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RESEARCH ARTICLE ISSN: 2213-5294 eISSN: 2213-5308
An Update on NLRP3 Inflammasome Activation by Engineered Nanomaterials1 Rob J Vandebriela,*, Susan Dekkersa, Wim H de Jonga and Flemming R Casseea,b a
National Institute for Public Health and the Environment, Bilthoven, The Netherlands; bInstitute for Risk Assessment Sciences, Utrecht University, Utrecht, The Netherlands
A R T I C L E H I S T O R Y Received: February 25, 2016 Revised: April 6, 2016 Accepted: April 6, 2016 DOI: 10.2174/22135294026661606011221 27
Abstract: The widespread and increasing use of engineered nanomaterials (ENMs) increases the risk of exposure by human beings. This notion has generated concern that ENMs may give rise to adverse health effects. Toxicity of ENMs is determined by both physical aspects and chemical composition. The immune system may respond to ENMs, amongst others by inflammatory reactions. Among the mechanisms underlying inflammation, inflammasome activation (especially the NLRP3 inflammasome) has drawn significant attention because it can be induced by a wide range of stimuli including ENMs and it is associated with various inflammatory diseases, including lung fibrosis, obesity and type 2 diabetes. Inflammasomes are intracellular multiprotein complexes that assemble upon stimulation, resulting in the activation of caspase-1 that in turn induces the production of interleukin (IL)-1 and IL-18. These cytokines are potent mediators of inflammation. This review summarizes the literature on NLRP3 inflammasome activation by ENMs published between 2013 and 2015. Newly identified mechanisms include a role for Endoplasmic Reticulum stress in NLRP3 inflammasome activation by Ag and ZnO ENMs and an initiating role for IL-1. Novel insights in surface functionalization with the aim of reducing NLRP3 inflammasome activation were obtained. In vitro assays for NLRP3 inflammasome activation may predict in vivo fibrogenic potency of ENMs; including such an assay in a test battery for hazard identification is advised.
Keywords: Engineered nanomaterial, NLRP3, inflammasome, inflammation, fibrosis, ER stress, surface functionalization, IL1alpha, IL-1beta, IL-18, caspase-1. MECHANISMS ACTIVATION
OF
NLRP3
INFLAMMASOME
One of the first forms of defense employed by the innate immune response is accomplished by pattern recognition receptors (PRRs) encoded in the germline to recognize pathogen-associated molecular patterns (PAMPs). These receptors may be either on the membrane surface e.g. Tolllike receptors (TLRs) or inside the cytoplasm e.g. Nod-like receptors (NLRs). Most studies on inflammasomes have focused on myeloid cells, such as macrophages and dendritic cells. However, also cells outside the myeloid compartment, such as keratinocytes, can activate inflammasomes. Basically there exist four types of inflammasomes: (a) NLR family pyrin domain-containing 1 (NLRP1), (b) NLRP3, (c) interleukin-1-converting enzyme (ICE)-protease activating factor (IPAF, also named NLRC4) and (d) absent in melanoma 2 (AIM2; the only type of inflammasome that is not a NLR). The NLRP3 inflammasome is the most fully characterized inflammasome (Fig. 1). It consists of a NLRP3 scaffold, an ASC adaptor, and pro-caspase-1. The NLRP3 *Address correspondence to this author at the Centre for Health Protection, National Institute for Public Health and the Environment (RIVM), PO Box 1, Bilthoven, The Netherlands; Tel: +31 30 274 2610; E-mail:
[email protected] 1 Supported by EU-funded projects nanoMILE (NMP4-LA-2013-310451) and FutureNanoNeeds (604602) 2213-5308/16 $58.00+.00
NLRP3
PYD
NACHT
LRR
P20
P10
PYD ASC CARD CASP1
CARD
Fig. (1). The NLRP3 inflammasome. Adapted from [1]. NLRP3, nucleotide-binding oligomerization domain (NOD)-like receptor family, pyrin domain-containing 3; ASC, apoptosis-associated speck-like protein containing a CARD; CASP1, caspase-1; PYD, pyrin domain; NACHT, central nucleotide binding and oligomerization domain; LRR, leucine-rich repeat; CARD, caspase recruitment domain.
