In vivo CRISPR screen reveals regulation of macrophage states in neuroinflammation

Area of Science:

• Neuroimmunology
• Genetics
• Cell Biology

Background:

• Macrophage polarization is critical in neuroinflammation and multiple sclerosis (MS).
• Understanding the in vivo regulation of macrophage states is essential for developing effective MS therapies.

Purpose of the Study:

• To establish an in vivo CRISPR screening pipeline to dissect macrophage regulation in mouse models of MS.
• To identify key cytokine regulators of macrophage polarization and function in the central nervous system (CNS).

Main Methods:

• Utilized genetically editable progenitor cells for in vivo CRISPR screening.
• Conducted high-throughput screening of over 100 cytokine receptors and signaling molecules.
• Employed single-cell transcriptomics and Perturb-seq for detailed cytokine action analysis.
• Integrated biosensor expression for real-time monitoring of myeloid cell functions.

Main Results:

• Identified interferon-γ, tumor necrosis factor, granulocyte-macrophage colony-stimulating factor, and transforming growth factor-β as essential in vivo regulators of macrophage polarization.
• Confirmed progenitor cell differentiation into all blood-derived CNS myeloid populations.
• Revealed conserved neuroinflammatory cytokine signatures across myeloid populations, CNS compartments, and species.
• Demonstrated monitoring of myeloid cell migration, phagocytosis, and oxidative activity.

Conclusions:

• The developed pipeline offers a scalable framework for high-resolution analysis of macrophage states in MS models.
• Uncovered critical cytokine signals regulating macrophage function in the CNS during neuroinflammation.
• Provides insights into conserved mechanisms of myeloid cell regulation relevant to human MS.