Granzymes, cytotoxic granules and cell death: the early work of Dr. Jurg Tschopp

Within the powerful legacy left by Jurg Tschopp, we should not forget his early work that helped to elucidate the molecular pathways responsible for the clearance of virus-infected and transformed cells by cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. Jurg's skilful biochemical approach formed a firm platform upon which the work of so many other biochemists, cell biologists and immunologists would come to rely. Jurg coined the shorthand term 'granzyme' to denote the individual members of a family of serine proteases sequestered in and secreted from the cytotoxic granules of CTL/NK cells. He was also one of the first to describe the lytic properties of purified perforin and to postulate the synergy of perforin and granzymes, which we now know to underpin target cell apoptosis. Jurg was a major protagonist in the debate that raged throughout the 1980's and early 1990's on the physiological relevance of the 'granule exocytosis' pathway. Ultimately, resolving this issue led Jurg and his colleagues to even greater and impactful discoveries in the broader field of apoptosis research. Jurg Tschopp ranks with other pioneers, particularly Gideon Berke, Chris Bleackley, Pierre Golstein, Pierre Henkart and Eckhard Podack for making seminal discoveries on our understanding of how the immune system eliminates dangerous cells.     

NLRP3 Inflammasome Activation by Paracoccidioides brasiliensis

Paracoccidioides brasiliensis is the etiologic agent of paracoccidioidomycosis (PCM), the most prevalent systemic mycosis that is geographically confined to Latin America. The pro-inflammatory cytokine IL-1β that is mainly derived from the activation of the cytoplasmic multiprotein complex inflammasome is an essential host factor against opportunistic fungal infections; however, its role in infection with a primary fungal pathogen, such as P. brasiliensis, is not well understood. In this study, we found that murine bone marrow-derived dendritic cells responded to P. brasiliensis yeast cells infection by releasing IL-1β in a spleen tyrosine kinase (Syk), caspase-1 and NOD-like receptor (NLR) family member NLRP3 dependent manner. In addition, P. brasiliensis-induced NLRP3 inflammasome activation was dependent on potassium (K+) efflux, reactive oxygen species production, phagolysosomal acidification and cathepsin B release. Finally, using mice lacking the IL-1 receptor, we demonstrated that IL-1β signaling has an important role in killing P. brasiliensis by murine macrophages. Altogether, our results demonstrate that the NLRP3 inflammasome senses and responds to P. brasiliensis yeast cells infection and plays an important role in host defense against this fungus.     

Nlrp3: An immune sensor of cellular stress and infection

Innate immune cells rely on pathogen recognition receptors such as the nucleotide-binding and oligomerization domain (NOD)-like receptor (NLR) family to mount an appropriate immune response against microbial threats. The NLR protein Nlrp3 senses microbial ligands, endogenous danger signals and crystalline substances in the cytosol to trigger the assembly of a large caspase-1-activating protein complex termed the Nlrp3 inflammasome. Autoproteolytic maturation of caspase-1 zymogens in the Nlrp3 inflammasome leads to maturation and extracellular release of the pro-inflammatory cytokines interleukin (IL)-1β and IL-18. Gain-of-function mutations in the NOD domain of Nlrp3 are associated with auto-inflammatory disorders characterized by skin rashes and prolonged episodes of fever. In addition, decreased Nlrp3 expression was recently linked with susceptibility to Crohn's disease in humans. In this review, we discuss recent developments on the role of the Nlrp3 inflammasome in innate immunity, its activation mechanisms and the auto-inflammatory disorders associated with deregulation of Nlrp3 inflammasome activity.     

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.     

The NLRP3 inflammasome instigates obesity-induced inflammation and insulin resistance

The emergence of chronic inflammation during obesity in the absence of overt infection or well-defined autoimmune processes is a puzzling phenomenon. The Nod-like receptor (NLR) family of innate immune cell sensors, such as the nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (Nlrp3, but also known as Nalp3 or cryopyrin) inflammasome are implicated in recognizing certain nonmicrobial originated 'danger signals' leading to caspase-1 activation and subsequent interleukin-1β (IL-1β) and IL-18 secretion. We show that calorie restriction and exercise-mediated weight loss in obese individuals with type 2 diabetes is associated with a reduction in adipose tissue expression of Nlrp3 as well as with decreased inflammation and improved insulin sensitivity. We further found that the Nlrp3 inflammasome senses lipotoxicity-associated increases in intracellular ceramide to induce caspase-1 cleavage in macrophages and adipose tissue. Ablation of Nlrp3 in mice prevents obesity-induced inflammasome activation in fat depots and liver as well as enhances insulin signaling. Furthermore, elimination of Nlrp3 in obese mice reduces IL-18 and adipose tissue interferon-γ (IFN-γ) expression, increases naive T cell numbers and reduces effector T cell numbers in adipose tissue. Collectively, these data establish that the Nlrp3 inflammasome senses obesity-associated danger signals and contributes to obesity-induced inflammation and insulin resistance.     

