Abstract
Toll-like receptor 9 (TLR9) agonists cause activation of nucleotide-binding domain, leucine-rich repeat protein 3 (NLRP3) inflammasomes but the mechanism is not clear. We found that there is a second signal downstream of TLR9 that induces NLRP3 inflammasome activation. Through screening, adenylate cyclase 7 (ADCY7) was found to be an essential regulator of this process. In cells with Adcy7 deficiency, TLR9 agonists were no longer able to activate the NLRP3 inflammasome. ADCY7 not only catalyzes the generation of cyclic adenosine monophosphate (cAMP) but also catalyzes the synthesis of its dimeric form (c-di-AMP). Moreover, c-di-AMP promotes assembly and maturation of the inflammasome by directly binding to NLRP3. Cells with Adcy7 deletion or mutations impacting enzymatic activity cannot produce c-di-AMP. The survival of Adcy7-deficient mice in acute liver injury was also improved. In summary, we found that ADCY7 is required for NLRP3 inflammasome activation downstream of TLR9 by catalyzing the generation of c-di-AMP, which may serve as a target for controlling inflammatory responses in sterile infections.
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Acknowledgements
This work was supported by the Beijing Natural Science Foundation (JG23028), National Natural Science Foundation of China (92369104, 82271790, 92169113 and 22174003), National Key R&D Program of China (2021YFA1300202, 2022YFC2302900 and 2022YFA1304500), Strategic Priority Research Programs of the CAS (XDB29020000), Key Research Program of Frontier Sciences of the CAS (ZDBS-LY-SM025), CAS Project for Young Scientists in Basic Research (YSBR-010), Fok Ying Tung Education Foundation (to P.X.) and Youth Innovation Promotion Association of the CAS (to S.W.).
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Q.L. and Z.T. designed and performed the experiments and analyzed the data. Y.Q., C.W., C.K., M.L., X.G., Y.Z., X.C., C.R., K.W., L.B., L.W., D.J., S.W. and X.L. performed the experiments and analyzed the data. P.X. initiated the study, designed and performed the experiments, analyzed the data and wrote the paper.
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Extended data
Extended Data Fig. 1 Ion flux is not required for TLR9-induced inflammasome.
(a) Tlr9+/+ and Tlr9–/– BMDM cells were stimulated with 1 μM control ODN or CpG-ODN for 6 h. Cell viability was determined by an ATP quantification assay in cell pellets. (b) Tlr9–/– BMDM cells were stimulated with 1 μM control ODN or CpG-ODN for 6 h. Otherwise, cells were stimulated with 1 μM control ODN or CpG-ODN and 1 μg/ml LPS for 6 h, followed by culture media replacement with PBS containing 10 μM nigericin for 30 min. Cell viability was determined by an ATP quantification assay in cell pellets. (c) Tlr9+/+ and Tlr9–/– BMDM cells were stimulated with 1 μM control ODN or CpG-ODN for 6 h. Intracellular potassium levels were determined through an inductively coupled plasma optical emission spectrometry and shown as relative levels. d–f, Wild-type BMDM cells primed with 1 μg/ml LPS for 3 h were stimulated with 10 μM nigericin for 30 min. 50 mM KCl was added to the medium 30 min before the addition of nigericin. Otherwise, cells were co-stimulated with 1 μM CpG-ODN and 50 mM KCl for 6 h. (d) Cell culture supernatants were collected for an ELISA assay to determine the secreted IL-1β protein levels. (e) Cytotoxicity was determined by LDH release assay in cell culture supernatants. (f) Cell viability was determined by an ATP quantification assay in cell pellets. g, h, Wild-type BMDM cells primed with 1 μg/ml LPS for 3 h were stimulated with 2 mM ATP for 30 min. 50 mM KCl was added to the medium 30 min before the addition of ATP. Otherwise, cells were co-stimulated with 1 μM CpG-ODN and 50 mM KCl for 6 h. (g) Cytotoxicity was determined by LDH release assay in cell culture supernatants. (h) Cell viability was determined by an ATP quantification assay in cell pellets. In a–h, data were shown as means ± SD (n = 4, biological replicates). Two-tailed Student’s t-test were used.
Extended Data Fig. 2 CpG-ODN stimulation activates NLRP3 inflammasome.
