Caspase‐1 inflammasome activity in patients with Staphylococcus aureus bacteremia

The inflammasome is a multiprotein complex that mediates caspase‐1 activation with subsequent maturation of the proinflammatory cytokines IL‐1β and IL‐18. The NLRP3 inflammasome is known to be activated by Staphylococcus aureus, one of the leading causes of bacteremia worldwide. Inflammasome activation and regulation in response to bacterial infection have been found to be of importance for a balanced host immune response. However, inflammasome signaling in vivo in humans initiated by S. aureus is currently sparsely studied. This study therefore aimed to investigate NLRP3 inflammasome activity in 20 patients with S. aureus bacteremia (SAB), by repeated measurement during the first week of bacteremia, compared with controls. Caspase‐1 activity was measured in monocytes and neutrophils by flow cytometry detecting FLICA (fluorescent‐labeled inhibitor of caspase‐1), while IL‐1β and IL‐18 was measured by Luminex and ELISA, respectively. As a measure of inflammasome priming, messenger RNA (mRNA) expression of NLRP3, CASP1 (procaspase‐1), and IL1B (pro‐IL‐1β) was analyzed by quantitative PCR. We found induced caspase‐1 activity in innate immune cells with subsequent release of IL‐18 in patients during the acute phase of bacteremia, indicating activation of the inflammasome. There was substantial interindividual variation in caspase‐1 activity between patients with SAB. We also found an altered inflammasome priming with low mRNA levels of NLRP3 accompanied by elevated mRNA levels of IL1B. This increased knowledge of the individual host immune response in SAB could provide support in the effort to optimize management and treatment of each individual patient.     

ATP‐dependent activation of an inflammasome in primary gingival epithelial cells infected by Porphyromonas gingivalis

Production of IL‐1β typically requires two‐separate signals. The first signal, from a pathogen‐associated molecular pattern, promotes intracellular production of immature cytokine. The second signal, derived from a danger signal such as extracellular ATP, results in assembly of an inflammasome, activation of caspase‐1 and secretion of mature cytokine. The inflammasome component, Nalp3, plays a non‐redundant role in caspase‐1 activation in response to ATP binding to P2X₇ in macrophages. Gingival epithelial cells (GECs) are an important component of the innate‐immune response to periodontal bacteria. We had shown that GECs express a functional P2X₇ receptor, but the ability of GECs to secrete IL‐1β during infection remained unknown. We find that GECs express a functional Nalp3 inflammasome. Treatment of GECs with LPS or infection with the periodontal pathogen, Porphyromonas gingivalis, induced expression of the il‐1β gene and intracellular accumulation of IL‐1β protein. However, IL‐1β was not secreted unless LPS‐treated or infected cells were subsequently stimulated with ATP. Conversely, caspase‐1 is activated in GECs following ATP treatment but not P. gingivalis infection. Furthermore, depletion of Nalp3 by siRNA abrogated the ability of ATP to induce IL‐1β secretion in infected cells. The Nalp3 inflammasome is therefore likely to be an important mediator of the inflammatory response in gingival epithelium.     

Fluorofenidone attenuates pulmonary inflammation and fibrosis via inhibiting the activation of NALP3 inflammasome and IL‐1β/IL‐1R1/MyD88/NF‐κB pathway

