Fatal hepatitis mediated by tumor necrosis factor TNFalpha requires caspase-8 and involves the BH3-only proteins Bid and Bim

Abstract

Apoptotic death of hepatocytes, a contributor to many chronic and acute liver diseases, can be a consequence of overactivation of the immune system and is often mediated by TNFalpha. Injection with lipopolysaccharide (LPS) plus the transcriptional inhibitor D(+)-galactosamine (GalN) or mitogenic T cell activation causes fatal hepatocyte apoptosis in mice, which is mediated by TNFalpha, but the effector mechanisms remain unclear. Our analysis of gene-targeted mice showed that caspase-8 is essential for hepatocyte killing in both settings. Loss of Bid, the proapoptotic BH3-only protein activated by caspase-8 and essential for Fas ligand-induced hepatocyte killing, resulted only in a minor reduction of liver damage. However, combined loss of Bid and another BH3-only protein, Bim, activated by c-Jun N-terminal kinase (JNK), protected mice from LPS+GalN-induced hepatitis. These observations identify caspase-8 and the BH3-only proteins Bid and Bim as potential therapeutic targets for treatment of inflammatory liver diseases.

Figure 1. LPS plus GalN-Induced Hepatitis Requires the Initiator Caspase, Caspase-8, Is Inhibited by a Pan-Caspase Inhibitor and Involves Cleavage of the Pro-Apoptotic BH3-Only Bcl-2 Family Member Bid

(A) Mice lacking caspase-8 in hepatocytes (albumin Cre transgenic caspase-8^flox/flox) and littermate controls (albumin Cre transgenic caspase-8^flox/wt mice) were injected with 100 ng LPS plus 20 mg GalN. At the time when the wt mice were terminally ill (~8 h), all animals were sacrificed and serum levels of the liver transaminases ALT and AST measured. Data shown represent means +/−SD of 3–5 mice for each genotype. (B) H&E stained histological liver sections from mice treated as in (A). Pictures shown are representative of the analysis of at least 3 mice for each treatment and genotype. (C) Nine mice of the indicated genotypes in each group were injected i.p. with LPS+GalN and followed over 72 h post injection. P values were calculated using a time to event analysis with a log rank test. No mortality was observed in mice of either genotype injected with 20 mg GalN alone (n = 3 per genotype). (D) Three control wt mice and 3 wt mice pre-treated with 20 mg/kg of the pan-caspase inhibitor Q-VD-oph (i.p. injection 30 min prior to treatment) were injected with 100 ng LPS plus 20 mg GalN. All animals were sacrificed after 6 h and serum levels of ALT and AST measured. (E) H&E stained histological liver sections from the same mice treated in (D). Pictures shown are representative of the analysis of 3 mice for each treatment. (F) Mice (wt) were injected with 100 ng LPS plus 20 mg GalN and sacrificed after the indicated time points. Processing of caspase-8, Bid and caspase-7 was examined by Western blot analysis. Probing with a monoclonal antibody to β-actin served as a loading control.

Figure 2. Bid Contributes to LPS plus GalN-Induced Hepatitis

(A) Bid-deficient mice and wt littermate controls were injected i.p. with 10 ng of LPS plus GalN (20 mg per mouse) or with GalN alone as control. Mice were sacrificed after 6 h, bled and sera analyzed for the liver transaminases ALT and AST. (B) Bid-deficient mice and wt littermate controls were injected i.p. with increasing doses (10, 100 and 1000 ng) of LPS, all in the presence of 20 mg GalN, and analyzed after 6 h as in (A). Data are presented as means +/−SD. (C) Histological examination of H&E stained liver sections of bid^−/− and wt mice subjected to the treatments indicated (scale bars = 50μm). Pictures shown are representative of the analysis of at least 3 mice for each treatment and genotype.

Figure 3. Loss of Bim Cooperates with Loss of Bid to Protect Mice from LPS plus GalN-Induced Hepatitis

(A) Mice lacking both Bid and Bim (bim^−/− bid^−/−) and control animals (wt, bid^−/− or bim^−/−) were injected with 100 ng LPS plus 20 mg GalN. At the time when the wt mice were sick (6 h), all animals were sacrificed and serum levels of ALT and AST measured. Data shown represent means +/−SD of 3–6 mice for each genotype and each treatment. (B) Histological examination of H&E stained liver sections of wt, bid^−/−, bim^−/−, and bim^−/− bid^−/− mice injected 6 h earlier with 100 ng LPS plus 20 mg GalN (scale bars = 50 μm). Pictures shown are representative of the analysis of at least 3 mice for each treatment and genotype. (C) Table summarizing the long-term (followed up to 5 days) survival of the indicated genotypes of mice treated with 10 ng or 100 ng LPS plus 20 mg GalN. Mice that did not survive this treatment all succumbed within 24 h.

Figure 4. Treatment with LPS plus GalN Causes a Post-Translational Modification of Bim that Does not Require Caspase-8 or other Caspases

(A) Mice (wt), with or without pre-treatment with 20 mg/kg of the pan-caspase inhibitor Q-VD-oph, were injected with LPS (100 ng) plus GalN (20 mg) and sacrificed 4 h later. Total protein extracts from the livers of these animals were probed by Western blotting for Bim, Caspase-8, Bid, Caspase-7 and active Caspase-3. (B) Mice lacking caspase-8 in hepatocytes (C8: albumin Cre transgenic caspase-8^flox/flox) and littermate controls (albumin Cre transgenic caspase-8^flox/wt) were injected with 100 ng LPS plus 20 mg GalN and sacrificed after 4 h. Total protein extracts derived from the livers of these animals were probed by Western blotting for Bim or β-actin (loading control).

