Single-cell imaging of inflammatory caspase dimerization reveals differential recruitment to inflammasomes

Abstract

The human inflammatory caspases, including caspase-1, -4, -5 and -12, are considered as key regulators of innate immunity protecting from sepsis and numerous inflammatory diseases. Caspase-1 is activated by proximity-induced dimerization following recruitment to inflammasomes but the roles of the remaining inflammatory caspases in inflammasome assembly are unclear. Here, we use caspase bimolecular fluorescence complementation to visualize the assembly of inflammasomes and dimerization of inflammatory caspases in single cells. We observed caspase-1 dimerization induced by the coexpression of a range of inflammasome proteins and by lipospolysaccharide (LPS) treatment in primary macrophages. Caspase-4 and -5 were only dimerized by select inflammasome proteins, whereas caspase-12 dimerization was not detected by any investigated treatment. Strikingly, we determined that certain inflammasome proteins could induce heterodimerization of caspase-1 with caspase-4 or -5. Caspase-5 homodimerization and caspase-1/-5 heterodimerization was also detected in LPS-primed primary macrophages in response to cholera toxin subunit B. The subcellular localization and organization of the inflammasome complexes varied markedly depending on the upstream trigger and on which caspase or combination of caspases were recruited. Three-dimensional imaging of the ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain)/caspase-1 complexes revealed a large spherical complex of ASC with caspase-1 dimerized on the outer surface. In contrast, NALP1 (NACHT leucine-rich repeat protein 1)/caspase-1 complexes formed large filamentous structures. These results argue that caspase-1, -4 or -5 can be recruited to inflammasomes under specific circumstances, often leading to distinctly organized and localized complexes that may impact the functions of these proteases.

Non-canonical roles of caspase-4 and caspase-5 in heme driven- IL-1β release and cell death

“…When we analyzed the appearance and localization of the fluorescent complexes produced by heme in each case, we noticed some differences ( Figure 2D). The caspase-1 complex appeared as a single green punctum typical of ASC specks and of ASC-induced caspase-1 BiFC that we previously reported (27,42). Caspase-4 and caspase-5 BiFC appeared as a series of punctate spots located throughout the cytoplasm of the cell that did not co-localize with mitochondria.…”

“…The pBiFC.VC155 and pBiFC.VN173 plasmids encoding C1-Pro, C4-Pro, or C5-Pro were described previously (27). Single mutations were introduced using QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA).…”

“…BiFC uses non-fluorescent fragments of the yellow fluorescent protein, Venus ("split Venus") that can associate to reform the fluorescent Venus complex when fused to interacting proteins (41). When the CARD-containing prodomain of caspase-1 is fused to each half of split Venus, recruitment of caspase-1 to inflammasomes, and the subsequent induced proximity results in enforced association of the two Venus halves (27). Thus, Venus fluorescence (BiFC) acts as a read-out for caspase induced proximity, the proximal step for activation.…”

“…Recruitment of caspases to inflammasomes is dependent on an intact CARD. The aspartate residue (D59) in caspase-1 is essential for the ASC-caspase-1 interaction and mutation of this D59 residue blocks ASC-induced caspase-1 BiFC (27,43). We previously showed that ASC overexpression does not induce caspase-4 or caspase-5 BiFC but NLRC4 does (27).…”

Inflammatory caspase regulation: maintaining balance between inflammation and cell death in health and disease

“…This suggests that while NLRC4 can recruit ASC and caspase‐1 to induce cytokine processing, it can also form as separate complex independent of caspase‐1 that induces cell death. We previously demonstrated that caspase‐4 or capase‐5 dimerization is induced upon NLRC4 overexpression . Thus, it is possible that pyroptosis induced by this logarithmic phase S. typhimurium assembles an NLRC4 inflammasome that recruits and activates caspase‐4 or caspase‐5 to induce pyroptosis.…”