Interleukin (IL)‐1β plays an important role in the pathogenesis of idiopathic pulmonary fibrosis. The production of IL‐1β is dependent upon caspase‐1‐containing multiprotein complexes called inflammasomes and IL‐1R1/MyD88/NF‐κB pathway. In this study, we explored whether a potential anti‐fibrotic agent fluorofenidone (FD) exerts its anti‐inflammatory and anti‐fibrotic effects through suppressing activation of NACHT, LRR and PYD domains‐containing protein 3 (NALP3) inflammasome and the IL‐1β/IL‐1R1/MyD88/NF‐κB pathway in vivo and in vitro. Male C57BL/6J mice were intratracheally injected with Bleomycin (BLM) or saline. Fluorofenidone was administered throughout the course of the experiment. Lung tissue sections were stained with haemotoxylin and eosin and Masson's trichrome. Cytokines were measured by ELISA, and α‐smooth muscle actin (α‐SMA), fibronectin, collagen I, caspase‐1, IL‐1R1, MyD88 were measured by Western blot and/or RT‐PCR. The human actue monocytic leukaemia cell line (THP‐1) were incubated with monosodium urate (MSU), with or without FD pre‐treatment. The expression of caspase‐1, IL‐1β, NALP3, apoptosis‐associated speck‐like protein containing (ASC) and pro‐caspase‐1 were measured by Western blot, the reactive oxygen species (ROS) generation was detected using the Flow Cytometry, and the interaction of NALP3 inflammasome‐associated molecules were measured by Co‐immunoprecipitation. RLE‐6TN (rat lung epithelial‐T‐antigen negative) cells were incubated with IL‐1β, with or without FD pre‐treatment. The expression of nuclear protein p65 was measured by Western blot. Results showed that FD markedly reduced the expressions of IL‐1β, IL‐6, monocyte chemotactic protein‐1 (MCP‐1), myeloperoxidase (MPO), α‐SMA, fibronectin, collagen I, caspase‐1, IL‐1R1 and MyD88 in mice lung tissues. And FD inhibited MSU‐induced the accumulation of ROS, blocked the interaction of NALP3 inflammasome‐associated molecules, decreased the level of caspase‐1 and IL‐1β in THP‐1 cells. Besides, FD inhibited IL‐1β‐induced the expression of nuclear protein p65. This study demonstrated that FD, attenuates BLM‐induced pulmonary inflammation and fibrosis in mice via inhibiting the activation of NALP3 inflammasome and the IL‐1β/IL‐1R1/MyD88/ NF‐κB pathway.     

Activation of CXCR7 promotes endothelial repair and reduces the carotid atherosclerotic lesions through inhibition of pyroptosis signaling pathways

Endothelial injuries, including cell pyroptosis, are ongoing inflammatory processes with key roles in atherosclerosis development. Our previous report showed that the chemokine CXCL12 and its receptor CXCR7 are associated with the proliferation and angiogenesis of endothelial cells. Nevertheless, the mechanism underlying these effects on atherosclerotic lesions, especially on endothelial dysfunction, remains unknown. Here, we demonstrated that CXCR7 was upregulated in human carotid atherosclerotic plaques, apolipoprotein E knockout (ApoE^?/?) mice fed with a high‐fat diet (HFD), and oxidized lipopolysaccharide‐treated (ox‐LDL) human umbilical vein endothelial cells (HUVECs). Further, the activation of CXCR7 reversed ox‐LDL‐induced HUVEC dysfunction, such as migration, tube formation, and cell pyroptosis; all of these protective effects were alleviated by inhibition of CXCR7. The NOD‐like receptor family pyrin domain‐containing 3 (NLRP3) inflammasomes were also elevated in human carotid atherosclerotic plaques, ApoE^?/? mice fed with HFD, and ox‐LDL‐injured HUVECs by regulation of caspase‐1 and interleukin (IL)‐1β expression. The activation of CXCR7 by TC14012 led to a decrease in atherosclerotic lesions in ApoE^?/? mice fed with HFD. TC14012 also inhibited the expression of the NLRP3 inflammasome signaling pathway in vivo. In conclusion, our study suggests that CXCR7 plays an important role in regulating NLRP3 inflammasome‐modulated pyroptosis in HUVECs, providing a potential novel therapy for atherosclerosis.     

