Amino-Functionalized Polystyrene Nanoparticles Activate the NLRP3 Inflammasome in Human Macrophages

Abstract

Specifically designed and functionalized nanoparticles hold great promise for biomedical applications. Yet, the applicability of nanoparticles is critically predetermined by their surface functionalization. Here we demonstrate that amino-functionalized polystyrene nanoparticles (PS-NH(2)) of ∼100 nm in diameter, but not carboxyl- or nonfunctionalized particles, trigger NLRP3 inflammasome activation and subsequent release of proinflammatory interleukin 1β (IL-1β) by human macrophages. PS-NH(2) induced time-dependent proton accumulation in lysosomes associated with lysosomal destabilization, release of cathepsin B, and damage of the mitochondrial membrane. Accumulation of mitochondrial reactive oxygen species was accompanied by oxidation of thioredoxin, a protein playing a central role in maintaining the cellular redox balance. Upon oxidation, thioredoxin dissociated from the thioredoxin-interacting protein (TXNIP). Liberated TXNIP, in turn, interacted with the NLRP3 protein, resulting in a conformational change of the pyrin domain of the NLRP3 protein, as was predicted by molecular modeling. Consequently, this prompted assembly of the NLRP3 inflammasome complex with recruitment and activation of caspase-1, inducing IL-1β release by cleavage of pro-IL-1β. The central role of the NLRP3 inflammasome for cytokine production was confirmed by in vitro knockdown of NLRP3 and of the adaptor protein ASC, confirming that other inflammasomes were not activated by PS-NH(2). The PS-NH(2)-mediated proinflammatory macrophage activation could be antagonized by the radical scavenger N-acetyl-L-cysteine, which prevented mitochondrial damage, caspase-1 activation, and the subsequent release of IL-1β. Our study reveals the molecular mechanism of NLRP3 inflammasome activation by amino-functionalized nanoparticles and suggests a strategy as to how such adverse effects could be antagonized.

Caspase-1 Activity in Microglia Stimulated by Pro-Inflammagen Nanocrystals

“…Inflammasome activators often cause formation of mitochondria-derived reactive oxygen species (ROS) 35 and lysosomal destabilization. 36,37 Several laboratories, including ours, have shown that QDs can induce excessive ROS formation 38À41 and activation of microglia, 42 and the release of cytokines including interleukin-1β (IL-1β). 43,44 However, these earlier studies did not provide experimental evidence for a mechanistic link between the activation of microglia, lysosomal status, and activation pattern of caspase-1 together with the subsequent release of IL-1β.…”

Applications of nanomaterials as vaccine adjuvants

“…12,13,21,22,[44][45][46]50,51 Their nanoscale size, with all three dimensions ranging from 0.1 to 100 nm, happens to be the size range of the fundamental building blocks of biology (including DNA, proteins, viruses, ultrastructures and organelles). Thus, the ways in which engineered nanomaterials may interfere with the function of the host immune system and how these immunomodulatory activities may affect vaccines are increasingly important questions.…”

“…In the following sections, the immunomodulatory activities of nanomaterials, including inflammasome activation, recruitment of immune cells and complement system activation, will be introduced. 12,13,21,22,33,34,[44][45][46][47][48][49][50][51][87][88][89][90] The potential mechanisms for these effects will also be discussed to facilitate an in-depth understanding of the inherent adjuvant activity of nanomaterials.…”

“…In addition to their delivery function, a great number of nanoparticles have shown immunomodulatory effects, particularly for innate immune signaling. For example, nanoparticles can induce inflammasome activation in macrophages and DCs, [44][45][46] enhance antigen presentation and APC maturation, 33,34 recruit immune cells, 21,22,47,48 direct T cell differentiation to a particular subtype, and activate the complement system. 12,13,[49][50][51] Therefore, nanomaterials constitute a multifunctional unit to integrate target delivery and immunomodulation effects for clinical use in vaccination.…”

The nanotoxicology revolution

“…It has been convincingly shown that some nanoparticles can accumulate in immune cells, such as macrophages (Buono et al 2009;Arnida et al 2011;Lunov et al 2011;Fedeli et al 2013;Gasparotto et al 2013). However, the degree of accumulation that occurs in humans after NP exposure is unknown.…”