The NLRP3 Inflammasome: An Important Driver of Neuroinflammation in Hemorrhagic Stroke

Hemorrhagic stroke is a devastating clinical event with no effective medical treatment. Neuroinflammation, which follows a hemorrhagic stroke, is an important element that involves both acute brain injury and subsequent brain rehabilitation. Therefore, delineating the key inflammatory mediators and deciphering their pathophysiological roles in hemorrhagic strokes is of great importance in the development of novel therapeutic targets for this disease. The NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome is a multi-protein complex that is localized within the cytoplasm. This NOD-like receptor orchestrates innate immune responses to pathogenic organisms and cell stress through the activation of caspase-1 and the maturation of the proinflammatory cytokines such as interleukin-1β (IL-1β) and IL-18. Mounting evidence has demonstrated that when the NLRP3 inflammasome is activated, it exerts harmful effects on brain tissue after a hemorrhagic stroke. This review article summarizes the current knowledge regarding the role and the underlying mechanisms of the NLRP3 inflammasome in the pathophysiological processes of hemorrhagic strokes. A better understanding of the function and regulation of the NLRP3 inflammasome in hemorrhagic strokes will provide clues for devising novel therapeutic strategies to fight this disease.     

Activation of the NALP3 inflammasome is triggered by low intracellular potassium concentration

Inflammasomes are Nod-like receptor(NLR)- and caspase-1-containing cytoplasmic multiprotein complexes, which upon their assembly, process and activate the proinflammatory cytokines interleukin (IL)-1beta and IL-18. The inflammasomes harboring the NLR members NALP1, NALP3 and IPAF have been best characterized. While the IPAF inflammasome is activated by bacterial flagellin, activation of the NALP3 inflammasome is triggered not only by several microbial components, but also by a plethora of danger-associated host molecules such as uric acid. How NALP3 senses these chemically unrelated activators is not known. Here, we provide evidence that activation of NALP3, but not of the IPAF inflammasome, is blocked by inhibiting K(+) efflux from cells. Low intracellular K(+) is also a requirement for NALP1 inflammasome activation by lethal toxin of Bacillus anthracis. In vitro, NALP inflammasome assembly and caspase-1 recruitment occurs spontaneously at K(+) concentrations below 90 mM, but is prevented at higher concentrations. Thus, low intracellular K(+) may be the least common trigger of NALP-inflammasome activation.     

Sensing bacterial infections by NAIP receptors in NLRC4 inflammasome activation

The inflammasome is an emerging new pathway in innate immune defense against microbial infection or endogenous danger signals. The inflammasome stimulates activation of inflammatory caspases, mainly caspase-1. Caspase-1 activation is responsible for processing and secretion of IL-1β and IL-18 as well as for inducing macrophage pyroptotic death. Assembly of the large cytoplasmic inflammasome complex is thought to be mediated by members of NOD-like receptor (NLR) family. While functions of most of the NLR proteins remain to be defined, several NLR proteins including NLRC4 have been shown to assemble distinct inflammasome complexes. These inflammasome pathways, particularly the NLRC4 inflammasome, play a critical role in sensing and restricting diverse types of bacterial infections. Here we review recent advances in defining the exact bacterial ligands and the ligand-binding receptors involved in NLRC4 inflammasome activation. Implications of the discovery of the NAIP family of inflammasome receptors for bacterial flagellin and type III secretion apparatus on future inflammasome and bacterial infection studies are also discussed.     

炎性体及其在流感病毒感染过程中的作用

炎性体是胞液中感受危险信号、启动介导下游免疫防御或细胞死亡（pyroptosis）的多分子复合物,是细胞内天然免疫的重要受承信号转导的中介体.炎性体识别流感病毒后诱导先天免疫反应甚至pyroptosis样细胞死亡.流感病毒高尔基体表达的M2蛋白和P2X7、ATP、ROS在炎性体的调节过程中发挥了重要作用,微生物也可以通过激活炎性体调节呼吸道粘膜免疫.炎性体的提出为最优疫苗的设计提供了新的思路.     

