FMDV Leader Protein Interacts with the NACHT and LRR Domains of NLRP3 to Promote IL-1β Production

Foot-and-mouth disease virus (FMDV) infection causes inflammatory clinical symptoms, such as high fever and vesicular lesions, even death of animals. Interleukin-1β (IL-1β) is an inflammatory cytokine that plays an essential role in inflammatory responses against viral infection. The viruses have developed multiple strategies to induce the inflammatory responses, including regulation of IL-1β production. However, the molecular mechanism underlying the induction of IL-1β by FMDV remains not fully understood. Here, we found that FMDV robustly induced IL-1β production in macrophages and pigs. Infection of Casp-1 inhibitor-treated cells and NOD-, LRR- and pyrin domain-containing 3 (NLRP3)-knockdown cells indicated that NLRP3 is essential for FMDV-induced IL-1β secretion. More importantly, we found that FMDV L^pro associates with the NACHT and LRR domains of NLRP3 to promote NLRP3 inflammasome assembly and IL-1β secretion. Moreover, FMDV L^pro induces calcium influx and potassium efflux, which trigger NLRP3 activation. Our data revealed the mechanism underlying the activation of the NLRP3 inflammasome after FMDV L^pro expression, thus providing insights for the control of FMDV infection-induced inflammation.     

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.     

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.     

Absent in melanoma 2 is required for innate immune recognition of Francisella tularensis

Macrophages respond to cytosolic nucleic acids by activating cysteine protease caspase-1 within a complex called the inflammasome. Subsequent cleavage and secretion of proinflammatory cytokines IL-1β and IL-18 are critical for innate immunity. Here, we show that macrophages from mice lacking absent in melanoma 2 (AIM2) cannot sense cytosolic double-stranded DNA and fail to trigger inflammasome assembly. Caspase-1 activation in response to intracellular pathogen Francisella tularensis also required AIM2. Immunofluorescence microscopy of macrophages infected with F. tularensis revealed striking colocalization of bacterial DNA with endogenous AIM2 and inflammasome adaptor ASC. By contrast, type I IFN (IFN-α and -β) secretion in response to F. tularensis did not require AIM2. IFN-I did, however, boost AIM2-dependent caspase-1 activation by increasing AIM2 protein levels. Thus, inflammasome activation was reduced in infected macrophages lacking either the IFN-I receptor or stimulator of interferon genes (STING). Finally, AIM2-deficient mice displayed increased susceptibility to F. tularensis infection compared with wild-type mice. Their increased bacterial burden in vivo confirmed that AIM2 is essential for an effective innate immune response.     

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.     

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.     

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.     

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.     

β-Catenin in dendritic cells exerts opposite functions in cross-priming and maintenance of CD8+ T cells through regulation of IL-10

