Volume 25, Issue 6, Pages 1525-1536.e7 (November 2018) Shiga Toxin/Lipopolysaccharide Activates Caspase-4 and Gasdermin D to Trigger Mitochondrial Reactive Oxygen Species Upstream of the NLRP3 Inflammasome  Jaye M. Platnich, Hyunjae Chung, Arthur Lau, Christina F. Sandall, Adom Bondzi-Simpson, Huey-Miin Chen, Takanori Komada, Aaron C. Trotman-Grant, Jeremy R. Brandelli, Justin Chun, Paul L. Beck, Dana J. Philpott, Stephen E. Girardin, May Ho, Roger P. Johnson, Justin A. MacDonald, Glen D. Armstrong, Daniel A. Muruve  Cell Reports  Volume 25, Issue 6, Pages 1525-1536.e7 (November 2018) DOI: 10.1016/j.celrep.2018.09.071 Copyright © 2018 The Author(s) Terms and Conditions

Cell Reports 2018 25, 1525-1536.e7DOI: (10.1016/j.celrep.2018.09.071) Copyright © 2018 The Author(s) Terms and Conditions

Figure 1 Stx2 Activates the Canonical Inflammasome in Human Macrophages (A) Immunoblot analysis of PMA-primed THP-1 cells treated with increasing concentrations of Stx2 (2, 20, and 200 ng/mL) for 6 hr, probing for caspase-1 (antibody directed against N-terminal, detects p11 CARD fragment following caspase-1 activation) in supernatant (SUP) and cell lysates (LYS). Nigericin (NGC) treatment (50 μM) for 1 hr was used as a positive control. Data are representative of n = 3 independent experiments. (B) ELISA for IL-1β in supernatants of PMA-primed THP-1 cells treated with Stx2 (treatment versus PMA priming alone; ∗∗∗p < 0.001; n = 3 independent experiments; one-way ANOVA, post hoc Dunnett). Nigericin (NGC) treatment (50 μM) for 1 hr was used as a positive control. (C) Time course of PMA-primed THP-1 cells treated with Stx2 (200 ng/mL). Immunoblot analysis probing for caspase-1 in supernatant (SUP) and cell lysates (LYS). Nigericin (NGC) treatment (50 μM) for 1 hr was used as a positive control. (D) Graphical ELISA data for IL-1β in PMA-primed THP-1 cells treated over a time course with Stx2 (200 ng/mL). Data are representative of n = 3 independent experiments, with bars representing mean ± SEM. Cell Reports 2018 25, 1525-1536.e7DOI: (10.1016/j.celrep.2018.09.071) Copyright © 2018 The Author(s) Terms and Conditions

Figure 2 Stx2 Triggers Non-canonical Inflammasome Activation and Pyroptosis in Human Macrophages (A and B) Immunoblot analysis of PMA-primed THP-1 cells treated with Stx2 over both a range of doses (A; 2, 20, and 200 ng/mL) and time points (B; 1, 3, and 6 hr with 200 ng/mL Stx2) probing for caspase-4, caspase-8, and gasdermin D (GSDMD) cleavage in the lysate (LYS) and GAPDH release into the supernatant (SUP). Nigericin (NGC) treatment (50 μM) for 1 hr was used as a positive control. (C and D) Lactate dehydrogenase (LDH) assay in PMA-primed THP-1 cells treated with increasing doses of Stx2 for 6 hr (C) and with Stx2 (200 ng/mL) over a 6-hr time course (D). Bars represent mean ± SEM. Data are representative of n = 3 independent experiments. (E) Immunoblot analysis of PMA-primed THP-1 cells treated with Stx2 (200 ng/mL) over a 6-hr time course, probing for caspase-8, caspase-9, and caspase-1. Data are representative of n = 3 independent experiments. (F). LDH assay of PMA-primed, Stx-treated THP-1 cells pre-treated with pan-caspase inhibitor zVAD (50 μM) or NLRP3 inhibitor glibenclamide (100 μM) at 6 hr. Positive control is nigericin (NGC) treatment (50 μM) for 1 hr. Bars represent mean ± SEM (Stx2 alone versus inhibitor; ∗∗∗∗p < 0.0001; n = 3 independent experiments; one-way ANOVA, post hoc Dunnett). Cell Reports 2018 25, 1525-1536.e7DOI: (10.1016/j.celrep.2018.09.071) Copyright © 2018 The Author(s) Terms and Conditions

