Antiviral type III CRISPR signalling via conjugation of ATP and SAM.

CRISPR systems are widespread in the prokaryotic world, providing adaptive immunity against mobile genetic elements1,2. Type III CRISPR systems, with the signature gene cas10, use CRISPR RNA to detect non-self RNA, activating the enzymatic Cas10 subunit to defend the cell against mobile genetic elements either directly, via the integral histidine-aspartate (HD) nuclease domain3-5 or indirectly, via synthesis of cyclic oligoadenylate second messengers to activate diverse ancillary effectors6-9. A subset of type III CRISPR systems encode an uncharacterized CorA-family membrane protein and an associated NrN family phosphodiesterase that are predicted to function in antiviral defence. Here we demonstrate that the CorA-associated type III-B (Cmr) CRISPR system from Bacteroides fragilis provides immunity against mobile genetic elements when expressed in Escherichia coli. However, B. fragilis Cmr does not synthesize cyclic oligoadenylate species on activation, instead generating S-adenosyl methionine (SAM)-AMP (SAM is also known as AdoMet) by conjugating ATP to SAM via a phosphodiester bond. Once synthesized, SAM-AMP binds to the CorA effector, presumably leading to cell dormancy or death by disruption of the membrane integrity. SAM-AMP is degraded by CRISPR-associated phosphodiesterases or a SAM-AMP lyase, potentially providing an 'off switch' analogous to cyclic oligoadenylate-specific ring nucleases10. SAM-AMP thus represents a new class of second messenger for antiviral signalling, which may function in different roles in diverse cellular contexts.

R-loop-derived cytoplasmic RNA-DNA hybrids activate an immune response.

R-loops are RNA-DNA-hybrid-containing nucleic acids with important cellular roles. Deregulation of R-loop dynamics can lead to DNA damage and genome instability1, which has been linked to the action of endonucleases such as XPG2-4. However, the mechanisms and cellular consequences of such processing have remained unclear. Here we identify a new population of RNA-DNA hybrids in the cytoplasm that are R-loop-processing products. When nuclear R-loops were perturbed by depleting the RNA-DNA helicase senataxin (SETX) or the breast cancer gene BRCA1 (refs. 5-7), we observed XPG- and XPF-dependent cytoplasmic hybrid formation. We identify their source as a subset of stable, overlapping nuclear hybrids with a specific nucleotide signature. Cytoplasmic hybrids bind to the pattern recognition receptors cGAS and TLR3 (ref. 8), activating IRF3 and inducing apoptosis. Excised hybrids and an R-loop-induced innate immune response were also observed in SETX-mutated cells from patients with ataxia oculomotor apraxia type 2 (ref. 9) and in BRCA1-mutated cancer cells10. These findings establish RNA-DNA hybrids as immunogenic species that aberrantly accumulate in the cytoplasm after R-loop processing, linking R-loop accumulation to cell death through the innate immune response. Aberrant R-loop processing and subsequent innate immune activation may contribute to many diseases, such as neurodegeneration and cancer.

Comprehensive characterization of tumor microenvironment and m6A RNA methylation regulators and its effects on PD-L1 and immune infiltrates in cervical cancer.

Understanding the role of N6-adenosine methylation (m6A) in the tumor microenvironment (TME) is important since it can contribute to tumor development. However, the research investigating the association between m6A and TME and cervical cancer is still in its early stages. The aim of this study was to discover the possible relationship between m6A RNA methylation regulators, TME, PD-L1 expression levels, and immune infiltration in cervical cancer. We gathered RNA-seq transcriptome data and clinical information from cervical cancer patients using The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases. To begin, researchers assessed the differences in m6A regulatory factor expression levels between cervical cancer and normal tissues. Clustering analysis was adapted to assess PD-L1 expression, immunological score, immune cell infiltration, TME, and probable pathways in cervical cancer samples. The majority of m6A regulators were found to be considerably overexpressed in cervical cancer tissues. Using consensus clustering of 21 m6A regulators, we identified two subtypes (clusters 1/2) of cervical cancer, and we found that WHO stage and grade were associated with the subtypes. PD-L1 expression increased dramatically in cervical cancer tissues and was significantly linked to ALKBH5, FTO, METTL3, RBM15B, YTHDF1, YTHDF3, and ZC3H13 expression levels. Plasma cells and regulatory T cells (Tregs) were considerably elevated in cluster 2. Cluster 1 is involved in numerous signature pathways, including basal transcription factors, cell cycle, RNA degradation, and the spliceosome. The prognostic signature-based riskscore (METTL16, YTHDF1, and ZC3H13) was found to be an independent prognostic indicator of cervical cancer. The tumor immune microenvironment (TIME) was linked to m6A methylation regulators, and changes in their copy number will affect the quantity of tumor-infiltrating immune cells dynamically. Overall, our research discovered a powerful predictive signature based on m6A RNA methylation regulators. This signature correctly predicted the prognosis of cervical cancer patients. The m6A methylation regulator could be a critical mediator of PD-L1 expression and immune cell infiltration, and it could have a significant impact on the TIME of cervical cancer.

