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The ROS scavenger, NAC, regulates hepatic Vα14iNKT cells signaling during Fas mAb-dependent fulminant liver failure.

Downs I, Liu J, Aw TY, Adegboyega PA, Ajuebor MN - PLoS ONE (2012)

Bottom Line: In addition, enrichment of the liver with GSH due to Vα14iNKT cells deficiency, induced an anti-inflammatory response in the liver of Jα18(-/-) mice that inhibited apoptosis, nitrotyrosine formation, IFN-γ signaling and effector functions.In summary, we propose a novel and previously unrecognized pro-inflammatory and pro-apoptotic role for endogenous ROS in stimulating Th1 signaling in Vα14iNKT cells to promote the development of FLF.Therefore, our study provides critical new insights into how NAC, a ROS scavenger, regulates Th1 signaling in intrahepatic Vα14iNKT cells to impact inflammatory and pathological responses.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center-Shreveport, Shreveport, Louisiana, United States of America.

ABSTRACT
Uncontrolled systemic activation of the immune system is an early initiating event that leads to development of acute fulminant liver failure (FLF) in mice after treatment with agonistic Fas mAb. In this study, we demonstrate that treatment of mice with N-acetylcysteine (NAC), an ROS scavenger and glutathione (GSH) precursor, almost completely abolished Fas mAb-induced FLF through suppression of Vα14iNKT cell activation, IFN-γ signaling, apoptosis and nitrotyrosine formation in liver. In addition, enrichment of the liver with GSH due to Vα14iNKT cells deficiency, induced an anti-inflammatory response in the liver of Jα18(-/-) mice that inhibited apoptosis, nitrotyrosine formation, IFN-γ signaling and effector functions. In summary, we propose a novel and previously unrecognized pro-inflammatory and pro-apoptotic role for endogenous ROS in stimulating Th1 signaling in Vα14iNKT cells to promote the development of FLF. Therefore, our study provides critical new insights into how NAC, a ROS scavenger, regulates Th1 signaling in intrahepatic Vα14iNKT cells to impact inflammatory and pathological responses.

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Th1 differentiating signaling in the liver is dysregulated by Vα14iNKT cells deficiency during Fas mAb-induced FLF.(a) Serum ALT levels of naïve WT mice, Fas mAb-treated WT and Jα18−/− mice at 4.5 h. (b–d) H & E staining of liver sections from naïve WT mice, Fas mAb-treated WT and Jα18−/− mice at 4.5 h. Livers from Fas mAb-treated WT mice (c) showed extensive damage with destruction of hepatocytes and distortion of normal liver architecture. The hepatocytes show hemorrhagic necrosis (white arrows) and characteristic signs of apoptosis (black arrows) including chromatin condensation and cellular shrinkage. In comparison, livers from Fas mAb-treated Jα18−/− mice showed only minimal damage and retained the normal architecture (d). Liver from a naïve WT mouse is illustrated in (b) for comparison. (e) Western blot analysis of active caspase 3, T-bet, pSTAT-1, nitrotyrosine and GAPDH expression in the liver of PBS-treated WT mice and agonistic Fas mAb-treated WT and Jα18−/− mice at 4.5 h. (f) TUNEL staining of liver sections from WT and Jα18−/− mice at 4.5 h after Fas mAb injection in which WT mice showed intense TUNEL staining characteristic of apoptosis whereas Jα18−/− mice showed less/reduced TUNEL staining. (g) HPLC measurement of hepatic GSH levels in PBS-treated WT mice and Fas mAb-treated WT and Jα18−/− mice at 4.5 h. Figure S1 in a and g are presented as mean ± s.e.m with n = 5 mice/group; *P<0.05 by one-way analysis of variance followed by Newman-Kuels post hoc test. All experiments were conducted twice.
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pone-0038051-g001: Th1 differentiating signaling in the liver is dysregulated by Vα14iNKT cells deficiency during Fas mAb-induced FLF.(a) Serum ALT levels of naïve WT mice, Fas mAb-treated WT and Jα18−/− mice at 4.5 h. (b–d) H & E staining of liver sections from naïve WT mice, Fas mAb-treated WT and Jα18−/− mice at 4.5 h. Livers from Fas mAb-treated WT mice (c) showed extensive damage with destruction of hepatocytes and distortion of normal liver architecture. The hepatocytes show hemorrhagic necrosis (white arrows) and characteristic signs of apoptosis (black arrows) including chromatin condensation and cellular shrinkage. In comparison, livers from Fas mAb-treated Jα18−/− mice showed only minimal damage and retained the normal architecture (d). Liver from a naïve WT mouse is illustrated in (b) for comparison. (e) Western blot analysis of active caspase 3, T-bet, pSTAT-1, nitrotyrosine and GAPDH expression in the liver of PBS-treated WT mice and agonistic Fas mAb-treated WT and Jα18−/− mice at 4.5 h. (f) TUNEL staining of liver sections from WT and Jα18−/− mice at 4.5 h after Fas mAb injection in which WT mice showed intense TUNEL staining characteristic of apoptosis whereas Jα18−/− mice showed less/reduced TUNEL staining. (g) HPLC measurement of hepatic GSH levels in PBS-treated WT mice and Fas mAb-treated WT and Jα18−/− mice at 4.5 h. Figure S1 in a and g are presented as mean ± s.e.m with n = 5 mice/group; *P<0.05 by one-way analysis of variance followed by Newman-Kuels post hoc test. All experiments were conducted twice.

