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Hepatocyte-specific NEMO deletion promotes NK/NKT cell- and TRAIL-dependent liver damage.

Beraza N, Malato Y, Sander LE, Al-Masaoudi M, Freimuth J, Riethmacher D, Gores GJ, Roskams T, Liedtke C, Trautwein C - J. Exp. Med. (2009)

Bottom Line: Furthermore, hepatocyte-specific NEMO deletion strongly sensitized the liver to concanavalin A (ConA)-mediated injury.The critical role of the NK cell/TRAIL axis in NEMO(Delta hepa) livers during ConA hepatitis was further confirmed by selective NK cell depletion and adoptive transfer of TRAIL-deficient(-/-) mononuclear cells.Our results uncover an essential mechanism of NEMO-mediated protection of the liver by preventing NK cell tissue damage via TRAIL/DR5 signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine III, University Hospital (RWTH) Aachen, Aachen 5205, Germany.

ABSTRACT
Nuclear factor kappaB (NF-kappaB) is one of the main transcription factors involved in regulating apoptosis, inflammation, chronic liver disease, and cancer progression. The IKK complex mediates NF-kappaB activation and deletion of its regulatory subunit NEMO in hepatocytes (NEMO(Delta hepa)) triggers chronic inflammation and spontaneous hepatocellular carcinoma development. We show that NEMO(Delta hepa) mice were resistant to Fas-mediated apoptosis but hypersensitive to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) as the result of a strong up-regulation of its receptor DR5 on hepatocytes. Additionally, natural killer (NK) cells, the main source of TRAIL, were activated in NEMO(Delta hepa) livers. Interestingly, depletion of the NK1.1(+) cells promoted a significant reduction of liver inflammation and an improvement of liver histology in NEMO(Delta hepa) mice. Furthermore, hepatocyte-specific NEMO deletion strongly sensitized the liver to concanavalin A (ConA)-mediated injury. The critical role of the NK cell/TRAIL axis in NEMO(Delta hepa) livers during ConA hepatitis was further confirmed by selective NK cell depletion and adoptive transfer of TRAIL-deficient(-/-) mononuclear cells. Our results uncover an essential mechanism of NEMO-mediated protection of the liver by preventing NK cell tissue damage via TRAIL/DR5 signaling. As this mechanism is important in human liver diseases, NEMO(Delta hepa) mice are an interesting tool to give insight into liver pathophysiology and to develop future therapeutic strategies.

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Hepatocyte-specific NEMO-deleted mice (NEMOΔhepa) are protected against Fas-mediated apoptosis. (A) Survival curve after i.p. injection of 0.5 µg/g Jo2, evidencing the resistance of NEMOΔhepa mice to Fas-mediated death. (B–D) Macroscopic view of livers 3 h after Jo2 (B), liver weight/body weight ratio (B), serum transaminases (C), and H&E staining (D) showed fulminant hepatitis in NEMOf/f mice after Jo2, whereas NEMOΔhepa remained unaffected. (E and F) TUNEL (E) and caspase 8 and 3 (F) activity analysis confirmed Jo2 apoptosis. Activity is represented in times versus untreated NEMOf/f. (G and H) Fas mRNA and IHC analysis in whole liver (G) and isolated primary hepatocytes (H) showed stronger expression in WT compared than in NEMOΔhepa mice. (I) TNF mRNA and cellular location indicated by IHC. (J) Western blotting showed phosphorylation of JNK1 (p45) and JNK2 (p54) in NEMOf/f mice after Jo2. JNK1 and GAPDH act as loading controls. Bars, 50 µm. Data are representative of three independent experiments. n = 4. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (NEMOf/f vs. NEMOΔhepa). §§, P < 0.01; §§§, P < 0.001 (NEMOf/f vs. Jo2/NEMOf/f). Error bars represent SD.
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fig1: Hepatocyte-specific NEMO-deleted mice (NEMOΔhepa) are protected against Fas-mediated apoptosis. (A) Survival curve after i.p. injection of 0.5 µg/g Jo2, evidencing the resistance of NEMOΔhepa mice to Fas-mediated death. (B–D) Macroscopic view of livers 3 h after Jo2 (B), liver weight/body weight ratio (B), serum transaminases (C), and H&E staining (D) showed fulminant hepatitis in NEMOf/f mice after Jo2, whereas NEMOΔhepa remained unaffected. (E and F) TUNEL (E) and caspase 8 and 3 (F) activity analysis confirmed Jo2 apoptosis. Activity is represented in times versus untreated NEMOf/f. (G and H) Fas mRNA and IHC analysis in whole liver (G) and isolated primary hepatocytes (H) showed stronger expression in WT compared than in NEMOΔhepa mice. (I) TNF mRNA and cellular location indicated by IHC. (J) Western blotting showed phosphorylation of JNK1 (p45) and JNK2 (p54) in NEMOf/f mice after Jo2. JNK1 and GAPDH act as loading controls. Bars, 50 µm. Data are representative of three independent experiments. n = 4. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (NEMOf/f vs. NEMOΔhepa). §§, P < 0.01; §§§, P < 0.001 (NEMOf/f vs. Jo2/NEMOf/f). Error bars represent SD.

