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. 2014 Sep 30:5:4977.
doi: 10.1038/ncomms5977.

IKKα negatively regulates ASC-dependent inflammasome activation

Bradley N Martin  1 , Chenhui Wang  2 , Jami Willette-Brown  3 , Tomasz Herjan  4 , Muhammet F Gulen  4 , Hao Zhou  4 , Katarzyna Bulek  4 , Luigi Franchi  5 , Takashi Sato  6 , Emad S Alnemri  7 , Goutham Narla  8 , Xiao-Ping Zhong  9 , James Thomas  10 , Dennis Klinman  6 , Katherine A Fitzgerald  11 , Michael Karin  12 , Gabriel Nuñez  5 , George Dubyak  13 , Yinling Hu  3 , Xiaoxia Li  4
Affiliations

IKKα negatively regulates ASC-dependent inflammasome activation

Bradley N Martin et al. Nat Commun. .

Abstract

The inflammasomes are multiprotein complexes that activate caspase-1 in response to infections and stress, resulting in the secretion of pro-inflammatory cytokines. Here we report that IκB kinase α (IKKα) is a critical negative regulator of apoptosis-associated specklike protein containing a C-terminal caspase-activation-andrecruitment (CARD) domain (ASC)-dependent inflammasomes. IKKα controls the inflammasome at the level of the adaptor ASC, which interacts with IKKα in the nucleus of resting macrophages in an IKKα kinase-dependent manner. Loss of IKKα kinase activity results in inflammasome hyperactivation. Mechanistically, the downstream nuclear effector IKK-related kinase (IKKi) facilitates translocation of ASC from the nucleus to the perinuclear area during inflammasome activation. ASC remains under the control of IKKα in the perinuclear area following translocation of the ASC/IKKα complex. Signal 2 of NLRP3 activation leads to inhibition of IKKα kinase activity through the recruitment of PP2A, allowing ASC to participate in NLRP3 inflammasome assembly. Taken together, these findings reveal a IKKi-IKKα-ASC axis that serves as a common regulatory mechanism for ASC-dependent inflammasomes.

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Conflict of interest statement

Competing financial interests statement

The authors have no competing financial interests to declare.

