Macrophages are critical immune cells in the activation and timely resolution of inflammation. Classically-activated macrophages (M1) perform hallmark roles in inflammation including mobilization to sites of injury and phagocytosis of foreign bodies. We used Terminal Amino Isotopic Labeling of Substrates (TAILS) N terminomics to identify targets of intracellular cysteine and serine proteases that are required for macrophage polarization.
THP-1 monocytes were polarized with phorbol myristate acetate (PMA) to M0 macrophages and further differentiated with interferon gamma (IFNγ) or interleukin-4 (IL-4) to M1 or M2-type macrophages, respectively. We profiled macrophages against 720 human proteases, homologs and inhibitors using the protease/inhibitor CLIP-CHIP cDNA microarray. Macrophages were grown in triplex SILAC media, lysates digested, fractionated and analysed by a Thermo LTQ-Velos Orbitrap. SILAC-labeled M1 macrophages were treated with E64 (broad cysteine protease inhibitor) or AEBSF (broad serine protease inhibitor) and their N-terminomes analysed by TAILS using trypsin and lysargiNase digestion on a Thermo Q-Exactive HF.
CLIP-CHIP cDNA microarray quantified 429 protease and homologs as being expressed in M0, M1 and M2 macrophages, with 14 genes upregulated in M1 macrophages, of interest Cathepsin-B, -L, -S, Caspase-1 and TNFAIP3. Proteomic SILAC analysis quantified 1421 proteins showing that interferon response regulators were the most abundant protein changes in M1 macrophages as well as CATL, LAP3, PSMB9 being M1 protease effectors. Serine and cysteine protease inhibition abolished cell migration whereas cysteine protease inhibition alone abrogated endocytosis. SILAC TAILS terminomics identified 19,363 N-terminal peptides that were otherwise undetected by preTAILS (shotgun) analysis. SILAC abundance of 460 N-dimethylated termini were significantly changed >2σ by inhibition. These clustered to networks related to actin-, tubulin- and phagosome dynamics and calcium signaling.
TAILS positional proteomics quantified 19,363 N-termini and transcriptomics revealing essential cysteine proteases in the cytoskeletal remodeling that is crucial for endocytosis in M1 macrophages.