Leveraging PhagoID to Define How Cytokines Reprogram the Phagosomal Proteome

Abstract

The ability of phagocytosing diverse cargoes and maintaining tissue homeostasis under different immune contexts and challenges is what lends tissue resident macrophages their identity as immune sentinels. Previous studies that were aimed towards isolating and studying the macrophage phagosome suffered from certain limitations due to the highly dynamic and interactive nature of this organelle, making its proteomic analysis an especially arduous task. In a recent study, we reported the development of a novel tool called PhagoID which uses a proximity labeling-based strategy for resolving phagosomal lumen proteins with high specificity and efficiency, facilitating their identification using mass spectrometry and downstream proteomic analyses. Here, we validate the use of epigallocatechin gallate (EGCG) as a ROS scavenger capable of minimizing extracellular labeling in the case of PhagoID. Next, we adapted PhagoID to quantify the phagosome lumen proteome of fetal-derived alveolar-like macrophages (FLAMs) under the effect of type I interferon signaling. The phagocytic activity of alveolar macrophages (AMs) is important both at baseline for routine cleaning of cellular debris, as well as to initiate a strong inflammatory responses to respiratory pathogens. We profiled the phagosomal proteome of FLAMs as a model for primary AMs and found proteins involved in the class I MHC pathway to be the only ER subset enriched in the phagosome on IFN-β activated FLAMs. Additionally, we identified the presence of proteins belonging to 3 families of interferon induced GTPases (IIGPs), including multiple GBPs and all IRGM proteins which have been well recognized for their role in cell autonomous immunity against bacterial infections like Mycobacterium tuberculosis and Chlamydia trachomatis. We also detected the enrichment of the enzyme aconitate decarboxylase (ACOD1/IRG1) involved in the production of the anti-microbial metabolite, itaconate. Going ahead, we propose the design of an itaconate biosensor targeted to the endosomal and lysosomal compartments to understand which host factors govern itaconate trafficking to phagosomes. Thus, using PhagoID enabled us to gain important insights into how IFN-β, a cytokine known for its highly context-dependent role in case of bacterial infections, can alter the phagosome proteome of AMs.