Innate immune inflammatory cell death: PANoptosis and PANoptosomes in host defense and disease

Regulated cell death (RCD) triggered by innate immune activation is an important strategy for host survival during pathogen invasion and perturbations of cellular homeostasis. There are two main categories of RCD, including nonlytic and lytic pathways. Apoptosis is the most well-characterized nonlytic RCD, and the inflammatory pyroptosis and necroptosis pathways are among the best known lytic forms. While these were historically viewed as independent RCD pathways, extensive evidence of cross-talk among their molecular components created a knowledge gap in our mechanistic understanding of RCD and innate immune pathway components, which led to the identification of PANoptosis. PANoptosis is a unique innate immune inflammatory RCD pathway that is regulated by PANoptosome complexes upon sensing pathogens, pathogen-associated molecular patterns (PAMPs), damage-associated molecular patterns (DAMPs) or the cytokines produced downstream. Cytosolic innate immune sensors and regulators, such as ZBP1, AIM2 and RIPK1, promote the assembly of PANoptosomes to drive PANoptosis. In this review, we discuss the molecular components of the known PANoptosomes and highlight the mechanisms of PANoptosome assembly, activation and regulation identified to date. We also discuss how PANoptosomes and mutations in PANoptosome components are linked to diseases. Given the impact of RCD, and PANoptosis specifically, across the disease spectrum, improved understanding of PANoptosomes and their regulation will be critical for identifying new therapeutic targets and strategies.     

Pretreatment with 3-methyladenine ameliorated Pseudomonas aeruginosa-induced acute pneumonia by inhibiting cell death of neutrophils in a mouse infection model

Pseudomonas aeruginosa is one of the leading causes of nosocomial infections worldwide. Clinical isolates that are resistant to multiple antimicrobials make it intractable. The interactions between P. aeruginosa and host cell death have multiple effects on bacterial clearance and inflammation; however, the potential intervention effects remain to be defined. Herein, we demonstrated that intravenous administration of 3-methyladenine before, but not after, P. aeruginosa infection enhanced autophagy-independent survival, which was accompanied by a decrease in the bacterial load, alleviation of pathology and reduction in inflammatory cytokines, in an acute pneumonia mouse model. Interestingly, these beneficial effects were not dependent on neutrophil recruitment or phagocytosis, but on the enhanced killing capacity induced by inhibiting the cell death of 3-MA pretreated neutrophils. These findings demonstrate a novel protective role of 3-MA pretreatment in P. aeruginosa-induced acute pneumonia.     

受体相互作用蛋白激酶调控非酒精性脂肪肝中细胞程序性坏死的研究进展

非酒精性脂肪性肝病（NAFLD）是一种常见的慢性肝病,如果得不到有效控制,则会进一步发展为非酒精性脂肪性肝炎（NASH）,进而引起肝纤维化、肝硬化,甚至癌变。程序性坏死是近年来发现的一种新型细胞程序性死亡方式,由受体相互作用蛋白激酶（RIPK）介导所致,最终可以导致细胞膜溶解破裂,引发炎症。RIPK家族作为细胞内和细胞外应激的重要传感器,诱导调控程序性坏死的发生,并参与炎症及其他免疫反应。近年来研究表明,RIPK调控的程序性坏死在非酒精性脂肪性肝病的发生发展中具有重要作用,在动物NAFLD/NASH模型中,RIPK的表达情况与肝脂肪变性程度密切相关。在一些临床研究中亦观察到,NAFLD/NASH患者比健康人RIPK表达水平上升。但程序性坏死到底是加速肝病进程的因素,还是肝病发展过程中的保护因素,仍然没有定论。有研究表明,RIPK抑制剂可能为NAFLD治疗提供方向。我们综述了程序性坏死的分子机制及与非酒精性脂肪性肝病的关系,以及RIPK在其中扮演的重要角色,并总结了其在NAFLD/NASH治疗方面的研究进展,为进一步探究其机制,探索新的治疗手段提供理论依据。     