Figure 3 Canonical and Non-canonical Inflammasome Activation by Stx2 Requires NLRP3 (A) Immunoblotting for caspase-1, caspase-4, GSDMD, and IL-1β cleavage in cell lysates (LYS) and GAPDH release in supernatants (SUP) derived from PMA-primed wild-type or NLRP3 −/− THP-1 macrophages treated with Stx2 (200 ng/mL) for 6 hr. Nigericin (NGC) treatment (50 μM) for 1 hr was used as a positive control. Data are representative of three independent experiments. (B) IL-1β ELISA in supernatants from wild-type and NLRP3 −/− PMA-primed THP-1 cells treated with Stx2 (200 ng/mL) for 6 hr. Bars represent mean ± SEM (NLRP3 +/+ THP-1 versus NLRP3 −/− THP cells; ∗∗p < 0.01, ∗∗∗p < 0.001, n.s., not significant; NLRP3 +/+, n = 4; NLRP3 −/−, n = 6 independent experiments; two-way ANOVA, post hoc Šidák). Nigericin (NGC) treatment (50 μM) for 1 hr was used as a positive control. (C) Quantification of caspase-4 cleavage (p30/p43) by densitometry, normalized to wild-type caspase-4 cleavage in response to Stx2 treatment (200 ng/mL) for 6 hr. Bars represent mean ± SEM (NLRP3 +/+ THP-1 versus NLRP3 −/− THP cells; ∗∗p < 0.01; n = 3 independent experiments; unpaired, two-tailed Student’s t test). (D) LDH assay in supernatants from PMA-primed wild-type and NLRP3 −/− THP-1 cells treated with Stx2 (200 ng/mL) for 6 hr. Bars represent mean ± SEM (NLRP3 +/+ THP-1 versus NLRP3 −/− THP cells; ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001; n = 3 independent experiments; two-way ANOVA, post hoc Šidák). Nigericin (50 μM) at 1 hr is used as a positive control. Cell Reports 2018 25, 1525-1536.e7DOI: (10.1016/j.celrep.2018.09.071) Copyright © 2018 The Author(s) Terms and Conditions

Figure 4 Stx2-Mediated Inflammasome Activation Is Dependent on Co-transported LPS (A) Immunoblotting for LPS in “low-endotoxin” wild-type and mutant Stx2 preparations (5 mg, 1 mg, 500 μg, 250 μg, and 125 μg). One milligram of standard preparation wild-type Stx2 was blotted as a positive control. (B) Immunoblot analysis at 6 hr probing for caspase cleavage in the cell lysate (LYS) and supernatants (SUP) of PMA-primed THP-1 cells treated with standard preparation Stx2 (WT Stx2), “low-endotoxin” Stx2 (Low LPS WT Stx2), and “low-endotoxin” mutant (enzyme inactive) Stx2 (Low LPS Mut Stx2) (all at 200 ng/mL) with or without pre-incubation for 30 min with O157:H7 LPS (100 ng/mL). Treatment with LPS alone (100 ng/mL) was used as a negative control. (C) ELISA for IL-1β at 6 hr in the supernatants of PMA-primed THP-1 cells treated with standard preparation Stx2 (WT Stx2), “low-endotoxin” Stx2 (Low LPS WT Stx2), and “low-endotoxin” mutant (enzyme inactive) Stx2 (Low LPS Mut Stx2) (all at 200 ng/mL) with or without pre-incubation for 30 min with O157:H7 LPS (100 ng/mL), prior to treatment. Treatment with LPS alone (100 ng/mL) was used as a negative control. Bars represent mean ± SEM (WT Stx2 versus Low LPS WT Stx2 and Low LPS Mut Stx2, ∗p < 0.05; LPS versus Low LPS Mut Stx2, n.s., not significant; n = 3 independent experiments; one-way ANOVA, post hoc Tukey). (D) Live cell imaging of PMA-primed THP-1 cells at 1 hr following treatment with LPS-FITC alone (1 μg/mL) and wild-type and mutant Stx2 (Low LPS WT Stx2, Low LPS Mut Stx2) (200 ng/mL) pre-incubated with LPS-FITC (1 μg/mL) for 30 min. Hoechst dye (blue) was used for nuclear staining, and CellTracker CM-DiI (red) was used for cell membrane staining. Scale bar, 10 μm. (E) Confocal microscopy of PMA-primed THP-1 cells 3 hr following treatment with LPS-FITC alone (1 μg/mL) and wild-type and mutant Stx2 (Low LPS WT Stx2, Low LPS Mut Stx2) (200 ng/mL) pre-incubated with LPS-FITC (1 μg/mL) for 30 min. Hoechst dye (blue) was used for nuclear staining, and CellTracker CM-DiI (red) was used for cell membrane staining. Scale bar, 10 μm. Data are representative of n = 3 independent experiments. Cell Reports 2018 25, 1525-1536.e7DOI: (10.1016/j.celrep.2018.09.071) Copyright © 2018 The Author(s) Terms and Conditions

