Abstract
Intracellular recognition of lipopolysaccharide (LPS) by mouse caspase-11 or human caspase-4 is a vital event for the activation of the noncanonical inflammasome. Whether negative regulators are involved in intracellular LPS sensing is still elusive. Here we show that adipose triglyceride lipase (ATGL) is a negative regulator of the noncanonical inflammasome. Through screening for genes participating in the noncanonical inflammasome, ATGL is identified as a negative player for intracellular LPS signaling. ATGL binds LPS and catalyzes the removal of the acylated side chains that contain ester bonds. LPS with under-acylated side chains no longer activates the inflammatory caspases. Cells with ATGL deficiency exhibit enhanced immune responses when encountering intracellular LPS, including an elevated secretion of interleukin-1β, decreased cell viability and increased cell cytotoxicity. Moreover, ATGL-deficient mice show exacerbated responses to endotoxin challenges. Our results uncover that ATGL degrades cytosolic LPS to suppress noncanonical inflammasome activation.
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Data availability
The authors confirm that the data supporting the findings of this study are available within the article and Supplementary Information. Source data are provided with this paper.
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Acknowledgements
We thank T. Li (Peking University) for technical help. We thank X. Qi (Shandong University) for providing F. novicida. We also thank C.H. Liu, J. Wang and Y. Chen (Institute of Microbiology, Chinese Academy of Sciences) for technical help and advice. This work was supported by Beijing Natural Science Foundation (JQ23028 and 7212067), the National Natural Science Foundation of China (92369104, 82271790, 92169113 and 31925021), the National Key R&D Program of China (2021YFA1300202 and 2022YFC2302900), Strategic Priority Research Programs of the Chinese Academy of Sciences (XDB29020000), Key Research Program of Frontier Sciences of Chinese Academy of Sciences (ZDBS-LY-SM025), Chinese Academy of Sciences (CAS) Project for Young Scientists in Basic Research (YSBR-010), Fok Ying Tung Education Foundation to P.X. and Youth Innovation Promotion Association of CAS to S.W.
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W.L. and Q.L. performed experiments and analyzed data. Y.Q., C.W. and C.K. supported the planning and conduction of protein purification experiments. L.L.S. and L.S. contributed to mass spectrometry experiments. H.L., Y.Z., D.J., C.J. and S.W. provided technical assistance and analyzed data. P.X. initiated the study, designed and performed experiments, analyzed data and wrote the paper.
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Extended data
Extended Data Fig. 1 ATGL deficiency promotes non-canonical inflammasome activation.
a,b, iBMDM cells were infected with lentiviruses carrying Cas9 and a sgRNA against Pnpla2, the gene encoding ATGL. Cells were primed overnight with 1 μg/ml Pam3CSK4, followed by transfection of 2 μg/ml LPS using DOTAP. (a) Cell culture supernatants were collected for an ELISA assay to determine the secreted IL-1β protein levels 16 h postinfection. (b) Cell viability was determined by an ATP quantification assay in cell pellets 16 h postinfection. c–e, iBMDM cells infected with lentiviruses carrying Cas9 and a sgRNA against Pnpla2 were primed overnight with 1 μg/ml Pam3CSK4 and incubated with E. coli strain at an MOI of 30 for 1.5 h. Cells were then supplemented with fresh medium containing 100 μg/ml Gentamycin. (c) Cell culture supernatants were collected for an ELISA assay to determine the secreted IL-1β protein levels 16 h postinfection. (d) Cell viability was determined by an ATP quantification assay in cell pellets 16 h postinfection. (e) Cells were fixed 16 h postinfection and stained with antibodies against ASC, followed by the examination of ASC specks inside cells (left). Percentages of cells with ASC specks were calculated (right). f–h, Macrophages derived from human peripheral blood mononuclear cells were infected with lentiviruses carrying Cas9 and a sgRNA against human PNPLA2, followed by priming overnight with 1 μg/ml Pam3CSK4. (f) Cell culture supernatants were collected for an ELISA assay to determine the secreted IL-1β protein levels 16 h post-transfection. (g) Cell viability was determined by an ATP quantification assay in cell pellets 16 h post-transfection. (h) Cells were fixed 16 h post-transfection and stained with antibodies against ASC, followed by the examination of ASC specks inside cells (left). Percentages of cells with ASC specks were calculated (right). For e,h, scale bars, 10 μm. For a–h, data were shown as means ± SD of n = 3 replicates from one representative of three independent experiments. Two-tailed Student’s t-tests were used.
