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. 2018 Feb 28;8(1):3788.
doi: 10.1038/s41598-018-21998-3.

VSports手机版 - ASC- and caspase-8-dependent apoptotic pathway diverges from the NLRC4 inflammasome in macrophages

Bettina L Lee  1 , Kathleen M Mirrashidi  1 , Irma B Stowe  1 , Sarah K Kummerfeld  2 , Colin Watanabe  2 , Benjamin Haley  3 , Trinna L Cuellar  3 , Michael Reichelt  4 , Nobuhiko Kayagaki  5
Affiliations

"V体育平台登录" ASC- and caspase-8-dependent apoptotic pathway diverges from the NLRC4 inflammasome in macrophages

Bettina L Lee et al. Sci Rep. .

Abstract

The NLRC4 inflammasome recognizes bacterial flagellin and components of the type III secretion apparatus. NLRC4 stimulation leads to caspase-1 activation followed by a rapid lytic cell death known as pyroptosis. NLRC4 is linked to pathogen-free auto-inflammatory diseases, suggesting a role for NLRC4 in sterile inflammation. Here, we show that NLRC4 activates an alternative cell death program morphologically similar to apoptosis in caspase-1-deficient BMDMs. By performing an unbiased genome-wide CRISPR/Cas9 screen with subsequent validation studies in gene-targeted mice, we highlight a critical role for caspase-8 and ASC adaptor in an alternative apoptotic pathway downstream of NLRC4. Furthermore, caspase-1 catalytically dead knock-in (Casp1 C284A KI) BMDMs genetically segregate pyroptosis and apoptosis, and confirm that caspase-1 does not functionally compete with ASC for NLRC4 interactions VSports手机版. We show that NLRC4/caspase-8-mediated apoptotic cells eventually undergo plasma cell membrane damage in vitro, suggesting that this pathway can lead to secondary necrosis. Unexpectedly, we found that DFNA5/GSDME, a member of the pore-forming gasdermin family, is dispensable for the secondary necrosis that follows NLRC4-mediated apoptosis in macrophages. Together, our data confirm the existence of an alternative caspase-8 activation pathway diverging from the NLRC4 inflammasome in primary macrophages. .

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Conflict of interest statement

The authors declare no competing interests.

Figures (VSports最新版本)

Figure 1
Figure 1
NLRC4 activates a caspase-1-independent cell death pathway in absence of TLR signaling. (a–c) BMDMs with or without Pam3CSK4 (1 μg ml−1) pre-stimulation were electroporated with flagellin (0.5 μg ml−1). (a) LDH release measured after 16 h. Data is represented as mean ± SD; n = 3. (b) % YOYO-1 positive BMDMs from two mice/genotype after flagellin electroporation. Live cell images taken every 30 min up to 16 h. (c) Transmission electron microscopy BMDMs 6 h after flagellin electroporation. Representative images captured at 1000× magnification or 2000× magnification. Black arrows (→) indicate free nuclei. White asterisks (*) indicate chromatin condensation.
Figure 2
Figure 2
ASC and caspase-8 are identified through a genome-wide CRISPR/Cas9 screen for caspase-1-independent NLRC4-mediated cell death. (a) % YOYO-1 positive iMac cell lines from live imaging taken every hour up to 16 h after flagellin electroporation. Data is represented as mean ± SD; n = 3 images. (b) Scatter plot showing relative fold-change enrichment of genes (x-axis) with their corresponding enrichment p value (y-axis) from n = 3 biological replicates. Counts are log2 transformed and normalized using median scaling. Top scoring genes are highlighted. (c) Box-plot showing the distribution of individual sgRNA frequencies of flagellin- over control-treated populations, ordered left-to-right by increasing p-value.
Figure 3
Figure 3
ASC and caspase-8 are required for caspase-1-independent NLRC4 activated cell death in BMDMs. (a–d) BMDMs with or without Pam3CSK4 (1 μg ml−1) pre-stimulation were electroporated with flagellin (0.5 μg ml−1) or cytochrome-c (50 μg ml−1). (a) and (c) LDH release after 16 h. Data is represented as mean ± SD; n = 3. (b) and (d) % YOYO-1 positive BMDMs from two mice/genotype after flagellin electroporation. Live cell images taken every 30 min up to 16 h. (e) Immunoblot of caspase-8, caspase-1, caspase-3 and GSDMD in combined cell extract (ext) and supernatant (sup) from BMDMs 3 hrs after flagellin electroporation (no pre-stimulation). Pro-forms (pro) and cleaved forms are represented in blots. (f) BMDCs and thioglycollate-elicited peritoneal macrophages with no pre-stimulation were electroporated with flagellin (0.5 μg ml−1), cytochrome-c (50 μg ml−1), or FasL (100 ng ml−1) and measured for LDH release after 16 h. Data is represented as mean ± SD; n = 3.
Figure 4
Figure 4
NF-κB signaling blocks NLRC4/caspase-8-dependent apoptosis pathway. Casp1−/−Casp11−/− BMDMs were pre-stimulated for with or without various stimuli – Pam3CSK4 (1 μg ml−1), LPS (1 μg ml−1), R837 (2 μg ml−1), TNFα (100 ng ml−1), IFN-α (100 U ml−1), or IFN-β (100 U ml−1), then electroporated with flagellin (0.5 μg ml−1) or stimulated with FasL (100 ng ml−1). (a) LDH release after 16 h. Data is represented as mean ± SD; n = 3. (b) % YOYO-1 positive BMDMs from live cell imaging taken every 45 min for 16 h.
Figure 5
Figure 5
Caspase-1 does not compete with ASC for interaction with NLRC4 in enzymatically inactive Casp1C284A/C284A BMDMs. (a) Immunoblot of pro-caspase-1 and actin from wt, Casp1−/−, Casp1C284A/C284A BMDMs. (b) BMDMs with Pam3CSK4 (1 μg ml−1) pre-stimulation were electroporated with flagellin (0.5 μg ml−1). IL-1β release and LDH release measured after 16 h. Data is expressed as mean ± SD; n = 3. (c) % YOYO-1 positive BMDMs from two mice/genotype after stimulation. BMDMs with no pre-stimulation were electroporated with flagellin (0.5 μg ml−1). Live cell images taken every 45 min for 16 h.
Figure 6
Figure 6
DFNA5 is dispensable for secondary necrosis after NLRC4/caspase-8-mediated cell death. (a) Immunoblot of mouse DFNA5 and actin from iMac whole cell lysates. (b–e) Casp1−/−Casp11−/− iMac expressing Dfna5, Asc or luciferase (control) sgRNA were subjected to electroporation with flagellin (0.5 μg ml−1) and cytochrome-c (50 μg ml−1), or treated with FasL (100 ng ml−1). (b) and (e) LDH release after 16 h. Data is expressed as mean ± SD; n = 3. (c) and (d) % YOYO-1 positive iMacs from live cell imaging taken every hour for 16 h. Data is expressed as mean ± SD; n = 3. (f) Immunoblot for FLAG-tagged DFNA5 (FLAG antibody) and caspase-3 in combined cell extract and supernatant from FLAG-tagged DFNA5 expressing iMacs 3 hrs after electroporation with flagellin (0.5 μg) and cytochrome-c (25 μg), or treated with FasL (100 ng ml−1).
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