Abstract
Nutritional status and pyroptosis are important for host defence against infections. However, the molecular link that integrates nutrient sensing into pyroptosis during microbial infection is unclear. Here, using metabolic profiling, we found that Yersinia pseudotuberculosis infection results in a significant decrease in intracellular glucose levels in macrophages. This leads to activation of the glucose and energy sensor AMPK, which phosphorylates the essential kinase RIPK1 at S321 during caspase-8-mediated pyroptosis. This phosphorylation inhibits RIPK1 activation and thereby restrains pyroptosis. Boosting the AMPK–RIPK1 cascade by glucose deprivation, AMPK agonists, or RIPK1-S321E knockin suppresses pyroptosis, leading to increased susceptibility to Y. pseudotuberculosis infection in mice. Ablation of AMPK in macrophages or glucose supplementation in mice is protective against infection. Thus, we reveal a molecular link between glucose sensing and pyroptosis, and unveil a mechanism by which Y. pseudotuberculosis reduces glucose levels to impact host AMPK activation and limit host pyroptosis to facilitate infection.
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Data availability
The raw data for mass spectrometry analysis of RIPK1 phosphorylation sites by AMPK have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD045126. The raw data for untargeted metabolomics have been deposited in figshare (https://doi.org/10.6084/m9.figshare.c.6888193.v1)61. Pathway enrichment analysis was performed using MetaboAnalyst (https://www.metaboanalyst.ca) embedded with the KEGG pathway database. Source data are provided with this paper.
Code availability
The code for metabolite annotation is available via MetDNA at http://metdna.zhulab.cn.
References
Kayagaki, N. et al. Caspase-11 cleaves gasdermin D for non-canonical inflammasome signalling. Nature 526, 666–671 (2015).
He, W. T. et al. Gasdermin D is an executor of pyroptosis and required for interleukin-1β secretion. Cell Res. 25, 1285–1298 (2015).
Shi, J. et al. Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death. Nature 526, 660–665 (2015).
Ding, J. et al. Pore-forming activity and structural autoinhibition of the gasdermin family. Nature 535, 111–116 (2016).
Liu, X. et al. Inflammasome-activated gasdermin D causes pyroptosis by forming membrane pores. Nature 535, 153–158 (2016).
Evavold, C. L. et al. The pore-forming protein gasdermin D regulates interleukin-1 secretion from living macrophages. Immunity 48, 35–44 e36 (2018).
Reddick, L. E. & Alto, N. M. Bacteria fighting back: how pathogens target and subvert the host innate immune system. Mol. Cell 54, 321–328 (2014).
Paquette, N. et al. Serine/threonine acetylation of TGFbeta-activated kinase (TAK1) by Yersinia pestis YopJ inhibits innate immune signaling. Proc. Natl Acad. Sci. USA 109, 12710–12715 (2012).
Philip, N. H. et al. Caspase-8 mediates caspase-1 processing and innate immune defense in response to bacterial blockade of NF-kappaB and MAPK signaling. Proc. Natl Acad. Sci. USA 111, 7385–7390 (2014).
Peterson, L. W. et al. RIPK1-dependent apoptosis bypasses pathogen blockade of innate signaling to promote immune defense. J. Exp. Med. 214, 3171–3182 (2017).
Sarhan, J. et al. Caspase-8 induces cleavage of gasdermin D to elicit pyroptosis during Yersinia infection. Proc. Natl Acad. Sci. USA 115, E10888–E10897 (2018).
Orning, P. et al. Pathogen blockade of TAK1 triggers caspase-8-dependent cleavage of gasdermin D and cell death. Science 362, 1064–1069 (2018).
Wickersham, M. et al. Metabolic stress drives keratinocyte defenses against Staphylococcus aureus infection. Cell Rep. 18, 2742–2751 (2017).
Zhang, Q. et al. AMPK directly phosphorylates TBK1 to integrate glucose sensing into innate immunity. Mol. Cell 82, 4519–4536.e7 (2022).