Extended Data Fig. 2 ATGL suppresses non-canonical inflammasome.
a,b, Pnpla2+/+ and Pnpla2−/− BMDM cells were primed overnight with 1 μg/ml Pam3CSK4, followed by transfection of 2 μg/ml LPS using DOTAP. (a) Cell culture supernatants were collected for an ELISA assay to determine the secreted IL-1β protein levels 16 h post-transfection. (b) Cell viability was determined by an ATP quantification assay in cell pellets 16 h post-transfection. c,d Wild-type BMDM cells were infected with lentiviruses carrying a control vector or full-length ATGL. Cells were primed overnight with 1 μg/ml Pam3CSK4, followed by transfection of 2 μg/ml LPS using DOTAP. (c) Cell culture supernatants were collected for an ELISA assay to determine the secreted IL-1β protein levels 16 h post-transfection. (d) Cell viability was determined by an ATP quantification assay in cell pellets 16 h post-transfection. e–i, Wild-type BMDM cells were infected with lentiviruses carrying a control vector or full-length ATGL. Cells were primed overnight with 1 μg/ml Pam3CSK4 and incubated with E. coli strain at an MOI of 30 for 1.5 h. Cells were then supplemented with fresh medium containing 100 μg/ml Gentamycin. (e) Cells were lysed and immunoblotted 16 h postinfection. (f) Cell culture supernatants were collected for an ELISA assay to determine the secreted IL-1β protein levels 16 h postinfection. (g) Cell viability was determined by an ATP quantification assay in cell pellets 16 h postinfection. (h) Cytotoxicity was determined by LDH release assay in cell culture supernatants 16 h postinfection. (i) Cells were fixed 16 h postinfection and stained with antibodies against ASC, followed by the examination of ASC specks inside cells (left). Percentages of cells with ASC specks were calculated (right). For i, scale bars, 10 μm. For a–d, f–i, data were shown as means ± SD of n = 3 replicates from one representative of three independent experiments. Two-tailed Student’s t-tests were used. For e, experiments were repeated three times with similar results.
Extended Data Fig. 3 ATGL doesn’t affect canonical inflammasome.
a, BMDM cells were generated from Pnpla2+/+ and Pnpla2−/− bone marrow cells, followed by quantification of lipid abundance using Nile Green staining. b–f, WT BMDM cells were cultured in medium containing 300 μM oleic acid for 24 h. (b) Lipid abundance was quantified by Nile Green staining. Cells were then primed overnight with 1 μg/ml Pam3CSK4, followed by transfection of 2 μg/ml LPS using DOTAP. (c) Cells were lysed and immunoblotted 16 h post-transfection. (d) Cell culture supernatants were collected for an ELISA assay to determine the secreted IL-1β protein levels 16 h post-transfection. (e) Cell viability was determined by an ATP quantification assay in cell pellets 16 h post-transfection. (f) Cytotoxicity was determined by LDH release assay in cell culture supernatants 16 h post-transfection. g–i, Pnpla2+/+ and Pnpla2−/− BMDM cells primed with 1 μg/ml LPS for 3 h were incubated with 10 μM nigericin for 30 min, Salmonella (log phase) at an MOI of 10 for 4 h, or transfected with 2 μg/ml poly(dA:dT) for 4 h. (g) Cells were lysed and immunoblotted as indicated. (h) Cytotoxicity was determined by LDH release assay in cell culture supernatants. (i) Cells were fixed and stained with antibodies against ASC, followed by the examination of ASC specks inside cells (left). Percentages of cells with ASC specks were calculated (right). For i, scale bars, 10 μm. For a, b, d–f, h, i, data were shown as means ± SD of n = 3 replicates from one representative of three independent experiments. Two-tailed Student’s t-tests were used. For c, g, experiments were repeated three times with similar results.