Necrostatin-1对小鼠脑外伤脑组织损害及行为学能力的影响

目的 探讨细胞程序性坏死特异性抑制剂Necrostatin-1(Nec-1)对脑外伤导致的组织损伤和行为学能力(运动功能、学习能力)损害的影响。方法 健康成熟雄性昆明小鼠(体重20～25 g),采用改良的自由落体装置制作定量右侧脑外伤(Traumatic Brain Injury,TBI)模型,建模15 min前同侧脑室分别注射溶剂二甲基亚砜(DMSO)和DMSO配制的Nec-1溶液分别建立脑外伤DMSO组和脑外伤Nec-1组,同时设立假手术组(Sham组),利用行为学试验(Motor Test和Morris水迷宫试验)评估TBI 2周内各组小鼠的运动功能和学习能力损害,利用脑组织缺损体积(Lesion Volume,LV)评价TBI 3周后脑皮质损伤程度。结果(1)脑外伤后1周内能观察到小鼠运动能力评分降低明显(P<0.01),伤后2～7 d各组运动评分均逐渐恢复。与DMSO组比较,Nec-1预给药能明显加快运动功能的恢复过程(P<0.05);(2)所有TBI小鼠与Sham组比较,在水迷宫定位航行试验中搜找平台时需要更长的潜伏期(P<0.05); Nec-1组在伤后第9～12 d表现较短的潜伏期(P<0.05);(3)脑外伤导致各组小鼠脑组织的缺失(P<0.01),与DMSO组比较,Nec-1预处理明显减少了脑外伤后的脑组织损伤体积(P<0.05)。结论 侧脑室预注射Nec-1抑制程序性坏死可减少小鼠脑外伤后脑组织缺损,促进活动功能、学习能力的恢复。     

人肾小管上皮细胞necroptosis模型的构建

 目的：以体外培养的人肾小管上皮细胞（HK-2细胞）为靶细胞,构建肾小管上皮细胞凋亡样坏死（necroptosis）的模型。方法：采用肿瘤坏死因子 α (tumor nercosis factor α, TNF-α)诱导细胞凋亡,同时采用抗霉素A (antimycin A)耗竭ATP,构建肾小管上皮细胞凋亡的模型,并以caspase-8抑制剂苄氧羰酰-缬氨酰-丙氨酰-天冬氨酰-氟甲基酮(benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, zVAD-fmk) 阻断凋亡,用necroptosis的特异性抑制剂necrostatin-1（Nec-1）阻断necroptosis,观察细胞在不同的处理下形态学的变化,同时检测细胞存活率及标志物微管相关蛋白1轻链3-Ⅱ（microtubule-associated protein 1 light chain 3-Ⅱ,LC3-Ⅱ）的表达。结果：（1）在TNF-α+zVAD- fmk+antimycin A处理1 h时细胞及细胞器膨胀,电镜下细胞膜碎裂,线粒体变圆、肿胀,嵴逐渐模糊,胞浆中出现大量自噬小体,而Nec-1预处理后细胞的坏死程度较对照组明显改善。（2）在TNF-α+zVAD-fmk+antimycin A 1 h实验组,Nec-1预处理后细胞的存活率显著增加（P<0.05）。（3）TNF-α+zVAD-fmk+antimycin A干预1 h实验组在Nec-1预处理后LC3-Ⅱ的表达量明显下降（P<0.05）。结论：凋亡环境中阻断凋亡可以诱导肾小管上皮细胞necroptosis,抑制剂Nec-1能特异性阻断肾小管上皮细胞发生坏死。     

坏死性凋亡介导高糖引起的人脐静脉内皮细胞损伤

目的:研究坏死性凋亡是否介导高糖(HG)诱导的人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)损伤。方法:CCK-8法检测细胞存活率;Western blot法测定受体相互作用蛋白3(RIP3)、cleaved caspase-3的蛋白水平;罗丹明123染色荧光显微镜照相法检测线粒体膜电位(mitochondrial membrane potential,MMP);双氯荧光素(DCFH-DA)染色荧光显微镜照相法测定胞内活性氧簇(reactive oxygen species,ROS)的水平。结果:应用不同浓度葡萄糖(10、20和40 mmol/L)处理HUVECs 24 h,RIP3的蛋白水平随葡萄糖剂量增加而升高,40 mmol/L时达高峰;应用40 mmol/L葡萄糖处理HUVECs 3 h、6 h、9 h、12 h和24 h能上调RIP3的蛋白水平,于9 h达最高峰;应用20μmol/L凋亡蛋白酶抑制剂Z-VAD-FMK预处理HUVECs 30 min促进RIP3表达;应用100μmol/L坏死性凋亡抑制剂necrostatin-1预处理HUVECs 1 h能抑制HG诱导HUVECs的细胞存活率降低,ROS过度生成及MMP丢失,但能升高cleaved caspase-3的蛋白水平。结论:坏死性凋亡介导高糖引起的人脐静脉内皮细胞损伤,但与内皮细胞凋亡存在负相关。     