Figures

Figure 1
Figure 1
IKKα is a negative regulator of the inflammasome (a) BMDMs from WT and IKKα K44A mice were either left untreated or treated with LPS (1 μg/ml) for 4 hours, 5 mM ATP (30 min), or LPS (1 μg/ml) 4 hours + 5 mM ATP (30 min). Cell lysates and supernatants were collected together and were immunoblotted with the indicated antibodies. (b) Cell-free supernatants were collected from BMDMs that were either left untreated, or treated with LPS (1 μg/ml) for 4 hours, Pam3CSK4 (1 μg/ml) for 4 hours, LPS (1 μg/ml) 4 hours + 5 mM ATP (30 min), or Pam3CSK4 (1 μg/ml) 4 hours + 5 mM ATP (30 min). Levels of IL-1β and TNF-α were measured by ELISA. (c) BMDMs from WT and IKKα K44A mice were left untreated or treated with LPS (1 μg/ml) for 4 hours, or LPS (1 μg/ml) 4 hours + 5 mM ATP (30 min). Cell lysates were collected and immunoprecipitated with anti-ASC antibody or control IgG antibody, followed by immunoblotting with the indicated antibodies. (d,e) BMDMs from WT and either IKKα K44A (d) or IKKα AA (e) mice were treated with LPS (1 μg/mL) for 0, 10, 30, or 60 minutes, followed by the addition of 5 mM ATP for 30 minutes. (f) BMDMs from WT and IKKα K44A mice were infected with Salmonella typhimurium at a multiplicity of infection (MOI) of 50 for 0, 0.5, 1, or 2 hours. Cell lysates and supernatants were then collected together and immunoblotted with the indicated antibodies. (g) BMDMs from WT, IKKα K44A (left panel) and IKKα AA mice (right panel) were transfected with poly(dA:dT) (1.5 μg/ml) using lipofectamine 2000. After indicated times, cell lysates and supernatants were collected together and immunoblotted with the indicated antibodies. N.D. indicates not detectable, and “UN” indicates left untreated. Data are representative of at least five independent experiments. Error bars represent s.e.m. of technical replicates. * P <0.05 by Mann-Whitney test.
Figure 1
Figure 1
IKKα is a negative regulator of the inflammasome (a) BMDMs from WT and IKKα K44A mice were either left untreated or treated with LPS (1 μg/ml) for 4 hours, 5 mM ATP (30 min), or LPS (1 μg/ml) 4 hours + 5 mM ATP (30 min). Cell lysates and supernatants were collected together and were immunoblotted with the indicated antibodies. (b) Cell-free supernatants were collected from BMDMs that were either left untreated, or treated with LPS (1 μg/ml) for 4 hours, Pam3CSK4 (1 μg/ml) for 4 hours, LPS (1 μg/ml) 4 hours + 5 mM ATP (30 min), or Pam3CSK4 (1 μg/ml) 4 hours + 5 mM ATP (30 min). Levels of IL-1β and TNF-α were measured by ELISA. (c) BMDMs from WT and IKKα K44A mice were left untreated or treated with LPS (1 μg/ml) for 4 hours, or LPS (1 μg/ml) 4 hours + 5 mM ATP (30 min). Cell lysates were collected and immunoprecipitated with anti-ASC antibody or control IgG antibody, followed by immunoblotting with the indicated antibodies. (d,e) BMDMs from WT and either IKKα K44A (d) or IKKα AA (e) mice were treated with LPS (1 μg/mL) for 0, 10, 30, or 60 minutes, followed by the addition of 5 mM ATP for 30 minutes. (f) BMDMs from WT and IKKα K44A mice were infected with Salmonella typhimurium at a multiplicity of infection (MOI) of 50 for 0, 0.5, 1, or 2 hours. Cell lysates and supernatants were then collected together and immunoblotted with the indicated antibodies. (g) BMDMs from WT, IKKα K44A (left panel) and IKKα AA mice (right panel) were transfected with poly(dA:dT) (1.5 μg/ml) using lipofectamine 2000. After indicated times, cell lysates and supernatants were collected together and immunoblotted with the indicated antibodies. N.D. indicates not detectable, and “UN” indicates left untreated. Data are representative of at least five independent experiments. Error bars represent s.e.m. of technical replicates. * P <0.05 by Mann-Whitney test.
Figure 2
Figure 2
IKKα is a critical regulator of NLRP3 inflammasome activation in vivo (a,b) Irradiated WT mice received bone marrow from either WT or IKKα K44A mice. After six weeks of engraftment, chimeric mice received 30 mg/kg Ultrapure LPS in 100 μl PBS (i.p). Serum was collected after 2 hours and levels of IL-1β and TNF-α were measured by ELISA (a). Splenocytes were collected and cultured for 2 hours with or without re-stimulation with LPS (1μg/ml). Cell-free supernatants were collected and levels of IL-1β and TNF-α were measured by ELISA (b). (c) Chimeric mice as described in (a) received 1 mg MSU in 100 μl PBS (i.p.). Five hours later, peritoneal lavage was performed and infiltrating neutrophils were quantified (left panel). Serum was collected and IL-1β levels were determined by ELISA (right panel). (d,e) Chimeric mice as described in (a) received 1 mg sterilized silica by intra-tracheal instillation. Thity-six hours later, lungs were harvested and lung sections were stained with hematoxylin and eosin (upper panels) or with anti-neutrophil antibody (NIMP-R14) (lower panels); scale bar represents 50 μm (d). Broncho-alveolar lavage (BAL) was performed and IL-1β levels in cell-free BAL fluid was assessed by ELISA (e). N.D. indicates not detectable, and “UN” indicates left untreated. Data are representative of two independent experiments. n = 5 mice per group in each experiment. Error bars represent s.e.m. of biological replicates. * P <0.05 by Mann-Whitney test.
