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Papers by Yusen Liu

The Journal of Immunology, 2021

We have previously shown that Mkp-1–deficient mice produce elevated TNF-α, IL-6, and IL-10 follow... more We have previously shown that Mkp-1–deficient mice produce elevated TNF-α, IL-6, and IL-10 following systemic Escherichia coli infection, and they exhibited increased mortality, elevated bacterial burden, and profound metabolic alterations. To understand the function of Mkp-1 during bacterial infection, we performed RNA-sequencing analysis to compare the global gene expression between E. coli–infected wild-type and Mkp-1−/− mice. A large number of IFN-stimulated genes were more robustly expressed in E. coli–infected Mkp-1−/− mice than in wild-type mice. Multiplex analysis of the serum cytokine levels revealed profound increases in IFN-β, IFN-γ, TNF-α, IL-1α and β, IL-6, IL-10, IL-17A, IL-27, and GMSF levels in E. coli–infected Mkp-1−/− mice relative to wild-type mice. Administration of a neutralizing Ab against the receptor for type I IFN to Mkp-1−/− mice prior to E. coli infection augmented mortality and disease severity. Mkp-1−/− bone marrow–derived macrophages (BMDM) produced hig...

Journal of Immunology, Apr 15, 2007

Journal of Immunology, May 1, 2018

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.

Journal of Immunology, May 1, 2017

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.

Journal of Immunology, May 1, 2017

Fungal infections are a serious threat to people with a compromised immune system. The recognitio... more Fungal infections are a serious threat to people with a compromised immune system. The recognition of fungal pathogens relies on pathways mediated by TLRs and C-type lectin receptors. Activation of these TLR and C-type lectin receptor pathways converge on both NF-κB and MAPKs to initiate cytokine and chemokine production, leading to phagocyte recruitment and pathogen elimination. Mkp-1 is a negative feedback regulator of the p38 and JNK MAPKs. To understand the role of Mkp-1 in immune defense against fungal pathogens, we assessed the effects of Mkp-1 deficiency on the immune response to C. albicans, the most common fungal pathogen. Contrary to our previous observation with E. coli or bacterial products such as LPS, Mkp1-/-mice were protected from a lethal dose of C. albicans. In response to C. albicans challenge, Mkp1-/-mice produced greater amounts of TNF-α, had lower kidney fungal burdens and smaller fungal lesions. In wildtype mice large number of neutrophils were found in the kidneys, forming clusters surrounding fungal lesions. In contrast, far less neutrophils were found in the kidneys of Mkp-1-/-mice, although these neutrophils appeared to express greater levels of neutrophil elastase and form tighter clusters surrounding the fungi. Upon stimulation with heat-killed C. albicans yeasts and hyphae Mkp-1-/-bone marrow-derived macrophages (BMDM) also produced greater amounts of TNF-α and had higher p38 and JNK activities than did WT BMDM. TNF-α production in both wildtype and Mkp-1-/-BMDM in response to heat-killed C. albicans was attenuated by the pharmacological inhibitors of MEK1, JNK, p38, and Syk. Taken together, our results suggest that during fungal infection Mkp-1 acts to hamper the immune response against fungal pathogens.

The Journal of Immunology

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.

ImmunoHorizons, 2020

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...

Biochemical Journal, 1998

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...

Apoptosis, 1997

The molecular events associated with apoptosis induced by two distinct triggers (1) serum withdra... more The molecular events associated with apoptosis induced by two distinct triggers (1) serum withdrawal and (2) etoposide treatment were investigated in the human lung carcinoma cell line A549. Although both serum withdrawal and etoposide treatment resulted in internucleosomal DNA fragmentation, the morphologic features were distinct. Serum deprived apoptotic cells appeared small, round and refractile, with little evidence of nuclear fragmentation; etoposide-induced apoptotic cells appeared enlarged and flattened and displayed prominent nuclear fragmentation. p53 and p21/waf1 protein levels were elevated in etoposidetreated cells, but not in cells subjected to serum withdrawal. Apoptosis induced by both treatments was accompanied by a significant reduction in Rb protein levels. However, etoposide treatment led to hypophosphorylation of Rb, while serum withdrawal did not alter the Rb phosphorylation pattern. Serum withdrawal-induced apoptosis was correlated with activation of JNK and suppression of ERK activities, while both JNK and ERK activities were slightly elevated during etoposid-induced apoptosis. Together, these results support the hypothesis that apoptosis induced by serum withdrawal and etoposide treatment occurs through different pathways and involves distinct mediators.

