scholarly journals Discovery of a caspase cleavage motif antibody reveals insights into noncanonical inflammasome function

2021 ◽  
Vol 118 (12) ◽  
pp. e2018024118
Author(s):  
Christopher W. Davies ◽  
Irma Stowe ◽  
Qui T. Phung ◽  
Hoangdung Ho ◽  
Corey E. Bakalarski ◽  
...  
Keyword(s):  
Inflammatory Cell ◽  
Direct Detection ◽  
Inflammasome Activation ◽  
Caspase Cleavage ◽  
Peptide Recognition ◽  
Recognition Motif ◽  
Caspase 7 ◽  
Caspase Substrates ◽  
Cleavage Motif ◽  
Inflammatory Caspases

Inflammasomes sense a number of pathogen and host damage signals to initiate a signaling cascade that triggers inflammatory cell death, termed pyroptosis. The inflammatory caspases (1/4/5/11) are the key effectors of this process through cleavage and activation of the pore-forming protein gasdermin D. Caspase-1 also activates proinflammatory interleukins, IL-1β and IL-18, via proteolysis. However, compared to the well-studied apoptotic caspases, the identity of substrates and therefore biological functions of the inflammatory caspases remain limited. Here, we construct, validate, and apply an antibody toolset for direct detection of neo-C termini generated by inflammatory caspase proteolysis. By combining rabbit immune phage display with a set of degenerate and defined target peptides, we discovered two monoclonal antibodies that bind peptides with a similar degenerate recognition motif as the inflammatory caspases without recognizing the canonical apoptotic caspase recognition motif. Crystal structure analyses revealed the molecular basis of this strong yet paradoxical degenerate mode of peptide recognition. One antibody selectively immunoprecipitated cleaved forms of known and unknown inflammatory caspase substrates, allowing the identification of over 300 putative substrates of the caspase-4 noncanonical inflammasome, including caspase-7. This dataset will provide a path toward developing blood-based biomarkers of inflammasome activation. Overall, our study establishes tools to discover and detect inflammatory caspase substrates and functions, provides a workflow for designing antibody reagents to study cell signaling, and extends the growing evidence of biological cross talk between the apoptotic and inflammatory caspases.

Abstract P295: The Inflammasome Is Involved in Myocardial Ischemia-Reperfusion Injury

2011 ◽  
Vol 109 (suppl_1) ◽  
Author(s):  
Masafumi Takahashi ◽  
Masanori Kawaguchi ◽  
Fumitake Usui ◽  
Hiroaki Kimura ◽  
Shun'ichiro Taniguchi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Myocardial Ischemia ◽  
Inflammatory Cell ◽  
Bone Marrow Cells ◽  
Cardiac Fibroblasts ◽  
Ischemia Reperfusion ◽  
Inflammatory Cell Infiltration ◽  
Inflammasome Activation ◽  
Caspase 1 ◽  
Myocardial Ischemia Reperfusion

Background: Accumulating evidence indicates that inflammation is involved in the pathophysiology of myocardial ischemia-reperfusion (I/R) injury. However, the mechanism of I/R-initiated inflammation remains to be determined. The inflammasome is a multiprotein complex consisting of nod-like receptor (NLR), apoptosis-associated speck-like adaptor protein (ASC), and caspase-1, and regulates caspase-1-dependent maturation of IL-1beta and IL-18. In the present study, we investigated the role of inflammasome in myocardial I/R injury. Methods and Results: Wild-type (WT), ASC−/−, and caspase-1−/− mice were subjected to 30 min LAD ligation, followed by reperfusion. ASC and caspase-1 were expressed at the site of myocardial I/R injury. Deficiency of ASC and caspase-1 reduced inflammatory responses, such as inflammatory cell infiltration and cytokine expression, and subsequent injuries such as infarct development, myocardial fibrosis, and dysfunction in myocardial I/R injury. To determine the contribution of inflammasome in bone marrow cells, we produced bone marrow transplant mice and found that inflammasome activation was critical not only in bone marrow cells but also in myocardial resident cells. Since myocardial damage was observed before the inflammatory cell infiltration after I/R, we hypothesized that myocardial resident cells are responsible for an initial activation of inflammasome. To test this hypothesis, we examined whether hypoxia/reoxygenation (H/R) stimuli could induce inflammasome activation in cardiac fibroblasts and cardiomyocytes in vitro. Interestingly, inflammasome activation was detected only in cardiac fibroblasts, but not in cardiomyocytes, and mediated through reactive oxygen species (ROS) and potassium efflux. Conclusion: These findings indicate that inflammasome activation in cardiac fibroblasts is essential for inflammation and injury after myocardial I/R, and suggest that the inflammasome is a potential novel therapeutic target for myocardial I/R injury.

