Structures and functions of the inflammasome engine.

Inflammasomes are molecular machines that carry out inflammatory responses on challenges by pathogens and endogenous dangers. Dysregulation of inflammasome assembly and regulation is associated with numerous human diseases from autoimmunity to cancer. In recent years, significant advances have been made in understanding the mechanism of inflammasome signaling using structural approaches. Here, we review inflammasomes formed by the NLRP1, NLRP3, and NLRC4 sensors, which are well characterized structurally, and discuss the structural and functional diversity among them.

Higher-order assemblies in immune signaling: supramolecular complexes and phase separation.

Signaling pathways in innate and adaptive immunity play vital roles in pathogen recognition and the functions of immune cells. Higher-order assemblies have recently emerged as a central principle that governs immune signaling and, by extension, cellular communication in general. There are mainly two types of higher-order assemblies: 1) ordered, solid-like large supramolecular complexes formed by stable and rigid protein-protein interactions, and 2) liquid-like phase-separated condensates formed by weaker and more dynamic intermolecular interactions. This review covers key examples of both types of higher-order assemblies in major immune pathways. By placing emphasis on the molecular structures of the examples provided, we discuss how their structural organization enables elegant mechanisms of signaling regulation.

Mechanism of filament formation in UPA-promoted CARD8 and NLRP1 inflammasomes.

NLRP1 and CARD8 are related cytosolic sensors that upon activation form supramolecular signalling complexes known as canonical inflammasomes, resulting in caspase-1 activation, cytokine maturation and/or pyroptotic cell death. NLRP1 and CARD8 use their C-terminal (CT) fragments containing a caspase recruitment domain (CARD) and the UPA (conserved in UNC5, PIDD, and ankyrins) subdomain for self-oligomerization, which in turn form the platform to recruit the inflammasome adaptor ASC (apoptosis-associated speck-like protein containing a CARD) or caspase-1, respectively. Here, we report cryo-EM structures of NLRP1-CT and CARD8-CT assemblies, in which the respective CARDs form central helical filaments that are promoted by oligomerized, but flexibly linked, UPAs surrounding the filaments. Through biochemical and cellular approaches, we demonstrate that the UPA itself reduces the threshold needed for NLRP1-CT and CARD8-CT filament formation and signalling. Structural analyses provide insights on the mode of ASC recruitment by NLRP1-CT and the contrasting direct recruitment of caspase-1 by CARD8-CT. We also discover that subunits in the central NLRP1CARD filament dimerize with additional exterior CARDs, which roughly doubles its thickness and is unique among all known CARD filaments. Finally, we engineer and determine the structure of an ASCCARD-caspase-1CARD octamer, which suggests that ASC uses opposing surfaces for NLRP1, versus caspase-1, recruitment. Together these structures capture the architecture and specificity of the active NLRP1 and CARD8 inflammasomes in addition to key heteromeric CARD-CARD interactions governing inflammasome signalling.

Role of Annexin A1 in NLRP3 Inflammasome Activation in Murine Neutrophils.

This study evaluated the role of endogenous and exogenous annexin A1 (AnxA1) in the activation of the NLRP3 inflammasome in isolated peritoneal neutrophils. C57BL/6 wild-type (WT) and AnxA1 knockout mice (AnxA1-/-) received 0.3% carrageenan intraperitoneally and, after 3 h, the peritoneal exudate was collected. WT and AnxA1-/- neutrophils were then stimulated with lipopolysaccharide, followed by the NLRP3 agonists nigericin or ATP. To determine the exogenous effect of AnxA1, the neutrophils were pretreated with the AnxA1-derived peptide Ac2-26 followed by the NLRP3 agonists. Ac2-26 administration reduced NLRP3-derived IL-1ß production by WT neutrophils after nigericin and ATP stimulation. However, IL-1ß release was impaired in AnxA1-/- neutrophils stimulated by both agonists, and there was no further impairment in IL-1ß release with Ac2-26 treatment before stimulation. Despite this, ATP- and nigericin-stimulated AnxA1-/- neutrophils had increased levels of cleaved caspase-1. The lipidomics of supernatants from nigericin-stimulated WT and AnxA1-/- neutrophils showed potential lipid biomarkers of cell stress and activation, including specific sphingolipids and glycerophospholipids. AnxA1 peptidomimetic treatment also increased the concentration of phosphatidylserines and oxidized phosphocholines, which are lipid biomarkers related to the inflammatory resolution pathway. Together, our results indicate that exogenous AnxA1 negatively regulates NLRP3-derived IL-1ß production by neutrophils, while endogenous AnxA1 is required for the activation of the NLRP3 machinery.