Cancer and Metastasis Reviews, 2007

The mitogen-activated protein kinase (MAPK) phosphatases (MKPs) are a family of dual-specificity ... more The mitogen-activated protein kinase (MAPK) phosphatases (MKPs) are a family of dual-specificity protein phosphatases that dephosphorylate both phosphothreonine and phospho-tyrosine residues in MAP kinases, including the c-Jun N-terminal protein kinase (JNK)/stressactivated protein kinase (SAPK), the p38 MAPK, and the extracellular signal-related kinase (ERK). Since phosphorylation is required for the activation of MAP kinases, dephosphorylation by MKPs inhibits MAPK activity, thereby negatively regulating MAPK signaling. It is known that deregulation of MAPK signaling is the most common alteration in human cancers. Recent studies have suggested that MKPs play an important role not only in the development of cancers, but also in the response of cancer cells to chemotherapy. Thus, understanding the roles of MKPs in the development of cancer and their impact on chemotherapy can be exploited for therapeutic benefits for the treatment of human cancer.

Journal of Biological Chemistry, 1996

Mitogen-activated protein (MAP) kinases can be grouped into three structural families, ERK, JNK, ... more Mitogen-activated protein (MAP) kinases can be grouped into three structural families, ERK, JNK, and p38, which are thought to carry out unique functions within cells. We demonstrate that ERK, JNK, and p38 are activated by distinct combinations of stimuli in T cells that simulate full or partial activation through the T cell receptor. These kinases are regulated by reversible phosphorylation on Tyr and Thr, and the dual specific phosphatases PAC1 and MKP-1 previously have been implicated in the in vivo inactivation of ERK or of ERK and JNK, respectively. Here we characterize a new MAP kinase phosphatase, MKP-2, that is induced in human peripheral blood T cells with phorbol 12-myristate 13-acetate and is expressed in a variety of nonhematopoietic tissues as well. We show that the in vivo substrate specificities of individual phosphatases are unique. PAC1, MKP-2, and MKP-1 recognize ERK and p38, ERK and JNK, and ERK, p38, and JNK, respectively. Thus, individual MAP kinase phosphatases can differentially regulate the potential for cross-talk between the various MAP kinase pathways. A hyperactive allele of ERK2 (D319N), analogous to the Drosophila sevenmaker gain-of-function mutation, has significantly reduced sensitivity to all three MAP kinase phosphatases in vivo.

Free Radical Biology and Medicine, 1996

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 ...

Free Radical Biology and Medicine, 2010

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.

Experimental Cell Research, 2001

The p53-regulated stress-inducible gene GADD45 has been shown to participate in cellular response... more The p53-regulated stress-inducible gene GADD45 has been shown to participate in cellular response to DNA damage, including cell cycle checkpoint, apoptosis, and DNA repair. However, the regulation of GADD45 expression is complex and may involve both p53-dependent and-independent pathways. Recent findings have demonstrated that the p53-independent induction of GADD45 is mainly regulated by the transcription factors Oct-1 and NF-YA, which directly bind to their consensus motifs located at the GADD45 promoter region. Here, we report that mitogen-activated protein (MAP) kinases are involved in the induction of the GADD45 promoter after DNA damage. Inhibition of JNK1 and ERK kinase activities either by expression of the dominant negative mutant JNK1 or by treatment with a selective chemical inhibitor of ERK (PD098059) substantially abrogates the UV induction of the GADD45 promoter. In contrast, a p38 kinase inhibitor (SB203580) has little effect on GADD45 induction by UV. In addition, the GADD45 promoter is strongly activated following expression of JNK1; Raf-1, which is an upstream activator of the ERK pathway; or MEK1, an upstream activator of both the ERK and the JNK pathways. Activation of the GADD45 promoter by MAP kinases does not require normal p53 function. Interestingly, the MAP kinase-regulatory effect appears to be mediated via OCT-1 and CAAT motifs since disruption of these sites abrogates activation of the GADD45 promoter by MAP kinases. Therefore, these findings indicate that the MAP kinase pathways are involved in the regulation of the p53-independent induction of the GADD45 promoter, probably via interaction with transcription factors that directly bind to OCT-1 and CAAT motifs.

