Abstract
Sepsis is the most common cause of death for patients in intensive care worldwide due to a dysregulated host response to infection. Here, we investigate the role of sequestosome-1 (SQSTM1/p62), an autophagy receptor that functions as a regulator of innate immunity, in sepsis. We find that lipopolysaccharide elicits gasdermin D-dependent pyroptosis to enable passive SQSTM1 release from macrophages and monocytes, whereas transmembrane protein 173-dependent TANK-binding kinase 1 activation results in the phosphorylation of SQSTM1 at Ser403 and subsequent SQSTM1 secretion from macrophages and monocytes. Moreover, extracellular SQSTM1 binds to insulin receptor, which in turn activates a nuclear factor kappa B-dependent metabolic pathway, leading to aerobic glycolysis and polarization of macrophages. Intraperitoneal injection of anti-SQSTM1-neutralizing monoclonal antibodies or conditional depletion of Insr in myeloid cells using the Cre–loxP system protects mice from lethal sepsis (caecal ligation and puncture or infection by Escherichia coli or Streptococcus pneumoniae) and endotoxaemia. We also report that circulating SQSTM1 and the messenger RNA expression levels of SQSTM1 and INSR in peripheral blood mononuclear cells are related to the severity of sepsis in 40 patients. Thus, extracellular SQSTM1 has a pathological role in sepsis and could be targeted to develop therapies for sepsis.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 digital issues and online access to articles
$119.00 per year
only $9.92 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Data availability
All data supporting the findings of this study are available within the article and its supplementary information or from the corresponding author upon reasonable request. Source data are provided with this paper.
References
Singer, M. et al. The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3). JAMA 315, 801–810 (2016).
Hotchkiss, R. S. et al. Sepsis and septic shock. Nat. Rev. Dis. Primers 2, 16045 (2016).
Deretic, V., Saitoh, T. & Akira, S. Autophagy in infection, inflammation and immunity. Nat. Rev. Immunol. 13, 722–737 (2013).
Levine, B., Mizushima, N. & Virgin, H. W. Autophagy in immunity and inflammation. Nature 469, 323–335 (2011).
Klionsky, D. J. & Emr, S. D. Autophagy as a regulated pathway of cellular degradation. Science 290, 1717–1721 (2000).
Levine, B. & Kroemer, G. Biological functions of autophagy genes: a disease perspective. Cell 176, 11–42 (2019).
Xie, Y. et al. Posttranslational modification of autophagy-related proteins in macroautophagy. Autophagy 11, 28–45 (2015).
Dikic, I. & Elazar, Z. Mechanism and medical implications of mammalian autophagy. Nat. Rev. Mol. Cell Biol. 19, 349–364 (2018).
Cho, S. J. et al. Plasma ATG5 is increased in Alzheimer’s disease. Sci. Rep. 9, 4741 (2019).
Castellazzi, M. et al. Correlation between auto/mitophagic processes and magnetic resonance imaging activity in multiple sclerosis patients. J. Neuroinflammation 16, 131 (2019).
Naguib, M. & Rashed, L. A. Serum level of the autophagy biomarker Beclin-1 in patients with diabetic kidney disease. Diabetes Res. Clin. Pract. 143, 56–61 (2018).
Wang, X. et al. Defective lysosomal clearance of autophagosomes and its clinical implications in nonalcoholic steatohepatitis. FASEB J. 32, 37–51 (2018).
Kim, J. Y. & Ozato, K. The sequestosome 1/p62 attenuates cytokine gene expression in activated macrophages by inhibiting IFN regulatory factor 8 and TNF receptor-associated factor 6/NF-κB activity. J. Immunol. 182, 2131–2140 (2009).
Ponpuak, M. et al. Delivery of cytosolic components by autophagic adaptor protein p62 endows autophagosomes with unique antimicrobial properties. Immunity 32, 329–341 (2010).
Zheng, Y. T. et al. The adaptor protein p62/SQSTM1 targets invading bacteria to the autophagy pathway. J. Immunol. 183, 5909–5916 (2009).