Heterotypic Supramolecular Hydrogels Formed by Noncovalent Interactions in Inflammasomes.

The advance of structural biology has revealed numerous noncovalent interactions between peptide sequences in protein structures, but such information is less explored for developing peptide materials. Here we report the formation of heterotypic peptide hydrogels by the two binding motifs revealed by the structures of an inflammasome. Specifically, conjugating a self-assembling motif to the positively or negatively charged peptide sequence from the ASCPYD filaments of inflammasome produces the solutions of the peptides. The addition of the peptides of the oppositely charged and complementary peptides to the corresponding peptide solution produces the heterotypic hydrogels. Rheology measurement shows that ratios of the complementary peptides affect the viscoelasticity of the resulted hydrogel. Circular dichroism indicates that the addition of the complementary peptides results in electrostatic interactions that modulate self-assembly. Transmission electron microscopy reveals that the ratio of the complementary peptides controls the morphology of the heterotypic peptide assemblies. This work illustrates a rational, biomimetic approach that uses the structural information from the protein data base (PDB) for developing heterotypic peptide materials via self-assembly.

Human Autoinflammatory Diseases Mediated by NLRP3-, Pyrin-, NLRP1-, and NLRC4-Inflammasome Dysregulation Updates on Diagnosis, Treatment, and the Respective Roles of IL-1 and IL-18.

Recent research has led to novel findings in inflammasome biology and genetics that altered the diagnosis and management of patients with autoinflammatory syndromes caused by NLRP3-, Pyrin-, NLRP1-, and NLRC4-inflammasomes and spurred the development of novel treatments. The use of next-generation sequencing in clinical practice allows for rapid diagnosis and the detection of somatic mutations that cause autoinflammatory diseases. Clinical differences in patients with NLRP3, pyrin, and NLRP1 inflammasomopathies, and the constitutive elevation of unbound free serum IL-18 that predisposes to the development of macrophage activation syndrome (MAS) in patients with gain-of function mutations in NLRC4 led to the screening and the characterization of novel diseases presenting with constitutively elevated serum IL-18 levels, and start to unravel the biology of "high IL-18 states" that translate into the use of biomarkers that improve diagnosis and monitoring of disease activity and investigations of treatments that target IL-18 and IFN-gamma which promise to improve the management and outcome of these conditions. Lastly, advances in structural modeling by cryo-electron microscopy (cryo-EM) of gasdermin, and of NLRP3- and NLRC4-inflammasome assembly, and the characterization of post-translational modifications (PTM) that regulate inflammasome activation, coupled with high-throughput screening (HTS) of libraries of inflammasome-inhibiting compounds, promise a new generation of treatments for patients with inflammasome-mediated diseases.

Plasma membrane damage causes NLRP3 activation and pyroptosis during Mycobacterium tuberculosis infection.

