Macrophages play critical roles in immunity, development, tissue repair, and cancer, but studies of their function have been hampered by poorly-differentiated tumor cell lines and genetically-intractable primary cells. Here we report a facile system for genome editing in non-transformed macrophages by differentiating ER-Hoxb8 myeloid progenitors from Cas9-expressing transgenic mice. These conditionally immortalized macrophages (CIMs) retain characteristics of primary macrophages derived from the bone marrow yet allow for easy genetic manipulation and a virtually unlimited supply of cells. We demonstrate the utility of this system for dissection of host genetics during intracellular bacterial infection using two important human pathogens: Listeria monocytogenes and Mycobacterium tuberculosis.

Macrophages are highly plastic cells with critical roles in immunity, cancer, and tissue homeostasis, but how these distinct cellular fates are triggered by environmental cues is poorly understood. To uncover how primary murine macrophages respond to bacterial pathogens, we globally assessed changes in post-translational modifications of proteins during infection with Mycobacterium tuberculosis, a notorious intracellular pathogen. We identified hundreds of dynamically regulated phosphorylation and ubiquitylation sites, indicating that dramatic remodeling of multiple host pathways, both expected and unexpected, occurred during infection. Most of these cellular changes were not captured by mRNA profiling, and included activation of ubiquitin-mediated autophagy, an evolutionarily ancient cellular antimicrobial system. This analysis also revealed that a particular autophagy receptor, TAX1BP1, mediates clearance of ubiquitylated Mtb and targets bacteria to LC3-positive phagophores. These studies provide a new resource for understanding how macrophages shape their proteome to meet the challenge of infection.

Whether or not autophagy has a role in defence against Mycobacterium tuberculosis infection remains unresolved. Previously, conditional knockdown of the core autophagy component ATG5 in myeloid cells was reported to confer extreme susceptibility to M. tuberculosis in mice, whereas depletion of other autophagy factors had no effect on infection. We show that doubling cre gene dosage to more robustly deplete ATG16L1 or ATG7 resulted in increased M. tuberculosis growth and host susceptibility in mice, although ATG5-depleted mice are more sensitive than ATG16L1- or ATG7-depleted mice. We imaged individual macrophages infected with M. tuberculosis and identified a shift from apoptosis to rapid necrosis in autophagy-depleted cells. This effect was dependent on phagosome permeabilization by M. tuberculosis. We monitored infected cells by electron microscopy, showing that autophagy protects the host macrophage by partially reducing mycobacterial access to the cytosol. We conclude that autophagy has an important role in defence against M. tuberculosis in mammals.

Correction to: Nature Microbiologyhttps://doi.org/10.1038/s41564-023-01354-6, published online 10 April 2023. In the version of the article initially published, the Acknowledgements section did not include the Host Pathogen Map Initiative grant U19AI135990 to Jeffery S. Cox from the National Institute of Allergy and Infectious Diseases. This has now been updated in the HTML and PDF versions of the article.