Fujita, K., Maeda, D., Xiao, Q. & Srinivasula, S. M. Nrf2-mediated induction of p62 controls Toll-like receptor-4-driven aggresome-like induced structure formation and autophagic degradation. Proc. Natl Acad. Sci. USA 108, 1427–1432 (2011).
Lee, H.-M. et al. Autophagy negatively regulates keratinocyte inflammatory responses via scaffolding protein p62/SQSTM1. J. Immunol. 186, 1248–1258 (2011).
Duran, A. et al. The signaling adaptor p62 is an important NF-κB mediator in tumorigenesis. Cancer Cell 13, 343–354 (2008).
Wooten, M. W. et al. The p62 scaffold regulates nerve growth factor-induced NF-κB activation by influencing TRAF6 polyubiquitination. J. Biol. Chem. 280, 35625–35629 (2005).
Wang, H. et al. HMG-1 as a late mediator of endotoxin lethality in mice. Science 285, 248–251 (1999).
Kang, R. et al. HMGB1 in health and disease. Mol. Aspects Med. 40, 1–116 (2014).
Hesse, D. G. et al. Cytokine appearance in human endotoxemia and primate bacteremia. Surg. Gynecol. Obstet. 166, 147–153 (1988).
Poltorak, A. et al. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282, 2085–2088 (1998).
Kawai, T. et al. Lipopolysaccharide stimulates the MyD88-independent pathway and results in activation of IFN-regulatory factor 3 and the expression of a subset of lipopolysaccharide-inducible genes. J. Immunol. 167, 5887–5894 (2001).
Vanlandingham, P. A. & Ceresa, B. P. Rab7 regulates late endocytic trafficking downstream of multivesicular body biogenesis and cargo sequestration. J. Biol. Chem. 284, 12110–12124 (2009).
Dong, X.-P. et al. The type IV mucolipidosis-associated protein TRPML1 is an endolysosomal iron release channel. Nature 455, 992–996 (2008).
Ubersax, J. A. & Ferrell, J. E. Jr. Mechanisms of specificity in protein phosphorylation. Nat. Rev. Mol. Cell Biol. 8, 530–541 (2007).
Matsumoto, G., Wada, K., Okuno, M., Kurosawa, M. & Nukina, N. Serine 403 phosphorylation of p62/SQSTM1 regulates selective autophagic clearance of ubiquitinated proteins. Mol. Cell 44, 279–289 (2011).
Pilli, M. et al. TBK-1 promotes autophagy-mediated antimicrobial defense by controlling autophagosome maturation. Immunity 37, 223–234 (2012).
Perry, A. K., Chow, E. K., Goodnough, J. B., Yeh, W.-C. & Cheng, G. Differential requirement for TANK-binding kinase-1 in type I interferon responses to Toll-like receptor activation and viral infection. J. Exp. Med. 199, 1651–1658 (2004).
Heipertz, E. L., Harper, J. & Walker, W. E. STING and TRIF contribute to mouse sepsis, depending on severity of the disease model. Shock 47, 621–631 (2017).
Hu, Q. et al. STING-mediated intestinal barrier dysfunction contributes to lethal sepsis. EBioMedicine 41, 497–508 (2019).
Zeng, L. ALK is a therapeutic target for lethal sepsis. Sci. Transl. Med. 9, eaan5689 (2017).
Zhang, H. et al. TMEM173 drives lethal coagulation in sepsis. Cell Host Microbe 27, 556–570 (2020).
Xia, P. et al. Sox2 functions as a sequence-specific DNA sensor in neutrophils to initiate innate immunity against microbial infection. Nat. Immunol. 16, 366–375 (2015).
Shi, J. et al. Inflammatory caspases are innate immune receptors for intracellular LPS. Nature 514, 187–192 (2014).
Orning, P. et al. Pathogen blockade of TAK1 triggers caspase-8-dependent cleavage of gasdermin D and cell death. Science 362, 1064–1069 (2018).
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).