Mycobacterium tuberculosis is a global health problem in part as a result of extensive cytotoxicity caused by the infection. Here, we show how M. tuberculosis causes caspase-1/NLRP3/gasdermin D-mediated pyroptosis of human monocytes and macrophages. A type VII secretion system (ESX-1) mediated, contact-induced plasma membrane damage response occurs during phagocytosis of bacteria. Alternatively, this can occur from the cytosolic side of the plasma membrane after phagosomal rupture in infected macrophages. This damage causes K+ efflux and activation of NLRP3-dependent IL-1ß release and pyroptosis, facilitating the spread of bacteria to neighbouring cells. A dynamic interplay of pyroptosis with ESCRT-mediated plasma membrane repair also occurs. This dual plasma membrane damage seems to be a common mechanism for NLRP3 activators that function through lysosomal damage.

Structural Mechanism for GSDMD Targeting by Autoprocessed Caspases in Pyroptosis.

The pyroptosis execution protein GSDMD is cleaved by inflammasome-activated caspase-1 and LPS-activated caspase-11/4/5. The cleavage unmasks the pore-forming domain from GSDMD-C-terminal domain. How the caspases recognize GSDMD and its connection with caspase activation are unknown. Here, we show site-specific caspase-4/11 autoprocessing, generating a p10 product, is required and sufficient for cleaving GSDMD and inducing pyroptosis. The p10-form autoprocessed caspase-4/11 binds the GSDMD-C domain with a high affinity. Structural comparison of autoprocessed and unprocessed capase-11 identifies a ß sheet induced by the autoprocessing. In caspase-4/11-GSDMD-C complex crystal structures, the ß sheet organizes a hydrophobic GSDMD-binding interface that is only possible for p10-form caspase-4/11. The binding promotes dimerization-mediated caspase activation, rendering a cleavage independently of the cleavage-site tetrapeptide sequence. Crystal structure of caspase-1-GSDMD-C complex shows a similar GSDMD-recognition mode. Our study reveals an unprecedented substrate-targeting mechanism for caspases. The hydrophobic interface suggests an additional space for developing inhibitors specific for pyroptotic caspases.

Alterations in plasma membrane ion channel structures stimulate NLRP3 inflammasome activation in APOL1 risk milieu.

We evaluated alterations in the structural configurations of channels and activation of nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) inflammasome formation in apolipoprotein L1 (APOL1) risk and nonrisk milieus. APOL1G1- and APOL1G2-expressing podocytes (PD) displayed enhanced K+ efflux, induction of pyroptosis, and escalated transcription of interleukin (IL)-1ß and IL-18. APOL1G1- and APOL1G2-expressing PD promoted the transcription as well as translation of proteins involved in the formation of inflammasomes. Since glyburide (a specific inhibitor of K+ efflux channels) inhibited the transcription of NLRP3, IL-1ß, and IL-18, the role of K+ efflux in the activation of inflammasomes in APOL1 risk milieu was implicated. To evaluate the role of structural alterations in K+ channels in plasma membranes, bioinformatics studies, including molecular dynamic simulation, were carried out. Superimposition of bioinformatics reconstructions of APOL1G0, G1, and G2 showed several aligned regions. The analysis of pore-lining residues revealed that Ser342 and Tyr389 are involved in APOL1G0 pore formation and the altered conformations resulting from the Ser342Gly and Ile384Met mutation in the case of APOLG1 and deletion of the Tyr389 residue in the case of APOL1G2 are expected to alter pore characteristics, including K+ ion selectivity. Analysis of multiple membrane (lipid bilayer) models of interaction with the peripheral protein, integral membrane protein, and multimer protein revealed that for an APOL1 multimer model, APOL1G0 is not energetically favorable while the APOL1G1 and APOL1G2 moieties favor the insertion of multiple ion channels into the lipid bilayer. We conclude that altered pore configurations carry the potential to facilitate K+ ion transport in APOL1 risk milieu.

An integrative protocol for the structure determination of the mouse ASC-PYD filament.

