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TRAIL-producing NK cells contribute to liver injury and related fibrogenesis in the context of GNMT deficiency.

Fernández-Álvarez S, Gutiérrez-de Juan V, Zubiete-Franco I, Barbier-Torres L, Lahoz A, Parés A, Luka Z, Wagner C, Lu SC, Mato JM, Martínez-Chantar ML, Beraza N - Lab. Invest. (2014)

Bottom Line: Glycine-N-methyltransferase (GNMT) is essential to preserve liver homeostasis.The aim of our study is to elucidate the implication of TRAIL-producing NK cells in the progression of chronic liver injury and fibrogenesis.Overall, our work demonstrates that TRAIL-producing NK cells actively contribute to liver injury and further fibrogenesis in the pathological context of GNMT deficiency, a molecular scenario characteristic of chronic human liver disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Metabolomics, CIC bioGUNE, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (Ciberehd), Derio, Spain.

ABSTRACT
Glycine-N-methyltransferase (GNMT) is essential to preserve liver homeostasis. Cirrhotic patients show low expression of GNMT that is absent in hepatocellular carcinoma (HCC) samples. Accordingly, GNMT deficiency in mice leads to steatohepatitis, fibrosis, cirrhosis, and HCC. Lack of GNMT triggers NK cell activation in GNMT(-/-) mice and depletion of TRAIL significantly attenuates acute liver injury and inflammation in these animals. Chronic inflammation leads to fibrogenesis, further contributing to the progression of chronic liver injury regardless of the etiology. The aim of our study is to elucidate the implication of TRAIL-producing NK cells in the progression of chronic liver injury and fibrogenesis. For this we generated double TRAIL(-/-)/GNMT(-/-) mice in which we found that TRAIL deficiency efficiently protected the liver against chronic liver injury and fibrogenesis in the context of GNMT deficiency. Next, to better delineate the implication of TRAIL-producing NK cells during fibrogenesis we performed bile duct ligation (BDL) to GNMT(-/-) and TRAIL(-/-)/GNMT(-/-) mice. In GNMT(-/-) mice, exacerbated fibrogenic response after BDL concurred with NK1.1(+) cell activation. Importantly, specific inhibition of TRAIL-producing NK cells efficiently protected GNMT(-/-) mice from BDL-induced liver injury and fibrogenesis. Finally, TRAIL(-/-)/GNMT(-/-) mice showed significantly less fibrosis after BDL than GNMT(-/-) mice further underlining the relevance of the TRAIL/DR5 axis in mediating liver injury and fibrogenesis in GNMT(-/-) mice. Finally, in vivo silencing of DR5 efficiently protected GNMT(-/-) mice from BDL-liver injury and fibrogenesis, overall underscoring the key role of the TRAIL/DR5 axis in promoting fibrogenesis in the context of absence of GNMT. Overall, our work demonstrates that TRAIL-producing NK cells actively contribute to liver injury and further fibrogenesis in the pathological context of GNMT deficiency, a molecular scenario characteristic of chronic human liver disease.

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TRAIL deficiency in GNMT−/− mice attenuates fibrogenesis after BDL but shifts cell death from apoptosis to necrosis(A) TRAIL−/−/GNMT−/− mice show significantly lower fibrosis than GNMT−/− mice after BDL as shown by IF using an αSMA Ab, Sirius Red staining and further quantification. (B) Kaplan-Meier curve showing an overall comparable death rate after BDL in TRAIL−/−/GNMT−/− compared to GNMT−/− animals. (C) H&E staining and (G) further quantification (upper panel), (D) TUNEL assay and (G) Caspase3 activity (lower panel) of liver extracts showing lower apoptotic cell death but profuse necrosis in TRAIL−/−/GNMT−/− after BDL. (H) WB analysis showing lower FADD in TRAIL−/−/GNMT−/− mice after BDL. (I) qPCR showing lower DR5 expression in TRAIL−/−/GNMT−/− mice 14d after BDL. n = 5–7. *p< 0.05; **p< 0.01; ***P < 0.001 (TRAIL−/−/GNMT−/−vs GNMT−/−).
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Figure 6: TRAIL deficiency in GNMT−/− mice attenuates fibrogenesis after BDL but shifts cell death from apoptosis to necrosis(A) TRAIL−/−/GNMT−/− mice show significantly lower fibrosis than GNMT−/− mice after BDL as shown by IF using an αSMA Ab, Sirius Red staining and further quantification. (B) Kaplan-Meier curve showing an overall comparable death rate after BDL in TRAIL−/−/GNMT−/− compared to GNMT−/− animals. (C) H&E staining and (G) further quantification (upper panel), (D) TUNEL assay and (G) Caspase3 activity (lower panel) of liver extracts showing lower apoptotic cell death but profuse necrosis in TRAIL−/−/GNMT−/− after BDL. (H) WB analysis showing lower FADD in TRAIL−/−/GNMT−/− mice after BDL. (I) qPCR showing lower DR5 expression in TRAIL−/−/GNMT−/− mice 14d after BDL. n = 5–7. *p< 0.05; **p< 0.01; ***P < 0.001 (TRAIL−/−/GNMT−/−vs GNMT−/−).

