Volume 46, Issue 4, Pages 649-659 (April 2017) NAIP-NLRC4 Inflammasomes Coordinate Intestinal Epithelial Cell Expulsion with Eicosanoid and IL-18 Release via Activation of Caspase-1 and -8  Isabella Rauch, Katherine A. Deets, Daisy X. Ji, Jakob von Moltke, Jeannette L. Tenthorey, Angus Y. Lee, Naomi H. Philip, Janelle S. Ayres, Igor E. Brodsky, Karsten Gronert, Russell E. Vance  Immunity  Volume 46, Issue 4, Pages 649-659 (April 2017) DOI: 10.1016/j.immuni.2017.03.016 Copyright © 2017 Elsevier Inc. Terms and Conditions

Immunity 2017 46, 649-659DOI: (10.1016/j.immuni.2017.03.016) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 Specific Activation of NLRC4 in Intestinal Epithelial Cells Protects from Pathogen Invasion but Can Lead to Intestinal Pathology (A) Schematic of the Rosa26 locus after successful gene targeting with iNLRC4-IRES-GFP. Yellow triangles represent LoxP sites. (B and C) Mice were injected with 0.8 μg/g PA and 0.4 μg/g LFn-Fla and monitored for (B) body temperature and (C) hematocrit (30 min, n = 3). (D and E) Mice were injected with 0.8 μg/g PA and 80 ng/g LFn-Fla166 and (D) wet/dry ratio of intestinal content and (E) PGE2 amounts of intestinal tissue from mice treated for 30 min were determined (n = 4–5). (F) Quantification of serum IL-18 using ELISA in mice treated as in (B), 60 min time point (n = 3). (G) H&E staining of small intestinal tissue from mice treated as in (B), 60 min after injection. Scale bars represent 100 μm. (H) CFU in cecum 18 hr after oral S. Typhimurium infection (n = 5). Data are representative of three independent experiments. Shown are (B, F) mean ± SD, (C–E, H) median, (B–E) unpaired t test (in B compared to Nlrc4–/–), (H) Mann-Whitney test, ∗p < 0.01, ∗∗p < 0.005, ∗∗∗p < 0.001. Please see also Figure S1. Immunity 2017 46, 649-659DOI: (10.1016/j.immuni.2017.03.016) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 Cell-Autonomous Expulsion and Lytic Death of IECs (A) Wild-type small intestinal organoid treated with 16 μg/mL of PA and 1 μg/mL LFn-Fla166 (top) or 100 ng/mL TNF-α (bottom) and stained with propidium iodide. (B) Blinded quantification of C57BL/6 small intestinal organoid cells positive for propidium iodide before expulsion in Fla166Tox versus TNF-α treatment as in (A). n = 5–6 movies/ treatment; mean ± SEM. (C and D) Propidium iodide and fluorescent phalloidin (actin) (C) or propidium iodide and GFP (D) in small intestines from mice treated with 0.2 μg/g of PA and 0.1 μg/g LFn-Fla for 60 min. Arrows indicate expulsing cells. Scale bars represent 40 (C) and 20 (D) μm. (E) Quantification of GFP signal in propidium iodide-positive and neighboring negative cells from samples as in (D) (n = 3; mean). (F) Quantification of actin purse strings in propidium iodide-positive cells from samples as in (C) (n = 7; mean ± SD). (G) Wild-type small intestinal organoid treated with 16 μg/mL of PA, 1 μg/mL LFn-Fla166, and 2.5 μM cytochalasin D and stained with propidium iodide. Data representative of at least two independent experiments, unpaired t test, ∗∗p < 0.005, ∗∗∗p < 0.001. Please see also Figure S2. Immunity 2017 46, 649-659DOI: (10.1016/j.immuni.2017.03.016) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 Caspase-1 Is Required for IEC Pyroptosis but Not IEC Expulsion (A) Propidium iodide and fluorescent phalloidin (actin) in small intestines from mice treated with 0.8 μg/g PA and 0.4 μg/g LFn-Fla for 60 min. Arrows indicate expulsing cells. Scale bars represent 20 μm. (B) Blinded quantification of propidium iodide-positive and -negative cells with actin purse strings in samples as in (A) (n = 3). (C) Mice were treated as in (A) and monitored for body temperature (n = 3). (D) Casp1–/– small intestinal organoid treated with 16 μg/mL of PA and 1 μg/mL LFn-Fla166 and stained with propidium iodide. Data representative of at least two independent experiments. Shown are (B, C) mean ± SD, (C) unpaired t test compared to Casp1–/–Casp11–/–, ∗∗∗p < 0.001. Please see also Figure S3. Immunity 2017 46, 649-659DOI: (10.1016/j.immuni.2017.03.016) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 Gasdermin D Is Required for IEC Pyroptosis but Not IEC Expulsion (A) Propidium iodide and fluorescent phalloidin (actin) in small intestines from mice treated with 0.8 μg/g PA and 0.4 μg/g LFn-Fla for 60 min. Arrows indicate expulsing cells. Scale bars represent 20 μm. (B) Blinded quantification of propidium iodide-positive and -negative cells with actin purse strings in samples as in (A) (n = 3). (C) Mice were injected with 0.8 μg/g PA and 0.4 μg/g LFn-Fla and monitored for body temperature (n = 3). (D) H&E staining of small intestinal tissue from mice treated as in (A), 90 min time point. Scale bars represent 100 μm. Data representative of at least two independent experiments. Shown are (B, C) mean ± SD, (C) unpaired t test compared to Nlrc4–/–,∗∗p < 0.005, ∗∗∗p < 0.001. Please see also Figure S4. Immunity 2017 46, 649-659DOI: (10.1016/j.immuni.2017.03.016) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 5 ASC Is Required for the Caspase1-Independent NLRC4 Signaling (A and B) Mice were injected with 0.8 μg/g PA and 0.4 μg/g LFn-Fla and monitored for (A) body temperature and (B) hematocrit (90 min, n = 3). (C) H&E staining of small intestinal tissue from mice treated as in (A), 90 min time point. Scale bars represent 100 μm. Data representative of two independent experiments. Shown are (A) mean ± SD, (B) median. Unpaired t test (in A compared to Nlrc4–/–), ∗∗p < 0.005, ∗∗∗p < 0.001. Please see also Figure S3. Immunity 2017 46, 649-659DOI: (10.1016/j.immuni.2017.03.016) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 6 A Caspase-8 Inflammasome Compensates for Loss of Caspase-1 (A) Mice were injected with 0.8 μg/g PA and 0.4 μg/g LFn-Fla and monitored for body temperature (n = 3), mean ± SD. (B) H&E staining of small intestinal tissue from mice treated as in (A), 90 min time point. Scale bars represent 100 μm. (C) PGE2 amounts of intestinal tissue from mice treated as in (A) for 30 min were determined (n = 4/group), median. Data representative of two independent experiments. (D) CFU in cecum 18 hr after oral S. Typhimurium infection (n = 10), combined data of two independent experiments, median. Shown are (A, C) unpaired t test (in A compared to Ripk3–/–Casp1–/–Casp8–/–), (D) Mann-Whitney test, ∗p < 0.01, ∗∗p < 0.005, ∗∗∗p < 0.001. Please see also Figures S5 and S6. Immunity 2017 46, 649-659DOI: (10.1016/j.immuni.2017.03.016) Copyright © 2017 Elsevier Inc. Terms and Conditions