scaffold consists of a PYD, a NACHT, and a LRR; the ASC adaptor consists of a PYD and a CARD; pro-caspase 1 consists of a CARD, p20, and p10. Upon activation, NLRP3 recruits ASC via PYD-PYD interaction. ASC then binds to pro-caspase-1, resulting in auto-cleavage of this pro-enzyme © 2016 Bentham Science Publishers
Volume 2, Issue 1
BENTHAM SCIENCE
An Update on NLRP3 Inflammasome Activation
Current Bionanotechnology, 2016, Vol. 2, No. 1
to become the active enzyme, caspase-1 (p10/p20 tetramer). Caspase-1 processes pro-IL-1 and pro-IL-18 to bioactive IL-1 and IL-18, respectively.
Table 1.
Summary of diseases in which inflammasome activation is implicated.
The NLRP3 inflammasome can be activated by a wide range of infectious agents, such as fungi (Candida albicans), yeasts (Saccharomyces cerevisiae), gram-positive (Staphylococcus aureus) and gram-negative (Listeria monocytogenes) bacteria, and various viruses (Sendai virus, adenovirus, influenza virus). Importantly, they also respond to host-derived molecules, such as extracellular ATP and hyaluronan that are released by injured cells, and xenogeneic agents such as silica, asbestos, UV-B irradiation, chemical sensitizers, and engineered nanomaterials (ENMs) [1]. Two signals are generally required to activate the NLRP3 inflammasome (Fig. 2). Signal 1 constitutes the recognition of pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide (LPS), by Toll-like receptor 4 (TLR4); this increases the production of pro-IL-1 and NLRP3. Signal 2 comprises the activation of the NLRP3 inflammasome such as by ENMs; as a result pro-caspase-1 is auto-cleaved to caspase-1 and this in turn leads to the processing of pro-IL-1 to IL-1. A similar scheme holds for IL-18. INVOLVEMENT INFLAMMASOME DISEASES
OF SUSTAINED NLRP3 ACTIVATION IN CHRONIC
Sustained NLRP3 inflammasome activation may result in chronic inflammation and subsequent tissue damage. This sustained activation is implicated in a range of chronic diseases (reviewed in [2,3]). For some of these diseases causality has been shown in mouse models, whereas for others proof of causality is currently lacking. Nonetheless, for all chronic diseases the contribution of sustained inflammasome activation in man remains to be established. In order to get a feel for the current evidence parts of the above-mentioned reviews are reiterated here (Table 1). Obesity The NLRP3 inflammasome senses obesity-associated “damage-associated molecular patterns” (DAMPs) and its activation is an important mechanism in the development of insulin resistance [4-7]. Caspase-1 activation in adipose tissue and liver impairs insulin signaling and glucose homeostasis [4]. Obesity is associated with increases in ceramides, saturated fatty acids, ROS, mitochondrial dysfunction and ATP; these factors activate the NLRP3 inflammasome in macrophages. Caspase-1 and IL-1 production are enhanced in obese mouse models. Next, IL1 directs adipocytes to a more insulin-resistant phenotype during differentiation, and caspase-1 inhibitors improve insulin sensitivity of adipocytes in diseased mice. Mice
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NLRP3
Obesity Type-2 Diabetes Atherosclerosis Multiple Sclerosis Type-1 Diabetes Occupational pulmonary fibrosis Allergic asthma
