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NLRP3 inflammasome inhibition is disrupted in a group of auto-inflammatory disease CAPS mutations

Nature Immunology volume 17, pages 1176–1186 (2016)Cite this article

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Abstract

Inflammasomes are positioned to rapidly escalate the intensity of inflammation by activating interleukin (IL)-1β, IL-18 and cell death by pyroptosis. However, negative regulation of inflammasomes remains poorly understood, as is the signaling cascade that dampens inflammasome activity. We found that rapid NLRP3 inflammasome activation was directly inhibited by protein kinase A (PKA), which was induced by prostaglandin E2 (PGE2) signaling via the PGE2 receptor E-prostanoid 4 (EP4). PKA directly phosphorylated the cytoplasmic receptor NLRP3 and attenuated its ATPase function. We found that Ser295 in human NLRP3 was critical for rapid inhibition and PKA phosphorylation. Mutations in NLRP3-encoding residues adjacent to Ser295 have been linked to the inflammatory disease CAPS (cryopyrin-associated periodic syndromes). NLRP3-S295A phenocopied the human CAPS mutants. These data suggest that negative regulation at Ser295 is critical for restraining the NLRP3 inflammasome and identify a molecular basis for CAPS-associated NLRP3 mutations.

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Figure 1: PGE2 suppresses activation of the NLRP3 inflammasome.
Figure 2: PGE2 selectively inhibits NLRP3 inflammasome activation.
Figure 3: PGE2 receptor EP4 and EP2 inhibit the NLRP3 inflammasome.
Figure 4: cAMP-PKA signaling mediates NLRP3 inflammasome inhibition.
Figure 5: Rapid PGE2 NLRP3 inflammasome inhibition by PKA.
Figure 6: cAMP-PKA induces NLRP3 phosphorylation.
Figure 7: NLRP3 Ser295 mediates rapid inhibition by cAMP.
Figure 8: Other CAPS mutations adjacent to Ser295 are unresponsive to rapid cAMP inhibition.

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Acknowledgements

We thank Y. Shi (University of Calgary) for MSU and silica. Supported by the Canadian Institute of Health Research (MOP-340405 to K.C.; Tier 1 Canada Research Chair in Gastrointestinal Inflammation to K.C.; Team Grant in Health Challenges in Chronic Inflammation to J.A.M.), the Natural Sciences and Engineering Research Council of Canada (RGPIN-04023 to K.C.; and Canada Graduate Scholarship to L.M.) and Alberta Innovates Health Solutions (J.A.M. and L.M.).

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Authors and Affiliations

  1. Department of Microbiology, Immunology and Infectious Diseases, Snyder Institute for Chronic Diseases, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada

    Leanne Mortimer, France Moreau & Kris Chadee

  2. Department of Biochemistry and Molecular Biology, Snyder Institute for Chronic Diseases, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada

    Justin A MacDonald

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Contributions

L.M., J.A.M. and K.C. conceived of and designed the experiments and wrote the paper; L.M., F.M. and J.A.M. performed the experiments and analyzed the data; and L.M., F.M., J.A.M. and K.C. contributed reagents, materials and analysis tools.

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Correspondence to Kris Chadee.

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Integrated supplementary information

Supplementary Figure 1 PGE2 does not induce degradation of NLRP3 inflammasome components but rapidly inhibits NLRP3 activation in macrophages.

(a-d) LPS-primed BMDM. (a) Lysates (LYS) from PGE2 (500 nM) treatment for 30 min. (b) The indicated concentration of PGE2 was added for 5 min followed by stimulation with (left) nigericin (5 μM for 30 min) or (right) ATP (5 mM for 45 min). (c) PGE2 (500 nM) was added 30 min after stimulation with nigericin and analyzed 50 min post initial nigericin stimulation. (d) 16,16-dimethyl PGE2 (16-DM, 500 nM) or 15-keto PGE2 (15-keto, 500 nM) were added 5 min prior to nigericin or ATP stimulation. (e-g) PMA-differentiated THP-1 cells were treated with (e,f) the indicated concentration of PGE2 for 5 min prior to stimulation with (e) nigericin (5 μM) and LPS (50 ng/ml) for 30 min or (f) ATP (5 mM) and LPS (50 ng/ml) for 45 min. (g) 16,16-dimethyl PGE2 (16-DM, 250 nM), or 15-keto PGE2 (15-keto, 250 nM) were added 5 min prior to nigericin. (b-g) Total IL-1β release in cell supernatants analyzed by ELISA. *P < 0.005 (unpaired two-tailed t test). (a-g) Data are from one experiment representative of three separate experiments with similar results, (a-d) BMDM were derived from 2 mice per experiment mixed together, (b-g) three replicates, each replicate is one well + s.e.m.