Recent studies have demonstrated that β-catenin in DCs serves as a key mediator in promoting both CD4⁺ and CD8⁺ T-cell tolerance, although how β-catenin exerts its functions remains incompletely understood. Here we report that activation of β-catenin in DCs inhibits cross-priming of CD8⁺ T cells by up-regulating mTOR-dependent IL-10, suggesting blocking β-catenin/mTOR/IL-10 signaling as a viable approach to augment CD8⁺ T-cell immunity. However, vaccination of DC–β-catenin^−/− (CD11c-specific deletion of β-catenin) mice surprisingly failed to protect them against tumor challenge. Further studies revealed that DC–β-catenin^−/− mice were deficient in generating CD8⁺ T-cell immunity despite normal clonal expansion, likely due to impaired IL-10 production by β-catenin^−/− DCs. Deletion of β-catenin in DCs or blocking IL-10 after clonal expansion similarly led to reduced CD8⁺ T cells, suggesting that β-catenin in DCs plays a positive role in CD8⁺ T-cell maintenance postclonal expansion through IL-10. Thus, our study has not only identified mTOR/IL-10 as a previously unidentified mechanism for β-catenin–dependent inhibition of cross-priming, but also uncovered an unexpected positive role that β-catenin plays in maintenance of CD8⁺ T cells. Despite β-catenin’s opposite functions in regulating CD8⁺ T-cell responses, selectively blocking β-catenin with a pharmacological inhibitor during priming phase augmented DC vaccine-induced CD8⁺ T-cell immunity and improved antitumor efficacy, suggesting manipulating β-catenin signaling as a feasible therapeutic strategy to improve DC vaccine efficacy.As the initiators of antigen-specific immune responses, dendritic cells (DCs) play a central role in regulating both T-cell immunity and tolerance (1). β-Catenin, a major component in Wnt signaling pathway, has emerged as a key factor in DC differentiation and function (2). Previous studies have shown that β-catenin regulates DC-mediated CD4⁺ T-cell responses and promotes CD4⁺ T-cell tolerance in murine models of autoimmune diseases (3, 4). Consistently, activation of β-catenin in DCs has recently been shown to suppress CD8⁺ T-cell immunity in a DC-targeted vaccine model (5), suggesting that β-catenin in DCs might similarly serve as a tolerizing signal that shifts the balance between CD8⁺ T-cell immunity and tolerance. Although the underlying mechanisms of how β-catenin mediates CD8⁺ T-cell tolerance remain largely unclear, we have shown that activation of β-catenin in DCs genetically or induced by tumors suppresses CD8⁺ T-cell immunity by inhibiting cross-priming (5). Exploiting their ability to potentiate host effector and memory CD8⁺ T-cell responses, DC vaccines have emerged as a leading strategy for cancer immunotherapy (6). However, one major obstacle for their success is host DC-mediated immunosuppression (7–9). Given that cross-priming plays a major role in generating antitumor CD8⁺ T-cell immunity (7, 10), activation of β-catenin in DCs might be a key mechanism for tumors to achieve immunosuppression. Thus, manipulating β-catenin function in cross-priming might be a viable approach to overcome DC-mediated immunosuppression and improve DC vaccine efficacy. However, The underlying mechanisms of how β-catenin in DCs achieves immunosuppression, in particular how β-catenin negatively regulates cross-priming to suppress CD8⁺ T-cell immunity, remain poorly understood.Although the mechanisms for DC-mediated priming of antitumor CD8⁺ T cells through cross-presentation remain incompletely understood, DC subsets, DC maturation status and cytokines have been shown to possibly affect their capacity in cross-priming (7, 10, 11). Although the role of cytokines in cross-priming has not been directly tested, cytokines as “signal 3” have been shown in principal to play a critical role in priming and effector differentiation of antitumor CD8⁺ T cells (12). β-Catenin in DCs has been shown to play a critical role in regulating cytokine induction (3, 4), thus suggesting that β-catenin might regulate DC cytokine production to achieve its effects on cross-priming.In this report we have identified mTOR/IL-10 signaling as a mechanism for β-catenin–dependent inhibition of cross-priming. Activation of β-catenin in DCs inhibited cross-priming of CD8⁺ T cells by up-regulating mTOR-dependent IL-10, and blocking mTOR or IL-10 led to restored cross-priming by β-catenin^active DCs. Surprisingly, mice with DC-specific deletion of β-catenin (DC–β-catenin^−/− mice) exhibited reduced antitumor immunity upon vaccination, despite the fact that deletion of β-catenin in DCs abrogated tumor-induced inhibition of cross-priming. Further studies showed that DC–β-catenin^−/− mice were deficient in generating CD8⁺ T-cell immunity despite normal clonal expansion, and β-catenin in DCs was required to maintain primed CD8⁺ T cells postclonal expansion. Thus, β-catenin in DCs exerts negative and positive functions in cross-priming and maintenance of CD8⁺ T cells, respectively. Importantly, we have demonstrated blocking β-catenin selectively at priming phase as a feasible strategy to improve DC vaccine efficacy.     