Activation of the NLRP3 inflammasome in microglia: the role of ceramide

Inflammation within the CNS is a major component of many neurodegenerative diseases. A characteristic feature is the generation of microglia‐derived factors that play an essential role in the immune response. IL‐1β is a pro‐inflammatory cytokine released by activated microglia, able to exacerbate injury at elevated levels. In the presence of caspase‐1, pro‐IL‐1β is cleaved to the mature cytokine following NOD‐like receptor pyrin domain containing 3 (NLRP3) inflammasome activation. Growing evidence suggests that ceramide plays a critical role in NLRP3 inflammasome assembly, however, the relationship between ceramide and inflammasome activation in microglia remains unknown. Here, we investigated potential mechanistic links between ceramide as a modulator of NLRP3 inflammasome assembly and the resulting secretion of IL‐1β using small bioactive enzyme stimulators and inhibitors of ceramide signaling in wild‐type and apoptosis‐associated speck‐like protein containing a CARD knockout (ASC^?/?) primary microglia. To induce the expression of inflammasome components, microglia were primed prior to experiments. Treatment with sodium palmitate (PA) induced de novo ceramide synthesis via modulation of its synthesizing protein serine palmitoyl transferase resulting in increased IL‐1β secretion in microglia. Exposure of microglia to the serine palmitoyl transferase‐inhibitor l ‐cycloserine significantly prevented PA‐induced IL‐1β secretion. Application of the ceramide analogue C2 and the sphingosine‐1‐phosphate‐receptor agonist Fingolimod (FTY720) up‐regulated levels of IL‐1β and cleaved caspase‐1 in wild‐type microglia, whereas ASC^?/? microglia were unaffected. HPA‐12 inhibition of ceramide transport did not affect inflammasome activation. Taken together, our findings reveal a critical role for ceramide as a positive modulator of NLRP3 inflammasome assembly and the resulting release of IL‐1β.

     

Inflammasome activation by Pseudomonas aeruginosa's ExlA pore‐forming toxin is detrimental for the host

During acute Pseudomonas aeruginosa infection, the inflammatory response is essential for bacterial clearance. Neutrophil recruitment can be initiated following the assembly of an inflammasome within sentinel macrophages, leading to activation of caspase‐1, which in turn triggers macrophage pyroptosis and IL‐1β/IL‐18 maturation. Inflammasome formation can be induced by a number of bacterial determinants, including Type III secretion systems (T3SSs) or pore‐forming toxins, or, alternatively, by lipopolysaccharide (LPS) via caspase‐11 activation. Surprisingly, previous studies indicated that a T3SS‐induced inflammasome increased pathogenicity in mouse models of P. aeruginosa infection. Here, we investigated the immune reaction of mice infected with a T3SS‐negative P. aeruginosa strain (IHMA879472). Virulence of this strain relies on ExlA, a secreted pore‐forming toxin. IHMA879472 promoted massive neutrophil infiltration in infected lungs, owing to efficient priming of toll‐like receptors, and thus enhanced the expression of inflammatory proteins including pro‐IL‐1β and TNF‐α. However, mature‐IL‐1β and IL‐18 were undetectable in wild‐type mice, suggesting that ExlA failed to effectively activate caspase‐1. Nevertheless, caspase‐1/11 deficiency improved survival following infection with IHMA879472, as previously described for T3SS+ bacteria. We conclude that the detrimental effect associated with the ExlA‐induced inflammasome is probably not due to hyperinflammation, rather it stems from another inflammasome‐dependent process.     

