Glutathione reductase (Gsr) catalyzes the reduction of glutathione disulfide (GSSG) to glutathion... more Glutathione reductase (Gsr) catalyzes the reduction of glutathione disulfide (GSSG) to glutathione (GSH), a major cellular antioxidant. We have previously shown that Gsr facilitates neutrophil bactericidal activities and is pivotal for host defense against bacterial pathogens. However, it is unclear whether Gsr is required for immune defense against fungal pathogens. It is also unclear whether Gsr plays a role in immunological functions outside of neutrophils. To address these questions, we studied the effect of Gsr knockout on immune defense against C. albicans. Upon infection by C. albicans, Gsr−/− mice displayed dramatically increased fungal burden in the kidneys, cytokine and chemokine storm, striking neutrophil infiltration and histological abnormalities in both the kidneys and hearts, and substantially elevated mortality. Large fungal foci surrounded by massive numbers of neutrophils were detected outside of glomerulus in the kidneys of Gsr−/− mice but were not found in wildtype mice. Examination of the neutrophils and macrophages of Gsr−/− mice also revealed several defects, including compromised phagocytosis, attenuated respiratory burst, and impaired fungicidal activity in Gsr−/− neutrophils in vitro. Moreover, upon C. albicans stimulation, Gsr−/− macrophages produced increased levels of inflammatory cytokines and exhibited elevated p38 and JNK activities, at least in part due to lower mitogen-activated protein kinase phosphatase (Mkp)-1 activity and greater Syk activity. Thus, Gsr-mediated redox regulation was found to be crucial for fungal clearance by neutrophils and the proper control of the inflammatory response by macrophages during host defense against fungal challenge.
Glutathione reductase (Gsr) catalyzes the reduction of glutathione disulfide to glutathione, whic... more Glutathione reductase (Gsr) catalyzes the reduction of glutathione disulfide to glutathione, which plays an important role in Redox regulation. We expressed Gsr as a GST-fusion protein and produced a rabbit polyclonal antibody against Gsr. Using this antibody we examined Gsr expression levels in various mouse tissues. We found that Gsr was highly expressed in a variety of tissues, including lung, kidney, eyes, spleen, thymus, and bone marrow, while Gsr expression was very low in muscle and heart. Importantly, Gsr was not detected in any tissues of the Gsr hypomorphic mice. Because Gsr is expressed in lymphoid tissues and is implicated in phagocytic functions, we assessed the effect of Gsr deficiency on immune defense against a fungal pathogen, Candida albicans. We report in this study that Gsr-deficient mice exhibited substantially enhanced susceptibility to C. albicans challenge. Upon C. albicans infection, Gsr null mice exhibited dramatically increased fungal burdens in the kidneys, cytokine and chemokine storm, striking neutrophil infiltration and histological abnormalities in both the kidneys and hearts, and substantially elevated mortality. Large fungal foci surrounded by massive numbers of neutrophils were detected outside of glomerulus in the kidneys of Gsr null mice but not in wildtype mice. Examination of the bactericidal functions of the neutrophils from Gsr-deficient mice in vitro revealed normal phagocytosis and yet attenuated oxidative burst activity. Thus, Gsr-mediated redox regulation is crucial for fungal clearance during host defense against fungal challenge.
Akt-1 and Akt-2 are the major isoforms of the serine/threonine Akt family that play a key role in... more Akt-1 and Akt-2 are the major isoforms of the serine/threonine Akt family that play a key role in controlling immune responses. However, the involvement of Akt-1 and Akt-2 isoforms in antifungal innate immunity is completely unknown. In this study, we show that Akt2−/−, but not Akt1−/−, mice are protected from lethal Candida albicans infection. Loss of Akt-2 facilitates the recruitment of neutrophils and macrophages to the spleen and increases reactive oxygen species expression in these cells. Treating C57BL/6 mice with a specific inhibitor for Akt-2, but not Akt-1, provides protection from lethal C. albicans infection. Our data demonstrate that Akt-2 inhibits antifungal innate immunity by hampering neutrophil and macrophage recruitment to spleens and suppressing oxidative burst, myeloperoxidase activity, and NETosis. We thus describe a novel role for Akt-2 in the regulation of antifungal innate immunity and unveil Akt-2 as a potential target for the treatment of fungal sepsis.
