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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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Specific NK cell inhibition with ASIALO protects the liver of GNMT−/− mice from BDL-liver injury and fibrosis(A) αSMA IF (left panels) and Sirius red staining (right panels), further quantification using FRIDA software and (B) qPCR of αSMA, collagen1A1 and TLR9 expression evidenced the significantly lower fibrosis in ASIALO/GNMT−/− after BDL. (C) Kaplan-Meier survival curve showing improved animal survival in ASIALO/GNMT−/− compared to GNMT−/− after BDL. (D) Analysis of serum transaminases and bilirubin, (E) H&E staining (left panels) and TUNEL assay (right panels) showed an evident reduction of liver injury in ASIALO/GNMT−/− mice after BDL. (F) Quantification of necrotic areas in H&E stained liver sections after BDL showing less necrosis in ASIALO/GNMT−/− mice. (G) qPCR analysis of DR5 expression on liver samples and (H) lower phosphorylation of JNK detected by western blot analysis evidenced lower activation of the DR5-mediated cell death response in ASIALO/GNMT−/− mice after BDL. n = 5–7. **p< 0.01 (GNMT−/−vs ASIALO/GNMT−/−). Error bars represent SD.
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Figure 5: Specific NK cell inhibition with ASIALO protects the liver of GNMT−/− mice from BDL-liver injury and fibrosis(A) αSMA IF (left panels) and Sirius red staining (right panels), further quantification using FRIDA software and (B) qPCR of αSMA, collagen1A1 and TLR9 expression evidenced the significantly lower fibrosis in ASIALO/GNMT−/− after BDL. (C) Kaplan-Meier survival curve showing improved animal survival in ASIALO/GNMT−/− compared to GNMT−/− after BDL. (D) Analysis of serum transaminases and bilirubin, (E) H&E staining (left panels) and TUNEL assay (right panels) showed an evident reduction of liver injury in ASIALO/GNMT−/− mice after BDL. (F) Quantification of necrotic areas in H&E stained liver sections after BDL showing less necrosis in ASIALO/GNMT−/− mice. (G) qPCR analysis of DR5 expression on liver samples and (H) lower phosphorylation of JNK detected by western blot analysis evidenced lower activation of the DR5-mediated cell death response in ASIALO/GNMT−/− mice after BDL. n = 5–7. **p< 0.01 (GNMT−/−vs ASIALO/GNMT−/−). Error bars represent SD.

Mentions: Next, in order to better delineate the implication of TRAIL-producing NK cells during fibrogenesis we specifically depleted this cell compartment using the anti-ASIALO-GM1 antibody (ASIALO). Similarly to what we found in NK1.1/GNMT−/−, liver fibrosis was significantly attenuated in ASIALO/GNMT−/− mice as evidenced by αSMA, Sirius Red staining and further quantification (Fig. 5A, B). qPCR of αSMA and collagen 1A1 expression confirmed the reduced fibrosis found in ASIALO/GNMT−/− mice after BDL. Also, the decreased expression of TLR9 found in ASIALO/GNMT−/− mice after BDL pointed to a lower activation of HSC (Fig. 5B). The beneficial impact of specific inhibition of NK cells was supported by the high survival rate in ASIALO/GNMT−/− mice compared to GNMT−/− animals (Fig. 5C). Further analysis of serum transaminases (Fig. 5D), H&E staining and TUNEL assay (Fig. 5E, F) confirmed the significant improvement of the liver parenchyma status and the attenuation of cell death in NK cell-depleted mice after BDL. ASIALO/GNMT−/− mice showed lower DR5 expression and attenuated activation of JNK 14d after BDL (Fig. 5G, H). Finally, ASIALO/GNMT−/− mice showed partial recovery of KC activation evidenced by F4/80 IHC and TNF ELISA (Suppl. Fig. 4A, B), supporting the regulation between cell compartments in the context of cholestatic-liver damage in GNMT−/− animals. In parallel, a control group receiving IgG was performed showing no significant differences in fibrogenesis and liver injury when compared to the uninjected GNMT−/− mice, overall proving that the latter group can be considered as a proper control for both NK1.1+ and ASIALO treated groups (Suppl. Fig. 5).