Mentions: Overall, in order to better delineate the implication of TRAIL during fibrogenesis in the pathological context of GNMT deficiency we next performed BDL in TRAIL−/−/GNMT−/− mice. Liver fibrosis was significantly attenuated in TRAIL−/−/GNMT−/− when compared to GNMT−/− mice 21 days after BDL (Fig. 6A–C, Suppl. Fig. 6A). Also TLR9 was significantly less expressed in the double-KO mice, supporting lower activation of HSC after BDL (Suppl. Fig. 6B). Surprisingly, despite the obvious attenuation in liver fibrosis exerted by TRAIL depletion, BDL led to a similar lethality of TRAIL−/−/GNMT−/− mice, mainly occurring within the first days after surgery (Fig. 6D). Further analysis showed that mortality of TRAIL−/−/GNMT−/− mice correlated with severe liver necrosis, especially at 3 days after BDL (Fig. 6E, G upper panel). Interestingly, hepatocyte apoptosis was attenuated at 3, 7 and 14 days after BDL in TRAIL−/−/GNMT−/− mice (Fig. 6F, G lower panel). Accordingly with increased necrosis and lower apoptosis, we found less FADD expression in TRAIL−/−/GNMT−/− mice after BDL when compared to GNMT−/− and WT mice (Fig. 6H). Also, we observed reduced expression of DR5 in livers from the double-KO mice after BDL (Fig. 6I).


TRAIL-producing NK cells contribute to liver injury and related fibrogenesis in the context of GNMT deficiency.

Fernández-Álvarez S, Gutiérrez-de Juan V, Zubiete-Franco I, Barbier-Torres L, Lahoz A, Parés A, Luka Z, Wagner C, Lu SC, Mato JM, Martínez-Chantar ML, Beraza N - Lab. Invest. (2014)

TRAIL deficiency in GNMT−/− mice attenuates fibrogenesis after BDL but shifts cell death from apoptosis to necrosis(A) TRAIL−/−/GNMT−/− mice show significantly lower fibrosis than GNMT−/− mice after BDL as shown by IF using an αSMA Ab, Sirius Red staining and further quantification. (B) Kaplan-Meier curve showing an overall comparable death rate after BDL in TRAIL−/−/GNMT−/− compared to GNMT−/− animals. (C) H&E staining and (G) further quantification (upper panel), (D) TUNEL assay and (G) Caspase3 activity (lower panel) of liver extracts showing lower apoptotic cell death but profuse necrosis in TRAIL−/−/GNMT−/− after BDL. (H) WB analysis showing lower FADD in TRAIL−/−/GNMT−/− mice after BDL. (I) qPCR showing lower DR5 expression in TRAIL−/−/GNMT−/− mice 14d after BDL. n = 5–7. *p< 0.05; **p< 0.01; ***P < 0.001 (TRAIL−/−/GNMT−/−vs GNMT−/−).
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Figure 6: TRAIL deficiency in GNMT−/− mice attenuates fibrogenesis after BDL but shifts cell death from apoptosis to necrosis(A) TRAIL−/−/GNMT−/− mice show significantly lower fibrosis than GNMT−/− mice after BDL as shown by IF using an αSMA Ab, Sirius Red staining and further quantification. (B) Kaplan-Meier curve showing an overall comparable death rate after BDL in TRAIL−/−/GNMT−/− compared to GNMT−/− animals. (C) H&E staining and (G) further quantification (upper panel), (D) TUNEL assay and (G) Caspase3 activity (lower panel) of liver extracts showing lower apoptotic cell death but profuse necrosis in TRAIL−/−/GNMT−/− after BDL. (H) WB analysis showing lower FADD in TRAIL−/−/GNMT−/− mice after BDL. (I) qPCR showing lower DR5 expression in TRAIL−/−/GNMT−/− mice 14d after BDL. n = 5–7. *p< 0.05; **p< 0.01; ***P < 0.001 (TRAIL−/−/GNMT−/−vs GNMT−/−).
Mentions: Overall, in order to better delineate the implication of TRAIL during fibrogenesis in the pathological context of GNMT deficiency we next performed BDL in TRAIL−/−/GNMT−/− mice. Liver fibrosis was significantly attenuated in TRAIL−/−/GNMT−/− when compared to GNMT−/− mice 21 days after BDL (Fig. 6A–C, Suppl. Fig. 6A). Also TLR9 was significantly less expressed in the double-KO mice, supporting lower activation of HSC after BDL (Suppl. Fig. 6B). Surprisingly, despite the obvious attenuation in liver fibrosis exerted by TRAIL depletion, BDL led to a similar lethality of TRAIL−/−/GNMT−/− mice, mainly occurring within the first days after surgery (Fig. 6D). Further analysis showed that mortality of TRAIL−/−/GNMT−/− mice correlated with severe liver necrosis, especially at 3 days after BDL (Fig. 6E, G upper panel). Interestingly, hepatocyte apoptosis was attenuated at 3, 7 and 14 days after BDL in TRAIL−/−/GNMT−/− mice (Fig. 6F, G lower panel). Accordingly with increased necrosis and lower apoptosis, we found less FADD expression in TRAIL−/−/GNMT−/− mice after BDL when compared to GNMT−/− and WT mice (Fig. 6H). Also, we observed reduced expression of DR5 in livers from the double-KO mice after BDL (Fig. 6I).

