of agonist-induced STING pathway activation while inducing supraphysiological (i.e. increasing the EMax) levels of pro-inflammatory cytokines/chemokines (e.g. Type I interferon and CXCL10) in cell line and primary cells. Mechanistically, ST317 sensitizes STING to cGAMP by accelerating and increasing STING translocation from the endoplasmic reticulum to the Golgi, resulting in earlier and longer activation of the STING pathway components, as determined by the phosphorylation kinetics of TBK1 and IRF3, as well as STING itself. Consistent with its in vitro pharmacological profile, ST317 can be safely administered parenterally without evidence of peripheral STING activation. In vivo experiments in immunocompetent syngeneic mouse models demonstrate that ST317 dosed I.V. was well tolerated and was associated with anti-tumor efficacy when used as a single agent or in combination with immune checkpoint blockers such as anti-PD1. In addition, ST317 improved the anti-tumor response to a pharmacological STING agonist at doses that do not cause immune cell death that result from over-dosing this type of agent. In summary, ST317 represents a new class of STING pathway activator, which enables safe systemic administration while giving rise to enhanced STING pathway activation only at sites at which STING agonists are present. The data presented here warrant the assessment of ST317 in cancer patients in a clinical trial. 1. Jønsson et al. 2017 DOI: 10.1038/ncomms14391 Citation Format: Claus Elsborg Olesen, Esben Axelgaard, Emil Nilsson, Anna H. Hansen, Isis Benoit-Lizon, Justin V. Joseph, Kristina Byskov, Magnus Strandh, Maja Myrcha, Pernille Rimmer Noer, Ulrich Sensfuss, Richard Charles Sainson, Richard C. Bethell, Martin R. Jakobsen. Systemic delivery of ST317, a cell penetrating STING pathway sensitizer, results in strong anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1852.

Abstract 6417: Deciphering aberrant STING pathway and exploring oncolytic viruses therapy in low grade serous ovarian carcinoma

Abstract There are two serous ovarian cancer histotypes, low and high grade (LGSOC and HGSOC), which are distinct clinical and biological entities. LGSOC is a rare histotype with a relatively stable genome, while HGSC is more common and genomically unstable. Somewhat surprisingly LGSOC expresses high levels of the stimulator of the interferon genes (STING). The STING pathway recognizes cytoplasmic double-stranded DNA and mounts innate cellular immunity through interferon-beta type I production. Our objective is to investigate the aberrant STING signaling in LGSOC and test the effectiveness of oncolytic viruses against LGSOC. We used immunohistochemistry on tissue microarrays (TMAs) to assess STING protein expression in different ovarian cancer histotypes. Whole proteome analysis was applied to identify differentially expressed proteins in LGSOC and HGSOC patient samples (both n=9). Further, a semi-targeted proteomics approach was used to evaluate the expression levels of the STING pathway-related proteins in LGSOC, HGSOC, and LGSOC precursor tumors (each subtype, n=20). To evaluate the key transcription, phosphorylation, and translocation events in STING signaling, we treated LGSOC cell lines with an agonist (dsDNA90) and performed qPCR, immunoblotting, and immunofluorescence experiments, respectively. We tested the viability of the LGSOC cell lines in response to Vaccinia Virus (VV), and Vesicular Stomatitis Virus (VSV) based oncolytic vectors with or without immunostimulatory transgenes. Our results show that STING protein levels were consistently higher in LGSOC TMAs relative to other histotypes. Proteomics analysis showed that the half of the 16 most differentially expressed proteins were the effectors of STING signaling with unexpectedly lower expression in LGSOC, suggesting that despite the robust levels of STING in LGSOC tumors, the pathway is not fully active. Attenuated STING translocation and expression of IFNB1 and other cytokines in LGSOC cell lines confirm the aberrancy in the STING pathway. Semi-targeted proteomics revealed the considerable overexpression of STIM1 in LGSOC patient tumors, which has previously been shown to sequester STING in the endoplasmic reticulum. The treatment with VV and VSV oncolytic viruses significantly reduced the proliferation of LGSOC cell lines. In summary, we find attenuated STING signaling in LGSOC, possibly due to overexpression of STIM1 preventing STING translocation. Although oncolytic viruses show promising results in LGSOC cell lines, more research is needed to determine the optimal treatment strategy, testing oncolytic viruses expressing various transgenes and combination therapies. Citation Format: Almira Zhantuyakova, Dawn Cochrane, Gian Negri, Sandra E. Spencer Miko, Taha Azad, Jutta Huvila, Marta Llaurado Fernandez, Mark Carey, Gregg B. Morin, John Bell, David Huntsman. Deciphering aberrant STING pathway and exploring oncolytic viruses therapy in low grade serous ovarian carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6417.

DNA sensing via the cGAS/STING pathway activates the immunoproteasome and adaptive T‐cell immunity

The immunoproteasome is a specialized type of proteasome involved in MHC class I antigen presentation, antiviral adaptive immunity, autoimmunity, and is also part of a broader response to stress. Whether the immunoproteasome is regulated by DNA stress, however, is not known. We here demonstrate that mitochondrial DNA stress upregulates the immunoproteasome and MHC class I antigen presentation pathway via cGAS/STING/type I interferon signaling resulting in cell autonomous activation of CD8+ T cells. The cGAS/STING‐induced adaptive immune response is also observed in response to genomic DNA and is conserved in epithelial and mesenchymal cells of mice and men. In patients with idiopathic pulmonary fibrosis, chronic activation of the cGAS/STING‐induced adaptive immune response in aberrant lung epithelial cells concurs with CD8+ T‐cell activation in diseased lungs. Genetic depletion of the immunoproteasome and specific immunoproteasome inhibitors counteract DNA stress induced cytotoxic CD8+ T‐cell activation. Our data thus unravel cytoplasmic DNA sensing via the cGAS/STING pathway as an activator of the immunoproteasome and CD8+ T cells. This represents a novel potential pathomechanism for pulmonary fibrosis that opens new therapeutic perspectives.

