Echovirus 11 infection induces pyroptotic cell death by facilitating NLRP3 inflammasome activation

Echovirus 11 (ECHO 11) is a positive-strand RNA virus belonging to the genus Enterovirus of the family Picornaviridae. ECHO 11 infections can cause severe inflammatory illnesses in neonates, including severe acute hepatitis with coagulopathy. The activation of NLRP3 inflammasome is important for host defense against invading viruses, which also contributes to viral pathogenicity. However, whether and how ECHO 11 induces NLRP3 inflammasome activation remains unclear. In this study, we isolated a clinical strain of ECHO 11 from stools of an ECHO 11-infected newborn patient with necrotizing hepatitis. This virus shared 99.95% sequence identity with the previously published ECHO 11 sequence. The clinically isolated ECHO 11 can efficiently infect liver cells and strongly induces inflammation. Moreover, we showed that ECHO 11 induced IL-1β secretion and pyroptosis in cells and mouse bone marrow-derived macrophages (BMDMs). Furthermore, ECHO 11 infection triggered NLRP3 inflammasome activation, as evidenced by cleavages of GSDMD, pro-IL-1β and pro-caspase-1, and the release of LDH. ECHO 11 2B protein was required for NLRP3 inflammasome activation via interacting with NLRP3 to facilitate the inflammasome complex assembly. In vivo, expression of ECHO 11 2B also activated NLRP3 inflammasome in the murine liver. Besides, 2Bs of multiple EVs can also interact with NLRP3 and induce NLRP3 inflammasome activation. Together, our findings demonstrate a mechanism by which ECHO 11 induces inflammatory responses by activating NLRP3 inflammasome, providing novel insights into the pathogenesis of ECHO 11 infection.     

Mycobacterium tuberculosis protein ESAT‐6 is a potent activator of the NLRP3/ASC inflammasome

Interleukin‐1β (IL‐1β) represents one of the most important mediators of inflammation and host responses to infection. Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, induces IL‐1β secretion at the site of infection, but the underlying mechanism(s) are poorly understood. In this work we show that Mtb infection of macrophages stimulates caspase‐1 activity and promotes the secretion of IL‐1β. This stimulation requires live intracellular bacteria expressing a functional ESX‐1 secretion system. ESAT‐6, an ESX‐1 substrate implicated in membrane damage, is both necessary and sufficient for caspase‐1 activation and IL‐1β secretion. ESAT‐6 promotes the access of other immunostimulatory agents such as AG85 into the macrophage cytosol, indicating that this protein may contribute to caspase‐1 activation largely by perturbing host cell membranes. Using a high‐throughput shRNA‐based screen we found that numerous NOD‐like receptors (NLRs) and CARD domain‐containing proteins (CARDs) were important for IL‐1β secretion upon Mtb infection. Most importantly, NLRP3, ASC and caspase‐1 form an infection‐inducible inflammasome complex that is essential for IL‐1β secretion. In summary, we show that recognition of Mtb infection by the NLRP3 inflammasome requires the activity of the bacterial virulence factor ESAT‐6, and the subsequent IL‐1β response is regulated by a number of NLR/CARD proteins.     

Critical role for NLRP3 in necrotic death triggered by Mycobacterium tuberculosis

Induction of necrotic death in macrophages is a primary virulence determinant of Mycobacterium tuberculosis. The ESX-1 secretion system and its substrate ESAT-6 are required for M. tuberculosis to induce necrosis, but host factors that mediate the ESAT-6-promoted necrosis remain unknown. Here we report that ESAT-6-promoted necrotic death in THP-1 human macrophages is dependent on the NLRP3 inflammasome, as shown by RNA interference and pharmacological inhibitions. Phagosomes containing ESAT-6-expressing M. tuberculosis recruit markers previously associated with damaged phagosomal membrane, such as galectin-3 and ubiquitinated protein aggregates. In addition, ESAT-6 promoted lysosomal permeabilization by M. tuberculosis. ESAT-6 mutants defective for ubiquitination were unable to trigger NLRP3 activation and necrotic death. Furthermore, Syk tyrosine kinase, recently implicated in NLRP3 activation during fungal and malarial infections, was necessary for mediating the ESAT-6-promoted necrosis and NLRP3 activation. Our results thus link phagosomal damage and Syk activity to NLRP3-mediated necrotic death triggered by M. tuberculosis ESAT-6 during infection.     

