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. 2020 Sep 29;23(10):101612.
doi: 10.1016/j.isci.2020.101612. eCollection 2020 Oct 23.

A Rapid Caspase-11 Response Induced by IFN γ Priming Is Independent of Guanylate Binding Proteins

Sky W Brubaker  1 , Susan M Brewer  1 , Liliana M Massis  1 , Brooke A Napier  2 , Denise M Monack  1
Affiliations

A Rapid Caspase-11 Response Induced by IFN γ Priming Is Independent of Guanylate Binding Proteins

Sky W Brubaker et al. iScience. .

Abstract

In mammalian cells, inflammatory caspases detect Gram-negative bacterial invasion by binding lipopolysaccharides (LPS). Murine caspase-11 binds cytosolic LPS, stimulates pyroptotic cell death, and drives sepsis pathogenesis. Extracellular priming factors enhance caspase-11-dependent pyroptosis. Herein we compare priming agents and demonstrate that IFNγ priming elicits the most rapid and amplified macrophage response to cytosolic LPS. Previous studies indicate that IFN-induced expression of caspase-11 and guanylate binding proteins (GBPs) are causal events explaining the effects of priming on cytosolic LPS sensing. We demonstrate that these events cannot fully account for the increased response triggered by IFNγ treatment. Indeed, IFNγ priming elicits higher pyroptosis levels in response to cytosolic LPS when macrophages stably express caspase-11. In macrophages lacking GBPs encoded on chromosome 3, IFNγ priming enhanced pyroptosis in response to cytosolic LPS as compared with other priming agents. These results suggest an unknown regulator of caspase-11-dependent pyroptosis exists, whose activity is upregulated by IFNγ.