Extended Data Fig. 4 ATGL C-terminus binds LPS.
a, Recombinant GST and GST–ATGL were purified from E. coli, followed by contaminated LPS removal using Pierce high-capacity endotoxin removal spin columns. Proteins were then incubated with LPS and precipitated by a glutathione sepharose resin, followed by an ELISA assay for the determination of LPS levels. b, Pnpla2−/− BMDM cells were infected with lentiviruses carrying control vectors, full-length ATGL, ATGL N-terminus and ATGL C-terminus. Cells were lysed and immunoblotted as indicated. c,d, Pnpla2−/− BMDM cells were infected with lentiviruses carrying control vectors, full-length ATGL, ATGL N-terminus and ATGL C-terminus. Cells were primed overnight with 1 μg/ml Pam3CSK4, followed by transfection of 2 μg/ml LPS using DOTAP. (c) Cell culture supernatants were collected for an ELISA assay to determine the secreted IL-1β protein levels 16 h post-transfection. (d) Cell viability was determined by an ATP quantification assay in cell pellets 16 h post-transfection. e–h, Pnpla2−/− BMDM cells were infected with lentiviruses carrying control vectors, full-length ATGL, ATGL N-terminus and ATGL C-terminus. Cells were primed overnight with 1 μg/ml Pam3CSK4 and incubated with E. coli strain at an MOI of 30 for 1.5 h. Cells were then supplemented with fresh medium containing 100 μg/ml Gentamycin. (e) Cell culture supernatants were collected for an ELISA assay to determine the secreted IL-1β protein levels 16 h postinfection. (f) Cell viability was determined by an ATP quantification assay in cell pellets 16 h postinfection. (g) Cytotoxicity was determined by LDH release assay in cell culture supernatants 16 h postinfection. (h) Cells were fixed 16 h postinfection and stained with antibodies against ASC, followed by the examination of ASC specks inside cells (left). Percentages of cells with ASC specks were calculated (right). For h, scale bars, 10 μm. For a, c–h, data were shown as means ± SD of n = 3 replicates from one representative of three independent experiments. Two-tailed Student’s t-tests were used. For b, experiments were repeated three times with similar results.
Extended Data Fig. 5 ATGL’s enzymatic activity is required for suppressing non-canonical inflammasome.
a–c, Pnpla2−/− BMDM cells were infected with lentiviruses carrying control vectors, full-length ATGL and the catalytic domain-disabled ATGLS47A. Cells were primed overnight with 1 μg/ml Pam3CSK4, followed by transfection of 2 μg/ml LPS using DOTAP. (a) Cell culture supernatants were collected for an ELISA assay to determine the secreted IL-1β protein levels 16 h post-transfection. (b) Cell viability was determined by an ATP quantification assay in cell pellets 16 h post-transfection. (c) Cells were fixed 16 h post-transfection and stained with antibodies against ASC, followed by the examination of ASC specks inside cells (top). Percentages of cells with ASC specks were calculated (bottom). d–g, Pnpla2−/− BMDM cells were infected with lentiviruses carrying control vectors, full-length ATGL and the catalytic domain-disabled ATGLS47A. Cells were primed overnight with 1 μg/ml Pam3CSK4 and incubated with E. coli strain at an MOI of 30 for 1.5 h. Cells were then supplemented with fresh medium containing 100 μg/ml Gentamycin. (d) Cell culture supernatants were collected for an ELISA assay to determine the secreted IL-1β protein levels 16 h postinfection. (e) Cell viability was determined by an ATP quantification assay in cell pellets 16 h postinfection. (f) Cytotoxicity was determined by LDH release assay in cell culture supernatants 16 h postinfection. (g) Cells were fixed 16 h postinfection and stained with antibodies against ASC, followed by the examination of ASC specks inside cells (left). Percentages of cells with ASC specks were calculated (right). For c, g, scale bars, 10 μm. For a–g, data were shown as means ± SD of n = 3 replicates from one representative of three independent experiments. Two-tailed Student’s t-tests were used.
