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. 2013 Sep;20(9):1149-60.
doi: 10.1038/cdd.2013.37. Epub 2013 May 3.

AIM2 and NLRP3 Inflammasomes Activate Both Apoptotic and Pyroptotic Death Pathways via ASC

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Free PMC article

AIM2 and NLRP3 Inflammasomes Activate Both Apoptotic and Pyroptotic Death Pathways via ASC

V Sagulenko et al. Cell Death Differ. .
Free PMC article

Abstract

Inflammasomes are protein complexes assembled upon recognition of infection or cell damage signals, and serve as platforms for clustering and activation of procaspase-1. Oligomerisation of initiating proteins such as AIM2 (absent in melanoma-2) and NLRP3 (NOD-like receptor family, pyrin domain-containing-3) recruits procaspase-1 via the inflammasome adapter molecule ASC (apoptosis-associated speck-like protein containing a CARD). Active caspase-1 is responsible for rapid lytic cell death termed pyroptosis. Here we show that AIM2 and NLRP3 inflammasomes activate caspase-8 and -1, leading to both apoptotic and pyroptotic cell death. The AIM2 inflammasome is activated by cytosolic DNA. The balance between pyroptosis and apoptosis depended upon the amount of DNA, with apoptosis seen at lower transfected DNA concentrations. Pyroptosis had a higher threshold for activation, and dominated at high DNA concentrations because it happens more rapidly. Gene knockdown showed caspase-8 to be the apical caspase in the AIM2- and NLRP3-dependent apoptotic pathways, with little or no requirement for caspase-9. Procaspase-8 localised to ASC inflammasome 'specks' in cells, and bound directly to the pyrin domain of ASC. Thus caspase-8 is an integral part of the inflammasome, and this extends the relevance of the inflammasome to cell types that do not express caspase-1.