Immune Infiltration of Ulcerative Colitis and Detection of the m6A Subtype.

Ulcerative colitis (UC) is an inflammatory bowel disease characterized by persistent colon inflammation. N6-methyladenosine (m6A) methylation is one of the most prevalent RNA modifications with key roles in both normal and illness, but m6A methylation in ulcerative colitis is unknown. This research investigated m6A methylation in UC. We examined the expression of known m6A RNA methylation regulators in UC using the Gene Expression Omnibus database (GEO database). First, we used m6A regulators to examine m6A change in UC samples. These two patient groups were created by clustering three m6A gene expression datasets. These genes were then utilized to build an m6A gene network using WGCNA and PPI. These networks were built using differentially expressed genes. The 12 m6A regulators were found to be dispersed throughout the chromosome. The study's data were then connected, revealing positive or negative relationships between genes or signaling pathways. Then, PCA of the 12 m6A-regulated genes indicated that the two patient groups could be discriminated in both PC1 and PC2 dimensions. The ssGSEA algorithm found that immune invading cells could be easily distinguished across diverse patient groups. Both groups had varied levels of popular cytokines. The differential gene analysis of the two samples yielded 517 genes like FTO and RFX7. It found 9 hub genes among 121 genes in the blue module, compared their expression in two groups of samples, and found that the differences in expression of these 9 genes were highly significant. The identification of 9 possible m6A methylation-dependent gene regulatory networks suggests that m6A methylation is involved in UC pathogenesis. Nine candidate genes have been identified as possible markers for assessing UC severity and developing innovative UC targeted therapeutic approaches.

Comprehensive Analysis of m6A RNA Methylation Regulators and the Immune Microenvironment to Aid Immunotherapy in Pancreatic Cancer.

Pancreatic cancer (PAAD) is one of the most malignant cancers and immune microenvironment has been proved to be involved in pathogenesis of PAAD. m6A modification, related to the expression of m6A regulators, participates in the development of multiple cancers. However, the correlation between m6A regulators and immune microenvironment was largely unknown in PAAD. And because of the small sample size of pancreatic cancer in the TCGA database, it is not enough to draw a convincing conclusion. In the present study, we downloaded seven pancreatic cancer datasets with survival data and removed batch effects among these datasets to be used as the PAAD cohort to analyze the immune landscape of PAAD and the expression pattern of m6A regulators and divided the integrated dataset into cluster 1 and cluster 2 by consensus clustering for m6A regulators. Lower m6A regulators were found to be related to higher immune cell infiltration and a better survival. Moreover, we identified six m6A regulators and constructed the prognostic signature of m6A regulators. Patients with low-risk score had a higher response to immune checkpoint inhibitor and a longer overall survival. To figure out the underlying mechanism, we analyzed the cancer immunity cycle, most altered genes, gene set enrichment analysis (GSEA) and gene set variation analysis (GSVA) in risk subtypes. In summary, the present study proved m6A regulators modulated the PAAD immune microenvironment. And risk scores served as predictive indicator for immunotherapy and played a prognostic role for PAAD patients. Our study provided novel therapeutic targets to improve immunotherapy efficacy.

Characterization of RNA Sensing Pathways in Hepatoma Cell Lines and Primary Human Hepatocytes.