Bishonokiol A Induces Multiple Cell Death in Human Breast Cancer MCF-7 Cells

Objective: A dimeric neolignan, bishonokiol A (BHNKA) isolated from Magnolia grandiflora, significantly inhibits the proliferation of human breast cancer cells. However, the exact mechanism of BHNKA induced breast cancer cell death is unknown. In this study, we investigated the pharmacological mechanism underlying BHNKA induced MCF-7 cell death. Methods: Cell viability measurement was performed by the MTT assay. Flow cytometry with PI staining, DAPI staining, and electron microscopy were used to analyze cellular death modes. In addition, western blotting, siRNA transfection, ATP assay, and fluorescence microscopy were used to determine the mechanism of BHNKA induced MCF-7 cell death. Results: BHNKA induced cell death by apoptosis, necroptosis and autophagy at the same concentration and time in MCF-7 cells, and electron microscopy confirmed these results. The mechanism of BHNKA triggered apoptosis and autophagy in MCF-7 cells was primarily due to an increase in the Bax/Bcl-2 ratio and simultaneous up-regulation of LC3-II protein expression, respectively. BHNKA induced necroptosis by activation of the RIP1-RIP3-MLKL necroptosis cascade, up-regulation of cyclophilin D (CypD) protein expression to stimulate ROS generation. We further demonstrated that siRNA-mediated down-regulation of CypD protected against BHNKA induced cell death. Conclusions: These results suggest that BHNKA may be a potential lead compound for development as an anti-breast cancer agent for induction of multiple cell death pathways.     

Key players of the necroptosis pathway RIPK1 and SIRT2 are altered in placenta from preeclampsia and fetal growth restriction

IntroductionPreeclampsia (PE) and fetal growth restriction (FGR) are among the leading causes of perinatal morbidity and mortality. Placental insufficiency is central to these conditions. The mechanisms underlying placental insufficiency are poorly understood. Apoptosis has long been considered the only form of regulated cell death, recent research has identified an alternate process of programmed cell death known as necroptosis [1]. Necroptosis is distinct from apoptosis, relying on the deacetylase sirtuin-2 [2], receptor interacting kinases RIPK1 and 3, and the pseudokinase MLKL [3]. We aimed to determine whether these key necroptosis effector molecules were present in human placenta and whether they are differentially expressed in severe preterm (PT) PE and FGR.MethodsPT placentas from severe early onset (<34 weeks) PE (n = 30), FGR (n = 12) and control (18) pregnancies were collected. SIRT2 and RIPK1 localization and quantitation was determined by immunohistochemistry and western blot. Immunocytochemistry was used to detect SIRT2 and RIPK1 in trophoblastic debris from first trimester, term control and PE pregnancies. Expression of SIRT2, RIPK1, RIPK3 and MLKL was examined by qPCR.ResultsSIRT2 and RIPK1 were localized to the syncytiotrophoblast, villous leukocytes and vasculature in all PT placentas. A significant reduction in SIRT2 protein expression in both PE and FGR placentas was identified. RIPK1 mRNA expression was significantly increased in PE placentas. Immunofluorescence identified both SIRT2 and RIPK1 in the cytotrophoblast cytoplasm.DiscussionWe have identified the presence of activators of necroptosis in human placenta. Interestingly, there is differential expression in major pregnancy complications. We conclude necroptosis may contribute to placental pathophysiology that underlies serious pregnancy complications.     

PMA and crystal‐induced neutrophil extracellular trap formation involves RIPK1‐RIPK3‐MLKL signaling

Neutrophil extracellular trap (NET) formation contributes to gout, autoimmune vasculitis, thrombosis, and atherosclerosis. The outside‐in signaling pathway triggering NET formation is unknown. Here, we show that the receptor‐interacting protein kinase (RIPK)‐1‐stabilizers necrostatin‐1 or necrostatin‐1s and the mixed lineage kinase domain‐like (MLKL)‐inhibitor necrosulfonamide prevent monosodium urate (MSU) crystal‐ or PMA‐induced NET formation in human and mouse neutrophils. These compounds do not affect PMA‐ or urate crystal‐induced production of ROS. Moreover, neutrophils of chronic granulomatous disease patients are shown to lack PMA‐induced MLKL phosphorylation. Genetic deficiency of RIPK3 in mice prevents MSU crystal‐induced NET formation in vitro and in vivo. Thus, neutrophil death and NET formation may involve the signaling pathway defining necroptosis downstream of ROS production. These data imply that RIPK1, RIPK3, and MLKL could represent molecular targets in gout or other crystallopathies.     

Non-apoptotic cell death-based cancer therapy:Molecular mechanism,pharmacological modulators,and nanomedicineOA

As an emerging cancer therapeutic target,non-apoptotic cell death such as ferroptosis,necroptosis and pyroptosis,etc.,has revealed significant potential in cancer treatment for bypassing apoptosis to enhance the undermined therapeutic efficacy triggered by apoptosis resistance.A variety of anticancer drugs,synthesized compounds and natural products have been proven recently to induce non-apoptotic cell death and exhibit excellent anti-tumor effects.Moreover,the convergence of nanotechnology with...