scholarly journals Caspase-8 mediates inflammation and disease in rodent malaria

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Larissa M. N. Pereira ◽  
Patrícia A. Assis ◽  
Natalia M. de Araújo ◽  
Danielle F. Durso ◽  
Caroline Junqueira ◽  
...  
Keyword(s):  
Septic Shock ◽  
Human Disease ◽  
Caspase 8 ◽  
Inflammasome Activation ◽  
Plasmodium Chabaudi ◽  
Experimental Cerebral Malaria ◽  
Rodent Malaria ◽  
Canonical Pathways ◽  
Inflammatory Caspases

Abstract Earlier studies indicate that either the canonical or non-canonical pathways of inflammasome activation have a limited role on malaria pathogenesis. Here, we report that caspase-8 is a central mediator of systemic inflammation, septic shock in the Plasmodium chabaudi-infected mice and the P. berghei-induced experimental cerebral malaria (ECM). Importantly, our results indicate that the combined deficiencies of caspases-8/1/11 or caspase-8/gasdermin-D (GSDM-D) renders mice impaired to produce both TNFα and IL-1β and highly resistant to lethality in these models, disclosing a complementary, but independent role of caspase-8 and caspases-1/11/GSDM-D in the pathogenesis of malaria. Further, we find that monocytes from malaria patients express active caspases-1, -4 and -8 suggesting that these inflammatory caspases may also play a role in the pathogenesis of human disease.

scholarly journals A Type III Effector NleF from EHEC Inhibits Epithelial Inflammatory Cell Death by Targeting Caspase-4

2017 ◽  
Vol 2017 ◽  
pp. 1-11 ◽  
Author(s):  
Ting Song ◽  
Kaiwu Li ◽  
Wei Zhou ◽  
Jing Zhou ◽  
Yuan Jin ◽  
...  
Keyword(s):  
Cell Death ◽  
Inflammatory Cell ◽  
Gastrointestinal Disease ◽  
Highly Pathogenic ◽  
E Coli ◽  
Caspase 1 ◽  
T3ss Effector ◽  
Inflammatory Caspases ◽  
Epithelial Cell Death ◽  
Host Inflammatory Response

EnterohemorrhagicE. coli(EHEC) is a highly pathogenic bacterial strain capable of inducing severe gastrointestinal disease. Here, we show that EHEC uses the T3SS effector NleF to counteract the host inflammatory response by dampening caspase-4-mediated inflammatory epithelial cell death and by preventing the production of IL-1β. The other two inflammatory caspases, caspase-1 and caspase-5, are not involved in EHEC ΔnleF-induced inflammatory cell death. We found that NleF not only interrupted the heterodimerization of caspase-4-p19 and caspase-4-p10, but also inhibited the interaction of caspase-1 and caspase-4. The last four amino acids of the NleF carboxy terminus are essential in inhibiting caspase-4-dependent inflammatory cell death.

scholarly journals Human polymorphisms in GSDMD alter the inflammatory response

2020 ◽  
Vol 295 (10) ◽  
pp. 3228-3238 ◽  
Author(s):  
Joseph K. Rathkey ◽  
Tsan S. Xiao ◽  
Derek W. Abbott
Keyword(s):  
Cell Death ◽  
Genetic Variation ◽  
Posttranslational Modifications ◽  
Apoptotic Cell ◽  
Gene Families ◽  
Inflammasome Activation ◽  
Caspase Cleavage ◽  
Immune Genes ◽  
Functional Consequences ◽  
Innate Immune Genes