Keywords: Cell Biology; Immunology.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
IFNγ is a Potent Priming Agent for the Response to Cytosolic LPS in Macrophages (A–D) WT BMDMs were primed for 16 h overnight with the following treatments: unprimed (N/A), IFNγ (100 U/mL), IFNβ (100 U/mL), or LPS (10 ng/mL). NLRP3 and CASP11 inflammasome activation was triggered with ATP (5 mM), Nigericin (10 μM), or by LPS (E. coli 0111:B4, 25 μg/mL) transfection with FuGENE HD (Fu/LPS). At 2 h following inflammasome activation, supernatants were collected to measure release of (A) lactate dehydrogenase (LDH) for percent cell death calculations, (B) IL-18, and/or (C) IL-1β. (D) Cell death kinetics were monitored over time following inflammasome activation by measuring the incorporation of SYTOX Green. (E) Cell lysates were collected from BMDMs treated for 16 h of priming as described above and separated by SDS-PAGE. Western blot analysis was performed to determine protein expression of critical inflammasome components: NLRP3, CASP11, GSDMD, pro-IL-18, pro-IL-1β, and the loading control ACTIN. (F) Quantitation of CASP11 expression was compared with CASP11-induced cell death by LPS transfection (Fu/LPS). Bar graphs in black correspond to the left axis, which represents CASP11 expression as band intensity normalized to ACTIN. The mean percent cell death at 2 h following LPS transfection (from the three independent experiments shown in A) are plotted in pink and correspond to the right axis. (G) WT BMDMs were primed and transfected with LPS (E. coli 0111:B4, 25 μg/mL) as described for (A–D), then supernatants and lysates were collected at 1, 2, 3 h post transfection to monitor for the cleavage and release of inflammasome-related proteins by SDS-PAGE and western blot. Molecular weight marker positions are shown to the left of each blot, and arrows indicate a cleavage product. Bar graphs show the mean value +/− SEM along with individual data points pooled from independent experiments depicted with different shapes (A–C and F). Line graphs show the mean ± SD of three technical replicates (D). Data were pooled from three (A and F) or two (B and C) independent experiments or are representative of three (D and E) or two (G) independent experiments. Statistical analysis performed using a two-way ANOVA and Tukey's multiple comparisons test; ∗∗∗∗ <0.0001; ∗∗∗ = 0.0002-0.0001; ∗ = 0.0463. See also Figure S1.
Figure 2
Figure 2
IFNγ Priming Specifically Promotes CASP11 Inflammasome Activation (A–D) WT, Casp1−/−, and Casp11−/− BMDMs were primed for 16 h overnight with the following treatments: unprimed (N/A), IFNγ (100 U/mL), IFNβ (100 U/mL), or LPS (10 ng/mL). CASP11 inflammasome activation was triggered by LPS (E. coli 0111:B4, 25ug/mL) transfection with FuGENE HD. At 3 h following inflammasome activation, supernatants were collected to measure release of (A) LDH for percent cell death calculations, (B) IL-18, and/or (C) IL-1β. (D) Cell death kinetics were monitored over time following inflammasome activation by measuring the incorporation of SYTOX Green. (E) Cell lysates were collected from WT, Casp1−/−, or Casp11−/− BMDMs treated for 16 h of priming as described above and separated by SDS-PAGE. Western blot analysis was performed to determine protein expression of critical inflammasome components: CASP11, CASP1, GSDMD, pro-IL-18, pro-IL-1β, and the loading control ACTIN. Molecular weight marker positions are shown to the left of each blot. Bar graphs show the mean value +/− SEM along with individual data points pooled from independent experiments depicted with different shapes (A–C). Line graphs show the mean value +/− SEM from pooled independent experiments with technical triplicates (D). Data were pooled from three (A and D) or two (B and C) independent experiments or are representative of three independent experiments (E). Statistical analysis performed using a two-way ANOVA and Tukey's multiple comparisons test; ∗∗∗∗ <0.0001. See also Figure S2.
Figure 3
Figure 3
Priming Promotes Cell Death in Response to Cytosolic LPS Independently of CASP11 Expression A constitutive CASP11-expressing cell line was generated by transducing a CASP11 expression vector into Casp1,Casp11-CRISPR/Cas9 DKO RAW cells (Casp1/11 DKO). (A) Cell lysates were collected from WT, Casp1/11 DKO, and the constitutive cell line (Casp1/11 DKO + Casp11) and analyzed by western blot to determine CASP1 and CASP11 expression. WT and Casp1/11 DKO were treated with IFNγ (100 U/mL) for 16 h. (B) The constitutive CASP11-expressing cell line was primed for 16 h overnight with the following treatments; unprimed (N/A), IFNγ (100 U/mL), IFNβ (1,000 U/mL), or LPS (10 ng/mL). Lysates were collected to determine the effects of priming on CASP11 expression by western blot. (C and D) The constitutive CASP11-expressing cell line was primed as described above and CASP11 inflammasome activation was triggered by LPS (E. coli 0111:B4, 50 μg/mL) transfection with FuGENE HD. (C) At 4 and 8 h following inflammasome activation, supernatants were collected to measure release of LDH for percent cell death calculations. (D) Alternatively, supernatants and lysates were collected 3 h post transfection to monitor for the cleavage and release of inflammasome-related proteins by SDS-PAGE and western blot. Molecular weight marker positions are shown to the left of each blot, and arrows indicate a cleavage product. Bar graphs show the mean value +/− SEM along with individual data points pooled from independent experiments depicted with different shapes (C). Data were pooled from two (C) independent experiments or are representative of three (A and B) or two (D) independent experiments. Statistical analysis performed using a two-way ANOVA and Tukey's multiple comparisons test; ∗∗∗∗ <0.0001. See also Figure S3.