Hypoxia-induced cell death of HepG2 cells involves a necrotic cell death mediated by calpain

To elucidate mechanism of cell death in response to hypoxia, we attempted to compare hypoxia-induced cell death of HepG2 cells with cisplatin-induced cell death, which has been well characterized as a typical apoptosis. Cell death induced by hypoxia turned out to be different from cisplatin-mediated apoptosis in cell viability and cleavage patterns of caspases. Hypoxia-induced cell death was not associated with the activation of p53 while cisplatin-induced apoptosis is p53 dependent. In order to explain these differences, we tested involvement of μ-calpain and m-calpain in hypoxia-induced cell death. Calpains, especially μ-calpain, were initially cleaved by hypoxia, but not by cisplatin. Interestingly, the treatment of a calpain inhibitor restored PARP cleavage that was absent during hypoxia, indicating the recovery of activated caspase-3. The inhibition of calpains prevented proteolysis induced by hypoxia. In addition, hypoxia resulted in a necrosis-like morphology while cisplatin induced an apoptotic morphology. The calpain inhibitor prevented necrotic morphology induced by hypoxia and converted partially to apoptotic morphology with nuclear segmentation. Our result suggests that calpains are involved in hypoxia-induced cell death that is likely to be necrotic in nature and the inhibition of calpain switches hypoxia-induced cell death to apoptotic cell death without affecting cell viability.     

Gypenosides improve diabetic cardiomyopathy by inhibiting ROS‐mediated NLRP3 inflammasome activation

NLRP3 inflammasome activation plays an important role in diabetic cardiomyopathy (DCM), which may relate to excessive production of reactive oxygen species (ROS). Gypenosides (Gps), the major ingredients of Gynostemma pentaphylla (Thunb.) Makino, have exerted the properties of anti‐hyperglycaemia and anti‐inflammation, but whether Gps improve myocardial damage and the mechanism remains unclear. Here, we found that high glucose (HG) induced myocardial damage by activating the NLRP3 inflammasome and then promoting IL‐1β and IL‐18 secretion in H9C2 cells and NRVMs. Meanwhile, HG elevated the production of ROS, which was vital to NLRP3 inflammasome activation. Moreover, the ROS activated the NLRP3 inflammasome mainly by cytochrome c influx into the cytoplasm and binding to NLRP3. Inhibition of ROS and cytochrome c dramatically down‐regulated NLRP3 inflammasome activation and improved the cardiomyocyte damage induced by HG, which was also detected in cells treated by Gps. Furthermore, Gps also reduced the levels of the C‐reactive proteins (CRPs), IL‐1β and IL‐18, inhibited NLRP3 inflammasome activation and consequently improved myocardial damage in vivo. These findings provide a mechanism that ROS induced by HG activates the NLRP3 inflammasome by cytochrome c binding to NLRP3 and that Gps may be potential and effective drugs for DCM via the inhibition of ROS‐mediated NLRP3 inflammasome activation.     

Cutting edge: inflammasome activation by alum and alum's adjuvant effect are mediated by NLRP3

Alum is the only adjuvant approved for routine use in humans, although the basis for its adjuvanticity remains poorly understood. We have recently shown that alum activates caspase-1 and induces secretion of mature IL-1beta and IL-18. In this study we show that, in human and mouse macrophages, alum-induced secretion of IL-1beta, IL-18, and IL-33 is mediated by the NLR (nucleotide-binding domain leucine-rich repeat-containing) protein NLRP3 and its adaptor ASC, but not by NLRC4. Other particulate adjuvants, such as QuilA and chitosan, induce inflammasome activation in a NLRP3-dependent fashion, suggesting that activation of the NLRP3-inflammasome may be a common mechanism of action of particulate adjuvants. Importantly, we demonstrate that Ag-specific Ab production elicited by vaccines that contain alum is significantly impaired in NLRP3-deficient mice. Our results demonstrate for the first time a role for the NLRP3-inflammasome during development of the immune response elicited by alum-enhanced vaccination and suggest that therapeutic intervention aimed at NLRP3 may improve adjuvant efficacy.     

