A role for mitochondria in NLRP3 inflammasome activation

An inflammatory response initiated by the NLRP3 inflammasome is triggered by a variety of situations of host 'danger', including infection and metabolic dysregulation. Previous studies suggested that NLRP3 inflammasome activity is negatively regulated by autophagy and positively regulated by reactive oxygen species (ROS) derived from an uncharacterized organelle. Here we show that mitophagy/autophagy blockade leads to the accumulation of damaged, ROS-generating mitochondria, and this in turn activates the NLRP3 inflammasome. Resting NLRP3 localizes to endoplasmic reticulum structures, whereas on inflammasome activation both NLRP3 and its adaptor ASC redistribute to the perinuclear space where they co-localize with endoplasmic reticulum and mitochondria organelle clusters. Notably, both ROS generation and inflammasome activation are suppressed when mitochondrial activity is dysregulated by inhibition of the voltage-dependent anion channel. This indicates that NLRP3 inflammasome senses mitochondrial dysfunction and may explain the frequent association of mitochondrial damage with inflammatory diseases.     

中枢神经系统来源的HMGB1表达模式和释放特征

目的:阐明生理病理状态下,中枢神经系统(CNS)来源的高迁移率族蛋白1(HMGB1)细胞定位、亚细胞定位和释放特征.方法:使用Western Blot和细胞免疫荧光双标记法检测HMGB1在细胞株(U87, BV2和N2a)和原代星形胶质细胞、神经元的表达模式和释放特征.用PTX和/或ATP处理培养细胞,Western Blot检测浓缩上清中HMGB1蛋白表达,台盼蓝拒染法检测细胞的生存率.结果:HMGB1定位于U87、BV2、N2a,原代星形胶质细胞和神经元的细胞核,位于目的条带25 KDa位置. PTX和/或ATP能诱导U87和BV2主动分泌HMGB1,N2a细胞被动释放HMGB1.结论:生理状态下,星形胶质细胞、小胶质细胞和神经元细胞核表达HMGB1,属于HMGB1来源,HMGB1具有CNS释放特征.     

Differential activation of the inflammasome by caspase-1 adaptors ASC and Ipaf

Specific adaptors regulate the activation of initiator caspases; for example, FADD and Apaf-1 engage caspases 8 and 9, respectively. The adaptors ASC, Ipaf and RIP2 have each been proposed to regulate caspase-1 (also called interleukin (IL)-1 converting enzyme), which is activated within the 'inflammasome', a complex comprising several adaptors. Here we show the impact of ASC-, Ipaf- or RIP2-deficiency on inflammasome function. ASC was essential for extracellular ATP-driven activation of caspase-1 in toll-like receptor (TLR)-stimulated macrophages. Accordingly, ASC-deficient macrophages exhibited defective maturation of IL-1beta and IL-18, and ASC-null mice were resistant to lipopolysaccharide-induced endotoxic shock. Furthermore, activation of caspase-1 in response to an intracellular pathogen (Salmonella typhimurium) was abrogated severely in ASC-null macrophages. Unexpectedly, Ipaf-deficient macrophages activated caspase-1 in response to TLR plus ATP stimulation but not S. typhimurium. Caspase-1 activation was not compromised by loss of RIP2. These data show that whereas ASC is key to caspase-1 activation within the inflammasome, Ipaf provides a special conduit to the inflammasome for signals triggered by intracellular pathogens. Notably, cell death triggered by stimuli that engage caspase-1 was ablated in macrophages lacking either ASC or Ipaf, suggesting a coupling between the inflammatory and cell death pathways.     

Endothelium-derived relaxing factor release on activation of NMDA receptors suggests role as intercellular messenger in the brain

In the vascular system, endothelium-derived relaxing factor (EDRF) is the name of the local hormone released from endothelial cells in response to vasodilators such as acetylcholine, bradykinin and histamine. It diffuses into underlying smooth muscle where it causes relaxation by activating guanylate cyclase, so producing a rise in cyclic GMP levels. It has been known for many years that in the central nervous system (CNS) the excitatory neurotransmitter glutamate can elicit large increases in cGMP levels, particularly in the cerebellum where the turnover rate of cGMP is low. Recent evidence indicates that cell-cell interactions are involved in this response. We report here that by acting on NMDA (N-methyl-D-aspartate) receptors on cerebellar cells, glutamate induces the release of a diffusible messenger with strikingly similar properties to EDRF. This messenger is released in a Ca2+-dependent manner and its activity accounts for the cGMP responses that take place following NMDA receptor activation. In the CNS, EDRF may link activation of postsynaptic NMDA receptors to functional modifications in neighbouring presynaptic terminals and glial cells.     

