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Caspase-11 activation requires lysis of pathogen-containing vacuoles by IFN-induced GTPases

Abstract

Lipopolysaccharide from Gram-negative bacteria is sensed in the host cell cytoplasm by a non-canonical inflammasome pathway that ultimately results in caspase-11 activation and cell death1,2,3. In mouse macrophages, activation of this pathway requires the production of type-I interferons4,5, indicating that interferon-induced genes have a critical role in initiating this pathway. Here we report that a cluster of small interferon-inducible GTPases, the so-called guanylate-binding proteins, is required for the full activity of the non-canonical caspase-11 inflammasome during infections with vacuolar Gram-negative bacteria. We show that guanylate-binding proteins are recruited to intracellular bacterial pathogens and are necessary to induce the lysis of the pathogen-containing vacuole. Lysis of the vacuole releases bacteria into the cytosol, thus allowing the detection of their lipopolysaccharide by a yet unknown lipopolysaccharide sensor. Moreover, recognition of the lysed vacuole by the danger sensor galectin-8 initiates the uptake of bacteria into autophagosomes, which results in a reduction of caspase-11 activation. These results indicate that host-mediated lysis of pathogen-containing vacuoles is an essential immune function and is necessary for efficient recognition of pathogens by inflammasome complexes in the cytosol.

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Figure 1: Caspase-11 activation by intracellular bacterial pathogens requires GBPs.
Figure 2: GBPs control bacterial replication.
Figure 3: Autophagy reduces caspase-11 activation.
Figure 4: GBP-mediated lysis of the PCV releases Salmonella into the cytosol.

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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.

Author information

Authors and Affiliations

Authors

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 to Petr Broz.

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Competing interests

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 Gbpchr3 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 Gbpchr3 KO mice is indicated. bd, Induction of pro-caspase-11, GBP2 and GBP5 expression in lysates of wild-type and Gbpchr3 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 Gbpchr3 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.

ac, 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. bd, 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 (bd, 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. be, 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 Gbpchr3 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. ce, 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 (ac, eg) 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 Gbpchr3 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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This file contains the sequences of siRNA pools used in this study. (PDF 95 kb)

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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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