Promoting tolerance to proteolipid protein-induced experimental autoimmune encephalomyelitis through targeting dendritic cells

In T cell-mediated autoimmune diseases, self-reactive T cells with known antigen specificity appear to be particularly promising targets for antigen-specific induction of tolerance without compromising desired protective host immune responses. Several lines of evidence suggest that delivery of antigens to antigen-presenting dendritic cells (DCs) in the steady state (i.e., to immature DCs) may represent a suitable approach to induce antigen-specific T-cell tolerance peripherally. Here, we report that anti-DEC205–mediated delivery of the self-peptide proteolipid protein (PLP)139–151 to DCs ameliorated clinical symptoms in the PLP-induced SJL model of experimental autoimmune encephalomyelitis. Splenocytes from treated mice were anergized to PLP139–151, and IL-17 secretion was markedly reduced. Moreover, we show directly, using transgenic CD4⁺ Vβ6⁺ TCR T cells specific for PLP139–151, that, under the conditions of the present experiments, these cells also became anergic. In addition, evidence for a CD4⁺ T cell-mediated suppressor mechanism was obtained.     

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.     

Cell stress increases ATP release in NLRP3 inflammasome-mediated autoinflammatory diseases,resulting in cytokine imbalance

Cell stress is implicated in triggering bouts of systemic inflammation in patients with autoinflammatory disorders. Blood monocytes from patients affected by NLRP3-mediated cryopyrin-associated periodic syndromes (CAPS) release greater amounts of IL-1β than monocytes from unaffected subjects. Here we show that stress lowers the threshold of activation; blood monocytes from CAPS patients maintain the high levels of secreted IL-1β (fivefold) and IL-18 (10-fold) when stimulated with 1,000-fold less LPS than that required for full IL-1β secretion in control subjects. Unexpectedly, IL-1α secretion is increased 10-fold, indicating that inflammatory episodes in CAPS may not be entirely a result of IL-1β but may also involve IL-1α. In CAPS monocytes, LPS induces the externalization of copious amounts of ATP (10-fold), which drive IL-1β, IL-18, and IL-1α release via activation of the P2X purinoceptor 7. This enhanced ATP release appears to be the link between cell stress and increased cytokine secretion in CAPS. In the later phase after LPS stimulation, CAPS monocytes undergo oxidative stress, which impairs production of the anti-inflammatory IL-1 receptor antagonist (IL-1Ra). Remarkably, IL-1Ra secretion is fully restored by treatment with antioxidants. In two patients with the same NLRP3 mutation, but different disease severity, monocytes from the mildly affected patient exhibited more efficient redox response, lower ATP secretion, and more balanced cytokine production. Thus, the robustness of the individual antioxidant response increases the tolerance to stress and reduces the negative effect of the disease. Pharmacologic block of P2X purinoceptor 7 and improved stress tolerance may represent novel treatment strategies in stress-associated inflammatory diseases.Cryopyrin-associated periodic syndromes (CAPS) are autoinflammatory diseases linked to mutations in the gene NLRP3; the disease is characterized by recurrent episodes of fever and systemic inflammation (1). The pathophysiology of CAPS is mainly caused by the dysregulated secretion of IL-1β, which has been validated by dramatic therapeutic responses to the blocking of the IL-1 receptor with anakinra or the neutralization of IL-1β with canakinumab (2, 3). The NLRP3 inflammasome is a multiprotein complex that requires activating signals to assemble and generate active caspase-1, which in turn converts the inactive IL-1β and IL-18 precursors into their mature active forms (4). Extracellular ATP is a common inflammasome-activating event (5). ATP is released during inflammation by activated platelets, dying leukocytes, and injured parenchymal cells and binds to P2X purinoceptor 7 (P2X7R) on inflammatory cells, triggering a series of intracellular processes, only partially understood, that nevertheless lead to inflammasome activation (6). Human monocytes from healthy subjects stimulated by Toll-like receptor (TLR) agonists secrete their endogenous ATP, which autocrinally activates P2X7R (7, 8), thereby partially liberating monocytes from requiring exogenous ATP. ATP secretion requires reactive oxygen species (ROS) production (9), which is induced in inflammatory cells by triggering TLR (10–12).Although in some cell types, including mouse macrophages or human myelomonocytic cell lines, ROS inhibitors such as diphenyleneiodonium [DPI (13)] impair pro-IL-1β synthesis and NLRP3 priming (14, 15) in primary human monocytes, pro-IL-1β biosynthesis and intracellular accumulation are unaffected by treatment with DPI (12, 15), indicating that ROS inhibition in monocytes hinders IL-1β secretion by acting at the posttranslational level.Primary monocytes from patients affected by autoinflammatory diseases are under stress (16–20). In CAPS patients, blood monocytes display levels of both ROS and antioxidants considerably higher than in healthy subjects (16); this basal stress causes an aberrant redox response to TLR stimulation, with further increase of ROS followed by collapse of intracellular antioxidant systems, resulting in net oxidative stress. A functional consequence of oxidative stress is the impaired production of cytokines downstream of IL-1, such as IL-1 receptor antagonist (IL-1Ra) and IL-6 (18).Here, we identify a series of events induced by stress in monocytes from CAPS patients that contribute to the pathogenesis of the disease. First, we provide evidence that increased ATP secretion secondary to LPS-induced ROS is significantly contributing to the increased secretion of IL-1β and IL-18 and, unexpectedly, IL-1α. We also show that stress at baseline lowers the threshold of activation, with secretion of these three cytokines induced by markedly lower concentrations of LPS, concentrations unable to activate secretion in monocytes from healthy subjects. Moreover, we demonstrate that the presence of antioxidants restores the secretion of the anti-inflammatory cytokine IL-1Ra, which is inhibited by oxidative stress. In addition, comparative studies in two patients from the same family, with the same NLRP3 mutation, suggest that the basal degree of stress and the efficiency of redox response of the individual patients influence the severity of the disease phenotype.     

