Importance of NLRP3 Inflammasome in Abdominal Aortic Aneurysms

Abdominal aortic aneurysm (AAA) is a chronic inflammatory degenerative aortic disease, which particularly affects older people. Nucleotide-binding oligomerization domain-like receptor family protein 3 (NLRP3) inflammasome is a multi-protein complex and mediates inflammatory responses by activating caspase 1 for processing premature interleukin (IL)-1β and IL-18. In this review, we first summarize the principle of NLRP3 inflammasome activation and the functionally distinct classes of small molecule NLRP3 inflammasome inhibitors. Next, we provide a comprehensive literature review on the expression of NLRP3 inflammasome effector mediators (IL-1β and IL-18) and components (caspase 1, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and NLRP3) in clinical and experimental AAAs. Finally, we discuss the influence of genetic deficiency or pharmacological inhibition of individual effector mediators and components of NLRP3 inflammasome on experimental AAAs. Accumulating clinical and experimental evidence suggests that NLRP3 inflammasome may be a promise therapeutic target for developing pharmacological strategies for clinical AAA management.     

NLRP1 haplotypes associated with vitiligo and autoimmunity increase interleukin-1β processing via the NLRP1 inflammasome

Nuclear localization leucine-rich-repeat protein 1 (NLRP1) is a key regulator of the innate immune system, particularly in the skin where, in response to molecular triggers such as pathogen-associated or damage-associated molecular patterns, the NLRP1 inflammasome promotes caspase-1–dependent processing of bioactive interleukin-1β (IL-1β), resulting in IL-1β secretion and downstream inflammatory responses. NLRP1 is genetically associated with risk of several autoimmune diseases including generalized vitiligo, Addison disease, type 1 diabetes, rheumatoid arthritis, and others. Here we identify a repertoire of variation in NLRP1 by deep DNA resequencing. Predicted functional variations in NLRP1 reside in several common high-risk haplotypes that differ from the reference by multiple nonsynonymous substitutions. The haplotypes that are high risk for disease share two substitutions, L155H and M1184V, and are inherited largely intact due to extensive linkage disequilibrium across the region. Functionally, we found that peripheral blood monocytes from healthy subjects homozygous for the predominant high-risk haplotype 2A processed significantly greater (P < 0.0001) amounts of the IL-1β precursor to mature bioactive IL-1β under basal (resting) conditions and in response to Toll-like receptor (TLR) agonists (TLR2 and TLR4) compared with monocytes from subjects homozygous for the reference haplotype 1. The increase in basal release was 1.8-fold greater in haplotype 2A monocytes, and these differences between the two haplotypes were consistently observed three times over a 3-mo period; no differences were observed for IL-1α or TNFα. NLRP1 RNA and protein levels were not altered by the predominant high-risk haplotype, indicating that altered function of the corresponding multivariant NLRP1 polypeptide predisposes to autoimmune diseases by activation of the NLRP1 inflammasome.     

Viral Glycoproteins Induce NLRP3 Inflammasome Activation and Pyroptosis in Macrophages

Infections with viral pathogens are widespread and can cause a variety of different diseases. In-depth knowledge about viral triggers initiating an immune response is necessary to decipher viral pathogenesis. Inflammasomes, as part of the innate immune system, can be activated by viral pathogens. However, viral structural components responsible for inflammasome activation remain largely unknown. Here we analyzed glycoproteins derived from SARS-CoV-1/2, HCMV and HCV, required for viral entry and fusion, as potential triggers of NLRP3 inflammasome activation and pyroptosis in THP-1 macrophages. All tested glycoproteins were able to potently induce NLRP3 inflammasome activation, indicated by ASC-SPECK formation and secretion of cleaved IL-1β. Lytic cell death via gasdermin D (GSDMD), pore formation, and pyroptosis are required for IL-1β release. As a hallmark of pyroptosis, we were able to detect cleavage of GSDMD and, correspondingly, cell death in THP-1 macrophages. CRISPR-Cas9 knockout of NLRP3 and GSDMD in THP-1 macrophages confirmed and strongly support the evidence that viral glycoproteins can act as innate immunity triggers. With our study, we decipher key mechanisms of viral pathogenesis by showing that viral glycoproteins potently induce innate immune responses. These insights could be beneficial in vaccine development and provide new impulses for the investigation of vaccine-induced innate immunity.     

IRAK-1 bypasses priming and directly links TLRs to rapid NLRP3 inflammasome activation