Phylogeography and introgression between Pinus kesiya and Pinus yunnanensis in Southeast Asia

AbstractSoutheast Asia (SEA) has seen strong climatic oscillations and fluctuations in sea levels during the Quaternary. The impact of past climate changes on the evolution and distribution of local flora in SEA is still poorly understood. Here we aim to infer how the Quaternary climate change affects the evolutionary process and range shifts in two pine species. We investigated the population genetic structure and diversity using cytoplasmic DNA markers, and performed ecological niche modeling to reconstruct the species past distribution and to project range shift under future climates. We found substantial gene flow across the continuous distribution of the subtropical Pinus yunnanensis. In contrast, the tropical Pinus kesiya showed a strong population structure in accordance with its disjunct distribution across montane islands in Indochina and the Philippines. A broad hybrid zone of the two species occurs in southern Yunnan. Asymmetric introgression from the two species was detected in this zone with dominant mitochondrial gene flow from P. yunnanensis and chloroplast gene flow from P. kesiya. The observed population structure suggests a typical postglaciation expansion in P. yunnanensis, and a glacial expansion and interglacial contraction in P. kesiya. Ecological niche modeling supports the inferred demographic history and predicts a decrease in range size for P. kesiya under future climates. Our results suggest that tropical pine species in SEA have undergone evolutionary trajectories different from high latitude species related to their Quaternary climate histories. We also illustrate the need for urgent conservation actions in this fragmented landscape.

STING directly recruits WIPI2 for autophagosome formation during STING-induced autophagy.

The cGAS-STING pathway plays an important role in host defense by sensing pathogen DNA, inducing type I IFNs, and initiating autophagy. However, the molecular mechanism of autophagosome formation in cGAS-STING pathway-induced autophagy is still unclear. Here, we report that STING directly interacts with WIPI2, which is the key protein for LC3 lipidation in autophagy. Binding to WIPI2 is necessary for STING-induced autophagosome formation but does not affect STING activation and intracellular trafficking. In addition, the specific interaction between STING and the PI3P-binding motif of WIPI2 leads to the competition of WIPI2 binding between STING and PI3P, and mutual inhibition between STING-induced autophagy and canonical PI3P-dependent autophagy. Furthermore, we show that the STING-WIPI2 interaction is required for the clearance of cytoplasmic DNA and the attenuation of cGAS-STING signaling. Thus, the direct interaction between STING and WIPI2 enables STING to bypass the canonical upstream machinery to induce LC3 lipidation and autophagosome formation.

CGAS-STING pathway as a potential trigger of immunosenescence and inflammaging.

Aging is associated with an increased incidence of autoimmune diseases, despite the progressive decline of immune responses (immunosenescence). This apparent paradox can be explained by the age-related chronic low-grade systemic inflammation (inflammaging) and progressive dysregulation of innate signaling. During cellular aging, there is an accumulation of damaged DNA in the cell's cytoplasm, which serves as ubiquitous danger-associated molecule, promptly recognized by DNA sensors. For instance, the free cytoplasmic DNA can be recognized, by DNA-sensing molecules like cGAS-STING (cyclic GMP-AMP synthase linked to a stimulator of interferon genes), triggering transcriptional factors involved in the secretion of pro-inflammatory mediators. However, the contribution of this pathway to the aging immune system remains largely unknown. Here, we highlight recent advances in understanding the biology of the cGAS-STING pathway, its influence on the senescence-associated secretory phenotype (SASP), and its modulation of the immune system during sterile inflammation. We propose that this important stress sensor of DNA damage is also a trigger of immunosenescence and inflammaging.

Actinobacillus pleuropneumoniae encodes multiple phase-variable DNA methyltransferases that control distinct phasevarions.

Actinobacillus pleuropneumoniae is the cause of porcine pleuropneumonia, a severe respiratory tract infection that is responsible for major economic losses to the swine industry. Many host-adapted bacterial pathogens encode systems known as phasevarions (phase-variable regulons). Phasevarions result from variable expression of cytoplasmic DNA methyltransferases. Variable expression results in genome-wide methylation differences within a bacterial population, leading to altered expression of multiple genes via epigenetic mechanisms. Our examination of a diverse population of A. pleuropneumoniae strains determined that Type I and Type III DNA methyltransferases with the hallmarks of phase variation were present in this species. We demonstrate that phase variation is occurring in these methyltransferases, and show associations between particular Type III methyltransferase alleles and serovar. Using Pacific BioSciences Single-Molecule, Real-Time (SMRT) sequencing and Oxford Nanopore sequencing, we demonstrate the presence of the first ever characterised phase-variable, cytosine-specific Type III DNA methyltransferase. Phase variation of distinct Type III DNA methyltransferase in A. pleuropneumoniae results in the regulation of distinct phasevarions, and in multiple phenotypic differences relevant to pathobiology. Our characterisation of these newly described phasevarions in A. pleuropneumoniae will aid in the selection of stably expressed antigens, and direct and inform development of a rationally designed subunit vaccine against this major veterinary pathogen.