Several inflammasomes that activate as part of the eukaryotic innate immune response contain long helical filaments of the adaptor protein ASC as a central structural element. Here, we describe a technical protocol that has enabled the structure determination of the filament of the ASC pyrin domain. The protocol integrates data from cryo-electron microscopy and solid-state NMR spectroscopy into a single simulated annealing protocol to determine structural coordinates that fit all input data optimally. The structure shows that the ASC pyrin domain filament is formed by helical stacking of individual pyrin domains forms and that the CARD domains are flexibly attached to the filament outside. An artificial perturbation of the input data shows that the integrated structure determination protocol can allow high quality structures even at resolutions of the electron density map as low 8Å. The protocol is extendable to other structural input data from biochemical or biophysical experiments.

Cryo-EM studies of NAIP-NLRC4 inflammasomes.

The NAIP-NLRC4 family of inflammasomes are components of the innate immune system that sound a molecular alarm in the presence of intracellular pathogens. In this chapter, we provide an in-depth guide to using cryo-electron microscopy (cryo-EM) to investigate these inflammasomes, focusing especially on the techniques we used in our recent structural analysis of the NAIP5-NLRC4 inflammasome. We explain how to circumvent specific obstacles we encountered at each step, from sample preparation through data processing. The methods described here will be useful for further studies of the NAIP5-NLRC4 inflammasome and related supracomplexes involved in innate immune surveillance; they may also be useful for unrelated complexes that present similar issues, such as preferential orientations and compositional heterogeneity.

Structural mechanism for NEK7-licensed activation of NLRP3 inflammasome.

The NLRP3 inflammasome can be activated by stimuli that include nigericin, uric acid crystals, amyloid-ß fibrils and extracellular ATP. The mitotic kinase NEK7 licenses the assembly and activation of the NLRP3 inflammasome in interphase. Here we report a cryo-electron microscopy structure of inactive human NLRP3 in complex with NEK7, at a resolution of 3.8 Å. The earring-shaped NLRP3 consists of curved leucine-rich-repeat and globular NACHT domains, and the C-terminal lobe of NEK7 nestles against both NLRP3 domains. Structural recognition between NLRP3 and NEK7 is confirmed by mutagenesis both in vitro and in cells. Modelling of an active NLRP3-NEK7 conformation based on the NLRC4 inflammasome predicts an additional contact between an NLRP3-bound NEK7 and a neighbouring NLRP3. Mutations to this interface abolish the ability of NEK7 or NLRP3 to rescue NLRP3 activation in NEK7-knockout or NLRP3-knockout cells. These data suggest that NEK7 bridges adjacent NLRP3 subunits with bipartite interactions to mediate the activation of the NLRP3 inflammasome.

Targeting NLRP3 inflammasome via acetylsalicylic acid: Role in suppressing hepatic dysfunction and insulin resistance induced by atorvastatin in naïve versus alcoholic liver in rats.

BACKGROUND: NLRP3 inflammasome is described in many pathological conditions and is also involved in drug induced liver injury. AIM OF THE WORK: To investigate the role of NLRP3 inflammasome in liver injury induced by chronic alcohol and/or atorvastatin ingestion. MATERIALS AND METHODS: Sixty male Wistar rats were used. They were divided into 5 groups: (I) control naïve (II) Alcoholic: given ethanol 8 g/kg/day, p.o (III) Atorvastatin: given atorvastatin 10 mg/kg/day, p.o. (IV) Alcoholic + atorvastatin (V) Acetylsalicylic acid (ASA): given ASA 10 mg/kg/day, p.o together with alcohol and atorvastatin. Isolated perfused liver, biochemical and histological studies were done. RESULTS: Atorvastatin and alcohol induced liver inflammation with increasing the expression of NLRP3, IL-1ß and caspase-8 immune-reaction. Atorvastatin and alcohol decreased the reduced form of glutathione in hepatic tissues and induced insulin resistance. ASA administration alleviated the hepatotoxic effects of alcohol and atorvastatin to a significant extent. CONCLUSIONS: Acetylsalicylic acid alleviated the hepatotoxic effects of alcohol and atorvastatin through decreasing the production of NLRP3 inflammasome in rats' liver.