Identification of bacterial muramyl dipeptide as activator of the NALP3/cryopyrin inflammasome

Activation of caspase-1 and subsequent processing and secretion of the pro-inflammatory cytokine IL-1beta is triggered upon assembly of the inflammasome complex. It is generally believed that bacterial lipopolysaccharides (LPS) are activators of the inflammasome through stimulation of Toll-like receptor 4 (TLR4). Like TLRs, NALP3/Cryopyrin, which is a key component of the inflammasome, contains Leucine-Rich-Repeats (LRRs). LRRs are frequently used to sense bacterial components, thus raising the possibility that bacteria directly activate the inflammasome. Here, we show that bacterial peptidoglycans (PGN), but surprisingly not LPS, induce NALP3-mediated activation of caspase-1 and maturation of proIL-1beta. Activation is independent of TLRs because the PGN degradation product muramyl dipeptide (MDP), which is not sensed by TLRs, is the minimal-activating structure. Macrophages from a patient with Muckle-Wells syndrome, an autoinflammatory disease associated with mutations in the NALP3/Cryopyrin gene, show increased IL-1beta secretion in the presence of MDP. The activation of the NALP3-inflammasome by MDP may be the basis of the potent adjuvant activity of MDP.     

Prion Pathogenesis in the Absence of NLRP3/ASC Inflammasomes

The accumulation of the scrapie prion protein PrP^Sc, a misfolded conformer of the cellular prion protein PrP^C, is a crucial feature of prion diseases. In the central nervous system, this process is accompanied by conspicuous microglia activation. The NLRP3 inflammasome is a multi-molecular complex which can sense heterogeneous pathogen-associated molecular patterns and culminates in the activation of caspase 1 and release of IL 1β. The NLRP3 inflammasome was reported to be essential for IL 1β release after in vitro exposure to the amyloidogenic peptide PrP^106-126 and to recombinant PrP fibrils. We therefore studied the role of the NLRP3 inflammasome in a mouse model of prion infection. Upon intracerebral inoculation with scrapie prions (strain RML), mice lacking NLRP3 (Nlrp3^-/-) or the inflammasome adaptor protein ASC (Pycard^-/-) succumbed to scrapie with attack rates and incubation times similar to wild-type mice, and developed the classic histologic and biochemical features of prion diseases. Genetic ablation of NLRP3 or ASC did not significantly impact on brain levels of IL 1β at the terminal stage of disease. Our results exclude a significant role for NLRP3 and ASC in prion pathogenesis and invalidate their claimed potential as therapeutic target against prion diseases.     

Distinct cathepsins control necrotic cell death mediated by pyroptosis inducers and lysosome-destabilizing agents