A hot‐water extract of Sanguisorba officinalis ameliorates endotoxin‐induced septic shock by inhibiting inflammasome activation

The inflammasome is a multiprotein signaling complex that mediates inflammatory innate immune responses through caspase 1 activation and subsequent IL‐1β secretion. However, because its aberrant activation often leads to inflammatory diseases, targeting the inflammasome holds promise for the treatment of inflammation‐related diseases. In this study, it was found that a hot‐water extract of Sanguisorba officinalis (HSO) suppresses inflammasome activation triggered by adenosine 5′‐triphosphate, nigericin, microbial pathogens, and double stranded DNA in bone marrow‐derived macrophages. HSO was found to significantly suppress IL‐1β production in a dose‐dependent manner; this effect correlated well with small amounts of caspase 1 and little ASC pyroptosome formation in HSO‐treated cells. The anti‐inflammatory activity of HSO was further confirmed in a mouse model of endotoxin‐induced septic shock. Oral administration of HSO reduced IL‐1β titers in the serum and peritoneal cavity, increasing the survival rate. Taken together, our results suggest that HSO is an inhibits inflammasome activation through nucleotide‐binding domain and leucine‐rich repeat pyrin domain 3, nucleotide‐binding domain and leucine‐rich repeat caspase recruitment domain 4 and absent in melanoma 2 pathways, and may be useful for treatment of inflammasome‐mediated diseases.     

Activation of nucleotide‐binding domain‐like receptor containing protein 3 inflammasome in dendritic cells and macrophages by Streptococcus sanguinis

Streptococcus sanguinis is frequently isolated from the blood of patients with infective endocarditis and contributes to the pathology of this disease through induction of interleukin (IL)‐1β responsible for the development of the disease. However, the mechanism of IL‐1β induction remains unknown. In this study, S. sanguinis activated a murine dendritic cell (DC) to induce IL‐1β and this activity was attenuated by silencing the mRNAs of nucleotide‐binding domain‐like receptor containing protein 3 (NLRP3) and caspase‐1. S. sanguinis induced IL‐1β production in murine bone marrow‐derived macrophage, but this activity was significantly reduced in bone marrow‐derived macrophages from NLRP3‐, apoptosis‐associated speck‐like protein containing a caspase‐recruitment domain‐, and caspase‐1‐deficient mice. DC phagocytosed S. sanguinis cells, followed by the release of adenosine triphosphate (ATP). The ATP‐degradating enzyme attenuated the release of ATP and IL‐1β. The inhibitors for ATP receptor reduced IL‐1β release in DC. These results strongly suggest that S. sanguinis has the activity to induce IL‐1β through the NLRP3 inflammasome in macrophage and DC and interaction of purinergic receptors with ATP released is involved in expression of the activity.     

Influence of Sae‐regulated and Agr‐regulated factors on the escape of Staphylococcus aureus from human macrophages

Although Staphylococcus aureus is not a classical intracellular pathogen, it can survive within phagocytes and many other cell types. However, the pathogen is also able to escape from cells by mechanisms that are only partially understood. We analysed a series of isogenic S. aureus mutants of the USA300 derivative JE2 for their capacity to destroy human macrophages from within. Intracellular S. aureus JE2 caused severe cell damage in human macrophages and could efficiently escape from within the cells. To obtain this full escape phenotype including an intermittent residency in the cytoplasm, the combined action of the regulatory systems Sae and Agr is required. Mutants in Sae or mutants deficient in the Sae target genes lukAB and pvl remained in high numbers within the macrophages causing reduced cell damage. Mutants in the regulatory system Agr or in the Agr target gene psmα were largely similar to wild‐type bacteria concerning cell damage and escape efficiency. However, these strains were rarely detectable in the cytoplasm, emphasizing the role of phenol‐soluble modulins (PSMs) for phagosomal escape. Thus, Sae‐regulated toxins largely determine damage and escape from within macrophages, whereas PSMs are mainly responsible for the escape from the phagosome into the cytoplasm. Damage of macrophages induced by intracellular bacteria was linked neither to activation of apoptosis‐related caspase 3, 7 or 8 nor to NLRP3‐dependent inflammasome activation.     

