RESUMO
Caspase-8-dependent pyroptosis has been shown to mediate host protection from Yersinia infection. For this mode of cell death, the kinase activity of receptor-interacting protein kinase 1 (RIPK1) is required, but the autophosphorylation sites required to drive caspase-8 activation have not been determined. Here, we show that non-canonical autophosphorylation of RIPK1 at threonine 169 (T169) is necessary for caspase-8-mediated pyroptosis. Mice with alanine in the T169 position are highly susceptible to Yersinia dissemination. Mechanistically, the delayed formation of a complex containing RIPK1, ZBP1, Fas-associated protein with death domain (FADD), and caspase-8 abrogates caspase-8 maturation in T169A mice and leads to the eventual activation of RIPK3-dependent necroptosis in vivo; however, this is insufficient to protect the host, suggesting that timely pyroptosis during early response is specifically required to control infection. These results position RIPK1 T169 phosphorylation as a driver of pyroptotic cell death critical for host defense.
Assuntos
Piroptose , Proteína Serina-Treonina Quinases de Interação com Receptores , Yersiniose , Animais , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Fosforilação , Yersiniose/metabolismo , Yersiniose/microbiologia , Camundongos , Caspase 8/metabolismo , Camundongos Endogâmicos C57BL , Yersinia/metabolismo , HumanosRESUMO
Caspase-8, an aspartate-specific cysteine protease that primarily functions as an initiator caspase to induce apoptosis, can downregulate innate immunity in part by cleaving RIPK1 and IRF3. However, patients with caspase-8 mutations or deficiency develop immunodeficiency and are prone to viral infections. The molecular mechanism underlying this controversy remains unknown. Whether caspase-8 enhances or suppresses antiviral responses against influenza A virus (IAV) infection remains to be determined. Here, we report that caspase-8 is readily activated in A549 and NL20 cells infected with the H5N1, H5N6, and H1N1 subtypes of IAV. Surprisingly, caspase-8 deficiency and two caspase-8 inhibitors, Z-VAD and Z-IETD, do not enhance but rather downregulate antiviral innate immunity, as evidenced by decreased TBK1, IRF3, IκBα, and p65 phosphorylation, decreased IL-6, IFN-ß, MX1, and ISG15 gene expression; and decreased IFN-ß production but increased virus replication. Mechanistically, caspase-8 cleaves and inactivates CYLD, a tumor suppressor that functions as a deubiquitinase. Caspase-8 inhibition suppresses CYLD cleavage, RIG-I and TAK1 ubiquitination, and innate immune signaling. In contrast, CYLD deficiency enhances IAV-induced RIG-I and TAK1 ubiquitination and innate antiviral immunity. Neither caspase-3 deficiency nor treatment with its inhibitor Z-DEVD affects CYLD cleavage or antiviral innate immunity. Our study provides evidence that caspase-8 activation in two human airway epithelial cell lines does not silence but rather enhances innate immunity by inactivating CYLD.
Assuntos
Caspase 8 , Proteína DEAD-box 58 , Enzima Desubiquitinante CYLD , Imunidade Inata , Vírus da Influenza A , Influenza Humana , MAP Quinase Quinase Quinases , Ubiquitinação , Humanos , Enzima Desubiquitinante CYLD/metabolismo , Enzima Desubiquitinante CYLD/genética , Caspase 8/metabolismo , Caspase 8/genética , MAP Quinase Quinase Quinases/metabolismo , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/imunologia , Vírus da Influenza A/imunologia , Proteína DEAD-box 58/metabolismo , Proteína DEAD-box 58/genética , Proteína DEAD-box 58/imunologia , Influenza Humana/imunologia , Influenza Humana/virologia , Células A549 , Animais , Transdução de Sinais/imunologia , Receptores ImunológicosAssuntos
Caspase 8 , Proteína Supressora de Tumor p53 , Regulação para Cima , Humanos , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Caspase 8/metabolismo , Caspase 8/genética , Regulação para Cima/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Antineoplásicos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Apoptose/efeitos dos fármacosRESUMO
PANoptosis is a newly discovered type of cell death characterized by pyroptosis, apoptosis and/or necroptosis and has been implicated in the inflammatory response. Piezo1 is a mechanosensitive ion channel that plays important roles in physiological development and various diseases. However, whether cardiomyocytes undergo PANoptosis during myocardial ischaemia/reperfusion (I/R) injury and the role of Piezo1 in this process remain largely unexplored. In this study, our results revealed that the expression levels of the main components of the PANoptosome, including caspase-8, caspase-3, NLRP3, caspase-1, GSDMD, RIPK1, RIPK3 and MLKL, were significantly upregulated in I/R heart tissues over time, indicating the occurrence of PANoptosis in I/R hearts. Accordingly, Piezo1 expression was significantly upregulated in I/R-injured hearts and hypoxia/reoxygenation (H/R)-treated cardiomyocytes. In contrast, pharmacological inhibition of Piezo1 by the inhibitor GsMTx4 in mice markedly attenuated the I/R-mediated decline in cardiac contractile function and increases in infarct size, apoptosis, oxidative stress and inflammation accompanied by the inhibition of PANoptosis-related mediators in I/R hearts. Consistently, the effects of Piezo1 on calcium influx and PANoptosis were further verified by GsMTx4 and Piezo1 activator Yoda1 in H/R-treated cardiomyocytes in vitro. Moreover, caspase-8 rather than calcium influx was required for H/R-induced PANoptosis in vitro. Mechanistically, Piezo1 interacts with caspase-8, a key initial activator of the PANoptosome complex, which subsequently activates cardiomyocyte PANoptosis, leading to cardiac dysfunction. In summary, these data suggest that Piezo1 is a new cardiac mechanosensor that promotes cardiac I/R injury possibly through the caspase-8-mediated activation of cardiomyocyte PANoptosis and highlight that Piezo1 may represent a new target for treating ischaemic heart disease.