Pathogenic infections and tissue injuries trigger the assembly of inflammasomes, cytosolic protein complexes that activate caspase-1, leading to cleavage of pro-IL-1β and pro-IL-18 and to pyroptosis, a proinflammatory cell death program. Although microbial recognition by Toll-like receptors (TLRs) is known to induce the synthesis of the major caspase-1 substrate pro-IL-1β, the role of TLRs has been considered limited to up-regulation of the inflammasome components. During infection with a virulent microbe, TLRs and nucleotide-binding oligomerization domain-like receptors (NLRs) are likely activated simultaneously. To examine the requirements and outcomes of combined activation, we stimulated TLRs and a specific NLR, nucleotide binding and oligomerization, leucine-rich repeat, pyrin domain-containing 3 (NLRP3), simultaneously and discovered that such activation triggers rapid caspase-1 cleavage, leading to secretion of presynthesized inflammatory molecules and pyroptosis. This acute caspase-1 activation is independent of new protein synthesis and depends on the TLR-signaling molecule IL-1 receptor-associated kinase (IRAK-1) and its kinase activity. Importantly, Listeria monocytogenes induces NLRP3-dependent rapid caspase-1 activation and pyroptosis, both of which are compromised in IRAK-1–deficient macrophages. Our results reveal that simultaneous sensing of microbial ligands and virulence factors by TLRs and NLRP3, respectively, leads to a rapid TLR- and IRAK-1–dependent assembly of the NLRP3 inflammasome complex, and that such activation is important for release of alarmins, pyroptosis, and early IFN-γ production by memory CD8 T cells, all of which could be critical for early host defense.Toll-like receptors (TLRs) recognize conserved molecules from pathogens and initiate signaling that activates NF-κB, MAP kinases, and IFN response factor proteins (1, 2). This signaling induces proinflammatory cytokines, chemokines, adhesion molecules, and inflammasome components, all of which facilitate effector responses (1, 2). A second family of receptors, nucleotide-binding oligomerization domain-like like receptors (NLRs), reside in the cytosol and are activated in response to either microbial ligands that gain access to the cytosol or virulence factors, such as bacterial toxins (3, 4).Activation of NLRs leads to assembly of an inflammasome complex, leading to activation and cleavage of cysteine protease, caspase-1, which in turns cleaves IL-1β and IL-18, leading to their secretion (5). The widely studied nucleotide binding and oligomerization, leucine-rich repeat, pyrin domain-containing 3 (NLRP3) inflammasome, composed of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and procaspase-1, undergoes assembly in response to stimulation by various stimuli, including ATP, nigericin, maitotoxin, uric acid crystals, silica, asbestos, and such pathogens as Staphylococcus aureus, Streptococcus pyogenes, Listeria monocytogenes, and Salmonella typhimurium (6).Inflammasome-mediated caspase-1 activation promotes inflammation and host defense by two principal avenues: secretion of mature cytokines (IL-1β and IL-18) and activation of pyroptosis (7), a proinflammatory cell death pathway that eliminates the infected cell and removes the niche for intracellular microbial replication (8). The current understanding of the biology of IL-1β synthesis and secretion holds that the TLR signaling pathway induces synthesis and accumulation of pro-IL-1β in the cytosol, and inflammasome ligands cause assembly of the respective inflammasome complexes, leading to cleavage of pro-IL-1β by active caspase-1. The role of TLR signaling is thus considered limited to synthesis of the substrates or up-regulation of levels of the components of the inflammasome complexes themselves.In the present study, we investigated whether TLRs play a direct role in activation of the NLRP3 inflammosome and discovered that there are at least two phases of NLRP3 inflammasome activation. The early phase, acute inflammasome activation, is independent of new protein synthesis, depends on simultaneous activation of TLRs and NLRP3, and is directly regulated by TLR signaling via the TLR-signaling molecule IL-1 receptor-associated kinase (IRAK-1). The late phase, involving priming-dependent activation of the NLRP3 inflammasome, occurs independent of direct participation of IRAK-1. We also found that the acute IRAK-1–dependent NLRP3 inflammasome activation pathway is critical for pyroptosis and secretion of inflammatory proteins presynthesized by the cell. Our findings provide evidence supporting a direct link between TLR signaling and NLRP3 inflammasome activation and ascribe a unique function to IRAK-1 in early innate responses.     

β-Hydroxybutyrate,One of the Three Main Ketone Bodies,Ameliorates Acute Pancreatitis in Rats by Suppressing the NLRP3 Inflammasome Pathway

BackgroundAcute pancreatitis (AP) is a widespread disease resulting from the inflammation of acinar cells in the pancreas. β-hydroxybutyrate (BHB) is a water-soluble main ketone body synthesized in the human liver. The purpose of this study was to examine the possible therapeutic effects of BHB in the experimentally-induced AP model in rats. MethodsIn our study, male rats were randomly allotted into 6 groups, as control (0.9% saline i.p.), BHB1 (200 mg/kg BHB i.p.), BHB2 (2 doses of 200 mg/kg BHB i.p.), AP (4 doses of 50 µg/kg cerulein i.p., 4 doses at 1 h intervals), AP+BHB1 and AP+BHB2 groups. In pancreatic tissue sections, immunohistochemistry staining and western blot analysis for the inflammasome complex (caspase-1, ASC, and NLRP3) and inflammation-associated proteins (TNF-α and NF-κB) and a histopathological examination were performed. The levels of lipase, amylase, interleukin (IL)-18 and IL-1β in serum were measured. ResultsSeveral pathological degenerations, including edema, inflammatory cell infiltration, acinus necrosis, and bleeding were observed in the AP group, while the histological architecture of the control and the sham BHB1 and BHB2 groups were regular. The AP-induced pathological changes were considerably alleviated in the AP+BHB1 and AP+BHB2 groups. In the AP group, a conspicuous increase in caspase-1, ASC, NLRP3, TNF-α, and NF-κB proteins, and in the levels of amylase, lipase, IL-18, and IL-1β were detected. BHB treatments after AP induction decreased those proteins to the level of control. ConclusionsWe demonstrated that BHB has the potential to cure AP by suppressing the NLRP3 inflammasome and can be used in the treatment of many diseases which progress through the NLRP3 inflammasome.     

TLR-induced PAI-2 expression suppresses IL-1β processing via increasing autophagy and NLRP3 degradation

The NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome, a multiprotein complex, triggers caspase-1 activation and maturation of the proinflammatory cytokines IL-1β and IL-18 upon sensing a wide range of pathogen- and damage-associated molecules. Dysregulation of NLRP3 inflammasome activity contributes to the pathogenesis of many diseases, but its regulation remains poorly defined. Here we show that depletion of plasminogen activator inhibitor type 2 (PAI-2), a serine protease inhibitor, resulted in NLRP3- and ASC (apoptosis-associated Speck-like protein containing a C-terminal caspase recruitment domain)‐dependent caspase-1 activation and IL-1β secretion in macrophages upon Toll-like receptor 2 (TLR2) and TLR4 engagement. TLR2 or TLR4 agonist induced PAI-2 expression, which subsequently stabilized the autophagic protein Beclin 1 to promote autophagy, resulting in decreases in mitochondrial reactive oxygen species, NLRP3 protein level, and pro–IL-1β processing. Likewise, overexpressing Beclin 1 in PAI-2–deficient cells rescued the suppression of NLRP3 activation in response to LPS. Together, our data identify a tier of TLR signaling in controlling NLRP3 inflammasome activation and reveal a cell-autonomous mechanism which inversely regulates TLR- or Escherichia coli-induced mitochondrial dysfunction, oxidative stress, and IL-1β–driven inflammation.Innate immunity, the first line of host defense against pathogen infection, is composed of diverse germ line-encoded pattern-recognition receptors, such as Toll-like receptors (TLRs) and NOD-like receptors (NLRs), that recognize pathogen-associated molecular patterns (PAMPs) from pathogens or danger-associated molecular patterns from damaged tissue (1, 2). TLRs recognize a variety of PAMPs from microbes to induce autophagy and cytokine production for host defense against microbial infections. Inflammasomes, multiple protein complexes containing NLR proteins or AIM2, mediate caspase-1 activation leading to the processing of the proinflammatory cytokines IL-1β and IL-18 (3). The inflammasome/caspase-1 complexes also may target additional effector molecules to regulate diverse physiological functions, such as pyroptosis and tissue repair (4). Among the identified inflammasomes, the NLRP3 inflammasome has been studied extensively and has been shown to be activated by a large variety of activators that share no structural similarity (2). For this reason, it has been suggested that the NLRP3 inflammasome is activated through a secondary mediator, such as potassium (K⁺) efflux, reactive oxygen species (ROS), or lysosomal proteases (1). The inflammasomes play a critical role in the clearance of microbial pathogens and tissue repair (2, 5). However, dysregulation of inflammasome activation has been associated with a variety of human diseases, including autoinflammatory diseases, metabolic disorders, and cancer (3, 6).Autophagy, an evolutionarily conserved cellular catabolic process, facilitates the recycling of damaged proteins and organelles (7). Increasing evidence indicates that autophagy is involved in the regulation of immune responses and inflammation (7). Macrophages treated with an autophagy inhibitor or with the deletion of several autophagic components, including Atg16L1, Beclin 1, and LC3, induced greater caspase-1 activation and IL-1β secretion in response to LPS or LPS plus an NLRP3 agonist (8, 9). These data strongly suggest that the NLRP3 inflammasome activity is negatively regulated by autophagy, but the underlying mechanism is poorly understood.Plasminogen activator inhibitor type 2 (PAI-2), a serine proteinase inhibitor (SERPIN), originally was identified as an inhibitor of the urokinase-type plasminogen activator (uPA) involved in cellular invasion and tissue remodeling (10). Recently, PAI-2 has been associated with newly identified uPA-independent biological functions, probably through targeting an as yet uncharacterized intracellular molecule (11). In addition, PAI-2 is one of the major molecules up-regulated in macrophages in response to TLR4 activators or inflammatory mediators, suggesting its function in the regulation of innate immunity (12, 13).Macrophages treated with LPS alone do not release mature IL-1β and IL-18 unless accompanied by a second stimulus, such as ATP or crystals (8, 14). LPS activates TLR4 to induce the synthesis of pro–IL-1β and the inflammasome component NLRP3 via IκB kinase (IKK)/NF-κB activation; a second stimulus is required for inflammasome assembly and caspase-1 activation to cleave pro–IL-1β and pro–IL-18 to their mature forms. Nevertheless, previous work showed that LPS alone is sufficient to induce mature IL-1β production in IKKβ-deficient macrophages because of enhanced pro–IL-1β processing (15). Additionally, LPS-induced PAI-2 expression is blunted in IKKβ-deficient macrophages, and reintroduction of PAI-2 blocks IL-1β maturation in a caspase-1–dependent manner, suggesting that PAI-2 inhibits pro–IL-1β processing upon LPS stimulation; however, the underlying mechanism is unknown.Here, we show that depletion of PAI-2 in macrophages induces caspase-1 activation and IL-1β production in response to TLR agonists and Escherichia coli with no need of a second stimulus. TLR engagement induced PAI-2 expression and enhanced association of PAI-2 with Beclin 1, leading to an increase in autophagy, which then caused reduced mitochondrial ROS (mROS) and increased NLRP3 degradation, resulting in decreased IL-1β maturation. Inflammatory cytokines and cellular ROS play vital roles in innate immunity, but prolonged and excess production of these mediators can be detrimental. Our results suggest that PAI-2 is a cell-autonomous mechanism that counteracts the detrimental effects caused by TLR2/4- and E. coli-triggered cellular stress by reducing ROS production and the inflammasome activation, thereby resulting in less inflammation and tissue damage.     

Influenza A Virus Infection Activates NLRP3 Inflammasome through Trans-Golgi Network Dispersion

The NLRP3 inflammasome consists of NLRP3, ASC, and pro-caspase-1 and is an important arm of the innate immune response against influenza A virus (IAV) infection. Upon infection, the inflammasome is activated, resulting in the production of IL-1β and IL-18, which recruits other immune cells to the site of infection. It has been suggested that in the presence of stress molecules such as nigericin, the trans-Golgi network (TGN) disperses into small puncta-like structures where NLRP3 is recruited and activated. Here, we investigated whether IAV infection could lead to TGN dispersion, whether dispersed TGN (dTGN) is responsible for NLRP3 inflammasome activation, and which viral protein is involved in this process. We showed that the IAV causes dTGN formation, which serves as one of the mechanisms of NLRP3 inflammasome activation in response to IAV infection. Furthermore, we generated a series of mutant IAVs that carry mutations in the M2 protein. We demonstrated the M2 proton channel activity, specifically His37 and Trp41 are pivotal for the dispersion of TGN, NLRP3 conformational change, and IL-1β induction. The results revealed a novel mechanism behind the activation and regulation of the NLRP3 inflammasome in IAV infection.     