Critical role for the AIM2 inflammasome during acute CNS bacterial infection

Interleukin‐1β (IL‐1β) is essential for eliciting protective immunity during the acute phase of Staphylococcus aureus (S. aureus) infection in the central nervous system (CNS). We previously demonstrated that microglial IL‐1β production in response to live S. aureus is mediated through the Nod‐like receptor protein 3 (NLRP3) inflammasome, including the adapter protein ASC (apoptosis‐associated speck‐like protein containing a caspase‐1 recruitment domain), and pro‐caspase 1. Here, we utilized NLRP3, ASC, and caspase 1/11 knockout (KO) mice to demonstrate the functional significance of inflammasome activity during CNS S. aureus infection. ASC and caspase 1/11 KO animals were exquisitely sensitive, with approximately 50% of mice succumbing to infection within 24 h. Unexpectedly, the survival of NLRP3 KO mice was similar to wild‐type animals, suggesting the involvement of an alternative upstream sensor, which was later identified as absent in melanoma 2 (AIM2) based on the similar disease patterns between AIM2 and ASC KO mice. Besides IL‐1β, other key inflammatory mediators, including IL‐6, CXCL1, CXCL10, and CCL2 were significantly reduced in the CNS of AIM2 and ASC KO mice, implicating autocrine/paracrine actions of IL‐1β, as these mediators do not require inflammasome processing for secretion. These studies demonstrate a novel role for the AIM2 inflammasome as a critical molecular platform for regulating IL‐1β release and survival during acute CNS S. aureus infection.

     

Caspase-1 Is an Apical Caspase Leading to Caspase-3 Cleavage in the AIM2 Inflammasome Response,Independent of Caspase-8

Canonical inflammasomes are multiprotein complexes that can activate both caspase-1 and caspase-8. Caspase-1 drives rapid lysis of cells by pyroptosis and maturation of interleukin (IL)-1β and IL-18. In caspase-1-deficient cells, inflammasome formation still leads to caspase-3 activation and slower apoptotic death, dependent on caspase-8 as an apical caspase. A role for caspase-8 directly upstream of caspase-1 has also been suggested, but here we show that caspase-8-deficient macrophages have no defect in AIM2 inflammasome-mediated caspase-1 activation, pyroptosis, and IL-1β cleavage. In investigating the inflammasome-induced apoptotic pathway, we previously demonstrated that activated caspase-8 is essential for caspase-3 cleavage and apoptosis in caspase-1-deficient cells. However, here we found that AIM2 inflammasome-initiated caspase-3 cleavage was maintained in Ripk3^?/? Casp8^?/? macrophages. Gene knockdown showed that caspase-1 was required for the caspase-3 cleavage. Thus inflammasomes activate a network of caspases that can promote both pyroptotic and apoptotic cell death. In cells where rapid pyroptosis is blocked, delayed inflammasome-dependent cell death could still occur due to both caspase-1- and caspase-8-dependent apoptosis. Initiation of redundant cell death pathways is likely to be a strategy for coping with pathogen interference in death processes.     

Soluble uric acid induces myocardial damage through activating the NLRP3 inflammasome

Uric acid crystal is known to activate the NLRP3 inflammasome and to cause tissue damages, which can result in many diseases, such as gout, chronic renal injury and myocardial damage. Meanwhile, soluble uric acid (sUA), before forming crystals, is also related to these diseases. This study was carried out to investigate whether sUA could also activate NLRP3 inflammasome in cardiomyocytes and to analyse the mechanisms. The cardiomyocyte activity was monitored, along with the levels of mature IL‐1β and caspase‐1 from H9c2 cells following sUA stimulus. We found that sUA was able to activate NLRP3 inflammasome, which was responsible for H9c2 cell apoptosis induced by sUA. By elevating TLR6 levels and then activating NF‐κB/p65 signal pathway, sUA promoted NLRP3, pro‐caspase 1 and pro‐IL‐1β production and provided the first signal of NLRP3 inflammasome activation. Meanwhile, ROS production regulated by UCP2 levels also contributed to NLRP3 inflammasome assembly and subsequent caspase 1 activation and mature IL‐1β secretion. In addition, the tlr6 knockdown rats suffering from hyperuricemia showed the lower level of IL‐1β and an ameliorative cardiac function. These findings suggest that sUA activates NLRP3 inflammasome in cardiomyocytes and they may provide one therapeutic strategy for myocardial damage induced by sUA.     