deficient in Nlrp3, ASC, and caspase-1 are resistant to the development of high-fat diet-induced obesity and are protected from obesity-induced insulin resistance [8]. Type-2 Diabetes Palmitate, one of the most abundant saturated fatty acids in the plasma of type-2 diabetes (T2D) patients, can induce activation of the NLRP3 inflammasome [7]. This in turn promotes hyperglycemia, insulin insensitivity and liver steatohepatitis. Palmitate may be converted to ceramide, which is known to impair pancreatic function and activate the NLRP3 inflammasome [6]. Islet amyloid polypeptide (IAPP) induces NLRP3 activation [9]. During T2D disease progression, IAPP forms amyloid deposits in the pancreas, which is a hallmark of disease. Caloric restriction and exercise–mediated weight loss in obese individuals reduces NLRP3 expression [6]. In cells, thioredoxin-interacting protein (TXNIP) is downregulated by insulin, and TXNIP expression is consistently higher in humans with T2D. NLRP3 inflammasome activation induces the binding of TXNIP to NLRP3 [5]. TXNIP deficiency impaired NLRP3 inflammasome activation; Txnip -/- mice and Nlrp3 -/- mice showed improved glucose tolerance and insulin sensitivity. Atherosclerosis Cholesterol crystals are an important factor in inflammation and tissue damage during atherosclerosis pathogenesis; they induce disease by NLRP3 inflammasome activation [10]. Multiple Sclerosis IFN- attenuates the course and severity of Multiple Sclerosis (MS); it markedly diminishes NLRP1 and NLRP3 inflammasome activation and IL-1 production in MS patients [11].
pro-IL-1 TLR (signal 1) NLRP3 inflammasome (signal 2) caspase-1 IL-1 Fig. (2). NLRP3 inflammasome-dependent IL-1 production requires two signals: TLR engagement and inflammasome activation. A similar scheme holds for IL-18.
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Vandebriel et al.
Type-1 Diabetes Two Single Nucleotide Polymorphisms (SNPs) in NLRP3 are predisposing factor for type-1 diabetes [12]. Occupational Pulmonary Fibrosis Lung exposure to silica and asbestos results in lysosomal disruption and intracellular ROS production by macrophages, which in turn leads to NLRP3 activationdependent IL-1 secretion. In a chronic situation, this results in lung fibrosis [13,14]. Allergic Asthma Serum amyloid A is found in asthma patients. In mice it can activate NLRP3 inflammasome and by that Th17 allergic inflammation [15]. NLRP3 INFLAMMASOME ACTIVATION ENGINEERED NANOMATERIALS
BY
In 2013, Sun et al. [16] published a comprehensive review on inflammasome activation by ENMs. The authors identified five physicochemical properties of ENMs that affect NLRP3 inflammasome activation: (1) the ability to induce reactive oxygen species (ROS), (2) the ratio between length and width or height (the aspect ratio); ENMs with a high aspect ratio may cause lysosomal damage and cathepsin B release; cathepsin B provides a signal for inflammasome activation, (3) the dispersion state, with dispersed ENMs inducing stronger inflammasome activation than nondispersed ones, (4) the size, with nanosized ENMs inducing stronger inflammasome activation than microsized ones, and (5) surface functionalization (adding e.g. amino or carboxy groups to the ENM surface), with the effect on inflammasome activation depending on the type of functionalization. In addition to ROS generation and lysosomal damage/cathepsin B release, other mechanisms have been found to induce inflammasome activation. K+-efflux may represent a common signal for NLRP3 inflammasome activation [17]. Extracellular ATP released from damaged cells may lower cytosolic K+. Autophagosomes deliver cytoplasmic constituents to lysosomes for degradation. Activated NLRP3 inflammasomes trigger autophagosome formation; blocking autophagy increases inflammasome activity whereas inducing autophagy limits this activity. Thus, under normal conditions, autophagy accompanies inflammasome activation to temper inflammation.
