Non-canonical inflammasome activation

Activation of inflammatory caspases is triggered by intracellular danger signals initiating a cascade that leads to pyroptosis, a lytic, proinflammatory form of cell death that occurs in response to infection or cellular damage (Shi J et al., 2014, 2015; Viganò E et al., 2015). CASP1 activation requires inflammasomes such as NLRP3 or NLRC4, whereas CASP4 and CASP5 are directly activated by binding cytosolic lipopolysaccharide (LPS), a key component of the outer membrane of Gram-negative bacteria, forming non-canonical inflammasomes (Shi J et al., 2014; Casson CN et al., 2015; Christgen S et al., 2020). The term “non-canonical inflammasome” was first introduced for CASP11, the murine ortholog of human CASP4 and CASP5, which mediates signaling distinct from the classical CASP1-driven inflammasome (Kayagaki N et al., 2011). Intracellular LPS activates Casp11 independently of Toll-like receptor 4 (TLR4) (Kayagaki N et al., 2013; Hagar JA et al., 2013), and Casp11, along with its human orthologs CASP4 and CASP5, functions as a direct receptor for cytosolic LPS (Shi J et al., 2014). Upon activation, inactive caspase monomers oligomerize to form dimeric structures where the protease domains of the two monomers are aligned in an anti-parallel orientation. This dimerization leads to a conformational change that exposes the catalytic site, autoproteolytically activating the caspase (reviewed by Boatright KM et al., 2003; Clark AC 2016; Ross C et al., 2022).

Activated CASP1, CASP4, CASP5 and murine Casp11 cleave the linker domain of gasdermin D (GSDMD), generating N- and C-terminal fragments (Shi J et al., 2015; Kayagaki N et al., 2015; Liu X et al., 2016). The N-terminal fragment of GSDMD oligomerizes and inserts into cellular membranes to form pores, leading to cell swelling and membrane rupture, a hallmark of pyroptosis (Liu X et al., 2016; Ding J et al., 2016; Sborgi L et al., 2016; Aglietti RA et al., 2016). Unlike CASP1, which efficiently processes pro-IL-1β and pro-IL-18 to generate their mature, biologically active forms, both CASP4 and CASP5 preferentially activate pro-IL-18 and inactivate IL-1β (Shi X et al., 2023; Exconde PM et al., 2023; Devant P et al., 2023; reviewed by Exconde PM 2024). The mature cytokines of IL-1 family are secreted through GSDMD pores, thereby amplifying the inflammatory response in mammals (Shi J et al., 2015; Kayagaki N et al., 2015; reviewed by Broz P et al., 2020; Liu X et al., 2021). CASP4- and CASP5-mediated pyroptosis can also lead to secondary activation of the NLRP3 inflammasome, further enhancing CASP1 activity (Schmid-Burgk JL et al., 2015; Baker PJ et al., 2015).

CASP5 expression is low under basal conditions but is strongly upregulated by inflammatory stimuli such as bacterial LPS and IFNγ, particularly in immune cells. In contrast, both CASP1 and CASP4 are constitutively expressed in a tissue and cell type specific manner, with high expression detected in lung, spleen, and colon, and can also be upregulated during inflammation (Lin XY et al., 2000; Feng Q et al., 2004; Eckhart L et al., 2006; Viganò E et al., 2015). In human peripheral blood mononuclear cells (PBMCs), LPS stimulation results in a >10-fold increase in CASP5 expression levels, a greater induction than that observed for CASP1 or CASP4 (Eckhart L et al., 2006). Although CASP4 and CASP5 share overlapping mechanisms, such as activation by cytosolic LPS and processing of similar substrates, their distinct expression patterns and potential differences in substrate affinity suggest non-redundant roles in innate immunity and disease-specific contexts (Ghait M et al., 2023; Cheng Y et al., 2023; Shi X et al., 2023; Exconde PM et al., 2023, 2024). CASP12 is also classified as an inflammatory caspase, though its physiological role remains poorly understood (Di Sano F et al., 2006; Salvamoser R et al., 2019).