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. 2017 Oct 3;8(5):e01188-17.
doi: 10.1128/mBio.01188-17.

Inflammasome Activation by Bacterial Outer Membrane Vesicles Requires Guanylate Binding Proteins

Ryan Finethy  1 , Sarah Luoma  1 , Nichole Orench-Rivera  2 , Eric M Feeley  1 , Arun K Haldar  1 , Masahiro Yamamoto  3 , Thirumala-Devi Kanneganti  4 , Meta J Kuehn  2 , Jörn Coers  5   6
Affiliations

Inflammasome Activation by Bacterial Outer Membrane Vesicles Requires Guanylate Binding Proteins

Ryan Finethy et al. mBio. .

Abstract

The Gram-negative bacterial cell wall component lipopolysaccharide (LPS) is recognized by the noncanonical inflammasome protein caspase-11 in the cytosol of infected host cells and thereby prompts an inflammatory immune response linked to sepsis. Host guanylate binding proteins (GBPs) promote infection-induced caspase-11 activation in tissue culture models, and yet their in vivo role in LPS-mediated sepsis has remained unexplored. LPS can be released from lysed bacteria as "free" LPS aggregates or actively secreted by live bacteria as a component of outer membrane vesicles (OMVs). Here, we report that GBPs control inflammation and sepsis in mice injected with either free LPS or purified OMVs derived from Gram-negative Escherichia coli In agreement with our observations from in vivo experiments, we demonstrate that macrophages lacking GBP2 expression fail to induce pyroptotic cell death and proinflammatory interleukin-1β (IL-1β) and IL-18 secretion when exposed to OMVs. We propose that in order to activate caspase-11 in vivo, GBPs control the processing of bacterium-derived OMVs by macrophages as well as the processing of circulating free LPS by as-yet-undetermined cell types.IMPORTANCE The bacterial cell wall component LPS is a strong inducer of inflammation and is responsible for much of the toxicity of Gram-negative bacteria. Bacteria shed some of their cell wall and its associated LPS in the form of outer membrane vesicles (OMVs). Recent work demonstrated that secreted OMVs deliver LPS into the host cell cytosol by an unknown mechanism, resulting in the activation of the proinflammatory LPS sensor caspase-11. Here, we show that activation of cytosolic caspase-11 by OMVs requires additional host factors, the so-called guanylate binding proteins (GBPs). The discovery of GBPs as regulators of OMV-mediated inflammation paves the way toward a mechanistic understanding of the host response toward bacterial OMVs and may lead to effective strategies to ameliorate inflammation induced by bacterial infections.

Keywords: GBP2; GBP5; LPS; OMVs; caspase-11; guanylate binding proteins; inflammasome; interferons; lipopolysaccharide; outer membrane vesicles; sepsis.

