Volume 38, Issue 1, Pages 27-40 (January 2013) Caspase-8 Blocks Kinase RIPK3-Mediated Activation of the NLRP3 Inflammasome  Tae-Bong Kang, Seung-Hoon Yang, Beata Toth, Andrew Kovalenko, David Wallach  Immunity  Volume 38, Issue 1, Pages 27-40 (January 2013) DOI: 10.1016/j.immuni.2012.09.015 Copyright © 2013 Elsevier Inc. Terms and Conditions

Immunity 2013 38, 27-40DOI: (10.1016/j.immuni.2012.09.015) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 1 Caspase-8 Deficiency in Dendritic Cells Facilitates LPS-Induced Increase in Serum IL-1β and Death in Mice in a Way that Depends on RIPK1 and RIPK3 Function (A–C) Survival of WT mice and of C8− mice after injection of LPS (5 mg/kg). (A) and (C) show comparison of the above to the lethal effect of LPS on WT mice injected with LPS (5 μg/kg) together with D-galactosamine (GalN, 800 mg/kg). (A) shows comparison of the effects of IL-1RA (25 mg/kg) and of TNF antibody (50 μg) on survival. (C) shows comparison of the histology (H&E staining, top) of livers in C8− and WT mice at the time of death and of the extents of apoptotic cell death (arrows) in them (TUNEL staining, bottom; NT, not treated). In all histological images, the scale bars correspond to 200 μM. (B) shows the effect of Nec1 (6.25 mg/kg) and of ubiquitous deficiency in RIPK3. (D–K) Serum IL-1β and TNF levels after LPS injection in mice of the indicated genotypes, and the effects of IL-1RA (F and G) and of Nec1 (H and I) injection on them. Unless otherwise indicated, sera were collected 3 hr after LPS injection. All data presented in this article are representative results of at least three independent experiments. In all diagrams, values correspond to mean values and error bars show SD. Each of the bars in (F)–(K) corresponds to the mean value in the sera obtained from three mice. Immunity 2013 38, 27-40DOI: (10.1016/j.immuni.2012.09.015) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 2 Impact of Caspase-8 Deficiency in Dendritic Cells on Leukocyte Levels and its Modulation by RIPK3 (A–G) Experiments in WT and C8− mice (10 weeks old) expressing RIPK3 as follows: (A) spleen weight; (B) spleen histology (H&E staining), showing conservation of normal spleen architecture in the C8− spleen; WP, white pulp; RP, red pulp; (C) spleen cell counts. (D–G) FACS analysis of various cell types as follows: (D) stromal cells (CD45−); (E) relative proportions of erythrocytes (Ter119+); (F) granulocytes (Ly6G+); (G) myeloid cells expressing CD11b and Gr1. (H–L) Experiments in Ripk3−/− and C8− Ripk3−/− mice (10 and 20 weeks old) as follows: (H) spleen weight; (I) spleen histology at 20 weeks, showing distortion of the parenchymal architecture due to marked expansion of the white pulp as a result of accumulation of less heavily stained cells (blasts); (J) total spleen cell count. (K) CD3+ B220+ cells in the spleen; (L) representative FACS cytogram of CD3 and B220 expression pattern in splenocytes and lymph node cells at 20 weeks. No consistent change in amount of any other leukocyte type could be observed in the C8− Ripk3−/− mice. Immunity 2013 38, 27-40DOI: (10.1016/j.immuni.2012.09.015) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 3 Caspase-8-Deficient Dendritic Cells Secrete IL-1β and IL-18 in Response to Treatment with Priming Agents Only, in a RIPK1- and RIPK3-Dependent Manner (A–E) Secretion of the indicated cytokines by DCs derived in vitro from bone-marrow progenitor cells of WT and C8− mice in response either to LPS or to LPS + ATP. (F) Knockdown of caspase-8 in WT DCs. Left shows the effect of the knockdown on generation of IL-1β in response to LPS; right shows the expression of caspase-8 mRNA in the knocked-down cells. (G) Secretion of IL-1β in response to the indicated inducers (which in this experiment were applied for 6 hr) and the effect of added Nec1. (H and I) Effect of Nec1 on secretion of IL-1β in response to LPS + ATP and on secretion of TNF in response to LPS. (J) Secretion of IL-1β by DCs derived from the indicated mouse strains in response to treatment with LPS. Unless otherwise indicated, in all ex-vivo experiments LPS was applied for 3 hr at 1 μg/ml and ATP (5 mM) was applied for the last 15 min of incubation. All ex-vivo assessments were done in triplicate wells. Immunity 2013 38, 27-40DOI: (10.1016/j.immuni.2012.09.015) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 4 Caspase-8 Deficiency in Dendritic Cells Facilitates IL-1β Generation by Promoting