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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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NAC therapy ameliorates agonistic Fas mAb-induced FLF during IFN-γ deficiency.(a) Serum ALT levels in WT and IFN-γ−/− mice after PBS or NAC treatment during agonistic Fas mAb-induced FLF. (b) H & E staining of liver sections of WT and IFN-γ−/− mice after PBS or NAC treatment during agonistic Fas mAb-induced FLF. As shown in top panel, livers from Fas mAb-treated WT and IFN-γ−/− mice displayed widespread hepatocyte damage including hemorrhagic necrosis (white arrows) and apoptosis (black arrows) that distorted normal liver architecture. In contrast, liver sections of WT and IFN-γ−/− mice treated with NAC during Fas mAb-induced FLF (bottom panel) showed reduced hepatocyte damage and retained near normal architecture. (c & e) Western blot analysis of hepatic active caspase 3, T-bet, pSTAT-1 expression levels and nitrotyrosine formation in WT and IFN-γ−/− mice after PBS or NAC treatment during Fas mAb-induced FLF. (d) TUNEL staining of liver sections from WT and IFN-γ−/− mice treated with PBS during Fas mAb-induced FLF showed intense TUNEL staining characteristic of apoptosis whereas WT and IFN-γ−/− mice treated with NAC mice showed minimal TUNEL staining. Figure S1 in a are presented as mean ± s.e.m with n = 3–6 mice/group. *P<0.05, ≠P<0.05 by one-way analysis of variance followed by Newman-Kuels post hoc test. All experiments were performed twice.
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pone-0038051-g003: NAC therapy ameliorates agonistic Fas mAb-induced FLF during IFN-γ deficiency.(a) Serum ALT levels in WT and IFN-γ−/− mice after PBS or NAC treatment during agonistic Fas mAb-induced FLF. (b) H & E staining of liver sections of WT and IFN-γ−/− mice after PBS or NAC treatment during agonistic Fas mAb-induced FLF. As shown in top panel, livers from Fas mAb-treated WT and IFN-γ−/− mice displayed widespread hepatocyte damage including hemorrhagic necrosis (white arrows) and apoptosis (black arrows) that distorted normal liver architecture. In contrast, liver sections of WT and IFN-γ−/− mice treated with NAC during Fas mAb-induced FLF (bottom panel) showed reduced hepatocyte damage and retained near normal architecture. (c & e) Western blot analysis of hepatic active caspase 3, T-bet, pSTAT-1 expression levels and nitrotyrosine formation in WT and IFN-γ−/− mice after PBS or NAC treatment during Fas mAb-induced FLF. (d) TUNEL staining of liver sections from WT and IFN-γ−/− mice treated with PBS during Fas mAb-induced FLF showed intense TUNEL staining characteristic of apoptosis whereas WT and IFN-γ−/− mice treated with NAC mice showed minimal TUNEL staining. Figure S1 in a are presented as mean ± s.e.m with n = 3–6 mice/group. *P<0.05, ≠P<0.05 by one-way analysis of variance followed by Newman-Kuels post hoc test. All experiments were performed twice.

Mentions: In view of our preceding findings, we next treated WT and IFN-γ−/− mice with agonistic Fas mAb to evaluate whether IFN-γ is an essential and direct participant in FLF. As shown in Figure 3A, both WT and IFN-γ−/− mice were similarly susceptible to acute FLF since serum ALT levels in both mice strains were comparable. In correlation, histological evaluation of liver sections showed that the degree of hepatic inflammation and hepatocyte damage in both strains of mice was severe (>50%; Figure 3B, top panels). Histological scoring criteria was based on the severity/magnitude of inflammation, and the degree of hepatocyte degenerative changes including hepatocyte necrosis, hemorrhage and frequency of acidophilic bodies [5], [35]. Likewise, active caspase 3 expression and tunnel staining in the liver during IFN-γ deficiency was comparable to levels in WT mice during Fas mAb-mediated acute FLF (Figure 3C and D). It is also notable that the strong expression levels of pSTAT-1 and T-bet in the liver of WT mice during FLF was not suppressed by IFN-γ deficiency (Figure 3C). Likewise, nitrotyrosine formation in the liver of WT mice was comparable to IFN-γ−/− mice during Fas mAb-mediated acute FLF (Figure 3E). These data strongly suggests that IFN-γ may not be a key and/or direct mediator of FLF in response to agonistic Fas mAb treatment.