The protein kinase PKR is required for macrophage apoptosis after activation of Toll-like receptor 4

Macrophages are pivotal constituents of the innate immune system, vital for recognition and elimination of microbial pathogens. Macrophages use Toll-like receptors (TLRs) to detect pathogen-associated molecular patterns--including bacterial cell wall components, such as lipopolysaccharide or lipoteichoic acid, and viral nucleic acids, such as double-stranded (ds)RNA--and in turn activate effector functions, including anti-apoptotic signalling pathways. Certain pathogens, however, such as Salmonella spp., Shigellae spp. and Yersiniae spp., use specialized virulence factors to overcome these protective responses and induce macrophage apoptosis. We found that the anthrax bacterium, Bacillus anthracis, selectively induces apoptosis of activated macrophages through its lethal toxin, which prevents activation of the anti-apoptotic p38 mitogen-activated protein kinase. We now demonstrate that macrophage apoptosis by three different bacterial pathogens depends on activation of TLR4. Dissection of anti- and pro-apoptotic signalling events triggered by TLR4 identified the dsRNA responsive protein kinase PKR as a critical mediator of pathogen-induced macrophage apoptosis. The pro-apoptotic actions of PKR are mediated both through inhibition of protein synthesis and activation of interferon response factor 3.     

Crucial role for the Nalp3 inflammasome in the immunostimulatory properties of aluminium adjuvants

Aluminium adjuvants, typically referred to as 'alum', are the most commonly used adjuvants in human and animal vaccines worldwide, yet the mechanism underlying the stimulation of the immune system by alum remains unknown. Toll-like receptors are critical in sensing infections and are therefore common targets of various adjuvants used in immunological studies. Although alum is known to induce the production of proinflammatory cytokines in vitro, it has been repeatedly demonstrated that alum does not require intact Toll-like receptor signalling to activate the immune system. Here we show that aluminium adjuvants activate an intracellular innate immune response system called the Nalp3 (also known as cryopyrin, CIAS1 or NLRP3) inflammasome. Production of the pro-inflammatory cytokines interleukin-1beta and interleukin-18 by macrophages in response to alum in vitro required intact inflammasome signalling. Furthermore, in vivo, mice deficient in Nalp3, ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain) or caspase-1 failed to mount a significant antibody response to an antigen administered with aluminium adjuvants, whereas the response to complete Freund's adjuvant remained intact. We identify the Nalp3 inflammasome as a crucial element in the adjuvant effect of aluminium adjuvants; in addition, we show that the innate inflammasome pathway can direct a humoral adaptive immune response. This is likely to affect how we design effective, but safe, adjuvants in the future.     

细胞自噬与高迁移率族蛋白B-1(HMGB1)介导肿瘤耐药的研究进展

恶性肿瘤可通过多种细胞机制,产生对抗癌药物和放疗的抗性,即所谓耐药现象.细胞自噬是肿瘤细胞耐药的一个重要原因.高迁移率族蛋白B-1(HMGB1)是高度保守的非组蛋白DNA结合蛋白,在DNA结构、基因转录、基因重组、DNA损伤修复以及细胞存活等方面发挥重要的调控作用;HMGB1在细胞内的功能,与其氧化还原状态和细胞定位息...     

