ABSTRACT
Identifying the early islet cellular processes of autoimmune type 1 diabetes (T1D) in humans is challenging given the absence of symptoms during this period and the inaccessibility of the pancreas for sampling. In this article, we study temporal events in pancreatic islets in LEW.1WR1 rats, in which autoimmune diabetes can be induced with virus infection, by performing transcriptional analysis of islets harvested during the prediabetic period. Single-cell RNA-sequencing and differential expression analyses of islets from prediabetic rats reveal subsets of ß- and α-cells under stress as evidenced by heightened expression, over time, of a transcriptional signature characterized by interferon-stimulated genes, chemokines including Cxcl10, major histocompatibility class I, and genes for the ubiquitin-proteasome system. Mononuclear phagocytes show increased expression of inflammatory markers. RNA-in situ hybridization of rat pancreatic tissue defines the spatial distribution of Cxcl10+ ß- and α-cells and their association with CD8+ T cell infiltration, a hallmark of insulitis and islet destruction. Our studies define early islet transcriptional events during immune cell recruitment to islets and reveal spatial associations between stressed ß- and α-cells and immune cells. Insights into such early processes can assist in the development of therapeutic and prevention strategies for T1D.
Subject(s)
Diabetes Mellitus, Type 1 , Islets of Langerhans , Prediabetic State , Humans , Rats , Animals , Diabetes Mellitus, Type 1/metabolism , Islets of Langerhans/metabolism , RNA/metabolism , Inflammation/genetics , Inflammation/metabolism , Rats, Inbred LewABSTRACT
Type 1 diabetes is a chronic autoimmune disease, characterized by the immune-mediated destruction of insulin-producing ß cells of pancreatic islets. Essential components of the innate immune antiviral response, including type I IFN and IFN receptor (IFNAR)-mediated signaling pathways, likely contribute to human type 1 diabetes susceptibility. We previously showed that LEW.1WR1 Ifnar1 -/- rats have a significant reduction in diabetes frequency following Kilham rat virus (KRV) infection. To delineate the impact of IFNAR loss on immune cell populations in KRV-induced diabetes, we performed flow cytometric analysis in spleens from LEW.1WR1 wild-type (WT) and Ifnar1 -/- rats after viral infection but before the onset of insulitis and diabetes. We found a relative decrease in CD8+ T cells and NK cells in KRV-infected LEW.1WR1 Ifnar1 -/- rats compared with KRV-infected WT rats; splenic regulatory T cells were diminished in WT but not Ifnar1 -/- rats. In contrast, splenic neutrophils were increased in KRV-infected Ifnar1 -/- rats compared with KRV-infected WT rats. Transcriptional analysis of splenic cells from KRV-infected rats confirmed a reduction in IFN-stimulated genes in Ifnar1 -/- compared with WT rats and revealed an increase in transcripts related to neutrophil chemotaxis and MHC class II. Single-cell RNA sequencing confirmed that MHC class II transcripts are increased in monocytes and macrophages and that numerous types of splenic cells harbor KRV. Collectively, these findings identify dynamic shifts in innate and adaptive immune cells following IFNAR disruption in a rat model of autoimmune diabetes, providing insights toward the role of type I IFNs in autoimmunity.
Subject(s)
Autoimmunity/genetics , Diabetes Mellitus, Type 1/immunology , Interferon Type I/metabolism , Parvoviridae Infections/immunology , Animals , CD8-Positive T-Lymphocytes/immunology , Chemotaxis/immunology , Diabetes Mellitus, Type 1/blood , Disease Models, Animal , Female , Gene Expression Regulation/immunology , Histocompatibility Antigens Class II/genetics , Histocompatibility Antigens Class II/metabolism , Humans , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Male , Neutrophils/immunology , Neutrophils/metabolism , Parvoviridae Infections/blood , Parvoviridae Infections/virology , Parvovirus/immunology , RNA-Seq , Rats , Rats, Transgenic , Receptor, Interferon alpha-beta/genetics , Receptor, Interferon alpha-beta/metabolismABSTRACT
Enteroviral infections are implicated in islet autoimmunity and type 1 diabetes (T1D) pathogenesis. Significant ß-cell stress and damage occur with viral infection, leading to cells that are dysfunctional and vulnerable to destruction. Human stem cell-derived ß (SC-ß) cells are insulin-producing cell clusters that closely resemble native ß cells. To better understand the events precipitated by enteroviral infection of ß cells, we investigated transcriptional and proteomic changes in SC-ß cells challenged with coxsackie B virus (CVB). We confirmed infection by demonstrating that viral protein colocalized with insulin-positive SC-ß cells by immunostaining. Transcriptome analysis showed a decrease in insulin gene expression following infection, and combined transcriptional and proteomic analysis revealed activation of innate immune pathways, including type I interferon (IFN), IFN-stimulated genes, nuclear factor-kappa B (NF-κB) and downstream inflammatory cytokines, and major histocompatibility complex (MHC) class I. Finally, insulin release by CVB4-infected SC-ß cells was impaired. These transcriptional, proteomic, and functional findings are in agreement with responses in primary human islets infected with CVB ex vivo. Human SC-ß cells may serve as a surrogate for primary human islets in virus-induced diabetes models. Because human SC-ß cells are more genetically tractable and accessible than primary islets, they may provide a preferred platform for investigating T1D pathogenesis and developing new treatments.
