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A Single-Stranded Oligonucleotide Inhibits Toll-Like Receptor 3 Activation and Reduces Influenza A (H1N1) Infection
Stockholm Univ, Wenner Gren Inst, Dept Mol Biosci, Stockholm, Sweden.
Stockholm Univ, Wenner Gren Inst, Dept Mol Biosci, Stockholm, Sweden.
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0001-8165-6439
Stockholm Univ, Wenner Gren Inst, Dept Mol Biosci, Stockholm, Sweden.
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2019 (English)In: Frontiers in Immunology, E-ISSN 1664-3224, Vol. 10, article id 10.3389/fimmu.2019.02161Article in journal (Refereed) Published
Abstract [en]

The initiation of an immune response is dependent on the activation and maturation of dendritic cells after sensing pathogen associated molecular patterns by pattern recognition receptors. However, the response needs to be balanced as excessive pro-inflammatory cytokine production in response to viral or stress-induced pattern recognition receptor signaling has been associated with severe influenza A virus (IAV) infection. Here, we use an inhibitor of Toll-like receptor (TLR)3, a single-stranded oligonucleotide (ssON) with the capacity to inhibit certain endocytic routes, or a TLR3 agonist (synthetic double-stranded RNA PolyI:C), to evaluate modulation of innate responses during H1N1 IAV infection. Since IAV utilizes cellular endocytic machinery for viral entry, we also assessed ssON's capacity to affect IAV infection. We first show that IAV infected human monocyte-derived dendritic cells (MoDC) were unable to up-regulate the co-stimulatory molecules CD80 and CD86 required for T cell activation. Exogenous TLR3 stimulation did not overcome the IAV-mediated inhibition of co-stimulatory molecule expression in MoDC. However, TLR3 stimulation using PolyI:C led to an augmented pro-inflammatory cytokine response. We reveal that ssON effectively inhibited PolyI:C-mediated pro-inflammatory cytokine production in MoDC, notably, ssON treatment maintained an interferon response induced by IAV infection. Accordingly, RNAseq analyses revealed robust up-regulation of interferon-stimulated genes in IAV cultures treated with ssON. We next measured reduced IAV production in MoDC treated with ssON and found a length requirement for its anti-viral activity, which overlapped with its capacity to inhibit uptake of PolyI:C. Hence, in cases wherein an overreacting TLR3 activation contributes to IAV pathogenesis, ssON can reduce this signaling pathway. Furthermore, concomitant treatment with ssON and IAV infection in mice resulted in maintained weight and reduced viral load in the lungs. Therefore, extracellular ssON provides a mechanism for immune regulation of TLR3-mediated responses and suppression of IAV infection in vitro and in vivo in mice.

Place, publisher, year, edition, pages
Frontiers Media S.A., 2019. Vol. 10, article id 10.3389/fimmu.2019.02161
Keywords [en]
influenza A, TLR3, single-stranded oligonucleotides, human monocyte-derived dendritic cells (MoDC), mice, cytokines, co-stimulatory molecules, clathrin-mediated endocytosis
National Category
Immunology Immunology in the medical area Microbiology in the medical area
Identifiers
URN: urn:nbn:se:kth:diva-289348DOI: 10.3389/fimmu.2019.02161ISI: 000485281600001PubMedID: 31572376Scopus ID: 2-s2.0-85072762910OAI: oai:DiVA.org:kth-289348DiVA, id: diva2:1522326
Note