purinergic receptor for IL-1 production induced by silica ENMs was confirmed by others [19]. In that study, 30 nm particles induced higher IL-1 production and ATP release than 70 nm and 300 nm particles; ATP induced similar IL-1 production, as did 30 nm particles. ROS production was induced only by the 30 nm particles. ATP release, and ATP, ADP, and adenosine receptor signaling were required for inflammasome activation after exposure of THP-1 cells to SiO2 ENMs [20]. Crystalline silica activated the inflammasome in bronchial human epithelial cells (BEAS-2B) and normal human bronchial epithelial cells [21]. Surface modification of Dörentruper quartz (DQ12) by polyvinyl pyridine N-oxide markedly attenuated inflammasome activation in BEAS-2B cells and PMA-activated THP-1 monocytes, suggesting a role for surface reactivity in this response [22]. For fumed silica ENMs a positive correlation of toxicity with hydroxyl radical concentrations and its potential to generate ROS was found [23]. Interactions of the silanol surfaces of fumed silica aggregates with the extracellular plasma membrane were suggested to cause membrane perturbations that are sensed by the NLRP3 inflammasome; hydroxyl radicals may via this route contribute to inflammasome activation. In a follow-up paper it was shown that Ti or Al doping of fumed silica reduced hydroxyl radical generation, membrane perturbation, K+ efflux, and inflammasome activation [24]. Ti or Al doping was achieved by adding Ti isopropoxide or Al secondary butotoxide, respectively, to tetraethylorthosilicate, prior to synthesis. IL-1 was found to induce pro-IL-1 in alveolar macrophages in vitro [25]. Neutralization of IL-1 reduced IL-1 production and neutrophil accumulation after DQ12 pharyngeal aspiration in mice. This may suggest that rapid release of (pre-existing) IL-1 promotes subsequent lung inflammation through stimulating IL-1 production. Next, for a series of four silica ENMs, IL-1 released by J774 macrophages correlated with the number of alveolar neutrophils in vivo. From this correlation, the authors conclude that IL-1 release from macrophages in vitro may predict the acute inflammatory activity of silica ENMs. Carbonaceous Materials In LPS-stimulated human primary monocytes inflammasome activation by double-walled (DW) CNT was shown to require K+ efflux but not ROS generation [26]. Inhibition of lysosomal acidification or cathepsin B reduced IL-1 production, suggesting that activation by DWCNT occurs via lysosomal destabilization.
Silica
Purification and oxidation of CNT increased inflammasome activation [27]. Next to CNT, also 6 nm spherical carbon nano onions (CNO) induced inflammasome activation. Functionalization using benzoic acid of both CNT and CNO reduced inflammasome activation.
As mentioned in the section above “Mechanisms of NLRP3 inflammasome activation”, extracellular ATP may stimulate the P2X7 purinergic receptor, resulting in NLRP3 activation. This receptor was shown to be involved in IL-1 production by A549 - THP-1 co-cultures exposed to 5-15 nm SiO2 ENMs [18]. Requirement of signaling via the P2X7
Functionalization of MWCNT with COOH reduced the inflammatory response in mice [28]. Lung lavage fluid of mice instilled with COOH-functionalized MWCNT showed reduced neutrophil numbers and levels of cathepsin B activity, IL-1, IL-18, and IL-33, compared to mice instilled with unmodified MWCNT.
This review summarizes the literature on NLRP3 inflammasome activation by ENMs published between 2013 and 2015.