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Figures

FIG 1
FIG 1
GBPs control noncanonical and canonical inflammasome activation in macrophages exposed to E. coli. Wild-type, GBPchr3−/−, and Casp11−/− BMDMs were primed with IFN-γ overnight or left unprimed and then exposed to E. coli strain K-12 at the indicated MOI or left uninfected. (A) LDH release was measured at 8 hpi. (B) IFN-γ-primed and unprimed BMDMs were infected with E. coli at an MOI of 25, and propidium iodide fluorescence resulting from nuclear incorporation in dead cells was measured at the indicated times following infection. (C) IFN-γ-primed BMDMs were either left untreated or infected with E. coli at an MOI of 25 for 8 h and subsequently stained with anti-ASC antibody and Hoechst stain (for DNA/nuclei). White arrows indicate ASC specks. The number of ASC puncta per cell was quantified. (D) IFN-γ-primed BMDMs were infected with E. coli at an MOI of 25, and at 8 hpi cell lysates and supernatants were collected. Protein levels in total cell lysates (caspase-1, pro-IL-1β, and actin) or supernatant (IL-1β and caspase-1 p20) were visualized via immunoblotting. (E and F) IL-1β (E) and IL-18 (F) concentrations were measured by ELISA in culture supernatants collected from BMDMs exposed to E. coli for 8 h (MOI of 25). Data shown are means ± SEM from 3 independent experiments. The statistical significance shown is relative to the wild type (A, C, E, and F) or relative to GBPchr3−/− BMDMs (B) of the same experimental groups, unless indicated otherwise. Significance is indicated as follows: ***, P < 0.001; **, P < 0.01; *, P < 0.05; N.S, not statistically significant (two-way ANOVA with Tukey’s multiple-comparison test).
FIG 2
FIG 2
GBPs control noncanonical and canonical inflammasome activation in macrophages exposed to OMVs. Wild-type, GBPchr3−/−, and Casp11−/− BMDMs were primed with 100 U/ml IFN-γ overnight or left unprimed and then treated with 1 µg/ml LPS or E. coli-derived OMVs at the indicated protein concentrations. (A) LDH release was measured at 8 h posttreatment. (B) IFN-γ-primed and unprimed BMDMs were treated with 2 µg/ml OMVs, and propidium iodide fluorescence was measured at the indicated times following treatment. (C) IFN-γ-primed BMDMs were either treated with OMVs (2 µg/ml) for 8 h or left untreated and were subsequently stained with anti-ASC antibody and Hoechst stain (nuclei), and the number of ASC puncta (white arrows) per cell was quantified. (D) IFN-γ-primed BMDMs were treated with OMVs (2 µg/ml), and at 8 h posttreatment, cell lysates and supernatants were collected. Protein levels in total cell lysates (caspase-1, pro-IL-1β, and actin) or supernatant (IL-1β and caspase-1 p20) were visualized via immunoblotting. (E and F) IL-1β (E) and IL-18 (F) concentrations were measured by ELISA in culture supernatants collected from BMDMs treated with OMVs (2 µg/ml) for 8 h. Data shown are means ± SEM from 3 independent experiments. The statistical significance shown is relative to the wild type (A, C, E, and F) or relative to GBPchr3−/− BMDMs (B) of the same experimental groups, unless indicated otherwise. Significance was defined as follows: ***, P < 0.001; **, P < 0.01; *, P < 0.05; N.S, not statistically significant (two-way ANOVA with Tukey’s multiple-comparison test).
FIG 3
FIG 3
GBP2 promotes OMV-induced pyroptosis and IL-1β/IL-18 secretion in macrophages. (A to C) Wild-type, GBPchr3−/−, GBP2−/−, and GBP5−/− BMDMs were primed with IFN-γ and infected with E. coli at an MOI of 25. At 8 hpi, LDH release (A) and IL-1β (B) and IL-18 (C) secretion were measured. IFN-γ-primed wild-type, GBPchr3−/−, GBP2−/−, and GBP5−/− BMDMs were treated with E. coli-derived OMVs for 8 h. (D to F) Subsequently, LDH release (D) and IL-1β (E) and IL-18 (F) secretion were measured. Data shown are means ± SEM from 3 independent experiments (A and D) or 6 independent experiments (B, C, E, and F). The statistical significance shown is relative to the wild type. Significance was defined as follows: ***, P < 0.001; **, P < 0.01; *, P < 0.05; N.S, not statistically significant (two-way ANOVA with Tukey’s multiple-comparison test).
FIG 4
FIG 4
GBP-deficient mice display reduced IL-1β/IL-18 serum levels and increased survival rates during endotoxemia. (A and B) Wild-type and GBPchr3−/− mice were injected i.p. with poly(I⋅C) at a dose of 2 mg/kg body weight and then 6 h later injected i.p. with 4 µg purified OMVs per mouse. Serum was obtained 6 h after OMV injection from OMV-injected wild-type (n = 16) or GBPchr3−/− (n = 16) mice or from wild-type (n = 6) or GBPchr3−/− (n = 7) mice injected with an equal volume of PBS, and IL-1β (A) and IL-18 (B) serum concentrations were measured by ELISA. (C and D) Wild-type (n = 10) and GBPchr3−/− (n = 15) mice were injected with LPS (8 mg/kg body weight) or PBS alone (n = 9 for both wild-type and GBPchr3−/− mice), and serum IL-1β (C) and IL-18 (D) were measured by ELISA 4 h postinjection. (E and F) Wild-type (n = 7) and GBP2−/− (n = 7) mice were injected with LPS (8 mg/kg body weight), and serum IL-1β (E) and IL-18 (F) levels were measured by ELISA 4 h postinjection. (G) Wild-type (n = 9), GBPchr3−/− (n = 7), and GBP2−/− (n = 9) mice were i.p. injected with 2 mg/kg body weight of poly(I⋅C) and then 6 h later i.p. injected with LPS (20 mg/kg body weight). Morbidity and mortality were observed for 42 h at 3-h intervals. For panels A to F, mean ± standard deviation is shown. Each symbol represents an individual mouse. Significance was defined as follows: ***, P < 0.001; **, P < 0.01; *, P < 0.05; N.S, not statistically significant. Significance was measured by two-way ANOVA with Sidak’s multiple-comparison test (A to D), unpaired t test (E and F), or log rank test (G).
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