Supplementary Figure 2 Particle-induced NLRP3 inflammasome activation is partially suppressed by PGE2, and cyclooxygenase inhibition blocks NLRP3 activation.

(a-c) BMDM were primed with LPS. (a,b) The indicated concentration of PGE2 was added 5 min before stimulation with (left) silica (250 μg/ml for 5 h) or (right) MSU (150 μg/ml for 4 h). (c) Indomethacin (50 μM) was added 30 min before stimulation with nigericin (5 μM for 30 min). (a,c) Total IL-1β release in cell supernatants analyzed by ELISA. (b,c) Immunoblot analysis of culture supernatants (SN) and lysates (LYS). *P < 0.005 (unpaired two-tailed t test). (a-c) Data are from one experiment representative of three separate experiments with similar results, (a-c) BMDM were derived from 2 mice per experiment mixed together, (a,c) three replicates, each replicate is one well + s.e.m.

Supplementary Figure 3 PGE2 receptors EP1 and EP3 do not inhibit, but EP4 does inhibit, the NLRP3 inflammasome.

(a-i) BMDMs primed for 3.5 h with LPS. (a,b) The indicated concentration of EP1 agonist ONO-DI-004 or (c,d) EP3 agonist ONO-AE-248 was added 5 min prior to (a,c) nigericin (5 μM for 30 min) or (b,d) ATP (5 mM for 45 min). (e-g) The indicated concentration of EP4 agonists (e left,f) CAY10598 and (e right,g) ONO-AE1-32 were added 5 min before (e) nigericin (5 μM for 30 min) or (f,g) ATP (5 mM for 45 min). (h,i) An EP4 antagonist ONO-AE3-208 (2 μM) was added for 5 min prior to addition of ONO-AE1-329 (500 nM) for 5 min and then treated with (h) nigericin (5 μM for 30 min) or (i) ATP (5 mM for 45 min). (a-i) Total IL-1β release in cell supernatants analyzed by ELISA. (a-d,f,g,i) Immunoblot analysis of culture supernatants (SN) and lysates (LYS). *P < 0.005 (unpaired two-tailed t test). (a-i) Data are from one experiment representative of three separate experiments with similar results, (a-i) BMDM were derived from 2 mice per experiment mixed together, (a-i) three replicates, each replicate is one well + s.e.m.

Supplementary Figure 4 PGE2 receptors EP2 and EP4 inhibit the NLRP3 inflammasome, but EP4 mediates PGE2-induced inhibition.

(a-h) BMDMs primed for 3.5 h with LPS. (a,b) The indicated concentration of EP2 agonist ONO-AE1-259-01 was added 5 min before treatment with (a) nigericin (5 μM for 30 min) or (b) ATP (5 mM 45 min). (c) The EP2 antagonist AH6809 (2 μM) was added 5 min prior to addition of ONO-AE1-259-01 followed by ATP (5 mM 45 min). (d) CAY10598 (500 nM) was added 30 min after stimulation with nigericin and analyzed 50 min post initial nigericin stimulation. (e,f) EP2 agonist ONO-AE1-259-01 or (g,h) EP4 antagonist ONO-AE3-208 was added 5 min before treatment with PGE2 (500 nM) followed by (e,g) nigericin (5 μM for 30 min) or (f,h) ATP (5 mM 45 min). (a-h). Total IL-1β release in cell supernatants analyzed by ELISA. (b,c,f,h) Immunoblot analysis of culture supernatants (SN) and lysates (LYS). *P < 0.005 (unpaired two-tailed t test). (a-h) Data are from one experiment representative of three separate experiments with similar results, (a-h) BMDM were derived from 2 mice per experiment mixed together, (a-h) three replicates, each replicate is one well + s.e.m.

Supplementary Figure 5 cAMP induces rapid NLRP3 inflammasome inhibition.

