Monosodium Urate Activates Src/Pyk2/PI3 Kinase and Cathepsin Dependent Unconventional Protein Secretion From Human Primary Macrophages

Abstract

Monosodium urate (MSU) is an endogenous danger signal that is crystallized from uric acid released from injured cells. MSU is known to activate inflammatory response in macrophages but the molecular mechanisms involved have remained uncharacterized. Activated macrophages start to secrete proteins to activate immune response and to recruit other immune cells to the site of infection and/or tissue damage. Secretome characterization after activation of innate immune system is essential to unravel the details of early phases of defense responses. Here, we have analyzed the secretome of human primary macrophages stimulated with MSU using quantitative two-dimensional gel electrophoresis based proteomics as well as high-throughput qualitative GeLC-MS/MS approach combining protein separation by SDS-PAGE and protein identification by liquid chromatography-MS/MS. Both methods showed that MSU stimulation induced robust protein secretion from lipopolysaccharide-primed human macrophages. Bioinformatic analysis of the secretome data showed that MSU stimulation strongly activates unconventional, vesicle mediated protein secretion. The unconventionally secreted proteins included pro-inflammatory cytokines like IL-1␤ and IL-18, interferon-induced proteins, and danger signal proteins. Also active forms of lysosomal proteases cathepsins were secreted on MSU stimulation, and cathepsin activity was essential for MSUinduced unconventional protein secretion. Additionally, proteins associated to phosphorylation events including Src family tyrosine kinases were increased in the secretome of MSU-stimulated cells. Our functional studies demonstrated that Src, Pyk2, and PI3 kinases act upstream of cathepsins to activate the overall protein secretion from macrophages. In conclusion, we provide the first comprehensive characterization of protein secretion pathways activated by MSU in human macrophages, and reveal a novel role for cathepsins and Src, Pyk2, PI3 ki-

Global Characterization of Protein Secretion from Human Macrophages Following Non-canonical Caspase-4/5 Inflammasome Activation

“…Recent system level characterizations using modern mass spectrometrybased proteomics approaches have provided important novel information how innate immune cells, including macrophages, activate much more global protein secretion than just secreting cytokines and chemokines and that this global protein secretion is an important part of the innate immune response to different activation stimuli. We have previously shown that activators of canonical NLRP3 inflammasome, including monosodium urate and ATP, induce robust unconventional vesicle-mediated protein secretion in human macrophages (19,20). Our present results show that also non-canonical caspase-4/5 inflammasome activates EV-mediated protein secretion in human macrophages.…”

“…These cells are much more heterogeneous starting material than cell lines. We have seen this in our previous studies (19,29), and here these differences manifest in quantification fold differences obtained from different biological replicates.…”

“…Our present results show that also non-canonical caspase-4/5 inflammasome activates EV-mediated protein secretion in human macrophages. In addition, both canonical and non-canonical inflammasomes activated the secretion of many danger signal proteins (Table I) (19,20).…”

“…Recent system-level studies of protein secretion by activated immune cells, including macrophages, have highlighted the importance of different secretory pathways in innate immune response (15)(16)(17)(18). We have shown that NLRP3 inflammasome activators ATP and crystallized monosodium urate induce robust protein secretion in human macrophages (19,20), but the effect of non-canonical inflammasome activation on global protein secretion has remained uncharacterized. Here, we studied protein secretion in human macrophages in response to non-canonical caspase-4/5 inflammasome activation using label-free quantitative proteomics combined with bioinformatics.…”

Monosodium Urate Crystals Induce Upregulation of NK1.1-Dependent Killing by Macrophages and Support Tumor-Resident NK1.1+ Monocyte/Macrophage Populations in Antitumor Therapy

“…Macrophages are among the key cells that rapidly respond to and coordinate the local inflammatory response to MSU crystals (5)(6)(7)(8)(9)(10). Cells of the monocyte/macrophage lineage are highly dynamic immune cells that readily alter their functional phenotype in response to the local inflammatory environment and in a stimulus-specific manner (11,12).…”