Extended Data Fig. 6 Mass spectrometry identification of LPS hydrolysates catalyzed by ATGL.
a, A scheme for the lipid A structure of Salmonella LPS was shown. Putative cleavage sites of ATGL were highlighted in red. b, Lipid A was incubated with ATGL at 37 °C for 4 h, followed by component identification through mass spectrometry. The fractions of interest were annotated with asterisks (left) and the corresponding spectrums were listed (right). Structures of lipid A variants were also shown as inserts. Experiments were repeated three times with similar results.
Extended Data Fig. 7 ATGL removes LPS side chains.
a,b Salmonella LPS was incubated with ATGL at 37 °C for 4 h to produce LPS variants. Wild-type BMDM cells were primed overnight with 1 μg/ml Pam3CSK4, followed by transfection of 2 μg/ml LPS and LPS variants using DOTAP. (a) Cell culture supernatants were collected for an ELISA assay to determine the secreted IL-1β protein levels 16 h post-transfection. (b) Cell viability was determined by an ATP quantification assay in cell pellets 16 h post-transfection. c,d LPSs purified from E. coli or F. novicida mutant strain XWK4 were incubated with ATGL at 37 °C for 4 h to produce LPS variants. Wild-type BMDM cells were primed overnight with 1 μg/ml Pam3CSK4, followed by transfection of 2 μg/ml LPS and LPS variants using DOTAP. (c) Cytotoxicity was determined by LDH release assay in cell culture supernatants 16 h post-transfection. (d) Cells were fixed 16 h post-transfection and stained with antibodies against ASC, followed by the examination of ASC specks inside cells (left). Percentages of cells with ASC specks were calculated (right). e–g, Pnpla2+/+ and Pnpla2−/− BMDM cells were primed overnight with 1 μg/ml Pam3CSK4, followed by transfection of 2 μg/ml LPS using DOTAP with or without the presence of 20 μM Atglistatin. (e) Cell culture supernatants were collected for an ELISA assay to determine the secreted IL-1β protein levels 16 h post-transfection. (f) Cell viability was determined by an ATP quantification assay in cell pellets 16 h post-transfection. (g) Cells were fixed 16 h post-transfection and stained with antibodies against ASC, followed by the examination of ASC specks inside cells (left). Percentages of cells with ASC specks were calculated (right). For d,g, scale bars, 10 μm. For a–g, data were shown as means ± SD of n = 3 replicates from one representative of three independent experiments. Two-tailed Student’s t-tests were used.
Extended Data Fig. 8 Rescue of ATGL enzymatic mutant boosts LPS signaling.
a–e, Wild-type BMDM cells infected with lentiviruses carrying a control vector, full-length ATGL and ATGLS47A were primed overnight with 1 μg/ml Pam3CSK4, followed by transfection of 2 μg/ml LPS using DOTAP. (a) Cells were lysed and immunoblotted 16 h post-transfection. (b) Cell culture supernatants were collected for an ELISA assay to determine the secreted IL-1β protein levels 16 h post-transfection. (c) Cell viability was determined by an ATP quantification assay in cell pellets 16 h post-transfection. (d) Cytotoxicity was determined by LDH release assay in cell culture supernatants 16 h post-transfection. (e) Cells were fixed 16 h post-transfection and stained with antibodies against ASC, followed by the examination of ASC specks inside cells (left). Percentages of cells with ASC specks were calculated (right). For e, scale bars, 10 μm. For b–e, data were shown as means ± SD of n = 3 replicates from one representative of three independent experiments. Two-tailed Student’s t-tests were used. For a, experiments were repeated three times with similar results.