Microglia in juvenile neuronal ceroid lipofuscinosis are primed toward a pro‐inflammatory phenotype

Juvenile neuronal ceroid lipofuscinosis (JNCL) is a lysosomal storage disease caused by an autosomal recessive mutation in CLN3. Regions of microglial activation precede and predict areas of neuronal loss in JNCL; however, the functional role of activated microglia remains to be defined. The inflammasome is a key molecular pathway for activating pro‐IL‐1β in microglia, and IL‐1β is elevated in the brains of JNCL patients and can induce neuronal cell death. Here, we utilized primary microglia isolated from CLN3^Δex7/8 mutant and wild‐type (WT) mice to examine the impact of CLN3 mutation on microglial activation and inflammasome function. Treatment with neuronal lysates and ceramide, a lipid intermediate elevated in the JNCL brain, led to inflammasome activation and IL‐1β release in CLN3^Δex7/8 microglia but not WT cells, as well as increased expression of additional pro‐inflammatory mediators. Similar effects were observed following either TNF‐α or IL‐1β treatment, suggesting that CLN3^Δex7/8 microglia exist in primed state and hyper‐respond to several inflammatory stimuli compared to WT cells. CLN3^Δex7/8 microglia displayed constitutive caspase‐1 activity that when blocked led to increased glutamate release that coincided with hemichannel opening. Conditioned medium from activated CLN3^Δex7/8 or WT microglia induced significant cell death in CLN3^Δex7/8 but not WT neurons, demonstrating that intrinsically diseased CLN3^Δex7/8 neurons are less equipped to withstand cytotoxic insults generated by activated microglia. Collectively, aberrant microglial activation may contribute to the pathological chain of events leading to neurodegeneration during later stages of JNCL.

     

Runx2‐mediated bcl‐2 gene expression contributes to nitric oxide protection against hydrogen peroxide‐induced osteoblast apoptosis

Nitric oxide (NO) can regulate osteoblast activities. This study was aimed to evaluate the protective effects of pretreatment with sodium nitroprusside (SNP) as a source of NO on hydrogen peroxide‐induced osteoblast insults and its possible mechanisms. Exposure of human osteosarcoma MG63 cells to hydrogen peroxide significantly increased cellular oxidative stress, but decreased ALP activity and cell viability, inducing cell apoptosis. Pretreatment with 0.3 mM SNP significantly lowered hydrogen peroxide‐induced cell insults. Treatment of human MG63 cells with hydrogen peroxide inhibited Bcl‐2 mRNA and protein production, but pretreatment with 0.3 mM SNP significantly ameliorated such inhibition. Sequentially, hydrogen peroxide decreased the mitochondrial membrane potential, but increased the levels of cytochrome c and caspase‐3 activity. Pretreatment with 0.3 mM SNP significantly lowered such alterations. Exposure to hydrogen peroxide decreased Runx2 mRNA and protein syntheses. However, pretreatment with 0.3 mM SNP significantly lowered the suppressive effects. Runx2 knockdown using RNA interference inhibited Bcl‐2 mRNA production in human MG63 cells. Protection of pretreatment with 0.3 mM SNP against hydrogen peroxide‐induced alterations in ALP activity, caspase‐3 activity, apoptotic cells, and cell viability were also alleviated after administration of Runx2 small interference RNA. Thus, this study shows that pretreatment with 0.3 mM SNP can protect human MG63 cells from hydrogen peroxide‐induced apoptotic insults possibly via Runx2‐involved regulation of bcl‐2 gene expression. J. Cell. Biochem. 108: 1084–1093, 2009. © 2009 Wiley‐Liss, Inc.     

