Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The VSports app下载. gov means it’s official. Federal government websites often end in . gov or . mil. Before sharing sensitive information, make sure you’re on a federal government site. .

Https

The site is secure V体育官网. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely. .

. 2020 Jul 28;32(4):107959.
doi: 10.1016/j.celrep.2020.107959.

"VSports最新版本" An Apoptotic Caspase Network Safeguards Cell Death Induction in Pyroptotic Macrophages

Nathalia Moraes de Vasconcelos  1 , Nina Van Opdenbosch  2 , Hanne Van Gorp  1 , Rosa Martín-Pérez  3 , Annalisa Zecchin  3 , Peter Vandenabeele  4 , Mohamed Lamkanfi  5
Affiliations

An Apoptotic Caspase Network Safeguards Cell Death Induction in Pyroptotic Macrophages

Nathalia Moraes de Vasconcelos et al. Cell Rep. .

Abstract

Pyroptosis has emerged as a key mechanism by which inflammasomes promote host defense against microbial pathogens and sterile inflammation. Gasdermin D (GSDMD)-mediated cell lysis is a hallmark of pyroptosis, but our understanding of cell death signaling during pyroptosis is fragmented VSports手机版. Here, we show that independently of GSDMD-mediated plasma membrane permeabilization, inflammasome receptors engage caspase-1 and caspase-8, both of which redundantly promote activation of apoptotic executioner caspase-3 and caspase-7 in pyroptotic macrophages. Impaired GSDMD pore formation downstream of caspase-1 and caspase-8 activation suffices to unmask the apoptotic phenotype of pyroptotic macrophages. Combined inactivation of initiator caspase-1 and caspase-8, or executioner caspase-3 and caspase-7, is required to abolish inflammasome-induced DEVDase activity during pyroptosis and in apoptotic Gsdmd-/- cells. Collectively, these results unveil a robust apoptotic caspase network that is activated in parallel to GSDMD-mediated plasma membrane permeabilization and safeguards cell death induction in pyroptotic macrophages. .

Keywords: GSDMD; apoptosis; caspase-1; caspase-3; caspase-7; caspase-8; inflammasome; pyroptosis. V体育安卓版.