ABSTRACT
Several key components of the RNA interference (RNAi) pathway were identified in genetic screens performed in non-mammalian model organisms. To identify components of the mammalian RNAi pathway, we developed a recessive genetic screen in mouse embryonic stem (ES) cells. Recessive genetic screens are feasible in ES cells that are Bloom-syndrome protein deficient (Blm-deficient). We constructed a reporter cell line in Blm-deficient ES cells to isolate RNAi mutants using a simple drug selection scheme. This chapter describes how we used retroviral gene-traps to mutagenize the reporter cell line and select for RNAi mutants. Putative RNAi mutants were confirmed using a separate functional assay. The location of the gene-trap was then identified using molecular techniques such as splinkerette PCR. Our screening strategy successfully isolated several mutant clones of Argonaute 2, a vital component of the RNAi pathway.
Subject(s)
Genes, Recessive , Genetic Testing , Mouse Embryonic Stem Cells/metabolism , RNA Interference , Animals , Gene Expression Regulation, Developmental , Genes, Reporter , Genetic Testing/methods , Genetic Vectors/genetics , Mice , Mouse Embryonic Stem Cells/cytology , Mutation , RecQ Helicases/genetics , Retroviridae/genetics , TransfectionABSTRACT
Influenza A virus (IAV) lacks the enzyme for adding 5' caps to its RNAs and snatches the 5' ends of host capped RNAs to prime transcription. Neither the preference of the host RNA sequences snatched nor the effect of cap-snatching on host processes is completely defined. Previous studies of influenza cap-snatching used poly(A)-selected RNAs from infected cells or relied on annotated host genes to define the snatched host RNAs, and thus lack details on many noncoding host RNAs including snRNAs, snoRNAs, and promoter-associated capped small (cs)RNAs, which are made by "paused" Pol II during transcription initiation. In this study, we used a nonbiased technique, CapSeq, to identify host and viral-capped RNAs including nonpolyadenylated RNAs in the same samples, and investigated the substrate-product correlation between the host RNAs and the viral RNAs. We demonstrated that noncoding host RNAs, particularly U1 and U2, are the preferred cap-snatching source over mRNAs or pre-mRNAs. We also found that csRNAs are highly snatched by IAV. Because the functions of csRNAs remain mostly unknown, especially in somatic cells, our finding reveals that csRNAs at least play roles in the process of IAV infection. Our findings support a model where nascent RNAs including csRNAs are the preferred targets for cap-snatching by IAV and raise questions about how IAV might use snatching preferences to modulate host-mRNA splicing and transcription.
Subject(s)
Influenza A Virus, H1N1 Subtype/genetics , RNA Caps/metabolism , Base Sequence , Cell Line, Tumor , Gene Expression Regulation, Viral , Genes, Viral , Humans , Influenza A Virus, H1N1 Subtype/metabolism , RNA Processing, Post-Transcriptional , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Viral/genetics , RNA, Viral/metabolismABSTRACT
Innate immune receptors detect Helicobacter pylori infection and trigger downstream signaling events that result in the production of cytokines and interferon-ß. This chapter gives an overview of the receptors and their roles in responding to H. pylori infection and details the downstream signaling events. The tools that have been developed to study the innate immune response to H. pylori are also discussed. Understanding the immune response to H. pylori is critical to develop better treatments for H. pylori-induced disease states including gastric malignancies and cancer.
Subject(s)
Helicobacter Infections/immunology , Helicobacter pylori/immunology , Immunity, Innate , Animals , Helicobacter pylori/pathogenicity , HumansABSTRACT
MicroRNAs (miRNAs) regulate gene expression through translation repression and mRNA destabilization. However, the molecular mechanisms of miRNA silencing are still not well defined. Using a genetic screen in mouse embryonic stem (ES) cells, we identify mammalian hyperplastic discs protein EDD, a known E3 ubiquitin ligase, as a key component of the miRNA silencing pathway. ES cells deficient for EDD are defective in miRNA function and exhibit growth defects. We demonstrate that E3 ubiquitin ligase activity is dispensable for EDD function in miRNA silencing. Instead, EDD interacts with GW182 family proteins in the Argonaute-miRNA complexes. The PABC domain of EDD is essential for its silencing function. Through the PABC domain, EDD participates in miRNA silencing by recruiting downstream effectors. Among the PABC-interactors, DDX6 and Tob1/2 are both required and sufficient for silencing mRNA targets. Taken together, these data demonstrate a critical function for EDD in miRNA silencing.