Assuntos
Caspase 8 , Canais Iônicos , Camundongos Endogâmicos C57BL , Traumatismo por Reperfusão Miocárdica , Miócitos Cardíacos , Animais , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Caspase 8/metabolismo , Caspase 8/genética , Canais Iônicos/metabolismo , Canais Iônicos/genética , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Camundongos , Masculino , Necroptose , Apoptose , Oligopeptídeos/farmacologia , Venenos de Aranha , Peptídeos e Proteínas de Sinalização IntercelularRESUMO
The interactions induced by RIP homotypic interaction motif (RHIM) are essential for the activation of inflammatory signaling and certain cell death pathways. In the present study, a RHIM-containing protein was identified from Pacific oyster Crassostrea gigas, which harbored a RHIM domain and a Death domain (designated CgRHIM-containing protein). The mRNA transcripts of CgRHIM-containing protein were constitutively expressed in all the examined tissues of oysters, with the highest expression level in mantle. The CgRHIM-containing protein was mainly distributed in the cytoplasm of oyster haemocytes. After high temperature stress, the expression levels of CgRel and CgBcl-2 increased significantly, and reached the peak level at 12 h, then decreased gradually. The transcripts of CgRHIM-containing protein, Cgcaspase-8 and Cgcaspase-3 in haemocytes up-regulated at 12 h after high temperature stress. Moreover, the protein abundance of CgRHIM-containing protein increased significantly, and the ubiquitination level of CgRHIM-containing protein in haemocytes showed an increasing trend at first and then decreased. After the expression of CgRHIM-containing protein was knocked down by siRNA, the mRNA expression levels of CgRel and CgBcl-2 decreased significantly at 6 h after high temperature stress, and those of CgFADD-like, Cgcaspase-8 and Cgcaspase-3, as well as the apoptosis rate of haemocytes also decreased significantly at 24 h. These results indicated that CgRHIM-containing protein might regulate haemocyte apoptosis in oysters upon high temperature stress via mediating the expression of Rel, Bcl-2 and caspase-8/3.
Assuntos
Apoptose , Crassostrea , Hemócitos , Animais , Hemócitos/metabolismo , Hemócitos/fisiologia , Crassostrea/imunologia , Crassostrea/genética , Resposta ao Choque Térmico , Estresse Fisiológico , Temperatura Alta , Caspase 8/metabolismo , Caspase 8/genética , Caspase 3/metabolismoRESUMO
Dendritic cells (DCs) are crucial for initiating the acquired immune response to infectious diseases such as tuberculosis. Mycobacterium tuberculosis has evolved strategies to inhibit activation of the NLRP3 inflammasome in macrophages via its serine/threonine protein kinase, protein kinase F (PknF). It is not known whether this pathway is conserved in DCs. In this study, we show that the pknF deletion mutant of M. tuberculosis (MtbΔpknF) compared with wild-type M. tuberculosis-infected cells induces increased production of IL-1ß and increased pyroptosis in murine bone marrow-derived DCs (BMDCs). As shown for murine macrophages, the enhanced production of IL-1ß postinfection of BMDCs with MtbΔpknF is dependent on NLRP3, ASC, and caspase-1/11. In contrast to macrophages, we show that MtbΔpknF mediates RIPK3/caspase-8-dependent IL-1ß production in BMDCs. Consistently, infection with MtbΔpknF results in increased activation of caspase-1 and caspase-8 in BMDCs. When compared with M. tuberculosis-infected cells, the IL-6 production by MtbΔpknF-infected cells was unchanged, indicating that the mutant does not affect the priming phase of inflammasome activation. In contrast, the activation phase was impacted because the MtbΔpknF-induced inflammasome activation in BMDCs depended on potassium efflux, chloride efflux, reactive oxygen species generation, and calcium influx. In conclusion, PknF is important for M. tuberculosis to evade NLRP3 inflammasome-mediated activation of caspase-1 and RIPK3/caspase-8 pathways in BMDCs.