Figure 4
Figure 4
IFNγ Receptor Signaling Enhances but is not Required for CASP11 Inflammasome Activation (A, C, and D) WT and Ifngr1−/− BMDMs were primed for 16 h overnight with the following treatments: unprimed (N/A), IFNγ (100 U/mL), IFNβ (100 U/mL), or LPS (10 ng/mL). CASP11 inflammasome activation was triggered by LPS (E. coli 0111:B4, 25 μg/mL) transfection with FuGENE HD. (A) At 2 h following inflammasome activation, supernatants were collected to measure release of LDH for percent cell death calculations. (B) Cell lysates were collected from BMDMs treated for 16 h of priming as described above and separated by SDS-PAGE. Western blot analysis was performed to determine protein expression of CASP11, GSDMD, and the loading control ACTIN. Molecular weight marker positions are shown to the left of each blot. (C and D) Cell death kinetics were monitored over time following inflammasome activation by measuring the incorporation of SYTOX Green. (C) Comparisons between different priming conditions are shown for BMDMs transfected with LPS. (D) Additionally, a comparison between unprimed WT and Ifngr1−/− BMDMs treated with or without transfection reagent are shown. Bar graphs show the mean value +/− SEM along with individual data points pooled from independent experiments depicted with different shapes (A). Line graphs show the mean ± SD of three technical replicates (C and D). Data were pooled from two (A) independent experiments or are representative of two (B–D) independent experiments. Statistical analysis performed using a two-way ANOVA and Tukey's multiple comparisons test; ∗∗∗∗ <0.0001; ∗∗∗ <0.002. See also Figure S4.
Figure 5
Figure 5
IFNγ Enhancement of CASP11-Dependent Cell Death Requires GSDMD and Is Independent of GSDME (A and B) WT, Gsdmd−/−, and Gsdme−/− BMDMs were primed for 16 h overnight with or without IFNγ (100 U/mL). CASP11 inflammasome activation was triggered by LPS (E. coli 0111:B4, 25 μg/mL) transfection with FuGENE HD. (A) At 4 h following inflammasome activation, supernatants were collected to measure release of LDH for percent cell death calculations. (B) Cell death kinetics were monitored over time following inflammasome activation by measuring the incorporation of SYTOX Green. (C) Cell lysates were collected from WT, Gsdmd−/−, or Gsdme−/− BMDMs treated for 16 h of priming as described above and separated by SDS-PAGE. Western blot analysis was performed to determine protein expression of CASP11, GSDMD, GSDME, and the loading control ACTIN. Molecular weight marker positions are shown to the left of each blot. Bar graphs show the mean value +/− SEM along with individual data points pooled from independent experiments depicted with different shapes (A). Line graphs show the mean ± SEM from pooled independent experiments (B). Data were pooled from two (A and B) independent experiments or are representative of two independent experiments (C). Statistical analysis performed using a two-way ANOVA and Tukey's multiple comparisons test; ∗∗∗∗ <0.0001. See also Figure S5.
Figure 6
Figure 6
GBPs Encoded on Chromosome 3 Do Not Fully Account for Enhanced CASP11-Dependent Cell Death Triggered by IFNγ Priming (A and B) WT, Gbpchr3−/−, and Casp11−/− BMDMs were primed for 16 h overnight with or without IFNγ (100U/mL). CASP11 inflammasome activation was triggered by treating cells with OMVs (E. coli DH5α), LPS (E. coli 0111:B4, 25 μg/mL) transfection with FuGENE HD, or LPS (E. coli 0111:B4, 25 μg/mL) mixed with CTB (Cholera Toxin B Subunit, 20 μg/mL). (A) At the indicated time points following inflammasome activation, supernatants were collected to measure release of LDH for percent cell death calculations. (B) Cell lysates were collected from WT, Gbpchr3−/−, and Casp11−/− BMDMs treated with or without IFNγ as described above and separated by SDS-PAGE. Western blot analysis was performed to determine protein expression of GBP2, CASP11, GSDMD, and the loading control ACTIN. (C–F) WT, Gbpchr3−/−, and Casp11−/− BMDMs were primed for 16 h overnight with the following treatments: unprimed (N/A), IFNγ (100 U/mL), IFNβ (100 U/mL), or LPS (10 ng/mL). CASP11 inflammasome activation was triggered by LPS transfection (C and E) or with CTB and LPS (D and F) as described above. Cell death was determined following inflammasome activation by collecting supernatants at 3 h to monitor LDH release (C and D), or cell death kinetics were monitored over time by measuring the incorporation of SYTOX Green (E and F). (G) Cell lysates were collected from WT and Gbpchr3−/− BMDMs treated for 16 h of priming as described above and separated by SDS-PAGE. Western blot analysis was performed to determine protein expression of GBP2, CASP11, GSDMD, pro-IL-18, and the loading control ACTIN. (H) WT and Gbpchr3−/− BMDMs were primed and transfected with LPS as described above. Supernatants and lysates were collected from untransfected, 1, 2, or 3 h post-transfection to monitor for the cleavage and release of inflammasome-related proteins by SDS-PAGE and western blot. Molecular weight marker positions are shown to the left of each blot, and arrows indicate a cleavage product. Bar graphs show the mean value +/− SEM along with individual data points pooled from independent experiments depicted with different shapes (A, C, and D). Line graphs show the mean value +/− SEM from pooled independent experiments with technical replicates (E and F). Data were pooled from four (A - Fu/LPS and CTB/LPS) or two (A - OMV, C, D, E, F) independent experiments or are representative of two (B and G) or one (H) independent experiment. Statistical analysis performed using a two-way ANOVA and Tukey's multiple comparisons test; ∗∗∗∗ <0.0001; ∗∗∗ for (C) = 0.0005; ∗ for (A) = 0.0453; ∗ for (D) = 0.0294. See also Figure S6.
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