Michael C. Heymann

Naturally Occurring Genetic Variants of Human Caspase‐1 Differ Considerably in Structure and the Ability to Activate Interleukin‐1β

Caspase‐1 (Interleukin‐1 Converting Enzyme, ICE) is a proinflammatory enzyme that plays pivotal roles in innate immunity and many inflammatory conditions such as periodic fever syndromes and gout. Inflammation is often mediated by enzymatic activation of interleukin (IL)‐1β and IL‐18. We detected seven naturally occurring human CASP1 variants with different effects on protein structure, expression, and enzymatic activity. Most mutations destabilized the caspase‐1 dimer interface as revealed by crystal structure analysis and homology modeling followed by molecular dynamics simulations. All variants demonstrated decreased or absent enzymatic and IL‐1β releasing activity in vitro, in a cell transfection model, and as low as 25% of normal ex vivo in a whole blood assay of samples taken from subjects with variant CASP1, a subset of whom suffered from unclassified autoinflammation. We conclude that decreased enzymatic activity of caspase‐1 is compatible with normal life and does not prevent moderate and severe autoinflammation.

Human Procaspase-1 Variants with Decreased Enzymatic Activity Are Associated with Febrile Episodes and May Contribute to Inflammation via RIP2 and NF-κB Signaling

The proinflammatory enzyme caspase-1 plays an important role in the innate immune system and is involved in a variety of inflammatory conditions. Rare naturally occurring human variants of the caspase-1 gene (CASP1) lead to different protein expression and structure and to decreased or absent enzymatic activity. Paradoxically, a significant number of patients with such variants suffer from febrile episodes despite decreased IL-1β production and secretion. In this study, we investigate how variant (pro)caspase-1 can possibly contribute to inflammation. In a transfection model, such variant procaspase-1 binds receptor interacting protein kinase 2 (RIP2) via Caspase activation and recruitment domain (CARD)/CARD interaction and thereby activates NF-κB, whereas wild-type procaspase-1 reduces intracellular RIP2 levels by enzymatic cleavage and release into the supernatant. We approach the protein interactions by coimmunoprecipitation and confocal microscopy and show that NF-κB activation is inhibited by anti–RIP2-short hairpin RNA and by the expression of a RIP2 CARD-only protein. In conclusion, variant procaspase-1 binds RIP2 and thereby activates NF-κB. This pathway could possibly contribute to proinflammatory signaling.

Contribution of the inflammasomes to autoinflammatory diseases and recent mouse models as research tools

Inflammasomes are multiprotein complexes that serve as activating platforms for the enzyme caspase-1 in response to various danger signals. Active caspase-1 can cleave the precursors of the pro-inflammatory cytokines IL-1β and IL-18 and thereby activate them. Deregulation of this cascade caused by mutations in genes coding for inflammasomal components and their interaction partners can lead to severe disease. This review summarizes the contribution of deregulated inflammasomes to the field of autoinflammatory syndromes. In addition, it gives insight into currently available mouse models that are used to study and characterize the role of the inflammasome components in the pathophysiology of these diseases.

Fluorescent tags influence the enzymatic activity and subcellular localization of procaspase-1

Subcellular localization studies and life cell imaging approaches usually benefit from fusion-reporter proteins, such as enhanced green fluorescent protein (EGFP) and mCherry to the proteins of interest. However, such manipulations have several risks, including protein misfolding, altered protein shuttling, or functional impairment when compared to the wild-type proteins. Here, we demonstrate altered subcellular distribution and function of the pro-inflammatory enzyme procaspase-1 as a result of fusion with the reporter protein mCherry. Our observations are of central importance to further investigations of subcellular behavior and possible protein-protein interactions of naturally occurring genetic variants of human procaspase-1 which have recently been linked to autoinflammatory disorders.