Necrotic cell death triggers a range of biological responses including a strong adaptive immune response, yet we know little about the cellular pathways that control necrotic cell death. Inhibitor studies suggest that proteases, and in particular cathepsins, drive necrotic cell death. The cathepsin B-selective inhibitor CA-074-Me blocks all forms of programmed necrosis by an unknown mechanism. We found that cathepsin B deficiency does not prevent induction of pyroptosis and lysosome-mediated necrosis suggesting that CA-074-Me blocks necrotic cell death by targeting cathepsins other than cathepsin B. A single cathepsin, cathepsin C, drives necrotic cell death mediated by the lysosome-destabilizing agent Leu-Leu-OMe (LLOMe). Here we present evidence that cathepsin C-deficiency and CA-074-Me block LLOMe killing in a distinct and cell type-specific fashion. Cathepsin C-deficiency and CA-074-Me block LLOMe killing of all myeloid cells, except for neutrophils. Cathepsin C-deficiency, but not CA-074-Me, blocks LLOMe killing of neutrophils suggesting that CA-074-Me does not target cathepsin C directly, consistent with inhibitor studies using recombinant cathepsin C. Unlike other cathepsins, cathepsin C lacks endoproteolytic activity, and requires activation by other lysosomal proteases, such as cathepsin D. Consistent with this theory, we found that lysosomotropic agents and cathepsin D downregulation by siRNA block LLOMe-mediated necrosis. Our findings indicate that a proteolytic cascade, involving cathepsins C and D, controls LLOMe-mediated necrosis. In contrast, cathepsins C and D were not required for pyroptotic cell death suggesting that distinct cathepsins control pyroptosis and lysosome-mediated necrosis.     

AIM2 and NLRP3 inflammasomes activate both apoptotic and pyroptotic death pathways via ASC

Inflammasomes are protein complexes assembled upon recognition of infection or cell damage signals, and serve as platforms for clustering and activation of procaspase-1. Oligomerisation of initiating proteins such as AIM2 (absent in melanoma-2) and NLRP3 (NOD-like receptor family, pyrin domain-containing-3) recruits procaspase-1 via the inflammasome adapter molecule ASC (apoptosis-associated speck-like protein containing a CARD). Active caspase-1 is responsible for rapid lytic cell death termed pyroptosis. Here we show that AIM2 and NLRP3 inflammasomes activate caspase-8 and -1, leading to both apoptotic and pyroptotic cell death. The AIM2 inflammasome is activated by cytosolic DNA. The balance between pyroptosis and apoptosis depended upon the amount of DNA, with apoptosis seen at lower transfected DNA concentrations. Pyroptosis had a higher threshold for activation, and dominated at high DNA concentrations because it happens more rapidly. Gene knockdown showed caspase-8 to be the apical caspase in the AIM2- and NLRP3-dependent apoptotic pathways, with little or no requirement for caspase-9. Procaspase-8 localised to ASC inflammasome ‘specks'' in cells, and bound directly to the pyrin domain of ASC. Thus caspase-8 is an integral part of the inflammasome, and this extends the relevance of the inflammasome to cell types that do not express caspase-1.     

Mycobacterium abscessus activates the NLRP3 inflammasome via Dectin-1-Syk and p62/SQSTM1

Numerous atypical mycobacteria, including Mycobacterium abscessus (Mabc), cause nontuberculous mycobacterial infections, which present a serious public health threat. Inflammasome activation is involved in host defense and the pathogenesis of autoimmune diseases. However, inflammasome activation has not been widely characterized in human macrophages infected with atypical mycobacteria. Here, we demonstrate that Mabc robustly activates the nucleotide binding and oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome via dectin-1/Syk-dependent signaling and the cytoplasmic scaffold protein p62/SQSTM1 (p62) in human macrophages. Both dectin-1 and Toll-like receptor 2 (TLR2) were required for Mabc-induced mRNA expression of pro-interleukin (IL)-1β, cathelicidin human cationic antimicrobial protein-18/LL-37 and β-defensin 4 (DEFB4). Dectin-1-dependent Syk signaling, but not that of MyD88, led to the activation of caspase-1 and secretion of IL-1β through the activation of an NLRP3/apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) inflammasome. Additionally, potassium efflux was required for Mabc-induced NLRP3/ASC inflammasome activation. Furthermore, Mabc-induced p62 expression was critically involved in NLRP3 inflammasome activation in human macrophages. Finally, NLRP3/ASC was critical for the inflammasome in antimicrobial responses to Mabc infection. Taken together, these data demonstrate the induction mechanism of the NLRP3/ASC inflammasome and its role in innate immunity to Mabc infection.     

