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

Area of Science:

• Immunology
• Molecular Biology
• Cell Biology

Background:

• The innate immune system is crucial for host defense against pathogens.
• Type I interferons (IFNs), particularly IFN-β, are key regulators of antiviral responses.
• Monitoring dynamic IFN-β gene expression at the single-cell level is challenging.

Purpose of the Study:

• To develop a robust reporter system for precise spatial and temporal monitoring of IFNB1 (IFN-β) gene transcription.
• To enable detection, quantification, and isolation of cells with active innate immune signaling.
• To facilitate the study of genetic variations influencing immune regulation.

Main Methods:

• Development of a reporter cassette with a destabilized eGFP (d2eGFP) reporter gene under the control of the IFNB1 promoter and 3'-untranslated region.
• Stable integration of the reporter cassette into monocytic cells and keratinocytes.
• Utilizing fluorescence-activated cell sorting (FACS) to quantify and select cells based on d2eGFP expression.

Main Results:

• The reporter system successfully mimics endogenous IFN-β signaling, allowing tracking of IFNB1 gene activation and repression.
• d2eGFP expression serves as a reliable readout for activated innate immune signaling at the single-cell level.
• The reporter enabled FACS-based selection of cells with genotypes supporting cGAS-STING signaling.

Conclusions:

• A novel reporter system provides a powerful tool for monitoring the on/off switching of innate immune signaling.
• This system facilitates the investigation of genotypes that affect immune regulation at the single-cell level.
• The reporter system advances the study of cellular responses to viral infections and immune system dynamics.