An Update on NLRP3 Inflammasome Activation
A library of functionalized MWCNT showed in both BEAS-2B bronchial epithelial cells and PMA-activated THP-1 cells that compared to unmodified MWCNT, anionic (COOH and PEG) surface functionalization decreased inflammasome activation, while neutral (NH2) surface functionalization showed little effect on inflammasome activation [29]. Strongly cationic (polyetherimide) surface functionalization, however, increased inflammasome activation. The observations in vitro could be replicated in vivo with COOH- and polyetherimide-functionalized MWCNT showing weaker and stronger lung fibrosis, respectively, compared to the parent compound. Thus, surface charge of MWCNT plays an important role in inflammasome activation and fibrogenic potency. In a comparison of three forms of MWCNTs, original (O), purified (P), and carboxylic acid functionalized (F), inflammasome activation in vitro using PMA-activated THP1 cells [30] showed the same ranking as neutrophilia and severity of histopathological alterations after oropharyngeal aspiration of mice [31], i.e. O-MWCNT > P-MWCNT > FMWCNT. A comparison of nine MWCNT (short/long, wide/narrow, original/purified/COOH-functionalized) in primary alveolar macrophages or THP-1 cells showed that increased length and width corresponded to increased inflammasome activation [32]. Lung pathology of mice correlated with IL-1 production. Next, using primary alveolar macrophages or THP-1 cells, COOH– functionalization was found to abrogate inflammasome activation [33]. A library of carbonaceous nanomaterials, comprising three MWCNT, graphene and graphene oxide (the latter in two sizes), was prepared and investigated for inflammasome activation [34]. Graphene was dispersed in BSA or PF108. The parent SWCNT, purified SWCNT, graphene dispersed in BSA, and both types of graphene oxide induced inflammasome activation. Oropharyngeal aspiration in mice confirmed for four of these materials the induction of lung fibrosis. Large graphene oxide was most fibrogenic. SWCNT and graphene, dispersed in PF108, did not show fibrogenic effects. Three high aspect ratio (HAR) materials (MWCNT, SWCNT and Ag nanowires) were compared with spherical ENMs (carbon black and Ag) for their ability to trigger oxygen burst activity [35]. The HAR materials but not the spherical ENMs induced NADPH oxidase activation and respiratory burst activity in THP-1 cells. Using cells deficient in p22phox, which is an essential part of NADPH oxidase, it was shown that this enzyme is involved in lysosomal damage by HAR materials, i.e. decreased cathepsin B activation and IL-1 production. Using macrophages from mice deficient in the NADPH oxidase subunit p47phox, reduced respiratory burst and reduced NLRP3 inflammasome activation was observed. Moreover, lung collagen deposition was decreased in this strain of animals. MWCNT induced NLRP3 inflammasome activation in primary human bronchial epithelial (HBE) cells, resulting in mediator release; the supernatant induced an increase in pro-
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fibrotic markers when added to MRC-5 human lung fibroblasts [36]. Gold The influence of shape of Au ENMs on inflammasome activation was investigated by comparing spherical (20 and 40 nm diameter), rod (40 x 10 nm) and cubic (40 x 40 x 40 nm) Au ENMs [37]. Importantly, only rod ENMs activated the inflammasome, measured as IL-1 and IL-18 production by bone marrow-derived dendritic cells, implicating a clear shape-dependency of inflammasome activation. Silver The influence of shape of Ag ENMs on inflammasome activation was investigated by comparing Ag wires (274 nm x 5.3 m) to 150 nm spherical Ag ENMs [38]. Ag wires induced higher IL-1 production in PMA-primed THP-1 cells than the spherical ENMs. Endoplasmic reticulum (ER) stress plays an important role in apoptosis after exposure to Ag ENMs [39], and this was found to be a highly sensitive parameter for cytotoxicity [40]. The ER stress sensor activating transcription factor-6 (ATF-6) was found to be degraded after exposure of THP-1 cells to 15 nm Ag ENMs [41]. Inhibition of ATF-6 degradation blocked the effect of these Ag ENMs on pyroptosis and IL-1 production. Next, caspase-4 was shown to regulate the production of pro-IL-1. At sub-cytotoxic concentrations, Ag ENMs induced autophagy and NLRP3 inflammasome activation, and this was size-dependent (10 nm > 50 nm > 100 nm) [42]. Also for ZnO ENMs, ER stress was found to be a highly sensitive parameter for cytotoxicity [43]. Titanium Dioxide The size, crystal structure, and shape of TiO2 particles influenced inflammasome activation [44]. In PMAstimulated THP-1 cells,