Exomic studies have demonstrated that innate immune genes exhibit an even higher degree of variation than the majority of other gene families. However, the phenotypic implications of this genetic variation are not well understood, with effects ranging from hypomorphic to silent to hyperfunctioning. In this work, we study the functional consequences of this variation by investigating polymorphisms in gasdermin D, the key pyroptotic effector protein. We find that, although SNPs affecting potential posttranslational modifications did not affect gasdermin D function or pyroptosis, polymorphisms disrupting sites predicted to be structurally important dramatically alter gasdermin D function. The manner in which these polymorphisms alter function varies from conserving normal pyroptotic function to inhibiting caspase cleavage to disrupting oligomerization and pore formation. Further, downstream of inflammasome activation, polymorphisms that cause loss of gasdermin D function convert inflammatory pyroptotic cell death into immunologically silent apoptotic cell death. These findings suggest that human genetic variation can alter mechanisms of cell death in inflammation.

scholarly journals Death Receptor-induced Apoptosis Reveals a Novel Interplay between the Chromosomal Passenger Complex and CENP-C during Interphase

2007 ◽  
Vol 18 (4) ◽  
pp. 1337-1347 ◽  
Author(s):  
Alison J. Faragher ◽  
Xiao-Ming Sun ◽  
Michael Butterworth ◽  
Nick Harper ◽  
Mike Mulheran ◽  
...  
Keyword(s):  
Death Receptor ◽  
Caspase 8 ◽  
Cleavage Sites ◽  
Caspase Activation ◽  
Functional Link ◽  
Chromosomal Passenger Complex ◽  
Caspase Cleavage ◽  
Chromosomal Passenger ◽  
Caspase 7 ◽  
Induced Apoptosis

Despite the fact that the chromosomal passenger complex is well known to regulate kinetochore behavior in mitosis, no functional link has yet been established between the complex and kinetochore structure. In addition, remarkably little is known about how the complex targets to centromeres. Here, in a study of caspase-8 activation during death receptor-induced apoptosis in MCF-7 cells, we have found that cleaved caspase-8 rapidly translocates to the nucleus and that this translocation is correlated with loss of the centromere protein (CENP)-C, resulting in extensive disruption of centromeres. Caspase-8 activates cytoplasmic caspase-7, which is likely to be the primary caspase responsible for cleavage of CENP-C and INCENP, a key chromosomal passenger protein. Caspase-mediated cleavage of CENP-C and INCENP results in their mislocalization and the subsequent mislocalization of Aurora B kinase. Our results demonstrate that the chromosomal passenger complex is displaced from centromeres as a result of caspase activation. Furthermore, mutation of the primary caspase cleavage sites of INCENP and CENP-C and expression of noncleavable CENP-C or INCENP prevent the mislocalization of the passenger complex after caspase activation. Our studies provide the first evidence for a functional interplay between the passenger complex and CENP-C.

Evasion of Necroptosis and Inflammasome Activation Promotes Myeloid Leukemogenesis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2856-2856
Author(s):  
Ulrike Höckendorf ◽  
Yabal Monica ◽  
Christian Peschel ◽  
Philipp J. Jost
Keyword(s):  
Stem Cells ◽  
Cell Death ◽  
Hematopoietic Stem Cells ◽  
Progenitor Cells ◽  
Protein Level ◽  
Inflammatory Cell ◽  
Leukemic Cells ◽  
Inflammasome Activation ◽  
Short Term ◽  
Hematopoietic Stem