LPS-Induced Activation of the Inflammasome (A–D) Cellular expression levels of IL-1β mRNA (A) and of the indicated proteins (B and C) and expression levels of the mature form of IL-1β in the culture media (D) of WT and C8 DCs stimulated with LPS or LPS +ATP. (WB, protein immunoblot.) (E and F) Effect of knockdown of NLRP3 or ASC on generation of IL-1β in response to stimulation of WT and C8− DCs by LPS (E) or LPS+ATP (F). (G) Levels of NLRP3 and ASC in the knocked-down cells. (H) Protein immunoblot depicting effects of LPS, ATP, and Nec1 on NP40 solubility of the inflammasome components in WT and C8− DCs. (I) Protein immunoblot depicting association of the inflammasome components within the NP40 insoluble residue, assessed by application of a reversible crosslinking agent to it, followed by solubilization in SDS and immunoprecipitation, as described in Experimental Procedures (HC, heavy chain). Immunity 2013 38, 27-40DOI: (10.1016/j.immuni.2012.09.015) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 5 Enhancement of Inflammasome Activation by Caspase-8 Deficiency Does Not Reflect Increased Necrotic Cell Death or Generation of Extracellular Activating Molecules (A–D) Kinetics of (A and C) the secretion of IL-1β by WT and C8− DCs and (B and D) death of these cells (assessed by measuring LDH release, staining of the cells with Topro-3 [50 nm] and determining their loss of mitochondrial membrane potential) in response to LPS, LPS + the pan-caspase inhibitor z-VAD (20 μM), TNF (1,000 IU/ml) combined with the SMAC mimetic compound BV6 (1 μM) or TNF combined with BV6 and z-VAD. BV6 was applied 1 hr and z-VAD 15 min prior to initiation of the experiment, and both remained present, together with TNF or LPS, for the indicated periods. (E) Inflammasome assembly in response to the various inducers applied in the experiments of panels (A)–(D). Amounts of the adaptor protein ASC incorporated into a detergent-insoluble compartment (middle lane), and the extent of its association within this compartment with the NLRP3 inflammasome (top lane), in response to treatment for the specified durations with the indicated agents, were assessed as described for Figures 4H and 4I. (F) Generation of reactive oxygen species (ROS) by WT and C8− DCs in response to treatment for 3 hr with the inducing agents applied in the experiments depicted in (A)–(D). Rotenone (10 μM) was applied as a positive control. Left shows results of H2DCFDA flow cytometry assay. Right shows percentage of cells with elevated fluorescence intensity (over the range defined by the horizontal bars in the left panels). (G) LPS-induced secretion of IL-1β by WT and C8− DCs incubated in culture media containing lysates of either of those cells. Lysates (3 × 105 cells) were applied to 1 × 106 cells in a final volume of 1 ml. (H) Permeability to the fluorescent dye ethidium bromide, assessed in WT (red line) and C8− (blue line) DCs treated for the indicated time periods with LPS and in WT cells incubated for 15 min with and without ATP (green and magenta lines, respectively). (I) Release of ATP from WT and C8− DCs during their incubation for 3 hr with LPS or without it (NT) in the presence of the ecto-ATPase inhibitor ARL 67156 (200 μM, as in Piccini et al. [2008]). (J) Secretion of IL-1β by 1 × 106 WT or C8− DCs in culture, or by a mixture of 5 × 105 cells from each of the two (WT/C8−), with (black bars) and without (gray bars, too small to be visible) stimulation for 3 hr by LPS. Immunity 2013 38, 27-40DOI: (10.1016/j.immuni.2012.09.015) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 6 Signaling Mechanisms Mediating Activation of the Inflammasome in LPS-Treated Dendritic Cells (A–D, H, and I) Coimmunoprecipitation of the indicated proteins from detergent extracts of WT and C8− DCs (in C, D and H – just WT cells) treated with LPS or TNF (1,000 IU/ml) for the indicated periods. BV6 and z-VAD were applied as in Figure 5. The samples analyzed in (H) are identical to those in (C), and those analyzed in (I) are identical to those in (A). (LC, light chain.) (E–G) Effects of knockdown of MLKL or PGAM5 in WT and C8− DCs on (E) death of cells in response to their treatment for 16 hr with LPS or LPS+z-VAD, and (F) secretion of IL-1β in response to their treatment for 3 hr with LPS. (G) Levels of MLKL and PGAM5 mRNA in the knocked-down cells. Immunity 2013 38, 27-40DOI: (10.1016/j.immuni.2012.09.015) Copyright © 2013 Elsevier Inc. Terms and Conditions