Live-cell visualization of gasdermin D-driven pyroptotic cell death

Rathkey, Joseph K.; Benson, Bryan L.; Chirieleison, Steven M.; Yang, Jie; Xiao, Tsan Sam; Dubyak, George R.; Huang, Alex Y.; Abbott, Derek W.

doi:10.1074/jbc.m117.797217

Cited by 56 publications

(57 citation statements)

References 32 publications

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“…The lethality of LPS-induced is largely dependent on the activated caspase-1 encoded by Casp1. [14][15][16] GSDMD is cleaved into pore-forming peptides by activating caspase-1 that subsequently leads to pyroptosis, a dissolved form of cell death releasing inflammatory cytokines like IL-1β and IL-18. [17][18][19] Previous research has revealed that GSDMD expression acted differently with upper gastrointestinal cancer and suppressed gastric cancer development.…”

Section: Introductionmentioning

confidence: 99%

<p>LPS Enhances the Chemosensitivity of Oxaliplatin in HT29 Cells via GSDMD-Mediated Pyroptosis</p>

Liu

Wang

et al. 2020

CMAR

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Introduction: Pyroptosis induced by lipopolysaccharide (LPS) is a dissolved form of cell death. The molecular marker gasdermin D, specifically GSDMD-N, is critically required for the induction of pyroptosis. Recently, there have been studies showing that LPS is closely related to tumor biology. Methods: Specimens from 40 patients with colorectal cancer (CRC) were collected. Eight-to twelve-week-old C57BL6 male mice (n=30) were raised. Immunohistochemistry and Western blot were performed to test the expression of GSDMD. Moreover, cytotoxicity assay, IL-18 and IL-1β ELISA, Annexin V and PI stain, and wound healing assay were also made. Gene Expression Profiling Interactive Analysis (GEPIA) was used to verify the expression of GSDMD and overall survival of CRC patients with a high/low expression of GSDMD. Results: In the research, we showed that the poor prognosis in CRC patients was significantly related to the GSDMD expression and significantly down-regulated in human colorectal cancer (CRC) tissues. Treatment with LPS, but not TNF-α, induced pyroptosis via promoting the expression of GSDMD and GSDMD-N membrane translocation and enhanced chemosensitivity in response to L-OHP in HT29 cells. Furthermore, the enforced expression of GSDMD in HT29 cells reduced cell survival and induced cell death. Discussion: These results of studies suggest that the low expression of GSDMD correlates with a poor CRC prognosis, and that pyroptosis induced by LPS may improve the anti-cancer effect of L-OHP, inhibiting the tumorigenesis of CRC by activating GSDMD. Our findings lay the foundation for further development of GSDMD serving as an important prognostic biomarker and a valid CRC therapeutic target.

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Section: Introductionmentioning

confidence: 99%

<p>LPS Enhances the Chemosensitivity of Oxaliplatin in HT29 Cells via GSDMD-Mediated Pyroptosis</p>

Liu

Wang

et al. 2020

CMAR

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“…We interpreted the stark contrasts between MOA groups on the basis of Ucp3 expression alone to be indicative of remodeled lipid store management in cells, perhaps to alleviate insults on mtDNA integrity or ROS imbalances. This interpretation is supported by the functions of all other biomarkers detected by the LSTNR method (Figure 4I ): (a) Sugct, an enzyme involved in lysine degradation that metabolizes glutarate (Marlaire et al, 2014 ); (b) Acaa1a and Acaa1b, both enzymes that participate in lipid metabolism in peroxisomal compartments (Schram et al, 1987 ; Ferdinandusse et al, 2001 ); (c) Hadhb, a critical subunit of the mitochondrial trifunctional protein that governs fatty acid beta-oxidation inside mitochondria (Spiekerkoetter et al, 2004 ); (d) the gene encoding for lysoplasmalogenase, Tmem86b (Braverman and Moser, 2012 ); (e) Gsdmd, or gasdermin D, a lipid-porating protein that effects pyroptotic cell death during inflammation (Rathkey et al, 2017 ); and (f) Tfam, the master transcription factor for mtDNA (Woo et al, 2012 ; Stiles et al, 2016 ).…”

Section: Resultsmentioning

confidence: 99%

A Leveraged Signal-to-Noise Ratio (LSTNR) Method to Extract Differentially Expressed Genes and Multivariate Patterns of Expression From Noisy and Low-Replication RNAseq Data