Mentions: Fas activation promotes acute hepatic failure (Ogasawara et al., 1993). Hence, stimulation with an anti-Fas mAb (Jo2, 0.5 µg/g) resulted in the death of all NEMOf/f mice within 10 h (Fig. 1 A). Unexpectedly, NEMOΔhepa animals were less sensitive to Jo2, as 90% of the mice survived (Fig. 1 A). Macroscopic view confirmed that NEMOΔhepa livers remained unaffected while NEMOf/f showed significant blood accumulation and a higher liver weight/body weight ratio (Fig. 1 B). Accordingly, NEMOf/f mice showed clear signs of fulminant hepatitis, as high ALT levels, infiltrating PMN and RBC, necrosis, and apoptotic bodies were observed (Fig. 1, C and D). In contrast, we failed to detect any significant effects of Jo2 in NEMOΔhepa mice, as only the preexisting elevated ALT levels and parenchymal damage, caused by the spontaneous phenotype of these mice, was observed (Fig. 1, C and D). TUNEL assay, caspase 8, and caspase 3 activity confirmed resistance to Fas-induced apoptosis in NEMOΔhepa mice (Fig. 1, E and F).


Hepatocyte-specific NEMO deletion promotes NK/NKT cell- and TRAIL-dependent liver damage.

Beraza N, Malato Y, Sander LE, Al-Masaoudi M, Freimuth J, Riethmacher D, Gores GJ, Roskams T, Liedtke C, Trautwein C - J. Exp. Med. (2009)

Hepatocyte-specific NEMO-deleted mice (NEMOΔhepa) are protected against Fas-mediated apoptosis. (A) Survival curve after i.p. injection of 0.5 µg/g Jo2, evidencing the resistance of NEMOΔhepa mice to Fas-mediated death. (B–D) Macroscopic view of livers 3 h after Jo2 (B), liver weight/body weight ratio (B), serum transaminases (C), and H&E staining (D) showed fulminant hepatitis in NEMOf/f mice after Jo2, whereas NEMOΔhepa remained unaffected. (E and F) TUNEL (E) and caspase 8 and 3 (F) activity analysis confirmed Jo2 apoptosis. Activity is represented in times versus untreated NEMOf/f. (G and H) Fas mRNA and IHC analysis in whole liver (G) and isolated primary hepatocytes (H) showed stronger expression in WT compared than in NEMOΔhepa mice. (I) TNF mRNA and cellular location indicated by IHC. (J) Western blotting showed phosphorylation of JNK1 (p45) and JNK2 (p54) in NEMOf/f mice after Jo2. JNK1 and GAPDH act as loading controls. Bars, 50 µm. Data are representative of three independent experiments. n = 4. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (NEMOf/f vs. NEMOΔhepa). §§, P < 0.01; §§§, P < 0.001 (NEMOf/f vs. Jo2/NEMOf/f). Error bars represent SD.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2722179&req=5