Figure 3
Figure 3
IKKα regulates the inflammasome via interaction with ASC (a) HEK 293 cells were transfected with ASC and either GFP- IKKα WT or GFP-IKKα K44A using lipofectamine 2000. Empty vector was used to normalize total plasmid transfected. Cells were then lysed, followed by immunoprecipitation with anti-ASC antibody and immunoblotting with the indicated antibodies. (b,c) BMDMs from WT and IKKα K44A mice (b) or WT mice only (c) were either left untreated or treated with LPS (1 μg/ml) for 4 hours, or LPS (1 μg/ml) 4 hours + 5 mM ATP (30 min) (b), or either left untreated or treated with LPS (1 μg/ml) for 4 hours, LPS (1 μg/ml) 4 hours + 5 mM ATP (30 min), or LPS (1 μg/ml) 4 hours + 1 uM gramicidin (30 min) (c). Cell lysates were then immunoprecipitated using anti-ASC antibody or control IgG antibody, and were immunoblotted with the indicated antibodies. (d) BMDMs from WT mice were transfected with poly(dA:dT) (1.5 μg/ml) using lipofectamine 2000 for 4 hours (left panel), or were infected with 50 MOI of S. typhimurium for indicated times (right panel). Cell lysates were then immunoprecipitated using anti-ASC antibody or control IgG antibody, and were immunoblotted with the indicated antibodies. (e) WT, IKKα K44A and IKKα AA BMDMs were either left untreated or treated with LPS (1 μg/ml) for 4 hours, or LPS (1 μg/ml) 4 hours + 5 mM ATP (30 min), then washed, fixed, and stained with anti-ASC antibody and caspase-1 FLICA (FAM-YVAD-FMK). Images were then acquired using confocal microscopy with a 60X objective and 4X enlargement; scale bars are 5 μm. Images were quantified by counting number of cells with ASC specks co-staining with caspase-1 FLICA as a percentage of total cells counted in three fields. (far right panel). “UN” indicates left untreated. Data are representative of at least four independent experiments. Error bars represent s.e.m. of technical replicates. * P <0.05 by Mann-Whitney test.
Figure 4
Figure 4
S193 and S16 are critical residues on ASC for interaction with IKKα (a) HEK 293 cells were transfected with HA-IKKα and either WT ASC, S16A ASC, or S193A ASC using lipofectamine 2000. Empty vector was used to normalize total plasmid transfected. Cells were then lysed, followed by immunoprecipitation with anti-ASC antibody and immunoblotting with the indicated antibodies. (b) HEK 293 cells were transfected with HA-IKKα or HA-IKKα K44A along with either WT ASC, S16A ASC, or S193A ASC using lipofectamine 2000. Empty vector was used to normalize total plasmid transfected. Cells were then lysed, followed by immunoprecipitation with anti-ASC antibody and in vitro kinase assay. (c,d) Immortalized ASC−/− macrophages retrovirally reconstituted with empty vector, WT ASC, S193A ASC, or S16A ASC were either left untreated or treated with LPS (1 μg/mL) for 4 hours, followed by 30 min 5 mM ATP when indicated. Cells were then lysed, followed by immunoprecipitation with anti-ASC antibody and immunoblotting with indicated antibodies (c), or cells were then washed, fixed, and stained with anti-ASC antibody and caspase-1 FLICA (FAM-YVAD-FMK). Images were then acquired using confocal microscopy with 60X magnification and 4X enlargement; scale bars are 5 μm (d). Images from were quantified by counting the number of cells with ASC specks co-staining with caspase-1 FLICA as a percentage of total cells counted in three fields. (e–g) Retrovirally reconstituted ASC−/− macrophages from (c) were either left untreated or treated with LPS (1 μg/ml) for 4 hours, followed by 30 minutes of 5 mM ATP when indicated (e,f), or were transfected with poly(dA:dT) (1.5 μg/ml) using lipofectamine 2000 for 4 hours (g). Cell lysates and supernatants were then collected together and immunoblotted with the indicated antibodies (e,g), or were analyzed by ELISA (f). N.D. indicates not detectable, and “UN” indicates left untreated. Data are representative of at least four independent experiments. Error bars represent s.e.m. of technical replicates. * P <0.05 by Mann-Whitney test.
Figure 4
Figure 4