IL‐1α cleavage by inflammatory caspases of the noncanonical inflammasome controls the senescence‐associated secretory phenotype

Interleukin‐1 alpha (IL‐1α) is a powerful cytokine that modulates immunity, and requires canonical cleavage by calpain for full activity. Mature IL‐1α is produced after inflammasome activation and during cell senescence, but the protease cleaving IL‐1α in these contexts is unknown. We show IL‐1α is activated by caspase‐5 or caspase‐11 cleavage at a conserved site. Caspase‐5 drives cleaved IL‐1α release after human macrophage inflammasome activation, while IL‐1α secretion from murine macrophages only requires caspase‐11, with IL‐1β release needing caspase‐11 and caspase‐1. Importantly, senescent human cells require caspase‐5 for the IL‐1α‐dependent senescence‐associated secretory phenotype (SASP) in vitro, while senescent mouse hepatocytes need caspase‐11 for the SASP‐driven immune surveillance of senescent cells in vivo. Together, we identify IL‐1α as a novel substrate of noncanonical inflammatory caspases and finally provide a mechanism for how IL‐1α is activated during senescence. Thus, targeting caspase‐5 may reduce inflammation and limit the deleterious effects of accumulated senescent cells during disease and Aging.     

POP1 might be recruiting its type‐Ia interface for NLRP3‐mediated PYD‐PYD interaction: Insights from MD simulation

Inflammasomes are multiprotein caspase‐activating complexes that enhance the maturation and release of proinflammatory cytokines (IL‐1β and IL‐18) in response to the invading pathogen and/or host‐derived cellular stress. These are assembled by the sensory proteins (viz NLRC4, NLRP1, NLRP3, and AIM‐2), adaptor protein (ASC), and effector molecule procaspase‐1. In NLRP3‐mediated inflammasome activation, ASC acts as a mediator between NLRP3 and procaspase‐1 for the transmission of signals. A series of homotypic protein‐protein interactions (NLRP3^PYD:ASC^PYD and ASC^CARD:CASP1^CARD) propagates the downstream signaling for the production of proinflammatory cytokines. Pyrin‐only protein 1 (POP1) is known to act as the regulator of inflammasome. It modulates the ASC‐mediated inflammasome assembly by interacting with pyrin domain (PYD) of ASC. However, despite similar electrostatic surface potential, the interaction of POP1 with NLRP3^PYD is obscured till date. Herein, to explore the possible PYD‐PYD interactions between NLRP3^PYD and POP1, a combined approach of protein‐protein docking and molecular dynamics simulation was adapted. The current study revealed that POP1's type‐Ia interface and type‐Ib interface of NLRP3^PYD might be crucial for 1:1 PYD‐PYD interaction. In addition to type‐I mode of interaction, we also observed type‐II and type‐III interaction modes in two different dynamically stable heterotrimeric complexes (POP1‐NLRP3‐NLRP3 and POP1‐NLRP3‐POP1). The inter‐residual/atomic distance calculation exposed several critical residues that possibly govern the said interaction, which need further investigation. Overall, the findings of this study will shed new light on hitherto concealed molecular mechanisms underlying NLRP3‐mediated inflammasome, which will have strong future therapeutic implications.     

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.     

Mitochondrial PKC‐ε deficiency promotes I/R‐mediated myocardial injury via GSK3β‐dependent mitochondrial permeability transition pore opening