The liver is targeted by several human pathogenic RNA viruses for viral replication and dissemination; despite this, the extent of innate immune sensing of RNA viruses by human hepatocytes is insufficiently understood to date. In particular, for highly human tropic viruses such as hepatitis C virus, cell culture models are needed to study immune sensing. However, several human hepatoma cell lines have impaired RNA sensing pathways and fail to mimic innate immune responses in the human liver. Here we compare the RNA sensing properties of six human hepatoma cell lines, namely Huh-6, Huh-7, HepG2, HepG2-HFL, Hep3B, and HepaRG, with primary human hepatocytes. We show that primary liver cells sense RNA through retinoic acid-inducible gene I (RIG-I) like receptor (RLR) and Toll-like receptor 3 (TLR3) pathways. Of the tested cell lines, Hep3B cells most closely mimicked the RLR and TLR3 mediated sensing in primary hepatocytes. This was shown by the expression of RLRs and TLR3 as well as the expression and release of bioactive interferon in primary hepatocytes and Hep3B cells. Our work shows that Hep3B cells partially mimic RNA sensing in primary hepatocytes and thus can serve as in vitro model to study innate immunity to RNA viruses in hepatocytes.

RNA-Containing Immune Complexes Formed by Anti-Melanoma Differentiation Associated Gene 5 Autoantibody Are Potent Inducers of IFN-α.

Objective: Anti-melanoma differentiation-associated gene 5 (MDA5) autoantibody is a distinctive serology hallmark of dermatomyositis (DM). As an autoantigen, MDA5 is a cytoplasmic RNA recognition receptor. The aim of this study was to address the question of whether the RNA-containing immune complex (IC) formed by MDA5 and anti-MDA5 could activate type I interferon (IFN) response. Method: Patients with anti-MDA5+ DM (n = 217), anti-MDA5- DM (n = 68), anti-synthase syndrome (ASyS, n = 57), systemic lupus erythematosus (SLE, n = 245), rheumatoid arthritis (RA, n = 89), and systemic sclerosis (SSc, n = 30) and healthy donors (HD, n = 94) were enrolled in our studies. Anti-MDA5 antibody was detected by line blotting, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, and Western blotting. Cytokine profiling was determined by multiplex flow cytometry, and IFN-α was further measured by ELISA. Type I IFN-inducible genes were detected by quantitative PCR (qPCR). RNA-IC binding was analyzed by RNA immunoprecipitation. Plasmacytoid dendritic cells (pDCs) derived from healthy donors were cultivated and stimulated with MDA5 ICs with or without RNase and Toll-like receptor 7 (TLR-7) agonist. The interaction between MDA5 ICs and TLR7 was evaluated by immunoprecipitation and confocal microscopy. Results: According to our in-house ELISA, the presence of anti-MDA5 antibody in 76.1% of DM patients, along with 14.3% of SLE patients who had a lower titer yet positive anti-MDA5 antibody, was related to the high level of peripheral IFN-α. ICs formed by MDA5 and anti-MDA5 were potent inducers of IFN-α via TLR-7 in an RNA-dependent manner in vitro. Conclusion: Our data provided evidence of the mechanistic relevance between the anti-MDA5 antibody and type I IFN pathway.

ImmReg: the regulon atlas of immune-related pathways across cancer types.

Immune system gene regulation perturbation has been found to be a major cause of the development of various types of cancer. Numbers of mechanisms contribute to gene expression regulation, thus, systematically identification of potential regulons of immune-related pathways is critical to cancer immunotherapy. Here, we comprehensively chart the landscape of transcription factors, microRNAs, RNA binding proteins and long noncoding RNAs regulation in 17 immune-related pathways across 33 cancers. The potential immunology regulons are likely to exhibit higher expressions in immune cells, show expression perturbations in cancer, and are significantly correlated with immune cell infiltrations. We also identify a panel of clinically relevant immunology regulons across cancers. Moreover, the regulon atlas of immune-related pathways helps prioritizing cancer-related genes (i.e. ETV7, miR-146a-5p, ZFP36 and HCP5). We further identified two molecular subtypes of glioma (cold and hot tumour phenotypes), which were characterized by differences in immune cell infiltrations, expression of checkpoints, and prognosis. Finally, we developed a user-friendly resource, ImmReg (http://bio-bigdata.hrbmu.edu.cn/ImmReg/), with multiple modules to visualize, browse, and download immunology regulation. Our study provides a comprehensive landscape of immunology regulons, which will shed light on future development of RNA-based cancer immunotherapies.