Mitochondria-targeted drugs enhance Nlrp3 inflammasome-dependent IL-1β secretion in association with alterations in cellular redox and energy status

The Nlrp3 inflammasome is activated in response to an array of environmental and endogenous molecules leading to caspase-1-dependent IL-1β processing and secretion by myeloid cells. Several identified Nlrp3 inflammasome activators also trigger reactive oxygen species (ROS) production. However, the initial concept that NADPH oxidases are the primary source of ROS production during inflammasome activation is becoming less accepted. Therefore, the importance of mitochondria-derived ROS has been recently explored. In this study, we explore the impact of mitochondria dysfunction and ROS production on Nlrp3 inflammasome stimulation and IL-1β secretion induced by serum amyloid A (SAA) in primary mouse peritoneal macrophages. To induce mitochondrial dysfunction, we utilized antimycin A, which blocks electron flow at complex III, and carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), a mitochondrial oxidative phosphorylation uncoupler. We also utilized a superoxide dismutase mimetic, MnTBAP, which targets the mitochondria, as well as the broad-spectrum antioxidants DPI (diphenyleneiodonium chloride) and ebselen. Our findings demonstrate that SAA alone induces mitochondrial ROS in a time-dependent manner. We observed that MnTBAP and ebselen blocked IL-1β secretion caused by SAA only when added before stimulation, and DPI augmented IL-1β secretion. Surprisingly, these effects were not directly related to intracellular or mitochondrial ROS levels. We also found that mitochondria-targeted drugs increased IL-1β secretion regardless of their impact on mitochondrial function and ROS levels, suggesting that mitochondrial ROS-dependent and -independent mechanisms play a role in the Nlrp3 inflammasome/IL-1β secretion axis in SAA-stimulated cells. Finally, we found that FCCP significantly sustained the association of the Nlrp3 inflammasome complex, which could explain the most robust effect among the drugs tested in enhancing IL-1β secretion in SAA-treated cells. Overall, our data suggest that the Nlrp3 inflammasome/IL-1β secretion axis is a very highly regulated inflammatory pathway that is susceptible not only to changes in mitochondrial or intracellular ROS, but also to changes in overall mitochondrial function.     

The inflammasome puts obesity in the danger zone

Obesity-induced inflammation is an important contributor to the induction of insulin resistance. Recently, the cytokine interleukin-1β (IL-1β) has emerged as a prominent instigator of the proinflammatory response in obesity. Several studies over the last year have subsequently deciphered the molecular mechanisms responsible for IL-1β activation in adipose tissue, liver, and macrophages and demonstrated a central role of the processing enzyme caspase-1 and of the protein complex leading to its activation called the inflammasome. These data suggest that activation of the inflammasome represents a crucial step in the road from obesity to insulin resistance and type 2 diabetes.     

Interaction Between Nonviral Reprogrammed Fibroblast Stem Cells and Trophic Factors for Brain Repair

Diabetic encephalopathy is one of the most common complications of diabetes. Inflammatory events during diabetes may be an important mechanism of diabetic encephalopathy. Inflammasome is a multiprotein complex consisting of Nod-like receptor proteins (NLRPs), apoptosis-associated speck-like protein (ASC), and caspase 1 or 5, which functions to switch on the inflammatory process and the release of inflammatory factors. The present study hypothesized that the formation and activation of NLRP1 inflammasome turns on neuroinflammation and neuron injury during hyperglycemia. The results demonstrated that the levels of interleukin-1 beta (IL-1β) were increased in the cortex of streptozocin (STZ)-induced diabetic rats. The levels of mature IL-1β and IL-18 were also elevated in culture medium of neurons treated with high glucose (50 mM). The expression of three essential components of the NLRP1 inflammasome complex, namely, NLRP1, ASC, and caspase 1, was also upregulated in vivo and in vitro under high glucose. Silencing the ASC gene prevented the caspase-1 activation, and inhibiting caspase 1 activity blocked hyperglycemia-induced release of inflammatory factors and neuron injury. Moreover, we found that pannexin 1 mediated the actvitation of NLRP1 inflammasome under high glucose. These results suggest that hyperglycemia induces neuroinflammation through activation of NLRP1 inflammasome, which represents a novel mechanism of diabetes-associated neuron injury.     