Figure 5. Bim Is Phosphorylated by JNK Kinase in the Livers of Mice Injected with LPS plus GalN

(A) Mice (wt) were injected with LPS (100 ng) plus GalN (20 mg) and sacrificed at the time points indicated. Levels and post-translational modifications of Bim were investigated in liver-derived total protein extracts by Western blotting. Probing with an antibody to β-actin was used as a loading control. (B) Mice (wt) were injected with LPS (100 ng) plus GalN (20 mg) and sacrificed after 3 h. Total protein extracts were prepared from the liver in the absence of phosphatase inhibitors and left untreated or treated in vitro with λ-phosphatase prior to analyzing Bim modifications by Western blotting. (C) Mice were treated with GalN (20 mg) alone (controls) or with LPS (100 ng) plus GalN (20 mg) and livers harvested after 4 h. Protein extracts were prepared and then subjected to subcellular fractionation into the dynein enriched pellet fraction (P), containing Bim_EL/L sequestered to microtubules, and a soluble fraction (S) containing free cytosolic Bim. Fractions were analyzed by Western blotting using antibodies to Bim or HSP70 (control for the purity of the fractions). (D) Liver extracts from wt mice treated with 100 ng LPS plus 20 mg GalN for 4 h were probed by Western blotting with an antibody specific for phosphorylated (activated) JNK and the membrane re-probed with an antibody to total JNK (loading control). (E) Wt mice that had either been left untreated or pre-treated for 30 min with the JNK inhibitory peptide D-JNKI1 (20 mg/kg, i.p.) were treated for 4 h with either 20 mg GalN or with 100 ng LPS plus 20 mg GalN. Total liver extracts were probed by Western blotting with antibodies to Bim (two blots from independent experiments shown), Bid, Mcl-1 and β-actin (loading control). Asterix indicate non-specific bands.

Figure 6. Inhibition of JNK Enhances Resistance of Bid^−/− Mice to LPS plus GalN-Induced Hepatocyte Destruction

(A) Bid^−/−, bim^−/− or wt mice were injected with JNK inhibitory peptide D-JNKI1 (30 mg/kg, i.p.) 30 min prior to treatment with 10 ng LPS plus GalN and were then monitored for survival for up to 72 h. Mice injected with LPS+GalN served as controls. P values were calculated using a time to event analysis using a log-rank test. (B) Table summarizing the long-term (followed up to 3 days) survival of wt, bid^−/−, bim^−/− and bim^−/− bid^−/− mice treated with 10 ng LPS+GalN, with or without pre-treatment with D-JNKI1. Mice that did not survive, all succumbed to the treatment within 24 h. (C) Serum levels of ALT and AST in bid^−/− mice treated with 10 ng LPS plus 20 mg GalN, with or without pre-treatment with D-JNKI1 (30 mg/kg), were measured after 6 h. P-values: wt vs. bid^−/−: p (ALT) = 0.09; p (AST) = 0.16. Data shown represent means +/−SD of 8–11 mice for each genotype and are derived from three independent experiments.

Figure 7. Caspase-8 is Essential and Bim a Contributor in ConA-Induced Hepatitis

(A) Mice lacking caspase-8 in hepatocytes (albumin Cre transgenic caspase-8^flox/flox) and littermate controls (albumin Cre transgenic caspase-8^flox/wt mice) were injected with 30 mg/kg ConA. At the time when the wt mice were sick (8 h), all animals were sacrificed and serum levels of ALT and AST measured. Data shown represent means +/−SD of 3–5 mice for each genotype and each treatment. (B) H&E stained histological liver sections from mice treated as in (A). Pictures shown are representative of the analysis of at least 3 mice for each treatment and genotype. (C) Liver extracts from wt mice treated with 30 mg/kg ConA for 4 h were probed by Western blotting with an antibody specific for phosphorylated (activated) JNK and the membrane re-probed with an antibody to total JNK (loading control). (D) Mice (wt) were injected with ConA (30 mg/kg) and sacrificed at the time points indicated. Bim levels and possible post-translational modifications were analyzed by Western blotting of liver extracts as described in Figure 5A. (E) Mice (wt) were injected with 30 mg/kg ConA and sacrificed after 3 h. Total protein extracts were prepared from the liver in the absence of phosphatase inhibitors and left untreated or treated in vitro with λ-phosphatase prior to analyzing Bim modifications by Western blotting. (F) Mice (wt), with or without pre-treatment with 20 mg/kg of the pan-caspase inhibitor Q-VD-oph, were injected with ConA (30 mg/kg) and sacrificed 5 h later. Total protein extracts from the livers of these animals were probed by Western blotting for Bim. (G) Mice lacking both Bid and Bim (bim^−/− bid^−/−) and control animals (wt, bid^−/− or bim^−/−) were injected with 30 mg/kg ConA. At the time when the wt mice were sick (6 h), all animals were sacrificed and serum levels of ALT and AST measured. Data shown represent means +/−SD (n=3 for wt, bid^−/−, bim^−/− and n=4 for bid^−/− bim^−/− mice). (H) Histological examination of H&E stained liver sections of mice of the indicated genotypes subjected for 6 h to treatment with 30 mg/kg ConA (bars = 50 μm). Pictures shown are representative of the analysis of at least 3 mice for each genotype.