Caspase-8 Acts in a Non-enzymatic Role as a Scaffold for Assembly of a Pro- inflammatory “FADDosome” Complex upon TRAIL Stimulation  Conor M. Henry, Seamus J. Martin  Molecular Cell  Volume 65, Issue 4, Pages 715-729.e5 (February 2017) DOI: 10.1016/j.molcel.2017.01.022 Copyright © 2017 Elsevier Inc. Terms and Conditions

Molecular Cell 2017 65, 715-729.e5DOI: (10.1016/j.molcel.2017.01.022) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 TRAIL-Induced Inflammatory Cytokine Production Is a General Property of TRAIL-Receptor Engagement (A) HeLa cells were stimulated with the indicated concentrations of TRAIL for 24 hr. Apoptosis was assessed based on cell morphology on counts of triplicate fields of 100 cells (300 cells per well). The cytokine concentrations in cell culture supernatants were determined by ELISA assay. (B) HeLa cells were stimulated with TRAIL (100 ng/mL) for 16 hr, followed by visualization by phase contrast microscopy. (C) Representative confocal images of HeLa cells treated with TRAIL (100 ng/mL) for 16 hr and immunostained for the indicated cytokines and chemokines. The nuclei were stained with Hoechst 33342 (20 μM). (D–G) PancTu-1 cells (D), HCT116 cells (E), HaCat cells (F), and mouse 3LL cells (G) were stimulated with TRAIL for 24 hr, followed by assessment of apoptosis and chemokine production as described in (A). The results shown are representative of at least three independent experiments. The error bars represent the mean ± SEM of triplicate determinations from representative experiments. See also Figure S1. Molecular Cell 2017 65, 715-729.e5DOI: (10.1016/j.molcel.2017.01.022) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 TRAIL Stimulation Induces Physiologically Relevant Quantities of Chemokines that Promote Chemotaxis of Monocytes and Neutrophils (A) HeLa cells were either left untreated or were treated with TRAIL (100 ng/mL) in RPMI, supplemented with 0.5% FCS for 24 hr. The chemotaxis assays using THP-1 cells were performed using the indicated dilutions of supernatant (S/N) from TRAIL-stimulated cells. ATP (500 nM) serves as a positive control for THP-1 chemotaxis. (B) CFSE-labeled THP-1 cells that had migrated into the bottom wells of the chemotaxis chamber from the experiment depicted in (A) were photographed under UV microscopy. (C) HeLa cells were either left untreated or were treated with the indicated concentrations of TRAIL for 24 hr. The culture supernatants were then used for chemotaxis assays with THP-1 cells as outlined in (A). (D) HeLa cells were left untreated or were treated with TRAIL (100 ng/mL) in RPMI, supplemented with 0.5% FCS for 24 hr. The chemotaxis assays using neutrophils were performed using the indicated dilutions of supernatant (S/N) from TRAIL-stimulated cells. IL-8 (100 ng/mL) serves as a positive control for THP-1 chemotaxis. (E) HeLa cells were either untreated or were treated with TRAIL (100 ng/mL) in RPMI, supplemented with 0.5% FCS over the indicated time points. Apoptosis was scored at indicated periods of time by cell morphology. The cytokine concentrations in culture supernatants were determined by ELISA. The culture supernatants were then used for chemotaxis assays with THP-1 cells as outlined in (A). (F) HeLa cells were treated as outlined in (A). The resulting supernatants were immunodepleted of the indicated combinations of cytokines, followed by the assessment of their ability to promote chemotaxis of THP-1 cells. (G) HeLa cells were treated as outlined in (A). The supernatants were immunodepleted as outlined in (F), followed by the assessment of their ability to promote chemotaxis of primary human neutrophils. The results shown are representative of at least three independent experiments. The error bars represent the mean ± SEM of triplicate cell counts from a representative experiment. The significance levels are ∗∗∗ = p < 