Non-redundant roles of cathepsins in promoting necrosis of human macrophages

Macrophages can undergo inflammatory cell death via pyroptosis, an inflammasome-driven process mediated by caspase-1 and the pore-forming protein gasdermin D, or through alternative necrotic mechanisms triggered by lysosomal disruption involving cysteine cathepsins. However, the contributions of individual cysteine cathepsins to these pathways remain poorly defined, with overlapping roles often presumed. Here, we investigated the non-redundant roles of cathepsins in human macrophage cell death in response to distinct stimuli. Using selective pharmacological inhibitors and gene knockouts, we delineated protease functions during lysosomal membrane permeabilization (LMP) and NLRP3 inflammasome activation. We found that cathepsin B promotes lytic cell death induced by the lysosomotropic agent L-leucyl-L-leucine methyl ester (LLOMe), as its inhibition provided significant protection against LLOMe-triggered necrosis. This form of cell death was independent of canonical pyroptotic effectors, as LLOMe still caused rapid membrane lysis in gasdermin D- or caspase-1-deficient macrophages. Calpain-1, a calcium-activated protease, also cooperated in driving LMP-induced death. Calpain-1 inhibition delayed and partially prevented LLOMe-mediated lysis, an effect most pronounced in cells lacking gasdermin D or caspase-1. On the other hand, in LPS-stimulated M0 THP-1 cells nigericin responses were dose-dependent. We found that low-dose stimulation required the canonical caspase-1/GSDMD axis and was sensitive to cathepsin blockade, whereas high-dose nigericin bypassed inflammasome control and produced rapid, cathepsin-independent lysis. Together, our findings demonstrate that cathepsin B and calpain-1 orchestrate a necrotic, lytic cell death pathway in human macrophages that operates independently of the canonical pyroptosis machinery. These results reveal discrete, non-redundant functions of cysteine proteases in inflammatory cell death and suggest new therapeutic targets for modulating macrophage-driven inflammation.