Figure 5 GSDMD p30 Localizes to Membrane and Mitochondrial Fractions of Human Macrophages following Stx2/LPS Treatment (A) Immunoblotting for caspase-4, GSDMD, and organelle/membrane markers following crude subcellular fractionation of PMA-primed wild-type THP-1 cells treated with Stx2/LPS (200 ng/mL) over a 6-hr time course. (B) Immunoblotting for caspase-4, GSDMD, and organelle/membrane markers following subcellular fractionation by sucrose gradient/ultracentrifugation of PMA-primed wild-type THP-1 cells treated with Stx2/LPS (200 ng/mL) at 6 hr. (C) Immunoblotting for GSDMD and organelle/membrane markers following a more stringent subcellular fractionation and mitochondrial isolation of wild-type PMA-primed THP-1 cells treated with Stx2/LPS (200 ng/mL) over a 5-hr time course. Untreated homogenate depleted of mitochondria was used as a positive control for membrane/cytoplasmic markers and a negative control for VDAC. CYTO, cytoplasmic fraction; HOMO, homogenate; MITO, mitochondrial fraction. Data are representative of n = 3 independent experiments. Cell Reports 2018 25, 1525-1536.e7DOI: (10.1016/j.celrep.2018.09.071) Copyright © 2018 The Author(s) Terms and Conditions

