ZBP1 promotes fungi-induced inflammasome activation and pyroptosis,apoptosis, and necroptosis (PANoptosis)

Candida albicans and Aspergillus fumigatus are dangerous fungal pathogens with high morbidity and mortality, particularly in immunocompromised patients. Innate immune-mediated programmed cell death (pyroptosis, apoptosis, necroptosis) is an integral part of host defense against pathogens. Inflammasomes, which are canonically formed upstream of pyroptosis, have been characterized as key mediators of fungal sensing and drivers of proinflammatory responses. However, the specific cell death pathways and key upstream sensors activated in the context of Candida and Aspergillus infections are unknown. Here, we report that C. albicans and A. fumigatus infection induced inflammatory programmed cell death in the form of pyroptosis, apoptosis, and necroptosis (PANoptosis). Further, we identified the innate immune sensor Z-DNA binding protein 1 (ZBP1) as the apical sensor of fungal infection responsible for activating the inflammasome/pyroptosis, apoptosis, and necroptosis. The Zα2 domain of ZBP1 was required to promote this inflammasome activation and PANoptosis. Overall, our results demonstrate that C. albicans and A. fumigatus induce PANoptosis and that ZBP1 plays a vital role in inflammasome activation and PANoptosis in response to fungal pathogens.     

Inactivation of inflammasomes by pathogens regulates inflammation

Inflammatory response is initiated and sustained by the action of quintessential pro-inflammatory cytokines of immune system namely IL-1β and IL-18. The maturation process of those cytokines is ensured by caspase-1 enzymatic activity, that is in turn is tightly controlled by multiprotein complexes called inflammasomes. Inflammasomes are activated in cells of innate immune system in response to recognition of conservative parts of microbes (pathogen-associated molecular patterns) or by sensing molecular signs of tissue damage (damage-associated molecular patterns). Inflammasome activation apart of cytokines secretion leads to pro-inflammatory cell death, so-called pyroptosis. That culminates in release of cytoplasmatic content of cells including cytokines and alarmins that boost immune response against pathogens, as well as pyroptosis destroys replicative niches of intracellular pathogens. During co-evolution with the host, bacterial and viral pathogens developed a range of molecular inhibitors targeting each step of inflammasome activation. In current review, we will discuss the latest knowledge of inflammasomes’ signaling pathways and tricks that pathogens use to avoid immune recognition and clearance. Our better understanding of inflammasome inhibition by pathogens can lead to better therapeutic approaches for the treatment of infectious diseases.     

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.     

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.     

Inflammasomes in T cells

The concept of non-self recognition through germ-line encoded pattern recognition receptors (PRRs) has been well-established for professional innate immune cells. However, there is growing evidence that also T cells employ PRRs and associated effector functions in response to certain non-self or damage signals. Inflammasomes constitute a special subgroup of PRRs that is hardwired to a signaling cascade that culminates in the activation of caspase-1. Active caspase-1 processes pro-inflammatory cytokines of the IL-1 family and also triggers a lytic programmed cell death pathway known as pyroptosis. An increasing body of literature suggests that inflammasomes are also functional in T cells. On the one hand, conventional inflammasome signaling cascades have been described that operate similarly to pathways characterized in innate immune cells. On the other hand, unconventional functions have been suggested, in which certain inflammasome components play a role in unrelated processes, such as cell fate decisions and functions of T helper cells. In this review, we discuss our current knowledge on inflammasome functions in T cells and the biological implications of these findings for health and disease.     

