Multiplexed proteomics of autophagy deficient macrophages reveals enhanced anti-microbial immunity via the oxidative stress response

Abstract Macrophages play a critical role in clearance of cytosolic pathogens. Autophagy functions at the intersection of antimicrobial innate immunity, metabolism and protein quality control; however, the impacts of infection and autophagy on shaping the macrophage proteome are poorly understood. Here, we describe a deep multi-dimensional proteomic analysis of primary murine macrophages infected with Shigella flexneri (S. flexneri). Tandem mass tagging (TMT) revealed dynamic genotype- and infection-dependent differences in host and pathogen proteins, phosphorylation and ubiquitination. These data catalogue the complex circuitry connecting autophagy, inflammatory signaling and the oxidative stress response. Loss of the autophagy gene Atg16l1 induced basal oxidative stress, activated the compensatory glutathione biosynthetic machinery, and surprisingly, enhanced clearance of S. flexneri. Pathogen clearance was similarly enhanced in wild type macrophages upon pharmacological inhibition of cysteine import. Our study provides a resource for innate immunity research and unexpectedly reveals that ATG16L1 dampens antimicrobial immunity by regulating oxidative stress.