Assuntos
Caspase 1 , Caspase 8 , Células Dendríticas , Inflamassomos , Interleucina-1beta , Mycobacterium tuberculosis , Proteína 3 que Contém Domínio de Pirina da Família NLR , Proteína Serina-Treonina Quinases de Interação com Receptores , Animais , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Camundongos , Inflamassomos/imunologia , Inflamassomos/metabolismo , Caspase 8/metabolismo , Caspase 8/imunologia , Células Dendríticas/imunologia , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/imunologia , Mycobacterium tuberculosis/imunologia , Caspase 1/metabolismo , Interleucina-1beta/metabolismo , Interleucina-1beta/imunologia , Camundongos Endogâmicos C57BL , Tuberculose/imunologia , Camundongos Knockout , Macrófagos/imunologia , Macrófagos/metabolismoRESUMO
Efficient cellular fusion of mononuclear precursors is the prerequisite for the generation of fully functional multinucleated bone-resorbing osteoclasts. However, the exact molecular factors and mechanisms controlling osteoclast fusion remain incompletely understood. Here we identify RANKL-mediated activation of caspase-8 as early key event during osteoclast fusion. Single cell RNA sequencing-based analyses suggested that activation of parts of the apoptotic machinery accompanied the differentiation of osteoclast precursors into mature multinucleated osteoclasts. A subsequent characterization of osteoclast precursors confirmed that RANKL-mediated activation of caspase-8 promoted the non-apoptotic cleavage and activation of downstream effector caspases that translocated to the plasma membrane where they triggered activation of the phospholipid scramblase Xkr8. Xkr8-mediated exposure of phosphatidylserine, in turn, aided cellular fusion of osteoclast precursors and thereby allowed generation of functional multinucleated osteoclast syncytia and initiation of bone resorption. Pharmacological blockage or genetic deletion of caspase-8 accordingly interfered with fusion of osteoclasts and bone resorption resulting in increased bone mass in mice carrying a conditional deletion of caspase-8 in mononuclear osteoclast precursors. These data identify a novel pathway controlling osteoclast biology and bone turnover with the potential to serve as target for therapeutic intervention during diseases characterized by pathologic osteoclast-mediated bone loss. Proposed model of osteoclast fusion regulated by caspase-8 activation and PS exposure. RANK/RANK-L interaction. Activation of procaspase-8 into caspase-8. Caspase-8 activates caspase-3. Active capase-3 cleaves Xkr8. Local PS exposure is induced. Exposed PS is recognized by the fusion partner. FUSION. PS is re-internalized.
Assuntos
Caspase 8 , Fusão Celular , Osteoclastos , Fosfatidilserinas , Proteínas de Transferência de Fosfolipídeos , Caspase 8/metabolismo , Caspase 8/genética , Animais , Osteoclastos/metabolismo , Fosfatidilserinas/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Proteínas de Transferência de Fosfolipídeos/genética , Camundongos , Camundongos Endogâmicos C57BL , Reabsorção Óssea/metabolismo , Reabsorção Óssea/patologia , Reabsorção Óssea/genética , Diferenciação Celular , Ligante RANK/metabolismoRESUMO
Polycystic ovarian syndrome (PCOS) is related to pro-apoptotic and pro-inflammatory conditions generated by Endoplasmic reticulum (ER) stress. This study aimed to determine the effect of Astaxanthin (ASX), as carotenoid with potent antioxidant and anti-inflammatory properties, on serum inflammatory markers, apoptotic factors and ER stress-apoptotic genes in peripheral blood mononuclear cells (PBMCs) of women with PCOS. This randomized, double-blind clinical trial included 56 PCOS patients aged 18-40. For 8 weeks, subjects were randomly assigned to one of two groups: either 12 mg ASX (n = 28) or placebo (n = 28). Real-time PCR was used to quantify gene expression associated with ER stress-apoptosis in PCOS women's PBMCs. The levels of TNF-α, IL18, IL6 and CRP were determined by obtaining blood samples from all patients before and after the intervention using Enzyme-linked immunosorbent assay (ELISA). Also, the levels of active caspase-3 and caspase-8 were detected in the PBMC by ELISA kit. Furthermore, we evaluated the efficacy of ASX on disease symptoms. Following the 8-week intervention, ASX supplementation was able to reduce the expression of GRP78 (p = 0.051), CHOP (p = 0.008), XBP1 (p = 0.002), ATF4 (0.038), ATF6 (0.157) and DR5 (0.016) when compared to the placebo. However, this decrease was not statistically significant for ATF6 (p = 0.067) and marginally significant for GRP78 (p = 0.051). The levels of TNF-α (p = 0.009), IL-18 (p = 0.003), IL-6 (p = 0.013) and active caspase-3 (p = 0.012) were also statistically significant lower in the therapy group. However, there was no significant difference in CRP (p = 0.177) and caspase-8 (p = 0.491) levels between the treatment and control groups. In our study, ASX had no significant positive effect on BMI, hirsutism, hair loss and regularity of the menstrual cycle. It appears that ASX may benefit PCOS by changing the ER stress-apoptotic pathway and reducing serum inflammatory markers; however, additional research is required to determine this compound's potential relevance.
