Damage control: Management of cellular stress by the NLRP3 inflammasome

Authors

  • Stefanie Haasken,

    1. Inflammation Program, Department of Internal Medicine, University of Iowa Carver College of Medicine, Iowa City, IA, USA
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  • Fayyaz S. Sutterwala

    Corresponding author
    1. Veterans Affairs Medical Center, Iowa City, IA, USA
    • Inflammation Program, Department of Internal Medicine, University of Iowa Carver College of Medicine, Iowa City, IA, USA
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Full correspondence Dr. Fayyaz S. Sutterwala, Inflammation Program, Department of Internal Medicine, University of Iowa Carver College of Medicine, 2501 Crosspark Road, D156 MTF, Coralville, IA 52241, USA

Fax: +1-319-335-4194

e-mail: fayyaz-sutterwala@uiowa.edu

See accompanying article by Licandro et al.

Abstract

The NLRP3 inflammasome plays a critical role in regulating inflammatory and cell death pathways in response to a diverse array of stimuli. Activation of the NLRP3 inflammasome results in activation of the cysteine protease caspase-1 and the subsequent processing and secretion of the proinflammatory cytokines IL-1β and IL-18. In this issue of the European Journal of Immunology, Licandro et al. [Eur. J. Immunol. 2013. 43, 2126–2137] show that the NLRP3 inflammasome contributes to oxidative DNA damage. In addition, activation of the NLRP3 inflammasome modulates a number of pathways involved in DNA damage repair, cell cycle, and apoptosis, suggesting a novel role for the NLRP3 inflammasome in DNA damage responses following cellular stress.

From microbes to radiation and other carcinogens, the environment in which we live can seem like a veritable minefield. Fortunately, the cells and molecules of the innate immune system have evolved, along with cell-intrinsic processes, to respond swiftly in defense of our cellular and genomic integrity. These multilayered and redundant mechanisms combat the potentially deleterious effects of diverse environmental stresses by promoting either resolution or cell death in an attempt to return to homeostasis.

An important component of the innate immune system is the NLRP3 inflammasome. Following detection of cellular damage, the cytoplasmic nucleotide-binding domain leucine-rich repeat containing (NLR) molecule NLRP3 forms a multiprotein complex, along with the adaptor molecule ASC and the cysteine protease caspase-1 [1]. This process culminates in the activation of caspase-1 and the subsequent maturation and secretion of the proinflammatory cytokines, IL-1β and IL-18 [2-5]. Interestingly, oligomerization and activation of the NLRP3 inflammasome can be induced by a heterogeneous collection of pathogen- and damage-associated molecular patterns (PAMPs and DAMPs, respectively), although the means by which this occurs is unclear. It has been proposed that these inflammasome activating signals actually work indirectly via a common downstream ligand, such as reactive oxygen species (ROS) [6, 7] generated following mitochondrial damage [8, 9]. Cellular cation fluxes, including a potassium efflux and a calcium influx, have also been shown to be critical for activation of the NLRP3 inflammasome [10, 11].

In addition to its role in immune surveillance, dysregulation of the NLRP3 inflammasome has been reported to contribute to the pathogenesis of a number of human diseases that have an underlying component of chronic inflammation, such as type 2 diabetes mellitus, atherosclerosis, and inflammatory bowel disease [12]. As well, mutations within the gene encoding NLRP3 have been associated with the autoinflammatory cryopyrin-associated periodic syndromes [13]. Such widespread effects underscore the complexity of pathways through which the well-studied NLRP3 inflammasome functions, and emerging literature on the subject indicates there is much left to learn.

In this issue of the European Journal of Immunology, Licandro et al. [14] explore noncanonical roles for the NLRP3 inflammasome, i.e. proinflammatory cytokine-independent effects under conditions of cellular stress. They first performed gene arrays to identify differentially expressed genes from WT or Nlrp3−/− bone marrow-derived murine DCs stimulated with monosodium urate (MSU) crystals to generate ROS and activate NLRP3 in the absence of a priming signal to bypass the production of IL-1β and IL-18 [4, 15]. This approach revealed differences in genes involved in DNA damage repair (DDR), cell cycle, and apoptosis/survival pathways (Fig. 1). The physiological relevance of these findings was then confirmed by a series of experiments demonstrating enhanced DNA damage but diminished repair due to the activation of the p53 pathway in NLRP3-sufficient DCs, suggesting that NLRP3 favors programed cell death following genotoxic stress.

Figure 1.

The NLRP3 inflammasome regulates the DNA damage response pathway. In the model proposed by Licandro et al. [14] oxidative DNA damage is induced in dendritic cells by MSU or high dose γ-radiation. Concurrent NLRP3 inflammasome activation results in (1) stabilization of p53 resulting in increased cell death, (2) downregulation of prosurvival genes Xiap and Birc3 and (3) suppression of double-strand and base-excision DNA repair pathways.