Mentions: We first confirmed our recent observation [5] that the presence of hepatic Vα14iNKT cells promote acute FLF in response to agonistic Fas mAb treatment. Specifically, we found that Fas mAb administration into WT mice caused a significant increase in serum ALT level whereas Jα18−/− mice were highly resistant to acute FLF as reflected by almost complete suppression (>90% reduction) of serum ALT (Figure 1A). In parallel, liver sections from WT mice exhibited extensive hepatocyte apoptosis and necrotic damage following Fas mAb treatment relative to livers from Jα18−/− mice which displayed mild hepatocyte damage (Figure 1C and D). Specifically, the degree of hepatic inflammation and hepatocyte damage in WT mice after Fas mAb treatment was graded as severe (>50%) relative to mild (<25%) in Jα18−/− mice. As expected, normal serum ALT levels was observed in both naive WT and J Jα18−/− mice (Figure 1A). In the present study, we provide new data demonstrating that resistance of Jα18−/− mice to FLF was associated with a dramatic decrease in hepatic apoptosis as revealed by reduced expression of active caspase 3 and TUNEL staining in the liver (Figure 1E and 1F). The finding that active caspase 3 expression was not completely suppressed in Jα18−/− mice after Fas mAb treatment suggests that other hepatic cells apart from intrahepatic Vα14iNKT cells may also contribute to apoptosis. It is notable that reduced susceptibility of Jα18−/− mice to FLF was also accompanied by striking reductions in hepatic expression of Th1 differentiating signaling molecules, pSTAT-1 and T-bet (Figure 1E). To determine whether oxidative and nitrosative stress may also contribute to the development of FLF, we measured nitrotyrosine formation (a product of nitrosative stress) and the ROS scavenger, GSH. We observed a striking increase in nitrotyrosine formation in the liver of WT mice but not Jα18−/− mice after Fas mAb administration (Figure 1E). Remarkably, we also found that Fas mAb-mediated FLF in WT mice caused a significant decrease in hepatic GSH (relative to PBS-treated WT mice), but GSH levels were restored in the absence of Vα14iNKT cells (i.e. in Jα18−/− mice) during mild FLF to levels seen in PBS-treated WT mice (Figure 1G).


The ROS scavenger, NAC, regulates hepatic Vα14iNKT cells signaling during Fas mAb-dependent fulminant liver failure.

Downs I, Liu J, Aw TY, Adegboyega PA, Ajuebor MN - PLoS ONE (2012)

Th1 differentiating signaling in the liver is dysregulated by Vα14iNKT cells deficiency during Fas mAb-induced FLF.(a) Serum ALT levels of naïve WT mice, Fas mAb-treated WT and Jα18−/− mice at 4.5 h. (b–d) H & E staining of liver sections from naïve WT mice, Fas mAb-treated WT and Jα18−/− mice at 4.5 h. Livers from Fas mAb-treated WT mice (c) showed extensive damage with destruction of hepatocytes and distortion of normal liver architecture. The hepatocytes show hemorrhagic necrosis (white arrows) and characteristic signs of apoptosis (black arrows) including chromatin condensation and cellular shrinkage. In comparison, livers from Fas mAb-treated Jα18−/− mice showed only minimal damage and retained the normal architecture (d). Liver from a naïve WT mouse is illustrated in (b) for comparison. (e) Western blot analysis of active caspase 3, T-bet, pSTAT-1, nitrotyrosine and GAPDH expression in the liver of PBS-treated WT mice and agonistic Fas mAb-treated WT and Jα18−/− mice at 4.5 h. (f) TUNEL staining of liver sections from WT and Jα18−/− mice at 4.5 h after Fas mAb injection in which WT mice showed intense TUNEL staining characteristic of apoptosis whereas Jα18−/− mice showed less/reduced TUNEL staining. (g) HPLC measurement of hepatic GSH levels in PBS-treated WT mice and Fas mAb-treated WT and Jα18−/− mice at 4.5 h. Figure S1 in a and g are presented as mean ± s.e.m with n = 5 mice/group; *P<0.05 by one-way analysis of variance followed by Newman-Kuels post hoc test. All experiments were conducted twice.
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Related In: Results  -  Collection

