Calpain Activity Is Essential for ATP-Driven Unconventional Vesicle-Mediated Protein Secretion and Inflammasome Activation in Human Macrophages

Abstract

Extracellular ATP is an endogenous danger signal that is known to activate inflammatory responses in innate immune cells, including 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 unconventional 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 cytokine IL-1β. Furthermore, ATP-driven NLRP3 inflammasome activation was also dependent on calpain activity. Interestingly, pro-IL-1β 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.

Calpain drives pyroptotic vimentin cleavage, intermediate filament loss, and cell rupture that mediates immunostimulation

“…Curiously, EGTA treatment increased full-length vimentin levels in cell extracts at 4 h; this observation was repeated several times. Furthermore, consistent with reports that Ng-induced inflammasome formation is independent of extracellular calcium (33) or calpain activity (34), neither EGTA nor MDL28170 prevented pyroptotic permeabilization or inflammasome formation (SI Appendix, Fig. S6).…”

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.…”

“…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.…”

Characterizing the contribution of inflammasome-derived exosomes in the activation of the immune response

“…Exosomes have been proposed to mediate one of the mechanisms involved in the stimulation of P2X purinoceptor 7 receptor (P2X7R) that rapidly triggers a key step of the inflammatory response: the induction of NLRP3/caspase-1 inflammasome signalling complexes that drive the proteolytic maturation and secretion of the pro-inflammatory cytokines interleukin-1β (IL-1β) and interleukin-18 (IL-18) (6). ATP stimulation through a P2X7R-dependent pathway activates robust vesicle-mediated unconventional protein secretion, including exosome release and membrane shedding, and drives NLRP3 inflammasome activation in a calpain activity dependent manner (7). Also related, central nervous system trauma induces inflammasome activation and secretion of exosomes containing inflammasome protein cargo into cerebral spinal fluid that fuse with target cells to activate the innate immune response in peripheral tissues (8).…”