A Genome-wide CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) Screen Identifies NEK7 as an Essential Component of NLRP3 Inflammasome Activation

Inflammasomes are high molecular weight protein complexes that assemble in the cytosol upon pathogen encounter. This results in caspase-1-dependent pro-inflammatory cytokine maturation, as well as a special type of cell death, known as pyroptosis. The Nlrp3 inflammasome plays a pivotal role in pathogen defense, but at the same time, its activity has also been implicated in many common sterile inflammatory conditions. To this effect, several studies have identified Nlrp3 inflammasome engagement in a number of common human diseases such as atherosclerosis, type 2 diabetes, Alzheimer disease, or gout. Although it has been shown that known Nlrp3 stimuli converge on potassium ion efflux upstream of Nlrp3 activation, the exact molecular mechanism of Nlrp3 activation remains elusive. Here, we describe a genome-wide CRISPR/Cas9 screen in immortalized mouse macrophages aiming at the unbiased identification of gene products involved in Nlrp3 inflammasome activation. We employed a FACS-based screen for Nlrp3-dependent cell death, using the ionophoric compound nigericin as a potassium efflux-inducing stimulus. Using a genome-wide guide RNA (gRNA) library, we found that targeting Nek7 rescued macrophages from nigericin-induced lethality. Subsequent studies revealed that murine macrophages deficient in Nek7 displayed a largely blunted Nlrp3 inflammasome response, whereas Aim2-mediated inflammasome activation proved to be fully intact. Although the mechanism of Nek7 functioning upstream of Nlrp3 yet remains elusive, these studies provide a first genetic handle of a component that specifically functions upstream of Nlrp3.     

The NLRP3 inflammasome detects encephalomyocarditis virus and vesicular stomatitis virus infection

Inflammasomes are cytosolic protein complexes that regulate caspase-1 activation and the secretion of interleukin-1β (IL-1β) and IL-18. Several different inflammasome complexes have been identified, but the NLRP3 inflammasome is particularly notable because of its central role in diseases of inflammation. Recent work has demonstrated an essential role for the NLRP3 inflammasome in host defense against influenza virus. We show here that two other RNA viruses, encephalomyocarditis virus (EMCV) and vesicular stomatitis virus (VSV), activate the NLRP3 inflammasome in dendritic cells and macrophages through a mechanism requiring viral replication. Inflammasome activation in response to both viruses does not require MDA5 or RIG-I signaling. Despite the ability of the NLRP3 inflammasome to detect EMCV and VSV, wild-type and caspase-1-deficient mice were equally susceptible to infection with both viruses. These findings indicate that the NLRP3 inflammasome may be a common pathway for RNA virus detection, but its precise role in the host response may be variable.     

The Microtubule-associated Protein EB1 Links AIM2 Inflammasomes with Autophagy-dependent Secretion

Inflammasomes are multi-protein complexes that regulate chronic inflammation-associated diseases by inducing interleukin-1 β (IL-1β) secretion. Numerous components involved in inflammasome activation have been identified, but the mechanisms of inflammasome-mediated IL-1β secretion have not yet been fully explored. Here, we demonstrate that end-binding protein 1 (EB1), which is required for activation of AIM2 inflammasome complex, links the AIM2 inflammasome to autophagy-dependent secretion. Imaging studies revealed that AIM2 inflammasomes colocalize with microtubule organizing centers and autophagosomes. Biochemical analyses showed that poly(dA-dT)-activated AIM2 inflammasomes induce autophagy and IL-1β secretion in an LC3-dependent fashion. Furthermore, depletion of EB1 decreases autophagic shedding and intracellular trafficking. Finally, we found that the 5′-AMP activated protein kinase may regulate this EB1-mediated autophagy-based inflammasome-induced secretion of IL-1β. These findings reveal a novel EB1-mediated pathway for the secretion of IL-1β.     

病原体与宿主炎症小体相互作用

炎症小体(Inflammasome)是细胞质中多种蛋白组装成的复合物,炎症小体的激活能活化半胱天冬酶-1(caspase-1),进而引起系列促炎细胞因子的成熟与分泌和诱导细胞焦亡。当病原体感染时,炎症小体的激活在宿主天然免疫应答中起重要作用。大量研究表明,多数情况下炎症小体对宿主起保护作用,仅少数情况下保护作用不明显或表现出有利于病原体生存的一面。在长期进化中,病原体也发展出逃避宿主炎症小体作用的策略。病原体可直接抑制炎症小体的激活或减弱炎症小体的作用。本文从病原体感染宿主中炎症小体的作用及病原体对宿主炎性症小体的逃避机制两方面对二者相互作用的最新研究进展进行综述。     

