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A transgenic in vitro cell model for the analysis of proinflammatory effects of naturally occurring genetic variants of caspase-1
© Schulze et al. 2015
Published: 28 September 2015
Caspase-1 (Interleukin-1 Converting Enzyme, ICE) is a proinflammatory enzyme mediating cleavage and secretion of the proinflammatory cytokines IL-1β and IL-18. Caspase-1 plays pivotal roles in the innate immune system and in inflammatory diseases like periodic fever syndromes, arthritis, or type-II-diabetes. In previous studies published by our group, genetic variants of procaspase-1 had been detected in patients suffering from autoinflammatory symptoms. Analyses of the procaspase-1 variants in HEK293T cells revealed reduced enzymatic activity of caspase-1 but enhanced ability to activate NF-κB signaling. The latter was mediated by enhanced CARD/CARD interactions of procaspase-1 with RIP2.
The primary objective of this study was to analyze the effects of procaspase-1 variants in a monocyte/macrophage cell model. Furthermore, protein expression and enzymatic activity of procaspase-1 with or without a C-terminal FLAG-tag was analyzed.
Materials and methods
Genetically engineered THP-1 monocytes were used for the in vitro study. First, THP-1 cells were transduced with lentiviral vectors expressing shRNA against procaspase-1 mRNA. Subsequently, procaspase-1 wildtype (wt) or variants with or without a C-terminal FLAG-tag were reconstituted using a second lentiviral transduction. THP-1 cells were differentiated into macrophages and stimulated with different inflammasome activators. Activation and release of caspase-1 and IL-1β was assessed using immunoblotting (caspase-1) or immunoblotting and cytometric bead arrays (IL-1β). The activation of NF-κB was estimated by measuring the NF-κB regulated cytokines IL-6 and IL-8. Cell death following inflammasome activation was analyzed by measuring LDH in the cell culture supernatant.
The protein expression level of reconstituted FLAG-tagged procaspase-1 variants was significantly reduced compared to expression of endogenous procaspase-1 or reconstituted procaspase-1 variants without FLAG-tag. In line with this data, the release of IL-1β after inflammasome stimulation was reduced in cells expressing FLAG-tagged procaspase-1 compared to cells expressing procaspase-1 without FLAG-tag. Interestingly, the mRNA expression of the FLAG-tagged procaspase-1 variants was not reduced compared to reconstituted procaspase-1 without FLAG-tag. Furthermore, cells expressing the procaspase-1 variants released reduced amounts of IL-1β and showed a reduced frequency of cell death following inflammasome stimulation. No differences in IL-6 or IL-8 secretion were detected when cells expressing wt or enzymatically inactive procaspase-1 were compared.
This study shows that even short protein-tags can influence protein expression significantly. Therefore, phenotypic rescue in knockdown studies can be complicated when using tagged proteins. Using the genetically engineered THP-1 cells we were able to show a reduced IL-1β release and reduced frequency of pyroptosis following inflammasome stimulation without detecting any differential regulation of the proinflammatory cytokines IL-6 or IL-8.
This study was supported by the German Research Foundation (DFG, KFO 249) and by a MeDDrive project (University of Technology, Medical Faculty) to SW.
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.