CASP1 variants influence subcellular caspase-1 localization, pyroptosome formation, pro-inflammatory cell death and macrophage deformability.

CASP1 variants result in reduced enzymatic activity of procaspase-1 and impaired IL-1ß release. Despite this, affected individuals can develop systemic autoinflammatory disease. These seemingly contradictory observations have only partially been explained by increased NF-κB activation through prolonged interaction of variant procaspase-1 with RIP2. To identify further disease underlying pathomechanisms, we established an in vitro model using shRNA-directed knock-down of procaspase-1 followed by viral transduction of human monocytes (THP-1) with plasmids encoding for wild-type procaspase-1, disease-associated CASP1 variants (p.L265S, p.R240Q) or a missense mutation in the active center of procaspase-1 (p.C285A). THP1-derived macrophages carrying CASP1 variants exhibited mutation-specific molecular alterations. We here provide in vitro evidence for abnormal pyroptosome formation (p.C285A, p.240Q, p.L265S), impaired nuclear (pro)caspase-1 localization (p.L265S), reduced pro-inflammatory cell death (p.C285A) and changes in macrophage deformability that may contribute to disease pathophysiology of patients with CASP1 variants. This offers previously unknown molecular pathomechanisms in patients with systemic autoinflammatory disease.

Enzymatically Inactive Procaspase 1 stabilizes the ASC Pyroptosome and Supports Pyroptosome Spreading during Cell Division.

Caspase-1 is a key player during the initiation of pro-inflammatory innate immune responses, activating pro-IL-1ß in so-called inflammasomes. A subset of patients with recurrent febrile episodes and systemic inflammation of unknown origin harbor mutations in CASP1 encoding caspase-1. CASP1 variants result in reduced enzymatic activity of caspase-1 and impaired IL-1ß secretion. The apparent paradox of reduced IL-1ß secretion but systemic inflammation led to the hypothesis that CASP1 mutations may result in variable protein interaction clusters, thus activating alternative signaling pathways. To test this hypothesis, we established and characterized an in vitro system of transduced immortalized murine macrophages expressing either WT or enzymatically inactive (p.C284A) procaspase-1 fusion reporter proteins. Macrophages with variant p.C284A caspase-1 did not secrete IL-1ß and exhibited reduced inflammatory cell death, referred to as pyroptosis. Caspase-1 and apoptosis-associated speck-like protein containing a CARD (ASC) formed cytosolic macromolecular complexes (so-called pyroptosomes) that were significantly increased in number and size in cells carrying the p.C284A caspase-1 variant compared with WT caspase-1. Furthermore, enzymatically inactive caspase-1 interacted with ASC longer and with increased intensity compared with WT caspase-1. Applying live cell imaging, we documented for the first time that pyroptosomes containing enzymatically inactive variant p.C284A caspase-1 spread during cell division. In conclusion, variant p.C284A caspase-1 stabilizes pyroptosome formation, potentially enhancing inflammation by two IL-1ß-independent mechanisms: pyroptosomes convey an enhanced inflammatory stimulus through the recruitment of additional proteins (such as RIP2, receptor interacting protein kinase 2), which is further amplified through pyroptosome and cell division.