TNFAIP3-DEPTOR complex regulates inflammasome secretion through autophagy in ankylosing spondylitis monocytes.

Ankylosing spondylitis (AS) is a chronic autoimmune inflammatory disease with severe inflammatory symptoms in the axial skeleton. The cause of ankylosing spondylitis is unknown. TNFAIP3, also named A20, uses ubiquitin-related functions to regulate immune activation, deficiency of which is highly related to autoimmune disease. However, the role of TNFAIP3 in human AS has not been reported. Our objective was to study the role and mechanism of TNFAIP3 in ankylosing spondylitis. TNFAIP3 expression on different types of immunocytes from AS peripheral blood was measured by flow cytometry. In vitro, monocytes were transfected with a TNFAIP3 shRNA lentivirus, and IL6 and IL1B activation was tested using real-time PCR and ELISA. The novel interaction complex TNFAIP3-DEPTOR was determined through GST pull-down, yeast two-hybrid system, confocal microscopy, and co-immunoprecipitation. Transmission electron microscopy, the RFP-GFP-LC3 adenovirus, and LC3 expression were used for autophagy detection. Here, we show that TNFAIP3 expression in AS peripheral blood non-classical monocytes was decreased. In normal monocytes, TNFAIP3 induced autophagy, which restricted inflammasome activation to the early stage of LPS stimulation. Zinc-finger domains of TNFAIP3 were able to interact and stabilize DEPTOR. TNFAIP3 and DEPTOR together rapidly promoted autophagy after LPS treatment to prevent NLRP3 inflammasome formation. Finally, TNFAIP3 and DEPTOR deficiency in AS non-classical monocytes facilitated inflammasome activation. Our study indicates that TNFAIP3-DEPTOR complex-induced early-onset autophagy is vital for immune inhibition in autoimmune disease.

The structural basis of flagellin detection by NAIP5: A strategy to limit pathogen immune evasion.

Robust innate immune detection of rapidly evolving pathogens is critical for host defense. Nucleotide-binding domain leucine-rich repeat (NLR) proteins function as cytosolic innate immune sensors in plants and animals. However, the structural basis for ligand-induced NLR activation has so far remained unknown. NAIP5 (NLR family, apoptosis inhibitory protein 5) binds the bacterial protein flagellin and assembles with NLRC4 to form a multiprotein complex called an inflammasome. Here we report the cryo-electron microscopy structure of the assembled ~1.4-megadalton flagellin-NAIP5-NLRC4 inflammasome, revealing how a ligand activates an NLR. Six distinct NAIP5 domains contact multiple conserved regions of flagellin, prying NAIP5 into an open and active conformation. We show that innate immune recognition of multiple ligand surfaces is a generalizable strategy that limits pathogen evolution and immune escape.

Massively parallel unsupervised single-particle cryo-EM data clustering via statistical manifold learning.

Structural heterogeneity in single-particle cryo-electron microscopy (cryo-EM) data represents a major challenge for high-resolution structure determination. Unsupervised classification may serve as the first step in the assessment of structural heterogeneity. However, traditional algorithms for unsupervised classification, such as K-means clustering and maximum likelihood optimization, may classify images into wrong classes with decreasing signal-to-noise-ratio (SNR) in the image data, yet demand increased computational costs. Overcoming these limitations requires further development of clustering algorithms for high-performance cryo-EM data processing. Here we introduce an unsupervised single-particle clustering algorithm derived from a statistical manifold learning framework called generative topographic mapping (GTM). We show that unsupervised GTM clustering improves classification accuracy by about 40% in the absence of input references for data with lower SNRs. Applications to several experimental datasets suggest that our algorithm can detect subtle structural differences among classes via a hierarchical clustering strategy. After code optimization over a high-performance computing (HPC) environment, our software implementation was able to generate thousands of reference-free class averages within hours in a massively parallel fashion, which allows a significant improvement on ab initio 3D reconstruction and assists in the computational purification of homogeneous datasets for high-resolution visualization.