(a) Tlr9+/+ and Tlr9–/– BMDM cells were stimulated with 1 μM control ODN or CpG-ODN for 6 h. Cells were stained with a labeling calcium indicator Fluo-4 and calcium levels were quantified and shown as relative to that of controls. b–d, Wild-type BMDM cells primed with 1 μg/ml LPS for 3 h were stimulated with 10 μM nigericin for 30 min. Otherwise, cells were stimulated with 1 μM CpG-ODN for 6 h. Cells were also treated with or without 100 μM 2-APB. (b) Cell culture supernatants were collected for an ELISA assay to determine the secreted IL-1β protein levels. (c) Cytotoxicity was determined by LDH release assay in cell culture supernatants. (d) Cell viability was determined by an ATP quantification assay in cell pellets. e–h, Wild-type BMDM cells were stimulated with 1 μM control ODN or CpG-ODN for 6 h. Cells were lysed and immunoprecipitated with antibody against NLRP3. Precipitates were thermal-inactivated through incubation at 80 °C for 20 min. Thermal-inactivated precipitates were transfected into LPS-primed BMDM cells using a cationic lipid transfection reagent DOTAP for 6 h. (e) Cells were lysed and immunoblotted. (f) Cell culture supernatants were collected for an ELISA assay to determine the secreted IL-1β protein levels. (g) Cytotoxicity was determined by LDH release assay in cell culture supernatants. (h) Cell viability was determined by an ATP quantification assay in cell pellets. In a–d and f–h, data were shown as means ± SD (n = 4, biological replicates). Two-tailed Student’s t-test were used.
Extended Data Fig. 3 ADCY7 is positively involved in the TLR9-induced inflammasome.
a–c, iBMDM cells infected with lentiviruses carrying Cas9 and an sgRNA against the indicated genes or a scrambled control one (Ctrl) were stimulated with 1 μM CpG-ODN for 6 h. (a) Cell culture supernatants were collected for an ELISA assay to determine the secreted IL-1β protein levels. (b) Cytotoxicity was determined by LDH release assay in cell culture supernatants. (c) Cell viability was determined by an ATP quantification assay in cell pellets. (d) Adcy7+/+ and Adcy7–/– BMDM cells were stimulated with 1 μM control ODN or CpG-ODN for 6 h. Cell viability was determined by an ATP quantification assay in cell pellets. (e) Adcy7+/+ and Adcy7–/– BMDM cells were stimulated with 1 μM control ODN or 1 μg/ml E. coli genomic DNA for 6 h. Cells were lysed and immunoblotted. In a–c, data were shown as means ± SD (n = 3, biological replicates). In d, data were shown as means ± SD (n = 4, biological replicates). Two-tailed Student’s t-test were used.
Extended Data Fig. 4 ADCY7 enzymatic activity is required for inflammasome activation.
a–c, Adcy7–/– BMDM cells were rescued with wild-type or enzymatic-mutant ADCY7 without transmembrane domains. Cells were stimulated with 1 μM control ODN or CpG-ODN for 6 h. (a) Cell culture supernatants were collected for an ELISA assay to determine the secreted IL-1β protein levels. (b) Cytotoxicity was determined by LDH release assay in cell culture supernatants. (c) Cell viability was determined by an ATP quantification assay in cell pellets. d–g, Macrophages derived from human peripheral blood mononuclear cells were infected with lentiviruses carrying Cas9 and an sgRNA against human ADCY7. (d) The efficiencies of gene deletion were examined through immunoblotting. ADCY7+/+ and ADCY7–/– human macrophages were stimulated with 1 µg/ml E. coli genomic DNA for 6 h. (e) Cell culture supernatants were collected for an ELISA assay to determine the secreted IL-1β protein levels. (f) Cytotoxicity was determined by LDH release assay in cell culture supernatants. (g) Cell viability was determined by an ATP quantification assay in cell pellets. In a–c and e–g, data were shown as means ± SD (n = 4, biological replicates). Two-tailed Student’s t-test were used.
Extended Data Fig. 5 cAMP is not the second messenger that activates the NLRP3 inflammasome.
(a) Mass spectrometry of a c-di-AMP standard was shown. (b) Wild-type BMDM cells were infected with lentiviruses carrying Cas9 and the indicated sgRNAs for 3 days. Cells were then stimulated with 1 μM control ODN or CpG-ODN for 6 h, followed by ELISA assays for c-di-AMP levels. (c) Recombinant GST or the indicated ADCY7 isoforms were incubated with 10 mM ATP for 2 h, followed by ELISA assays for c-di-AMP levels. (d) Plasmids encoding GST-tagged dacA from Listeria and ADCY7 were transfected into E. coli strain BL21, followed by protein expression induction with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 6 h. Bacteria were lysed and subjected to ELISA assays for c-di-AMP levels. (e) Recombinant dgcN from E. coli and ADCY7 were incubated with saline or 10 mM GTP for 2 h, followed by ELISA assays for c-di-GMP levels. (f) Protein structures of Listeria dacA and ATP (PDB: 4rv7) were aligned with mouse ADCY7 (Alphafold: AF-P51829-F1-v4) (left panel). c-di-AMP was docked into the mouse ADCY7 structure (right panel). In b–e, data were shown as means ± SD (n = 4, biological replicates). Two-tailed Student’s t-test were used.