0.0001, ∗∗ = p < 0.001, and ∗ = p < 0.01, by Student’s t test. See also Figure S2. Molecular Cell 2017 65, 715-729.e5DOI: (10.1016/j.molcel.2017.01.022) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 TRAIL-Induced Inflammatory Responses Can Be Uncoupled from Cell Death (A) HeLa cells were stimulated with TRAIL (50 ng/mL) in the presence or absence of z-VAD-FMK (10 μM) for 6 hr. The cells were visualized by phase contrast microscopy. (B) HeLa cells were treated with indicated concentrations of TRAIL in the presence or absence of z-VAD-FMK (10 μM) for 24 hr. The proteins were analyzed by western blot. Apoptosis was scored based on cell morphology. (C) HeLa cells were stimulated with TRAIL at the indicated concentrations in the presence or absence of z-VAD-FMK (10 μM) for 24 hr. Apoptosis was scored based on cell morphology. The cytokine concentrations in the culture supernatants were determined by ELISA. (D) HT-29 cells were stimulated with TRAIL (50 ng/mL) for 24 hr. The cells were visualized by phase contrast microscopy. (E) HT-29 cells were stimulated with TRAIL at the indicated concentrations for 24 hr. Apoptosis was scored based on cell morphology. The data represent triplicate counts of a minimum of 300 cells per treatment. The cytokine concentrations in the culture supernatants were determined by ELISA. (F) Representative confocal images of HT-29 cells treated with TRAIL (50 ng/mL) for 24 hr and immunostained for IL-8 and counterstained nuclei with Hoechst 33342 (20 μM). (G) HT-29 cells were treated with indicated concentrations of TRAIL in the presence or absence of z-VAD-FMK or Q-VD-Oph (10 μM) for 24 hr. The proteins were analyzed by western blot. (H) Apoptosis was scored based on cell morphology. The cytokine concentrations in the culture supernatants were determined by ELISA. The results shown are representative of at least three independent experiments. The error bars represent the mean ± SEM of triplicate determinations from a representative experiment. See also Figure S3. Molecular Cell 2017 65, 715-729.e5DOI: (10.1016/j.molcel.2017.01.022) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 TRAIL-Induced Cytokine Production Is Regulated via FADD, RIPK1, and Caspase-8 (A) HeLa cells were treated with TRAIL (50 ng/mL) over the indicated period of time in the presence or absence of z-VAD-FMK (10 μM). The proteins were analyzed by western blot. (B) HeLa cells were nucleofected with non-targeting control siRNA or siRNA targeted against TRADD, FADD, caspase-8, or RIPK1 for 48 hr. The knockdown efficiency was analyzed by western blotting. The cells were treated with TRAIL (12 ng/mL) and TNFα (1 ng/mL) for a further 24 hr. (C) Cytokine concentrations in the culture supernatants from (B) were determined by ELISA. (D and E) HeLa cells were nucleofected with non-targeting control siRNA or siRNA targeted against RIPK1, TAK1, NEMO, and p65 for 48 hr, followed by stimulation with TRAIL (12 ng/ml) or TNFα (1 ng/ml) for a further 24 hr. Knockdown efficiency was analyzed by western blot (D) and cytokine concentrations in the culture supernatants were determined by ELISA (E). (F) HeLa cells were treated with TRAIL (25 ng/mL) over 24 hr in the presence or absence of indicated doses of z-VAD-FMK. Apoptosis was scored based on Annexin V/PI staining by flow cytometry. Cytokine concentrations in the culture supernatants were determined by ELISA (top). HeLa cells were nucleofected with non-targeting control siRNA or siRNA targeted against caspase-8 for 48 hr and analyzed as described in (B) (lower). (G) HeLa cells were nucleofected with non-targeting control siRNA or siRNA targeted against caspase-8 for 48 hr. The cells were treated with TRAIL (50 ng/mL) for the indicated period of time. The proteins were analyzed by western blot. The results shown are representative of at least three independent experiments. The