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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 deletion of NEMO promotes spontaneous activation of liver NK/NKT cells, and administration of NK1.1-depleting mAb attenuates the damaging phenotype in NEMOΔhepa mice. (A) FACS analysis revealed a lower number of NK1.1+CD3− and NK1.1+CD3+ cells in NEMOΔhepa mice. The graph represents the percentage of NK1.1+ cells related to the percentage of CD45+ cells in the liver (B) FACS analysis of NK1.1+/annexin V+ related to % of CD45+ cells revealed strong apoptosis of NK1.1+ cells in livers from NEMOΔhepa mice. (C) RT-PCR showed strong expression of IL-12 (p35), IFN-γ, and CCL5 but lower IL-4 mRNA in livers. Data are presented as times versus NEMOf/f untreated. (D) mRNA analysis of isolated NK1.1+ cells confirmed cell activation and increased cytokine expression. (E) FACS analysis showed stronger TRAIL expression on NK1.1+ cells from livers from NEMOΔhepa mice. mRNA analysis on isolated NK1.1+ cells confirmed this. (F and G) FACS analysis proved effective depletion of NK1.1+ cells (F) 40 h after NK1.1 mAb administration that reduced IFN-γ and CCL5 mRNA levels (G). (H–J) Serum ALT (H), H&E staining (I), and FACS analysis (J; CD11b+ cells) were used as markers of liver damage and inflammation. Bars, 50 µm. All 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 (NEMOΔhepa vs. Nk1.1mAb/NEMOΔhepa). Error bars represent SD.
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fig3: Hepatocyte-specific deletion of NEMO promotes spontaneous activation of liver NK/NKT cells, and administration of NK1.1-depleting mAb attenuates the damaging phenotype in NEMOΔhepa mice. (A) FACS analysis revealed a lower number of NK1.1+CD3− and NK1.1+CD3+ cells in NEMOΔhepa mice. The graph represents the percentage of NK1.1+ cells related to the percentage of CD45+ cells in the liver (B) FACS analysis of NK1.1+/annexin V+ related to % of CD45+ cells revealed strong apoptosis of NK1.1+ cells in livers from NEMOΔhepa mice. (C) RT-PCR showed strong expression of IL-12 (p35), IFN-γ, and CCL5 but lower IL-4 mRNA in livers. Data are presented as times versus NEMOf/f untreated. (D) mRNA analysis of isolated NK1.1+ cells confirmed cell activation and increased cytokine expression. (E) FACS analysis showed stronger TRAIL expression on NK1.1+ cells from livers from NEMOΔhepa mice. mRNA analysis on isolated NK1.1+ cells confirmed this. (F and G) FACS analysis proved effective depletion of NK1.1+ cells (F) 40 h after NK1.1 mAb administration that reduced IFN-γ and CCL5 mRNA levels (G). (H–J) Serum ALT (H), H&E staining (I), and FACS analysis (J; CD11b+ cells) were used as markers of liver damage and inflammation. Bars, 50 µm. All 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 (NEMOΔhepa vs. Nk1.1mAb/NEMOΔhepa). Error bars represent SD.

Mentions: NEMOΔhepa livers had significantly reduced numbers of NK and NKT cells compared with NEMOf/f littermates (Fig. 3 A). As NK cells' activation is associated with their apoptosis, the lower NK1.1+ cell number found in NEMOΔhepa livers could reflect their activation. Hence, NK1.1+ cells from NEMOΔhepa mice exhibited significantly higher levels of annexin V when compared with NEMOf/f mice (Fig. 3 B). Activation of NK cells involves strong production of proinflammatory cytokines, like IFN-γ and IL-12, promoting their cytotoxic activity and apoptosis (Vivier et al., 2008). Accordingly, we found higher IFN-γ and IL-12 (p35) expression associated with lower IL-4 levels in NEMOΔhepa mice compared with WT littermates. Additionally, higher CCL5 (RANTES) levels found in NEMOΔhepa livers (Fig. 3 C) may reflect a compensatory response to restore liver NK cells number (Morris and Ley, 2004). These data were confirmed on isolated NK1.1+ cells from livers of NEMOΔhepa mice showing higher IFN-γ and CCL5 but lower IL4 levels (Fig. 3 D). FACS and mRNA analysis evidenced strong TRAIL expression on liver NK1.1+ cells from NEMOΔhepa compared with WT mice (Fig. 3 E).