Tucey, T. M. et al. Glucose homeostasis is important for immune cell viability during Candida challenge and host survival of systemic fungal infection. Cell Metab. 27, 988–1006.e7 (2018).
Thaker, S. K. et al. Differential metabolic reprogramming by Zika virus promotes cell death in human versus mosquito cells. Cell Metab. 29, 1206–1216.e4 (2019).
Wang, A. et al. Opposing effects of fasting metabolism on tissue tolerance in bacterial and viral inflammation. Cell 166, 1512–1525.e2 (2016).
Hardie, D. G., Ross, F. A. & Hawley, S. A. AMPK: a nutrient and energy sensor that maintains energy homeostasis. Nat. Rev. Mol. Cell Biol. 13, 251–262 (2012).
Garcia, D. & Shaw, R. J. AMPK: mechanisms of cellular energy sensing and restoration of metabolic balance. Mol. Cell 66, 789–800 (2017).
Lin, S. C. & Hardie, D. G. AMPK: sensing glucose as well as cellular energy status. Cell Metab. 27, 299–313 (2018).
Zhang, C. S. et al. Fructose-1,6-bisphosphate and aldolase mediate glucose sensing by AMPK. Nature 548, 112–116 (2017).
Zhang, C. S. et al. The lysosomal v-ATPase-Ragulator complex is a common activator for AMPK and mTORC1, acting as a switch between catabolism and anabolism. Cell Metab. 20, 526–540 (2014).
Bhutta, M. S., Gallo, E. S. & Borenstein, R. Multifaceted role of AMPK in viral infections. Cells 10, 1118 (2021).
Silwal, P., Kim, J. K., Yuk, J. M. & Jo, E. K. AMP-activated protein kinase and host defense against infection. Int. J. Mol. Sci. 19, 3495 (2018).
Jo, E. K., Silwal, P. & Yuk, J. M. AMPK-targeted effector networks in mycobacterial infection. Front. Microbiol. 10, 520 (2019).
Zhang, T. et al. Metabolic orchestration of cell death by AMPK-mediated phosphorylation of RIPK1. Science 380, 1372–1380 (2023).
Lachmandas, E. et al. Microbial stimulation of different Toll-like receptor signalling pathways induces diverse metabolic programmes in human monocytes. Nat. Microbiol. 2, 16246 (2016).
Galvan-Pena, S. & O’Neill, L. A. Metabolic reprograming in macrophage polarization. Front. Immunol. 5, 420 (2014).
Saxton, R. A. & Sabatini, D. M. mTOR signaling in growth, metabolism, and disease. Cell 168, 960–976 (2017).
Battaglioni, S., Benjamin, D., Walchli, M., Maier, T. & Hall, M. N. mTOR substrate phosphorylation in growth control. Cell 185, 1814–1836 (2022).
Sag, D., Carling, D., Stout, R. D. & Suttles, J. Adenosine 5′-monophosphate-activated protein kinase promotes macrophage polarization to an anti-inflammatory functional phenotype. J. Immunol. 181, 8633–8641 (2008).
Zheng, Z. et al. The lysosomal rag-ragulator complex licenses RIPK1 and caspase-8-mediated pyroptosis by Yersinia. Science 372, eabg0269 (2021).
Calabrese, M. F. et al. Structural basis for AMPK activation: natural and synthetic ligands regulate kinase activity from opposite poles by different molecular mechanisms. Structure 22, 1161–1172 (2014).
Cool, B. et al. Identification and characterization of a small molecule AMPK activator that treats key components of type 2 diabetes and the metabolic syndrome. Cell Metab. 3, 403–416 (2006).
Lai, Y. C. et al. A small-molecule benzimidazole derivative that potently activates AMPK to increase glucose transport in skeletal muscle: comparison with effects of contraction and other AMPK activators. Biochem. J. 460, 363–375 (2014).