Sepsis is the leading cause of death in the world. Recent reports suggest that in response to sep... more Sepsis is the leading cause of death in the world. Recent reports suggest that in response to sepsis, metabolism of macrophages switches from oxidative phosphorylation to aerobic glycolysis. MAPK phosphatase (MKP)–1 (also known as DUSP1) localized in the nucleus and preferentially dephosphorylates p38 and JNK. MKP-1 controls the expression of numerous inflammatory genes and transcription factors, thereby regulating innate and adaptive immunity. MKP-1–deficient animals exhibit aberrant metabolic responses following bacterial infections with a markedly increased mortality in response to sepsis. Because metabolic reprogramming modulates immune responses to TLR-4 activation, we investigated the effect of MKP-1 deficiency on mitochondrial electron transport chains involved in oxidative phosphorylation and transcription factors regulating mitochondrial biogenesis. Mitochondrial biogenesis is regulated by three nuclear-encoded proteins, including transcription factor A (TFAM), nuclear resp...
The mammalian response to stress is complex, often involving multiple signalling pathways that ac... more The mammalian response to stress is complex, often involving multiple signalling pathways that act in concert to influence cell fate. To examine potential interactions between the signalling cascades, we have focused on the effects of a model oxidant stress in a single cell type through an examination of the relative influences of mitogen-activated protein kinases (MAPKs) as well as two proposed apoptosis regulators, nuclear factor κB (NF-κB) and Bcl-2, in determining cell survival. Treatment of HeLa cells with H2O2 resulted in a time- and dose-dependent induction of apoptosis accompanied by sustained activation of all three MAPK subfamilies: extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38. This H2O2-induced apoptosis was markedly enhanced when ERK2 activation was selectively inhibited by PD098059. Apoptosis decreased when JNK/SAPK activation was inhibited by expression of a dominant negative mutant form...
Exposure of cells to either proliferative or stressful stimuli elicits a complex response involvi... more Exposure of cells to either proliferative or stressful stimuli elicits a complex response involving one or more distinct phosphorylation cascades culminating in the activation of multiple members of the mitogen-activated protein kinase (MAPK) family, including ...
Vascular endothelial growth factor (VEGF) is required for proper lung development and is transcri... more Vascular endothelial growth factor (VEGF) is required for proper lung development and is transcriptionally regulated in alveolar epithelial cells by hypoxia-inducible factor (HIF). Previous findings in a newborn mouse model of bronchopulmonary dysplasia (BPD) suggest that thioredoxin-interacting protein (Txnip) is a novel regulator of VEGF expression. The present studies were designed to test the hypothesis that Txnip negatively regulates VEGF through effects on HIF-mediated gene expression. To test this hypothesis, we first examined the levels of VEGF and Txnip protein in the lungs of 1-day-old newborn mice and E19 embryos and detected a significant inverse correlation. To elucidate the mechanisms underlying this relationship, we studied the effects of Txnip overexpression on HIF-mediated transcription using murine lung epithelial (MLE-12) cells. Overexpression of Txnip inhibited HIF-mediated reporter activity in both hypoxia and room air. Suppression of HIF activity by Txnip seemed to be independent of the ability of Txnip to bind to thioredoxin. Thus, our studies support a model in which Txnip is a potentially critical regulator of HIF-mediated gene transcription in the murine lung. Alterations in Txnip expression could alter lung VEGF expression in prematurely born human infants and contribute to the development of BPD.