PubMed Disclaimer

Conflict of interest statement

Declaration of Interests N. V. O. , R. M. -P. , A. Z. , and M VSports最新版本. L. are/were employees of Janssen Pharmaceutica. The authors declare no other competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Pyroptosis Features a Caspase-3/7 Signature (A and C) Macrophages of the indicated genotypes were left untreated or stimulated with LeTx (A) or FlaTox (C) in media containing the caspase-3/7 activity (DEVD) probe and imaged on an Incucyte platform. (B) Macrophages of the indicated genotypes were left untreated or pretreated with MG132 (10 μM) for 30 min prior to being stimulated with LeTx in media containing the DEVD probe. Cells were imaged over time on an Incucyte platform. (D) B6Nlrp1b+ macrophages (upper panel) or macrophages of the indicated genotypes (lower panel) were treated with LeTx, FlaTox, staurosporine, or TNF+CHX for 2 h, and cell lysates were immunoblotted for the indicated proteins. Results from Incucyte experiments are plotted as the number of positive cells relative to a PI-stained, Triton-x100-treated well (considered 100%). Values represent mean ± SD of technical duplicates of a representative experiment from three biological repeats.
Figure 2
Figure 2
Caspase-1 and Caspase-8 promote Activation of Caspase-3 and Caspase-7 during Pyroptosis (A and B) Macrophages of the indicated genotypes were left untreated or received TAT-Cre (as described in STAR Methods), and subsequently received FlaTox (A) or LeTx (B) or were left untreated in media containing the caspase-3/7 activity (DEVD) probe, and were imaged on an Incucyte platform. (C–F) Macrophages of the indicated genotypes were left untreated or stimulated with LeTx (C), FlaTox (D, E), or log-phase S. Typhimurium (F) in media containing the caspase-3/7 activity (DEVD) probe and imaged on an Incucyte platform. Results from Incucyte experiments are plotted as the number of positive cells relative to a PI-stained, Triton-x100-treated well (considered 100%). Values represent mean ± SD of technical duplicates of a representative experiment from three biological repeats.
Figure 3
Figure 3
Defective GSDMD Pore Formation Unveils Activation of an Apoptotic Caspase Network by the Nlrp1b and Nlrc4 Inflammasomes (A, C, E, and J) Macrophages of the indicated genotypes were left untreated or stimulated with LeTx for 2 h. Culture supernatants were analyzed for LDH activity (A), cells were imaged under a confocal microscope (C) or analyzed by fluorescence-activated cell sorting (FACS) for Annexin-V/PI positivity (E), and cell lysates were immunoblotted for the indicated proteins (J). (B, D, F, and J) Macrophages of the indicated genotypes were left untreated or stimulated with FlaTox for 2 h. Culture supernatants were analyzed for LDH activity (B), cells were imaged under a confocal microscope (D) or analyzed by FACS for Annexin-V/PI positivity (F), and cell lysates were immunoblotted for the indicated proteins (J). (G and H) Macrophages of the indicated genotypes were left untreated or stimulated with LeTx (G) or FlaTox (H) in media containing DEVD probe and PI and imaged on an Incucyte platform. (I) GSDMDI105N knockin homozygous (GSDMD I105Nki/ki) or wild-type (GSDMD I105N+/+) macrophages were treated with FlaTox in media containing DEVD probe and PI and imaged on an Incucyte platform. Percentages of all Incucyte experiments were calculated as the number of positive cells relative to a PI-stained, Triton-x100-treated well (considered 100%). Values represent mean ± SD of technical duplicates of a representative experiment from three biological repeats. All scale bars represent 10 μm.
Figure 4
Figure 4
Caspase-1 and Caspase-8 Act Redundantly in GSDMD-Deficient Macrophages for Activation of Caspase-3 and Caspase-7 (A–C) Macrophages of the indicated genotypes were left untreated or stimulated with either LeTx or FlaTox for 2 h. Cells were imaged under a confocal microscope (A) or analyzed by FACS for Annexin-V/PI positivity (B), and cell lysates were immunoblotted for the indicated proteins (C). (D and E) Macrophages of the indicated genotypes were left untreated or stimulated with either LeTx (D) or FlaTox (E) in media containing DEVD probe and PI and imaged on an Incucyte platform. (F and G) Macrophages of the indicated genotypes were left untreated or stimulated with either LeTx (F) or FlaTox (G) in media containing DEVD probe and PI and imaged on an Incucyte platform. (H) Macrophages of the indicated genotypes were left untreated or stimulated with either LeTx or FlaTox for 2 h, and cell lysates were immunoblotted for the indicated proteins. Percentages of all Incucyte experiments were calculated as the number of positive cells relative to a PI-stained, Triton-x100-treated well (considered 100%). Values represent mean ± SD of technical duplicates of a representative experiment from three biological repeats. All scale bars represent 10 μm.
Figure 5
Figure 5
Nlrp3 Activation Promotes Apoptosis in GSDMD-Deficient Macrophages (A and D) Macrophages of the indicated genotypes were primed with LPS (100 ng/mL) for 3 h and left untreated or stimulated with ATP or nigericin (nig) for 2 h. Cells were imaged under a confocal microscope (A), and cell lysates were immunoblotted for the indicated proteins (D). (B and C) Macrophages of the indicated genotypes were primed with LPS (100 ng/mL) for 3 h, left untreated or stimulated with ATP (B) or nigericin (C) in DEVD and PI-containing media, and imaged on an Incucyte platform. Percentages of all Incucyte experiments were calculated as the number of positive cells relative to a PI-stained, Triton-x100-treated well (considered 100%). Values represent mean ± SD of technical duplicates of a representative experiment from three biological repeats. All scale bars represent 10 μm.
Figure 6
Figure 6
Caspase-3 and Caspase-7 Are Critical for Inflammasome-Induced Apoptosis of GSDMD-Deficient Macrophages (A) Immortalized macrophages of the indicated genotypes were left untreated or stimulated with either LeTx or FlaTox for 2 h, and cell lysates were immunoblotted for the indicated proteins. (B and C) Immortalized macrophages of the indicated genotypes were left untreated or stimulated with either LeTx (B) or FlaTox (C) in media containing DEVD probe and PI and imaged on an Incucyte platform. Percentages of all Incucyte experiments were calculated as the number of positive cells relative to a PI-stained, Triton-x100-treated well (considered 100%). Values represent mean ± SD of technical duplicates of a representative experiment from three biological repeats.
See this image and copyright information in PMC

References

    1. Aftab O., Nazir M., Fryknäs M., Hammerling U., Larsson R., Gustafsson M.G. Label free high throughput screening for apoptosis inducing chemicals using time-lapse microscopy signal processing. Apoptosis. 2014;19:1411–1418. - PubMed
    1. Aglietti R.A., Estevez A., Gupta A., Ramirez M.G., Liu P.S., Kayagaki N., Ciferri C., Dixit V.M., Dueber E.C. GsdmD p30 elicited by caspase-11 during pyroptosis forms pores in membranes. Proc. Natl. Acad. Sci. USA. 2016;113:7858–7863. - PMC - PubMed
    1. Bauernfeind F.G., Horvath G., Stutz A., Alnemri E.S., MacDonald K., Speert D., Fernandes-Alnemri T., Wu J., Monks B.G., Fitzgerald K.A. Cutting edge: NF-kappaB activating pattern recognition and cytokine receptors license NLRP3 inflammasome activation by regulating NLRP3 expression. J. Immunol. 2009;183:787–791. - PMC - PubMed
    1. Boyden E.D., Dietrich W.F. Nalp1b controls mouse macrophage susceptibility to anthrax lethal toxin. Nat. Genet. 2006;38:240–244. - PubMed
    1. Broz P., Dixit V.M. Inflammasomes: mechanism of assembly, regulation and signalling. Nat. Rev. Immunol. 2016;16:407–420. - PubMed

Publication types

MeSH terms

"V体育官网入口" Grants and funding