A Genome-wide CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) Screen Identifies NEK7 as an Essential Component of NLRP3 Inflammasome Activation

Abstract

Inflammasomes are high molecular weight protein complexes that assemble in the cytosol upon pathogen encounter. This results in caspase-1-dependent pro-inflammatory cytokine maturation, as well as a special type of cell death, known as pyroptosis. The Nlrp3 inflammasome plays a pivotal role in pathogen defense, but at the same time, its activity has also been implicated in many common sterile inflammatory conditions. To this effect, several studies have identified Nlrp3 inflammasome engagement in a number of common human diseases such as atherosclerosis, type 2 diabetes, Alzheimer disease, or gout. Although it has been shown that known Nlrp3 stimuli converge on potassium ion efflux upstream of Nlrp3 activation, the exact molecular mechanism of Nlrp3 activation remains elusive. Here, we describe a genome-wide CRISPR/Cas9 screen in immortalized mouse macrophages aiming at the unbiased identification of gene products involved in Nlrp3 inflammasome activation. We employed a FACS-based screen for Nlrp3-dependent cell death, using the ionophoric compound nigericin as a potassium efflux-inducing stimulus. Using a genome-wide guide RNA (gRNA) library, we found that targeting Nek7 rescued macrophages from nigericininduced lethality. Subsequent studies revealed that murine macrophages deficient in Nek7 displayed a largely blunted Nlrp3 inflammasome response, whereas Aim2-mediated inflammasome activation proved to be fully intact. Although the mechanism of Nek7 functioning upstream of Nlrp3 yet remains elusive, these studies provide a first genetic handle of a component that specifically functions upstream of Nlrp3.Employing an evolutionary conserved set of pattern recognition receptors (PRRs), 2 the innate immune system senses the presence of microbial pathogens (1). Although PRRs can directly detect microbe-associated molecular patterns, some PRRs also respond to endogenous, host-derived signals that are formed or released upon perturbation or damage that is caused by microbial infections. These signals, which are commonly referred to as damage-associated molecular patterns, can also trigger PRR activation in the context of sterile inflammatory conditions (2). At the cell-autonomous level, PRR engagement and associated signaling cascades can result in a diverse set of responses, such as the induction of pro-inflammatory gene expression, the control of cytoskeletal rearrangement (e.g. in the context of phagocytosis), as well as the activation of proteolytic cascades, such as the inflammasome pathway. The inflammasome is a cytosolic multiprotein complex that regulates the activation and processing of caspase-1 (3). Inflammasome sensor proteins employ the adapter protein ASC to recruit caspase-1, which in turn results in the proximity-induced autoprocessing and activation of caspase-1. Active caspase-1 cleaves and thereby matures pro-inflammatory cytokines such as IL-1␤ and IL-18, and at the same time, it triggers an inflammatory type of cell death known as pyroptosis (3). The exact mechanisms of this caspase-1-dependent ce...

Manipulation of Interleukin-1β and Interleukin-18 Production by Yersinia pestis Effectors YopJ and YopM and Redundant Impact on Virulence

“…YopJ appears to be a major driver of cell death, with some inhibitory effects mediated by YopM. Very recent findings on cell death mediated by caspase-11 and caspase-1 via gasdermin D (43,44) and by NEK7 via NLRP3/caspase-1 (45,46) suggest that there are a number of additional players involved in regulation of inflammasome and pyroptosis pathways. Furthermore, other bacterial components, such as YopK (47), could differently impact the various death pathways.…”

AIM2 inflammasome is activated by pharmacological disruption of nuclear envelope integrity

“…For example, it is unclear how AIM2 activation is prevented during mitosis. One possibility is that AIM2 is regulated by mitotic factors as reported for NLRP3 (69,70). We previously showed that treatment with HIV-PIs triggers a transcriptional and translational reprogramming via the integrated stress response.…”

Inflammasomes: mechanism of assembly, regulation and signalling

“…CARD, caspase recruitment domain; CC, coiled-coil; PYD, pyrin domain. New insights into the mechanism of NLRP3 activation might be provided by three recent reports that have identified an unexpected role for NIMA-related kinase 7 (NEK7) in this process [14][15][16] . These studies found that NEK7 is essential for NLRP3 activation in response to both canonical and non-canonical stimuli and that it acts downstream of potassium efflux.…”

“…b | Potassium efflux is a common event that is associated with a diverse array of NLRP3 stimuli 13 . NLRP3 activation also requires NIMA-related kinase 7 (NEK7), which binds to the NLRP3 leucine-rich repeats (LRRs) and is required for its oligomerization [14][15][16] . c | NLR family, apoptosis inhibitory proteins (NAIPs) function as direct receptors for bacterial flagellin and the type 3 secretion system (T3SS) needle and rod subunits 21,22 .…”