Emerging Roles for the Death Adaptor FADD in Death Receptor Avidity and Cell Cycle Regulation

The Fas-associated death domain protein FADD is best known as an adaptor protein that senses a signal received at a death receptor and nucleates the assembly of the death-inducing signaling complex. Recent work reveals unexpected properties for this signaling protein, suggesting new roles for FADD in apoptotic signaling and in non-apoptotic functions linked to chemical modification of the FADD C-terminus. These new studies suggest novel types of high valency complexes may form in the plasma membrane and in the nucleus, raising intriguing questions as to how FADD senses the environment and responds to different signaling inputs to promote a biochemical response. In particular, we discuss the role of FADD in death receptor avidity and examine the relationship between FADD phosphorylation and subcellular localization with respect to various biological functions. Since FADD serves to modulate both apoptosis and cell cycle progression, these new findings promote the concept that differential complex assembly dictates disparate cellular processes mediated by this adaptor molecule.     

The inflammasome mediates UVB-induced activation and secretion of interleukin-1beta by keratinocytes

It has long been known that human keratinocytes are a potent source of the proinflammatory cytokines proIL-1alpha and -1beta[1], which are activated and released in response to UV irradiation [2]. However, the intracellular pathways, which regulate maturation and secretion of IL-1 in keratinocytes, are unknown. Here we show that the UVB-mediated enhancement of cytoplasmic Ca(2+) is required for activation of the IL-1beta-converting enzyme caspase-1 by the inflammasome, a multiprotein innate immune complex [3, 4]. Caspase-1 in turn activates proIL-1beta, and keratinocytes secrete the cytokine as well as inflammasome components. These results demonstrate the presence of a proIL-1beta-processing inflammasome in nonprofessional immune cells and the necessity of inflammasome components for the UVB-induced secretion of IL-1beta. This supports the concept that keratinocytes are important immuno-competent cells under physiological and pathological conditions [5].     

Pseudomonas aeruginosa pilin activates the inflammasome

IL-1β is produced from inactive pro-IL-1β by activation of caspase-1 brought about by a multi-subunit protein platform called the inflammasome. Many bacteria can trigger inflammasome activity through flagellin activation of the host protein NLRC4. However, strains of the common human pathogen Pseudomonas aeruginosa lacking flagellin can still activate the inflammasome. We set out to identify what non-flagellin components could produce this activation. Using mass spectroscopy, we identified an inflammasome-activating factor from P. aeruginosa as pilin, the major component of the type IV bacterial pilus. Purified pilin introduced into mouse macrophages by liposomal delivery activated caspase-1 and led to secretion of mature IL-1β, as did recombinant pilin purified from Escherichia coli. This was dependent on caspase-1 but not on the host inflammasome proteins NLRC4, NLRP3 or ASC. Mutants of P. aeruginosa strain PA103 lacking pilin did not activate the inflammasome following infection of macrophages with live bacteria. Type III secretion remained intact in the absence of pili, showing this was not due to a lack of effector delivery. Our observations show pilin is a novel activator of the inflammasome in addition to flagellin and the recently described PrgJ protein family, the basal body rod component of the type III apparatus.     

Endoplasmic reticulum stress and NLRP3 inflammasome: Crosstalk in cardiovascular and metabolic disorders

When endoplasmic reticulum (ER) homeostasis is disrupted, known as ER stress (ERS), the ER generates an adaptive signaling pathway called the unfolded protein response to maintain the homeostasis of this organelle. However, if homeostasis is not restored, the ER initiates death signaling pathways, which contribute to the pathogenesis of various disorders. The activation of inflammatory mechanisms is also emerging as a crucial component of cardiovascular and metabolic disorders. Furthermore, the nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome has attracted more attention than others and is the best-characterized member of the NLR family of inflammasomes to date. ERS intersects with many different inflammatory pathways, particularly the NLRP3 inflammasome. In this review, we focus on the interactions between ERS and the NLRP3 inflammasome. The pharmacologic and nonpharmaceutical manipulation of these two processes may offer novel opportunities for the treatment of cardiovascular and metabolic disorders.     