S193 and S16 are critical residues on ASC for interaction with IKKα (a) HEK 293 cells were transfected with HA-IKKα and either WT ASC, S16A ASC, or S193A ASC using lipofectamine 2000. Empty vector was used to normalize total plasmid transfected. Cells were then lysed, followed by immunoprecipitation with anti-ASC antibody and immunoblotting with the indicated antibodies. (b) HEK 293 cells were transfected with HA-IKKα or HA-IKKα K44A along with either WT ASC, S16A ASC, or S193A ASC using lipofectamine 2000. Empty vector was used to normalize total plasmid transfected. Cells were then lysed, followed by immunoprecipitation with anti-ASC antibody and in vitro kinase assay. (c,d) Immortalized ASC−/− macrophages retrovirally reconstituted with empty vector, WT ASC, S193A ASC, or S16A ASC were either left untreated or treated with LPS (1 μg/mL) for 4 hours, followed by 30 min 5 mM ATP when indicated. Cells were then lysed, followed by immunoprecipitation with anti-ASC antibody and immunoblotting with indicated antibodies (c), or cells were then washed, fixed, and stained with anti-ASC antibody and caspase-1 FLICA (FAM-YVAD-FMK). Images were then acquired using confocal microscopy with 60X magnification and 4X enlargement; scale bars are 5 μm (d). Images from were quantified by counting the number of cells with ASC specks co-staining with caspase-1 FLICA as a percentage of total cells counted in three fields. (e–g) Retrovirally reconstituted ASC−/− macrophages from (c) were either left untreated or treated with LPS (1 μg/ml) for 4 hours, followed by 30 minutes of 5 mM ATP when indicated (e,f), or were transfected with poly(dA:dT) (1.5 μg/ml) using lipofectamine 2000 for 4 hours (g). Cell lysates and supernatants were then collected together and immunoblotted with the indicated antibodies (e,g), or were analyzed by ELISA (f). N.D. indicates not detectable, and “UN” indicates left untreated. Data are representative of at least four independent experiments. Error bars represent s.e.m. of technical replicates. * P <0.05 by Mann-Whitney test.
Figure 5
Figure 5
ATP signaling throμgh P2X7R inhibits IKKα and releases ASC (a) BMDMs from WT mice were pre-treated with either DMSO or the P2X7R antagonist AZ10606120 (AZ) at a concentration of 10 nM for 30 min. Cells were then left untreated or treated with LPS (1 μg/ml) for 4 hours, or LPS (1 μg/ml) 4 hours + 5 mM ATP (30 min). Cell lysates were immunoprecipitated with anti-ASC antibody or control IgG antibody, and immunoblotted with the indicated antibodies (upper panel). Some cell lysates that were not used for immunoprecipitation were immunoblotted with other antibodies as indicated (lower panel). (b,c) BMDMs from WT and P2X7R−/− mice were either left untreated or treated with LPS (1 μg/ml) for 4 hours, or LPS (1 μg/ml) 4 hours plus 5 mM ATP (30 min). Cell lysates were immunoprecipitated with anti-ASC antibody or control IgG antibody, and were then immunoblotted with the indicated antibodies (b). Cell lysates not used for immunoprecipitation were immunoblotted with the indicated antibodies (c). (d) BMDMs from WT mice were pre-treated with 5 mM ATP 15 minutes prior to treatment with LPS (1 μg/ml) for 0, 15, 30, 60, or 120 minutes. Cells were then lysed and immunoprecipitated with anti-IKKα antibody. Immunoprecipitated proteins were eluted and immunoblotted for p-IKKα/β and IKKα. Lysates obtained before immunoprecipitated were immunoblotted for p-IκBα and Actin. (e) WT BMDMs were pre-treated with 5 mM ATP or 10 μM nigericin for 5 minutes, then treated with LPS (1 μg/ml) for 0, 15, or 30 minutes. Cells were then lysed and immunoprecipitated with anti-IKKα antibody, followed by in vitro kinase assay using GST-IκBα (1–317) as substrate. Data are representative of at least four independent experiments. Error bars represent s.e.m. of technical replicates. * P <0.05 by Mann-Whitney test.
Figure 6
Figure 6
PP2A activity is required for NLRP3 inflammasome activation (a,d) BMDMs from WT mice were pre-treated with DMSO, 100 nm okadaic acid (OA), or 10 nM AZ10606120 for 15 minutes. Cells were then left untreated or treated with LPS (1 μg/ml) for 4 hours, LPS (1 μg/ml) 4 hours plus 5 mM ATP (30 min), LPS (1 μg/ml) 4 hours plus 5 mM ATP (30 min) plus OA (100nM) or LPS (1 μg/ml) 4 hours plus 5 mM ATP (30 min) plus AZ10606120 (10 nM) (a) or 10 uM nigericin (30 min) (d). Cell lysates were immunoprecipitated using anti-IKKα antibody, and immunoblotted with the indicated antibodies. (b,c,e) BMDMs from WT mice were pre-treated with either DMSO or 10 nM or 100 nM okadaic acid for 15 minutes. Cells were then left untreated or treated with LPS (1 μg/ml) for 4 hours, or LPS (1 μg/ml) 4 hours + 5 mM ATP (30 min) (b, c) or 10 uM nigericin (30 min) (e). Cell lysates and supernatants were collected together and were immunoblotted with the indicated antibodies (b,e), and cell-free supernatants were collected and IL-1β and TNF-α levels were assessed by ELISA (c). (f,g) WT BMDMs were infected with lentivirus containing either control or anti-PP2A-C shRNA. Cells were either left untreated or treated with LPS (1 μg/ml) for 4 hours, followed by 5 mM ATP for 30 min when indicated (f), or were transfected with poly(dA:dT) (1.5 μg/ml) using lipofectamine 2000 for 4 hours (g). After the indicated time, cell lysates and supernatants were collected together and immunoblotted with indicated antibodies. N.D. indicates not detectable, and “UN” indicates left untreated. Data are representative of at least four independent experiments. Error bars represent s.e.m. of technical replicates. * P <0.05 by Mann-Whitney test.