To examine the impact of NLRP3 on the DDR response following stimulation of DCs with MSU and H2O2, the authors first employed single-cell gel electrophoresis, also known as a comet assay, to separate fragmented DNA from whole DNA. The quantification of these data convincingly demonstrates an increase in DNA breaks in the presence of NLRP3. Next, immunoblots were performed to assay for H2AX histone phosphorylation on serine 139 (γH2AX), which is a hallmark of DNA damage and is required to provoke DDR. In line with the results of the comet assay, the authors found high levels of γH2AX in WT and Nlrp3−/− DCs early after stimulation, however these levels were sustained for at least 24 h in the WT samples, in contrast to the Nlrp3−/− samples in which the levels of γH2AX decreased over time. This effect could be reproduced using rotenone or γ-radiation in place of MSU, but not when DCs were stimulated with camptothecin, which causes DNA damage in the absence of ROS [16]. DCs lacking caspase-1 showed a similar trend to that seen in the Nlrp3−/− DCs, suggesting that NLRP3 alone is not responsible for this phenotype and a functional NLRP3 inflammasome is required. Despite the increase in DNA damage seen in WT DCs following stimulation, the authors found lower levels of 8-oxoG DNA glycosylase 1 (Ogg1) and decreased phosphorylation of NBS1, both components of the DNA repair pathway, in WT DCs compared with those in Nlrp3−/− DCs. These data indicate that although NLRP3 activators lead to DNA damage, the NLRP3 inflammasome is also involved in the negative regulation of the DDR pathway.

To elucidate the mechanism by which the NLRP3 inflammasome may be influencing the DDR response, Licandro et al. turned their attention to the cell cycle, due to the differential gene expression they had noted in their initial array as well as the convergence of the DDR and cell cycle at discrete checkpoints [14]. Specifically, the authors sought to determine whether the p53 pathway was differentially activated in WT versus Nlrp3−/− DCs following cellular stress. Indeed, early p53 phosphorylation at Ser15 and Ser20 was noted in WT, Nlrp3−/−, and caspase-1−/− DCs, however only the WT DCs demonstrated sustained activation of p53 over time. Considered together with the previous experiments looking at Ogg1 and NBS1, the prolonged activation of p53 in NLRP3-sufficient cells suggested that the NLRP3 inflammasome and p53 might be promoting cell death. In support of this hypothesis, they found that stimulation of DCs with MSU caused upregulation of p21, which is protective against p53-driven cell death in Nlrp3−/− cells, but not WT DCs. Furthermore, WT DCs exhibited a significant increase in MSU-induced cell death, as measured by propidium iodide staining and lactate dehydrogenase release, with decreased expression of the prosurvival genes Xiap and Birc3, when compared with those in Nlrp3−/− DCs. Although the authors assert that this form of programed cell death is pyroptosis, the data do not confirm caspase-1 dependence and the lack of proinflammatory cytokines in the model precludes that label as yet. Thus, these data represent a novel mechanism by which the NLRP3 inflammasome, together with the p53 pathway, restricts DNA repair and promotes cell death following oxidative and genotoxic stress.

That the novel NLRP3 inflammasome pathway described by Licandro et al. proceeds independently of IL-1β and IL-18 is intriguing considering the glut of literature on the topic asserting that proinflammatory cytokine production is the main means by which the inflammasome exerts its effector function. Although infrequent, other reports proposing noncanonical pathways for caspase-1 exist. For example, Shao et al. [17] identified glycolytic enzymes as additional substrates for caspase-1, demonstrating that caspase-1 causes a reduction in the cellular glycolytic rate during conditions of endotoxic shock or infection with Salmonella typhimurium, which contributes to pyroptosis.

Of particular interest for future studies is the connection between the NLRP3 inflammasome and the tumor suppressor p53, which is thought to be mutated in greater than 50% of human cancers [18]. The authors propose that the NLRP3 inflammasome and the p53 pathway might intersect at the inflammasome adaptor molecule ASC, as it has been shown to colocalize at the mitochondria with apoptosis-inducing molecule, Bax [19]. The data presented by Licandro et al., taken together with the widely accepted concept of inflammation as a hallmark of cancer [20], are certain to inspire exciting new lines of investigation. Indeed, a few studies have begun to look into the relationship between NLRP3 inflammasome-driven inflammation and cancer, however the results are conflicting at present [21-25]. Further exploration into the molecular interactions between these two networks will yield a better understanding of the maintenance of homeostasis following assaults on genomic integrity.

Acknowledgments

NIH grants R01 AI087630 (F.S.S.) and T32 AI007511 (S.H.) supported this work.

Conflict of interest

The authors declare no financial or commercial conflict of interest.

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