Dynamics of in vivo ASC speck formation.

Activated danger or pathogen sensors trigger assembly of the inflammasome adaptor ASC into specks, large signaling platforms considered hallmarks of inflammasome activation. Because a lack of in vivo tools has prevented the study of endogenous ASC dynamics, we generated a live ASC reporter through CRISPR/Cas9 tagging of the endogenous gene in zebrafish. We see strong ASC expression in the skin and other epithelia that act as barriers to insult. A toxic stimulus triggered speck formation and rapid pyroptosis in keratinocytes in vivo. Macrophages engulfed and digested that speck-containing, pyroptotic debris. A three-dimensional, ultrastructural reconstruction, based on correlative light and electron microscopy of the in vivo assembled specks revealed a compact network of highly intercrossed filaments, whereas pyrin domain (PYD) or caspase activation and recruitment domain alone formed filamentous aggregates. The effector caspase is recruited through PYD, whose overexpression induced pyroptosis but only after substantial delay. Therefore, formation of a single, compact speck and rapid cell-death induction in vivo requires a full-length ASC.

Unified polymerization mechanism for the assembly of ASC-dependent inflammasomes.

Inflammasomes elicit host defense inside cells by activating caspase-1 for cytokine maturation and cell death. AIM2 and NLRP3 are representative sensor proteins in two major families of inflammasomes. The adaptor protein ASC bridges the sensor proteins and caspase-1 to form ternary inflammasome complexes, achieved through pyrin domain (PYD) interactions between sensors and ASC and through caspase activation and recruitment domain (CARD) interactions between ASC and caspase-1. We found that PYD and CARD both form filaments. Activated AIM2 and NLRP3 nucleate PYD filaments of ASC, which, in turn, cluster the CARD of ASC. ASC thus nucleates CARD filaments of caspase-1, leading to proximity-induced activation. Endogenous NLRP3 inflammasome is also filamentous. The cryoelectron microscopy structure of ASC(PYD) filament at near-atomic resolution provides a template for homo- and hetero-PYD/PYD associations, as confirmed by structure-guided mutagenesis. We propose that ASC-dependent inflammasomes in both families share a unified assembly mechanism that involves two successive steps of nucleation-induced polymerization. PAPERFLICK:

Triggering of inflammasome by aggregated α-synuclein, an inflammatory response in synucleinopathies.

Parkinson's disease (PD) is one of the most common neurodegenerative diseases. It is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta of the brain. Another feature is represented by the formation in these cells of inclusions called Lewy bodies (LB), principally constituted by fibrillar α-synuclein (αSyn). This protein is considered a key element in the aetiology of a group of neurodegenerative disorders termed synucleinopathies, which include PD, but the cellular and molecular mechanisms involved are not completely clear. It is established that the inflammatory process plays a crucial role in the pathogenesis and/or progression of PD; moreover, it is known that aggregated αSyn, released by neurons, activates microglia cells to produce pro-inflammatory mediators, such as IL-1ß. IL-1ß is one of the strongest pro-inflammatory cytokines; it is produced as an inactive mediator, and its maturation and activation requires inflammasome activation. In particular, the NLRP3 inflammasome is activated by a wide variety of stimuli, among which are crystallized and particulate material. In this work, we investigated the possibility that IL-1ß production, induced by fibrillar αSyn, is involved the inflammasome activation. We demonstrated the competence of monomeric and fibrillar αSyn to induce synthesis of IL-1ß, through TLR2 interaction; we found that the secretion of the mature cytokine was a peculiarity of the fibrillated protein. Moreover, we observed that the secretion of IL-1ß involves NLRP3 inflammasome activation. The latter relies on the phagocytosis of fibrillar αSyn, followed by increased ROS production and cathepsin B release into the cytosol. Taken together, our data support the notion that fibrillar αSyn, likely released by neuronal degeneration, acts as an endogenous trigger inducing a strong inflammatory response in PD.