Freemerman, A. J. et al. Metabolic reprogramming of macrophages: glucose transporter 1 (GLUT1)-mediated glucose metabolism drives a proinflammatory phenotype. J. Biol. Chem. 289, 7884–7896 (2014).
Lu, Q. & Lemke, G. Homeostatic regulation of the immune system by receptor tyrosine kinases of the Tyro 3 family. Science 293, 306–311 (2001).
Chaudhuri, A. Regulation of macrophage polarization by RON receptor tyrosine kinase signaling. Front. Immunol. 5, 546 (2014).
Liu, C.-P. et al. NF-κB pathways are involved in M1 polarization of RAW 264.7 macrophage by polyporus polysaccharide in the tumor microenvironment. PLoS ONE 12, e0188317 (2017).
Mauro, C. et al. NF-κB controls energy homeostasis and metabolic adaptation by upregulating mitochondrial respiration. Nat. Cell Biol. 13, 1272–1279 (2011).
Spec, A. et al. T cells from patients with Candida sepsis display a suppressive immunophenotype. Crit. Care 20, 15 (2016).
Moscat, J., Karin, M. & Diaz-Meco, M. T. p62 in cancer: signaling adaptor beyond autophagy. Cell 167, 606–609 (2016).
Sánchez-Martín, P., Saito, T. & Komatsu, M. p62/SQSTM1: ‘jack of all trades’ in health and cancer. FEBS J. 286, 8–23 (2019).
Barber, G. N. STING: infection, inflammation and cancer. Nat. Rev. Immunol. 15, 760–770 (2015).
Gaidt, M. M. et al. The DNA inflammasome in human myeloid cells is initiated by a STING-cell death program upstream of NLRP3. Cell 171, 1110–1124 (2017).
Li, N. et al. STING-IRF3 contributes to lipopolysaccharide-induced cardiac dysfunction, inflammation, apoptosis and pyroptosis by activating NLRP3. Redox Biol. 24, 101215 (2019).
Han, S. et al. Macrophage insulin receptor deficiency increases ER stress-induced apoptosis and necrotic core formation in advanced atherosclerotic lesions. Cell Metab. 3, 257–266 (2006).
Mauer, J. et al. Myeloid cell-restricted insulin receptor deficiency protects against obesity-induced inflammation and systemic insulin resistance. PLoS Genet. 6, e1000938 (2010).
Kang, R. et al. Lipid peroxidation drives gasdermin D-mediated pyroptosis in lethal polymicrobial sepsis. Cell Host Microbe 24, 97–108 (2018).
Peng, T. et al. Disruption of phospholipase Cγ1 signalling attenuates cardiac tumor necrosis factor-α expression and improves myocardial function during endotoxemia. Cardiovasc. Res. 78, 90–97 (2008).
Weischenfeldt, J. & Porse, B. Bone marrow-derived macrophages (BMM): isolation and applications. Cold Spring Harb. Protoc. https://doi.org/10.1101/pdb.prot5080 (2008).
Kang, R. et al. A novel PINK1- and PARK2-dependent protective neuroimmune pathway in lethal sepsis. Autophagy 12, 2374–2385 (2016).
Deng, W. et al. The circadian clock controls immune checkpoint pathway in sepsis. Cell Rep. 24, 366–378 (2018).
Yang, L. et al. PKM2 regulates the Warburg effect and promotes HMGB1 release in sepsis. Nat. Commun. 5, 4436 (2014).
Chen, R. et al. cAMP metabolism controls caspase-11 inflammasome activation and pyroptosis in sepsis. Sci. Adv. 5, eaav5562 (2019).
Hagar, J. A., Powell, D. A., Aachoui, Y., Ernst, R. K. & Miao, E. A. Cytoplasmic LPS activates caspase-11: implications in TLR4-independent endotoxic shock. Science 341, 1250–1253 (2013).
Kayagaki, N. et al. Noncanonical inflammasome activation by intracellular LPS independent of TLR4. Science 341, 1246–1249 (2013).
Taylor, F. B. Jr. et al. Towards definition, clinical and laboratory criteria, and a scoring system for disseminated intravascular coagulation. Thromb. Haemost. 86, 1327–1330 (2001).