Assuntos
Apoptose , Biomarcadores , Suplementos Nutricionais , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático , Leucócitos Mononucleares , Síndrome do Ovário Policístico , Xantofilas , Humanos , Feminino , Síndrome do Ovário Policístico/tratamento farmacológico , Síndrome do Ovário Policístico/sangue , Síndrome do Ovário Policístico/genética , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Xantofilas/farmacologia , Xantofilas/administração & dosagem , Xantofilas/uso terapêutico , Adulto , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Biomarcadores/sangue , Adulto Jovem , Adolescente , Método Duplo-Cego , Regulação da Expressão Gênica/efeitos dos fármacos , Fator de Necrose Tumoral alfa/sangue , Interleucina-18/sangue , Interleucina-18/genética , Inflamação/sangue , Inflamação/tratamento farmacológico , Inflamação/genética , Interleucina-6/sangue , Interleucina-6/genética , Caspase 8/genética , Caspase 8/metabolismoRESUMO
Pathogen infection of host cells triggers an inflammatory cell death termed pyroptosis via activation of inflammatory caspases. However, blockade of immune signaling kinases by the Yersinia virulence factor YopJ triggers cell death involving both apoptotic caspase-8 and pyroptotic caspase-1. While caspase-1 is normally activated within inflammasomes, Yersinia-induced caspase-1 activation is independent of known inflammasome components. We report that caspase-8 is an essential initiator, while caspase-1 is an essential amplifier of its own activation through two feed-forward loops involving caspase-1 auto-processing and caspase-1-dependent activation of gasdermin D and NLPR3. Notably, while Yersinia-induced caspase-1 activation and cell death are inflammasome-independent, IL-1ß release requires NLPR3 inflammasome activation. Mechanistically, caspase-8 is rapidly activated within multiple foci throughout the cell, followed by assembly of a canonical inflammasome speck, indicating that caspase-8 and canonical inflammasome complex assemblies are kinetically and spatially distinct. Our findings reveal that functionally interconnected but distinct death complexes mediate pyroptosis and IL-1ß release in response to pathogen blockade of immune signaling.
Assuntos
Caspase 1 , Caspase 8 , Inflamassomos , Interleucina-1beta , Proteínas de Ligação a Fosfato , Piroptose , Transdução de Sinais , Yersinia , Interleucina-1beta/metabolismo , Caspase 8/metabolismo , Animais , Caspase 1/metabolismo , Inflamassomos/metabolismo , Yersinia/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Camundongos , Humanos , Proteínas de Bactérias/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Yersiniose/imunologia , Yersiniose/microbiologia , Yersiniose/metabolismo , GasderminasRESUMO
c-FLIP functions as a dual regulator of apoptosis and inflammation, yet its implications in Zika virus (ZIKV) infection remain partially understood, especially in the context of ZIKV-induced congenital Zika syndrome (CZS) where both apoptosis and inflammation play pivotal roles. Our findings demonstrate that c-FLIP promotes ZIKV infection in placental cells and myeloid-derived macrophages, involving inflammation and caspase-8/3-mediated apoptosis. Moreover, our observations reveal that c-FLIP augments ZIKV infection in multiple tissues, including blood cell, spleen, uterus, testis, and the brain of mice. Notably, the partial deficiency of c-FLIP provides protection to embryos against ZIKV-induced CZS, accompanied by a reduction in caspase-3-mediated apoptosis. Additionally, we have found a distinctive parental effect of c-FLIP influencing ZIKV replication in fetal heads. In summary, our study reveals the critical role of c-FLIP as a positive regulator in caspase-8/3-mediated apoptosis during ZIKV infection, significantly contributing to the development of CZS.