The RNA sensor MDA5 detects SARS-CoV-2 infection.

Human cells respond to infection by SARS-CoV-2, the virus that causes COVID-19, by producing cytokines including type I and III interferons (IFNs) and proinflammatory factors such as IL6 and TNF. IFNs can limit SARS-CoV-2 replication but cytokine imbalance contributes to severe COVID-19. We studied how cells detect SARS-CoV-2 infection. We report that the cytosolic RNA sensor MDA5 was required for type I and III IFN induction in the lung cancer cell line Calu-3 upon SARS-CoV-2 infection. Type I and III IFN induction further required MAVS and IRF3. In contrast, induction of IL6 and TNF was independent of the MDA5-MAVS-IRF3 axis in this setting. We further found that SARS-CoV-2 infection inhibited the ability of cells to respond to IFNs. In sum, we identified MDA5 as a cellular sensor for SARS-CoV-2 infection that induced type I and III IFNs.

Revealing immune responses in the Mycobacterium avium subsp. paratuberculosis-infected THP-1 cells using single cell RNA-sequencing.

Mycobacterium avium subsp. paratuberculosis (MAP) is a causative agent of Johne's disease, which is a chronic and debilitating disease in ruminants. MAP is also considered to be a possible cause of Crohn's disease in humans. However, few studies have focused on the interactions between MAP and human macrophages to elucidate the pathogenesis of Crohn's disease. We sought to determine the initial responses of human THP-1 cells against MAP infection using single-cell RNA-seq analysis. Clustering analysis showed that THP-1 cells were divided into seven different clusters in response to phorbol-12-myristate-13-acetate (PMA) treatment. The characteristics of each cluster were investigated by identifying cluster-specific marker genes. From the results, we found that classically differentiated cells express CD14, CD36, and TLR2, and that this cell type showed the most active responses against MAP infection. The responses included the expression of proinflammatory cytokines and chemokines such as CCL4, CCL3, IL1B, IL8, and CCL20. In addition, the Mreg cell type, a novel cell type differentiated from THP-1 cells, was discovered. Thus, it is suggested that different cell types arise even when the same cell line is treated under the same conditions. Overall, analyzing gene expression patterns via scRNA-seq classification allows a more detailed observation of the response to infection by each cell type.

ceRNA network development and tumor-infiltrating immune cell analysis in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is among the primary causes of cancer deaths globally. Despite efforts to understand liver cancer, its high morbidity and mortality remain high. Herein, we constructed two nomograms based on competing endogenous RNA (ceRNA) networks and invading immune cells to describe the molecular mechanisms along with the clinical prognosis of HCC patients. RNA maps of tumors and normal samples were downloaded from The Cancer Genome Atlas database. HTseq counts and fragments per megapons per thousand bases were read from 421 samples, including 371 tumor samples and 50 normal samples. We established a ceRNA network based on differential gene expression in normal versus tumor subjects. CIBERSORT was employed to differentiate 22 immune cell types according to tumor transcriptomes. Kaplan-Meier along with Cox proportional hazard analyses were employed to determine the prognosis-linked factors. Nomograms were constructed based on prognostic immune cells and ceRNAs. We employed Receiver operating characteristic (ROC) and calibration curve analyses to estimate these nomogram. The difference analysis found 2028 messenger RNAs (mRNAs), 128 micro RNAs (miRNAs), and 136 long non-coding RNAs (lncRNAs) to be significantly differentially expressed in tumor samples relative to normal samples. We set up a ceRNA network containing 21 protein-coding mRNAs, 12 miRNAs, and 3 lncRNAs. In Kaplan-Meier analysis, 21 of the 36 ceRNAs were considered significant. Of the 22 cell types, resting dendritic cell levels were markedly different in tumor samples versus normal controls. Calibration and ROC curve analysis of the ceRNA network, as well as immune infiltration of tumor showed restful accuracy (3-year survival area under curve (AUC): 0.691, 5-year survival AUC: 0.700; 3-year survival AUC: 0.674, 5-year survival AUC: 0.694). Our data suggest that Tregs, CD4 T cells, mast cells, SNHG1, HMMR and hsa-miR-421 are associated with HCC based on ceRNA immune cells co-expression patterns. On the basis of ceRNA network modeling and immune cell infiltration analysis, our study offers an effective bioinformatics strategy for studying HCC molecular mechanisms and prognosis.