Extended Data Fig. 6 ADCY7 catalyzes c-di-AMP production.
(a) c-di-AMP was docked into the mouse ADCY7 or mouse ADCY4 structure (Alphafold: AF-Q91WF3-F1-v4) and vacuum electrostatics was shown. (b) Adcy7–/– BMDM cells were rescued with wild-type or enzymatic-mutant ADCY7 without transmembrane domains. Cells were stimulated with 1 μM control ODN or 1 μg/ml E. coli genomic DNA for 6 h. Cells were lysed and the intracellular c-di-AMP levels were determined by an ELISA assay. (c) Human macrophages were infected with lentiviruses carrying Cas9 and an sgRNA against human ADCY7. ADCY7+/+ and ADCY7–/– human macrophages were stimulated with 1 μM control ODN or CpG-ODN for 6 h. Cells were lysed and the intracellular c-di-AMP levels were determined by an ELISA assay using an antibody specific for c-di-AMP. (d) ADCY7+/+ and ADCY7–/– human macrophages were stimulated with 1 μM control ODN or 1 μg/ml E. coli genomic DNA for 6 h. Cells were lysed and the intracellular c-di-AMP levels were determined by an ELISA assay using an antibody specific for c-di-AMP. (e) ADCY7–/– human macrophages were rescued with wild-type or enzymatic-mutant ADCY7 without transmembrane domains. Cells were stimulated with 1 μM control ODN or 1 μg/ml E. coli genomic DNA for 6 h. Cells were lysed and the intracellular c-di-AMP levels were determined by an ELISA assay using an antibody specific for c-di-AMP. (f) Wild-type BMDM cells primed with 1 μg/ml LPS for 3 h were stimulated with 10 μM nigericin for 30 min. Otherwise, cells were stimulated with 1 μM CpG-ODN for 6 h. Cells were lysed and the intracellular c-di-AMP levels were determined by an ELISA assay using an antibody specific for c-di-AMP. (g) Wild-type human macrophages primed with 1 μg/ml LPS for 3 h were stimulated with 10 μM nigericin for 30 min. Otherwise, cells were stimulated with 1 μM CpG-ODN for 6 h. Cells were lysed and the intracellular c-di-AMP levels were determined by an ELISA assay using an antibody specific for c-di-AMP. In b–g, data were shown as means ± SD (n = 4, biological replicates). Two-tailed Student’s t-test were used.
Extended Data Fig. 7 c-di-AMP binds NLRP3.
(a) Biotin or biotin-labelled c-di-AMP were incubated with BMDM cell lysates for 4 h, followed by precipitation using streptavidin beads. Precipitates were immunoblotted with antibodies against NLRP3 and two known NLRP3 binding partners. (b) Wild-type BMDM cells were stimulated with 1 μM control ODN or CpG-ODN for 6 h. Cells were lysed and immunoprecipitated with a control IgG or antibody against NLRP3. Precipitates were subjected to an ELISA assay for determining the c-di-AMP levels. (c) BMDM cells from Nlrp3+/+ and Nlrp3–/– mice were primed with 1 μg/ml LPS for 3 h, followed by transfection of 10 μg/ml c-di-AMP using DOTAP for 6 h. Cells were lysed and pellets were crosslinked with 2 mM disuccinimidyl suberate (DSS) for 30 min at room temperature. Crosslinked pellets were dissolved in 1 X Laemmli buffer and immunoblotted with antibody against ASC. Cell lysates were also immunoblotted as indicated. (d) Biotin-labelled c-di-AMP were incubated with recombinant NLRP3 protein for 2 h and crosslinked by ultraviolet, followed by precipitation using antibody against NLRP3. Precipitates were immunoblotted with antibody against biotin. (e) Wild-type BMDM cells were transfected with saline or 10 μg/ml c-di-AMP using DOTAP for 6 h, followed by immunostaining with antibody against NLRP3. Nuclei were stained with DAPI. (f) Plasmids encoding NLRP3 and ADCY7 without transmembrane domains were co-transfected into HEK293T cells for 24 h, followed by immunoprecipitation with a control IgG or antibody against NLRP3. Precipitates were immunoblotted as indicated. (g) Plasmids encoding FLAG-tagged NLRP3 PYD domain (aa 1–134) and ADCY7 without transmembrane domains were co-transfected into HEK293T cells for 24 h, followed by immunoprecipitation with a control IgG or antibody against FLAG. Precipitates were immunoblotted as indicated. In e, scale bar, 10 μm. In b, data were shown as means ± SD (n = 4, biological replicates). Two-tailed Student’s t-test were used.