error bars represent the mean ± SEM of triplicate cell counts from a representative experiment. The significance levels are ∗∗∗ = p < 0.0001, ∗∗ = p < 0.001, and ∗ = p < 0.01, by Student’s t test. (H) Primary prostate epithelial cells were nucleofected with non-targeting control siRNA or siRNA targeted against FADD or caspase-8 for 48 hr. The knockdown efficiency was analyzed by western blotting. The cells were treated with the indicated concentrations of TRAIL for a further 24 hr. (I) Cytokine concentrations in the culture supernatants from (H) were determined by ELISA. Molecular Cell 2017 65, 715-729.e5DOI: (10.1016/j.molcel.2017.01.022) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 5 Caspase-8 Is Required as a Scaffold for TRAIL-Induced Inflammatory Signaling (A) HeLa and HeLa CASP8−/− cells were treated with TRAIL (50 ng/mL) over the indicated period of time. The proteins were analyzed by western blot. (B) HeLa and HeLa CASP8−/− cells were treated as in (A). Apoptosis was scored based on Annexin V/PI staining by flow cytometry, and cytokine concentrations in the culture supernatants were determined by ELISA. (C) HeLa and HeLa CASP8−/− cells were treated with indicated concentrations of TRAIL or TNF. After 24 hr, apoptosis was scored based on Annexin V/PI staining by flow cytometry, and cytokine concentrations in the culture supernatants were determined by ELISA. (D) HeLa and HeLa CASP8−/− cells were treated with TRAIL (50 ng/mL) and TNFα (10 ng/mL) over the indicated period of time. The proteins were analyzed by western blot. Apoptosis was scored based on Annexin V/PI staining by flow cytometry, and cytokine concentrations in the culture supernatants were determined by ELISA. (E) HeLa CASP8−/− cells were transfected with plasmids encoding empty vector (100 ng), caspase-8 (100 ng), caspase-8 F122G and L123G (100 ng), caspase-8 C360A (100 ng), or caspase-8 C360A, F122G, and L123G for 12 hr. The proteins levels of reconstituted cells were analyzed by western blot. HeLa were used as a positive control. (F) HeLa CASP8−/− cells were transfected with plasmids encoding eGFP (50 ng), caspase-8 (500 ng), and caspase-8 C360A (500 ng) for 12 hr in the presence or absence of z-VAD-FMK (10 μM). TRAIL (50 ng/mL) was added at 12 hr post-transfection. At the indicated period of time post-TRAIL treatment, cell death was counted among GFP-positive cells based on cell morphology, and cytokine concentrations in the culture supernatants were determined by ELISA. (G) HeLa CASP8−/− cells were transfected with plasmids encoding eGFP (50 ng), caspase-8 (100 ng), caspase-8 F122G and L123G (100 ng), caspase-8 C360A (100 ng), or caspase-8 C360A, F122G, and L123G for 12 hr in the presence or absence of z-VAD-FMK (10 μM). TRAIL (50 ng/mL) was added at 12 hr post-transfection. At the indicated period of time post-TRAIL treatment, cell death was counted among GFP-positive cells based on cell morphology, and cytokine concentrations in the culture supernatants were determined by ELISA. The results shown are representative of at least three independent experiments. The error bars represent the mean ± SEM of triplicate cell counts from a representative experiment. The significance levels are ∗∗∗ = p < 0.0001, ∗∗ = p < 0.001, and ∗ = p < 0.01, by Student’s t test. See also Figure S4. Molecular Cell 2017 65, 715-729.e5DOI: (10.1016/j.molcel.2017.01.022) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 6 TRAIL-R Engagement Assembles a Caspase-8-FADD-RIPK1 NFκB-Activating Complex (A) HeLa cells were treated with iz-TRAIL (1.5 μg/mL) for the indicated periods of time in the presence or absence of z-VAD-FMK (20 μM), followed by caspase-8 immunoprecipitation. The proteins were analyzed by western blot. (B) Schematic representation of immunoprecipitation strategies. (C) HeLa cells were treated with biotin-iz-TRAIL (1.5 μg/mL) for the indicated