QC 20210126

Available from: 2021-01-26 Created: 2021-01-26 Last updated: 2024-01-17Bibliographically approved
In thesis
1. Exploring the transcriptional space
Open this publication in new window or tab >>Exploring the transcriptional space
2021 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Transcriptomics promises biological insight into gene regulation, cell diversity, and mechanistic understanding of dysfunction. Driven by technological advancements in sequencing technologies, the field has witnessed an exponential growth in data output. Not only has the amount of raw data increased tremendously but it’s granularity as well. From only being able to obtain aggregated transcript information from large tissue samples, we can now pinpoint the precise origin of transcripts within the tissue, sometimes even within the confines of individual cells. This thesis focuses on the different aspects of how to use these emergent technologies to obtain a greater understanding of biological mechanisms. The work conducted here spans only a few years of the much longer history of spatially resolved transcriptomics, which started with the early in situ hybridization techniques and will continue to a potential future with complete molecular profiling ofevery cell in their natural, active state. Thus, at the same time the workpresented here introduces and demonstrates the use of the latest techniques within spatial transcriptomics, it also deals with the shortcomings of the current state of the field, which undoubtedly will see extensive improvements in the not too distant future. Article I is part of a series of articles where we mechanistically examine the biological underpinnings of a serendipitous finding that single-stranded nucleic acids have immunomodulatory effects. In particular, we look at influenza-infected innate immune cells and the ability of the oligonucleotide to inhibit viral entry. The oligonucleotides prevent the cells from responding to certain types of pattern recognitionand cause a decrease in viral load. Our hypothesis is that the administration of oligonucleotides blocks certain endocytic routes. While the invivo experiments suggest that the influenza virus is still able to infect and promote disease in the host, changes in signaling response due to the inhibition of the endocytotic routes could represent an avenue for future therapeutics. The conclusions were drawn by combining protein labeling and conventional methods for RNA profiling in the form of quantitative realtime PCR and bulk RNA sequencing. As a transition into the concept of spatial RNA profiling, the thesis includes an Additional material review article on spatial transcriptomics, where we give an overview of the current state of the field, as it looked like in the beginning of 2020. In Article II, we report on the development of an R package for analyzing spatial transcriptomics datasets. The package offers visualization features and an automated pipeline for masking tissue images and aligning serially sectioned experiments. The tool is extensively used throughout the rest of the articles where spatial transcript information is analyzed and is available for all scientists that use the supported spatial transcriptomics platforms in their research. In Article III, we propose a method to spatially map long-read sequencing data. While previously described methods for high-throughput spatial transcriptomics produce short-read data, full-length transcript information allows us to spatially profile alternatively spliced transcripts. Using the proposed method, we find alternatively spliced transcripts and find isoforms of the same gene to be differentially expressed in different regions of the mouse brain. Furthermore, we profile RNA editing across the full-length transcripts and find certain parts of the mouse left hemisphere to display a substantially higher degree of editing events compared to the rest of the brain. The proposed method is based on readily available reagents and does not require advanced instrumentation. We believe full-length transcript information obtained in this manner could help scientists obtain a deeper understanding from transcriptome data. Finally, in Article IV, we explore how the latest technologies for spatial transcriptomics can be used to characterize the expression landscape of respiratory syncytial virus infections by comparing infected and non-infected mouse lungs. By integration of annotated single-cell data and spatially resolved transcriptomics, we map the location of the single cells onto the spatial grid to localize immune cell populations across the tissue sections. By correlating the locations to gene expression, we profile locally confined cellular processes and immune responses. We believe that high-throughput spatial information obtained without predefined targets will become an important tool for exploratory analysis and hypothesis generation, which in turn could unlock mechanistic knowledge of the differences between experimental models that are important for translational research.

Abstract [sv]