Experimental Cell Research, 1998

Following mitogenic stimulation, progression through dressed at: Gene Expression and Aging Sectio... more Following mitogenic stimulation, progression through dressed at: Gene Expression and Aging Section, Laboratory of Biologthe cell cycle is governed by the sequential activation ical Chemistry, National Institute on Aging, Gerontology Research of cyclin-dependent kinases (CDKs) through their as

Endocrinology, 2008

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...

Cytokine, 2012

Glucocorticoids acting through the glucocorticoid receptor (GR) inhibit TNF-induced lethal inflam... more Glucocorticoids acting through the glucocorticoid receptor (GR) inhibit TNF-induced lethal inflammation. Here, we demonstrate that GR dimerization plays a role in reducing TNF sensitivity. In mutant mice unable to dimerize GR, we found that TNF failed to induce MAPK phosphatase 1 (MKP1). We assessed TNF sensitivity in Mkp1-/mice and found increased inflammatory gene induction in livers, increased circulating cytokines, cell death in intestinal epithelium, severe intestinal inflammation, hypothermia, and death. Mkp1-/mice had increased levels of phosphorylated JNK, which promotes apoptosis, in liver tissue. We further examined JNK-deficient mice for their response to TNF. Although Jnk1-/mice showed no change in sensitivity to TNF, Jnk2-/mice were significantly protected against TNF, identifying JNK2 as an essential player in inflammation induced by TNF. Furthermore, we found that loss of Jnk2 partially rescued the increased sensitivity of Mkp1-/and mutant GR mice to TNF. Our data show that GR dimerization inhibits JNK2 through MKP1 and protects from TNF-induced apoptosis and lethal inflammation. Conflict of interest: The authors have declared that no conflict of interest exists.

Circulation Research, 2010

Rationale: Multiple protein kinases have been implicated in cardiovascular disease; however, litt... more Rationale: Multiple protein kinases have been implicated in cardiovascular disease; however, little is known about the role of their counterparts: the protein phosphatases. Objective: To test the hypothesis that mitogen-activated protein kinase phosphatase (MKP)-1 is actively involved in atherogenesis. Methods and Results: Mice with homozygous deficiency in MKP-1 (MKP-1 ؊/؊) were bred with apolipoprotein (Apo)E-deficient mice (ApoE ؊/؊) and the 3 MKP-1 genotypes (MKP-1 ؉/؉ /ApoE ؊/؊ ; MKP-1 ؉/؊ /ApoE ؊/؊ and MKP-1 ؊/؊ /ApoE ؊/؊) were maintained on a normal chow diet for 16 weeks. The 3 groups of mice exhibited similar body weight and serum lipid profiles; however, both MKP-1 ؉/؊ and MKP-1 ؊/؊ mice had significantly less aortic root atherosclerotic lesion formation than MKP-1 ؉/؉ mice. Less en face lesion was observed in 8-month-old MKP-1 ؊/؊ mice. The reduction in atherosclerosis was accompanied by decreased plasma levels of interleukin-1␣ and tumor necrosis factor ␣, and preceded by increased antiinflammatory cytokine interleukin-10. In addition, MKP-1-null mice had higher levels of plasma stromal cell-derived factor-1a, which negatively correlated with atherosclerotic lesion size. Immuno-histochemical analysis revealed that MKP-1 expression was enriched in macrophage-rich areas versus smooth muscle cell regions of the atheroma. Furthermore, macrophages isolated from MKP-1-null mice showed dramatic defects in their spreading/migration and impairment in extracellular signal-regulated kinase, but not c-Jun N-terminal kinase and p38, pathway activation. In line with this, MKP-1-null atheroma exhibited less macrophage content. Finally, transplantation of MKP-1-intact bone marrow into MKP-1-null mice fully rescued the wild-type atherosclerotic phenotype. Conclusion: These findings demonstrate that chronic deficiency of MKP-1 leads to decreased atherosclerosis via mechanisms involving impaired macrophage migration and defective extracellular signal-regulated kinase signaling. (Circ Res. 2010;106:902-910.

