Calpain Activity Is Essential for ATP-Driven Unconventional Vesicle-Mediated Protein Secretion and Inflammasome Activation in Human Macrophages This information is current as of October 1, 2021. Elina Välimäki, Wojciech Cypryk, Juhani Virkanen, Katariina Nurmi, Pauli M. Turunen, Kari K. Eklund, Karl E. Åkerman, Tuula A. Nyman and Sampsa Matikainen J Immunol published online 16 September 2016 http://www.jimmunol.org/content/early/2016/09/15/jimmun Downloaded from ol.1501840 Supplementary http://www.jimmunol.org/content/suppl/2016/09/15/jimmunol.150184 Material 0.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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Published September 16, 2016, doi:10.4049/jimmunol.1501840 The Journal of Immunology Calpain Activity Is Essential for ATP-Driven Unconventional Vesicle-Mediated Protein Secretion and Inflammasome Activation in Human Macrophages Elina Va¨lima¨ki,*,† Wojciech Cypryk,* Juhani Virkanen,‡ Katariina Nurmi,x Pauli M. Turunen,{ Kari K. Eklund,x Karl E. A˚ kerman,{ Tuula A. Nyman,* and Sampsa Matikainen†,x Extracellular ATP is an endogenous danger signal that is known to activate inflammatory responses in innate immune cells, in- cluding macrophages. Activated macrophages start to secrete proteins to induce an immune response, as well as to recruit other immune cells to the site of infection and tissue damage. In this study, we characterized the secretome (i.e., the global pattern of secreted proteins) of ATP-stimulated human macrophages. We show that ATP stimulation activates robust vesicle-mediated un- Downloaded from conventional protein secretion, including exosome release and membrane shedding, from human macrophages. Pathway analysis of the identified secreted proteins showed that calpain-related pathways were overrepresented in the secretome of ATP-stimulated cells. In accordance with this, calpains, which are calcium-dependent nonlysosomal cysteine proteases, were activated upon ATP stimulation through a P2X purinoceptor 7 receptor–dependent pathway. Functional studies demonstrated that calpain activity is essential for the P2X purinoceptor 7 receptor–mediated activation of unconventional protein secretion. Unconventional protein secretion was followed by cell necrosis and NLRP3 inflammasome–mediated secretion of the mature form of the proinflammatory http://www.jimmunol.org/ cytokine IL-1b. Furthermore, ATP-driven NLRP3 inflammasome activation was also dependent on calpain activity. Interestingly, pro–IL-1b and inflammasome components ASC and caspase-1 were released by ATP-activated macrophages through a vesicle- mediated secretion pathway. In conclusion, to our knowledge, we provide the first global characterization of proteins secreted by ATP-activated human macrophages and show a pivotal role for calpains in the activation of the inflammatory response during ATP exposure. The Journal of Immunology, 2016, 197: 000–000. he innate immune system is activated during the early Extracellular ATP is a well-recognized damage-associated molec- response to microbial infection and tissue damage. Mac- ular pattern by the innate immune system. During normal homeostasis, T rophages are the central effector cells of innate immunity, extracellular ATP is rapidly hydrolyzed by ectonucleotidases; however, by guest on October 1, 2021 and their pattern recognition receptors detect the presence of pathogen- ATP released upon tissue damage exceeds the amount readily hy- associated molecular patterns and damage-associated molecular pat- drolyzed, resulting in the detection of ATP by tissue-resident mac- terns (1, 2). This recognition results in the activation of macrophages. rophages. The receptor for extracellular ATP is P2X purinoceptor They start to secrete cytokines to mount an inflammatory response, 7 (P2X7), which is an ATP-gated ion channel located on the plasma chemokines to recruit other immune cells to the site of infection or membrane of many cell types (4). Binding of ATP causes the channel inflammation, and other proteins that induce antimicrobial defense to dilate, allowing small cations to move across the plasma membrane and tissue regeneration (3). and resulting in changes in the ion balance of the cells. Activation of P2X7 initiates multiple, probably cell-type–specific downstream sig- *Institute of Biotechnology, University of Helsinki, 00014 