Figure 6 Stx2/LPS-Mediated Non-canonical Inflammasome Activation Induces mitoROS Production to Trigger the Canonical NLRP3 Inflammasome (A) Quantification of MitoSOX Red fluorescence (using Incucyte Zoom automated fluorescent microscopy) in PMA-primed THP-1 cells pre-treated with the caspase-4 inhibitor LEVD (100 μM), the pan-caspase inhibitor zVAD (100 μM), or the antioxidant N-acetylcysteine (NAC) (25 mM) followed by challenge with Stx2/LPS for 6 hr (200 ng/mL). Stimulations ± inhibitors were performed in conjunction with the same NT and Stx2 controls but separated in the panels for clarity. Bars represent mean ± SEM (Stx2/LPS treatment alone versus Stx2/LPS + inhibitor; ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001; n = 3 independent experiments; repeated-measures [RM] two-way ANOVA, post hoc Dunnett). NT, PMA-primed THP-1 cells without additional stimulation. (B) Quantification of MitoSOX Red fluorescence (using Incucyte Zoom automated fluorescent microscopy) in PMA-primed wild-type and NLRP3 −/− THP-1 cells challenged with Stx2/LPS for 6 hr (200 ng/mL). Bars represent mean ± SEM (Stx2/LPS-treated NLRP3 +/+ THP-1 versus Stx2/LPS-treated NLRP3 −/− THP-1; ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001; n = 4 independent experiments; RM two-way ANOVA, post hoc Dunnett). NT, PMA-primed THP-1 cells without additional stimulation. (C) Quantification of MitoSOX Red fluorescence (using Incucyte Zoom automated fluorescent microscopy) in PMA-primed wild-type and GSDMD −/− THP-1 cells challenged with Stx2/LPS for 6 hr (200 ng/mL). Bars represent mean ± SEM (Stx/LPS-treated GSDMD +/+ THP-1 versus Stx/LPS-treated GSDMD −/− THP-1; ∗∗p < 0.01 and ∗∗∗∗p < 0.0001; n = 3 independent experiments; RM two-way ANOVA, post hoc Dunnett). NT, PMA-primed THP-1 cells without additional stimulation. (D) Immunoblotting for caspase-1, caspase-4, GSDMD, and IL-1β cleavage and GAPDH release in cell lysates (LYS) and supernatants (SUP) derived from PMA-primed wild-type, NLRP3 −/−, or GSDMD −/− THP-1 macrophages treated with Stx2/LPS (200 ng/mL) for 6 hr. (E and F) IL-1β ELISA (E) and LDH assay (F) of supernatants from PMA-primed wild-type and GSDMD −/− THP-1 cells treated with Stx2/LPS (200 ng/mL) for 6 hr. For the ELISA, bars represent mean ± SEM (GSDMD +/+ THP-1 versus GSDMD −/− THP-1; ∗∗∗∗p < 0.0001, n.s., not significant; n = 3 independent experiments; two-way ANOVA, post hoc Šidák). For the LDH assay, bars represent mean ± SEM (GSDMD +/+ THP-1 versus GSDMD −/− THP-1 ∗∗p < 0.01; n = 3 independent experiments; unpaired, two-tailed Student’s t test). Cell Reports 2018 25, 1525-1536.e7DOI: (10.1016/j.celrep.2018.09.071) Copyright © 2018 The Author(s) Terms and Conditions

Figure 7 NLRP3 and GSDMD Are Required for Stx2-Induced ROS Generation (A) Immunoblot analysis probing for caspase and GSDMD cleavage in the cell lysates (LYS) and GAPDH release in the supernatants (SUP) of PMA-primed THP-1 cells pre-treated with increasing concentrations of NAC (1, 5, and 25 mM) prior to challenge with Stx2/LPS (200 ng/mL) for 6 hr. Nigericin (NGC) treatment (50 μM) for 1 hr was used as a positive control. (B) Immunoblot analysis probing for caspase and GSDMD cleavage in the cell lysates and GAPDH release in the supernatants (SUP) of PMA-primed THP-1 cells pre-treated with the caspase-4 inhibitor LEVD (100 μM), the pan-caspase inhibitor zVAD (100 μM), or the antioxidant NAC (25 mM) followed by challenge with Stx2/LPS for 6 hr (200 ng/mL). (C) IL-1β ELISA in supernatants from Stx2/LPS (200 ng/mL) stimulated PMA-primed THP-1 cells pre-treated with the caspase-4 inhibitor LEVD (100 μM), the pan-caspase inhibitor zVAD (100 μM), or the antioxidant N-acetylcysteine (NAC) (25 mM) at 6 hr. Data are representative of n = 3 independent experiments, with bars representing mean ± SEM (Stx2 alone versus Stx2 + inhibitor; ∗∗p < 0.01 and ∗∗∗p < 0.001; n = 3 independent experiments; one-way ANOVA, post hoc Dunnett). (D) Immunoblotting for caspase-4, GSDMD, and mitochondrial membrane marker VDAC following crude subcellular fractionation of NLRP3 −/− PMA-primed THP-1 cells treated with Stx2/LPS alone (200 ng/mL) or Stx2/LPS (200 ng/mL) + LEVD-FMK (5 μM) for 6 hr. CYTO, cytoplasmic fraction; HOMO, homogenate; MEMB, crude membrane fraction. Data are representative of n = 3 independent experiments. Cell Reports 2018 25, 1525-1536.e7DOI: (10.1016/j.celrep.2018.09.071) Copyright © 2018 The Author(s) Terms and Conditions