fig1: Hepatocyte-specific NEMO-deleted mice (NEMOΔhepa) are protected against Fas-mediated apoptosis. (A) Survival curve after i.p. injection of 0.5 µg/g Jo2, evidencing the resistance of NEMOΔhepa mice to Fas-mediated death. (B–D) Macroscopic view of livers 3 h after Jo2 (B), liver weight/body weight ratio (B), serum transaminases (C), and H&E staining (D) showed fulminant hepatitis in NEMOf/f mice after Jo2, whereas NEMOΔhepa remained unaffected. (E and F) TUNEL (E) and caspase 8 and 3 (F) activity analysis confirmed Jo2 apoptosis. Activity is represented in times versus untreated NEMOf/f. (G and H) Fas mRNA and IHC analysis in whole liver (G) and isolated primary hepatocytes (H) showed stronger expression in WT compared than in NEMOΔhepa mice. (I) TNF mRNA and cellular location indicated by IHC. (J) Western blotting showed phosphorylation of JNK1 (p45) and JNK2 (p54) in NEMOf/f mice after Jo2. JNK1 and GAPDH act as loading controls. Bars, 50 µm. Data are representative of three independent experiments. n = 4. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (NEMOf/f vs. NEMOΔhepa). §§, P < 0.01; §§§, P < 0.001 (NEMOf/f vs. Jo2/NEMOf/f). Error bars represent SD.
Mentions: Fas activation promotes acute hepatic failure (Ogasawara et al., 1993). Hence, stimulation with an anti-Fas mAb (Jo2, 0.5 µg/g) resulted in the death of all NEMOf/f mice within 10 h (Fig. 1 A). Unexpectedly, NEMOΔhepa animals were less sensitive to Jo2, as 90% of the mice survived (Fig. 1 A). Macroscopic view confirmed that NEMOΔhepa livers remained unaffected while NEMOf/f showed significant blood accumulation and a higher liver weight/body weight ratio (Fig. 1 B). Accordingly, NEMOf/f mice showed clear signs of fulminant hepatitis, as high ALT levels, infiltrating PMN and RBC, necrosis, and apoptotic bodies were observed (Fig. 1, C and D). In contrast, we failed to detect any significant effects of Jo2 in NEMOΔhepa mice, as only the preexisting elevated ALT levels and parenchymal damage, caused by the spontaneous phenotype of these mice, was observed (Fig. 1, C and D). TUNEL assay, caspase 8, and caspase 3 activity confirmed resistance to Fas-induced apoptosis in NEMOΔhepa mice (Fig. 1, E and F).

Bottom Line: Furthermore, hepatocyte-specific NEMO deletion strongly sensitized the liver to concanavalin A (ConA)-mediated injury.The critical role of the NK cell/TRAIL axis in NEMO(Delta hepa) livers during ConA hepatitis was further confirmed by selective NK cell depletion and adoptive transfer of TRAIL-deficient(-/-) mononuclear cells.Our results uncover an essential mechanism of NEMO-mediated protection of the liver by preventing NK cell tissue damage via TRAIL/DR5 signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine III, University Hospital (RWTH) Aachen, Aachen 5205, Germany.

ABSTRACT
Nuclear factor kappaB (NF-kappaB) is one of the main transcription factors involved in regulating apoptosis, inflammation, chronic liver disease, and cancer progression. The IKK complex mediates NF-kappaB activation and deletion of its regulatory subunit NEMO in hepatocytes (NEMO(Delta hepa)) triggers chronic inflammation and spontaneous hepatocellular carcinoma development. We show that NEMO(Delta hepa) mice were resistant to Fas-mediated apoptosis but hypersensitive to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) as the result of a strong up-regulation of its receptor DR5 on hepatocytes. Additionally, natural killer (NK) cells, the main source of TRAIL, were activated in NEMO(Delta hepa) livers. Interestingly, depletion of the NK1.1(+) cells promoted a significant reduction of liver inflammation and an improvement of liver histology in NEMO(Delta hepa) mice. Furthermore, hepatocyte-specific NEMO deletion strongly sensitized the liver to concanavalin A (ConA)-mediated injury. The critical role of the NK cell/TRAIL axis in NEMO(Delta hepa) livers during ConA hepatitis was further confirmed by selective NK cell depletion and adoptive transfer of TRAIL-deficient(-/-) mononuclear cells. Our results uncover an essential mechanism of NEMO-mediated protection of the liver by preventing NK cell tissue damage via TRAIL/DR5 signaling. As this mechanism is important in human liver diseases, NEMO(Delta hepa) mice are an interesting tool to give insight into liver pathophysiology and to develop future therapeutic strategies.

Show MeSH
Related in: MedlinePlus