Abstract
The NLRP3 (NLR family, Pyrin domain containing 3) inflammasome is a multiprotein complex comprised of NLRP3, pro-caspase-1, the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC), and the protein kinase NIMA related kinase 7 (NEK7) (Shi et al., 2016; He et al., 2016; Schmid-Burgk et al., 2016). When cells are exposed to microbes and/or danger signals, the inflammasome assembles and serves as a platform for the activation of caspase-1. Caspase-1 activation promotes the processing and secretion of the pro-inflammatory cytokines interleukin-1β (IL-1β), IL-18, and IL-33 as well as pyroptosis induction (Gross et al., 2011; Arend et al., 2008), which elicit inflammatory responses. Here, we describe how to co-transfect the NLRP3 inflammasome components into HEK293T cells, which enables inflammasome activation and the production of IL-1β upon stimulation with nigericin.
Background
Inflammasomes are multiprotein complexes that control inflammatory responses and coordinate immune responses against invading microbes. Reconstitution of the NLRP3 inflammasome in vitro provides an easy and efficient way to study the regulation of inflammasome activation. In this protocol, NEK7 was introduced into the in vitro NLRP3 inflammasome system and the ratio among the NLRP3 inflammasome components was optimized, making the reconstituted NLRP3 inflammasome more similar to the physiological inflammasome in vivo. Nigericin was used to activate the inflammasome as we have observed that it induces a rapid rate of IL-1β secretion compared to other inflammasome activators. Using this protocol, the levels of IL-1β can be assayed to determine NLRP3 inflammasome function under physiological conditions as well as after gene knockdown or overexpression.
Materials and Reagents
Equipment
Software
Procedure
Data analysis
ELISA data should be collected from a minimum of two independent experiments with at least 3 replicates per treatment. Figure 1 shows representative ELISA data after transfection of the indicated plasmids into HEK293T cells. The statistical significance of the differences between samples can be determined with GraphPad Prism 6 software and Student’s t-test (unpaired, two-tailed). A P value of < 0.05 is considered statistically significant. Figure 1. Levels of secreted IL-1β from reconstituted HEK293T cells. Transfected components in each sample are indicated below the X-axis. 50 μl supernatants of each HEK293T cell culture sample were used for the IL-1β ELISA. Representative data were graphed using GraphPad Prism 6 software.
Recipes
Acknowledgments
This protocol was adapted from the previously published study, Lu et al. (2012) and was performed by Shi et al. (2016). This work was supported by the US National Institutes of Health (U19 AI100627).
References
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