NLRP3炎性小体研究新进展

NLRP3炎性小体是一种分子量约为700Kda的大分子多蛋白复合体,能被多种病原相关的分子模式或损伤相关的分子模式活化,对固有免疫系统免疫功能的发挥具有极其重要的作用。但如果其被过度激活则可通过活化的半胱天冬酶-1持续地将pro-IL-1β和pro-IL-18剪切为成熟的IL-1β和IL-18,进而激活下游信号转导通路,产生大量的炎性介质,引起机体发生严重的炎症反应,最终促进多种炎症性疾病的发生与发展,如Muckle—wells综合征、2型糖尿病、非酒精性脂肪肝、动脉粥样硬化、炎症性肠病和阿尔兹海默病等。因此,对NLRP3炎性小体进行深入的研究不仅有助于阐释固有免疫系统如何有效地发挥其免疫功能,而且作为系列炎症反应的核心,NLRP3炎性小体：还可能成为多种炎症性疾病防治的新靶点。我们就NLRP3炎性小体的结构与功能,激活与调控,分布与疾病的近期研究作一综：违。     

Negative regulation of NLRP3 inflammasome signaling

Inflammasomes are multiprotein complexes that serve as a platform for caspase-1 activation and interleukin-1β (IL-1β) maturation as well as pyroptosis. Though a number of inflammasomes have been described, the NLRP3 inflammasome is the most extensively studied. NLRP3 inflammasome is triggered by a variety of stimuli, including infection, tissue damage and metabolic dysregulation, and then activated through an integrated cellular signal. Many regulatory mechanisms have been identifi ed to attenuate NLRP3 inflammasome signaling at multiple steps. Here, we review the developments in the negative regulation of NLRP3 inflammasome that protect host from inflammatory damage.     

Sirtuin 3-induced macrophage autophagy in regulating NLRP3 inflammasome activation

Defective autophagy of monocytes or macrophages might result in NLRP3 inflammasome activation and cause vascular metabolic inflammation. However, the mechanism underlying the initiation of the autophagy response to hyperlipidaemia remains unclear. Sirtuin 3 (SIRT3), an NAD-dependent deacetylase, is sensitive to the metabolic status and mediates adaptation responses. In this study, we investigated the role of SIRT3-mediated autophagy in regulating NLRP3 inflammasome activation. We determined that the inhibition of autophagy and the activation of the NLRP3 inflammasome were concomitant with reduced SIRT3 levels both in peripheral blood monocytes from obese humans and in palmitate-treated THP-1 cells. Furthermore, we demonstrated that SIRT3 could form a molecular complex with ATG5, while SIRT3 overexpression altered the acetylation of endogenous ATG5. ATG5 acetylation inhibited autophagosome maturation and induced NLRP3 inflammasome activation. In parallel, SIRT3 overexpression in THP-1 cells decreased the palmitate-induced generation of mitochondrial reactive oxygen species, restored autophagy, and attenuated NLRP3 inflammasome activation. The incubation of human aortic endothelial cells (HAECs) with macrophage-conditioned medium (MCM) induced HAEC expression of vascular cell adhesion molecule-1, intercellular adhesion molecule 1, α-smooth muscle actin, and collagen-1. The effect of MCM could be reversed by the addition of neutralizing anti-IL-1β antibody or the overexpression of SIRT3. Consistent with this, en face analyses displayed a marked increase in α-SMC-positive endothelial cells in SIRT3^?/? mice with acute hyperlipidaemia. Taken together, these findings revealed that SIRT3-deficient macrophages displayed impaired autophagy and accelerated NLRP3 inflammasome activation and endothelial dysfunction.     

P2X4 Assembles with P2X7 and Pannexin-1 in Gingival Epithelial Cells and Modulates ATP-induced Reactive Oxygen Species Production and Inflammasome Activation

We have previously reported that Porphyromonas gingivalis infection of gingival epithelial cells (GEC) requires an exogenous danger signal such as ATP to activate an inflammasome and caspase-1, thereby inducing secretion of interleukin (IL)-1β. Stimulation with extracellular ATP also stimulates production of reactive oxygen species (ROS) in GEC. However, the mechanism by which ROS is generated in response to ATP, and the role that different purinergic receptors may play in inflammasome activation, is still unclear. In this study, we revealed that the purinergic receptor P2X₄ is assembled with the receptor P2X₇ and its associated pore, pannexin-1. ATP induces ROS production through a complex consisting of the P2X₄, P2X_7, and pannexin-1. P2X₇−mediated ROS production can activate the NLRP3 inflammasome and caspase-1. Furthermore, separate depletion or inhibition of P2X₄, P2X₇, or pannexin-1 complex blocks IL-1β secretion in P. gingivalis-infected GEC following ATP treatment. However, activation via P2X₄ alone induces ROS generation but not inflammasome activation. These results suggest that ROS is generated through stimulation of a P2X₄/P2X₇/pannexin-1 complex, and reveal an unexpected role for P2X₄, which acts as a positive regulator of inflammasome activation during microbial infection.