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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 reverts the spontaneous damaging phenotype in GNMT−/− mice, which show attenuated NK cell activation(A) H&E staining and (B) Sirius Red staining and further quantification using FRIDA software showing attenuation of liver injury and fibrogenesis in TRAIL−/−/GNMT−/− mice compared to GNMT−/− at 3 and 9 months of age. (D) qRTPCR analysis showing increased Collagen1A1, αSMA and TGFβ expression in 9 months old GNMT−/− mice compared to TRAIL−/−/GNMT−/− mice that show comparable levels than WT animals (E) Macroscopic view of livers from GNMT−/− and TRAIL−/−/GNMT−/− mice evidencing apparent regression of the damaging phenotype in the 9 months of age double KO animals (F) Dot plot of FACS analysis and bar-plot showing restoration of the number of NK1.1+ cells in TRAIL−/−/GNMT−/− compared to GNMT−/− animals, all 3 months old. (G) pPCR analysis on FACSaria isolated NK1.1+ cells confirming low activation in TRAIL−/−/GNMT−/− cells compared to GNMT−/−. n= 5–10. *p<0.05; **p< 0.01, ***<0.001 (GNMT−/− vs TRAIL−/−/GNMT−/−). Error bars represent SD.
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Figure 2: TRAIL deficiency reverts the spontaneous damaging phenotype in GNMT−/− mice, which show attenuated NK cell activation(A) H&E staining and (B) Sirius Red staining and further quantification using FRIDA software showing attenuation of liver injury and fibrogenesis in TRAIL−/−/GNMT−/− mice compared to GNMT−/− at 3 and 9 months of age. (D) qRTPCR analysis showing increased Collagen1A1, αSMA and TGFβ expression in 9 months old GNMT−/− mice compared to TRAIL−/−/GNMT−/− mice that show comparable levels than WT animals (E) Macroscopic view of livers from GNMT−/− and TRAIL−/−/GNMT−/− mice evidencing apparent regression of the damaging phenotype in the 9 months of age double KO animals (F) Dot plot of FACS analysis and bar-plot showing restoration of the number of NK1.1+ cells in TRAIL−/−/GNMT−/− compared to GNMT−/− animals, all 3 months old. (G) pPCR analysis on FACSaria isolated NK1.1+ cells confirming low activation in TRAIL−/−/GNMT−/− cells compared to GNMT−/−. n= 5–10. *p<0.05; **p< 0.01, ***<0.001 (GNMT−/− vs TRAIL−/−/GNMT−/−). Error bars represent SD.

Mentions: In order to investigate the contribution of TRAIL to chronic liver disease we generated TRAIL- and GNMT-deficient mice (TRAIL−/−/GNMT−/−). TRAIL−/−/GNMT−/− mice showed an apparent remission of the spontaneous damaging phenotype found in GNMT−/− mice3. H&E staining evidenced a clear attenuation of the liver parenchymal damage, especially in 9 month-old TRAIL−/−/GNMT−/− mice compared to GNMT−/− animals (Fig. 2A). Moreover, we observed a significant reduction of liver fibrogenesis in 3 and 9 month-old TRAIL−/−/GNMT−/− compared to GNMT−/− mice as evidenced by Sirius red staining (Fig. 2B) and further quantification using Frida software (Fig. 2C). Further analysis by qRTPCR confirmed the beneficial impact of TRAIL deficiency in the spontaneous fibrogenesis observed in GNMT−/− animals as evidenced by lower expression of collagen 1A1, αSMA and TGFβ found in 9 months-old TRAIL−/−/GNMT−/− mice (Fig. 2D). Macroscopical analysis further confirmed the important beneficial impact of TRAIL deficiency in GNMT deficient mice in the attenuation of the cirrhotic/tumorigenic phenotype found in GNMT−/− mice at 9m of age (Fig. 2E). Importantly, the improved phenotype found in TRAIL−/−/GNMT−/− mice correlated with a significantly lower activation of NK cells. Hence, we found a significant higher number of NK1.1+/CD3− (NK) cells in TRAIL−/−/GNMT−/− compared to GNMT−/− mice at 3 months of age (Fig. 2F). Restoration of the presence of NK cells to a percentage comparable to WT animals, correlated with a lower expression of NK cell activation markers such as IFNγ, Granzyme and CCL5 determined by qRTPCR analysis in FACSaria isolated NK1.1+ cells (Fig. 2G). Overall, TRAIL deficiency efficiently restored the number and activation status of NK cells; NK1.1+/CD3− cells in GNMT−/− mice that reached comparable levels to WT animals (Fig. 2F, G). These data support the implication of TRAIL-producing NK cells in mediating liver injury and fibrogenesis in the pathological context of reduced GNMT expression.