Muendlein, H. I. et al. ZBP1 promotes LPS-induced cell death and IL-1beta release via RHIM-mediated interactions with RIPK1. Nat. Commun. 12, 86 (2021).
Muendlein, H. I. et al. cFLIPl protects macrophages from LPS-induced pyroptosis via inhibition of complex II formation. Science 367, 1379–1384 (2020).
Mounier, R. et al. AMPKα1 regulates macrophage skewing at the time of resolution of inflammation during skeletal muscle regeneration. Cell Metab. 18, 251–264 (2013).
Ofengeim, D. et al. Activation of necroptosis in multiple sclerosis. Cell Rep. 10, 1836–1849 (2015).
Degterev, A. et al. Identification of RIP1 kinase as a specific cellular target of necrostatins. Nat. Chem. Biol. 4, 313–321 (2008).
Sun, L. et al. Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase. Cell 148, 213–227 (2012).
Najjar, M. et al. RIPK1 and RIPK3 kinases promote cell-death-independent inflammation by Toll-like receptor 4. Immunity 45, 46–59 (2016).
Kaiser, W. J. et al. Toll-like receptor 3-mediated necrosis via TRIF, RIP3, and MLKL. J. Biol. Chem. 288, 31268–31279 (2013).
Mariathasan, S. et al. Cryopyrin activates the inflammasome in response to toxins and ATP. Nature 440, 228–232 (2006).
Crute, B. E., Seefeld, K., Gamble, J., Kemp, B. E. & Witters, L. A. Functional domains of the α1 catalytic subunit of the AMP-activated protein kinase. J. Biol. Chem. 273, 35347–35354 (1998).
Hardie, D. G., Schaffer, B. E. & Brunet, A. AMPK: an energy-sensing pathway with multiple inputs and outputs. Trends Cell Biol. 26, 190–201 (2016).
Geng, J. et al. Regulation of RIPK1 activation by TAK1-mediated phosphorylation dictates apoptosis and necroptosis. Nat. Commun. 8, 359 (2017).
Jaco, I. et al. MK2 phosphorylates RIPK1 to prevent TNF-induced cell death. Mol. Cell 66, 698–710.e5 (2017).
Van den Bossche, J., O’Neill, L. A. & Menon, D. Macrophage immunometabolism: where are we (going)? Trends Immunol. 38, 395–406 (2017).
Kelly, B. & O’Neill, L. A. Metabolic reprogramming in macrophages and dendritic cells in innate immunity. Cell Res. 25, 771–784 (2015).
Peterson, L. W. et al. Cell-extrinsic TNF collaborates with TRIF signaling to promote Yersinia-induced apoptosis. J. Immunol. 197, 4110–4117 (2016).
Chu, X. et al. Gasdermin D-mediated pyroptosis is regulated by AMPK-mediated phosphorylation in tumor cells. Cell Death Dis. 14, 469 (2023).
Li, Y., Pu, D., Huang, J., Zhang, Y. & Yin, H. Protein phosphatase 1 regulates phosphorylation of gasdermin D and pyroptosis. Chem. Commun. 58, 11965–11968 (2022).
Ai, Y. L. et al. Mannose antagonizes GSDME-mediated pyroptosis through AMPK activated by metabolite GlcNAc-6P. Cell Res. 33, 904–922 (2023).
Wen, H. et al. Fatty acid-induced NLRP3-ASC inflammasome activation interferes with insulin signaling. Nat. Immunol. 12, 408–415 (2011).
O’Neill, L. A., Golenbock, D. & Bowie, A. G. The history of Toll-like receptors—redefining innate immunity. Nat. Rev. Immunol. 13, 453–460 (2013).
van den Berghe, G. et al. Intensive insulin therapy in critically ill patients. N. Engl. J. Med. 345, 1359–1367 (2001).
NICE-SUGAR Study Investigators et al. Intensive versus conventional glucose control in critically ill patients. N. Engl. J. Med. 360, 1283–1297 (2009).