Extended Data Fig. 8 ADCY7 deficiency hampers NLRP3 inflammasome.
(a) Adcy7–/– BMDM cells were rescued with lentiviruses carrying the indicated ADCY7 isoforms for 3 days. Cells were then stimulated with 1 μM CpG-ODN for 6 h and immunostained with antibodies against TLR9 and ADCY7. Nuclei were stained with DAPI. (b) Adcy7–/– BMDM cells rescued with wild-type or enzymatic-mutant ADCY7 were stimulated with 1 μM CpG-ODN for 6 h. Cells were lysed and immunoprecipitated with a control IgG or antibody against TLR9. Precipitates were immunoblotted as indicated. (c) Adcy7–/– BMDM cells rescued with the indicated ADCY7 truncations were stimulated with 1 μM CpG-ODN for 6 h. Cells were lysed and immunoprecipitated with a control IgG or antibody against TLR9. Precipitates were immunoblotted as indicated. (d) Wild-type BMDM cells were stimulated with 1 μM control ODN or CpG-ODN for 6 h. Otherwise, cells were transfected with 10 μg/ml cGAMP using DOTAP for 6 h. Cells were lysed and run on a Native-PAGE for STING dimerization. Cells lysates were also immunoblotted as indicated. (e) Wild-type, Adcy7–/– and Tlr9–/– mice (n = 4 for each group) were intraperitoneally injected with D-GalN (20 mg per mice) and CpG-ODN (20 nmol per mice). Meanwhile, mice were injected with 100 nmol c-di-AMP through hydrodynamic tail vein injection. Serum levels of IL-1β were determined 6 h post injection. In a, scale bar, 10 µm. In e, data were shown as means ± SD (n = 4, biological replicates). Two-tailed Student’s t-test were used.
Extended Data Fig. 9 ADCY7 catalyzes c-di-AMP for inflammasome activation.
(a) Tlr9+/+ and Tlr9–/– BMDM cells were stimulated with 2 μg/ml nuclear non-CpG DNA, nuclear CpG DNA, mitochondrial DNA from HEK293T cells or E. coli genomic DNA for 6 h, followed by ELISA assays to determine the secreted IL-1β protein levels. (b) Adcy7–/– BMDM cells rescued with the indicated ADCY7 truncations were stimulated with 1 μM CpG-ODN for 6 h. Cells were lysed and immunoprecipitated with a control IgG or antibody against TLR9. Precipitates were subjected to an ELISA assay for determining c-di-AMP levels. c–e, Adcy7–/– BMDM cells rescued with the indicated ADCY7 truncations were stimulated with 1 μM control ODN or CpG-ODN for 6 h. (c) Cells were lysed and immunoblotted. (d) Cell culture supernatants were collected for an ELISA assay to determine the secreted IL-1β protein levels. (e) Cytotoxicity was determined by LDH release assay in cell culture supernatants. (f) Adcy7–/– BMDM cells rescued with the indicated ADCY7 truncations were stimulated with 1 μM control ODN or CpG-ODN for 6 h. Cell viability was determined by an ATP quantification assay in cell pellets. In a, b and d–f, data were shown as means ± SD (n = 4, biological replicates). Two-tailed Student’s t-test were used.
Extended Data Fig. 10 ADCY7 is involved in sterile inflammation.
(a) Wild-type, Adcy7–/–, Nlrp3–/– and Tlr9–/– mice were intraperitoneally injected with CpG-ODN (20 nmol per mice) for 6 h. Cell viability was determined by an ATP quantification assay in cell pellets. (b) Peritoneal macrophages were collected from wild-type, Adcy7–/–, Nlrp3–/– and Tlr9–/– mice. Cells were stimulated with 1 μM control ODN or CpG-ODN for 6 h. Cell culture supernatants were collected for an ELISA assay to determine the secreted IL-1β protein levels. (c) A scheme for APAP stimulation was shown. d–f, Wild-type, Adcy7–/–, Nlrp3–/– and Tlr9–/– mice (n = 12) were intraperitoneally injected with 500 mg/kg APAP post a starvation of 15 h. (d) Survivals of mice were monitored for the indicated time points. Serum levels of (e) IL-1β and (f) transaminase (ALT) were determined 12 h post injection. In a, b and e, f, data were shown as means ± SD (n = 4, biological replicates). Two-tailed Student’s t-test were used.
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Liu, Q., Tang, Z., Qian, Y. et al. Eukaryotic ADCY7 catalyzes the production of c-di-AMP to activate the NLRP3 inflammasome. Nat Chem Biol 21, 1283–1291 (2025). https://doi.org/10.1038/s41589-025-01919-y
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DOI: https://doi.org/10.1038/s41589-025-01919-y
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