2018

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To life scientists, one important feature offered by RNAseq, a next-generation sequencing tool used to estimate changes in gene expression levels, lies in its unprecedented resolution. It can score countable differences in transcript numbers among thousands of genes and between experimental groups, all at once. However, its high cost limits experimental designs to very small sample sizes, usually N = 3, which often results in statistically underpowered analysis and poor reproducibility. All these issues are compounded by the presence of experimental noise, which is harder to distinguish from instrumental error when sample sizes are limiting (e.g., small-budget pilot tests), experimental populations exhibit biologically heterogeneous or diffuse expression phenotypes (e.g., patient samples), or when discriminating among transcriptional signatures of closely related experimental conditions (e.g., toxicological modes of action, or MOAs). Here, we present a leveraged signal-to-noise ratio (LSTNR) thresholding method, founded on generalized linear modeling (GLM) of aligned read detection limits to extract differentially expressed genes (DEGs) from noisy low-replication RNAseq data. The LSTNR method uses an agnostic independent filtering strategy to define the dynamic range of detected aggregate read counts per gene, and assigns statistical weights that prioritize genes with better sequencing resolution in differential expression analyses. To assess its performance, we implemented the LSTNR method to analyze three separate datasets: first, using a systematically noisy in silico dataset, we demonstrated that LSTNR can extract pre-designed patterns of expression and discriminate between “noise” and “true” differentially expressed pseudogenes at a 100% success rate; then, we illustrated how the LSTNR method can assign patient-derived breast cancer specimens correctly to one out of their four reported molecular subtypes (luminal A, luminal B, Her2-enriched and basal-like); and last, we showed the ability to retrieve five different modes of action (MOA) elicited in livers of rats exposed to three toxicants under three nutritional routes by using the LSTNR method. By combining differential measurements with resolving power to detect DEGs, the LSTNR method offers an alternative approach to interrogate noisy and low-replication RNAseq datasets, which handles multiple biological conditions at once, and defines benchmarks to validate RNAseq experiments with standard benchtop assays.

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“…In the later stages of pyroptosis, the cell membrane ruptures, leaving a relatively intact nucleus and diffuse GSDMD-immunopositive cellular debris [13]. Live cell imaging has recapitulated these findings, demonstrating that diffuse cytoplasmic GSDMD immunoreactivity gives way to localized aggregates at the plasma membrane within 15 min of nigericin exposure, which corresponds to the appearance of bleb-like membrane protrusions (i.e., pyroptotic bodies) at the cell surface [17]. Likewise, Salmonella typhimurium and other NLRC4 inflammasome activators trigger pyroptotic body formation, and these can be seen bursting to release cellular contents using time-lapse confocal microscopy [15].…”

Section: Introductionmentioning

confidence: 86%

Activation of the executioner caspases-3 and -7 promotes microglial pyroptosis in models of multiple sclerosis

McKenzie

Fernandes

Doan

et al. 2020

J Neuroinflammation

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Background: Pyroptosis is a type of proinflammatory regulated cell death (RCD) in which caspase-1 proteolytically cleaves gasdermin D (GSDMD) to yield a cytotoxic pore-forming protein. Recent studies have suggested that additional cell death pathways may interact with GSDMD under certain circumstances to execute pyroptosis. Microglia/macrophages in the central nervous system (CNS) undergo GSDMD-associated pyroptosis in multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE) but the contribution of other cell death pathways to this phenomenon is unknown. Herein, we tested the hypothesis that multiple RCD pathways underlie microglial pyroptosis in the context of neuroinflammation. Methods: A siRNA screen of genes with known RCD functions was performed in primary human microglia to evaluate their role in nigericin-induced pyroptosis using supernatant lactate dehydrogenase activity as a read-out of cell lysis. Activation of apoptotic executioner proteins and their contribution to pyroptosis was assessed using semiquantitative confocal microscopy, high-sensitivity ELISA, immunoblot, cell lysis assays, and activity-based fluorescent probes. Quantification of pyroptosis-related protein expression was performed in CNS lesions from patients with progressive MS and mice with MOG 35-55-induced EAE, and in matched controls. Results: Among progressive MS patients, activated caspase-3 was detected in GSDMD immunopositive pyroptotic microglia/macrophages within demyelinating lesions. In the siRNA screen, suppression of caspase-3/7, caspase-1, or GSDMD expression prevented plasma membrane rupture during pyroptosis. Upon exposure to pyroptotic stimuli (ATP or nigericin), human microglia displayed caspase-3/7 activation and cleavage of caspase-3/7-specific substrates (e.g., DFF45, ROCK1, and PARP), with accompanying features of pyroptosis including GSDMD immunopositive pyroptotic bodies, IL-1β release, and membrane rupture. Pyroptosis-associated nuclear condensation and pyroptotic body formation were suppressed by caspase-3/7 inhibition. Pharmacological and siRNA-mediated inhibition of caspase-1 diminished caspase-3/7 activation during pyroptosis. In mice with EAE-associated neurological deficits, activated caspase-3 colocalized with GSDMD immunopositivity in lesion-associated macrophages/microglia.

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Live-cell visualization of gasdermin D-driven pyroptotic cell death

Cited by 56 publications

References 32 publications

<p>LPS Enhances the Chemosensitivity of Oxaliplatin in HT29 Cells via GSDMD-Mediated Pyroptosis</p>

<p>LPS Enhances the Chemosensitivity of Oxaliplatin in HT29 Cells via GSDMD-Mediated Pyroptosis</p>

A Leveraged Signal-to-Noise Ratio (LSTNR) Method to Extract Differentially Expressed Genes and Multivariate Patterns of Expression From Noisy and Low-Replication RNAseq Data

Activation of the executioner caspases-3 and -7 promotes microglial pyroptosis in models of multiple sclerosis

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