Emerging insights into molecular mechanisms underlying pyroptosis and functions of inflammasomes in diseases

Pyroptosis is a form of necrotic and inflammatory programmed cell death, which could be characterized by cell swelling, pore formation on plasma membranes, and release of proinflammatory cytokines (IL-1β and IL-18). The process of pyroptosis presents as dual effects: protecting multicellular organisms from microbial infection and endogenous dangers; leading to pathological inflammation if overactivated. Two pathways have been found to trigger pyroptosis: caspase-1 mediated inflammasome pathway with the involvement of NLRP1-, NLRP3-, NLRC4-, AIM2-, pyrin-inflammasome (canonical inflammasome pathway) and caspase-4/5/11-mediated inflammasome pathway (noncanonical inflammasome pathway). Gasdermin D (GSDMD) has been proved to be a substrate of inflammatory caspases (caspase-1/4/5/11), and the cleaved N-terminal domain of GSDMD oligomerizes to form cytotoxic pores on the plasma membrane. Here, we mainly reviewed the up to date mechanisms of pyroptosis, and began with the inflammasomes as the activator of caspase-1/caspase-11, 4, and 5. We further discussed these inflammasomes functions in diseases, including infectious diseases, sepsis, inflammatory autoimmune diseases, and neuroinflammatory diseases.     

Plasma-derived exosomes contribute to pancreatitis-associated lung injury by triggering NLRP3-dependent pyroptosis in alveolar macrophages

Progression of acute pancreatitis (AP) into a severe form usually results in a life-threatening condition with multiple organ dysfunction, and in particular acute lung injury (ALI), often contributes to the majority of AP-associated deaths. Increasing evidence has shown that uncontrolled activation of the immune system with rapid production of inflammatory cytokines play a dominant role in this process. As an intracellular inflammatory signaling platform, the NOD-like receptor protein 3 (NLRP3) inflammasome, is recently reported to be involved in the pathogenesis of AP progression, however, the relationship between NLRP3 inflammasome activation and AP-associated lung injury remains unclear yet. Here, we show that NLRP3 inflammasome activation and subsequent pyroptosis in alveolar macrophages (AMs) is responsible for the lung injury secondary to AP. In addition, plasma-derived exosomes from AP mice is capable of triggering NLRP3-dependent pyroptosis in AMs. Inhibition of exosome release or uptake in vivo by inhibitors substantially suppresses AMs pyroptosis and thereby alleviates AP-induced pulmonary lesion. Collectively, the current work reveals for the first time the involvement of NLRP3-dependent pyroptosis induced by plasma exosomes in the pathogenesis of AP-induced ALI, suggesting that the exosome-mediated NLRP3 inflammatory pathway is a potential therapeutic target for the treatment of lung injury during AP.     

Checks and Balances between Autophagy and Inflammasomes during Infection

Autophagy and inflammasome complex assembly are physiological processes that control homeostasis, inflammation, and immunity. Autophagy is a ubiquitous pathway that degrades cytosolic macromolecules or organelles, as well as intracellular pathogens. Inflammasomes are multi-protein complexes that assemble in the cytosol of cells upon detection of pathogen- or danger-associated molecular patterns. A critical outcome of inflammasome assembly is the activation of the cysteine protease caspase-1, which activates the pro-inflammatory cytokine precursors pro-IL-1β and pro-IL-18. Studies on chronic inflammatory diseases, heart diseases, Alzheimer's disease, and multiple sclerosis revealed that autophagy and inflammasomes intersect and regulate each other. In the context of infectious diseases, however, less is known about the interplay between autophagy and inflammasome assembly, although it is becoming evident that pathogens have evolved multiple strategies to inhibit and/or subvert these pathways and to take advantage of their intricate crosstalk. An improved appreciation of these pathways and their subversion by diverse pathogens is expected to help in the design of anti-infective therapeutic interventions.     

