Pan-Caspase Inhibitor zVAD Induces Necroptotic and Autophagic Cell Death in TLR3/4-Stimulated Macrophages

Abstract

In addition to inducing apoptosis, caspase inhibition contributes to necroptosis and/or autophagy depending on the cell type and cellular context. In macrophages, necroptosis can be induced by co-treatment with Toll-like receptor (TLR) ligands (lipopolysaccharide [LPS] for TLR4 and polyinosinic-polycytidylic acid [poly I:C] for TLR3) and a cell-permeable pan-caspase inhibitor zVAD. Here, we elucidated the signaling pathways and molecular mechanisms of cell death. We showed that LPS/zVAD- and poly I:C/zVAD-induced cell death in bone marrow-derived macrophages (BMDMs) was inhibited by receptor-interacting protein kinase 1 (RIP1) inhibitor necrostatin-1 and autophagy inhibitor 3-methyladenine. Electron microscopic images displayed autophagosome/autolysosomes, and immunoblotting data revealed increased LC3II expression. Although zVAD did not affect LPS- or poly I:C-induced activation of IKK, JNK, and p38, it enhanced IRF3 and STAT1 activation as well as type I interferon (IFN) expression. In addition, zVAD inhibited ERK and Akt phosphorylation induced by LPS and poly I:C. Of note, zVAD-induced enhancement of the IRF3/IFN/STAT1 axis was abolished by necrostatin-1, while zVAD-induced inhibition of ERK and Akt was not. Our data further support the involvement of autocrine IFNs action in reactive oxygen species (ROS)-dependent necroptosis, LPS/zVAD-elicited ROS production was inhibited by necrostatin-1, neutralizing antibody of IFN receptor (IFNR) and JAK inhibitor AZD1480. Accordingly, both cell death and ROS production induced by TLR ligands plus zVAD were abrogated in STAT1 knockout macrophages. We conclude that enhanced TRIF-RIP1-dependent autocrine action of IFNβ, rather than inhibition of ERK or Akt, is involved in TLRs/zVAD-induced autophagic and necroptotic cell death via the JAK/STAT1/ROS pathway.

Keywords: JAK/STAT1; autophagy; interferon; macrophage; necrosis; zVAD.

Fig. 1. zVAD co-treatment with TLR3 and TLR4 ligands induces the RIP1-dependent necroptosis in BMDMs.

BMDMs were pre-treated with vehicle (veh) or necrostatin-1 (Nec-1) (10 µM) for 30 min, followed by zVAD (20 µM for most experiments or at the indicated concentrations) for another 30 min. Cells were then stimulated with LPS (1 µg/ml) or poly I:C (20 µg/ml) for 24 h, and cell viability was assessed by MTT assay (A) or PI uptake (B). Data were presented as the mean ± SEM from at least three independent experiments. *P < 0.05, indicating significant cytotoxic effects. ^#P < 0.05, indicating a significant effect of Nec-1 in inhibiting cytotoxicity induced by zVAD/TLR ligands.

Fig. 2. zVAD/LPS and zVAD/poly I:C induce autophagic cell death in BMDMs.

(A and B) BMDMs were pretreated with zVAD (20 µM) for 30 min prior to stimulation with LPS (1 µg/ml) or poly I:C (20 µg/ml) for different periods. The total cell lysates were subjected to SDS-PAGE and immunoblotting with LC3 antibody (A). In electron micrographs, BMDMs were conducted with 8 h drug treatment, either LPS/zVAD or poly I:C/zVAD, compared to control group. The white arrows indicate autolysosome, and the black arrow indicates the double membrane components, autophagosome. “N” indicates nuclei, “M” indicates mitochondria and the scale bar is 500 nm (B). (C) BMDMs were pretreated with zVAD (20 µM), 3-MA (1 mM), and/or bafilomycin A1 (100 nM) for 30 min, followed by the stimulation with LPS (1 µg/ml) or poly I:C (20 µg/ml). After treatment for 24 h, the cell viability was assessed by MTT assay. veh, vehicle. Data were the mean ± SEM from at least three independent experiments. *P < 0.05, indicating significant cytotoxic effects. ^#P < 0.05, indicating significant effect of 3-MA and bafilomycin A1 to inhibit cytotoxicity induced by zVAD/TLR ligands.

Fig. 3. zVAD differentially affects LPS- and poly I:C-induced signaling pathways: Inhibition of ERK and Akt, and enhancement of IRF3-STAT1 pathway, but no effects on IKK, JNK, and p38.

BMDMs were pretreated with zVAD (20 µM) for 30 min prior to stimulation with LPS (1 µg/ml) (A and C) or poly I:C (20 µg/ml) (B and D). After incubation for different periods as indicated, the total cell lysates were subjected to SDS-PAGE followed by immunoblotting for JNK, p38, IKK, ERK, Akt (A and B), IRF3, and STAT1 (C and D). The results were representative traces that were repeated in three independent experiments.

Fig. 4. Effects of zVAD on LPS- and poly I:C-induced inflammatory gene expression and phagocytosis.

(A-C) BMDMs were pretreated with zVAD (20 µM) prior to treatment with LPS (1 µg/ml) or poly I:C (20 µg/ml) for 6 h. Real time-PCR was conducted to measure gene expression. (D) Cells were treated with agents as mentioned above for 4 h and 8 h. E. coli bioparticles were treated at the last one hour. Phagocytosis was determined by flow cytometry. Data were the mean ± SEM from at least three independent experiments. *P < 0.05, indicating significant effects of TLR ligands. ^#P < 0.05, indicating significant effect of zVAD to inhibit the effects of TLR ligands.

