Cargando…

Single-Cell Monitoring of Activated Innate Immune Signaling by a d2eGFP-Based Reporter Mimicking Time-Restricted Activation of IFNB1 Expression

The innate immune system represents a balanced first line of defense against infection. Type I interferons (IFNs) are key regulators of the response to viral infections with an essential early wave of IFN-β expression, which is conditional, time-restricted, and stochastic in its nature. The possibil...

Descripción completa

Detalles Bibliográficos
Autores principales: Thomsen, Emil Aagaard, Andersen, Sofie, Marqvorsen, Mikkel Haarslev Schröder, Skipper, Kristian Alsbjerg, Paludan, Søren R., Mikkelsen, Jacob Giehm
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8803904/
https://www.ncbi.nlm.nih.gov/pubmed/35118008
http://dx.doi.org/10.3389/fcimb.2021.784762
_version_ 1784642971232108544
author Thomsen, Emil Aagaard
Andersen, Sofie
Marqvorsen, Mikkel Haarslev Schröder
Skipper, Kristian Alsbjerg
Paludan, Søren R.
Mikkelsen, Jacob Giehm
author_facet Thomsen, Emil Aagaard
Andersen, Sofie
Marqvorsen, Mikkel Haarslev Schröder
Skipper, Kristian Alsbjerg
Paludan, Søren R.
Mikkelsen, Jacob Giehm
author_sort Thomsen, Emil Aagaard
collection PubMed
description The innate immune system represents a balanced first line of defense against infection. Type I interferons (IFNs) are key regulators of the response to viral infections with an essential early wave of IFN-β expression, which is conditional, time-restricted, and stochastic in its nature. The possibility to precisely monitor individual cells with active IFNB1 transcription during innate signaling requires a robust reporter system that mimics the endogenous IFN-β signal. Here, we present a reporter system based on expression of a destabilized version of eGFP (d2eGFP) from a stably integrated reporter cassette containing the IFNB1 promoter and 3’-untranslated region, enabling both spatial and temporal detection of regulated IFNB1 expression. Specifically, this reporter permits detection, quantification, and isolation of cells actively producing d2eGFP in a manner that fully mimics IFN-β production allowing tracking of IFNB1 gene activation and repression in monocytic cells and keratinocytes. Using induced d2eGFP expression as a readout for activated immune signaling at the single-cell level, we demonstrate the application of the reporter for FACS-based selection of cells with genotypes supporting cGAS-STING signaling. Our studies provide a novel approach for monitoring on/off-switching of innate immune signaling and form the basis for investigating genotypes affecting immune regulation at the single-cell level.
format Online
Article
Text
id pubmed-8803904
institution National Center for Biotechnology Information
language English
publishDate 2022
publisher Frontiers Media S.A.
record_format MEDLINE/PubMed
spelling pubmed-88039042022-02-02 Single-Cell Monitoring of Activated Innate Immune Signaling by a d2eGFP-Based Reporter Mimicking Time-Restricted Activation of IFNB1 Expression Thomsen, Emil Aagaard Andersen, Sofie Marqvorsen, Mikkel Haarslev Schröder Skipper, Kristian Alsbjerg Paludan, Søren R. Mikkelsen, Jacob Giehm Front Cell Infect Microbiol Cellular and Infection Microbiology The innate immune system represents a balanced first line of defense against infection. Type I interferons (IFNs) are key regulators of the response to viral infections with an essential early wave of IFN-β expression, which is conditional, time-restricted, and stochastic in its nature. The possibility to precisely monitor individual cells with active IFNB1 transcription during innate signaling requires a robust reporter system that mimics the endogenous IFN-β signal. Here, we present a reporter system based on expression of a destabilized version of eGFP (d2eGFP) from a stably integrated reporter cassette containing the IFNB1 promoter and 3’-untranslated region, enabling both spatial and temporal detection of regulated IFNB1 expression. Specifically, this reporter permits detection, quantification, and isolation of cells actively producing d2eGFP in a manner that fully mimics IFN-β production allowing tracking of IFNB1 gene activation and repression in monocytic cells and keratinocytes. Using induced d2eGFP expression as a readout for activated immune signaling at the single-cell level, we demonstrate the application of the reporter for FACS-based selection of cells with genotypes supporting cGAS-STING signaling. Our studies provide a novel approach for monitoring on/off-switching of innate immune signaling and form the basis for investigating genotypes affecting immune regulation at the single-cell level. Frontiers Media S.A. 2022-01-18 /pmc/articles/PMC8803904/ /pubmed/35118008 http://dx.doi.org/10.3389/fcimb.2021.784762 Text en Copyright © 2022 Thomsen, Andersen, Marqvorsen, Skipper, Paludan and Mikkelsen https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Cellular and Infection Microbiology
Thomsen, Emil Aagaard
Andersen, Sofie
Marqvorsen, Mikkel Haarslev Schröder
Skipper, Kristian Alsbjerg
Paludan, Søren R.
Mikkelsen, Jacob Giehm
Single-Cell Monitoring of Activated Innate Immune Signaling by a d2eGFP-Based Reporter Mimicking Time-Restricted Activation of IFNB1 Expression
title Single-Cell Monitoring of Activated Innate Immune Signaling by a d2eGFP-Based Reporter Mimicking Time-Restricted Activation of IFNB1 Expression
title_full Single-Cell Monitoring of Activated Innate Immune Signaling by a d2eGFP-Based Reporter Mimicking Time-Restricted Activation of IFNB1 Expression
title_fullStr Single-Cell Monitoring of Activated Innate Immune Signaling by a d2eGFP-Based Reporter Mimicking Time-Restricted Activation of IFNB1 Expression
title_full_unstemmed Single-Cell Monitoring of Activated Innate Immune Signaling by a d2eGFP-Based Reporter Mimicking Time-Restricted Activation of IFNB1 Expression
title_short Single-Cell Monitoring of Activated Innate Immune Signaling by a d2eGFP-Based Reporter Mimicking Time-Restricted Activation of IFNB1 Expression
title_sort single-cell monitoring of activated innate immune signaling by a d2egfp-based reporter mimicking time-restricted activation of ifnb1 expression
topic Cellular and Infection Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8803904/
https://www.ncbi.nlm.nih.gov/pubmed/35118008
http://dx.doi.org/10.3389/fcimb.2021.784762
work_keys_str_mv AT thomsenemilaagaard singlecellmonitoringofactivatedinnateimmunesignalingbyad2egfpbasedreportermimickingtimerestrictedactivationofifnb1expression
AT andersensofie singlecellmonitoringofactivatedinnateimmunesignalingbyad2egfpbasedreportermimickingtimerestrictedactivationofifnb1expression
AT marqvorsenmikkelhaarslevschroder singlecellmonitoringofactivatedinnateimmunesignalingbyad2egfpbasedreportermimickingtimerestrictedactivationofifnb1expression
AT skipperkristianalsbjerg singlecellmonitoringofactivatedinnateimmunesignalingbyad2egfpbasedreportermimickingtimerestrictedactivationofifnb1expression
AT paludansørenr singlecellmonitoringofactivatedinnateimmunesignalingbyad2egfpbasedreportermimickingtimerestrictedactivationofifnb1expression
AT mikkelsenjacobgiehm singlecellmonitoringofactivatedinnateimmunesignalingbyad2egfpbasedreportermimickingtimerestrictedactivationofifnb1expression