Release of chromatin protein HMGB1 by necrotic cells triggers inflammation

High mobility group 1 (HMGB1) protein is both a nuclear factor and a secreted protein. In the cell nucleus it acts as an architectural chromatin-binding factor that bends DNA and promotes protein assembly on specific DNA targets. Outside the cell, it binds with high affinity to RAGE (the receptor for advanced glycation end products) and is a potent mediator of inflammation. HMGB1 is secreted by activated monocytes and macrophages, and is passively released by necrotic or damaged cells. Here we report that Hmgb1(-/-) necrotic cells have a greatly reduced ability to promote inflammation, which proves that the release of HMGB1 can signal the demise of a cell to its neighbours. Apoptotic cells do not release HMGB1 even after undergoing secondary necrosis and partial autolysis, and thus fail to promote inflammation even if not cleared promptly by phagocytic cells. In apoptotic cells, HMGB1 is bound firmly to chromatin because of generalized underacetylation of histone and is released in the extracellular medium (promoting inflammation) if chromatin deacetylation is prevented. Thus, cells undergoing apoptosis are programmed to withhold the signal that is broadcast by cells that have been damaged or killed by trauma.     

The role of sediment resuspension duration in release of PAHs

Polycyclic aromatic hydrocarbons （PAHs） are ubiquitous environmental pollutants. Due to their low water solubility and high hydrophobicity, PAHs are rapidly sorbed onto particles and subsequently deposit in sediments once introduced into aquatic environment. In such a way, sediments become a huge sink for PAHs. During sediment resuspension, the potential exists for PAHs to be released from sediments into water. Sediment resuspension plays an important role in the transportation and fate of PAHs in the aquatic environment. In this study, release behavior of PAHs on Yangtze River sediment during resuspension was investigated using a particle entrainment simulator （PES）. The role of resuspension duration on release of 16 PAHs was measured by resuspending sediment for 12 h at 0.2 and 0.5N/m^2, respectively. Results indicated that PAH concentrations in TSS increase over time with more increase of phenanthrene and 4-ring PAHs. Comparing with 0.2 N/m2 （30%）, TPAHs concentrations in TSS demonstrated remarkable increase during 0.5 N/m2 resuspension （37%）. Dissolved PAH concentrations increased throughout the duration with more increase of 2-3 ring PAHs （50%-88%）. Dissolved PAH concentrations showed remarkable increase during 0.5 N/m^2 resuspension （50%）. Moreover, PAH concentrations in overlying water throughout the duration of resuspension were higher than toxic effects threshold values in drinking water developed by WHO, which may cause toxic effect on ecosystem.     

HMGB1对心肌成纤维细胞增殖和Ⅰ、Ⅲ胶原蛋白分泌的影响及意义

摘要：目的：通过观察HMGB1（ High mobility group protein-B1）对新生SD（Sprague-Dawley）大鼠心肌成纤维细胞(CFs)增殖和胶原合成的影响,探讨HMGB1在心梗后心脏重构过程中的作用。方法：以培养的新生SD（Sprague-Dawley）大鼠CFs为实验对象,分别给予0(对照组)、0.01、0.1、和1 mg/L 浓度的HMGB1进行干预,6、12、18、24和48小时后进行四氮唑盐(MTT)比色法检测CFs增殖水平,采用荧光实时定量PCR法检测Ⅰ、Ⅲ型胶原蛋白mRNA表达。结果：1.与对照组相比,48小时后,不同浓度的HMGB1组新生SD大鼠CFs增殖水平和Ⅰ、Ⅲ型胶原mRNA的表达水平均较对照组有所增高(p<0.05)。2.HMGB1浓度为0.1mg/L时CFs增殖水平和I、Ⅲ型胶原mRNA表达水平较其他处理组均升高(p<0.05)。结论：HMGB1能促进SD大鼠CFs增殖及Ⅰ、Ⅲ型胶原蛋白mRNA的表达,其作用显示在低水平时可能呈浓度依赖性,并且浓度为0.1mg/L时效应最明显,结果显示HMGB1参与了心梗后的心脏重构过程并发挥了重要作用。     

Critical role of Toll-like receptor signaling in NK cell activation

Toll-like receptors (TLRs) and NK cell receptors are the most important receptor superfamilies in innate immunity. TLRs act as the sensor of external pathogens, while NK cells detect alterations in endogenous protein expression on target cells through activating and inhibitory receptors. Accumulating data has demonstrated that TLRs and NK cell receptors can coordinate and regulate each other during immune responses, which contributes to the initiation of innate response and the priming of adaptive responses. TLRs can activate NK cell function directly or with the help of accessory cells in a cytokine or cell-to-cell contact dependent manner. More understanding of the recognition of innate receptors and interactions between them may provide important insights into the design of effective strategies to combat tumor and microbial infections. In this review, we summarize how TLRs and NK cells discriminate the self or non-self components respectively. And importantly, we pay more attention to the role of TLR sig-naling in induction of NK cell activation, responses and the crosstalk between them.     

