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

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-1β 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 MSU-induced 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 kinases in the activation of unconventional protein secretion.The innate immune system is activated in response to microbial infection and tissue damage. Macrophages are the central players of the innate immunity and detect the presence of pathogen-associated molecular patterns (PAMPs)¹ and damage-associated molecular patterns (DAMPs) with their pattern recognition receptors. This recognition results in the activation of antimicrobial defense, inflammatory response, tissue regeneration, and recruitment of other inflammatory cells to the site of infection and/or tissue damage (1). Proper innate immune response is essential for the activation of the adaptive immune system. At present it is thought that the activation of innate immunity is most effective when both signals of microbial origin and damage are perceived at the same time (2, 3).Monosodium urate (MSU) is an endogenous DAMP that is crystallized from uric acid released by injured cells (4). Uric acid is a byproduct of purine degradation, and abnormally high levels of uric acid in serum, or hyperuricemia, is a hallmark of metabolic disorders where balance between intake of purines via food and excretion of uric acid is distorted. A well-known disease associated to hyperuricemia is gouty arthritis, in which deposits of MSU can be found in synovial fluid of peripheral joints, and MSU-induced inflammation is the initial trigger of symptoms (5). Hyperuricemia is also linked to other inflammatory diseases, like metabolic syndrome (6, 7), type 2 diabetes (8), and cardiovascular disease (9). MSU-induced inflammation is driven by the innate immune system. MSU engages antigen-presenting cells, macrophages, and dendritic cells. It is a potent adjuvant, initiating a robust adaptive immune response (4). Recently it has been shown that the adjuvant properties of alum are dependent on release of uric acid in vivo (10).It is unclear how cells detect the presence of MSU. It has been suggested that MSU activates intracellular signaling pathways in dendritic cells by directly engaging cellular membranes, particularly the cholesterol-rich components of the plasma membrane (11). Recently Uratsuji and coworkers showed that MSU activates inflammatory response in keratinocytes and monocytic THP-1 cells through membrane-associated P2Y6 (12). It is also well-documented that MSU activates the NLRP3 inflammasome in macrophages (13). The NLRP3 inflammasome is a multiprotein complex comprising of NACHT, LRR, and PYD domains-containing protein 3 (NLRP3), Apoptosis-associated speck-like protein containing a CARD (ASC) and cysteine protease Caspase-1. Activation of NLRP3 inflammasome results in the autocleavage of Caspase-1. The activated Caspase-1 then in turn cleaves pro-inflammatory cytokines IL-1β and IL-18 into their biologically active forms, which are then readily secreted (14–17). However, the signaling pathways that are involved in MSU-induced NLRP3 inflammasome activation have remained only partially characterized.Macrophages respond to activating stimuli by producing inflammatory mediators that are delivered to neighboring cells through multiple protein secretion pathways including both conventional and unconventional protein secretion (18). Conventionally secreted proteins contain an N-terminal signal peptide, which directs their transport to the plasma membrane through the well-characterized endoplasmic reticulum (ER)-Golgi pathway. In contrast, mediators and regulators of unconventional protein secretion pathways are less well understood. At present, different proteomic techniques allow for in-depth analysis of the secretome, the global pattern of secreted proteins. Secretome analysis is important in revealing complex cellular processes that require communication and signaling between the cells, and it has recently been applied to analyze the signaling pathways related to cell differentiation (19, 20), cancer (21, 22), and immune responses (23–25). In the present work we have characterized the secretome of human primary macrophages that have been activated simultaneously by microbial signal lipopolysaccharide (LPS) and endogenous danger signal MSU to get a global view of macrophage response to combined PAMP and DAMP stimulation.