Subject(s)
Gene Silencing/physiology , MicroRNAs/physiology , Ubiquitin-Protein Ligases/physiology , Animals , Carrier Proteins/chemistry , Carrier Proteins/metabolism , DEAD-box RNA Helicases/chemistry , DEAD-box RNA Helicases/metabolism , Humans , Intracellular Signaling Peptides and Proteins , Mice , Protein Structure, Tertiary , Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins/metabolism , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/metabolismABSTRACT
Several key components of the RNA interference (RNAi) pathway were identified in genetic screens performed in nonmammalian model organisms. To identify components of the mammalian RNAi pathway, we developed a recessive genetic screen in mouse embryonic stem (ES) cells. Recessive genetic screens are feasible in ES cells that are Bloom-syndrome protein (Blm-) deficient. Therefore, we constructed a reporter cell line in Blm-deficient ES cells to isolate RNAi mutants through a simple drug-selection scheme. This chapter describes how we used retroviral gene traps to mutagenize the reporter cell line and select for RNAi mutants. Putative RNAi mutants were confirmed using a separate functional assay. The location of the gene trap was then identified using molecular techniques such as Splinkerette PCR. Our screening strategy successfully isolated several mutant clones of Argonaute2, a vital component of the RNAi pathway.
Subject(s)
Embryonic Stem Cells/metabolism , RNA Interference/physiology , Animals , Cell Line , Mice , Models, Genetic , Polymerase Chain Reaction , Retroviridae/genetics , Signal Transduction/genetics , Signal Transduction/physiologyABSTRACT
Genetic screens performed in model organisms have helped identify key components of the RNA interference (RNAi) pathway. Recessive genetic screens have recently become feasible through the use of mouse embryonic stem (ES) cells that are Bloom's syndrome protein (Blm) deficient. Here, we developed and performed a recessive genetic screen to identify components of the mammalian RNAi pathway in Blm-deficient ES cells. Genome-wide mutagenesis using a retroviral gene trap strategy resulted in the isolation of putative homozygous RNAi mutant cells. Candidate clones were confirmed by an independent RNAi-based reporter assay and the causative gene trap integration site was identified using molecular techniques. Our screen identified multiple mutant cell lines of Argonaute 2 (Ago2), a known essential component of the RNAi pathway. This result demonstrates that true RNAi components can be isolated by this screening strategy. Furthermore, Ago2 homozygous mutant ES cells provide a null genetic background to perform mutational analyses of the Ago2 protein. Using genetic rescue, we resolve an important controversy regarding the role of two phenylalanine residues in Ago2 activity.
Subject(s)
Mutagenesis , RNA Interference , RecQ Helicases/genetics , Animals , Argonaute Proteins , Cells, Cultured , Embryonic Stem Cells/metabolism , Eukaryotic Initiation Factor-2/genetics , Gene Deletion , Genes, Recessive , Loss of Heterozygosity , Mice , Mutation , Protein Structure, Tertiary/genetics , RecQ Helicases/deficiencyABSTRACT
MicroRNA (miRNA) silencing fine-tunes protein output and regulates diverse biological processes. Argonaute (Ago) proteins are the core effectors of the miRNA pathway. In lower organisms, multiple Agos have evolved specialized functions for distinct RNA silencing pathways. However, the roles of mammalian Agos have not been well characterized. Here we show that mouse embryonic stem (ES) cells deficient for Ago1-4 are completely defective in miRNA silencing and undergo apoptosis. In miRNA silencing-defective ES cells, the proapoptotic protein Bim, a miRNA target, is increased, and up-regulation of Bim is sufficient to induce ES cell apoptosis. Expression of activated Akt inhibits Bim expression and partially rescues the growth defect in Ago-deficient ES cells. Furthermore, reintroduction of any single Ago into Ago-deficient cells is able to rescue the endogenous miRNA silencing defect and apoptosis. Consistent with this, each Ago is functionally equivalent with bulged miRNA duplexes for translational repression, whereas Ago1 and Ago2 appear to be more effective at utilizing perfectly matched siRNAs. Thus, our results demonstrate that mammalian Agos all contribute to miRNA silencing, and individual Agos have largely overlapping functions in this process.