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 reverts the spontaneous damaging phenotype in GNMT−/− mice, which show attenuated NK cell activation(A) H&E staining and (B) Sirius Red staining and further quantification using FRIDA software showing attenuation of liver injury and fibrogenesis in TRAIL−/−/GNMT−/− mice compared to GNMT−/− at 3 and 9 months of age. (D) qRTPCR analysis showing increased Collagen1A1, αSMA and TGFβ expression in 9 months old GNMT−/− mice compared to TRAIL−/−/GNMT−/− mice that show comparable levels than WT animals (E) Macroscopic view of livers from GNMT−/− and TRAIL−/−/GNMT−/− mice evidencing apparent regression of the damaging phenotype in the 9 months of age double KO animals (F) Dot plot of FACS analysis and bar-plot showing restoration of the number of NK1.1+ cells in TRAIL−/−/GNMT−/− compared to GNMT−/− animals, all 3 months old. (G) pPCR analysis on FACSaria isolated NK1.1+ cells confirming low activation in TRAIL−/−/GNMT−/− cells compared to GNMT−/−. n= 5–10. *p<0.05; **p< 0.01, ***<0.001 (GNMT−/− vs TRAIL−/−/GNMT−/−). Error bars represent SD.
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Figure 2: TRAIL deficiency reverts the spontaneous damaging phenotype in GNMT−/− mice, which show attenuated NK cell activation(A) H&E staining and (B) Sirius Red staining and further quantification using FRIDA software showing attenuation of liver injury and fibrogenesis in TRAIL−/−/GNMT−/− mice compared to GNMT−/− at 3 and 9 months of age. (D) qRTPCR analysis showing increased Collagen1A1, αSMA and TGFβ expression in 9 months old GNMT−/− mice compared to TRAIL−/−/GNMT−/− mice that show comparable levels than WT animals (E) Macroscopic view of livers from GNMT−/− and TRAIL−/−/GNMT−/− mice evidencing apparent regression of the damaging phenotype in the 9 months of age double KO animals (F) Dot plot of FACS analysis and bar-plot showing restoration of the number of NK1.1+ cells in TRAIL−/−/GNMT−/− compared to GNMT−/− animals, all 3 months old. (G) pPCR analysis on FACSaria isolated NK1.1+ cells confirming low activation in TRAIL−/−/GNMT−/− cells compared to GNMT−/−. n= 5–10. *p<0.05; **p< 0.01, ***<0.001 (GNMT−/− vs TRAIL−/−/GNMT−/−). Error bars represent SD.
Mentions: In order to investigate the contribution of TRAIL to chronic liver disease we generated TRAIL- and GNMT-deficient mice (TRAIL−/−/GNMT−/−). TRAIL−/−/GNMT−/− mice showed an apparent remission of the spontaneous damaging phenotype found in GNMT−/− mice3. H&E staining evidenced a clear attenuation of the liver parenchymal damage, especially in 9 month-old TRAIL−/−/GNMT−/− mice compared to GNMT−/− animals (Fig. 2A). Moreover, we observed a significant reduction of liver fibrogenesis in 3 and 9 month-old TRAIL−/−/GNMT−/− compared to GNMT−/− mice as evidenced by Sirius red staining (Fig. 2B) and further quantification using Frida software (Fig. 2C). Further analysis by qRTPCR confirmed the beneficial impact of TRAIL deficiency in the spontaneous fibrogenesis observed in GNMT−/− animals as evidenced by lower expression of collagen 1A1, αSMA and TGFβ found in 9 months-old TRAIL−/−/GNMT−/− mice (Fig. 2D). Macroscopical analysis further confirmed the important beneficial impact of TRAIL deficiency in GNMT deficient mice in the attenuation of the cirrhotic/tumorigenic phenotype found in GNMT−/− mice at 9m of age (Fig. 2E). Importantly, the improved phenotype found in TRAIL−/−/GNMT−/− mice correlated with a significantly lower activation of NK cells. Hence, we found a significant higher number of NK1.1+/CD3− (NK) cells in TRAIL−/−/GNMT−/− compared to GNMT−/− mice at 3 months of age (Fig. 2F). Restoration of the presence of NK cells to a percentage comparable to WT animals, correlated with a lower expression of NK cell activation markers such as IFNγ, Granzyme and CCL5 determined by qRTPCR analysis in FACSaria isolated NK1.1+ cells (Fig. 2G). Overall, TRAIL deficiency efficiently restored the number and activation status of NK cells; NK1.1+/CD3− cells in GNMT−/− mice that reached comparable levels to WT animals (Fig. 2F, G). These data support the implication of TRAIL-producing NK cells in mediating liver injury and fibrogenesis in the pathological context of reduced GNMT expression.

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