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.     

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.     

From the Cover: Mutant superoxide dismutase 1-induced IL-1β accelerates ALS pathogenesis

ALS is a fatal motor neuron disease of adult onset. Neuroinflammation contributes to ALS disease progression; however, the inflammatory trigger remains unclear. We report that ALS–linked mutant superoxide dismutase 1 (SOD1) activates caspase-1 and IL-1β in microglia. Cytoplasmic accumulation of mutant SOD1 was sensed by an ASC containing inflammasome and antagonized by autophagy, limiting caspase-1–mediated inflammation. Notably, mutant SOD1 induced IL-1β correlated with amyloid-like misfolding and was independent of dismutase activity. Deficiency in caspase-1 or IL-1β or treatment with recombinant IL-1 receptor antagonist (IL-1RA) extended the lifespan of G93A-SOD1 transgenic mice and attenuated inflammatory pathology. These findings identify microglial IL-1β as a causative event of neuroinflammation and suggest IL-1 as a potential therapeutic target in ALS.     

Peroxisome proliferator-activated receptor-delta agonist ameliorated inflammasome activation in nonalcoholic fatty liver disease

AIM: To evaluate the inflammasome activation and the effect of peroxisome proliferator-activated receptors (PPAR)-δ agonist treatment in nonalcoholic fatty liver disease (NAFLD) models.METHODS: Male C57BL/6J mice were classified according to control or high fat diet (HFD) with or without PPAR-δ agonist (GW) over period of 12 wk [control, HFD, HFD + lipopolysaccharide (LPS), HFD + LPS + GW group]. HepG2 cells were exposed to palmitic acid (PA) and/or LPS in the absence or presence of GW.RESULTS: HFD caused glucose intolerance and hepatic steatosis. In mice fed an HFD with LPS, caspase-1 and interleukin (IL)-1β in the liver were significantly increased. Treatment with GW ameliorated the steatosis and inhibited overexpression of pro-inflammatory cytokines. In HepG2 cells, PA and LPS treatment markedly increased mRNA of several nucleotide-binding and oligomerization domain-like receptor family members (NLRP3, NLRP6, and NLRP10), caspase-1 and IL-1β. PA and LPS also exaggerated reactive oxygen species production. All of the above effects of PA and LPS were reduced by GW. GW also enhanced the phosphorylation of AMPK-α.CONCLUSION: PPAR-δ agonist reduces fatty acid-induced inflammation and steatosis by suppressing inflammasome activation. Targeting the inflammasome by the PPAR-δ agonist may have therapeutic implication for NAFLD.