Zhou, Z. et al. Metabolite annotation from knowns to unknowns through knowledge-guided multi-layer metabolic networking. Nat. Commun. 13, 6656 (2022).
Shen, X. et al. Metabolic reaction network-based recursive metabolite annotation for untargeted metabolomics. Nat. Commun. 10, 1516 (2019).
Yang, Y. et al. Raw data for untargeted metabolomics. figshare https://doi.org/10.6084/m9.figshare.c.6888193.v1 (2024).
Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372 (2008).
Acknowledgements
We thank J. Yuan of the Interdisciplinary Research Center on Biology and Chemistry (IRCBC) for providing Ripk1S321E/S321E mice, T. Zhang of Shanghai Jiao Tong University (SJTU) for providing Ripk1S415A/S415A mice, Y. Xu of SJTU for providing Yersinia pseudotuberculosis (strain IP2666) and staff members of the Animal Facility at the National Facility for Protein Science in Shanghai (NFPS) for providing technical support. The work of D.X. was supported in part by grants from the STI2030-Major Projects (2022ZD0213200), the National Natural Science Foundation of China (32350022, 32070737 and 92049303), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB39030600), the Shanghai Science and Technology Development Funds (20JC1411600, 20QA1411500 and 22JC1410400), the CAS Youth Interdisciplinary Team (JCTD-2022-10), the Shanghai Key Laboratory of Aging Studies (19DZ2260400) and the Shanghai Municipal Science and Technology Major Project (2019SHZDZX02). The work of Z-J.Z. was supported in part by grants from the National Key R&D Program of China (2022YFC3400700) and the National Natural Science Foundation of China (22022411). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
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This project was conceived, designed and directed by D.X. Y.Y. and H.F. designed and conducted the majority of the experiments. Z.-J.Z., Z.X. and F.R. helped with mass spectrometry-based metabolomics. B.S. and M.Z. helped with mass spectrometry analysis of protein phosphorylation. G.X. bred the Ripk1-S321E mice. L.Y. helped with cell death experiments. Z.S. and Z.C. helped with mouse Yersinia infection experiments. W.S. helped with the expression of recombinant proteins. D.X. wrote the manuscript.
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Extended data
Extended Data Fig. 1 Yersinia infection results in a decrease in intracellular glucose levels.
a-d, Volcano plot of metabolites (a and b) and pathway enrichment analysis of significantly regulated metabolites (c and d) in primary BMDMs with or without S. Typhimurium (S.t.) infection (MOI 100) or LPS (100 ng/ml) stimulation for 1 hour. The hits were selected based on the fold change of the n = 6 biologically independent samples in each group with cut-offs set at log2(S.t./UT) or log2(LPS/Veh) > 0.5 or < −0.5 and P value < 0.05. Glucose is labeled in red. ATP, ADP, and AMP are labeled in blue. e, Heatmap showing the degree of metabolite level changes in primary BMDMs stimulated with Y. pseudotuberculosis (Y.p., MOI 40), S. Typhimurium (MOI 100) and LPS (100 ng/ml) for 1 hour relative to the untreated control. f-h, Relative abundances of glucose in primary BMDMs stimulated with S. Typhimurium (MOI 100) (f), LPS (100 ng/ml) (g), WT or YopJ-deficient Y. pseudotuberculosis (h) for 1 hour were measured. i, j, Volcano plot of metabolites (i) and pathway enrichment analysis of significantly regulated metabolites (j) in primary lymphocytes with or without Y. pseudotuberculosis (MOI 40) infection for 1 hour. The hits were selected based on the fold change of the n = 6 biologically independent samples in each group with cut-offs set at log2(Y.p./UT) > 0.5 or < −0.5 and P value < 0.05. Glucose is labeled in red. ATP, ADP, and AMP are labeled in blue. k, Relative abundances of glucose in primary lymphocytes infected with Y. pseudotuberculosis for the indicated time were measured. l, Food consumption was monitored for 8-week-old male mice infected with Y. pseudotuberculosis (1 × 108 CFU). Mean ± s.e.m. of n = 6 biologically independent samples (f-h and k) or mice (l) in each group. Unpaired two-tailed Student’s t-test (a-d and f-i). One-way ANOVA post hoc Dunnett’s tests (k). Two-way ANOVA post hoc Bonferroni’s tests (l). UT, untreated. Veh, vehicle. Ctrl, control.
