Caspase-11 activation requires lysis of pathogen-containing vacuoles by IFN-induced GTPases (original) (raw)

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Acknowledgements

We thank N. Mizushima and S. Virgin for _Atg5_-deficient BMDMs, K. Pfeffer for _Gbp2_-deficient BMDMs, J. Frey for B. thailandensis, the Biozentrum Proteomics and Imaging Core Facilities for technical assistance, K. Anderson, T. Soukup, R. Schwingendorf, J. C. Cox, V. M. Dixit for reagents and N. Personnic for discussions. This work was supported by an SNSF Professorship PP00P3_139120/1, University of Basel project grant ID2153162 to P.B. and a Marie Heim-Voegtlin Fellowship 145516 to D.K.B.

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Author notes

  1. Mathias S. Dick and Roland F. Dreier: These authors contributed equally to this work.

Authors and Affiliations

  1. Focal Area Infection Biology, Biozentrum, University of Basel, CH-4056 Basel, Switzerland,
    Etienne Meunier, Mathias S. Dick, Roland F. Dreier, Nura Schürmann, Dirk Bumann & Petr Broz
  2. Department Biomedicine, University of Basel, CH-4056 Basel, Switzerland,
    Daniela Kenzelmann Broz
  3. Genentech Inc., South San Francisco, 94080, California, USA
    Søren Warming, Merone Roose-Girma & Nobuhiko Kayagaki
  4. Department of Microbiology and Immunology, Osaka University, Yamadaoka, Suita, Osaka 565-0871, Japan,
    Kiyoshi Takeda & Masahiro Yamamoto

Authors

  1. Etienne Meunier
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  2. Mathias S. Dick
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  3. Roland F. Dreier
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  4. Nura Schürmann
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  5. Daniela Kenzelmann Broz
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  6. Søren Warming
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  7. Merone Roose-Girma
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  8. Dirk Bumann
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  9. Nobuhiko Kayagaki
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  10. Kiyoshi Takeda
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  11. Masahiro Yamamoto
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  12. Petr Broz
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Contributions

E.M. and P.B. designed the study and wrote the manuscript. E.M., R.F.D., M.S.D., N.S. and P.B. performed the experiments and analysed data; D.K.B., D.B., S.W., M.R.-G., N.K., M.Y. and K.T. contributed reagents.

Corresponding author

Correspondence toPetr Broz.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Type-I-interferon signalling is required to induce caspase-11-dependent cell death in response to bacterial infection, but not in response to LPS transfection.

a, LDH release from unprimed BMDMs infected for 16 h with wild-type (WT) S. typhimurium or ΔSPI-2 S. typhimurium grown to stationary phase. b, LDH release from primed BMDMs transfected with LPS O111:B4. Graphs show the mean and s.d. of quadruplicate wells and are representative of three independent experiments.

Extended Data Figure 2 BMDMs from Gbp chr3 KO mice have normal responses to priming stimuli, but fail to activate the non-canonical inflammasome during bacterial infections.

a, Schematic representation of the GBP locus on murine chromosome 3. The extent of the deletion in Gbp chr3 KO mice is indicated. b–d, Induction of pro-caspase-11, GBP2 and GBP5 expression in lysates of wild-type and Gbp chr3 KO BMDMs stimulated for 16 h with the indicated amounts of murine IFN-β, murine IFN-γ or LPS O111:B4. e, TNF-α release from BMDMs stimulated for 16 h with LPS O111:B4. f, g, LDH release and IL-1β secretion from wild-type and Gbp chr3 KO BMDMs infected for 16 h with wild-type (WT) S. typhimurium, ΔSPI-2 S. typhimurium, V. cholerae, E. cloacae or C. koseri grown to stationary phase. Cells were primed overnight with LPS (f) or poly(I:C) (g). *Indicates background band. Graphs show the mean and s.d. of quadruplicate wells and data are representative of two independent experiments. **P < 0.01, NS, not significant (two-tailed _t_-test).

Extended Data Figure 3 GBPs assist the detection of bacteria that escape into the cytosol only in primed macrophages.

a–c, LDH release, IL-1β secretion and immunoblots for processed caspase-1 and caspase-11 released from unprimed BMDMs infected for 8–16 h with ΔsifA S. typhimurium or B. thailandensis grown to stationary phase. d, LDH release and IL-1β secretion from unprimed or IFN-γ-primed BMDMs infected for 16 h with ΔsifA S. typhimurium grown to stationary phase. Ext, extract; SN, supernatant. Graphs show the mean and s.d. of quadruplicate wells and data are representative of two independent experiments. *P < 0.05; **P < 0.01; NS, not significant (two-tailed _t_-test).

Extended Data Figure 4 Murine GBP2 controls non-canonical inflammasome activation during Salmonella infection, but is dispensable for direct LPS sensing and canonical inflammasomes.

a, Schematic drawing of the inflammasome pathways activated by flagellin-deficient Salmonella. b–d, LDH release, IL-1β secretion and immunoblots for processed caspase-1 and processed IL-1β released from unprimed BMDMs infected for 17 h with Δflag S. typhimurium grown to stationary phase. BMDMs were treated with the indicated siRNA for 56 h before infection. e, Immunoblots for processed caspase-1, IL-18 and caspase-11 released from unprimed BMDMs infected for 16 h with ΔSPI-2 S. typhimurium, E. cloacae or C. koseri grown to stationary phase. f, g, LDH release and IL-1β secretion from primed wild-type and Gbp2 −/− BMDMs transfected with the indicated types of LPS for 16 h, treated with nigericin for 1 h, infected with SPI-1 T3SS expressing logarithmic phase wild-type S. typhimurium for 1 h, or transfected with poly(dA:dT) for 6 h. Cell were primed with PAM3CSK4 in f or LPS g. Graphs show the mean and s.d. of quadruplicate wells and data are representative of two (e) and three (b–d, f, g) independent experiments. NT, non-targeting siRNA; GM, GenMute transfection reagent; NS, not significant (two-tailed _t_-test).