Läran om genuttryck tros kunna ge kunskap kring celldiversitet och en ökad mekanistisk förståelse för dysregulation. Detta fält, benämnt transkriptomik, har sett exponentiell tillväxt i mån av genererad data på senare år, till stor del drivet av teknologiska framsteg. Inte bara den råa mängden data har ökat, utan även förmågan att särskilja vilka celler som informationen om generna kommer ifrån. Historiskt har sådan information endast observerats utifrån större vävnadsbitar, och således har ett medelvärde över flertalet celler observerats, utan att veta från vilka celler de individuella observationerna härstammar eller cellernas inbördes lokalisation. Denna avhandling kretsar kring de nya metoderna för spatiell analys av transkriptomet, vilka möjliggör positionering av vart någonstans i vävnaden genuttrycket sker och på så vis ger den granularitet som verklig mekanistisk förståelse ofta kräver. Det arbete som presenteras här spänner endast över några år av den längre bana som utvecklingen av spatiell transkriptomik befinner sig på, från de tidiga experimenten av in situ hybridisering till en potentiell framtid med komplett molekylär profilering av varje cell i deras naturliga miljö. Då det senare än ej är realiserat idag, behandlar avhandlingen och de inkluderade arbeten även tillkortakommanden i dagens teknik. Detta fält är under mycket snabb utveckling, och flera av de svagheter som finns idag tros vara kraftigt förminskade inom en relativt snart framtid. Artikel I är en del av en serie av artiklar där vi mekanistiskt undersöker ett fenomen där enkelsträngade nukleinsyror medför immunomodulativa effekter. Mer specifikt undersöker vi i den aktuella artikeln hur oligonukleotider av särskild längd påverkar influenzainfekterade dendritiska celler och viruspartiklarnas möjlighet att ta sig in i dessa celler. Vi finner inhibering av cellernas förmåga att respondera till särskild mönsterigenkänning samt minskade virusmängder direkt efter administration av oligonukleotider. Vår hypotes är att detta är en effekt av blockering av särskilda endocytotiska vägar. Experiment i möss tyder på att influensaviruset fortfarande är kapabelt att infektera och medföra sjukdom hos djuren, men resultatet av att blockera de endocytotiska upptagsvägarna för viruset medför förändrad signalering, vilket kan utgöra en intressant möjlighet för terapeutiska interventioner. Slutsatserna dras genom att kombinera protein-infärgning och konventionella metoder för analys av transkriptomet, i form av kvantitativ realtids-PCR och bulk-RNA-sekvensering. En övergång till spatiell analys görs sedan, där en review på ämnet är inkluderad i avhandlingen som en bilaga, och fungerar som översikt över alla de metoder som tagits fram för att möjliggöra denna typ av analys, så som det såg ut i början av 2020. I Artikel II visar vi utvecklingen av en mjukvara skriven i R för sekvensbaserad spatiell transkriptomik. Mer specifikt adderar vi visualiseringsmöjligheter och en automatiserad pipeline för bildhantering. Verktyget är öppet tillgängligt för alla som använder de spatiella transkriptomik-plattformarna som stöds. I Artikel III vidareutvecklar vi protokollet för spatiell transkriptomik för att kunna utnyttja de teknologiska framstegen som skett inom sekvensering av fullängds-transkriptomik. Genom att läsa av hela transkript istället för endast kortare bitar, som är standard idag, kan transkriptomets fulla komplexitet analyseras. Exempelvis visar vi hur kvantiteter av olika isoformer av en och samma gen skiljer sig markant mellan olika regioner i mushjärnan samt hur vissa typer av RNA-förändringar är vanligare i olika regioner. Det föreslagna protokollet använder enkelt tillgängliga reagenser och kräver ingen avancerad mätutrustning. Vi tror att fullängds-information kommer att vara avgörande för att uppnå komplett biologisk förståelse utifrån transkriptomdata. Slutligen, i Artikel IV, använder vi de senaste metoderna för spatiell transkriptomik för att undersöka hur den lokala miljön i lungan påverkas av en viral infektion genom att jämföra genuttrycket mellan infekterade och icke-infekterade möss. Genom att integrera publikt tillgänglig annoterad data från enskilda celler me spatiell transkriptomdata, kartlägger vi hur olika typer av immunceller lokaliserar sig över vävnadssnitten. Genom att korrelera genuttryck och celltypernas position, skapar vi en uttömmande bild över hur olika cellulära processer och immunresponser uppvisar lokala anpassningar. Vi tror att storskalig spatial information utan fördefinierade val kring vilka gener som undersöks kommer att utgöra ett viktigt verktyg för explorativ analys och hypotesgenerering.  

Place, publisher, year, edition, pages
KTH Royal Institute of Technology, 2021. p. 59
Series
TRITA-CBH-FOU ; 2021:3
Keywords
Transcriptomics Spatial
National Category
Natural Sciences
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-289365 (URN)978-91-7873-761-1 (ISBN)
Public defence
2021-02-19, https://kth-se.zoom.us/w/68188432057, 10:00 (English)
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Supervisors
Note

Remote defense due to ongoing pandemic

QC 2021-01-29

Available from: 2021-01-29 Created: 2021-01-26 Last updated: 2022-06-25Bibliographically approved

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Bergenstråhle, JosephLundeberg, Joakim

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