Cellular Signalling, 2007

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.

Cellular Signalling, 2007

Mitogen-activated protein (MAP) § kinase cascades are signal transduction pathways that play pivo... more Mitogen-activated protein (MAP) § kinase cascades are signal transduction pathways that play pivotal regulatory roles in the biosynthesis of proinflammatory cytokines. MAP kinase phosphatase (MKP)-1, an archetypal member of the MKP family, is essential for the dephosphorylation/ deactivation of MAP kinases p38 and JNK. Earlier studies conducted using cultured immortalized macrophages provided compelling evidence indicating that MKP-1 deactivates p38 and JNK, thereby limiting pro-inflammatory cytokine biosynthesis in innate immune cells exposed to microbial components. Recent studies employing MKP-1 knockout mice have confirmed the central function of MKP-1 in the feedback control of p38 and JNK activity as well as the crucial physiological function of MKP-1 as a negative regulator of the synthesis of pro-inflammatory cytokines in vivo. MKP-1 was shown to be a major feedback regulator of the innate immune response and to play a critical role in preventing septic shock and multi-organ dysfunction during pathogenic infection. In this review, we will update the studies on the biochemical properties and the regulation of MKP-1, and summarize our understanding on the physiological function of this key phosphatase in the innate immune response.

The Journal of Immunology, 2021

We have previously shown that Mkp-1–deficient mice produce elevated TNF-α, IL-6, and IL-10 follow... more We have previously shown that Mkp-1–deficient mice produce elevated TNF-α, IL-6, and IL-10 following systemic Escherichia coli infection, and they exhibited increased mortality, elevated bacterial burden, and profound metabolic alterations. To understand the function of Mkp-1 during bacterial infection, we performed RNA-sequencing analysis to compare the global gene expression between E. coli–infected wild-type and Mkp-1−/− mice. A large number of IFN-stimulated genes were more robustly expressed in E. coli–infected Mkp-1−/− mice than in wild-type mice. Multiplex analysis of the serum cytokine levels revealed profound increases in IFN-β, IFN-γ, TNF-α, IL-1α and β, IL-6, IL-10, IL-17A, IL-27, and GMSF levels in E. coli–infected Mkp-1−/− mice relative to wild-type mice. Administration of a neutralizing Ab against the receptor for type I IFN to Mkp-1−/− mice prior to E. coli infection augmented mortality and disease severity. Mkp-1−/− bone marrow–derived macrophages (BMDM) produced hig...

Journal of Immunology, Apr 15, 2007

Journal of Immunology, May 1, 2018

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.

Journal of Immunology, May 1, 2017

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.