Assuntos
Apoptose , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD , Caspase 3 , Caspase 8 , Infecção por Zika virus , Zika virus , Infecção por Zika virus/virologia , Infecção por Zika virus/metabolismo , Infecção por Zika virus/patologia , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Animais , Camundongos , Caspase 8/metabolismo , Feminino , Humanos , Caspase 3/metabolismo , Gravidez , Placenta/virologia , Placenta/metabolismo , Placenta/patologia , Camundongos Endogâmicos C57BL , Replicação Viral , Camundongos KnockoutRESUMO
The main causative factors of diabetic nephropathy (DN), a common complication of diabetes mellitus, are metabolic abnormalities and hemodynamic changes. However, studies have shown that the immune-inflammatory response also plays an important role in DN pathogenesis. Therefore, in this study, we analyzed the causal relationship and immune infiltration between inflammatory factors and DN using Mendelian randomization (MR) and bioinformatics techniques. We analyzed the causal relationship between 91 inflammatory factors and DN using two-sample MR dominated by the results of inverse variance-weighted analysis. Based on the MR analysis, the immune mechanism of inflammatory factors in DN was further explored using immune cell infiltration analysis. MR analysis indicated a positive causal relationship between DN and IL1A, caspase 8 (CASP8), macrophage colony-stimulating factor 1, IL10, STAM-binding protein, and tumor necrosis factor ligand superfamily member 12 (TNFSF12) and a negative causal relationship between DN and cystatin D, fibroblast growth factor 19, neurturin, and TNFSF14. The pathogenic mechanism of CASP8 may involve the recruitment of CD4+ T cells and macrophages for DN infiltration. In this study, we found a causal relationship between DN and IL1A, CASP8, macrophage colony-stimulating factor 1, IL10, STAM-binding protein, TNFSF12, cystatin D, fibroblast growth factor 19, neurturin, and TNFSF14. Bioinformatic immune infiltration analysis further revealed that CASP8 regulates DN by influencing the infiltration of immune cells, such as T cells and macrophages.
Assuntos
Biologia Computacional , Diabetes Mellitus Tipo 2 , Nefropatias Diabéticas , Análise da Randomização Mendeliana , Humanos , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/sangue , Nefropatias Diabéticas/imunologia , Biologia Computacional/métodos , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/complicações , Fatores de Risco , Inflamação/genética , Inflamação/sangue , Caspase 8/genéticaRESUMO
We aimed to elucidate the toxic effects and biological activities of 3-phenoxybenzoic acid (3PBA) and its metabolite products.Numerous in silico methods were used to identify the toxic effects and biological activities of 3PBA, including PASS online, molecular docking, ADMETlab 2.0, ADMESWISS, MetaTox, and molecular dynamic simulation.Ten metabolite products were identified via Phase II reactions (O-glucuronidation, O-sulfation, and methylation).All of the investigated compounds were followed by Lipinski's rule, indicating that they were stimulants or inducers of hazardous processes.Because of their high gastrointestinal absorption and ability to reach the blood-brain barrier, the studied compounds' physicochemical and pharmacokinetic properties matched existing evidence of harmful effects, including haematemesis, reproductive dysfunction, allergic dermatitis, toxic respiration, and neurotoxicity.The studied compounds have been linked to the apoptotic pathway, the reproductivity system, neuroendocrine disruptors, phospholipid-translocating ATPase inhibitors, and JAK2 expression.An O-glucuronidation metabolite product demonstrated higher binding affinity and interaction with CYP2C9, CYP3A4, caspase 3, and caspase 8 than 3PBA and other metabolite products, whereas metabolite products from methylation were predominant and more toxic.Our in silico findings partly meet the 3Rs principle by minimizing animal testing before more study is needed to identify the detrimental effects of 3PBA on other organs (liver, kidneys).Future research directions may involve experimental validation of in silico predictions, elucidation of molecular mechanisms, and exploration of therapeutic interventions.These findings contribute to our understanding of the toxicological profile of 3PBA and its metabolites, which has implications for risk assessment and regulatory decisions.
Key properties & pharmacokinetics of 3PBA & its metabolites were reportedMetabolite products from methylation were predominant and more toxicMain toxics: haematemesis, reproductive dysfunction, toxic respiration, dermatitis.
Assuntos
Benzoatos , Simulação por Computador , Benzoatos/química , Benzoatos/metabolismo , Benzoatos/toxicidade , Modelos Moleculares , Conformação Molecular , Fenômenos Químicos , Caspase 3/química , Caspase 3/metabolismo , Caspase 8/química , Caspase 8/metabolismo , Sítios de Ligação de Anticorpos , Citocromo P-450 CYP3A/química , Citocromo P-450 CYP3A/metabolismoRESUMO
Perturbation of the apoptosis and necroptosis pathways critically influences embryogenesis. Receptor-associated protein kinase-1 (RIPK1) interacts with Fas-associated via death domain (FADD)-caspase-8-cellular Flice-like inhibitory protein long (cFLIPL) to regulate both extrinsic apoptosis and necroptosis. Here, we describe Ripk1-mutant animals (Ripk1R588E [RE]) in which the interaction between FADD and RIPK1 is disrupted, leading to embryonic lethality. This lethality is not prevented by further removal of the kinase activity of Ripk1 (Ripk1R588E K45A [REKA]). Both Ripk1RE and Ripk1REKA animals survive to adulthood upon ablation of Ripk3. While embryonic lethality of Ripk1RE mice is prevented by ablation of the necroptosis effector mixed lineage kinase-like (MLKL), animals succumb to inflammation after birth. In contrast, Mlkl ablation does not prevent the death of Ripk1REKA embryos, but animals reach adulthood when both MLKL and caspase-8 are removed. Ablation of the nucleic acid sensor Zbp1 largely prevents lethality in both Ripk1RE and Ripk1REKA embryos. Thus, the RIPK1-FADD interaction prevents Z-DNA binding protein-1 (ZBP1)-induced, RIPK3-caspase-8-mediated embryonic lethality, affected by the kinase activity of RIPK1.