Extended Data Fig. 2 Yersinia infection induces a high increase in glycolysis.
a, Ratio of AMP/ATP (left) and ADP/ATP (right) in primary lymphocytes with Y. pseudotuberculosis infection (MOI 40) for the indicated time were measured. b, c, Ratio of AMP/ATP (left) and ADP/ATP (right) in primary BMDMs with S. Typhimurium (MOI 100) infection (b) or LPS (100 ng/ml) stimulation (c) for 1 hour were measured. d, Ratio of AMP/ATP in the blood of mice infected with Y. pseudotuberculosis (1 × 108 CFU) for the indicated time. e, Seahorse analysis of the oxygen consumption rate (OCR) in primary BMDMs infected with WT Y. pseudotuberculosis (MOI 40) or YopJ-deficient Y. pseudotuberculosis (MOI 40) for the indicated time. f, g, Relative abundances of glucose-6-phosphate in primary BMDMs with S. Typhimurium (MOI 100) (f) or LPS (100 ng/ml) (g) stimulation for 1 hour were measured. Mean ± s.e.m. of n = 6 biologically independent samples in each group (a-c, f and g). Mean ± s.e.m. of n = 5 mice in each group (d). Mean ± s.d. of n = 3 (WT and Y.p.-WT) or n = 4 (Y.p.-ΔYopJ) biologically independent samples (e). One-way ANOVA post hoc Dunnett’s tests (a). Unpaired two-tailed Student’s t-test (b-d, f and g).
Extended Data Fig. 3 Yersinia-induced glucose drop is detrimental to the host.
a, b, Eight-week-old male mice challenged with Y. pseudotuberculosis (1 × 108 CFU) were subjected to intragastric gavage of glucose (6 gkg−1) at 6, 24 and 48 hours post infection. Blood glucose levels were measured (a). At 72 hpi, mice were provided with 20% glucose in drinking water, survival ratio was monitored (b). Mean ± s.e.m. of n = 6 (Saline-0/Glucose-30 hpi), 4 (Saline-54 hpi) or 5 (Glucose-54 hpi) mice (a). c, Schematic diagram of experimental design. d, Survival ratio of eight-week-old male mice treated in (c). e, Bacterial burdens in spleen/liver of mice treated in (c) were determined three days after infection. f, Eight-week-old male mice challenged with YopJ-deficient Y. pseudotuberculosis were subjected to intragastric gavage with glucose at 6, 24 and 48 hours post infection. At 72 hpi, mice were provided with 20% glucose in drinking water. g, Blood glucose levels in eight-week-old male mice fasted for 12 hours. h-k, Eight-week-old male mice were subjected to fasting for 12 hours before intragastric gavage (h and i) or intraperitoneal (i.p.) injection (j and k) with WT (h, j and k) or YopJ-deficient (i) Y. pseudotuberculosis. Six hours after infection, mice were provided with ad libitum access to food. l, m, Eight-week-old male mice were intraperitoneally injected with 2-DG (250 mgkg−1) for 8 hours after intragastric gavage with WT (l) or YopJ-deficient (m) Y. pseudotuberculosis, then mice were injected continuous with 2-DG at 24, 32, 48 and 56 hours after infection. Survival ratio (h, j and l) and bacterial burdens in spleen/liver (f, i, k and m) were determined. n = 6 (b, d-f, i and k-m) or 5 (g, h and j) mice. Mean ± s.e.m. (e-g, i, k and m). Two-way ANOVA post hoc Bonferroni’s tests (a). Two-sided log-rank (Mantel-Cox) test (b, d, h, j and l). Unpaired two-tailed Student’s t-test (e, g and k).