Journal of Immunology, May 1, 2017

Fungal infections are a serious threat to people with a compromised immune system. The recognitio... more Fungal infections are a serious threat to people with a compromised immune system. The recognition of fungal pathogens relies on pathways mediated by TLRs and C-type lectin receptors. Activation of these TLR and C-type lectin receptor pathways converge on both NF-κB and MAPKs to initiate cytokine and chemokine production, leading to phagocyte recruitment and pathogen elimination. Mkp-1 is a negative feedback regulator of the p38 and JNK MAPKs. To understand the role of Mkp-1 in immune defense against fungal pathogens, we assessed the effects of Mkp-1 deficiency on the immune response to C. albicans, the most common fungal pathogen. Contrary to our previous observation with E. coli or bacterial products such as LPS, Mkp1-/-mice were protected from a lethal dose of C. albicans. In response to C. albicans challenge, Mkp1-/-mice produced greater amounts of TNF-α, had lower kidney fungal burdens and smaller fungal lesions. In wildtype mice large number of neutrophils were found in the kidneys, forming clusters surrounding fungal lesions. In contrast, far less neutrophils were found in the kidneys of Mkp-1-/-mice, although these neutrophils appeared to express greater levels of neutrophil elastase and form tighter clusters surrounding the fungi. Upon stimulation with heat-killed C. albicans yeasts and hyphae Mkp-1-/-bone marrow-derived macrophages (BMDM) also produced greater amounts of TNF-α and had higher p38 and JNK activities than did WT BMDM. TNF-α production in both wildtype and Mkp-1-/-BMDM in response to heat-killed C. albicans was attenuated by the pharmacological inhibitors of MEK1, JNK, p38, and Syk. Taken together, our results suggest that during fungal infection Mkp-1 acts to hamper the immune response against fungal pathogens.

The Journal of Immunology

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.

ImmunoHorizons, 2020

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...

Biochemical Journal, 1998

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...

Apoptosis, 1997

The molecular events associated with apoptosis induced by two distinct triggers (1) serum withdra... more The molecular events associated with apoptosis induced by two distinct triggers (1) serum withdrawal and (2) etoposide treatment were investigated in the human lung carcinoma cell line A549. Although both serum withdrawal and etoposide treatment resulted in internucleosomal DNA fragmentation, the morphologic features were distinct. Serum deprived apoptotic cells appeared small, round and refractile, with little evidence of nuclear fragmentation; etoposide-induced apoptotic cells appeared enlarged and flattened and displayed prominent nuclear fragmentation. p53 and p21/waf1 protein levels were elevated in etoposidetreated cells, but not in cells subjected to serum withdrawal. Apoptosis induced by both treatments was accompanied by a significant reduction in Rb protein levels. However, etoposide treatment led to hypophosphorylation of Rb, while serum withdrawal did not alter the Rb phosphorylation pattern. Serum withdrawal-induced apoptosis was correlated with activation of JNK and suppression of ERK activities, while both JNK and ERK activities were slightly elevated during etoposid-induced apoptosis. Together, these results support the hypothesis that apoptosis induced by serum withdrawal and etoposide treatment occurs through different pathways and involves distinct mediators.

Cancer and Metastasis Reviews, 2007

The mitogen-activated protein kinase (MAPK) phosphatases (MKPs) are a family of dual-specificity ... more The mitogen-activated protein kinase (MAPK) phosphatases (MKPs) are a family of dual-specificity protein phosphatases that dephosphorylate both phosphothreonine and phospho-tyrosine residues in MAP kinases, including the c-Jun N-terminal protein kinase (JNK)/stressactivated protein kinase (SAPK), the p38 MAPK, and the extracellular signal-related kinase (ERK). Since phosphorylation is required for the activation of MAP kinases, dephosphorylation by MKPs inhibits MAPK activity, thereby negatively regulating MAPK signaling. It is known that deregulation of MAPK signaling is the most common alteration in human cancers. Recent studies have suggested that MKPs play an important role not only in the development of cancers, but also in the response of cancer cells to chemotherapy. Thus, understanding the roles of MKPs in the development of cancer and their impact on chemotherapy can be exploited for therapeutic benefits for the treatment of human cancer.

