Caspase-8 and FADD prevent spontaneous ZBP1 expression and necroptosis

Abstract

The absence of Caspase-8 or its adapter, Fas-associated death domain (FADD), results in activation of receptor interacting protein kinase-3 (RIPK3)- and mixed-lineage kinase-like (MLKL)-dependent necroptosis in vivo. Here, we show that spontaneous activation of RIPK3, phosphorylation of MLKL, and necroptosis in Caspase-8- or FADD-deficient cells was dependent on the nucleic acid sensor, Z-DNA binding protein-1 (ZBP1). We genetically engineered a mouse model by a single insertion of FLAG tag onto the N terminus of endogenous MLKL (Mlkl^FLAG/FLAG), creating an inactive form of MLKL that permits monitoring of phosphorylated MLKL without activating necroptotic cell death. Casp8^-/-Mlkl^FLAG/FLAG mice were viable and displayed phosphorylated MLKL in a variety of tissues, together with dramatically increased expression of ZBP1 compared to Casp8^+/+ mice. Studies in vitro revealed an increased expression of ZBP1 in cells lacking FADD or Caspase-8, which was suppressed by reconstitution of Caspase-8 or FADD. Ablation of ZBP1 in Casp8^-/-Mlkl^FLAG/FLAG mice suppressed spontaneous MLKL phosphorylation in vivo. ZBP1 expression and downstream activation of RIPK3 and MLKL in cells lacking Caspase-8 or FADD relied on a positive feedback mechanism requiring the nucleic acid sensors cyclic GMP-AMP synthase (cGAS), stimulator of interferon genes (STING), and TBK1 signaling pathways. Our study identifies a molecular mechanism whereby Caspase-8 and FADD suppress spontaneous necroptotic cell death.

Keywords: MLKL; ZBP1; caspase-8; interferon; necroptosis.

Fig. 1.

Fadd or Casp8 absence promotes constitutive RIPK3-MLKL pathway activity. (A) Fadd^−/−Mlkl^−/− or Casp8^−/−Mlkl^−/− MEF were reconstituted with empty vector (CTRL), FADD, or Caspase-8 (Casp8), respectively. Cell lysates were collected and phosphorylation of RIPK1, RIPK3, and all reconstituted components were assessed by immunoblotting. (B) Cells as shown in A were reconstituted with Dox-inducible MLKL-FLAG (C terminal; iMLKL-FLAG) and incubated with Dox (1 μg/mL) in the absence or presence of GSK′872 (1 μM) or TNF (10 ng/mL) plus zVAD (50 μM). Kinetics of cell death was monitored by propidium iodide (PI⁺ %) uptake using an Incucyte Kinetic Live Cell Imager. (C) Cells as shown in A were reconstituted with Dox-inducible FLAG-MLKL (N terminal). Cell lysates were analyzed by immunoblotting using indicated antibodies. (D) Expected Mendelian ratios and observed numbers of offspring from Mlkl^FLAG/FLAGCasp8^+/− intercrosses. All observed genotypes reached adulthood. (E) Tissues from 4- to 6-wk-old Mlkl^FLAG/FLAGCasp8^+/+ or Mlkl^FLAG/FLAGCasp8^−/− mice were examined by immunoblotting using indicated antibodies. (F) Immunofluorescence analysis of phospho-MLKL (pMLKL) from cryopreserved lung sections from animals as shown in E. (Scale bars, 10 μm.)

Fig. 2.

Fadd or Casp8 absence results in increased Zbp1 expression. (A) RNA-seq analysis from Fadd^−/−Mlkl^−/− or Casp8^−/−Mlkl^−/− MEF reconstituted with empty vector (CTRL), FADD, or Caspase-8 (Casp8), respectively. The red bold dot is Zbp1. (B) Detection of Zbp1 mRNA by qRT-PCR from cells as described in A. Zbp1 expression was normalized using Gapdh mRNA levels and standardized against control (CTRL). (C) Cell lysates from cells described in A, (D) immortalized WT, Ripk3^−/−, or Fadd^−/−Ripk3^−/− MEF, and (E) ZBP1 expression in liver, kidney, lung, or heart tissues from Casp8^+/−Ripk3^−/− intercrosses. Casp8^+/+(C8^+/+); Casp8^+/−(C8^+/−) or Casp8^−/−(C8^−/−), respectively. (F) ZBP1 expression in kidney, liver, heart, or lung tissues from Mlkl^FLAG/FLAGCasp8^+/+ or Mlkl^FLAG/FLAGCasp8^−/− mice was examined by immunoblotting. Numbers above panels (1 to 3) denote individual animals. (G) Primary Ripk3^−/−, Ripk1^−/− Ripk3^−/−, Casp8^−/−Ripk3^−/−, Ripk1^−/− Ripk3^−/−Casp8^−/−, and Fadd^−/−Mlkl^−/− MEF were analyzed by immunoblotting using the indicated antibodies. (H) Immunoblotting in lung tissues from Mlkl^FLAG/FLAGZbp1^+/+Casp8^+/+, Mlkl^FLAG/FLAGZbp1^+/+Casp8^−/−, Mlkl^FLAG/FLAGZbp1^−/−Casp8^+/+, and Mlkl^FLAG/FLAGZbp1^−/−Casp8^−/− mice (Left). Densitometric quantification of pMLKL/MLKL ratios from three separate animals (numbers 1 to 3) (Right). Significance in B was calculated with unpaired Student’s t test or (H) two-way ANOVA analysis including a Tukey’s multiple comparison test, ***P < 0.001.