Nitration products of unsaturated fatty acids are formed via NO-dependent oxidative reactions and... more Nitration products of unsaturated fatty acids are formed via NO-dependent oxidative reactions and appear to be a new class of endogenous antiinflammatory mediators. Nitroalkene derivatives of nitrated linoleic acid (LNO2) and nitrated oleic acid (OA-NO2) alleviate inflammatory responses in macrophages, but the underlying mechanisms remain to be fully defined. Herein we report that LNO2 and OA-NO2 suppress proinflammatory signal transducer and activator of transcription (STAT) signaling in macrophages. In RAW264.7 cells, a murine macrophage cell line, LNO2 and OA-NO2 inhibited the lipopolysaccharide (LPS)-induced STAT1 phosphorylation and the STAT1-dependent transcriptional activity, thereby suppressing expression of its target gene such as iNOS and MCP-1. The nitroalkene-mediated inhibition of STAT1 activity was not affected by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (a NO scavenger), GW9662 (a peroxisome proliferator-activated receptor-γ-specific antagonis...
Mitogen-activated protein (MAP) kinases play a critical role in innate immune responses to microb... more Mitogen-activated protein (MAP) kinases play a critical role in innate immune responses to microbial infection through eliciting the biosynthesis of proinflammatory cytokines. MAP phosphatases (MKP)-1 is an archetypical member of the dual-specificity phosphatase family that deactivates MAP kinases. Induction of MKP-1 has been implicated in attenuating the lipopolysaccharide (LPS) and Peptidoglycan (PGN) responses, but how the expression of the MKP-1 is regulated is still not fully understood. Here, we show that inhibition of p38 MAP kinase by specific inhibitor SB 203580 or RNA interference (RNAi) markedly reduced the expression of MKP-1 in LPS or PGN-treated macrophages, which is correlated with prolonged activation of p38 and JNK. Depletion of MAPKAP kinase 2 (MK2), a downstream substrate of p38, by RNAi also inhibited the expression of MKP-1. The mRNA level of MKP-1 is not affected by inhibition of p38, but the expression of MKP-1 is inhibited by treatment of cycloheximide. Thus, p38 MAPK plays a critical role in mediating expression of MKP-1 at a post-transcriptional level. Furthermore, inhibition of p38 by SB 203580 prevented the expression of MKP-1 in LPS-tolerized macrophages, restored the activation of MAP kinases after LPS restimulation. These results indicate a critical role of p38-MK2-dependent induction of MKP-1 in innate immune responses.
Glutathione reductase (Gsr) catalyzes the reduction of glutathione disulfide (GSSG) to glutathion... more Glutathione reductase (Gsr) catalyzes the reduction of glutathione disulfide (GSSG) to glutathione (GSH), a major cellular antioxidant. We have previously shown that Gsr facilitates neutrophil bactericidal activities and is pivotal for host defense against bacterial pathogens. However, it is unclear whether Gsr is required for immune defense against fungal pathogens. It is also unclear whether Gsr plays a role in immunological functions outside of neutrophils. To address these questions, we studied the effect of Gsr knockout on immune defense against C. albicans. Upon infection by C. albicans, Gsr−/− mice displayed dramatically increased fungal burden in the kidneys, cytokine and chemokine storm, striking neutrophil infiltration and histological abnormalities in both the kidneys and hearts, and substantially elevated mortality. Large fungal foci surrounded by massive numbers of neutrophils were detected outside of glomerulus in the kidneys of Gsr−/− mice but were not found in wildtype mice. Examination of the neutrophils and macrophages of Gsr−/− mice also revealed several defects, including compromised phagocytosis, attenuated respiratory burst, and impaired fungicidal activity in Gsr−/− neutrophils in vitro. Moreover, upon C. albicans stimulation, Gsr−/− macrophages produced increased levels of inflammatory cytokines and exhibited elevated p38 and JNK activities, at least in part due to lower mitogen-activated protein kinase phosphatase (Mkp)-1 activity and greater Syk activity. Thus, Gsr-mediated redox regulation was found to be crucial for fungal clearance by neutrophils and the proper control of the inflammatory response by macrophages during host defense against fungal challenge.