Zhu, S. et al. HSPA5 regulates ferroptotic cell death in cancer cells. Cancer Res. 77, 2064–2077 (2017).
Song, X. et al. AMPK-mediated BECN1 phosphorylation promotes ferroptosis by directly blocking system Xc− activity. Curr. Biol. 28, 2388–2399 (2018).
Tang, D. et al. Endogenous HMGB1 regulates autophagy. J. Cell Biol. 190, 881–892 (2010).
Tang, D. et al. High-mobility group box 1 is essential for mitochondrial quality control. Cell Metab. 13, 701–711 (2011).
Tan, J. M. J. et al. An ATG16L1-dependent pathway promotes plasma membrane repair and limits Listeria monocytogenes cell-to-cell spread. Nat. Microbiol. 3, 1472–1485 (2018).
Acknowledgements
We thank D. Primm for his critical reading of the manuscript. G.K. is supported by the Ligue Contre le Cancer (équipe labellisée), Agence National de la Recherche (ANR)—Projets blancs, ANR under the frame of E-Rare-2, ERA-Net for Research Programmes on Rare Diseases, Association pour la Recherche sur le Cancer, Cancéropôle Ile-de-France, Chancelerie des Universités de Paris (Legs Poix), Fondation pour la Recherche Médicale, a donation by Elior, the European Research Area Network on Cardiovascular Diseases (MINOTAUR), Gustave Roussy Odyssea, the European Union Horizon 2020 Project Oncobiome, Fondation Carrefour, High-end Foreign Expert Program in China (nos. GDW20171100085 and GDW20181100051), Institut National du Cancer, Inserm (HTE), Institut Universitaire de France, LeDucq Foundation, LabEx Immuno-Oncology, RHU Torino Lumière, the Seerave Foundation, the SIRIC Stratified Oncology Cell DNA Repair and Tumor Immune Elimination and the SIRIC Cancer Research and Personalized Medicine. J.L. is supported by grants from the National Natural Science Foundation of China (nos. 31671435, 81400132 and 81772508). J.J. is supported by a grant from the National Natural Science Foundation of China (no. 81530063). L.Z. is supported by an Excellent Youth Grant of the State Key Laboratory of Trauma, Burns and Combined Injury of China (no. SKLYQ201901).
Author information
Authors and Affiliations
Contributions
B.Z., D.T., R.K. and J.J. conceived and planned the experiments. B.Z., J.L., L.Z., S.Z., D.T. and R.K. carried out the simulations, sample preparation and analysed the data. T.R.B. provided the mouse strains. H.W., T.R.B., G.K., D.J.K. and H.J.Z. edited the manuscript and contributed to the interpretation of the results. D.T. and R.K. wrote the manuscript. All authors provided critical feedback and helped shape the research, analysis and manuscript.
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data
Extended Data Fig. 1 LPS induces SQSTM1 secretion in monocytes and macrophages.
a, ELISA analysis of SQSTM1 release in THP1 (a human monocytic cell line), primary human blood monocyte-derived macrophages (HPBMs), primary mouse peritoneal macrophages (MPMs), primary mouse lung macrophages (MLMs), and primary mouse hepatic macrophages (MHMs) following treatment with LPS (200 ng/ml) for 3-48 h (n = 5 well /group; two-tailed t test, versus untreated group). b, LDH analysis of cytotoxicity in the indicated cells following treatment with LPS (200 ng/ml) for 3-48 h (n = 5 well /group). c, The indicated BMDMs were treated with LPS (200 ng/ml) for 24 h and the level of Sqstm1 mRNA was assayed by qPCR (n = 3 well /group; two-tailed t test, versus control LPS group). Data in (a–c) are presented as mean ± SD. Data in (a–b) are from two independent experiments. Data in (c) are from three independent experiments.