Assuntos
Caspase 8 , Proteína de Domínio de Morte Associada a Fas , Inflamação , Proteína Serina-Treonina Quinases de Interação com Receptores , Animais , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Proteína de Domínio de Morte Associada a Fas/metabolismo , Inflamação/metabolismo , Inflamação/patologia , Camundongos , Caspase 8/metabolismo , Proteínas Quinases/metabolismo , Apoptose , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Necroptose , Ligação Proteica , Camundongos Endogâmicos C57BLRESUMO
Z-DNA binding protein 1 (ZBP1) has important functions in anti-viral immunity and in the regulation of inflammatory responses. ZBP1 induces necroptosis by directly engaging and activating RIPK3, however, the mechanisms by which ZBP1 induces inflammation and in particular the role of RIPK1 and the contribution of cell death-independent signaling remain elusive. Here we show that ZBP1 causes skin inflammation by inducing RIPK3-mediated necroptosis and RIPK1-caspase-8-mediated apoptosis in keratinocytes. ZBP1 induced TNFR1-independent skin inflammation in mice with epidermis-specific ablation of FADD by triggering keratinocyte necroptosis. Moreover, transgenic expression of C-terminally truncated constitutively active ZBP1 (ZBP1ca) in mouse epidermis caused skin inflammation that was only partially inhibited by abrogation of RIPK3-MLKL-dependent necroptosis and fully prevented by combined deficiency in MLKL and caspase-8. Importantly, ZBP1ca induced caspase-8-mediated skin inflammation by RHIM-dependent but kinase activity-independent RIPK1 signaling. Furthermore, ZBP1ca-induced inflammatory cytokine production in the skin was completely prevented by combined inhibition of apoptosis and necroptosis arguing against a cell death-independent pro-inflammatory function of ZBP1. Collectively, these results showed that ZBP1 induces inflammation by activating necroptosis and RIPK1 kinase activity-independent apoptosis.
Assuntos
Apoptose , Caspase 8 , Inflamação , Queratinócitos , Necroptose , Proteínas de Ligação a RNA , Proteína Serina-Treonina Quinases de Interação com Receptores , Animais , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Camundongos , Caspase 8/metabolismo , Inflamação/patologia , Inflamação/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Queratinócitos/metabolismo , Proteína de Domínio de Morte Associada a Fas/metabolismo , Transdução de Sinais , Humanos , Proteínas Quinases/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
Osteoclasts are multinucleated cells of hematopoietic origin, with a pivotal role in bone development and remodeling. Failure in osteoclast differentiation and activation leads to various bone disorders; thus, attention has focused on a search of molecules involved in osteoclast regulatory pathways. Caspase-8 appears to be an interesting candidate for further exploration, due to its potential function in bone development and homeostasis. Mouse bone marrow cells were differentiated into osteoclasts by RANKL stimulation. Increased activation of caspase-8 and its downstream executioner caspases (caspase-3 and caspase-6) was found during osteoclastogenesis. Subsequent inhibition of caspase-8, caspase-3, or caspase-6, respectively, during osteoclast differentiation showed distinct changes in the formation of TRAP-positive multinucleated cells and reduced expression of osteoclast markers including Acp5, Ctsk, Dcstamp, and Mmp9. Analysis of bone matrix resorption confirmed significantly reduced osteoclast function after caspase inhibition. The results clearly showed the role of caspases in the proper development of osteoclasts and contributed new knowledge about non-apoptotic function of caspases.