(a-c,e,f) BMDMs primed for 3.5 h with LPS. (a,b) The indicated concentration of forskolin was added 5 min prior to (a) nigericin (5 μM for 30 min) or (b) ATP (5 mM for 45 min). (c) db-cAMP (1 μM) was added 5 min prior to nigericin (5 μM for 30 min) or ATP (5 mM for 45 min). (d) PMA-differentiated THP-1 cells were treated for 5 min with indicated concentration of forskolin or db-cAMP prior to addition of nigericin (5 μM) and LPS (50 ng/ml) for 30 min. (e) Forskolin (50 μM) was added 30 min after stimulation with nigericin and analyzed 50 min post initial nigericin stimulation. (f) Forskolin (50 μM) or db-cAMP (1 μM) was added for 5 min prior to nigericin (5 μM for 30 min). (a-e) Total IL-1β release in cell supernatants analyzed by ELISA. (b-d,f) Immunoblot analysis of culture supernatants (SN) and lysates (LYS). *P < 0.005 (unpaired two-tailed t test). (a-f) Data are from one experiment representative of three separate experiments with similar results, (a-c,e,f) BMDM were derived from 2 mice per experiment mixed together, (a-e) three replicates, each replicate is one well + s.e.m.

Supplementary Figure 6 PKA inhibits the NLRP3 inflammasome in response to extracellular agonists but not particulate agonists.

(a-h) BMDMs primed for 3.5 h with LPS. (a,c) The indicated concentration of 6-Bnz-cAMP or (b) 8-CPT-2-O-Me-cAMP was added 5 min before nigericin (5 μM for 30 min). (d) 6-Bnz-cAMP (50 μM) or 8-CPT-2-O-Me-cAMP (50 μM) was added 5 min prior to treatment with MSU (125 μg/ml for 4h) or silica (250 μg/ml for 5h). (e) Rp-cAMP was added 5 min before or (f) PKI was added 30 min before (e,f) 5 min treatment with db-cAMP (1 μM) or forskolin (50 μM) and followed by nigericin (5 μM for 30 min). (g) Rp-cAMP was added 5 min before or (h) PKI was added 30 min before (g,h) 5 min treatment with PGE2 (500 nM) followed by nigericin (5 μM for 30 min). (a,b,d-h) Total IL-1β release in cell supernatants analyzed by ELISA. (c-e) Immunoblot analysis of culture supernatants (SN) and lysates (LYS). *P < 0.005 (unpaired two-tailed t test). (a-h) Data are from one experiment representative of three separate experiments with similar results, (a-h) BMDM were derived from 2 mice per experiment mixed together, (a,b,d-h) three replicates, each replicate is one well + s.e.m.

Supplementary Figure 7 PGE2-induced suppression of nigericin-triggered NLRP3 inflammasomes is mediated by PKA, but particulate NLRP3 agonists are not.

(a-f) BMDMs primed for 3.5 h with LPS. (a) Rp-cAMP was added 5 min before or (b) PKI was added 30 min before treatment with EP2 agonist ONO-AE1-259-01 (50 nM) for 5 min followed by nigericin (5 μM for 30 min). (c) Rp-cAMP was added 5 min before or (d) PKI was added 30 min before treatment with EP4 agonist ONO-AE1-329 (500 nM) for 5 min followed by nigericin (5 μM for 30 min). (e) PKI was added 30 min before or (f) Rp-cAMP was added 5 min before (e,f) PGE2 (500 nM) treatment for 5 min followed by addition of MSU (125 μg/ml for 4h) or silica (250 μg/ml for 5h). (a,c,e,f) Immunoblot analysis of culture supernatants (SN) and lysates (LYS). (a-f) Total IL-1β release in cell supernatants analyzed by ELISA. *P < 0.005 (unpaired two-tailed t test). (a-f) Data are from one experiment representative of three separate experiments with similar results, (a-f) BMDM were derived from 2 mice per experiment mixed together, (a-f) three replicates, each replicate is one well + s.e.m.

Supplementary Figure 8 PKA C-α and NLRP3 interact.

(a) Flow of NLRP3 ATPase assay used in Figure 6e and Figure 7e. (b,c) Immunoprecipitation of indicated protein of BMDMs primed for 3.5 h with LPS and treated for 5 min with forskolin (50 μM) in the presence or absence of PKI (1 μM). (b,c) Data are from one experiment representative of three separate experiments with similar results, (b,c) BMDM were derived from 2 mice per experiment mixed together.

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Mortimer, L., Moreau, F., MacDonald, J. et al. NLRP3 inflammasome inhibition is disrupted in a group of auto-inflammatory disease CAPS mutations. Nat Immunol 17, 1176–1186 (2016). https://doi.org/10.1038/ni.3538

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