Fig. 5. RIP1 positively regulates IFN expression and STAT1 signaling.

(A) BMDMs were pretreated with zVAD (20 µM) prior to treatment with LPS (1 µg/ml) or poly I:C (20 µg/ml) for 5 or 8 h. After incubation soluble IFNβ in culture medium was measured with commercial mouse IFNβ ELISA kit. (B) BMDMs were pretreated with zVAD (20 µM) either in the absence or presence of Nec-1 (10 µM) or 3-MA (1 mM) prior to the treatment with LPS (1 µg/ml) or poly I:C (20 µg/ml). After incubation for 3 h, mRNA was extracted and reversely transcribed for qPCR analyses of IFNβ and IFNα gene expression. Values were normalized to β-actin gene expression and expressed relative to the control group. Data were the mean ± SEM from at least three independent experiments. *P < 0.05, indicating significant enhancement of IFNs expression in the presence of zVAD. ^#P < 0.05, indicating significant effects of 3-MA and Nec-1 to inhibit IFNs expression induced by zVAD/TLR ligands. (C) Similar procedure as (B) was applied, and STAT1 phosphorylation at different time points was determined by immunobloting.

Fig. 6. IFNβ mediates cell necroptosis under LPS/zVAD and poly I:C/zVAD treatment.

(A-C) BMDMs were pretreated with zVAD (20 µM), AZD1480 (1 µM), IFNR-Ab (1 µg/ml) or Embrel (10 ng/ml) for 30 min and stimulated with LPS (1 µg/ml) or poly I:C (20 µg/ml). After incubation for 24 h, cell viability was determined by PI uptake (A) and MTT assay (B). In some experiments, STAT1 phosphorylation was determined by immunobloting at different periods (C). (D) The wild type and STAT1 knockout BMDMs were pretreated with zVAD (20 µM) for 30 min prior to LPS (1 µg/ml) or poly I:C (20 µg/ml) stimulation. After 24 h incubation, the cell viability was assessed by MTT assay. (E) BMDMs were pretreated with zVAD (20 µM) for 30 min prior to IFNβ (10 ng/ml) stimulation. Cell viability was determined by MTT assay after 24 h incubation (upper panel), and STAT1 activation status was determined at different periods of time as indicated (lower panel). veh, vehicle. Data were the mean ± SEM from at least three independent experiments. *P < 0.05, indicating significant effects of zVAD/TLR ligands on cell viability. ^#P < 0.05, indicating significant reversal effects of AZD1480, IFNR-Ab and STAT1 KO on cytotoxicity induced by zVAD/TLR ligands.

Fig. 7. ERK and Akt inhibition are not mediated by RIP1 and not involved in necroptosis.

(A) BMDMs were pretreated with zVAD (20 µM) in the absence or presence of 3-MA (1 mM) or Nec-1 (10 µM) for 30 min and then treated with poly I:C (20 µg/ml). After incubation for different periods as indicated, the total cell lysates were subjected to SDS-PAGE followed by immunoblotting for ERK and Akt. (B) BMDMs were pretreated with U0126 (3 µM) or PD98059 (10 µM) for 30 min and then treated with LPS (1 µg/ml) and/or zVAD (20 µM). After 24 h, the cell viability was determined by MTT assay. veh, vehicle. Data were the mean ± SEM from at least three independent experiments. *P < 0.05, indicating synergistic cell death caused by LPS and zVAD.

Fig. 8. ROS production resulting from STAT1 activation contributes to necroptosis.

(A) BMDMs were pretreated with BHA (100 µM) and/or zVAD (20 µM) for 30 min prior to LPS (1 µg/ml) or poly I:C (20 µg/ml) treatment. After incubation for 24 h, the cell viability was assessed by MTT assay. (B) Cells were pretreated with zVAD, LPS (1 µg/ml) and/or poly I:C for different periods, and then intracellular ROS was measured by DCFH₂-DA fluorescence. (C) Similar experiments as (B) were conducted in cells treated with Nec-1 (10 µM), AZD1480 (1 µM) or IFNR-Ab (1 µg/ml). veh, vehicle. (D) BMDMs from WT and STAT1 KO mice were treated with zVAD and/or LPS, and ROS levels at 8 h and 18 h were measured. Data were the mean ± SEM from at least three independent experiments. *P < 0.05, indicating enhancement effects of zVAD on cytotoxicity and ROS production. ^#P < 0.05, indicating reversal effects of BHA, Nec-1, AZD1480, IFNR-Ab, and STAT1 knockout on zVAD-induced cytotoxicity and ROS production.

Fig. 9. RIP1-dependent ROS production and STAT1 activation, but not ERK, are involved in autophagy initiation.

BMDMs were pretreated with BHA (100 µM) (A), U0126 (3 µM), or Nec-1 (10 µM) (B) for 30 min followed by treatment with zVAD (20 µM), LPS (1 µg/ml), poly I:C (20 µg/ml), or different periods as indicated. In (C), STAT1 knockout BMDMs were treated in a similar manner. Total cell lysates were subjected to SDS-PAGE and immunoblotting with an LC3 antibody.

Fig. 10. Signaling pathway and molecular mechanisms underlying TLR/zVAD-induced autophagic cell necroptosis.

In TLR3/4 activated macrophages, TRIF-mediated RIP1/IRF3 and RIP1/RIP3 signaling pathways are enhanced by the caspase inhibitor zVAD, leading to IFN production and necrosome formation, respectively. Autocrine IFNR-JAK-STAT1 action further increases ROS production, which is a prerequisite for autophagic cell necroptosis. zVAD also inhibits LPS- and poly I:C-elicited ERK and Akt activation, both of which are not involved in the cell death mechanism.