Current Knowledge on Procaspase-1 Variants with Reduced or Abrogated Enzymatic Activity in Autoinflammatory Disease

Caspase-1 is a proinflammatory enzyme that is essential in many inflammatory conditions including infectious, autoimmune, and autoinflammatory disorders. The inflammation is mainly mediated by the generation of inflammasomes that activate caspase-1 and subsequently interleukin (IL)-1β and IL-18. In addition, homotypic CARD/CARD interaction of procaspase-1 with RIP2 and thereby activation of the NF-κB pathways may play some role in the inflammation. However, normally, this pathway seems to become downregulated rapidly by the cleavage and excretion of RIP2 by active (pro-)caspase-1. In patients with unexplained recurrent systemic inflammation, CASP1 variants were detected, which often destabilized the caspase-1 dimer interface. Obviously, the resulting decreased or abrogated enzymatic activity and IL-1β production did not prevent the febrile episodes. As an unexpected finding, the inactive procaspase-1 variants significantly enhanced proinflammatory signaling by increasing RIP2 mediated NF-κB activation in an in vitro cell transfection model. A likely reason is the failure of inactive procaspase-1 to cleave bound RIP2 and also to mediate its excretion out of the intracelluar space thereby keeping the RIP2-NF-κB pathway upregulated. Hence, proinflammatory effects of enzymatically inactive procaspase-1 variants may partially explain the inflammatory episodes of the patients.

Recent Advances in Autoinflammatory Diseases and Animal Models

Autoinflammatory disorders are a fast growing group of human diseases which have provided unique insights into key mechanisms of inflammatory pathways. With the recognition of inflammasomes as an important factor in causing ongoing inflammatory responses, the innate immune system experienced a renaissance in the field of immunological research. Here we summarize recent advances in the understanding of the pathogenesis of autoinflammatory diseases and review selected mouse models available to study such diseases.

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-1β 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-1β secretion. The apparent paradox of reduced IL-1β 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-1β 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-1β-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.

Influence of the naturally occurring human CASP1 variant L265S on subcellular distribution and pyroptosis.

Patients with unexplained recurrent febrile episodes and CASP1 variants suffer from systemic sterile inflammation despite altered enzymatic activity of procaspase-1 and reduced IL-1β release. Most recent findings from our group indicate that the proinflammatory effects of CASP1 variants with reduced or abrogated enzymatic activity could be due to receptor interacting protein kinase 2 (RIP2) mediated increase of NF-kB activation. These findings are additionally supported by a trend to elevated IL-6 and TNF-α expression in patients with CASP1 variants.

Generation of inducible immortalized bone marrow derived cell lines expressing mutant procaspase-1 C284A on a caspase-1 knock-out background

Caspase-1, belonging to the family of cystein proteases, is well-known for its involvement in IL-1β and IL-18 maturation. Although, caspase-1 is one of the best described caspase-family-members, open questions concerning autoinflammatory diseases, caused by procaspase-1 variants (e.g. ICE-Fever), remain. ICE-Fever patients suffer from chronic febrile episodes, although procaspase-1 variants show reduced enzymatic activity leading to limited IL-1β secretion. The paradox of reduced IL-1β secretion but increased inflammation led to the hypothesis, that CASP1-variants enhance alternative signaling pathways. However, the role of procaspase-1 variants and their interaction partners during IL-1β maturation or NF-κB activation is still poorly understood.

PW03-027 - CASP1 variants and live cell imaging

Patients with unexplained recurrent febrile episodes and CASP1 variants suffer from systemic sterile inflammation despite reduced secretion of IL-1s. As previously demonstrated by our group CASP1 variants lead to reduced enzymatic activity of procaspase-1 by destabilizing the tertiary structure of the caspase-1 tetramer. A possible explanation for an alternative pro-inflammatory pathway has been provided by Lamkanfi and colleagues indicating an association between enzymatically inactive procaspase-1 and receptor interacting protein kinase 2 (RIP2) leading to NF-kB activation.