B cell subset composition segments clinically and serologically distinct groups in chronic cutaneous lupus erythematosus.

OBJECTIVE: While the contribution of B-cells to SLE is well established, its role in chronic cutaneous lupus erythematosus (CCLE) remains unclear. Here, we compare B-cell and serum auto-antibody profiles between patients with systemic lupus erythematosus (SLE), CCLE, and overlap conditions. METHODS: B-cells were compared by flow cytometry amongst healthy controls, CCLE without systemic lupus (CCLE+/SLE-) and SLE patients with (SLE+/CCLE+) or without CCLE (SLE+/CCLE-). Serum was analyed for autoreactive 9G4+, anti-double-stranded DNA, anti-chromatin and anti-RNA antibodies by ELISA and for anti-RNA binding proteins (RBP) by luciferase immunoprecipitation. RESULTS: Patients with CCLE+/SLE- share B-cell abnormalities with SLE including decreased unswitched memory and increased effector B-cells albeit at a lower level than SLE patients. Similarly, both SLE and CCLE+/SLE- patients have elevated 9G4+ IgG autoantibodies despite lower levels of anti-nucleic acid and anti-RBP antibodies in CCLE+/SLE-. CCLE+/SLE- patients could be stratified into those with SLE-like B-cell profiles and a separate group with normal B-cell profiles. The former group was more serologically active and more likely to have disseminated skin lesions. CONCLUSION: CCLE displays perturbations in B-cell homeostasis and partial B-cell tolerance breakdown. Our study demonstrates that this entity is immunologically heterogeneous and includes a disease segment whose B-cell compartment resembles SLE and is clinically associated with enhanced serological activity and more extensive skin disease. This picture suggests that SLE-like B-cell changes in primary CCLE may help identify patients at risk for subsequent development of SLE. B-cell profiling in CCLE might also indentify candidates who would benefit from B-cell targeted therapies.

A novel nano therapeutic using convalescent plasma derived exosomal (CPExo) for COVID-19: A combined hyperactive immune modulation and diagnostics.

Extracellular vesicles like exosomes are important therapeutic tactics for treating COVID -19. By utilizing convalescent plasma derived exosomes (CPExo) from COVID-19 recovered persistence could accelerate the treatment strategies in the current state of affairs. Adequate literature has shown that administering the exosome to the in vivo system could be beneficial and could target the pathogens in an effective and precise manner. In this hypothesis we highlight the CPExo instead of convalescent plasma (CP), perhaps to dispense of exosomes are gratified and it's more effectively acquired immune response conferral through antibodies. COVID-19 convalescent plasma has billions of exosomes and it has aptitudes to carry molecular constituents like proteins, lipids, RNA and DNA, etc. Moreover, exosomes are capable of recognizing antigens with adequate sensitivity and specificity. Many of these derivatives could trigger an immune modulation into the cells and act as an epigenetic inheritor response to target pathogens through RNAs. COIVID-19 resistance activated plasma-derived exosomes are either responsible for the effects of plasma beyond the contained immune antibodies or could be inhibitory. The proposed hypothesis suggests that preselecting the plasma-derived antibodies and RNAs merged exosomes would be an optimized therapeutic tactic for COVID-19 patients. We suggest that, the CPExo has a multi-potential effect for treatment efficacy by acting as immunotherapeutic, drug carrier, and diagnostic target with noncoding genetic materials as a biomarker.

