Recent insights into the molecular mechanisms of the NLRP3 inflammasome activation

Abstract

Inflammasomes are high-molecular-weight protein complexes that are formed in the cytosolic compartment in response to danger- or pathogen-associated molecular patterns. These complexes enable activation of an inflammatory protease caspase-1, leading to a cell death process called pyroptosis and to proteolytic cleavage and release of pro-inflammatory cytokines interleukin (IL)-1β and IL-18. Along with caspase-1, inflammasome components include an adaptor protein, ASC, and a sensor protein, which triggers the inflammasome assembly in response to a danger signal. The inflammasome sensor proteins are pattern recognition receptors belonging either to the NOD-like receptor (NLR) or to the AIM2-like receptor family. While the molecular agonists that induce inflammasome formation by AIM2 and by several other NLRs have been identified, it is not well understood how the NLR family member NLRP3 is activated. Given that NLRP3 activation is relevant to a range of human pathological conditions, significant attempts are being made to elucidate the molecular mechanism of this process. In this review, we summarize the current knowledge on the molecular events that lead to activation of the NLRP3 inflammasome in response to a range of K (+) efflux-inducing danger signals. We also comment on the reported involvement of cytosolic Ca (2+) fluxes on NLRP3 activation. We outline the recent advances in research on the physiological and pharmacological mechanisms of regulation of NLRP3 responses, and we point to several open questions regarding the current model of NLRP3 activation.

Keywords: Inflammasome; NLRP3; autoinflammatory disease; pyroptosis.

Figure 1.. Canonical NLRP3 inflammasome activation by K ⁺ efflux

Under basal conditions, high intracellular K ⁺ concentration is maintained by the activity of Na ⁺/K ⁺-ATPase, which actively imports K ⁺ ions into the cell and generates an electrical gradient that favors movement of cations into the cytoplasm. Together with leak K ⁺ channels, Na ⁺/K ⁺-ATPase contributes to the transmembrane potential, characterized by a slight excess of negative charges inside the cell. Under conditions of NLRP3 stimulation, this equilibrium is disturbed. ATP increases the open probability of P2X7R, a cation channel that allows for net exchange of intracellular K ⁺ ions for extracellular Na ⁺ or Ca ²⁺ ions. This produces a net K ⁺ efflux that acts as an NLRP3 activator. Activation of P2X7R is also accompanied by opening of pannexin-1 channels. During hypotonic stimulation, the regulatory volume decrease (RVD) response causes opening of K ⁺ and Cl ^- channels, driving an efflux of K ⁺ and Cl ^- ions to balance the intracellular and extracellular osmolarity values. To induce NLRP3 activation, this mechanism of K ⁺ ions depletion additionally requires an influx of Ca ²⁺ through TRP channels and activation of the kinase TAK1. NLRP3-activating K ⁺ ionophores produce a net K ⁺ efflux through different mechanisms. The peptide gramicidin can insert itself into plasma membranes, forming pores that are permeable to monovalent cations. This enables an exchange of intracellular K ⁺ for extracellular Na ⁺. Valinomycin, a neutral ionophore, is a cell-permeant compound that can bind to K ⁺ ions, replacing the hydration shell of this cation. Consequently, K ⁺ ions shielded by valinomycin molecules can pass across the plasma membrane without a requirement for opening a K ⁺-permeable pore. Nigericin is a carboxylic ionophore that can bind to H ⁺ or to K ⁺. Both the H ⁺- and K ⁺-bound forms of nigericin are plasma membrane permeant. In this way, nigericin mediates K ⁺ transport from the compartment with higher K ⁺ concentration to the compartment with lower K ⁺ concentration, concomitantly leading to a transient acidification of cytosol. In further stages, the increased cytosolic [H ⁺] can stimulate Na ⁺/H ⁺ exchangers to extrude H ⁺ ions from the cytosol, which is accompanied by Na ⁺ influx . Lysosomal damage caused by particulate materials or by other factors requires K ⁺ efflux to induce NLRP3 activation, but it is unknown which factors are involved in this K ⁺ depletion pathway.

Figure 2.. Noncanonical NLRP3 activation by cytosolic LPS.

Upon recognition of LPS in the cytosol, caspase-11 cleaves pannexin-1 and gasdermin D. Cleavage of pannexin-1 leads to opening of the channel and leakage of K ⁺ and ATP from the cell into the extracellular space. This efflux of K ⁺ ions activates the NLRP3 inflammasome, causing proteolytic processing and secretion of IL-1β. Simultaneously, ATP acts as an agonist for the P2X7R, leading to NLRP3 inflammasome-independent pyroptotic cell death. Proteolytic cleavage of gasdermin D produces a highly toxic N-terminal fragment of this protein, which mediates both activation of the NLRP3 inflammasome (with subsequent IL-1β processing and secretion) and NLRP3-independent pyroptotic cell death. The relationship between two caspase-11 effectors, pannexin-1 and gasdermin D, is currently not understood.

Figure 3.. Modulation of the NLRP3 inflammasome by cAMP and autophagy

Several physiological mechanisms regulate NLRP3 responses on the cellular level. Agonists of G _S-coupled GPCRs stimulate the generation of cAMP by transmembrane adenylyl cyclases. cAMP is believed to bind to the nucleotide-binding domain of NLRP3. This formed NLRP3-cAMP complex recruits the ubiquitin ligase MARCH7 that polyubiquitinates NLRP3, targeting it for autophagosomal degradation. Autophagosomes are also the organelles responsible for degradation of pro-IL-1β (the inactive pro-form of the proinflammatory cytokine IL-1β), which is a more general mechanism controlling the inflammatory responses mediated by a range of inflammasomes. Finally, mitophagy is a way to dispose of damaged mitochondria that starts with sequestering them in autophagosomes. Autophagosomal degradation of dysfunctional mitochondria curbs the inflammasome responses, possibly by removing the source of direct NLRP3 activators.