Abstract Acute myeloid leukemia (AML) is a heterogeneous group of hematopoietic neoplasms driven partly by the loss of differentiation and theblockade of cell death. AML is sustained by leukemia-initiating cells (LICs) that arise from pre-leukemic hematopoietic stem and progenitor cells (HSPCs) that carry genetic alterations being selected for during leukemogenesis. The resistance of LICs to standard chemotherapies presents a major clinical challenge as they eventually cause disease relapse and death. Understanding the mechanisms of LIC resistance to undergoing cell death is therefore critical for a curative therapy of AML. While the regulatory factors that maintain HSPC proliferation and differentiation under normal conditions are well understood, significantly less is known about how LIC fate is regulated. As many hematopoietic disorders are characterized by the overproduction of pro-inflammatory cytokines, we hypothesized that necroptosis controlled cytokine secretion and inflammatory cell death might influence AML development. We therefore addressed the role of MLKL and XIAP in AML and tested whether deletion of Mlkl or Xiap would affect disease progression. Here we show that MLKL limits oncogene-mediated leukemogenesis by promoting the inflammatory cell death of common myeloid progenitors (CMPs) and short-term hematopoietic stem cells (HSCs) in experimental mice. Upon oncogenic stress MLKL-dependent necroptosis and subsequent inflammasome activation were triggered, promoting the production of IL-1β, a potent stimulator of HSPC differentiation and maturation, thus, suppressing the emergence of LICs and limiting leukemogenesis. In a murine bone marrow transplantation model of AML the absence of MLKL accelerated AML development significantly. The enhanced disease was due to the expansion of common myeloid progenitors (CMPs) and short-term hematopoietic stem cells (ST-HSCs), being the cellular compartments to contain LICs. The survival advantage of Mlkl-/- HSPCs became apparent in colony-forming assays and liquid cultures specifically within the CMP and ST-HSC compartments. Sorted ST-HSCs from Mlkl-/- produced more GEMM colonies than WT, the colony type harboring the multipotential myeloid progenitor cells, and both ST-HSCs and CMPs retained significantly more lineage-negative cells in liquid culture. In addition, Mlkl-/- colonies showed a reduction in propidium iodide (PI)-positive dead cells compared with WT colonies. Importantly, WT cells showed caspase activation and produced substantial amounts of the inflammatory cytokine IL-1β which was severely blunted by Mlkl deficiency. We also observed reduced expression of MLKL in leukemic cells on both mRNA and protein level, implying that suppression of cell death was beneficial for the survival of LICs. In contrast, deletion of Xiap did not alter survival or differentiation of leukemic cells when compared with WT cells. Furthermore, XIAP was not differentially expressed on mRNA or protein level compared with WT, indicating that XIAP does not play a critical role in leukemogenesis. In agreement with the murine data, gene expression analysis from primary leukemia cells from two large patient cohorts newly diagnosed with AML showed significantly lower expression of MLKL, but not XIAP, in a variety of AML subtypes compared to healthy controls. Overall, our data demonstrate a key role for MLKL-mediated cell death and activation of the inflammasome in AML and represents a novel tumor-suppressive mechanism. Disclosures Peschel: MophoSys: Honoraria.

scholarly journals Assessment of Caspase Activities in Intact Apoptotic Thymocytes Using Cell-Permeable Fluorogenic Caspase Substrates

2000 ◽  
Vol 191 (11) ◽  
pp. 1819-1828 ◽  
Author(s):  
Akira Komoriya ◽  
Beverly Z. Packard ◽  
Martin J. Brown ◽  
Ming-Lei Wu ◽  
Pierre A. Henkart
Keyword(s):  
Apoptotic Cell ◽  
Apoptotic Cells ◽  
Caspase Activation ◽  
Peptide Recognition ◽  
Caspase Substrates ◽  
Intracellular Target ◽  
Recognition Motifs ◽  
In Vitro Treatment ◽  
Fluoromethyl Ketone

To detect caspase activities in intact apoptotic cells at the single cell level, cell-permeable fluorogenic caspase substrates were synthesized incorporating the optimal peptide recognition motifs for caspases 1, 3/7, 6, 8, and 9. Caspase activities were then assessed at various times after in vitro treatment of mouse thymocytes with dexamethasone or anti-Fas antibody. Dexamethasone induced the following order of appearance of caspase activities as judged by flow cytometry: LEHDase, WEHDase, VEIDase, IETDase, and DEVDase. Since the relative order of caspases 3 (DEVDase) and 6 (VEIDase) in the cascade has been controversial, this caspase activation order was reexamined using confocal microscopy. The VEIDase activity appeared before DEVDase in every apoptotic cell treated with dexamethasone. In contrast, anti-Fas stimulation altered this sequence: IETDase was the first measurable caspase activity and DEVDase preceded VEIDase. In an attempt to determine the intracellular target of the potent antiapoptotic agent carbobenzoxy-valyl-alanyl-aspartyl(β-methyl ester)-fluoromethyl ketone (Z-VAD[OMe]-FMK), we examined its ability to inhibit previously activated intracellular caspases. However, no significant reductions of these activities were observed. These fluorogenic caspase substrates allow direct observation of the caspase cascade in intact apoptotic cells, showing that the order of downstream caspase activation is dependent on the apoptotic stimulus.

scholarly journals MyD88 in Macrophages Enhances Liver Fibrosis by Activation of NLRP3 Inflammasome in HSCs

