Unveiling the Reaction of Macrophages Towards Pathogens Through CRISPR Analysis

In a groundbreaking study, researchers at the CeMM Research Center for Molecular Medicine and Medical University of Vienna have combined CRISPR gene editing and machine learning to dissect the intricate regulatory programs in mouse macrophages. The findings, published in Cell Systems, offer a comprehensive understanding of the molecular processes in macrophages when encountering various pathogens and infection-triggering stimuli.

Macrophages, one of the first immune cells to respond to invading pathogens, play a crucial role in the body's innate immunity. These cells sense pathogens via pattern recognition receptors and promptly release signals to recruit other immune cells, trigger inflammation, and present digested fragments of pathogens on their surface. This, in turn, guides the adaptive immune system to develop long-term immunity.

The study, led by Christoph Bock and Matthias Farlik, employed a combined CROP-seq and CITE-seq method to perform high-throughput functional dissection of the macrophage response to Listeria. The team identified regulatory proteins that orchestrate these programs using CRISPR genome editing and single-cell RNA sequencing.

The researchers investigated six immune stimuli—Listeria, LCMV, Candida, LPS, IFN-β, and IFN-γ—over a dense multiomics time course. They tracked changes inside murine macrophages by measuring gene activity and DNA accessibility every few hours, establishing a molecular timeline of how regulatory programs unfold step by step when exposed to various immune stimuli that mimic bacterial or viral infections.

One of the key findings of the study is that Spi1/PU.1 is a key regulator in the macrophage response to Listeria. This discovery adds to our understanding of how macrophages initiate a tailored immune response quickly when encountering a pathogen. Both a late or insufficient response, and an overshooting immune response, are damaging.

Senior author Bock stated, "It's impressive how much complexity there is in the ancient part of the immune system that we studied, which is shared with sponges, jellyfish, and corals." Thanks to the advances in CRISPR screening technology, the team can systematically study the underlying regulatory programs in macrophages.

The study establishes a time-resolved regulatory map of pathogen response in macrophages, offering a broadly applicable method for dissecting immune-regulatory programs through integrative time-series analysis and high-content CRISPR screening. The team believes this study marks a significant step forward in understanding the intricacies of the immune system and paves the way for future research in this field.