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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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Exacerbated fibrogenesis in GNMT−/− mice concurs with activation of NK1.1+ cells(A) IF using an αSMA Ab and (B) Sirius Red staining showed increased fibrosis in GNMT−/− mice. (C) qPCR analysis of mRNA expression of αSMA, collagen1A1 and TLR9 confirmed HSC activation. (D) Dot plot of FACS analysis on isolated liver mononuclear cells (MNCs) showing lower number of CD45+/NK1.1.+ cells in GNMT−/− mice. (E) Histogram showing higher presence of TRAIL in the cell surface of NK1.1+ cells in GNMT−/− liver-MNCs. Bar-plot representing the data obtained by FACS analysis showing low presence of NK1.1+ cells correlating with high expression of TRAIL in GNMT−/− liver-MNCs. (F) qPCR analysis showing increased DR5 mRNA expression in GNMT−/− mice. (H) Western blot analysis confirmed that GNMT−/− mice have higher expression of DR5 after BDL correlating with increased phosphorylation of JNK after surgery. n = 5–7. *p< 0.05; **p< 0.01; ***P < 0.001 (GNMT+/+ vs GNMT−/−) Error bars represent SD.
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Figure 3: Exacerbated fibrogenesis in GNMT−/− mice concurs with activation of NK1.1+ cells(A) IF using an αSMA Ab and (B) Sirius Red staining showed increased fibrosis in GNMT−/− mice. (C) qPCR analysis of mRNA expression of αSMA, collagen1A1 and TLR9 confirmed HSC activation. (D) Dot plot of FACS analysis on isolated liver mononuclear cells (MNCs) showing lower number of CD45+/NK1.1.+ cells in GNMT−/− mice. (E) Histogram showing higher presence of TRAIL in the cell surface of NK1.1+ cells in GNMT−/− liver-MNCs. Bar-plot representing the data obtained by FACS analysis showing low presence of NK1.1+ cells correlating with high expression of TRAIL in GNMT−/− liver-MNCs. (F) qPCR analysis showing increased DR5 mRNA expression in GNMT−/− mice. (H) Western blot analysis confirmed that GNMT−/− mice have higher expression of DR5 after BDL correlating with increased phosphorylation of JNK after surgery. n = 5–7. *p< 0.05; **p< 0.01; ***P < 0.001 (GNMT+/+ vs GNMT−/−) Error bars represent SD.

Mentions: First, we found that only 25% of GNMT−/− mice survived the BDL up to 21 days, whereas survival was 60% in GNMT WT animals (GNMT+/+) (Suppl. Fig. 1A). The increased activation of hepatic stellate cells (HSC) and stronger collagen deposition found in GNMT−/− were evidenced by αSMA IF (Fig. 3A) and Sirius Red staining (Fig. 3B) in liver sections and further quantification at 14d and 21d after BDL (Suppl. Fig. 1B). qPCR analysis of αSMA and collagen1A1 expression confirmed the increased pro-fibrogenic response in GNMT−/− mice after BDL in comparison with WT animals (Fig. 3C). Interestingly, BDL led to significant increase of TLR9 in GNMT−/− mice (Fig. 3C), a receptor involved in the activation of HSC after engulfment of apoptotic hepatocyte-DNA7.


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)

Exacerbated fibrogenesis in GNMT−/− mice concurs with activation of NK1.1+ cells(A) IF using an αSMA Ab and (B) Sirius Red staining showed increased fibrosis in GNMT−/− mice. (C) qPCR analysis of mRNA expression of αSMA, collagen1A1 and TLR9 confirmed HSC activation. (D) Dot plot of FACS analysis on isolated liver mononuclear cells (MNCs) showing lower number of CD45+/NK1.1.+ cells in GNMT−/− mice. (E) Histogram showing higher presence of TRAIL in the cell surface of NK1.1+ cells in GNMT−/− liver-MNCs. Bar-plot representing the data obtained by FACS analysis showing low presence of NK1.1+ cells correlating with high expression of TRAIL in GNMT−/− liver-MNCs. (F) qPCR analysis showing increased DR5 mRNA expression in GNMT−/− mice. (H) Western blot analysis confirmed that GNMT−/− mice have higher expression of DR5 after BDL correlating with increased phosphorylation of JNK after surgery. n = 5–7. *p< 0.05; **p< 0.01; ***P < 0.001 (GNMT+/+ vs GNMT−/−) Error bars represent SD.
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Related In: Results  -  Collection

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Figure 3: Exacerbated fibrogenesis in GNMT−/− mice concurs with activation of NK1.1+ cells(A) IF using an αSMA Ab and (B) Sirius Red staining showed increased fibrosis in GNMT−/− mice. (C) qPCR analysis of mRNA expression of αSMA, collagen1A1 and TLR9 confirmed HSC activation. (D) Dot plot of FACS analysis on isolated liver mononuclear cells (MNCs) showing lower number of CD45+/NK1.1.+ cells in GNMT−/− mice. (E) Histogram showing higher presence of TRAIL in the cell surface of NK1.1+ cells in GNMT−/− liver-MNCs. Bar-plot representing the data obtained by FACS analysis showing low presence of NK1.1+ cells correlating with high expression of TRAIL in GNMT−/− liver-MNCs. (F) qPCR analysis showing increased DR5 mRNA expression in GNMT−/− mice. (H) Western blot analysis confirmed that GNMT−/− mice have higher expression of DR5 after BDL correlating with increased phosphorylation of JNK after surgery. n = 5–7. *p< 0.05; **p< 0.01; ***P < 0.001 (GNMT+/+ vs GNMT−/−) Error bars represent SD.
Mentions: First, we found that only 25% of GNMT−/− mice survived the BDL up to 21 days, whereas survival was 60% in GNMT WT animals (GNMT+/+) (Suppl. Fig. 1A). The increased activation of hepatic stellate cells (HSC) and stronger collagen deposition found in GNMT−/− were evidenced by αSMA IF (Fig. 3A) and Sirius Red staining (Fig. 3B) in liver sections and further quantification at 14d and 21d after BDL (Suppl. Fig. 1B). qPCR analysis of αSMA and collagen1A1 expression confirmed the increased pro-fibrogenic response in GNMT−/− mice after BDL in comparison with WT animals (Fig. 3C). Interestingly, BDL led to significant increase of TLR9 in GNMT−/− mice (Fig. 3C), a receptor involved in the activation of HSC after engulfment of apoptotic hepatocyte-DNA7.

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