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pone-0038051-g001: Th1 differentiating signaling in the liver is dysregulated by Vα14iNKT cells deficiency during Fas mAb-induced FLF.(a) Serum ALT levels of naïve WT mice, Fas mAb-treated WT and Jα18−/− mice at 4.5 h. (b–d) H & E staining of liver sections from naïve WT mice, Fas mAb-treated WT and Jα18−/− mice at 4.5 h. Livers from Fas mAb-treated WT mice (c) showed extensive damage with destruction of hepatocytes and distortion of normal liver architecture. The hepatocytes show hemorrhagic necrosis (white arrows) and characteristic signs of apoptosis (black arrows) including chromatin condensation and cellular shrinkage. In comparison, livers from Fas mAb-treated Jα18−/− mice showed only minimal damage and retained the normal architecture (d). Liver from a naïve WT mouse is illustrated in (b) for comparison. (e) Western blot analysis of active caspase 3, T-bet, pSTAT-1, nitrotyrosine and GAPDH expression in the liver of PBS-treated WT mice and agonistic Fas mAb-treated WT and Jα18−/− mice at 4.5 h. (f) TUNEL staining of liver sections from WT and Jα18−/− mice at 4.5 h after Fas mAb injection in which WT mice showed intense TUNEL staining characteristic of apoptosis whereas Jα18−/− mice showed less/reduced TUNEL staining. (g) HPLC measurement of hepatic GSH levels in PBS-treated WT mice and Fas mAb-treated WT and Jα18−/− mice at 4.5 h. Figure S1 in a and g are presented as mean ± s.e.m with n = 5 mice/group; *P<0.05 by one-way analysis of variance followed by Newman-Kuels post hoc test. All experiments were conducted twice.
Mentions: We first confirmed our recent observation [5] that the presence of hepatic Vα14iNKT cells promote acute FLF in response to agonistic Fas mAb treatment. Specifically, we found that Fas mAb administration into WT mice caused a significant increase in serum ALT level whereas Jα18−/− mice were highly resistant to acute FLF as reflected by almost complete suppression (>90% reduction) of serum ALT (Figure 1A). In parallel, liver sections from WT mice exhibited extensive hepatocyte apoptosis and necrotic damage following Fas mAb treatment relative to livers from Jα18−/− mice which displayed mild hepatocyte damage (Figure 1C and D). Specifically, the degree of hepatic inflammation and hepatocyte damage in WT mice after Fas mAb treatment was graded as severe (>50%) relative to mild (<25%) in Jα18−/− mice. As expected, normal serum ALT levels was observed in both naive WT and J Jα18−/− mice (Figure 1A). In the present study, we provide new data demonstrating that resistance of Jα18−/− mice to FLF was associated with a dramatic decrease in hepatic apoptosis as revealed by reduced expression of active caspase 3 and TUNEL staining in the liver (Figure 1E and 1F). The finding that active caspase 3 expression was not completely suppressed in Jα18−/− mice after Fas mAb treatment suggests that other hepatic cells apart from intrahepatic Vα14iNKT cells may also contribute to apoptosis. It is notable that reduced susceptibility of Jα18−/− mice to FLF was also accompanied by striking reductions in hepatic expression of Th1 differentiating signaling molecules, pSTAT-1 and T-bet (Figure 1E). To determine whether oxidative and nitrosative stress may also contribute to the development of FLF, we measured nitrotyrosine formation (a product of nitrosative stress) and the ROS scavenger, GSH. We observed a striking increase in nitrotyrosine formation in the liver of WT mice but not Jα18−/− mice after Fas mAb administration (Figure 1E). Remarkably, we also found that Fas mAb-mediated FLF in WT mice caused a significant decrease in hepatic GSH (relative to PBS-treated WT mice), but GSH levels were restored in the absence of Vα14iNKT cells (i.e. in Jα18−/− mice) during mild FLF to levels seen in PBS-treated WT mice (Figure 1G).

Bottom Line: In addition, enrichment of the liver with GSH due to Vα14iNKT cells deficiency, induced an anti-inflammatory response in the liver of Jα18(-/-) mice that inhibited apoptosis, nitrotyrosine formation, IFN-γ signaling and effector functions.In summary, we propose a novel and previously unrecognized pro-inflammatory and pro-apoptotic role for endogenous ROS in stimulating Th1 signaling in Vα14iNKT cells to promote the development of FLF.Therefore, our study provides critical new insights into how NAC, a ROS scavenger, regulates Th1 signaling in intrahepatic Vα14iNKT cells to impact inflammatory and pathological responses.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center-Shreveport, Shreveport, Louisiana, United States of America.

ABSTRACT
Uncontrolled systemic activation of the immune system is an early initiating event that leads to development of acute fulminant liver failure (FLF) in mice after treatment with agonistic Fas mAb. In this study, we demonstrate that treatment of mice with N-acetylcysteine (NAC), an ROS scavenger and glutathione (GSH) precursor, almost completely abolished Fas mAb-induced FLF through suppression of Vα14iNKT cell activation, IFN-γ signaling, apoptosis and nitrotyrosine formation in liver. In addition, enrichment of the liver with GSH due to Vα14iNKT cells deficiency, induced an anti-inflammatory response in the liver of Jα18(-/-) mice that inhibited apoptosis, nitrotyrosine formation, IFN-γ signaling and effector functions. In summary, we propose a novel and previously unrecognized pro-inflammatory and pro-apoptotic role for endogenous ROS in stimulating Th1 signaling in Vα14iNKT cells to promote the development of FLF. Therefore, our study provides critical new insights into how NAC, a ROS scavenger, regulates Th1 signaling in intrahepatic Vα14iNKT cells to impact inflammatory and pathological responses.

Show MeSH
Related in: MedlinePlus