periods of time in the presence or absence of z-VAD-FMK (20 μM), followed by strepavidin-Sepharose immunoprecipitation. The proteins were analyzed by western blot. (D and E) (D) HeLa cells were nucleofected with non-targeting control siRNA or siRNA targeted against caspase-8, A20, and CYLD for 48 hr. The knockdown efficiency was analyzed by western blotting. (E) Cells silenced for expression of caspase-8, A20, or CYLD were then treated with TRAIL (12 ng/mL) or TNFα (1 ng/mL) for a further 24 hr. Apoptosis was scored based on Annexin V/PI staining by flow cytometry. The cytokine concentrations in the culture supernatants were determined by ELISA. The results shown are representative of at least three independent experiments. The error bars represent the mean ± SEM of triplicate cell counts from a representative experiment. See also Figure S5. Molecular Cell 2017 65, 715-729.e5DOI: (10.1016/j.molcel.2017.01.022) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 7 The Scaffold Function of Caspase-8 Is Critical for Recruitment of RIPK1 Downstream of TRAIL-R Engagement (A) HeLa cells were nucleofected with non-targeting control siRNA or siRNA targeted against FADD, caspase-8, caspase-10, and RIPK1 for 48 hr. The knockdown efficiency was analyzed by western blotting. The cells were treated with TRAIL (12 ng/mL) and TNFα (1 ng/mL) for a further 24 hr. The cytokine concentrations in the culture supernatants were determined by ELISA. (B) HeLa and HeLa CASP8−/− cells were treated with biotin-iz-TRAIL (1.5 μg/mL) for the indicated periods of time in the presence of z-VAD-FMK (20 μM), followed by strepavidin-Sepharose immunoprecipitation. The proteins were analyzed by western blot. (C) TRAIL-R signaling promotes formation of a FADDosome complex to promote NFκB activation and pro-inflammatory cytokine/chemokine production. The binding of TRAIL ligand to DR4/5 receptors leads to receptor trimerization and formation of the death-inducing signaling complex (DISC). FADD is recruited to the receptor complex via its death domain (DD), which in turn recruits caspase-8 via death-effector domains (DEDs), namely DED1. RIPK1 associates with the TRAIL-R through binding with FADD via mutual DD domains. The oligomerization of caspase-8 is facilitated through DED2, which recruits additional caspase-8 molecules to form a molecular scaffold. The oligomerized caspase-8 scaffold forms a soluble complex that stabilizes FADD and facilitates the recruitment of RIPK1. The ubiquitin linkages generated by the cIAPs then lead to the recruitment of LUBAC. Modified RIPK1 can recruit two protein complexes, the TAB/TAK complex and the IKK complexes, which leads to downstream pro-inflammatory gene activation and cytokine/chemokine production. (D) HeLa cells were nucleofected with non-targeting control siRNA or siRNA targeted against caspase-8 and caspase-10 for 48 hr. HeLa cells were then treated with biotin-iz-TRAIL (1.5 μg/mL) for the indicated periods of time in the presence of z-VAD-FMK (20 μM), followed by strepavidin-Sepharose immunoprecipitation. The proteins were analyzed by western blot. (E) HeLa cells were nucleofected with non-targeting control siRNA or siRNA targeted against caspase-8, caspase-10, and RIPK1 for 48 hr. HeLa cells were then treated with iz-TRAIL (1.5 μg/mL) for the indicated periods of time in the presence of z-VAD-FMK (20 μM), followed by FADD immunoprecipitation. The proteins were analyzed by western blot. The results shown are representative of at least three independent experiments. The error bars represent the mean ± SEM of triplicate cell counts from a representative experiment. The significance levels are ∗∗∗ = p < 0.0001, ∗∗ = p < 0.001, and ∗ = p < 0.01, by Student’s t test. See also Figure S6. Molecular Cell 2017 65, 715-729.e5DOI: (10.1016/j.molcel.2017.01.022) Copyright © 2017 Elsevier Inc. Terms and Conditions