Extended Data Fig. 2 The activation of the GSDMD-dependent pyroptosis pathway induces SQSTM1 release.
a, Analysis of cytotoxicity, HMGB1 release, and SQSTM1 release in the indicated poly (I:C)-primed BMDMs following LPS electroporation (LPSe) or E. coli (MOI = 25) infection for 24 h (n = 5 well/group; two-tailed t test, versus wild type group). b, Analysis of cytotoxicity, HMGB1 release, and SQSTM1 release in the indicated LPS-primed BMDMs following ATP (5 mM, 3 h) or nigericin (10 µM, 3 h) treatment (n = 5 well/group; two-tailed t test, versus wild type group). c, Analysis of cytotoxicity, HMGB1 release, and SQSTM1 release in the indicated BMDMs following Y. pestis (MOI = 25) infection or LPS5Z7 (LPS [50 ng/ml] + 5z7 [400 nM]) (n = 5 well/group; two-tailed t test, versus wild type group). Data in (a–c) are presented as mean ± SD from two independent experiments.
Extended Data Fig. 3 Exogenous SQSTM1 promotes glucose uptake and lactate production.
a,b, Indicated primary macrophages were stimulated with rSQSTM1 (10, 100, and 1000 ng/ml) or boiled rSQSTM1 (BrSQSTM1) for 24 h. The glucose uptake (a) and lactate production (b) were assayed (n = 3 well/group; one-way ANOVA test, versus control group). c, TMs and HPBMs were treated with rSQSTM1 (100 ng/ml) and IL4 (50 ng/ml) for 48 h and the mRNA expression of Il10 and Arg1 were assayed (n = 3 well/group; two-tailed t test, versus control group). HPBMs, primary human blood monocyte-derived macrophages; MPMs, primary mouse peritoneal macrophages; MLMs, primary mouse lung macrophages; MHMs, primary mouse hepatic macrophages. Data in (a–c) are presented as mean ± SD from two independent experiments.
Extended Data Fig. 4 Effects of TLRs on rSQSTM1-induced NFKB activity.
The indicated TLR-knockdown THP1 cells were stimulated with (a) pam3CSK4 (1 ng/ml), (b) HKLM (107 cells/ml), (c) poly(I:C) (10 µg/ml), (d) LPS (200 ng/ml), (e) FLA-ST (100 ng/ml), (f) FSL1 (0.1 ng/ml), (g) imiquimod (5 µg/ml), (h) ssRNA40 (5 µg/ml), (i) ODN2006 (10 µg/ml), or (a–i) rSQSTM1 (100 ng/ml) for 24 h and the levels of NFKB-induced Lucia luciferase were assessed (n = 3 well/group; two-tailed t test, versus control group). Data in (a–i) are presented as mean ± SD from two independent experiments.
Extended Data Fig. 5 PLCG1 and PTK2B mediate the activity of SQSTM1.
a, Heatmap of kinase phosphorylation in the indicated TMs after rSQSTM1 (100 ng/ml) stimulation for 24 h. b, Western blot analysis of protein expression in the indicated TMs following treatment with rSQSTM1 (100 ng/ml) for 24 h. c, Indicated TMs (control, SLC2A1KD, or SLC2A4KD) were stimulated with insulin (100 nM) for 24 h. The glucose uptake was assayed (n = 3 well/group; two-tailed t test, versus control group). AU, arbitrary units. d,e, Analysis of protein expression and NFKB activity in TMs following treatment with insulin (100 nM) for 24 h (n = 3 well/group). f, Analysis of interaction between PTK2B and INSR in membrane protein extraction from TMs following treatment with insulin (100 nM) or rSQSTM1 (100 ng/ml) for 24 h. g, Analysis of interaction between PLCG1 and RELA in whole protein extraction from indicated TMs following treatment with rSQSTM1 (100 ng/ml) for 24 h. h, Indicated cells were treated with rSQSTM1 (100 ng/ml) for 24 h, and the levels of lactate and IL6 mRNA were assayed (n = 3 well/group). Data in (c), (e), and (h) are presented as mean ± SD. Data in (a) is from one independent experiment. Data in (b), (d), (f), and (g) are from two independent experiments. Data in (c), (e), and (h) are from three independent experiments.