Figures

Figure 1
Figure 1
Cell death induced by cytosolic DNA in mouse macrophages is ASC-dependent, but has caspase-1-dependent and -independent phases. BMMs from WT and corresponding knockout mice were electroporated with 20 μg of CT DNA. Cell viability was determined by MTT reduction, a measure of mitochondrial activity, at the indicated times post electroporation. Results from each time point were normalised to samples electroporated without DNA. The dotted line indicates no loss of viability relative to this control. Each data point shown is the result of an independent experiment and is the average of 2–3 separate electroporations within that experiment. (a) Viability of BMMs from Casp1−/− and WT littermate mice in four independent experiments. (b) Viability of BMMs from Casp11−/− and WT littermate mice in three independent experiments. (c) Viability of BMMs from Asc−/− and WT littermate mice in three independent experiments
Figure 2
Figure 2
The DNA inflammasome activates both apoptosis and pyroptosis in a DNA dose-dependent manner. (a) Analysis of the cell death phenotype by Annexin-V–PI staining. BMMs from WT, Casp1−/− and Asc−/− mice were electroporated with or without 20 μg of CT DNA. One hour post electroporation, cells were stained with Annexin-V and PI, and analysed by flow cytometry. Live cells are negative for both stains, whereas early apoptotic cells are characterised by a high Annexin-V signal in the absence of PI staining. Necrotic, pyroptotic and late-apoptotic cells have a high PI signal. The results shown are typical of four independent experiments. (b) Cells from the experiment shown in panel a were assessed for sub-G0/G1 DNA content by flow cytometry at 6 h post electroporation. Examples of primary data are shown, with a graph showing data from three independent experiments. (c) WT C57BL/6 BMMs die by apoptosis after electroporation with low DNA concentrations. BMMs were electroporated with the indicated amounts of DNA and analysed by Annexin-V–PI staining after 6 h. The percentage of Annexin-V-positive/PI-negative cells was used to indicate apoptosis. The percentage of PI-positive cells was used to indicate pyroptotic and late-apoptotic cells. The results shown are the mean and range of duplicate electroporations. Apoptosis of WT cells with low DNA concentration was observed in four independent experiments. (d) The pan-caspase inhibitor Z-VAD-FMK prevents caspase-1-independent cell death. BMMs from Casp1−/− mice were pretreated for 1 h with 20 or 50 μM Z-VAD-FMK, or a DMSO vehicle control, then electroporated with or without 10 or 20 μg of CT DNA in the continued presence of Z-VAD-FMK. Cell viability was assayed by MTT reduction 3 h after electroporation. Results were normalised to samples electroporated without DNA, for each pretreatment regime. The dotted line indicates no loss of viability relative to this control. The results of four independent experiments are shown, with different symbols used for each experiment to show the consistent trend. (e) Cleavage of caspase-3 and -8 in response to cytosolic DNA in Casp1−/− BMMs was effectively inhibited by Z-VAD-FMK. Cells were treated in parallel with the experiment shown in panel d, except they were lysed at 30 min post electroporation, and the levels of cleaved caspase-3 (p17) and caspase-8 (p18) were analysed by western blot using α-tubulin as a loading control
Figure 3
Figure 3
Cytosolic DNA activates apoptotic caspases independently of caspase-1 and -11, but depending on ASC. (a) Caspase-1 and -11 are dispensable for cleavage of apoptotic caspases in response to transfected DNA. BMMs from Casp1−/− (naturally null for caspase-11) and C57BL/6 WT mice were electroporated with 10 or 20 μg of CT DNA. Cell lysates and proteins released into the culture medium were collected 30 min post electroporation, and analysed by western blot. (b) ASC is essential for activation of the apoptotic cascade in response to cytosolic DNA. BMMs from Asc−/− and C57BL/6 WT mice were electroporated with 10 or 20 μg of CT DNA. Cell lysates and proteins released into the tissue culture medium were collected 30 min post electroporation, and analysed by western blot
Figure 4
Figure 4
Caspase-8 is the apical apoptotic caspase activated by the DNA inflammasome. (a) Caspase-2 is not involved in DNA-dependent caspase-3 cleavage. WT C57BL/6 and Casp2−/− BMMs were electroporated with either 20 μg of CT DNA (CT) or 2 μg of poly(dA):(dT) (AT), and harvested after 30 min. Some cells were subjected to incubation at 43 °C for 1 h to induce a heat-shock response as a positive control for caspase-2 activation. Cleaved caspase-3 and total caspase-2 were detected by western blotting. (b) No evidence for the intrinsic pathway in DNA-dependent caspase-3 cleavage. C57BL/6 (WT) and Bcl-2-overexpressing BMMs were electroporated with the stated doses of either CT DNA (CT) or poly(dA):(dT) (AT), or left untreated. Cell extracts were prepared 30 min after electroporation and the level of cleaved caspase-3 was detected by western blotting. As shown in the lower panel, the effect of Bcl-2 overexpression was confirmed by inhibition of the response to STS. Cells were either untreated (cont.) or treated with 1μM STS or a DMSO vehicle control for 3 h prior to preparation of extracts for western blotting. (c) Knockdown of caspase-8 for 28 h, using three different siRNAs in Casp1−/− iBMMs, assessed by western blot. (d) Caspase-8 is essential for DNA-dependent caspase-3 cleavage. Casp1−/− iBMMs with caspase-8 knocked down, from the experiment shown in panel c, were electroporated with 10 μg of DNA and lysed after 30 min for analysis by western blot. (e) Knockdown of caspase-9 using three different siRNAs, and caspase-8 