Journal of Biological Chemistry, 1996

Mitogen-activated protein (MAP) kinases can be grouped into three structural families, ERK, JNK, ... more Mitogen-activated protein (MAP) kinases can be grouped into three structural families, ERK, JNK, and p38, which are thought to carry out unique functions within cells. We demonstrate that ERK, JNK, and p38 are activated by distinct combinations of stimuli in T cells that simulate full or partial activation through the T cell receptor. These kinases are regulated by reversible phosphorylation on Tyr and Thr, and the dual specific phosphatases PAC1 and MKP-1 previously have been implicated in the in vivo inactivation of ERK or of ERK and JNK, respectively. Here we characterize a new MAP kinase phosphatase, MKP-2, that is induced in human peripheral blood T cells with phorbol 12-myristate 13-acetate and is expressed in a variety of nonhematopoietic tissues as well. We show that the in vivo substrate specificities of individual phosphatases are unique. PAC1, MKP-2, and MKP-1 recognize ERK and p38, ERK and JNK, and ERK, p38, and JNK, respectively. Thus, individual MAP kinase phosphatases can differentially regulate the potential for cross-talk between the various MAP kinase pathways. A hyperactive allele of ERK2 (D319N), analogous to the Drosophila sevenmaker gain-of-function mutation, has significantly reduced sensitivity to all three MAP kinase phosphatases in vivo.

Free Radical Biology and Medicine, 1996

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 ...

Free Radical Biology and Medicine, 2010

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.

Experimental Cell Research, 2001

The p53-regulated stress-inducible gene GADD45 has been shown to participate in cellular response... more The p53-regulated stress-inducible gene GADD45 has been shown to participate in cellular response to DNA damage, including cell cycle checkpoint, apoptosis, and DNA repair. However, the regulation of GADD45 expression is complex and may involve both p53-dependent and-independent pathways. Recent findings have demonstrated that the p53-independent induction of GADD45 is mainly regulated by the transcription factors Oct-1 and NF-YA, which directly bind to their consensus motifs located at the GADD45 promoter region. Here, we report that mitogen-activated protein (MAP) kinases are involved in the induction of the GADD45 promoter after DNA damage. Inhibition of JNK1 and ERK kinase activities either by expression of the dominant negative mutant JNK1 or by treatment with a selective chemical inhibitor of ERK (PD098059) substantially abrogates the UV induction of the GADD45 promoter. In contrast, a p38 kinase inhibitor (SB203580) has little effect on GADD45 induction by UV. In addition, the GADD45 promoter is strongly activated following expression of JNK1; Raf-1, which is an upstream activator of the ERK pathway; or MEK1, an upstream activator of both the ERK and the JNK pathways. Activation of the GADD45 promoter by MAP kinases does not require normal p53 function. Interestingly, the MAP kinase-regulatory effect appears to be mediated via OCT-1 and CAAT motifs since disruption of these sites abrogates activation of the GADD45 promoter by MAP kinases. Therefore, these findings indicate that the MAP kinase pathways are involved in the regulation of the p53-independent induction of the GADD45 promoter, probably via interaction with transcription factors that directly bind to OCT-1 and CAAT motifs.

Experimental Cell Research, 1998

Following mitogenic stimulation, progression through dressed at: Gene Expression and Aging Sectio... more Following mitogenic stimulation, progression through dressed at: Gene Expression and Aging Section, Laboratory of Biologthe cell cycle is governed by the sequential activation ical Chemistry, National Institute on Aging, Gerontology Research of cyclin-dependent kinases (CDKs) through their as

Endocrinology, 2008

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...

Cytokine, 2012

Glucocorticoids acting through the glucocorticoid receptor (GR) inhibit TNF-induced lethal inflam... more Glucocorticoids acting through the glucocorticoid receptor (GR) inhibit TNF-induced lethal inflammation. Here, we demonstrate that GR dimerization plays a role in reducing TNF sensitivity. In mutant mice unable to dimerize GR, we found that TNF failed to induce MAPK phosphatase 1 (MKP1). We assessed TNF sensitivity in Mkp1-/mice and found increased inflammatory gene induction in livers, increased circulating cytokines, cell death in intestinal epithelium, severe intestinal inflammation, hypothermia, and death. Mkp1-/mice had increased levels of phosphorylated JNK, which promotes apoptosis, in liver tissue. We further examined JNK-deficient mice for their response to TNF. Although Jnk1-/mice showed no change in sensitivity to TNF, Jnk2-/mice were significantly protected against TNF, identifying JNK2 as an essential player in inflammation induced by TNF. Furthermore, we found that loss of Jnk2 partially rescued the increased sensitivity of Mkp1-/and mutant GR mice to TNF. Our data show that GR dimerization inhibits JNK2 through MKP1 and protects from TNF-induced apoptosis and lethal inflammation. Conflict of interest: The authors have declared that no conflict of interest exists.