NLRP3炎症体与炎症性疾病

炎症体是胱天蛋白酶的活化平台,并促进一些前炎症细胞因子如IL-1β、IL-18和IL-33的成熟,启动机体的先天性免疫防御功能。炎症体的激活和失调与人类先天及后天的炎症性疾病都密切相关。通过对NLRP1、NL-RP3、IPAF和AIM2炎症体调节机制的研究,可为家族性周期性自身炎症反应、痛风、II型糖尿病等的治疗提供新的靶点。主要就NLRP3炎症体的组成、分布和调节机制及与NLRP3炎症体相关的炎症性疾病进行了简要介绍。     

Anthrax Lethal Factor Cleavage of Nlrp1 Is Required for Activation of the Inflammasome

NOD-like receptor (NLR) proteins (Nlrps) are cytosolic sensors responsible for detection of pathogen and danger-associated molecular patterns through unknown mechanisms. Their activation in response to a wide range of intracellular danger signals leads to formation of the inflammasome, caspase-1 activation, rapid programmed cell death (pyroptosis) and maturation of IL-1β and IL-18. Anthrax lethal toxin (LT) induces the caspase-1-dependent pyroptosis of mouse and rat macrophages isolated from certain inbred rodent strains through activation of the NOD-like receptor (NLR) Nlrp1 inflammasome. Here we show that LT cleaves rat Nlrp1 and this cleavage is required for toxin-induced inflammasome activation, IL-1 β release, and macrophage pyroptosis. These results identify both a previously unrecognized mechanism of activation of an NLR and a new, physiologically relevant protein substrate of LT.     

食源性致病菌激活NLRP3炎症小体及食源性功能物质的抑制机理研究进展

食源性致病菌感染是引起食源性疾病的首要因素,严重影响人类健康。炎症小体通过识别受体感知入侵宿主的危险信号进而组装形成多聚蛋白复合物,从而诱导炎症反应,是先天免疫系统中识别食源性病原菌感染和清除病原体的重要防线。NLRP3炎症小体是位于胞内的炎症反应平台,可以感知多种病原微生物的侵袭,在先天性免疫反应中起着至关重要的作用。食源性致病菌感染常引起NLRP3炎症小体的异常激活,介导多种炎症性疾病的发生和发展,因此,许多抗炎研究中常常以NLRP3炎症小体作为靶点。本文总结了食源性致病菌及其代谢产物激活NLRP3炎症小体的分子机制,以及天然产物和膳食功能物质抑制NLRP3炎症小体激活的机理,为治疗炎症性疾病、开发缓解致病菌诱导的炎症反应的功能化合物提供新的思路。     

Activated human CD4+CD45RO+ memory T-cells indirectly inhibit NLRP3 inflammasome activation through downregulation of P2X7R signalling

Inflammasomes are multi-protein complexes that control the production of pro-inflammatory cytokines such as IL-1β. Inflammasomes play an important role in the control of immunity to tumors and infections, and also in autoimmune diseases, but the mechanisms controlling the activation of human inflammasomes are largely unknown. We found that human activated CD4+CD45RO+ memory T-cells specifically suppress P2X7R-mediated NLRP3 inflammasome activation, without affecting P2X7R-independent NLRP3 or NLRP1 inflammasome activation. The concomitant increase in pro-IL-1β production induced by activated memory T-cells concealed this effect. Priming with IFNβ decreased pro-IL-1β production in addition to NLRP3 inflammasome inhibition and thus unmasked the inhibitory effect on NLRP3 inflammasome activation. IFNβ suppresses NLRP3 inflammasome activation through an indirect mechanism involving decreased P2X7R signaling. The inhibition of pro-IL-1β production and suppression of NLRP3 inflammasome activation by IFNβ-primed human CD4+CD45RO+ memory T-cells is partly mediated by soluble FasL and is associated with down-regulated P2X7R mRNA expression and reduced response to ATP in monocytes. CD4+CD45RO+ memory T-cells from multiple sclerosis (MS) patients showed a reduced ability to suppress NLRP3 inflammasome activation, however their suppressive ability was recovered following in vivo treatment with IFNβ. Thus, our data demonstrate that human P2X7R-mediated NLRP3 inflammasome activation is regulated by activated CD4+CD45RO+ memory T cells, and provide new information on the mechanisms mediating the therapeutic effects of IFNβ in MS.     