using a single siRNA, in Casp1−/− iBMMs, assessed by western blot after 28 h. (f) Caspase-9 has little or no role in DNA-dependent caspase-3 cleavage. Casp1−/− iBMMs with caspase-9 or -8 knocked down, from the experiment shown in panel e, were treated and analysed as per panel d. (g) Knockdown of caspase-8 prevents DNA-dependent apoptosis. The degree of caspase-8 and -9 knockdown in this experiment, as assessed by western blot, is shown in the lower right-hand panel. Casp1−/− iBMMs with caspase-8 or -9 knocked down were analysed for DNA-dependent apoptosis by Annexin-V–PI staining and sub-G0/G1 DNA content at 1.5 and 6 h, respectively, post electroporation using 10 μg of DNA. Representative primary data are shown, and the graphs show the mean and range of duplicate electroporations. Inhibition of apoptosis by caspase-8 knockdown was shown in three separate experiments
Figure 5
Figure 5
NLRP3 inflammasome agonist nigericin induces apoptosis in a caspase-8-dependent manner. (a) Nigericin-induced cell death is delayed in Casp1−/− BMMs. BMMs of the indicated genotypes were primed for 4 h with 10 ng/ml LPS prior to treatment for 1–6 h with nigericin at doses 0, 2.5, 5 and 10 μM. The degree of cell death was determined by PI staining followed by flow cytometry. The results shown are the means±S.E. of the data of three independent experiments. (b) Nigericin causes pyroptotic death of WT cells and apoptotic death of Casp1−/− BMMs. BMMs were treated with 10 ng/ml LPS, followed by 10 μM nigericin for 2 h. Annexin-V–PI staining was used to detect apoptotic cells by flow cytometry. (c) Apoptotic death of Casp1−/− BMMs was detected by the presence of sub-G0/G1 DNA content. Cells were primed with 10 ng/ml LPS for 4 h, followed by nigericin for 6 h. Each data point shows the result of an independent experiment. (d) Nigericin activates apoptotic caspases in an ASC-dependent and caspase-1-independent manner. BMMs from C57BL/6 (WT), Asc−/− and Casp1−/− mice were pretreated with 10 ng/ml LPS and after 4 h nigericin was added to the cells at a final concentration of 10 μM. Control cells were treated with a methanol vehicle control. Cell protein extracts and proteins released into the culture medium were collected after 2.5 h of incubation at 37 °C, and analysed by western blot. (e) Caspase-8 is the apical apoptotic caspase activated by nigericin. Casp1−/− iBMMs with caspase-8 or -9 knocked down were treated with 100 ng/ml LPS for 4 h followed by 10 μM nigericin for 75 min. Cell extracts were analysed by western blot. The degree of knockdown in this experiment is shown in Figure 4g. (f) Nigericin-dependent apoptosis requires caspase-8. Casp1−/− iBMMs with caspase-8 or -9 knocked down were treated with 10 ng/ml LPS for 4 h followed by 10 μM nigericin for 4 h. The results shown are the mean and range of duplicate cell treatments from a single experiment. An effect of caspase-8 knockdown was shown in four independent experiments. The left-hand panel shows cell death measured by flow cytometry of PI-stained cells; the middle panel shows sub-G0/G1 cells assessed by flow cytometry as an indication of apoptosis; and the right hand panel shows the degree of caspase-8 and -9 knockdown, assessed by western blot. Example primary data from flow cytometry are shown in Supplementary Figure 4
Figure 6
Figure 6
Procaspase-8 colocalises with ASC and directly interacts with the ASC pyrin domain. (a) Procaspase-8 colocalises with ASC upon inflammasome induction with nigericin. Casp1−/− BMMs were pre-treated with LPS and Z-VAD-FMK prior to inflammasome stimulation with 5 μM nigericin (upper panel) or treatment with an ethanol vehicle control (lower panel). Cells were stained with anti-ASC (red) and anti-caspase-8 (green), and nuclei were counterstained with DAPI (blue). The arrows indicate examples of caspase-8 specks colocalised with ASC. The data shown are representative of two independent experiments and represent the appearance of the majority of the cells under the stated conditions. (b) Pull-down of procaspase-8 by ASC pyrin and ASC CARD domains. Bead-immobilised recombinant GST-ASC-pyrin, GST-ASC-CARD and a GST control were used to pull down [35S]-labelled in vitro translated procaspase-8 (catalytic site mutant). Loading of beads with GST proteins was shown by SDS-PAGE and protein stain (lower panel), and the pulled down procaspase-8 was detected with an X-ray film (upper panel). Similar results were seen in three experiments. Lack of binding to other DDs is shown in Supplementary Figure 6
Figure 7
Figure 7
A model for the AIM2 and NLRP3 inflammasomes, activating parallel apoptotic and pyroptotic death pathways. DNA in the cytosol is recognised by the HIN domain of AIM2., , , Binding of AIM2 along the length of the DNA then recruits ASC via homotypic pyrin domain interactions. This then nucleates the formation of a cluster of ASC, which self-associates via both pyrin and CARD domain interactions. The ASC cluster may lose association with AIM2, as it has been observed in cells adjacent to the AIM2–DNA complex. Alternatively, nigericin leads to NLRP3 clustering and nucleation of an ASC speck. ASC is an adapter molecule that recruits procaspase-1 via homotypic CARD domain interactions. Clustering leads to activation of procaspase-1, which promotes pyroptosis. The alternative pathway elucidated here involves recruitment of procaspase-8 via the pyrin domain of ASC and its activation, leading to downstream caspase-3 cleavage and apoptotic cell death. Whether both procaspase-1 and -8 can be recruited to the same ASC molecule as shown here has not been established, although since they bind to different domains, this is feasible

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