Assuntos
Células da Medula Óssea , Inibidores de Caspase , Diferenciação Celular , Osteoclastos , Ligante RANK , Animais , Camundongos , Células da Medula Óssea/metabolismo , Reabsorção Óssea/metabolismo , Caspase 3/metabolismo , Caspase 6/metabolismo , Caspase 8/metabolismo , Inibidores de Caspase/farmacologia , Células Cultivadas , Camundongos Endogâmicos C57BL , Osteoclastos/metabolismo , Ligante RANK/metabolismo , Fosfatase Ácida Resistente a Tartarato/metabolismoRESUMO
Cardiac ischemia results in anaerobic metabolism and lactic acid accumulation and with time, intracellular and extracellular acidosis. Ischemia and subsequent reperfusion injury (IRI) lead to various forms of programmed cell death. Necroptosis is a major form of programmed necrosis that worsens cardiac function directly and also promotes inflammation by the release of cellular contents. Potential effects of increasing acidosis on programmed cell death and their specific components have not been well studied. While apoptosis is caspase-dependent, in contrast, necroptosis is mediated by the receptor-interacting protein kinases 1 and 3 (RIPK1/3). In our study, we observed that at physiological pH = 7.4, caspase-8 inhibition did not prevent TNFα-induced cell death in mouse cardiac vascular endothelial cells (MVECs) but promoted necroptotic cell death. As expected, necroptosis was blocked by RIPK1 inhibition. However, at pH = 6.5, TNFα induced an apoptosis-like pattern which was inhibited by caspase-8 inhibition. Interestingly phosphorylation of necroptotic molecules RIPK1, RIPK3, and mixed lineage kinase domain-like protein (MLKL) was enhanced in an acidic pH environment. However, RIPK3 and MLKL phosphorylation was self-limited which may have limited their participation in necroptosis. In addition, an acidic pH promoted apoptosis-inducing factor (AIF) cleavage and nuclear translocation. AIF RNA silencing inhibited cell death, supporting the role of AIF in this cell death. In summary, our study demonstrated that the pH of the micro-environment during inflammation can bias cell death pathways by altering the function of necroptosis-related molecules and promoting AIF-mediated cell death. Further insights into the mechanisms by which an acidic cellular micro-environment influences these and perhaps other forms of regulated cell death, may lead to therapeutic strategies to attenuate IRI.
Assuntos
Apoptose , Necroptose , Proteína Serina-Treonina Quinases de Interação com Receptores , Fator de Necrose Tumoral alfa , Animais , Concentração de Íons de Hidrogênio , Apoptose/efeitos dos fármacos , Necroptose/efeitos dos fármacos , Camundongos , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Fator de Necrose Tumoral alfa/metabolismo , Caspase 8/metabolismo , Proteínas Quinases/metabolismo , Proteínas Quinases/genética , Células Cultivadas , Fosforilação , Células Endoteliais/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologiaRESUMO
Coxiella burnetii is an obligate intracellular bacteria that causes the global zoonotic disease Q Fever. Treatment options for chronic infection are limited, and the development of novel therapeutic strategies requires a greater understanding of how C. burnetii interacts with immune signaling. Cell death responses are known to be manipulated by C. burnetii, but the role of caspase-8, a central regulator of multiple cell death pathways, has not been investigated. In this research, we studied bacterial manipulation of caspase-8 signaling and the significance of caspase-8 to C. burnetii infection, examining bacterial replication, cell death induction, and cytokine signaling. We measured caspase, RIPK, and MLKL activation in C. burnetii-infected tumor necrosis factor alpha (TNFα)/cycloheximide-treated THP-1 macrophage-like cells and TNFα/ZVAD-treated L929 cells to assess apoptosis and necroptosis signaling. Additionally, we measured C. burnetii replication, cell death, and TNFα induction over 12 days in RIPK1-kinase-dead, RIPK3-kinase-dead, or RIPK3-kinase-dead-caspase-8-/- bone marrow-derived macrophages (BMDMs) to understand the significance of caspase-8 and RIPK1/3 during infection. We found that caspase-8 is inhibited by C. burnetii, coinciding with inhibition of apoptosis and increased susceptibility to necroptosis. Furthermore, C. burnetii replication was increased in BMDMs lacking caspase-8, but not in those lacking RIPK1/3 kinase activity, corresponding with decreased TNFα production and reduced cell death. As TNFα is associated with the control of C. burnetii, this lack of a TNFα response may allow for the unchecked bacterial growth we saw in caspase-8-/- BMDMs. This research identifies and explores caspase-8 as a key regulator of C. burnetii infection, opening novel therapeutic doors.