Extended Data Fig. 6 The SQSTM1-INSR pathway mediates CLP-induced polymicrobial sepsis.
a–f, The serum level of IL6 (a), IL1B (b), TNF (c), HMGB1 (d), LDH (e), and lactate (f) were assayed in indicated CLP-induced mice with or without anti-SQSTM1 monoclonal antibodies (20 mg/kg) treatment or depletion of INSR in myeloid cells (n = 5 mice/group; two-tailed t test, versus control group). g, Administration of anti-SQSTM1 monoclonal antibodies (20 mg/kg) and/or depletion of INSR in myeloid cells in mice prevented CLP-induced animal death (n = 10 mice/group; Log-rank test). h–j, Survival of the indicated mice after E. coli (h) or S. pneumoniae (i) infection or (j) CLP-induced sepsis (n = 10 mice/group; Log-rank test). Data in (a–f) are presented as mean ± SD from three independent experiments. Data in (g–j) are from two independent experiments.
Extended Data Fig. 7 The inhibition of the SQSTM1-INSR pathway protects mice against LPS-induced endotoxemia.
a, Administration of anti-SQSTM1 monoclonal antibodies (20 mg/kg) or depletion of INSR in myeloid cells in mice prevented LPS (10 mg/kg)-induced animal death (n = 10 mice/group; Log-rank test). b–k, In parallel, the serum levels of CK (b), AMY (c), BUN (d), GPT/ALT I (e), IL6 (f), IL1B (g), TNF (h), HMGB1 (i), LDH (j), and lactate (k) were assayed (n = 5 mice/group; two-tailed t test, versus control group). Data in (b–k) are presented as mean ± SD. Data in (a–k) are from two independent experiments.
Extended Data Fig. 8 The inhibition of the SQSTM1-INSR pathway protects mice against bacterial sepsis.
a–c, Survival of the indicated mice in E. coli (a) or S. pneumoniae (b) infection or (c) clinically relevant moderate CLP-induced sepsis (n = 10 mice/group; Log-rank test). d,e, In parallel, the levels of apoptosis (d) and Pdcd1 mRNA (e) were assayed at 72 h (n = 5 mice/group; two-tailed t test, versus control group). Data in (d, e) are presented as mean ± SD. Data in (a–e) are from two independent experiments.
Extended Data Fig. 9 The SQSTM1-INSR pathway promotes systemic coagulation in bacterial sepsis.
The levels of blood markers of DIC (a–e) were assayed at 72 h in the indicated mice in E. coli or S. pneumoniae infection or clinically relevant moderate CLP-induced sepsis (n = 5 mice/group; two-tailed t test, versus control group). Data in (a–e) are presented as mean ± SD from two independent experiments.
Extended Data Fig. 10 Association of the SQSTM1-INSR axis with the severity of sepsis in patients.
a–f, The correlation assay between SQSTM1, INSR, SOFA, and DIC score in patients with sepsis and septic shock (n=40 cases). g, Pearson correlation heatmap of immune mediators.
Supplementary information
Supplementary Information
Supplementary Tables 1 and 2.
Source data
Source Data Fig. 1
Unprocessed western blots.
Source Data Fig. 2
Numerical data.
Rights and permissions
About this article
Cite this article
Zhou, B., Liu, J., Zeng, L. et al. Extracellular SQSTM1 mediates bacterial septic death in mice through insulin receptor signalling. Nat Microbiol 5, 1576–1587 (2020). https://doi.org/10.1038/s41564-020-00795-7
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41564-020-00795-7
This article is cited by
-
The multifunctional protein HMGB1: 50 years of discovery
Nature Reviews Immunology (2023)
-
Sepsis-induced immunosuppression: mechanisms, diagnosis and current treatment options
Military Medical Research (2022)
-
Metabolic reprogramming consequences of sepsis: adaptations and contradictions
Cellular and Molecular Life Sciences (2022)
-
The STING1 network regulates autophagy and cell death
Signal Transduction and Targeted Therapy (2021)