Circulation Research, 2010

Rationale: Multiple protein kinases have been implicated in cardiovascular disease; however, litt... more Rationale: Multiple protein kinases have been implicated in cardiovascular disease; however, little is known about the role of their counterparts: the protein phosphatases. Objective: To test the hypothesis that mitogen-activated protein kinase phosphatase (MKP)-1 is actively involved in atherogenesis. Methods and Results: Mice with homozygous deficiency in MKP-1 (MKP-1 ؊/؊) were bred with apolipoprotein (Apo)E-deficient mice (ApoE ؊/؊) and the 3 MKP-1 genotypes (MKP-1 ؉/؉ /ApoE ؊/؊ ; MKP-1 ؉/؊ /ApoE ؊/؊ and MKP-1 ؊/؊ /ApoE ؊/؊) were maintained on a normal chow diet for 16 weeks. The 3 groups of mice exhibited similar body weight and serum lipid profiles; however, both MKP-1 ؉/؊ and MKP-1 ؊/؊ mice had significantly less aortic root atherosclerotic lesion formation than MKP-1 ؉/؉ mice. Less en face lesion was observed in 8-month-old MKP-1 ؊/؊ mice. The reduction in atherosclerosis was accompanied by decreased plasma levels of interleukin-1␣ and tumor necrosis factor ␣, and preceded by increased antiinflammatory cytokine interleukin-10. In addition, MKP-1-null mice had higher levels of plasma stromal cell-derived factor-1a, which negatively correlated with atherosclerotic lesion size. Immuno-histochemical analysis revealed that MKP-1 expression was enriched in macrophage-rich areas versus smooth muscle cell regions of the atheroma. Furthermore, macrophages isolated from MKP-1-null mice showed dramatic defects in their spreading/migration and impairment in extracellular signal-regulated kinase, but not c-Jun N-terminal kinase and p38, pathway activation. In line with this, MKP-1-null atheroma exhibited less macrophage content. Finally, transplantation of MKP-1-intact bone marrow into MKP-1-null mice fully rescued the wild-type atherosclerotic phenotype. Conclusion: These findings demonstrate that chronic deficiency of MKP-1 leads to decreased atherosclerosis via mechanisms involving impaired macrophage migration and defective extracellular signal-regulated kinase signaling. (Circ Res. 2010;106:902-910.

Cellular Signalling, 2007

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.

Cellular Signalling, 2007

Mitogen-activated protein (MAP) § kinase cascades are signal transduction pathways that play pivo... more Mitogen-activated protein (MAP) § kinase cascades are signal transduction pathways that play pivotal regulatory roles in the biosynthesis of proinflammatory cytokines. MAP kinase phosphatase (MKP)-1, an archetypal member of the MKP family, is essential for the dephosphorylation/ deactivation of MAP kinases p38 and JNK. Earlier studies conducted using cultured immortalized macrophages provided compelling evidence indicating that MKP-1 deactivates p38 and JNK, thereby limiting pro-inflammatory cytokine biosynthesis in innate immune cells exposed to microbial components. Recent studies employing MKP-1 knockout mice have confirmed the central function of MKP-1 in the feedback control of p38 and JNK activity as well as the crucial physiological function of MKP-1 as a negative regulator of the synthesis of pro-inflammatory cytokines in vivo. MKP-1 was shown to be a major feedback regulator of the innate immune response and to play a critical role in preventing septic shock and multi-organ dysfunction during pathogenic infection. In this review, we will update the studies on the biochemical properties and the regulation of MKP-1, and summarize our understanding on the physiological function of this key phosphatase in the innate immune response.