Assuntos
Caspase 8 , Coxiella burnetii , Macrófagos , Febre Q , Fator de Necrose Tumoral alfa , Caspase 8/metabolismo , Animais , Fator de Necrose Tumoral alfa/metabolismo , Macrófagos/microbiologia , Macrófagos/metabolismo , Macrófagos/imunologia , Camundongos , Febre Q/microbiologia , Febre Q/imunologia , Febre Q/metabolismo , Humanos , Apoptose , Transdução de Sinais , Linhagem Celular , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Células THP-1RESUMO
Epidemiological evidence showed that serum high perfluorooctane sulfonate (PFOS) levels are associated with multiple eye related diseases, but the potential underlying molecular mechanisms remain poorly understood. Zebrafish and photoreceptor cell (661w) models were used to investigate the molecular mechanism of PFOS induced eye development defects. Our results showed a novel molecular mechanism of PFOS-induced inflammation response-mediated photoreceptor cell death associated with eye development defects. Inhibition of Caspase-8 activation significantly decreased photoreceptor cell death in PFOS exposure. Mechanistically, Toll-like receptor 4 (TLR4) mediates activation of Caspase-8 promote activation of NLR family pyrin domain-containing 3 (NLRP3) inflammasome to elicit maturation of interleukin-1 beta (IL-1ß) via Caspase-1 activation, facilitating photoreceptor cell inflammation damage in PFOS exposure. In addition, we also made a novel finding that Caspase-3 activation was increased via Caspase-8 activation and directly intensified cell death. Our results show the important role of Caspase-8 activation in PFOS induced eye development defects and highlight Caspase-8 mediated activation of the NLRP3 inflammation triggers activation of Caspase-1 and promote the maturation of IL-1ß in retinal inflammatory injury.
Assuntos
Ácidos Alcanossulfônicos , Caspase 8 , Fluorocarbonos , Inflamassomos , Larva , Proteína 3 que Contém Domínio de Pirina da Família NLR , Peixe-Zebra , Animais , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Inflamassomos/metabolismo , Caspase 8/metabolismo , Caspase 8/genética , Ácidos Alcanossulfônicos/toxicidade , Fluorocarbonos/toxicidade , Larva/efeitos dos fármacos , Interleucina-1beta/metabolismo , Olho/efeitos dos fármacos , Poluentes Químicos da Água/toxicidadeRESUMO
Necroptosis is a caspase-independent modality of cell death implicated in many inflammatory pathologies. The execution of this pathway requires the formation of a cytosolic platform that comprises RIPK1 and RIPK3 which, in turn, mediates the phosphorylation of the pseudokinase MLKL (S345 in mouse). The activation of this executioner is followed by its oligomerisation and accumulation at the plasma-membrane where it leads to cell death via plasma-membrane destabilisation and consequent permeabilisation. While the biochemical and cellular characterisation of these events have been amply investigated, the study of necroptosis involvement in vivo in animal models is currently limited to the use of Mlkl-/- or Ripk3-/- mice. Yet, even in many of the models in which the involvement of necroptosis in disease aetiology has been genetically demonstrated, the fundamental in vivo characterisation regarding the question as to which tissue(s) and specific cell type(s) therein is/are affected by the pathogenic necroptotic death are missing. Here, we describe and validate an immunohistochemistry and immunofluorescence-based method to reliably detect the phosphorylation of mouse MLKL at serine 345 (pMLKL-S345). We first validate the method using tissues derived from mice in which Caspase-8 (Casp8) or FADD are specifically deleted from keratinocytes, or intestinal epithelial cells, respectively. We next demonstrate the presence of necroptotic activation in the lungs of SARS-CoV-infected mice and in the skin and spleen of mice bearing a Sharpin inactivating mutation. Finally, we exclude necroptosis occurrence in the intestines of mice subjected to TNF-induced septic shock. Importantly, by directly comparing the staining of pMLKL-345 with that of cleaved Caspase-3 staining in some of these models, we identify spatio-temporal and functional differences between necroptosis and apoptosis supporting a role of RIPK3 in inflammation independently of MLKL versus the role of RIPK3 in activation of necroptosis.
Assuntos
Necroptose , Proteínas Quinases , Animais , Proteínas Quinases/metabolismo , Proteínas Quinases/genética , Camundongos , Fosforilação , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Caspase 8/metabolismo , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Genetic perturbations influencing early eye development can result in microphthalmia, anophthalmia, and coloboma (MAC). Over 100 genes are associated with MAC, but little is known about common disease mechanisms. In this study, we generated induced pluripotent stem cell (iPSC)-derived optic vesicles (OVs) from two unrelated microphthalmia patients and healthy controls. At day 20, 35, and 50, microphthalmia patient OV diameters were significantly smaller, recapitulating the "small eye" phenotype. RNA sequencing (RNA-seq) analysis revealed upregulation of apoptosis-initiating and extracellular matrix (ECM) genes at day 20 and 35. Western blot and immunohistochemistry revealed increased expression of lumican, nidogen, and collagen type IV, suggesting ECM overproduction. Increased apoptosis was observed in microphthalmia OVs with reduced phospho-histone 3 (pH3+) cells confirming decreased cell proliferation at day 35. Pharmacological inhibition of caspase-8 activity with Z-IETD-FMK decreased apoptosis in one patient model, highlighting a potential therapeutic approach. These data reveal shared pathophysiological mechanisms contributing to a microphthalmia phenotype.