Uptake of particulate vaccine adjuvants by dendritic cells activates the NALP3 inflammasome

Many currently used and candidate vaccine adjuvants are particulate in nature, but their mechanism of action is not well understood. Here, we show that particulate adjuvants, including biodegradable poly(lactide-co-glycolide) (PLG) and polystyrene microparticles, dramatically enhance secretion of interleukin-1β (IL-1β) by dendritic cells (DCs). The ability of particulates to promote IL-1β secretion and caspase 1 activation required particle uptake by DCs and NALP3. Uptake of microparticles induced lysosomal damage, whereas particle-mediated enhancement of IL-1β secretion required phagosomal acidification and the lysosomal cysteine protease cathepsin B, suggesting a role for lysosomal damage in inflammasome activation. Although the presence of a Toll-like receptor (TLR) agonist was required to induce IL-1β production in vitro, injection of the adjuvants in the absence of TLR agonists induced IL-1β production at the injection site, indicating that endogenous factors can synergize with particulates to promote inflammasome activation. The enhancement of antigen-specific antibody production by PLG microparticles was independent of NALP3. However, the ability of PLG microparticles to promote antigen-specific IL-6 production by T cells and the recruitment and activation of a population of CD11b⁺Gr1⁻ cells required NALP3. Our data demonstrate that uptake of microparticulate adjuvants by DCs activates the NALP3 inflammasome, and this contributes to their enhancing effects on innate and antigen-specific cellular immunity.     

Reactive oxygen species–independent activation of the IL-1β inflammasome in cells from patients with chronic granulomatous disease

Humans with chronic granulomatous diseases (CGDs) due to mutations in p47-phox have defective NADPH activity and thus cannot generate NADPH-dependent reactive oxygen species (ROS). The role of ROS in inflammation is controversial; some in vitro studies suggest that ROS are crucial for secretion of IL-1β via inflammasome activation, whereas mice defective for ROS and patients with CGD have a proinflammatory phenotype. In this study, we evaluated activation of the IL-1β inflammasome in cells from CGD patients. In contrast to previous studies using the small molecule diphenylene iodonium (DPI) as a ROS inhibitor, we found no decrease in either caspase-1 activation or secretion of IL-1β and IL-18 in primary CGD monocytes. Moreover, activation of CGD monocytes by uric acid crystals induced a 4-fold higher level of IL-1β secretion compared with that seen in monocytes from unaffected subjects, and this increase was not due to increased synthesis of the IL-1β precursor. In addition, Western blot analysis of CGD cells revealed that caspase-1 activation was not decreased, but rather was increased compared with control cells. Examination of the effects exerted by the inhibition of ROS activity by DPI revealed that the decrease in IL-1β secretion by DPI was actually due to inhibition of IL-1β gene expression. Thus, inconsistent with the proinflammatory role of ROS, the present findings support the concept that ROS likely dampen inflammasome activation. The absence of ROS in CGD monocytes may explain the presence of an inflammatory phenotype characterized by granulomas and inflammatory bowel disease occurring in CGD patients.     

Mitochondrial protein mitofusin 2 is required for NLRP3 inflammasome activation after RNA virus infection

Nod-like receptor family, pyrin domain-containing 3 (NLRP3), is involved in the early stages of the inflammatory response by sensing cellular damage or distress due to viral or bacterial infection. Activation of NLRP3 triggers its assembly into a multimolecular protein complex, termed “NLRP3 inflammasome.” This event leads to the activation of the downstream molecule caspase-1 that cleaves the precursor forms of proinflammatory cytokines, such as interleukin 1 beta (IL-1β) and IL-18, and initiates the immune response. Recent studies indicate that the reactive oxygen species produced by mitochondrial respiration is critical for the activation of the NLRP3 inflammasome by monosodium urate, alum, and ATP. However, the precise mechanism by which RNA viruses activate the NLRP3 inflammasome is not well understood. Here, we show that loss of mitochondrial membrane potential [ΔΨ(m)] dramatically reduced IL-1β secretion after infection with influenza, measles, or encephalomyocarditis virus (EMCV). Reduced IL-1β secretion was also observed following overexpression of the mitochondrial inner membrane protein, uncoupling protein-2, which induces mitochondrial proton leakage and dissipates ΔΨ(m). ΔΨ(m) was required for association between the NLRP3 and mitofusin 2, a mediator of mitochondrial fusion, after infection with influenza virus or EMCV. Importantly, the knockdown of mitofusin 2 significantly reduced the secretion of IL-1β after infection with influenza virus or EMCV. Our results provide insight into the roles of mitochondria in NLRP3 inflammasome activation.Nod-like receptor family, pyrin domain-containing 3 (NLRP3) can be activated by a wide variety of stimuli, such as endogenous danger signals from damaged cells, bacterial components, environmental irritants, and DNA and RNA viruses (1). It forms a multiprotein complex called the NLRP3 inflammasome, which includes an adaptor protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and procaspase-1. The NLRP3 inflammasome-mediated cytokine release requires two signaling pathways (2). The first signal is induced by Toll-like receptors (TLRs), interleukin 1 receptor (IL-1R), or tumor necrosis factor receptor, and leads to the synthesis of inactive NLRP3, pro–IL-1β, and pro–IL-18 in the cytosol. The second signal is triggered by NLRP3 agonists, which induce the activation of caspase-1. Caspase-1 catalyzes the proteolytic processing of pro–IL-1β and pro–IL-18, and their conversion to mature forms, and stimulates their secretion across the plasma membrane (1). These inflammasome-dependent cytokines play a key role in the induction of adaptive immunity and the initiation of tissue healing after influenza virus infection (3–5). Migration of dendritic cells (DCs) to the draining lymph nodes and priming of CD8 T cells during influenza virus infection require IL-1R signaling in respiratory DCs (6). By contrast, chronic activation of the NLRP3 inflammasome has been linked to many inflammatory diseases (7, 8). Therefore, increasing the number of studies dedicated to the investigation of the molecular mechanisms of NLRP3 inflammasome activation will be crucial for improving our understanding of the pathogenesis of infectious and autoinflammatory diseases.Mitochondria are compartmentalized by two membrane bilayers (outer and inner membranes) and are involved in a wide variety of functions in eukaryotic cells. Within the past decade, novel functions of mitochondria have been discovered demonstrating their crucial role in innate antiviral immunity in vertebrates (9). A direct link between mitochondria and innate immunity was first highlighted with the finding that an adaptor protein, mitochondrial antiviral signaling (MAVS; also known as IPS-1, VISA, or Cardif) (10–13), triggered retinoic acid-inducible gene 1 (RIG-I) and melanoma differentiation-associated protein 5-mediated type I interferon (IFN) induction. In addition to their role in antiviral immunity, mitochondria also function as a platform for the activation of the NLRP3 inflammasome by producing mitochondrial reactive oxygen species (mROS) (14, 15). In this context, NLRP3 agonists trigger the generation of mROS from damaged mitochondria, resulting in the dissociation of thioredoxin (TRX) from TRX-interacting protein, which associates with NLRP3 to facilitate inflammasome formation (16). Furthermore, cytosolic mitochondrial DNA (mtDNA) released from damaged mitochondria has been reported to activate the NLRP3 inflammasome (17) and absent in melanoma 2 inflammasome (15), recently identified as a cytoplasmic DNA sensor for the inflammasome (18–21). Although mitochondria are essential for host-cell defense, the mechanism of their involvement in the activation of the NLRP3 inflammasome is still unclear. In the present study, we demonstrate that the mitofusin 2 (Mfn2) is required for the full activation of the NLRP3 inflammasomes in macrophages.     