Extended Data Figure 5 Normal activation of non-canonical and canonical inflammasomes in Gbp5 −/− BMDMs.

a, Expression of GBP5 in wild-type and two lines of Gbp5 −/− BMDMs (1 and 2). *Indicates a cross-reactive band. b–e, LDH release and IL-1β secretion from BMDMs infected for 16 h with wild-type (WT) S. typhimurium, ΔSPI-2 S. typhimurium, V. cholerae, E. cloacae or C. koseri grown to stationary phase (b), transfected with the indicated LPS for 16 h (c) infected for 1 h with SPI-1 T3SS expressing logarithmic phase wild-type S. typhimurium (d), or treated with 5 mM ATP or 20 mM nigericin for 4 h (e). Cells were left unprimed (b) or primed with PAM3CSK4 in (c) or LPS (d, e). Graphs show the mean and s.d. of triplicate or quadruplicate wells and data are representative of three independent experiments.

Extended Data Figure 6 GBPs control bacterial replication.

c.f.u.s at 16 h post-infection in wild-type and Gbp chr3 KO BMDMs infected with the indicated bacterial strains. Experiments are representative of two independent experiments.

Extended Data Figure 7 Inhibition of ROS and NO production does not affect non-canonical inflammasome activation.

a, b, ROS levels, LDH release and IL-1β secretion in unprimed BMDMs left uninfected or infected for 16 h with wild-type S. typhimurium grown to stationary phase. c–e, LDH release, IL-1β secretion, ROS levels and immunoblots for processed caspase-1 and caspase-11 released from unprimed BMDMs infected for 16 h with wild-type (WT) S. typhimurium or E. cloacae grown to stationary phase in the presence of the ROS inhibitor (apocynin) or a vehicle control (DMSO). f, g, LDH release, IL-1β secretion and immunoblots for processed caspase-1 and caspase-11 released from unprimed BMDMs infected for 16 h with wild-type S. typhimurium or E. cloacae grown to stationary phase in the presence of the iNOS inhibitor (l-NAME) or a vehicle control (DMSO). h, NO release from unprimed or IFN-γ-primed BMDMs infected for 16 h with S. typhimurium in presence of the iNOS inhibitor (l-NAME) or a vehicle control (DMSO). Ext, extract; SN, supernatant. Graphs show the mean and s.d. of quadruplicate wells and data are representative of two (a–c, e–g) and three (d, h) independent experiments. NS, not significant (two-tailed _t_-test).

Extended Data Figure 8 Colocalization of GBPs and autophagy proteins on intracellular bacteria.

a, Colocalization of LC3 with GBPs in unprimed wild-type BMDMs infected with E. cloacae or C. koseri for 4 h and stained for LC3, GBP2 and DNA. b, Colocalization of galectin-8 and NDP52 in unprimed wild-type BMDMs infected with wild-type S. typhimurium for 4 h and stained for galectin-8, NDP52 and DNA. c, Colocalization of p62 and LC3 in unprimed wild-type BMDMs infected with wild-type S. typhimurium for 4 h and stained for LC3, p62 and DNA. d, Quantification of p62 and LC3 co-staining in wild-type and Gbp chr3 KO BMDMs at 4 h post-infection with Salmonella. Arrowheads indicate region shown in insets. Scale bars, 1 μm (a) and 10 μm (b, c). Graph shows the mean and s.d. of triplicate counts and images and graph are representative of at least two independent experiments. NS, not significant (two-tailed _t_-test).

Extended Data Figure 9 Digitonin-based quantification of cytoplasmic bacteria.

a, Immunostaining for calnexin and PDI (protein disulphide isomerase) in wild-type BMDMs left untreated or permeabilized with digitonin or saponin. b, Differentially permeabilized macrophages stained for cytosolic and vacuolar Salmonella at 4 h post-infection. c, Schematic representation of FACS-based analysis of cytosolic and vacuolar bacterial populations of Salmonella. Scale bars, 10 μm.

Extended Data Figure 10 Model for the role of GBPs and autophagy in caspase-11 activation.

The pathogen-containing vacuole of vacuolar bacterial pathogens is recognized by interferon-induced GBPs in an unknown manner. GBPs promote the lysis of the PCV either directly or indirectly, resulting in the release of the bacteria into the cytosol and activation of caspase-11 by bacterial LPS. β-galactosides of the lysed vacuole serve as danger signals upon exposure to the cytosol and are recognized by galectin-8 leading to the recruitment of the autophagy machinery. p62 participates in this process by recognizing ubiquitin-chains on the vacuole or the bacterium. Uptake of the bacterium and the lysed vacuole into autophagosomes reduces caspase-11 activation by removing the source of LPS from the cytosol.

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Meunier, E., Dick, M., Dreier, R. et al. Caspase-11 activation requires lysis of pathogen-containing vacuoles by IFN-induced GTPases.Nature 509, 366–370 (2014). https://doi.org/10.1038/nature13157

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