Figure 7
Figure 7
IKKi is required for ASC nuclear-to-cytosolic translocation and inflammasome activation (a) BMDMs from WT and IKKi−/− mice were treated with LPS (1 μg/ml) for 4 hours, followed by 30 min 5 mM ATP when indicated. Cell lysates and supernatants were then collected together and immunoblotted with the indicated antibodies. (b) Cell-free supernatants from (s) were analyzed by ELISA to determine levels of IL-1β (left panel) and TNF-α (right panel). (c) BMDMs from WT and IKKi−/− mice were transfected with poly(dA:dT) (1.5 μg/ml) using lipofectamine 2000 for 4 hours. Cell lysates and supernatants were then collected together and immunoblotted with the indicated antibodies. (d) BMDMs from WT and IKKi−/− mice were left untreated or treated with LPS (1 μg/ml) for 4 hours, LPS 4 hours + 5 mM ATP (30 min), or were transfected with poly(dA:dT) (1.5 μg/ml) using lipofectamine 2000 for 4 hours. Cells were then washed and fixed, and stained with anti-ASC antibody and anti-IKKi antibody. Images were acquired using confocal microscopy with 60X magnification and 4X enlargement; scale bars are 5 μm (upper panels). LPS+ATP- and poly(dA:dT)-treated samples were quantified by counting the number of cells with ASC specks as a percentage of total cells counted in three fields. (lower panels) (e) BMDMs from WT and IKKi−/− mice were left untreated or treated with LPS (1 μg/ml) for 15 minutes, 30 mins, 4 hours or LPS 15 mins and 4 hours + 5 mM ATP (30 min). Cells were then lysed and immunoprecipitated with anti-ASC antibody, followed by western blotting with indicated antibodies. N.D. indicates not detectable, and “UN” indicates left untreated. Data are representative of at least four independent experiments. Error bars represent s.e.m. of technical replicates. * P <0.05 by Mann-Whitney test.
Figure 8
Figure 8
S58 of ASC is a critical residue for IKKi-mediated nuclear-to-cytosolic translocation and inflammasome activation (a) HEK 293 cells were transfected with FLAG-IKKi WT or FLAG-IKKi KD, along with either WT ASC or S58A ASC using lipofectamine 2000. Empty vector was used to normalize total plasmid transfected. Cells were then lysed, followed by immunoprecipitation with anti-ASC antibody, and in vitro kinase assay. (b,c) Immortalized ASC−/− macrophages were retrovirally reconstituted with WT ASC or S58A ASC, then either left untreated, treated with LPS (1 μg/ml) for 4 hours followed by 5 mM ATP (30 mins), or were transfected with poly(dA:dT) (1.5 μg/ml) for 4 hours using lipofectamine 2000. Cells were then washed and fixed, and stained with anti-ASC antibody and caspase-1 FLICA (FAM-YVAD-FMK), and images were acquired using confocal microscopy with 60X magnification and 4X enlargement; scale bars are 5 μm. Samples were quantified by counting the number of cells with ASC specks as a percentage of total cells counted in three fields (b). Or, lysates were immunoprecipitated with anti-ASC antibody and immunoblotted with the indicated antibodies (c). (d,e) Immortalized ASC−/− macrophages were retrovirally reconstituted with S193A ASC, S16A ASC, S193A/S58A ASC, or S16A/S58A ASC. Cells were treated with LPS (1 μg/ml) for 4 hours or with LPS (1 μg/ml) for 4 hours followed by 5 mM ATP (30 mins). Lysates and supernatants were then collected together and immunoblotted with the indicated antibodies (d), or cells were washed and fixed, stained with anti-ASC antibody and caspase-1 FLICA (FAM-YVAD-FMK), and images were acquired using confocal microscopy with 60X magnification and 4X enlargement; scale bars are 5 μm. Samples were quantified by counting the number of cells with ASC specks as a percentage of total cells counted in three fields (e). (f) WT ASC, S58A ASC, S193A/S58A ASC, and S16A/S58A ASC retrovirally reconstituted cells were transfected with poly(dA:dT) (1.5 μg/ml) using lipofectamine 2000 for 4 hours. Lysates and supernatants were then collected together and immunoblotted with the indicated antibodies. “UN” indicates left untreated. Data are representative of at least four independent experiments. Error bars represent s.e.m. of technical replicates. * P <0.05 by Mann-Whitney test.
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