Bottom Line: Glycine-N-methyltransferase (GNMT) is essential to preserve liver homeostasis.The aim of our study is to elucidate the implication of TRAIL-producing NK cells in the progression of chronic liver injury and fibrogenesis.Overall, our work demonstrates that TRAIL-producing NK cells actively contribute to liver injury and further fibrogenesis in the pathological context of GNMT deficiency, a molecular scenario characteristic of chronic human liver disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Metabolomics, CIC bioGUNE, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (Ciberehd), Derio, Spain.

ABSTRACT
Glycine-N-methyltransferase (GNMT) is essential to preserve liver homeostasis. Cirrhotic patients show low expression of GNMT that is absent in hepatocellular carcinoma (HCC) samples. Accordingly, GNMT deficiency in mice leads to steatohepatitis, fibrosis, cirrhosis, and HCC. Lack of GNMT triggers NK cell activation in GNMT(-/-) mice and depletion of TRAIL significantly attenuates acute liver injury and inflammation in these animals. Chronic inflammation leads to fibrogenesis, further contributing to the progression of chronic liver injury regardless of the etiology. The aim of our study is to elucidate the implication of TRAIL-producing NK cells in the progression of chronic liver injury and fibrogenesis. For this we generated double TRAIL(-/-)/GNMT(-/-) mice in which we found that TRAIL deficiency efficiently protected the liver against chronic liver injury and fibrogenesis in the context of GNMT deficiency. Next, to better delineate the implication of TRAIL-producing NK cells during fibrogenesis we performed bile duct ligation (BDL) to GNMT(-/-) and TRAIL(-/-)/GNMT(-/-) mice. In GNMT(-/-) mice, exacerbated fibrogenic response after BDL concurred with NK1.1(+) cell activation. Importantly, specific inhibition of TRAIL-producing NK cells efficiently protected GNMT(-/-) mice from BDL-induced liver injury and fibrogenesis. Finally, TRAIL(-/-)/GNMT(-/-) mice showed significantly less fibrosis after BDL than GNMT(-/-) mice further underlining the relevance of the TRAIL/DR5 axis in mediating liver injury and fibrogenesis in GNMT(-/-) mice. Finally, in vivo silencing of DR5 efficiently protected GNMT(-/-) mice from BDL-liver injury and fibrogenesis, overall underscoring the key role of the TRAIL/DR5 axis in promoting fibrogenesis in the context of absence of GNMT. Overall, our work demonstrates that TRAIL-producing NK cells actively contribute to liver injury and further fibrogenesis in the pathological context of GNMT deficiency, a molecular scenario characteristic of chronic human liver disease.

Show MeSH
Related in: MedlinePlus