NLRP3 inflammasome activation induced by engineered nanomaterials

Abstract

Engineered nanomaterials (ENMs) continue to attract significant attention because they have novel physicochemical properties that can improve the functions of products that will benefit human lives. However, the physicochemical properties that make ENMs attractive could interact with biological systems and induce cascades of events that cause toxicological effects. Recently, there have been more studies suggesting inflammasome activation may play an important role in ENM-induced biological responses. Inflammasomes are a family of multiprotein complexes that are increasingly recognized as major mediators of the host immune system. Among these, NLRP3 inflammasome is the most studied that could directly interact with ENMs to generate inflammatory responses. In this review, the ENM physicochemical properties are linked to NLRP3 inflammasome activation. An understanding of the mechanisms of ENM-NLRP3 inflammasome interactions will provide us with strategies for safer nanomaterial design and therapy.

Figure 1. Inflammasomes and inflammasome activation

Four inflammasomes including NLRP1, NLRP3, IPAF and AIM2 inflammasomes have been identified. Each type of inflammasome contains distinctive domains. NLRP1 contains a pyrin domain (PYD), a nucleotide-binding domain (NBD), a carboxy-terminal leucine-rich repeat (LRR) domain, a caspase activation and recruitment domain (CARD) and a domain with unknown function (FIIND). NLRP3 contains a PYD, a central NBD and a LRR domain. IPAF contains an N-terminal CARD, a central NBD, and a C-terminal LRR domain. AIM2 contains PYD and HIN domains. Different inflammasomes are responsive to specific stimuli. Upon activation, inflammasome complexes are assembled and active caspase-1 is produced, which processes pro-IL-1β and pro-IL-18 to mature IL-1β and IL-18.

Figure 2. Stimuli for NLRP3 inflammasome activation and NLRP3 inflammasome-associated diseases

NLRP3 inflammasome is activated through a wide range of stimuli including pathogen-associated molecular patterns (PAMPs), damage-associated molecular patterns (DAMPs), metabolic products, environmental hazards including engineered nanomaterials (ENMs). The activation of NLRP3 inflammasome and subsequent secretion of IL-1β have been associated with many diseases including asbestosis, silicosis, gout, Alzheimer's disease, inflammatory bowel disease, type II diabetes, and cryopyrin-associated periodic syndrome (CAPS).

Figure 3. Major mechanisms of NLRP3 inflammasome activation

NLRP3 inflammasome activation requires two signals in vitro. For signal 1, pathogen-associated molecular patterns (PAMPs) is recognized by Toll-like receptor 4 (TLR4) residing on cell membrane, which further leads to NF-κB activation through adaptor protein MyD88, and the production of pro-IL-1β. For signal 2, upon the activation of NLRP3 inflammasome complex by various stimuli including metabolic products, environmental hazards, vaccine adjuvants and ENMs, the pro-IL-1β is further processed to mature IL-1β. NLRP3 inflammasome activation mechanisms include ROS generation, potassium efflux, lysosomal damage and cathepsin B release. After phagocytosis of particles, fibers, and ENMs, NADPH oxidase is activated to generate ROS. The over-production of ROS may cause the destabilization and permeabilization of lysosomes and cathepsin B release, which will initiate the inflammasome activation cascade. ROS production could also lead to oxidation of the redox-active thioredoxin (TXN) and its subsequent dissociation from thioredoxin-interacting protein (TXNIP). The freed TXNIP could interact with NLRP3 and trigger a conformational change of NLRP3 and subsequent binding of adaptor protein ASC, which lead to the inflammasome activation and IL-1β release. Mitochondrion is another important source of ROS in cells. Over-production of mitochondrial ROS could cause the destabilization of lysosomes and release of cathepsin B, which then activates caspase-2 to cause mitochondrial permeabilization and cytochrome c release. Similarly, lysosomal enzymes such as cathepsin B can cleave Bid to its active form tBid; and another lysosomal protease, cathepsin D activates Bax to cause the mitochondrial rupture and cytochrome c release. Cytochrome c release will further induce mitochondrial ROS production and promote NLRP3 inflammasome activation. In addition, potassium efflux induced by particles and fibers could also induce cellular ROS production and NLRP3 inflammasome activation.

Figure 4. NLRP3 inflammasome activation induced by MWCNTs and CeO₂ nanowires

NLRP3 inflammasome activation studies induced by MWCNTs and CeO₂ nanorods have been performed in UC Center of Environmental Implications of Nanotechnology (UC CEIN) and the UCLA Center for Nanobiology and Predictive Toxicology (CNPT). Well-dispersed MWCNTs by BSA and DPPC could induce lysosomal damage, cathepsin B release and NLRP3 inflammasome activation without causing frustrated phagocytosis. However, MWCNTs coated by Pluronic F108 (PF108) decreases lysosomal damages, cathepsin B release and IL-1β production in THP-1 cells as compared to BSA-dispersed MWCNTs. The mechanism of the protective effects involves decrease in surface reactivity of CNTs by PF108 coating. For CeO₂ nanorods, when their length is ≥200 nm and aspect ratio is ≥ 22, the CeO₂ nanorods progressively induce more lysosomal damage, cathepsin B release and IL-1β production compared to spherical or short CeO₂ nanorods. TEM and SEM showed that CeO₂ nanorods formed stacking bundles, which could pierce through cell membrane, a feature that is known as “frustrated phagocytosis”. The CeO₂ stacking bundles could also pierce through lysosomes and cause cathepsin B release and induce NLRP3 inflammasome activation.

Figure 5. Intervention strategies for inflammasome activation associated biological effects

Understanding of inflammasome activation mechanisms has been utilized to develop intervention strategies for inflammasome activation associated diseases. Canakinumab is a human monoclonal antibody that binds to IL-1β. Rilonacept is a fusion protein consisting of human IL-1 receptor extracellular domain and human IgG1 FC domain that binds and neutralizes IL-1. Glibenclamide is a potassium efflux inhibitor. Anakinra is an IL-1 receptor antagonist. VX-765 is caspase-1 inhibitor. N-acetyl-cysteine (NAC) is an antioxidant that can neutralize ROS, and is a potential candidate for the treatment of inflammasome-related diseases. In addition, modification of ENM properties including aspect ratio, dispersion state, size and surface functionalization (R= COOH, NH₂, PEG, SO₃H) could lead to safer ENMs with lower NLRP3 inflammasome activation potentials.