Extended Data Fig. 9 ATGL deficiency affects IL-1β secretion.
a,b Pnpla2+/+ and Pnpla2−/− mice were intraperitoneally challenged with poly(I:C) at a dose of 2 mg/kg body weight for 6 h and then intraperitoneally injected with LPS at a dose of 8 mg/kg body weight. Serum levels of TNF-α (a) and IL-6 (b) were determined through ELISA assays 4 h later. c–f, Pnpla2+/+ and Pnpla2−/− mice were intraperitoneally challenged with poly(I:C) at a dose of 2 mg/kg body weight for 6 h and then intraperitoneally injected with LPS at a dose of 8 mg/kg body weight. Peritoneal macrophages were collected 4 h later and plated on culture plates for 6 h. (c) Cell culture supernatants were collected for an ELISA assay to determine the secreted IL-1β protein levels. (d) Cell viability was determined by an ATP quantification assay in cell pellets. (e) Cytotoxicity was determined by LDH release assay in cell culture supernatants. (f) Cells were fixed and stained with antibodies against ASC, followed by the examination of ASC specks inside cells (left). Percentages of cells with ASC specks were calculated (right). For f, scale bars, 10 μm. For a–f, data were shown as means ± SD of n = 3 (for a, b, n = 5) replicates from one representative of three independent experiments. Two-tailed Student’s t-tests were used.
Extended Data Fig. 10 ATGL overexpression protects host from endotoxins.
a,b, Wild-type mice orally administrated with 2 mM/kg diet Atglistatin for 5 days were intraperitoneally challenged with poly(I:C) at a dose of 2 mg/kg body weight for 6 h and then intraperitoneally injected with LPS at a dose of 8 mg/kg body weight. Serum levels of TNF-α (a) and IL-6 (b) were determined through ELISA assays 4 h later. c–e, AAV viral particles containing ATGL gene were delivered into WT mice through the tail vein for 2 weeks. (c) Peritoneal macrophages were collected and immunoblotted. Mice were intraperitoneally challenged with poly(I:C) at a dose of 2 mg/kg body weight for 6 h and then intraperitoneally injected with LPS at a dose of 16 mg/kg body weight and monitored for survival. (d) Survivals were calculated. (e) Serum levels of IL-1β were determined through ELISA assays 4 h later. f–h, Human macrophages were primed overnight with 1 μg/ml Pam3CSK4, followed by transfection of 2 μg/ml LPS using DOTAP with or without the presence of 40 μM NG-497. (f) Cell culture supernatants were collected for an ELISA assay to determine the secreted IL-1β protein levels 16 h post-transfection. (g) Cell viability was determined by an ATP quantification assay in cell pellets 16 h post-transfection. (h) Cells were fixed 16 h post-transfection and stained with antibodies against ASC, followed by the examination of ASC specks inside cells (left). Percentages of cells with ASC specks were calculated (right). For h, scale bars, 10 μm. For d, data were shown as means ± SD of n = 10. Gehan-Breslow-Wilcoxon test was used. For a, b, e–h, data were shown as means ± SD of n = 5 (for f–h, n = 3) replicates from one representative of three independent experiments. Two-tailed Student’s t-tests were used. For c, experiments were repeated three times with similar results.
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Li, W., Liu, Q., Qian, Y. et al. Adipose triglyceride lipase suppresses noncanonical inflammasome by hydrolyzing LPS. Nat Chem Biol (2024). https://doi.org/10.1038/s41589-024-01569-6
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DOI: https://doi.org/10.1038/s41589-024-01569-6
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