Multiplexed proteomics of autophagy-deficient murine macrophages reveals enhanced antimicrobial immunity via the oxidative stress response

Abstract

Defective autophagy is strongly associated with chronic inflammation. Loss-of-function of the core autophagy gene Atg16l1 increases risk for Crohn’s disease in part by enhancing innate immunity through myeloid cells such as macrophages. However, autophagy is also recognized as a mechanism for clearance of certain intracellular pathogens. These divergent observations prompted a re-evaluation of ATG16L1 in innate antimicrobial immunity. In this study, we found that loss of Atg16l1 in myeloid cells enhanced the killing of virulent Shigella flexneri (S.flexneri), a clinically relevant enteric bacterium that resides within the cytosol by escaping from membrane-bound compartments. Quantitative multiplexed proteomics of murine bone marrow-derived macrophages revealed that ATG16L1 deficiency significantly upregulated proteins involved in the glutathione-mediated antioxidant response to compensate for elevated oxidative stress, which simultaneously promoted S.flexneri killing. Consistent with this, myeloid-specific deletion of Atg16l1 in mice accelerated bacterial clearance in vitro and in vivo. Pharmacological induction of oxidative stress through suppression of cysteine import enhanced microbial clearance by macrophages. Conversely, antioxidant treatment of macrophages permitted S.flexneri proliferation. These findings demonstrate that control of oxidative stress by ATG16L1 and autophagy regulates antimicrobial immunity against intracellular pathogens.

MSstatsPTM: Statistical Relative Quantification of Posttranslational Modifications in Bottom-Up Mass Spectrometry-Based Proteomics

“…Maculins et al . ( 21 ) studied primary murine macrophages infected with S. flexneri . The experiment quantified the abundance of total protein and of phosphorylation in wildtype (WT) and in ATG16L1-deficient (cKO) samples, uninfected and infected with S. flexneri .…”

MSstatsShiny: A GUI for Versatile, Scalable, and Reproducible Statistical Analyses of Quantitative Proteomic Experiments

“…Masculins et al investigated primary murine macrophages infected with Shigella flexneri (S. flexneri).…”

“…Data set 2: Mouse -Phosphorylation -2mix-TMT Experimental Overview. Masculins et al 40 investigated primary murine macrophages infected with Shigella flexneri (S. flexneri). The experiment included both a total proteome (i.e., global profiling run) and a phosphopeptide enrichment run.…”

“…The instrument used, search parameters, and detailed data acquisition are described in the paper. 40 The original statistical analysis was performed using the command line version of MSstatsTMT. The raw data is available in MassIVE ID MSV000085565 and the input data for MSstatsShiny is available in reanalysis container RMSV000000357.2.…”

MSstatsPTM: Statistical relative quantification of post-translational modifications in bottom-up mass spectrometry-based proteomics

“…Experimental Design: Maculins et al [24] studied primary murine macrophages infected with S. Flexneri. The experiment quantified the abundance of total protein and of phosphorylation in wild type (WT), and in ATG16L1-deficient (cKO) samples, uninfected and infected with S. Flexneri.…”