Membrane potential has no direct role in evoking neurotransmitter release

Neurons communicate by secreting a transmitter that excites or inhibits other neurons at synapses. The role of presynaptic membrane potential in triggering transmitter release is still controversial. In one view, presynaptic action potentials trigger the release by the entry of calcium ions into presynaptic terminals through voltage-dependent calcium channels. Calcium acts at high local concentrations at release sites near channel mouths to cause neurosecretion. An opposing view is that, in addition to elevating presynaptic calcium, presynaptic potential stimulates transmitter release by a distinct direct action. The relative importance of depolarization and calcium entry in neurosecretion cannot be determined because the two events are tightly linked. To delineate the roles of presynaptic potential and calcium entry in transmitter release, we have used nitr-5, a photolabile calcium chelator, and a voltage-clamp technique to control intracellular calcium and membrane potential independently at a synapse formed between cell bodies of cultured neurons of the fresh water snail Helisoma trivolvis. We found transmitter release occurred when presynaptic calcium levels were elevated to concentrations of a few micromolar, and that presynaptic voltage had no direct effect on neurosecretion.     

The second messenger linking receptor activation to internal Ca release in liver

The increase in cytosolic [Ca2+] induced by Ca-mobilizing hormones in liver is mainly due to release of Ca from intracellular stores. For Ca to be released from internal sites a messenger must be formed at the plasma membrane which diffuses into the cytosol to signal Ca release from the intracellular organelles. One of the first actions of these hormones is to cause breakdown of the polyphosphoinositides to form soluble inositol phosphates. Some evidence for the idea that these substances could be the second messenger has been obtained in pancreatic acinar cells. Here we have found that hormone activation of hepatocytes causes rapid breakdown of phosphatidylinositol 4,5-bisphosphate [ PtdIns (4,5)P2] to form inositol trisphosphate ( InsP3 ). When applied to permeabilized hepatocytes, InsP3 releases Ca from non-mitochondrial ATP-dependent pools. This suggests that InsP3 could be the messenger linking Ca-mobilizing receptor activation to intracellular Ca release in liver.     

Cryopyrin activates the inflammasome in response to toxins and ATP

A crucial part of the innate immune response is the assembly of the inflammasome, a cytosolic complex of proteins that activates caspase-1 to process the proinflammatory cytokines interleukin (IL)-1beta and IL-18. The adaptor protein ASC is essential for inflammasome function, binding directly to caspase-1 (refs 3, 4), but the triggers of this interaction are less clear. ASC also interacts with the adaptor cryopyrin (also known as NALP3 or CIAS1). Activating mutations in cryopyrin are associated with familial cold autoinflammatory syndrome, Muckle-Wells syndrome and neonatal onset multisystem inflammatory disease, diseases that are characterized by excessive production of IL-1beta. Here we show that cryopyrin-deficient macrophages cannot activate caspase-1 in response to Toll-like receptor agonists plus ATP, the latter activating the P2X7 receptor to decrease intracellular K+ levels. The release of IL-1beta in response to nigericin, a potassium ionophore, and maitotoxin, a potent marine toxin, was also found to be dependent on cryopyrin. In contrast to Asc-/- macrophages, cells deficient in the gene encoding cryopyrin (Cias1-/-) activated caspase-1 and secreted normal levels of IL-1beta and IL-18 when infected with Gram-negative Salmonella typhimurium or Francisella tularensis. Macrophages exposed to Gram-positive Staphylococcus aureus or Listeria monocytogenes, however, required both ASC and cryopyrin to activate caspase-1 and secrete IL-1beta. Therefore, cryopyrin is essential for inflammasome activation in response to signalling pathways triggered specifically by ATP, nigericin, maitotoxin, S. aureus or L. monocytogenes.