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)

NAC therapy ameliorates agonistic Fas mAb-induced FLF during IFN-γ deficiency.(a) Serum ALT levels in WT and IFN-γ−/− mice after PBS or NAC treatment during agonistic Fas mAb-induced FLF. (b) H & E staining of liver sections of WT and IFN-γ−/− mice after PBS or NAC treatment during agonistic Fas mAb-induced FLF. As shown in top panel, livers from Fas mAb-treated WT and IFN-γ−/− mice displayed widespread hepatocyte damage including hemorrhagic necrosis (white arrows) and apoptosis (black arrows) that distorted normal liver architecture. In contrast, liver sections of WT and IFN-γ−/− mice treated with NAC during Fas mAb-induced FLF (bottom panel) showed reduced hepatocyte damage and retained near normal architecture. (c & e) Western blot analysis of hepatic active caspase 3, T-bet, pSTAT-1 expression levels and nitrotyrosine formation in WT and IFN-γ−/− mice after PBS or NAC treatment during Fas mAb-induced FLF. (d) TUNEL staining of liver sections from WT and IFN-γ−/− mice treated with PBS during Fas mAb-induced FLF showed intense TUNEL staining characteristic of apoptosis whereas WT and IFN-γ−/− mice treated with NAC mice showed minimal TUNEL staining. Figure S1 in a are presented as mean ± s.e.m with n = 3–6 mice/group. *P<0.05, ≠P<0.05 by one-way analysis of variance followed by Newman-Kuels post hoc test. All experiments were performed twice.
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pone-0038051-g003: NAC therapy ameliorates agonistic Fas mAb-induced FLF during IFN-γ deficiency.(a) Serum ALT levels in WT and IFN-γ−/− mice after PBS or NAC treatment during agonistic Fas mAb-induced FLF. (b) H & E staining of liver sections of WT and IFN-γ−/− mice after PBS or NAC treatment during agonistic Fas mAb-induced FLF. As shown in top panel, livers from Fas mAb-treated WT and IFN-γ−/− mice displayed widespread hepatocyte damage including hemorrhagic necrosis (white arrows) and apoptosis (black arrows) that distorted normal liver architecture. In contrast, liver sections of WT and IFN-γ−/− mice treated with NAC during Fas mAb-induced FLF (bottom panel) showed reduced hepatocyte damage and retained near normal architecture. (c & e) Western blot analysis of hepatic active caspase 3, T-bet, pSTAT-1 expression levels and nitrotyrosine formation in WT and IFN-γ−/− mice after PBS or NAC treatment during Fas mAb-induced FLF. (d) TUNEL staining of liver sections from WT and IFN-γ−/− mice treated with PBS during Fas mAb-induced FLF showed intense TUNEL staining characteristic of apoptosis whereas WT and IFN-γ−/− mice treated with NAC mice showed minimal TUNEL staining. Figure S1 in a are presented as mean ± s.e.m with n = 3–6 mice/group. *P<0.05, ≠P<0.05 by one-way analysis of variance followed by Newman-Kuels post hoc test. All experiments were performed twice.
Mentions: In view of our preceding findings, we next treated WT and IFN-γ−/− mice with agonistic Fas mAb to evaluate whether IFN-γ is an essential and direct participant in FLF. As shown in Figure 3A, both WT and IFN-γ−/− mice were similarly susceptible to acute FLF since serum ALT levels in both mice strains were comparable. In correlation, histological evaluation of liver sections showed that the degree of hepatic inflammation and hepatocyte damage in both strains of mice was severe (>50%; Figure 3B, top panels). Histological scoring criteria was based on the severity/magnitude of inflammation, and the degree of hepatocyte degenerative changes including hepatocyte necrosis, hemorrhage and frequency of acidophilic bodies [5], [35]. Likewise, active caspase 3 expression and tunnel staining in the liver during IFN-γ deficiency was comparable to levels in WT mice during Fas mAb-mediated acute FLF (Figure 3C and D). It is also notable that the strong expression levels of pSTAT-1 and T-bet in the liver of WT mice during FLF was not suppressed by IFN-γ deficiency (Figure 3C). Likewise, nitrotyrosine formation in the liver of WT mice was comparable to IFN-γ−/− mice during Fas mAb-mediated acute FLF (Figure 3E). These data strongly suggests that IFN-γ may not be a key and/or direct mediator of FLF in response to agonistic Fas mAb treatment.

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