Mitochondrial fission is critically involved in cardiomyocyte apoptosis, which has been considered as one of the leading causes of ischaemia/reperfusion (I/R)‐induced myocardial injury. In our previous works, we demonstrate that aldehyde dehydrogenase‐2 (ALDH2) deficiency aggravates cardiomyocyte apoptosis and cardiac dysfunction. The aim of this study was to elucidate whether ALDH2 deficiency promotes mitochondrial injury and cardiomyocyte death in response to I/R stress and the underlying mechanism. I/R injury was induced by aortic cross‐clamping for 45 min. followed by unclamping for 24 hrs in ALDH2 knockout (ALDH2^?/?) and wild‐type (WT) mice. Then myocardial infarct size, cell apoptosis and cardiac function were examined. The protein kinase C (PKC) isoform expressions and their mitochondrial translocation, the activity of dynamin‐related protein 1 (Drp1), caspase9 and caspase3 were determined by Western blot. The effects of N‐acetylcysteine (NAC) or PKC‐δ shRNA treatment on glycogen synthase kinase‐3β (GSK‐3β) activity and mitochondrial permeability transition pore (mPTP) opening were also detected. The results showed that ALDH2^?/? mice exhibited increased myocardial infarct size and cardiomyocyte apoptosis, enhanced levels of cleaved caspase9, caspase3 and phosphorylated Drp1. Mitochondrial PKC‐ε translocation was lower in ALDH2^?/? mice than in WT mice, and PKC‐δ was the opposite. Further data showed that mitochondrial PKC isoform ratio was regulated by cellular reactive oxygen species (ROS) level, which could be reversed by NAC pre‐treatment under I/R injury. In addition, PKC‐ε inhibition caused activation of caspase9, caspase3 and Drp1Ser⁶¹⁶ in response to I/R stress. Importantly, expression of phosphorylated GSK‐3β (inactive form) was lower in ALDH2^?/? mice than in WT mice, and both were increased by NAC pre‐treatment. I/R‐induced mitochondrial translocation of GSK‐3β was inhibited by PKC‐δ shRNA or NAC pre‐treatment. In addition, mitochondrial membrane potential (?Ψ_m) was reduced in ALDH2^?/? mice after I/R, which was partly reversed by the GSK‐3β inhibitor (SB216763) or PKC‐δ shRNA. Collectively, our data provide the evidence that abnormal PKC‐ε/PKC‐δ ratio promotes the activation of Drp1 signalling, caspase cascades and GSK‐3β‐dependent mPTP opening, which results in mitochondrial injury‐triggered cardiomyocyte apoptosis and myocardial dysfuction in ALDH2^?/? mice following I/R stress.     

Lysosomal storage and impaired autophagy lead to inflammasome activation in Gaucher macrophages

Gaucher disease, the inherited deficiency of lysosomal glucocerebrosidase, is characterized by the presence of glucosylcer‐amide macrophages, the accumulation of glucosylceramide in lysosomes and the secretion of inflammatory cytokines. However, the connection between this lysosomal storage and inflammation is not clear. Studying macrophages derived from peripheral monocytes from patients with type 1 Gaucher disease with genotype N370S/N370S, we confirmed an increased secretion of interleukins IL‐1β and IL‐6. In addition, we found that activation of the inflammasome, a multiprotein complex that activates caspase‐1, led to the maturation of IL‐1β in Gaucher macrophages. We show that inflammasome activation in these cells is the result of impaired autophagy. Treatment with the small‐molecule glucocerebrosidase chaperone NCGC758 reversed these defects, inducing autophagy and reducing IL‐1β secretion, confirming the role of the deficiency of lysosomal glucocerebrosidase in these processes. We found that in Gaucher macrophages elevated levels of the autophagic adaptor p62 prevented the delivery of inflammasomes to autophagosomes. This increase in p62 led to activation of p65‐NF‐kB in the nucleus, promoting the expression of inflammatory cytokines and the secretion of IL‐1β. This newly elucidated mechanism ties lysosomal dysfunction to inflammasome activation, and may contribute to the massive organomegaly, bone involvement and increased susceptibility to certain malignancies seen in Gaucher disease. Moreover, this link between lysosomal storage, impaired autophagy, and inflammation may have implications relevant to both Parkinson disease and the aging process. Defects in these basic cellular processes may also provide new therapeutic targets.