Extracellular SQSTM1 mediates bacterial septic death in mice through insulin receptor signalling

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.

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Fig. 1: Activation of MYD88-dependent TLR4 signalling induces the expression and secretion of SQSTM1.
Fig. 2: TMEM173-dependent TBK1 activation mediates SQSTM1 phosphorylation, expression and secretion.
Fig. 3: Activation of the GSDMD-dependent pyroptosis pathway induces passive release of SQSTM1.
Fig. 4: Exogenous SQSTM1 promotes metabolic programming and macrophage polarization.
Fig. 5: INSR is the major receptor for the immunometabolic activity of SQSTM1.
Fig. 6: The SQSTM1–INSR pathway mediates CLP-induced polymicrobial sepsis.

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.

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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).

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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.

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Correspondence to Jianxin Jiang or Daolin Tang or Rui Kang.

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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 (ac) are presented as mean ± SD. Data in (ab) 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 (ac) 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 (ac) 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 (ai) 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 (ai) 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.

af, 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). hj, 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 (af) are presented as mean ± SD from three independent experiments. Data in (gj) 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). bk, 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 (bk) are presented as mean ± SD. Data in (ak) are from two independent experiments.

Extended Data Fig. 8 The inhibition of the SQSTM1-INSR pathway protects mice against bacterial sepsis.

ac, 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 (ae) 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 (ae) 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 (ae) 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.

af, 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.

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Source Data Fig. 1

Unprocessed western blots.

Source Data Fig. 2

Numerical data.

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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

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