Altered redox state of monocytes from cryopyrin-associated periodic syndromes causes accelerated IL-1β secretion

In healthy monocytes, Toll-like receptor (TLR) engagement induces production of reactive oxygen species (ROS), followed by an antioxidant response involved in IL-1β processing and secretion. Markers of the antioxidant response include intracellular thioredoxin and extracellular release of reduced cysteine. Cryopyrin-associated periodic syndromes (CAPS) are autoinflammatory diseases in which Nod-like receptor family pyrin domain–containing 3 (NLRP3) gene mutations lead to increased IL-1β secretion. We show in a large cohort of patients that IL-1β secretion by CAPS monocytes is much faster than that by healthy monocytes. This accelerated kinetics is caused by alterations in the basal redox state, as well as in the redox response to TLR triggering displayed by CAPS monocytes. Indeed, unstimulated CAPS monocytes are under a mild oxidative stress, with elevated levels of both ROS and antioxidants. The redox response to LPS is quickened, with early generation of the reducing conditions favoring IL-1β processing and secretion, and then rapidly exhausted. Therefore, secretion of IL-1β is accelerated, but reaches a plateau much earlier than in healthy controls. Pharmacologic inhibition of the redox response hinders IL-1β release, confirming the functional link between redox impairment and altered kinetics of secretion. Monocytes from patients with juvenile idiopathic arthritis display normal kinetics of redox response and IL-1β secretion, excluding a role of chronic inflammation in the alterations observed in CAPS. We conclude that preexisting redox alterations distinct from CAPS monocytes anticipate the pathogen-associated molecular pattern molecule–induced generation of the reducing environment favorable to inflammasome activation and IL-1β secretion.     

Soluble α-synuclein–antibody complexes activate the NLRP3 inflammasome in hiPSC-derived microglia

Parkinson’s disease is characterized by accumulation of α-synuclein (αSyn). Release of oligomeric/fibrillar αSyn from damaged neurons may potentiate neuronal death in part via microglial activation. Heretofore, it remained unknown if oligomeric/fibrillar αSyn could activate the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family pyrin domain-containing 3 (NLRP3) inflammasome in human microglia and whether anti-αSyn antibodies could prevent this effect. Here, we show that αSyn activates the NLRP3 inflammasome in human induced pluripotent stem cell (hiPSC)-derived microglia (hiMG) via dual stimulation involving Toll-like receptor 2 (TLR2) engagement and mitochondrial damage. In vitro, hiMG can be activated by mutant (A53T) αSyn secreted from hiPSC-derived A9-dopaminergic neurons. Surprisingly, αSyn–antibody complexes enhanced rather than suppressed inflammasome-mediated interleukin-1β (IL-1β) secretion, indicating these complexes are neuroinflammatory in a human context. A further increase in inflammation was observed with addition of oligomerized amyloid-β peptide (Aβ) and its cognate antibody. In vivo, engraftment of hiMG with αSyn in humanized mouse brain resulted in caspase-1 activation and neurotoxicity, which was exacerbated by αSyn antibody. These findings may have important implications for antibody therapies aimed at depleting misfolded/aggregated proteins from the human brain, as they may paradoxically trigger inflammation in human microglia.