The networks of m6A-SARS-CoV-2 related genes and immune infiltration patterns in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disease with a poor prognosis. The current coronavirus disease 2019 (COVID-19) shares some similarities with IPF. SARS-CoV-2 related genes have been reported to be broadly regulated by N6-methyladenosine (m6A) RNA modification. Here, we identified the association between m6A methylation regulators, COVID-19 infection pathways, and immune responses in IPF. The characteristic gene expression networks and immune infiltration patterns of m6A-SARS-CoV-2 related genes in different tissues of IPF were revealed. We subsequently evaluated the influence of these related gene expression patterns and immune infiltration patterns on the prognosis/lung function of IPF patients. The IPF cohort was obtained from the Gene Expression Omnibus dataset. Pearson correlation analysis was performed to identify the correlations among genes or cells. The CIBERSORT algorithm was used to assess the infiltration of 22 types of immune cells. The least absolute shrinkage and selection operator (LASSO) and proportional hazards model (Cox model) were used to develop the prognosis prediction model. Our research is pivotal for further understanding of the cellular and genetic links between IPF and SARS-CoV-2 infection in the context of the COVID-19 pandemic, which may contribute to providing new ideas for prognosis assessment and treatment of both diseases.

DUSP11-mediated control of 5'-triphosphate RNA regulates RIG-I sensitivity.

Deciphering the mechanisms that regulate the sensitivity of pathogen recognition receptors is imperative to understanding infection and inflammation. Here we demonstrate that the RNA triphosphatase dual-specificity phosphatase 11 (DUSP11) acts on both host and virus-derived 5'-triphosphate RNAs rendering them less active in inducing a RIG-I-mediated immune response. Reducing DUSP11 levels alters host triphosphate RNA packaged in extracellular vesicles and induces enhanced RIG-I activation in cells exposed to extracellular vesicles. Virus infection of cells lacking DUSP11 results in a higher proportion of triphosphorylated viral transcripts and attenuated virus replication, which is rescued by reducing RIG-I expression. Consistent with the activity of DUSP11 in the cellular RIG-I response, mice lacking DUSP11 display lower viral loads, greater sensitivity to triphosphorylated RNA, and a signature of enhanced interferon activity in select tissues. Our results reveal the importance of controlling 5'-triphosphate RNA levels to prevent aberrant RIG-I signaling and demonstrate DUSP11 as a key effector of this mechanism.

Nucleic Acid Sensors as Therapeutic Targets for Human Disease.

Innate immune sensors that detect nucleic acids are attractive targets for therapeutic intervention because of their diverse roles in many disease processes. In detecting RNA and DNA from either self or non-self, nucleic acid sensors mediate the pathogenesis of many autoimmune and inflammatory conditions. Despite promising pre-clinical data and investigational use in the clinic, relatively few drugs targeting nucleic acid sensors are approved for therapeutic use. Nevertheless, there is growing appreciation for the untapped potential of nucleic acid sensors as therapeutic targets, driven by the need for better therapies for cancer, infectious diseases, and autoimmune disorders. This review highlights the diverse mechanisms by which nucleic acid sensors are activated and exert their biological effects in the context of various disease settings. We discuss current therapeutic strategies utilizing agonists and antagonists targeting nucleic acid sensors to treat infectious disease, cancer, and autoimmune and inflammatory disorders.

Rate of replenishment and microenvironment contribute to the sexually dimorphic phenotype and function of peritoneal macrophages.

Macrophages reside in the body cavities where they maintain serosal homeostasis and provide immune surveillance. Peritoneal macrophages are implicated in the etiology of pathologies including peritonitis, endometriosis, and metastatic cancer; thus, understanding the factors that govern their behavior is vital. Using a combination of fate mapping techniques, we have investigated the impact of sex and age on murine peritoneal macrophage differentiation, turnover, and function. We demonstrate that the sexually dimorphic replenishment of peritoneal macrophages from the bone marrow, which is high in males and very low in females, is driven by changes in the local microenvironment that arise upon sexual maturation. Population and single-cell RNA sequencing revealed marked dimorphisms in gene expression between male and female peritoneal macrophages that was, in part, explained by differences in composition of these populations. By estimating the time of residency of different subsets within the cavity and assessing development of dimorphisms with age and in monocytopenic Ccr2 -/- mice, we demonstrate that key sex-dependent features of peritoneal macrophages are a function of the differential rate of replenishment from the bone marrow, whereas others are reliant on local microenvironment signals. We demonstrate that the dimorphic turnover of peritoneal macrophages contributes to differences in the ability to protect against pneumococcal peritonitis between the sexes. These data highlight the importance of considering both sex and age in susceptibility to inflammatory and infectious diseases.