Extended Data Fig. 4 Yersinia infection activates AMPK.
a, Primary BMDMs were infected with Y. pseudotuberculosis (MOI 40) for the indicated time. Activation of mTORC1 was indicated by p-T389 S6K1 immunoblotting. b, c, Primary BMDMs were stimulated with S. Typhimurium (MOI 100) (b) or LPS (100 ng/ml) (c) for the indicated time. Activation of AMPK was indicated by p-S79 ACC immunoblotting. d, e, Activation of endogenous AMPK in mouse liver (d) and peritoneal macrophages (e) was indicated by p-T172-AMPK and p-S79ACC immunoblotting in response to Y. pseudotuberculosis infection (1 × 108 CFU) for 6 hours. n = 2 mice for each group. The relative levels of p-T172 AMPK and p-S79 ACC normalized to Tubulin levels are indicated below the respective blot. f, Immunohistochemical staining for CD163 as a marker of M2 macrophages in the liver and spleen of 8-week-old mice with or without Y. pseudotuberculosis infection (1 × 108 CFU) for two days. Representative images of n = 3 mice for each group are shown. g, Immunoblotting for CD163 as a marker of M2 macrophages in peritoneal macrophages isolated from 8-week-old mice with or without Y. pseudotuberculosis infection (1 × 108 CFU) for two days. n = 3 mice for each group.
Extended Data Fig. 5 AMPK activation suppresses LPS/5z7-induced pyroptosis.
a-c, Volcano plot of metabolites (a), relative abundances of glucose (b) and AMP/ATP (left) and ADP/ATP (right) ratios (c) in primary BMDMs with or without LPS (100 ng/ml) and/or 5z7 (200 nM) stimulation for 1 hour. The hits were selected based on the fold change of the n = 6 biologically independent samples in each group with cut-offs set at log2(LPS/5z7 vs Veh) > 0.5 or < −0.5 and P value < 0.05. Glucose is labeled in red. ATP, ADP, and AMP are labeled in blue. Unpaired two-tailed Student’s t test (a). d, e, Primary BMDMs were glucose-starved (d) or treated with LPS (50 ng/ml)/5z7 (200 nM) in the presence of 25 mM glucose or galactose (e). Cell death was measured by SytoxGreen positivity assay (d and e). f-h, Primary BMDMs were pretreated with 2-DG (5 mM) (f and g), Torin-1 (250 nM) or Rapamycin (100 nM) with or without glucose (h) for 1 hour followed by treatment with LPS (50 ng/ml)/5z7 (200 nM). Cell death was measured by SytoxGreen positivity assay (f and h). Activation of pyroptosis was indicated by cleaved-GSDMD immunoblotting (g). The relative levels of GSDMD p30 normalized to Tubulin are indicated below the GSDMD blot (g). i-l, Primary BMDMs were pretreated with AICAR (1 mM) (i and j) or Compound 991 (40 μM) (k and l) for 1 hour followed by treatment with LPS (50 ng/ml)/5z7 (200 nM). Cell death was measured by SytoxGreen positivity assay (i and k). Activation of pyroptosis was indicated by cleaved-GSDMD immunoblotting (j and l). The relative levels of GSDMD p30 normalized to Tubulin are indicated below the GSDMD blot (j and l). Mean ± s.e.m. of n = 6 biologically independent samples (b and c). Mean ± s.d. of n = 6 (d-f, h and i) or 4 (k) independent wells of one representative experiment. GD, glucose deprivation.