Parkinson’s disease (PD) is characterized by accumulation of α-synuclein (αSyn; encoded by the SNCA gene) (1). Release of oligomeric/fibrillar αSyn from damaged neurons may potentiate neuronal cell death in part via microglial activation (2, 3). Moreover, misfolded proteins in general are thought to interact with brain microglia, triggering microglial activation that contributes to neurodegenerative disorders, although microglial phagocytosis may also initially clear aberrant proteins to afford some degree of protection (2, 4). Additionally, in Alzheimer’s disease (AD), amyloid-β peptide (Aβ) is thought to trigger similar processes in microglia (5–7); however, the mechanism for this trigger is still poorly understood.Microglial cells contribute to neuroinflammation, specifically that mediated by the inflammasome. In particular, the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family pyrin domain-containing 3 (NLRP3) inflammasome has been associated with several neurodegenerative disorders, although other types of inflammation may also be important in this regard (8). The NLRP3 inflammasome is a multiprotein complex that responds to cell stress and pathogenic stimuli to promote activation of caspase-1, which in turn mediates maturation and release of proinflammatory cytokines, including interleukin-1β (IL-1β) and IL-18 (9–11). NLRP3 inflammasome activation is a two-step process, involving an initial priming step and a secondary trigger. Priming involves a proinflammatory stimulus, such as endotoxin, a ligand for Toll-like receptor 4 (TLR4), that increases the abundance of NLRP3 and promotes de novo synthesis of pro–IL-1β via nuclear factor κB (11). The secondary trigger promotes inflammasome complex assembly and caspase-1 activation that in turn mediates the cleavage of pro–IL-1β and subsequent release of mature IL-1β. There are various secondary triggers, including adenosine triphosphate (ATP), microparticles, and bacterial toxins, all of which somehow lead to mitochondrial damage and release of oxidized mitochondrial DNA (11). Neuroinflammation has been reported in both human PD and AD brains (12–15), and NLRP3 inflammasome activation in particular has been observed in mouse models of PD and AD (7, 16). Importantly, in these PD models, dopaminergic (DA) neurons in the substantia nigra are resistant to damage in NLRP3-deficient mice compared with wild-type (WT) mice (16). Interestingly, a recent report identified an NLRP3 polymorphism that confers decreased risk in PD (17). Several groups have reported that fibrillar αSyn can activate the NLRP3 inflammasome in mice and in human monocytes (18–22), but it remains unknown if human brain microglia can be activated in this manner. Critically, antibodies targeting misfolded proteins are being tested in human clinical trials for several neurodegenerative diseases, including AD and PD; however, it is still unclear how antibodies to αSyn might affect this inflammatory response. In this study, we characterized the response of human induced pluripotent stem cell (hiPSC)-derived microglia (hiMG) to oligomeric/fibrillar αSyn in vitro and in vivo, using engraftment of hiMG in humanized mice. We used these immunocompromised mice because they prevent human cell rejection and express three human genes that support human cell engraftment (23). We show that αSyn and, even more so, αSyn–antibody complexes activate the NLRP3 inflammasome. Moreover, this process is further sensitized by the presence of Aβ and its cognate antibodies. These observations are of heightened interest because recent studies have shown that both misfolded Aβ and αSyn are present in several neurodegenerative disorders such as AD and Lewy body dementia (LBD), a form of dementia that can occur in the setting of PD (24–26).     

Targeting tumor-derived NLRP3 reduces melanoma progression by limiting MDSCs expansion

Interleukin-1β (IL-1β)–mediated inflammation suppresses antitumor immunity, leading to the generation of a tumor-permissive environment, tumor growth, and progression. Here, we demonstrate that nucleotide-binding domain, leucine-rich containing family, pyrin domain-containing-3 (NLRP3) inflammasome activation in melanoma is linked to IL-1β production, inflammation, and immunosuppression. Analysis of cancer genome datasets (TCGA and GTEx) revealed greater NLRP3 and IL-1β expression in cutaneous melanoma samples (n = 469) compared to normal skin (n = 324), with a highly significant correlation between NLRP3 and IL-1β (P < 0.0001). We show the formation of the NLRP3 inflammasome in biopsies of metastatic melanoma using fluorescent resonance energy transfer analysis for NLRP3 and apoptosis-associated speck-like protein containing a CARD. In vivo, tumor-associated NLRP3/IL-1 signaling induced expansion of myeloid-derived suppressor cells (MDSCs), leading to reduced natural killer and CD8⁺ T cell activity concomitant with an increased presence of regulatory T (Treg) cells in the primary tumors. Either genetic or pharmacological inhibition of tumor-derived NLRP3 by dapansutrile (OLT1177) was sufficient to reduce MDSCs expansion and to enhance antitumor immunity, resulting in reduced tumor growth. Additionally, we observed that the combination of NLRP3 inhibition and anti–PD-1 treatment significantly increased the antitumor efficacy of the monotherapy by limiting MDSC-mediated T cell suppression and tumor progression. These data show that NLRP3 activation in melanoma cells is a protumor mechanism, which induces MDSCs expansion and immune evasion. We conclude that inhibition of NLRP3 can augment the efficacy of anti–PD-1 therapy.

Tumorigenesis is initiated by genomic alterations, leading to cell transformation, proliferation, and resistance to apoptotic signals, which ultimately lead to metastasis and tissue invasion. Tumor progression is also linked to dysregulated inflammation, which is characterized by cytokine signaling between cancer and noncancer cells (1, 2). The proinflammatory cytokine interleukin-1β (IL-1β) mediates several inflammatory diseases and is a pivotal cytokine in initiating inflammatory responses (3). In the context of malignancy, IL-1β is a validated target in mouse models of cancer, including melanoma, where the cytokine contributes to immunosuppression, angiogenesis, metastasis, and regulation of myeloid-derived suppressor cells (MDSCs) (1, 2, 4, 5). In humans, IL-1β is overexpressed in biopsies from metastatic melanoma patients, suggesting a possible role in the melanoma-induced inflammation (6).Processing of IL-1β is largely governed by inflammasomes, cytosolic macromolecular complexes responsible for the conversion of biologically inactive IL-1β and IL-18 precursors into their active forms via caspase-1 cleavage (7). The nucleotide-binding domain, leucine-rich containing family, pyrin domain-containing-3 (NLRP3) is the most studied of the inflammasome sensors driving IL-1β–mediated conditions from sterile inflammation to rare hereditary syndromes (8, 9). NLRP3 is particularly relevant to the processing of IL-1β in melanoma because NLRP3 is constitutively expressed in melanoma cell lines (6) and NLRP3 polymorphisms are linked to increased risk to develop melanoma (10). Whereas these studies indicate a possible role for NLRP3 in melanoma progression, the biological function for NLRP3 in melanoma remains unclear. Furthermore, although it is well known that inflammation participates in the development and progression of melanoma (11–13), the inflammatory pathways that drive this process are still poorly characterized and no therapy developed to date is actually designed to specifically target inflammatory pathways in melanoma. Here, using genetic models of NLRP3 depletion and a specific pharmacological inhibitor of NLRP3 (14), we show that NLRP3 represents a melanoma intrinsic pathway exploited for tumor-mediated immune escape. We demonstrate that tumor-derived NLRP3 activation induces MDSC expansion, which suppresses recruitment and activation of antitumor immunity.From a clinical standpoint, immune checkpoint therapy (ICT) has significantly improved the outcome for melanoma patients, and numerous studies have demonstrated that expression of PD-1/PD-L1 and CTLA4 are often predictors for efficacy of immunotherapy. However, the number of patients that are unresponsive to ICT or relapse continues to rise, and clinical data show that expression of immune checkpoints do not always correlate with responses (15). The limited response to monotherapy in some patients suggests that intrinsic pathways in melanoma cells, such as expression of checkpoint ligands, are not the only mechanisms that drive tumor progression. Identification of other tumor-specific strategies provides an opportunity to interrupt the oncogenic process and improve survival in this population. For example, melanoma-associated inflammation facilitates tumor progression (11, 16) and, specifically, is linked to IL-1β activity (4, 17). An approach for reducing IL-1β activity is via inhibition of NLRP3. Here, we show that disruption of the NLRP3 signaling in combination with ICT increases antitumor activity. The data support the concept that tumor NLRP3 activation represents an intrinsic pathway that favors tumor immune escape. Thus, targeting NLRP3 represents an innovative strategy for treating melanoma, especially in the context of immunotherapy resistance tumors.     

