Complete lysosomal disruption: A route to necrosis, not to the inflammasome

Lysosomes and their most abundant hydrolases, the cathepsins, have been implicated in several modes of cell death, including necrosis and apoptosis.¹ Indeed, lysosomal membrane permeabilization with release of lysosomal enzymes into the cytosol is a feature in many cell death cascades and can either act as a primary trigger or as an amplifier of the death signaling.² What determines the mode of cell death, or if the cell ultimately survives the leakage of the lysosomal enzymes, is not completely understood, but it is likely that multiple factors influence the final outcome. For example, an extensive and complete lysosome rupture has been shown to induce necrosis, whereas a partial, selective lysosomal permeabilization leads to apoptosis. In addition, the presence of cytosolic endogenous cathepsin inhibitors (cystatins and some serpins) and the relative expression and stability of each cathepsin at neutral pH may all contribute to the preferential activation of one death pathway vs. another.¹ Recently, several studies have postulated that lysosomal disruption may also signal the activation of the NLRP3 inflammasome, resulting in secretion of the pro-inflammatory cytokines IL-1β and IL-18, and pyroptosis, a novel pro-inflammatory form of cell death initially described in macrophages ingesting microbes, especially Salmonella species.³

NLRP3 (also known as NALP3 or cryopyrin) is an intracellular surveillance receptor regulating immune signaling in response to a variety of ligands, including bacterial toxins, viral particles, organic crystals and inorganic particulate compounds. NLRP3 has been implicated in bacterial and viral pathogenesis, autoimmune disorders, chronic inflammatory conditions and vaccine adjuvant activities. Engagement of NLRP3 triggers the formation of a large cytoplasmic complex (inflammasome), resulting in activation of the cysteine protease caspase-1, which leads to caspase-1-mediated cell death (pyroptosis), and processing and release of the pro-inflammatory cytokines IL-1β and IL-18. Given the great structural diversity of NLRP3 inducers, it is unlikely that these agents interact directly with the receptor. Instead, these heterogeneous agents are thought to activate NLRP3 by inducing a common upstream stress signal. Based on the observations that the adjuvant alum and other particulate NLRP3 inducers effectively destabilize lysosomes, and that inhibitors of lysosomal cathepsins block NLRP3 signaling, it has been hypothesized that lysosomal disruption may be the common upstream trigger. However, a direct assessment of the role of lysosome rupture in NLRP3 activation has been lacking.

In the June 15, 2013 issue of Cell Cycle,⁴ Lima et al. performed a side-by-side comparison of the effect of lysosome-disrupting agents (alum and LLOMe) and prototypical NLRP3 inducers (ATP and nigericin) on mouse macrophages. As expected, induction of the NLRP3 inflammasome resulted in caspase-1 activation, caspase-1-mediated pyroptosis and processing and secretion of IL-1β and IL-18. On the contrary, the lysosome-disrupting agents induced caspase-1-independent cell death with only minimal IL-1β release. The study provides evidence that alum and LLOMe trigger a cascade of events initiated by rapid and complete lysosome rupture, followed by cathepsin-dependent degradation of inflammatory proteins (including caspase-1) with inhibition of the NLRP3 signaling, and necrotic cell death (Fig. 1). This was remarkably different from the cellular pathways mediated by ATP or nigericin, which triggered significant IL-1β release, caspase-1-dependent pyroptosis and no protein degradation. Importantly, in cells treated with ATP or nigericin, lysosome rupture occurred only after caspase-1 activation and induction of pyroptosis, suggesting that lysosome dysfunction is not required for inflammasome activation. Based on these results, the authors conclude that upstream signals, such as potassium efflux, are likely more effective stress signals for NLRP3 activation than lysosome disruption. Taken together, their findings confirm that complete lysosome rupture is a catastrophic event leading to necrotic cell death; this cell death is independent of NLRP3 signaling and distinct from pyroptosis triggered by inflammasome-inducers, and can therefore explain the different immune response associated with these compounds. These observations complement another recent article published by the same group, where the authors showed that alum and LLOMe trigger cathepsin-mediated, caspase-1 and RIP-1-independent necrosis that is essential for their function as immunologic adjuvants.⁵ Together these papers provide insight into the mechanism by which the cell death phenotype of lysosome-disrupting agents contributes to the unique immunologic response generated by these compounds when used as adjuvants. More broadly, these studies provide strong proof for a danger theory of adjuvancy suggesting that our immune system has evolved to respond to agents that trigger cytotoxic events.

Figure 1. Involvement of lysosomes in different pro-inflammatory molecular pathways. Structurally different NLRP3-inducers stimulate the assembly of the NLRP3 inflammasome, which comprises the NOD-like receptor (NLR) NLRP3, the adaptor ASC and pro-caspase-1. Interaction of ASC with pro-caspase-1 leads to caspase-1 activation, which, in turn, results in (1) process and activation the pro-inflammatory cytokines pro-IL-1β and pro-IL-18 and extracellular secretion of mature IL-1β and IL-18 and (2) caspase-1-dependent cell death (pyroptosis). In this scenario, lysosome rupture is a late event that follows the induction of pyroptosis. Conversely, lysosome-disrupting adjuvants induce early and effective lysosome rupture with complete release of lysosomal cathepsins into the cytosol, which, in turn, leads to (1) inhibition of the NLRP3 inflammasome signaling through cathepsin-dependent degradation of pro-inflammatory proteins, including caspase-1, IL-1β and IL-18 and (2) cathepsin-mediated necrosis. Recent studies also suggest that selected chemotherapeutic drugs (i.e., gemcitabine, 5-fluorouracil) activate the NLRP3 inflammasome by causing limited lysosomal permeabilization and release of cathepsin B, which directly binds to NLRP3 and drives caspase-1 activation.⁶

Lima H, Jacobson LS, Goldberg MF, Chandran K, Diaz-Griffero F, Lisanti MP, et al. Role of lysosome rupture in controlling Nlrp3 signaling and necrotic cell death. Cell Cycle. 2013;12