Development of RNA/DNA Hydrogel Targeting Toll-Like Receptor 7/8 for Sustained RNA Release and Potent Immune Activation.

Guanosine- and uridine-rich single-stranded RNA (GU-rich RNA) is an agonist of Toll-like receptor (TLR) 7 and TLR8 and induces strong immune responses. A nanostructured GU-rich RNA/DNA assembly prepared using DNA nanotechnology can be used as an adjuvant capable of improving the biological stability of RNA and promoting efficient RNA delivery to target immune cells. To achieve a sustained supply of GU-rich RNA to immune cells, we developed a GU-rich RNA/DNA hydrogel (RDgel) using nanostructured GU-rich RNA/DNA assembly, from which GU-rich RNA can be released in a sustained manner. A hexapod-like GU-rich RNA/DNA nanostructure, or hexapodRD6, was designed using a 20-mer phosphorothioate-stabilized GU-rich RNA and six phosphodiester DNAs. Two sets of hexapodRD6 were mixed to obtain RDgel. Under serum-containing conditions, GU-rich RNA was gradually released from the RDgel. Fluorescently labeled GU-rich RNA was efficiently taken up by DC2.4 murine dendritic cells and induced a high level of tumor necrosis factor-α release from these cells when it was incorporated into RDgel. These results indicate that the RDgel constructed using DNA nanotechnology can be a useful adjuvant in cancer therapy with sustained RNA release and high immunostimulatory activity.

Regulation of cGAS- and RLR-mediated immunity to nucleic acids.

Pathogen-derived nucleic acids are crucial signals for innate immunity. Despite the structural similarity between those and host nucleic acids, mammalian cells have been able to evolve powerful innate immune signaling pathways that originate from the detection of cytosolic nucleic acid species, one of the most prominent being the cGAS-STING pathway for DNA and the RLR-MAVS pathway for RNA, respectively. Recent advances have revealed a plethora of regulatory mechanisms that are crucial for balancing the activity of nucleic acid sensors for the maintenance of overall cellular homeostasis. Elucidation of the various mechanisms that enable cells to maintain control over the activity of cytosolic nucleic acid sensors has provided new insight into the pathology of human diseases and, at the same time, offers a rich and largely unexplored source for new therapeutic targets. This Review addresses the emerging literature on regulation of the sensing of cytosolic DNA and RNA via cGAS and RLRs.

Identification of U11snRNA as an endogenous agonist of TLR7-mediated immune pathogenesis.

The activation of innate immune receptors by pathogen-associated molecular patterns (PAMPs) is central to host defense against infections. On the other hand, these receptors are also activated by immunogenic damage-associated molecular patterns (DAMPs), typically released from dying cells, and the activation can evoke chronic inflammatory or autoimmune disorders. One of the best known receptors involved in the immune pathogenesis is Toll-like receptor 7 (TLR7), which recognizes RNA with single-stranded structure. However, the causative DAMP RNA(s) in the pathogenesis has yet to be identified. Here, we first developed a chemical compound, termed KN69, that suppresses autoimmunity in several established mouse models. A subsequent search for KN69-binding partners led to the identification of U11 small nuclear RNA (U11snRNA) as a candidate DAMP RNA involved in TLR7-induced autoimmunity. We then showed that U11snRNA robustly activated the TLR7 pathway in vitro and induced arthritis disease in vivo. We also found a correlation between high serum level of U11snRNA and autoimmune diseases in human subjects and established mouse models. Finally, by revealing the structural basis for U11snRNA's ability to activate TLR7, we developed more potent TLR7 agonists and TLR7 antagonists, which may offer new therapeutic approaches for autoimmunity or other immune-driven diseases. Thus, our study has revealed a hitherto unknown immune function of U11snRNA, providing insight into TLR7-mediated autoimmunity and its potential for further therapeutic applications.