NLRP3-Inflammasome Inhibition during Respiratory Virus Infection Abrogates Lung Immunopathology and Long-Term Airway Disease Development

Respiratory syncytial virus (RSV) infects most infants by two years of age. It can cause severe disease leading to an increased risk of developing asthma later in life. Previously, our group has shown that RSV infection in mice and infants promotes IL-1β production. Here, we characterized the role of NLRP3-Inflammasome activation during RSV infection in adult mice and neonates. We observed that the inhibition of NLRP3 activation using the small molecule inhibitor, MCC950, or in genetically modified NLRP3 knockout (Nlrp3−/−) mice during in vivo RSV infection led to decreased lung immunopathology along with a reduced expression of the mucus-associated genes and reduced production of innate cytokines (IL-1β, IL-33 and CCL2) linked to severe RSV disease while leading to significant increases in IFN-β. NLRP3-inflammasome inhibition or deletion diminished Th2 cytokines and inflammatory cell infiltration into the lungs. Furthermore, NLRP3 inhibition or deletion during early-life RSV infection led to reducing viral-exacerbated allergic response in a mouse model of RSV-induced allergy exacerbation. Here, we demonstrated the critical role of NLRP3-inflammasome activation in RSV immunopathology and the related long-term airway alteration. Moreover, these findings suggest the NLRP3-inflammasome as a potential therapeutic target to attenuate severe RSV disease and limit childhood asthma development.     

The NLRP3 inflammasome inhibitor OLT1177 rescues cognitive impairment in a mouse model of Alzheimer’s disease

Numerous studies demonstrate that neuroinflammation is a key player in the progression of Alzheimer’s disease (AD). Interleukin (IL)-1β is a main inducer of inflammation and therefore a prime target for therapeutic options. The inactive IL-1β precursor requires processing by the the nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome into a mature and active form. Studies have shown that IL-1β is up-regulated in brains of patients with AD, and that genetic inactivation of the NLRP3 inflammasome improves behavioral tests and synaptic plasticity phenotypes in a murine model of the disease. In the present study, we analyzed the effect of pharmacological inhibition of the NLRP3 inflammasome using dapansutrile (OLT1177), an oral NLRP3-specific inhibitor that is safe in humans. Six-month-old WT and APP/PS1 mice were fed with standard mouse chow or OLT1177-enriched chow for 3 mo. The Morris water maze test revealed an impaired learning and memory ability of 9-mo-old APP/PS1 mice (P = 0.001), which was completely rescued by OLT1177 fed to mice (P = 0.008 to untreated APP/PS1). Furthermore, our findings revealed that 3 mo of OLT1177 diet can rescue synaptic plasticity in this mouse model of AD (P = 0.007 to untreated APP/PS1). In addition, microglia were less activated (P = 0.07) and the number of plaques was reduced in the cortex (P = 0.03) following NLRP3 inhibition with OLT1177 administration. We also observed an OLT1177 dose-dependent normalization of plasma metabolic markers of AD to those of WT mice. This study suggests the therapeutic potential of treating neuroinflammation with an oral inhibitor of the NLRP3 inflammasome.

