TY - JOUR
T1 - Single-Cell Monitoring of Activated Innate Immune Signaling by a d2eGFP-Based Reporter Mimicking Time-Restricted Activation of IFNB1 Expression
AU - Thomsen, Emil Aagaard
AU - Andersen, Sofie
AU - Marqvorsen, Mikkel Haarslev Schröder
AU - Skipper, Kristian Alsbjerg
AU - Paludan, Søren R.
AU - Mikkelsen, Jacob Giehm
N1 - Publisher Copyright:
Copyright © 2022 Thomsen, Andersen, Marqvorsen, Skipper, Paludan and Mikkelsen.
PY - 2022/1
Y1 - 2022/1
N2 - 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.
AB - 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.
KW - flow cytometry
KW - IFNB1 reporter
KW - IFNB1 transcription
KW - innate immunity
KW - single-cell
UR - http://www.scopus.com/inward/record.url?scp=85123982757&partnerID=8YFLogxK
U2 - 10.3389/fcimb.2021.784762
DO - 10.3389/fcimb.2021.784762
M3 - Journal article
C2 - 35118008
AN - SCOPUS:85123982757
SN - 2235-2988
VL - 11
JO - Frontiers in cellular and infection microbiology
JF - Frontiers in cellular and infection microbiology
M1 - 784762
ER -