Glutathione reductase (Gsr) catalyzes the reduction of glutathione disulfide to glutathione, whic... more Glutathione reductase (Gsr) catalyzes the reduction of glutathione disulfide to glutathione, which plays an important role in Redox regulation. We expressed Gsr as a GST-fusion protein and produced a rabbit polyclonal antibody against Gsr. Using this antibody we examined Gsr expression levels in various mouse tissues. We found that Gsr was highly expressed in a variety of tissues, including lung, kidney, eyes, spleen, thymus, and bone marrow, while Gsr expression was very low in muscle and heart. Importantly, Gsr was not detected in any tissues of the Gsr hypomorphic mice. Because Gsr is expressed in lymphoid tissues and is implicated in phagocytic functions, we assessed the effect of Gsr deficiency on immune defense against a fungal pathogen, Candida albicans. We report in this study that Gsr-deficient mice exhibited substantially enhanced susceptibility to C. albicans challenge. Upon C. albicans infection, Gsr null mice exhibited dramatically increased fungal burdens in the kidneys, cytokine and chemokine storm, striking neutrophil infiltration and histological abnormalities in both the kidneys and hearts, and substantially elevated mortality. Large fungal foci surrounded by massive numbers of neutrophils were detected outside of glomerulus in the kidneys of Gsr null mice but not in wildtype mice. Examination of the bactericidal functions of the neutrophils from Gsr-deficient mice in vitro revealed normal phagocytosis and yet attenuated oxidative burst activity. Thus, Gsr-mediated redox regulation is crucial for fungal clearance during host defense against fungal challenge.
Akt-1 and Akt-2 are the major isoforms of the serine/threonine Akt family that play a key role in... more Akt-1 and Akt-2 are the major isoforms of the serine/threonine Akt family that play a key role in controlling immune responses. However, the involvement of Akt-1 and Akt-2 isoforms in antifungal innate immunity is completely unknown. In this study, we show that Akt2−/−, but not Akt1−/−, mice are protected from lethal Candida albicans infection. Loss of Akt-2 facilitates the recruitment of neutrophils and macrophages to the spleen and increases reactive oxygen species expression in these cells. Treating C57BL/6 mice with a specific inhibitor for Akt-2, but not Akt-1, provides protection from lethal C. albicans infection. Our data demonstrate that Akt-2 inhibits antifungal innate immunity by hampering neutrophil and macrophage recruitment to spleens and suppressing oxidative burst, myeloperoxidase activity, and NETosis. We thus describe a novel role for Akt-2 in the regulation of antifungal innate immunity and unveil Akt-2 as a potential target for the treatment of fungal sepsis.
Sepsis is the leading cause of death in the world. Recent reports suggest that in response to sep... more Sepsis is the leading cause of death in the world. Recent reports suggest that in response to sepsis, metabolism of macrophages switches from oxidative phosphorylation to aerobic glycolysis. MAPK phosphatase (MKP)–1 (also known as DUSP1) localized in the nucleus and preferentially dephosphorylates p38 and JNK. MKP-1 controls the expression of numerous inflammatory genes and transcription factors, thereby regulating innate and adaptive immunity. MKP-1–deficient animals exhibit aberrant metabolic responses following bacterial infections with a markedly increased mortality in response to sepsis. Because metabolic reprogramming modulates immune responses to TLR-4 activation, we investigated the effect of MKP-1 deficiency on mitochondrial electron transport chains involved in oxidative phosphorylation and transcription factors regulating mitochondrial biogenesis. Mitochondrial biogenesis is regulated by three nuclear-encoded proteins, including transcription factor A (TFAM), nuclear resp...
The mammalian response to stress is complex, often involving multiple signalling pathways that ac... more The mammalian response to stress is complex, often involving multiple signalling pathways that act in concert to influence cell fate. To examine potential interactions between the signalling cascades, we have focused on the effects of a model oxidant stress in a single cell type through an examination of the relative influences of mitogen-activated protein kinases (MAPKs) as well as two proposed apoptosis regulators, nuclear factor κB (NF-κB) and Bcl-2, in determining cell survival. Treatment of HeLa cells with H2O2 resulted in a time- and dose-dependent induction of apoptosis accompanied by sustained activation of all three MAPK subfamilies: extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38. This H2O2-induced apoptosis was markedly enhanced when ERK2 activation was selectively inhibited by PD098059. Apoptosis decreased when JNK/SAPK activation was inhibited by expression of a dominant negative mutant form...