Alzheimer’s disease (AD) and other related neurodegenerative diseases leading to dementia represent an enormous burden for the society and health economies. AD patients suffer progressive cognitive and functional deficits often for many years, which result in a heavy burden to patients, families, and the public health system. In fact, in 2015 an estimated 46.8 million people worldwide were living with dementia, which could extend to 131.5 million by 2050 (1). Rising prevalence and mortality rates in combination with a lack of effective treatments lead to enormous costs to society. Research on AD in the last decades has focused on the pathological hallmarks and cellular deposits of amyloid-β (Aβ) peptides and neurofibrils (2). Recently, there has been increased evidence supporting a central role of the immune system in the progression or even the origin of the disease (3–5). In this respect, it is noteworthy that it has been known since 1989 that levels of interleukin (IL)-1β, one of the main mediators of innate immune response, are elevated in brains of patients with AD and can be associated with the progression and onset of AD (6–11). Additionally, it was shown that the nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome (12, 13), a multisubunit complex important for the maturation of IL-1β, is activated by Aβ peptides, leading to an overproduction of IL-1β, neuroinflammation, and cognitive impairment (14, 15). Inhibition of the NLRP3 inflammasome and the subsequent reduced IL-1β production can be linked to a change in the phenotype of microglia, the innate immune cells in the brain. Heneka et al. (16) pointed out the important role of the NLRP3 inflammasome/caspase-1 axis in AD pathogenesis by demonstrating significant improvements (e.g., in cognition) in APP/PS1 mice (a mouse model for AD) when crossed with NLRP3^−/− animals. The APP/PS1 mice express a human amyloid precursor protein (APP) and human presenilin-1 (PS1), leading to the accumulation of Aβ peptides, neuroinflammation, and cognitive impairment (17).OLT1177 (rINN: dapansutrile) is a new chemical entity small molecule that specifically targets the NLRP3 inflammasome and prevents the activation of caspase-1 and the maturation and release of IL-1β (18). OLT1177 has been shown to be well tolerated in animals and humans (18) and is currently in phase 2 clinical studies for the treatment of inflammatory conditions, such as osteoarthritis (topical gel dosage form) and inflammatory diseases, such as acute gout flare (oral capsule dosage form), among other diseases (19).In this study, we used the APP/PS1 mouse model of AD to investigate the effects of OLT1177 as an acute, oral pharmacological intervention (17). Six-month-old WT and APP/PS1ΔE9 mice consumed ad libitum OLT1177 in feed pellets (∼0, 500, or 1,000 mg/kg/d based on feed concentrations of 0, 3.75 or 7.5 g of OLT1177 per kilogram of feed; hereafter referred to as 3.75 or 7.5 g/kg OLT1177) for the treatment duration of 3 mo. APP/PS1 mice treated with OLT1177 showed rescue effects in various assessments, ranging from improved cognitive function to overall reduction in proinflammatory cytokines in the brain, suggesting the potential benefits of pharmaceutically blocking NLRP3 signaling in AD.     

Human NLRP3 inflammasome senses multiple types of bacterial RNAs

Inflammasomes are multiprotein platforms that activate caspase-1, which leads to the processing and secretion of the proinflammatory cytokines IL-1β and IL-18. Previous studies demonstrated that bacterial RNAs activate the nucleotide-binding domain, leucine-rich-repeat-containing family, pyrin domain-containing 3 (NLRP3) inflammasome in both human and murine macrophages. Interestingly, only mRNA, but neither tRNA nor rRNAs, derived from bacteria could activate the murine Nlrp3 inflammasome. Here, we report that all three types of bacterially derived RNA (mRNA, tRNA, and rRNAs) were capable of activating the NLRP3 inflammasome in human macrophages. Bacterial RNA’s 5′-end triphosphate moieties, secondary structure, and double-stranded structure were dispensable; small fragments of bacterial RNA were sufficient to activate the inflammasome. In addition, we also found that 20-guanosine ssRNA can activate the NLRP3 inflammasome in human macrophages but not in murine macrophages. Therefore, human and murine macrophages may have evolved to recognize bacterial cytosolic RNA differently during bacterial infections.The innate immune system is the first line of defense against microbial infections. Germ-line–encoded pattern-recognition receptors (PRRs) of the innate immune system recognize the presence of invariant evolutionarily conserved microbial components called “pathogen-associated molecular patterns” (1–3). In response to microbial infections, PRRs rapidly initiate signal-transduction pathways to induce type 1 IFN production, proinflammatory cytokine production, and inflammasome activation. The inflammasome is a cytosolic large caspase-1–containing multiprotein complex that enables autocatalytic activation of caspase-1. Once caspase-1 is activated, it starts to cleave prointerleukin-1β (pro–IL-1β) and prointerleukin-18 (pro–IL-18) proteolytically into bioactive IL-1β and IL-18 (4–7). The mature forms of IL-1β and IL-18 play roles in a variety of infectious and inflammatory processes.Cytosolic microbial nucleic acids are important activators of the innate immune system against both bacterial and viral infections, which induce type 1-IFN and proinflammatory cytokine responses as well as inflammasome activation. The role of microbial nucleic acids in inflammasome activation has been studied mostly in murine bone marrow-derived dendritic cells (BMDCs) or bone marrow-derived macrophages (BMDMs). AIM2 has been identified as a specific cytosolic dsDNA sensor that directly binds ASC (apoptosis-associated speck-like protein containing a carboxyl-terminal CARD-like domain) and forms inflammasome complexes in human and murine cells (8–11).Viral dsRNA was found to activate the nucleotide-binding domain, leucine-rich-repeat-containing family, pyrin domain-containing 3 (NLRP3) inflammasome in human and murine cells (12–15). Several groups have reported that cytosolic bacterial RNA activate the Nlrp3 inflammasome in murine macrophages (13, 16, 17). Our group also has reported that human THP-1–derived macrophages recognize cytosolic bacterial RNA and induce NLRP3 inflammasome activation (12). Bacterial RNA is composed of mRNA, tRNA, and three different sizes of rRNA (23s, 16s, and 5s). Sander et al. (18) reported that, of the different types of Escherichia coli RNA, only E. coli mRNA induced the secretion of IL-1β by murine BMDMs, but E. coli tRNA and E. coli rRNAs did not.We aimed to study (i) whether a variety of cytosolic bacterial RNAs could activate the inflammasome in human myeloid cells and (ii) what types of bacterial RNA activate the inflammasome in human and murine myeloid cells. Here, we demonstrate that a broad spectrum of cytosolic bacterial RNAs strongly induce the cleavage of caspase-1 and the secretion of IL-1β and IL-18 in human macrophages. Human macrophages can sense mRNA, tRNA, rRNAs, and small synthetic ssRNA through NLRP3, but murine macrophages can sense only the mRNA component. Bacterial RNA’s 5′-end triphosphate moieties, secondary structure, and double-stranded structure were dispensable, but small fragments of bacterial RNA were sufficient to activate the inflammasome. These findings suggest that upon bacterial infections the human and murine NLRP3 inflammasomes sense cytosolic bacterial RNAs differently.