Rutin modulates ASC expression in NLRP3 inflammasome: a study in alcohol and cerulein-induced rat model of pancreatitis

Inflammasomes are protein complexes formed in response to tissue injury and inflammation to regulate the formation of proinflammatory cytokines. Nod-like receptor pyrin domain containing 3 (NLRP3) is one such inflammasome involved in pancreatic inflammation. Caspase activation recruitment domain (CARD) is an interaction motif found in all the major components of NLRP3 inflammasome such as apoptosis associated speck-like CARD containing protein (ASC) and procaspase-1. NLRP3 activates procaspase-1 with the concerted action of CARD domain of ASC. In the present study, the effect of rutin, a natural flavonoid on the expression of ASC of NLRP3, was investigated in rats treated with ethanol (EtOH) and cerulein (Cer). Male albino Wistar rats were divided into four groups. Groups 1 and 2 rats were fed normal diet, whereas groups 3 and 4 rats were fed EtOH (36 % of total calories) containing diet for a total period of 5 weeks and also administered Cer (20 µg/kg body weight i.p.) thrice weekly for the last 3 weeks. In addition, groups 2 and 4 rats received daily 100 mg/kg body weight of rutin from third week. Rutin co-administration significantly decreased the level of pancreatic marker enzymes, oxidative stress markers, inflammatory markers, mRNA expression of caspase-1, cytokines, ASC–NLRP3, and protein expression of caspase-1 and ASC in rats received EtOH–Cer. The results of the study revealed that rutin can reduce inflammation in pancreas probably by influencing the down regulation of ASC–NLRP3 which might result in the reduced activation of caspase-1 and controlled cytokine production.     

Necroptosis signaling and NLRP3 inflammasome cross-talking in epithelium facilitate Pseudomonas aeruginosa mediated lung injury

Pseudomonas aeruginosa induced acute lung injury is such a serious risk to public health, but the pathological regulation remains unclear. Here, we reported that PA mediated epithelial necroptosis plays an important role in pathological process. Pharmacological and genomic ablation of necroptosis signaling ameliorate PA mediated ALI and pulmonary inflammation. Our results further proved NLRP3 inflammasome to involve in the process. Mechanism investigation revealed the cross-talking between inflammasome activation and necroptosis that MLKL-dependent necroptosis signaling promotes the change of mitochondrial membrane potential for the release of reactive oxygen species (ROS), which is the important trigger for functional inflammasome activation. Furthermore, antioxidants such as Mito-TEMPO was confirmed to significantly restrain inflammasome activation in epithelium, resulting in a reduction in PA induced pulmonary inflammation. Taken together, our findings revealed that necroptosis-triggered NLRP3 inflammasome in epithelium plays a crucial role in PA mediated injury, which could be a potential therapeutic target for pulmonary inflammation.     

Human pathogenic fungus Trichophytonschoenleinii activates the NLRP3 inflammasome

The fungus Trichophyton schoenleinii (T. schoenleinii) is the causative agent of Trichophytosis and Tinea favosa of the scalp in certain regions of Eurasia and Africa. Human innate immune system plays an important role in combating with various pathogens including fungi. The inflammasome is one of the most critical arms of host innate immunity, which is a protein complex controlling maturation of IL-1β. To clarify whether T. schoenleinii is able to activate the inflammasome, we analyzed human monocytic cell line THP-1 for IL-1β production upon infection with T. schoenleinii strain isolated from Tinea favosa patients, and rapid IL-1β secretion from THP-1 cells was observed. Moreover, applying competitive inhibitors and gene specific silencing with shRNA, we found that T. schoenleinii induced IL-1β secretion, ASC pyroptosome formation as well as caspase-1 activation were all dependent on NLRP3. Cathepsin B activity, ROS production and K⁺ efflux were required for the inflammasome activation by T. schoenleinii. Our data thus reveal that the NLRP3 inflammasome plays an important role in host defense against T. schoenleinii, and suggest that manipulating NLRP3 signaling can be a novel approach for control of diseases caused by T. schoenleinii infection.