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 deletion of NEMO promotes spontaneous activation of liver NK/NKT cells, and administration of NK1.1-depleting mAb attenuates the damaging phenotype in NEMOΔhepa mice. (A) FACS analysis revealed a lower number of NK1.1+CD3− and NK1.1+CD3+ cells in NEMOΔhepa mice. The graph represents the percentage of NK1.1+ cells related to the percentage of CD45+ cells in the liver (B) FACS analysis of NK1.1+/annexin V+ related to % of CD45+ cells revealed strong apoptosis of NK1.1+ cells in livers from NEMOΔhepa mice. (C) RT-PCR showed strong expression of IL-12 (p35), IFN-γ, and CCL5 but lower IL-4 mRNA in livers. Data are presented as times versus NEMOf/f untreated. (D) mRNA analysis of isolated NK1.1+ cells confirmed cell activation and increased cytokine expression. (E) FACS analysis showed stronger TRAIL expression on NK1.1+ cells from livers from NEMOΔhepa mice. mRNA analysis on isolated NK1.1+ cells confirmed this. (F and G) FACS analysis proved effective depletion of NK1.1+ cells (F) 40 h after NK1.1 mAb administration that reduced IFN-γ and CCL5 mRNA levels (G). (H–J) Serum ALT (H), H&E staining (I), and FACS analysis (J; CD11b+ cells) were used as markers of liver damage and inflammation. Bars, 50 µm. All 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 (NEMOΔhepa vs. Nk1.1mAb/NEMOΔhepa). Error bars represent SD.
© Copyright Policy - openaccess
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fig3: Hepatocyte-specific deletion of NEMO promotes spontaneous activation of liver NK/NKT cells, and administration of NK1.1-depleting mAb attenuates the damaging phenotype in NEMOΔhepa mice. (A) FACS analysis revealed a lower number of NK1.1+CD3− and NK1.1+CD3+ cells in NEMOΔhepa mice. The graph represents the percentage of NK1.1+ cells related to the percentage of CD45+ cells in the liver (B) FACS analysis of NK1.1+/annexin V+ related to % of CD45+ cells revealed strong apoptosis of NK1.1+ cells in livers from NEMOΔhepa mice. (C) RT-PCR showed strong expression of IL-12 (p35), IFN-γ, and CCL5 but lower IL-4 mRNA in livers. Data are presented as times versus NEMOf/f untreated. (D) mRNA analysis of isolated NK1.1+ cells confirmed cell activation and increased cytokine expression. (E) FACS analysis showed stronger TRAIL expression on NK1.1+ cells from livers from NEMOΔhepa mice. mRNA analysis on isolated NK1.1+ cells confirmed this. (F and G) FACS analysis proved effective depletion of NK1.1+ cells (F) 40 h after NK1.1 mAb administration that reduced IFN-γ and CCL5 mRNA levels (G). (H–J) Serum ALT (H), H&E staining (I), and FACS analysis (J; CD11b+ cells) were used as markers of liver damage and inflammation. Bars, 50 µm. All 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 (NEMOΔhepa vs. Nk1.1mAb/NEMOΔhepa). Error bars represent SD.
Mentions: NEMOΔhepa livers had significantly reduced numbers of NK and NKT cells compared with NEMOf/f littermates (Fig. 3 A). As NK cells' activation is associated with their apoptosis, the lower NK1.1+ cell number found in NEMOΔhepa livers could reflect their activation. Hence, NK1.1+ cells from NEMOΔhepa mice exhibited significantly higher levels of annexin V when compared with NEMOf/f mice (Fig. 3 B). Activation of NK cells involves strong production of proinflammatory cytokines, like IFN-γ and IL-12, promoting their cytotoxic activity and apoptosis (Vivier et al., 2008). Accordingly, we found higher IFN-γ and IL-12 (p35) expression associated with lower IL-4 levels in NEMOΔhepa mice compared with WT littermates. Additionally, higher CCL5 (RANTES) levels found in NEMOΔhepa livers (Fig. 3 C) may reflect a compensatory response to restore liver NK cells number (Morris and Ley, 2004). These data were confirmed on isolated NK1.1+ cells from livers of NEMOΔhepa mice showing higher IFN-γ and CCL5 but lower IL4 levels (Fig. 3 D). FACS and mRNA analysis evidenced strong TRAIL expression on liver NK1.1+ cells from NEMOΔhepa compared with WT mice (Fig. 3 E).

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