Extended Data Fig. 6 AMPK activation suppresses Yersinia-induced pyroptosis.
a, b, Primary BMDMs were pretreated with A-769662 (50 μM) for 1 hour followed by WT (a) or YopJ-deficient (b) Y. pseudotuberculosis (MOI 40) challenge for 1 hour (a) or the indicated time (b). The amount of Y. pseudotuberculosis taken up by cells was quantified by counting colony-forming units (CFUs) (a). Cell death was measured by LDH release (b). c, Immunoblotting of p-S79 ACC in spleen/peritoneal macrophages of mice intraperitoneally injected with A-769662 (25 mg/kg) for 12 hours. The relative levels of p-S79 ACC normalized to Tubulin are indicated below. d, Eight-week-old male mice were intraperitoneally injected with A-769662 for 4 hours followed by intragastric gavage with Y. pseudotuberculosis while injected continuously with A-769662 at 6, 24 and 48 hours after infection. Survival ratio was monitored. e, Schematic diagram of the experimental design. f, Survival ratio of mice treated in (e) was monitored. g, Bacterial burden in spleen/liver of mice treated in (e) were determined three days after infection. h, Eight-week-old male mice were intraperitoneally injected with A-769662 for 4 hours followed by intragastric gavage with Y. pseudotuberculosis while injected continuously with A-769662 at 6, 24 and 48 hours after infection. Survival ratio was monitored. i, LDH release in primary BMDMs infected with YopJ-deficient Y. pseudotuberculosis (MOI 40) for the indicated time. j, The amount of bacteria taken up by primary BMDMs challenged with Y. pseudotuberculosis (MOI 40) for 1 hour. k-m, Eight-week-old mice of the indicated genotypes were subjected to intragastric gavage with Y. pseudotuberculosis. Survival ratio was monitored. Mean ± s.d. of n = 3 (a and j) or 5 (b and i) independent wells of one representative experiment. Mean ± s.e.m of n = 2 (c), 6 (d, f and g) or 5 (h and k-m) mice. Unpaired two-tailed Student’s t-test (a, g and j). Two-sided log-rank (Mantel-Cox) test (d, f, h and k-m).
Extended Data Fig. 7 Glucose and AMPK regulate RIPK1-dependent necroptosis but not RIPK1-independent pyroptosis.
a, Primary BMDMs were pretreated with 2-DG (5 mM) for 1 hour followed by treatment with LPS (50 ng/ml)/5z7 (200 nM) in the presence of Nec-1s (10 μM) for the indicated time. Activation of RIPK1 was indicated by p-S166 RIPK1 immunoblotting and activation of caspase-8 was indicated by cleaved-caspase-8 (CC8) immunoblotting. The relative levels of p-S166 RIPK1 and CC8 p18 normalized to Tubulin are indicated below the respective blot. b-d, Primary BMDMs were treated with LPS (50 ng/ml)/zVAD (50 μM) in the presence or absence of Nec-1s (10 μM), A-769662 (50 μM) or glucose for the indicated time. Cell death was measured by SytoxGreen positivity assay (b). Activation of RIPK1 was indicated by immunoblotting for p-S166 RIPK1 and activation of necroptosis was indicated by immunoblotting for p-T231/S232 RIPK3 and p-S345 MLKL (c and d). The relative levels of p-S166 RIPK1, p-T231/S232 RIPK3 and p-S345 MLKL normalized to Tubulin levels are indicated below the respective blot (c and d). e, Primary BMDMs were infected with S. Typhimurium (MOI 100) in the presence or absence of 2-DG (5 mM) or glucose for 2 hours. Cell death was measured by LDH release. f, Primary BMDMs were primed with LPS (100 ng/ml) with or without 2-DG (5 mM) or glucose for 3 hours before stimulation with nigericin (Nig, 10 μM) for the indicated time. Cell death was measured by SytoxGreen positivity assay. g, h, Primary BMDMs were primed with LPS (100 ng/ml) for 3 hours before stimulation with nigericin (Nig, 10 μM) for the indicated time. Cell death was measured by SytoxGreen positivity assay (g). Activation of pyroptosis was indicated by cleaved GSDMD immunoblotting (h). Mean ± s.d. of n = 3 (b), 6 (e) or 4 (f) independent wells of one representative experiment. Mean ± s.d. of n = 4 (Ampkα1+/+) or 3 (Ampkα1−/−) independent wells of one representative experiment (g).