Shigella hijacks the glomulin–cIAPs–inflammasome axis to promote inflammation

Shigella deploys a unique mechanism to manipulate macrophage pyroptosis by delivering the IpaH7.8 E3 ubiquitin ligase via its type III secretion system. IpaH7.8 ubiquitinates glomulin (GLMN) and elicits its degradation, thereby inducing inflammasome activation and pyroptotic cell death of macrophages. Here, we show that GLMN specifically binds cellular inhibitor of apoptosis proteins 1 and 2 (cIAP1 and cIAP2), members of the inhibitor of apoptosis (IAP) family of RING‐E3 ligases, which results in reduced E3 ligase activity, and consequently inflammasome‐mediated death of macrophages. Importantly, reducing the levels of GLMN in macrophages via IpaH7.8, or siRNA‐mediated knockdown, enhances inflammasome activation in response to infection by Shigella, Salmonella, or Pseudomonas, stimulation with NLRP3 inflammasome activators (including SiO₂, alum, or MSU), or stimulation of the AIM2 inflammasome by poly dA:dT. GLMN binds specifically to the RING domain of both cIAPs, which inhibits their self‐ubiquitination activity. These findings suggest that GLMN is a negative regulator of cIAP‐mediated inflammasome activation, and highlight a unique Shigella stratagem to kill macrophages, promoting severe inflammation.     

GSDMD介导的细胞焦亡在感染性疾病中的研究进展

细胞焦亡是细胞感染时由炎症小体介导,以裂解细胞为特点的程序性死亡形式。其激活途径分为依赖半胱氨酸蛋白酶-1或半胱氨酸蛋白酶-4/5/11活化的经典与非经典途径。目前的研究表明细胞焦亡过程中主要效应蛋白是具有膜成孔活性的gasdermin(也作GSDM)家族成员。因此,细胞焦亡也被称为gasdermin介导的程序性坏死。当宿主受到感染时,细胞焦亡与宿主自身其他免疫防御机制存在互相调节机制,保证宿主在清除感染的同时降低自身损伤程度。本文笔者将从研究最为广泛的GSDMD在细胞焦亡途径中的作用机制、细胞焦亡在感染性疾病中的研究进展以及细胞焦亡与其他程序性死亡在感染性疾病中的相互作用这三个方面作系统叙述,期望为今后研究如何通过细胞焦亡途径治疗感染性疾病提供理论基础。     

Shikonin Suppresses NLRP3 and AIM2 Inflammasomes by Direct Inhibition of Caspase-1

Shikonin is a highly lipophilic naphtoquinone found in the roots of Lithospermum erythrorhizon used for its pleiotropic effects in traditional Chinese medicine. Based on its reported antipyretic and anti-inflammatory properties, we investigated whether shikonin suppresses the activation of NLRP3 inflammasome. Inflammasomes are cytosolic protein complexes that serve as scaffolds for recruitment and activation of caspase-1, which, in turn, results in cleavage and secretion of proinflammatory cytokines IL-1β and IL-18. NLRP3 inflammasome activation involves two steps: priming, i.e. the activation of NF-κB pathway, and inflammasome assembly. While shikonin has previously been reported to suppress the priming step, we demonstrated that shikonin also inhibits the second step of inflammasome activation induced by soluble and particulate NLRP3 instigators in primed immortalized murine bone marrow-derived macrophages. Shikonin decreased NLRP3 inflammasome activation in response to nigericin more potently than acetylshikonin. Our results showed that shikonin also inhibits AIM2 inflammasome activation by double stranded DNA. Shikonin inhibited ASC speck formation and caspase-1 activation in murine macrophages and suppressed the activity of isolated caspase-1, demonstrating that it directly targets caspase-1. Complexing shikonin with β-lactoglobulin reduced its toxicity while preserving the inhibitory effect on NLRP3 inflammasome activation, suggesting that shikonin with improved bioavailability might be interesting for therapeutic applications in inflammasome-mediated conditions.