2021 ◽  
Vol 22 (22) ◽  
pp. 12413
Author(s):  
Shuang Ge ◽  
Wei Yang ◽  
Haiqiang Chen ◽  
Qi Yuan ◽  
Shi Liu ◽  
...  
Keyword(s):  
Liver Fibrosis ◽  
Nlrp3 Inflammasome ◽  
Inflammatory Cell ◽  
Myeloid Cells ◽  
Inflammasome Activation ◽  
Potential Candidate ◽  
Pyrin Domain ◽  
Nlrp3 Inflammasome Activation ◽  
Candidate Target

Chronic liver disease mediated by the activation of hepatic stellate cells (HSCs) leads to liver fibrosis. The signal adaptor MyD88 of Toll-like receptor (TLR) signaling is involved during the progression of liver fibrosis. However, the specific role of MyD88 in myeloid cells in liver fibrosis has not been thoroughly investigated. In this study, we used a carbon tetrachloride (CCl4)-induced mouse fibrosis model in which MyD88 was selectively depleted in myeloid cells. MyD88 deficiency in myeloid cells attenuated liver fibrosis in mice and decreased inflammatory cell infiltration. Furthermore, deficiency of MyD88 in macrophages inhibits the secretion of CXC motif chemokine 2 (CXCL2), which restrains the activation of HSCs characterized by NLR Family Pyrin Domain Containing 3 (NLRP3) inflammasome activation. Moreover, targeting CXCL2 by CXCR2 inhibitors attenuated the activation of HSCs and reduced liver fibrosis. Thus, MyD88 may represent a potential candidate target for the prevention and treatment of liver fibrosis.

scholarly journals SARS-CoV-2 induces inflammasome-dependent pyroptosis and downmodulation of HLA-DR in human monocytes

2020 ◽  
Author(s):  
André C. Ferreira ◽  
Vinicius Cardoso Soares ◽  
Isaclaudia G. de Azevedo-Quintanilha ◽  
Suelen da Silva Gomes Dias ◽  
Natalia Fintelman-Rodrigues ◽  
...  
Keyword(s):  
Immune Response ◽  
Cell Death ◽  
Severe Acute Respiratory Syndrome ◽  
Inflammatory Response ◽  
Inflammatory Cell ◽  
Viral Infections ◽  
Relevant Information ◽  
Inflammasome Activation ◽  
Human Monocytes ◽  
Hla Dr

AbstractInfection by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been associated with leukopenia and uncontrolled inflammatory response in critically ill patients. A better comprehension of SARS-CoV-2-induced monocyte death is essential for the identification of therapies capable to control the hyper-inflammation and reduce viral replication in patients with COVID-19. Here, we show that SARS-CoV-2 induces inflammasome activation and cell death by pyroptosis in human monocytes, experimentally infected and from patients under intensive care. Pyroptosis was dependent on caspase-1 engagement, prior to IL-1ß production and inflammatory cell death. Monocytes exposed to SARS-CoV-2 downregulate HLA-DR, suggesting a potential limitation to orchestrate the immune response. Our results originally describe mechanisms by which monocytes, a central cellular component recruited from peripheral blood to respiratory tract, succumb to control severe 2019 coronavirus disease (COVID-19).Author summarySince its emergence in China in late 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused thousands of deaths worldwide. Currently, the number of individuals infected with SARS-CoV-2 and in need of antiviral, anti-inflammatory, anticoagulant and more invasive treatments has overwhelmed the health systems worldwide. In our study, we found that SARS-CoV-2 is capable of inducing inflammatory cell death in human monocytes, one of the main cell types responsible for anti-SARS-CoV-2 immune response. As a consequence of this intracellular inflammatory mechanism (inflammasome engagement), an exacerbated production of inflammatory mediators occurs. The infection also decreases the expression of HLA-DR in monocytes, a molecule related to the orchestration of the immune response in case of viral infections. We also demonstrated that the HIV-1 protease inhibitor, atazanavir (ATV), prevented the uncontrolled inflammatory response, cell death and reduction in HLA-DR expression in SARS-CoV-2-infected monocytes. Our study provides relevant information on the effects of SARS-CoV-2 infection on human monocytes, as well as on the effect of ATV in preventing these pathological effects on the host.

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