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)

Specific NK cell inhibition with ASIALO protects the liver of GNMT−/− mice from BDL-liver injury and fibrosis(A) αSMA IF (left panels) and Sirius red staining (right panels), further quantification using FRIDA software and (B) qPCR of αSMA, collagen1A1 and TLR9 expression evidenced the significantly lower fibrosis in ASIALO/GNMT−/− after BDL. (C) Kaplan-Meier survival curve showing improved animal survival in ASIALO/GNMT−/− compared to GNMT−/− after BDL. (D) Analysis of serum transaminases and bilirubin, (E) H&E staining (left panels) and TUNEL assay (right panels) showed an evident reduction of liver injury in ASIALO/GNMT−/− mice after BDL. (F) Quantification of necrotic areas in H&E stained liver sections after BDL showing less necrosis in ASIALO/GNMT−/− mice. (G) qPCR analysis of DR5 expression on liver samples and (H) lower phosphorylation of JNK detected by western blot analysis evidenced lower activation of the DR5-mediated cell death response in ASIALO/GNMT−/− mice after BDL. n = 5–7. **p< 0.01 (GNMT−/−vs ASIALO/GNMT−/−). Error bars represent SD.
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Figure 5: Specific NK cell inhibition with ASIALO protects the liver of GNMT−/− mice from BDL-liver injury and fibrosis(A) αSMA IF (left panels) and Sirius red staining (right panels), further quantification using FRIDA software and (B) qPCR of αSMA, collagen1A1 and TLR9 expression evidenced the significantly lower fibrosis in ASIALO/GNMT−/− after BDL. (C) Kaplan-Meier survival curve showing improved animal survival in ASIALO/GNMT−/− compared to GNMT−/− after BDL. (D) Analysis of serum transaminases and bilirubin, (E) H&E staining (left panels) and TUNEL assay (right panels) showed an evident reduction of liver injury in ASIALO/GNMT−/− mice after BDL. (F) Quantification of necrotic areas in H&E stained liver sections after BDL showing less necrosis in ASIALO/GNMT−/− mice. (G) qPCR analysis of DR5 expression on liver samples and (H) lower phosphorylation of JNK detected by western blot analysis evidenced lower activation of the DR5-mediated cell death response in ASIALO/GNMT−/− mice after BDL. n = 5–7. **p< 0.01 (GNMT−/−vs ASIALO/GNMT−/−). Error bars represent SD.
Mentions: Next, in order to better delineate the implication of TRAIL-producing NK cells during fibrogenesis we specifically depleted this cell compartment using the anti-ASIALO-GM1 antibody (ASIALO). Similarly to what we found in NK1.1/GNMT−/−, liver fibrosis was significantly attenuated in ASIALO/GNMT−/− mice as evidenced by αSMA, Sirius Red staining and further quantification (Fig. 5A, B). qPCR of αSMA and collagen 1A1 expression confirmed the reduced fibrosis found in ASIALO/GNMT−/− mice after BDL. Also, the decreased expression of TLR9 found in ASIALO/GNMT−/− mice after BDL pointed to a lower activation of HSC (Fig. 5B). The beneficial impact of specific inhibition of NK cells was supported by the high survival rate in ASIALO/GNMT−/− mice compared to GNMT−/− animals (Fig. 5C). Further analysis of serum transaminases (Fig. 5D), H&E staining and TUNEL assay (Fig. 5E, F) confirmed the significant improvement of the liver parenchyma status and the attenuation of cell death in NK cell-depleted mice after BDL. ASIALO/GNMT−/− mice showed lower DR5 expression and attenuated activation of JNK 14d after BDL (Fig. 5G, H). Finally, ASIALO/GNMT−/− mice showed partial recovery of KC activation evidenced by F4/80 IHC and TNF ELISA (Suppl. Fig. 4A, B), supporting the regulation between cell compartments in the context of cholestatic-liver damage in GNMT−/− animals. In parallel, a control group receiving IgG was performed showing no significant differences in fibrogenesis and liver injury when compared to the uninjected GNMT−/− mice, overall proving that the latter group can be considered as a proper control for both NK1.1+ and ASIALO treated groups (Suppl. Fig. 5).

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