Helsinki, Finland; naling events, such as reactive oxygen species formation, cell pro- †Finnish Institute of Occupational Health, 00250 Helsinki, Finland; ‡Department of x liferation, activation of transcription, and induction of apoptosis (5). Geosciences and Geography, University of Helsinki, 00014 Helsinki, Finland; Di- vision of Rheumatology, Helsinki University Hospital, Helsinki University, 00015 In macrophages, an important P2X7 downstream signaling event is Helsinki, Finland; and {Department of Physiology, Institute of Biomedicine, Biomedicum activation of the NLRP3 inflammasome, which is a protein complex Helsinki, University of Helsinki, 00014 Helsinki, Finland consisting of NLRP3, adaptor protein ASC, and cysteine protease ˚ ORCIDs: 0000-0002-3129-1678 (P.M.T.); 0000-0003-4625-3081 (K.E.A.). caspase-1 (6). Assembly of the NLRP3 inflammasome results in Received for publication August 20, 2015. Accepted for publication August 21, 2016. activation of caspase-1. Activated caspase-1 cleaves pro–IL-1b and This work was supported by Academy of Finland Grants 135628, 140950, 272931, pro–IL-18 to biologically active cytokines IL-1b and IL-18, which and 255826, the Sigrid Juse´lius Foundation, the National Doctoral Programme in Informational and Structural Biology (to E.V.), and the Integrative Life Science are secreted and initiate a strong inflammatory response (7, 8). The Doctoral Program (to W.C.). P2RX7 gene is highly polymorphic, and the activation of P2X7 is Address correspondence and reprint requests to Dr. Sampsa Matikainen, Division of associated with many inflammatory, immune, neurologic, and mus- Rheumatology, Helsinki University Hospital, Helsinki University, P.O. Box 63, culoskeletal diseases. 00014 Helsinki, Finland. E-mail address: sampsa.matikainen@helsinki.fi Calpains are calcium-dependent nonlysosomal cysteine prote- The online version of this article contains supplemental material. ases that are expressed ubiquitously in mammals and many other Abbreviations used in this article: BMDM, bone marrow–derived macrophage; DAVID, Database for Annotation, Visualization and Integrated Discovery; EV, extracellular ves- organisms. The calpain superfamily in humans consists of 18 genes icle; GO, Gene Ontology; ITGAX, integrin a-X; LC-MS/MS, liquid chromatography– coding for ubiquitously expressed and tissue-specific proteases (9). tandem mass spectrometry; LDH, lactate dehydrogenase; MSU, monosodium urate; Two ubiquitously expressed major species of calpains, calpain-1 NTA, nanoparticle tracking analysis; P2X7, P2X purinoceptor 7. (m-calpain) and calpain-2 (m-calpain), are the well-studied pro- Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 teases among the calpain family. They form heterodimers with www.jimmunol.org/cgi/doi/10.4049/jimmunol.1501840 2 CALPAINS AND ATP-DRIVEN PROTEIN SECRETION calpain small subunit-1 (also calpain-4), which is required for urate (MSU) was purchased from Enzo Life Sciences (Farmingdale, NY). catalytic activity of the enzyme. The activity of calpains is regulated The pharmacological inhibitors CA-074 Me (25 mM), MDL 28170 (Calpain by binding of Ca2+ and phospholipids and by phosphorylation. In Inhibitor III; 10–100 mM), PD 150606 (100 mM), calpeptin (100 mM), and Z-YVAD-FMK (Caspase-1 Inhibitor VI; 25–50 mM) (all from Calbiochem), addition, calpastatin is an endogenous calpain-specific inhibitor (10). as well as BAPTA-AM (10 mM; Enzo Life Sciences) and AZ 10606120 2+ Binding of Ca to calpain promotes translocation of the calpain (10 mM; Tocris Bioscience) were added to the cells 1 h prior to stimulation. heterodimer to cell membranes, after which both subunits are au- Secretome characterization tolyzed, and the enzyme becomes fully active and is released into cytosol (11). A well-known substrate for calpains is proinflammatory For secretome analysis, macrophages were stimulated in serum-free RPMI cytokine IL-1a, and the cleavage and secretion of IL-1a are de- 1640 (supplemented with L-glutamine and antibiotics; all from Life Technologies) with 3 mM ATP for 15 min, after which cell culture su- pendent on calpain activity (10, 12). However, there is little infor- pernatants were collected. Supernatants from cells of three donors were mation whether calpains regulate protein secretion in general. always pooled together to reduce individual variation among blood