Exposure of cells to either proliferative or stressful stimuli elicits a complex response involvi... more Exposure of cells to either proliferative or stressful stimuli elicits a complex response involving one or more distinct phosphorylation cascades culminating in the activation of multiple members of the mitogen-activated protein kinase (MAPK) family, including ...
Vascular endothelial growth factor (VEGF) is required for proper lung development and is transcri... more Vascular endothelial growth factor (VEGF) is required for proper lung development and is transcriptionally regulated in alveolar epithelial cells by hypoxia-inducible factor (HIF). Previous findings in a newborn mouse model of bronchopulmonary dysplasia (BPD) suggest that thioredoxin-interacting protein (Txnip) is a novel regulator of VEGF expression. The present studies were designed to test the hypothesis that Txnip negatively regulates VEGF through effects on HIF-mediated gene expression. To test this hypothesis, we first examined the levels of VEGF and Txnip protein in the lungs of 1-day-old newborn mice and E19 embryos and detected a significant inverse correlation. To elucidate the mechanisms underlying this relationship, we studied the effects of Txnip overexpression on HIF-mediated transcription using murine lung epithelial (MLE-12) cells. Overexpression of Txnip inhibited HIF-mediated reporter activity in both hypoxia and room air. Suppression of HIF activity by Txnip seemed to be independent of the ability of Txnip to bind to thioredoxin. Thus, our studies support a model in which Txnip is a potentially critical regulator of HIF-mediated gene transcription in the murine lung. Alterations in Txnip expression could alter lung VEGF expression in prematurely born human infants and contribute to the development of BPD.
Nitration products of unsaturated fatty acids are formed via NO-dependent oxidative reactions and... more Nitration products of unsaturated fatty acids are formed via NO-dependent oxidative reactions and appear to be a new class of endogenous antiinflammatory mediators. Nitroalkene derivatives of nitrated linoleic acid (LNO2) and nitrated oleic acid (OA-NO2) alleviate inflammatory responses in macrophages, but the underlying mechanisms remain to be fully defined. Herein we report that LNO2 and OA-NO2 suppress proinflammatory signal transducer and activator of transcription (STAT) signaling in macrophages. In RAW264.7 cells, a murine macrophage cell line, LNO2 and OA-NO2 inhibited the lipopolysaccharide (LPS)-induced STAT1 phosphorylation and the STAT1-dependent transcriptional activity, thereby suppressing expression of its target gene such as iNOS and MCP-1. The nitroalkene-mediated inhibition of STAT1 activity was not affected by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (a NO scavenger), GW9662 (a peroxisome proliferator-activated receptor-γ-specific antagonis...
Mitogen-activated protein (MAP) kinases play a critical role in innate immune responses to microb... more Mitogen-activated protein (MAP) kinases play a critical role in innate immune responses to microbial infection through eliciting the biosynthesis of proinflammatory cytokines. MAP phosphatases (MKP)-1 is an archetypical member of the dual-specificity phosphatase family that deactivates MAP kinases. Induction of MKP-1 has been implicated in attenuating the lipopolysaccharide (LPS) and Peptidoglycan (PGN) responses, but how the expression of the MKP-1 is regulated is still not fully understood. Here, we show that inhibition of p38 MAP kinase by specific inhibitor SB 203580 or RNA interference (RNAi) markedly reduced the expression of MKP-1 in LPS or PGN-treated macrophages, which is correlated with prolonged activation of p38 and JNK. Depletion of MAPKAP kinase 2 (MK2), a downstream substrate of p38, by RNAi also inhibited the expression of MKP-1. The mRNA level of MKP-1 is not affected by inhibition of p38, but the expression of MKP-1 is inhibited by treatment of cycloheximide. Thus, p38 MAPK plays a critical role in mediating expression of MKP-1 at a post-transcriptional level. Furthermore, inhibition of p38 by SB 203580 prevented the expression of MKP-1 in LPS-tolerized macrophages, restored the activation of MAP kinases after LPS restimulation. These results indicate a critical role of p38-MK2-dependent induction of MKP-1 in innate immune responses.
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Papers by Yusen Liu