Fig. 3.

ZBP1 nucleic acid sensing is required to activate RIPK3 in the absence of FADD. (A–E) Fadd^−/−Mlkl^−/− or Fadd^−/−Mlkl^−/− Zbp1^−/− MEF were reconstituted with iMLKL-FLAG (C terminal). (A) Fadd^−/−Mlkl^−/− MEF reconstituted with empty vector or FADD, and Fadd^−/−Mlkl^−/−Zbp1^−/− MEF reconstituted with ZBP1. Immunoblot analysis of pRIPK1 or pRIPK3 (Left). Densitometric quantification of pRIPK3 (Upper Right) and pRIPK1 (Lower Right). Ratios (pRIPKs/RIPKs) were normalized to Fadd^−/−Mlkl^−/− cells, which was set at 100%. (B) Structure of mouse ZBP1 wild-type or mutants (WT, RHIM mutant [mutRHIM], Zα2 mutant [mutZα2], and deleted Zα1/2 domains [ΔZα1/2]). (C) Immunoblot analysis of reconstituted ZBP1 constructs shown in B in Fadd^−/−Mlkl^−/−Zbp1^−/−MEF. (D) Cells described in C were incubated with Dox (1 μg/mL) or in combination with GSK′872 (1 μM). Kinetics of cell death was monitored with Sytoxgreen (Sytoxgreen⁺, %) uptake. (E) Immunoprecipitation of FLAG-ZBP1 in MEF. Immunoblot of pRIPK3, RIPK3, and RIPK1 coprecipitates. Representation of two (C and E) or three independent experiments (A and D).

Fig. 4.

Cells lacking FADD or Caspase-8 contain constitutive expression of Z-RNA. (A) Fadd^−/−Mlkl^−/− or (B) Casp8^−/−Mlkl^−/− MEF were reconstituted with empty vector (CTRL), FADD, or Caspase-8 (Casp8), respectively. Representative images of Z-RNA (red) or A-RNA (green) for Fadd^−/−Mlkl^−/− (A) and Casp8^−/−Mlkl^−/−(B). Columns are quantification of MFI compiled from three independent experiments. Dots represent individual cells (SI Appendix, SI Materials and Methods). Significance was calculated with two-way ANOVA analysis including a Tukey’s multiple comparison test, ***P < 0.001.

Fig. 5.

The absence of FADD or Caspase-8 promotes a positive feedback loop involving the cGAS-STING and TBK1 pathways inducing ZBP1 expression. (A) Fadd^−/−Mlkl^−/− MEF expressing empty vector (CTRL) or FADD, and Fadd^−/−Mlkl^−/−Zbp1^−/−MEF expressing WT, or ΔZα1/2-ZBP1 were harvested under untreated conditions. Cell lysates were examined by immunoblotting analysis using the indicated antibodies. (B) Fadd^−/−Mlkl^−/− MEF were transiently transfected with the indicated siRNA. Forty-eight hours after transfection, cell lysates were immunoblotted for ZBP1 and actin as loading control. (C) Immunoprecipitation of FLAG-ZBP1 in Zbp1^−/− MEF reconstituted with empty vector (CTRL) or a Dox-inducible FLAG-ZBP1 (used as internal control), and Casp8^−/−Mlkl^−/− MEF expressing empty vector (CTRL) or Casp8. Endogenous coprecipitated proteins were assessed by immunoblotting with the indicated antibodies. (D and E) Fadd^−/−Mlkl^−/− or Casp8^−/−Mlkl^−/− MEF reconstituted with empty vector (CTRL), FADD, or Casp8, respectively, were harvested for liquid-chromatography/mass spectrometry quantification of cGAMP (SI Appendix, SI Materials and Methods). (D) Immunoblots of cells in (E, Left). (E) Dots represent cGAMP peak relative to its respective control cells (SI Appendix, SI Materials and Methods). Compiled from three independent experiments. (F) Quantification of cell death and (G) immunoblot analysis of endogenous components in Casp8^−/−Mlkl^−/− or Cgas^−/−Casp8^−/−Mlkl^−/− MEF versus reconstituted cells with control (CTRL), Casp8, or GFP-cGAS, respectively.