Extended Data Fig. 8 AMPK phosphorylates RIPK1 S321 to inhibit its activation.
a, HEK293T cells were transfected with expression vectors of HA-tagged AMPKα1 with or without Flag-tagged RIPK1 for 24 hours. The interaction between RIPK1 and AMPKα1 was revealed by immunoprecipitation-immunoblotting assay as indicated. b, Representative mass spectrometry spectra of p-S321 RIPK1 from overexpressed RIPK1 in the presence of constitutively active AMPKα1(1-312). c, Alignment of the amino acid sequences of mouse RIPK1 amino acids 47 to 384 with orthologues in humans. S321 but not T49 and S382, as highlighted by red, is conserved between mice and humans. d, Peritoneal macrophages were isolated from 8-week-old mice intraperitoneally injected with A-769662 (25 mg/kg) for the indicated time. The levels of p-S321 RIPK1 were determined by immunoblotting. The relative levels of p-S321 RIPK1 normalized to Tubulin levels are indicated below the p-S321 RIPK1 blot. One mouse was used for each time point. e, Primary Ampkα1+/+ and Ampkα1-/- BMDMs were treated with A-769662 (50 μM) for the indicated time. The levels of p-S321 RIPK1 were determined by immunoblotting. The relative levels of p-S321 RIPK1 normalized to Tubulin levels are indicated below the p-S321 RIPK1 blot. f, Primary BMDMs were pretreated with 5z7 (200 nM) for 0.5 hours followed by treatment with A-769662 (50 μM) for 2 hours. The levels of p-S321 RIPK1 were determined by immunoblotting. The relative levels of p-S321 RIPK1 normalized to Tubulin levels are indicated below the p-S321 RIPK1 blot.
Extended Data Fig. 9 RIPK1 S321 phosphorylation is important for inhibiting LPS/5z7-induced pyroptosis.
a, Confirmation of the expression of WT RIPK1 and different mutants in Ripk1-KO BMDMs by immunoblotting. b, RIPK1-WT or mutant-reconstituted BMDMs were treated with LPS (50 ng/ml) and 5z7 (200 nM) for the indicated time. Cell death was measured by SytoxGreen positivity assay. c, Primary Ripk1WT/WT and Ripk1S321E/S321E BMDMs were challenged with YopJ-deficient Y. pseudotuberculosis (MOI 40) for 4 hours. Cell death was measured by LDH release. d, Primary Ripk1WT/WT and Ripk1S321E/S321E BMDMs were challenged with Y. pseudotuberculosis (MOI 40) for 1 hour. The amount of bacteria taken up by cells was quantified by CFUs. e, Primary Ripk1WT/WT and Ripk1S415A/S415A BMDMs were treated with LPS (50 ng/ml) and 5z7 (200 nM) for the indicated time. Cell death was measured by SytoxGreen positivity assay. f, Primary Ripk1WT/WT and Ripk1S415A/S415A BMDMs were treated with LPS (50 ng/ml) and zVAD (50 μM) for the indicated time. Cell death was measured by SytoxGreen positivity assay. g, Eight-week-old male Ripk1WT/WT and Ripk1S321E/S321E mice were subjected to intragastric gavage with Y. pseudotuberculosis (1 × 107 CFU). The survival ratio of mice was monitored at desired stages. n = 5 mice for each group. Two-sided log-rank (Mantel-Cox) test. Mean ± s.d. of n = 3 (b, d and f), 5 (c) or 4 (e) independent wells of one representative experiment.
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Yang, Y., Fang, H., Xie, Z. et al. Yersinia infection induces glucose depletion and AMPK-dependent inhibition of pyroptosis in mice. Nat Microbiol 9, 2144–2159 (2024). https://doi.org/10.1038/s41564-024-01734-6
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DOI: https://doi.org/10.1038/s41564-024-01734-6
