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Loss of Nrf2 in bone marrow derived macrophages (BMDMΦ) impairs antigen-driven CD8(+) T cell function by limiting GSH and Cys availability.
Abstract
NF-E2-related factor 2 (Nrf2), known to protect against reactive oxygen species (ROS), recently emerged to resolve acute inflammatory responses in activated macrophages. Consequently, disruption of Nrf2 promotes a pro-inflammatory macrophage phenotype. In the current study, we addressed the impact of this macrophage phenotype on CD8+ T cell activation by using an antigen-driven co-culture model consistent of Nrf2-/- and Nrf2+/+ bone marrow derived macrophages (BMDMΦ) and transgenic OT-1 CD8+ T cells. OT-1 CD8+ T cells encode a T cell receptor that specifically recognizes MHCI-presented OVA(257-264) peptide, thereby causing a downstream T cell activation. Interestingly, co-culture of OVA(257-264)-pulsed Nrf2-/- BMDMΦ with transgenic OT-1 CD8+ T cells attenuated CD8+ T cell activation, proliferation, and cytotoxic function, compared to Nrf2+/+ BMDMΦ.Since the provision of low molecular thiols such as glutathione (GSH) or cysteine (Cys) by macrophages limits antigen-driven CD8+ T cell activation, we quantified the amount of intracellular and extracellular GSH and Cys in both co-cultures. Indeed, GSH levels were strongly decreased in Nrf2-/- co-cultures compared to wildtype counterparts. Supplementation of thiols in Nrf2-/- co-cultures via addition of glutathione ester, N-acetylcysteine, β-mercaptoethanol, or cysteine itself restored T cell proliferation as well as cytotoxicity by increasing intracellular GSH. Mechanistically, we identified two potential Nrf2-regulated genes involved in thiol synthesis in BMDMΦ: the cystine transporter subunit xCT and the modulatory subunit of the GSH synthesizing enzyme γ-GCS (GCLM). Pharmacological inhibition of γ-GCS-dependent GSH synthesis as well as knockdown of the cystine importer xCT in Nrf2+/+ BMDMΦ mimicked the effect of Nrf2-/- BMDMΦ on CD8+ T cell function. Our findings demonstrate, that reduced levels of GCLM as well as xCT in Nrf2-/- BMDMΦ limit GSH availability, thereby inhibiting antigen-induced CD8+ T cell function.
... Nrf2 is involved in the functional development of CD8 + T cells through Glutathione (GSH) production [135,136]. Since the naïve T CD8 + cells cannot produce GSH, macrophages produce GSH to help T cells develop their function. Accordingly, it has been shown that when Nrf2 has not expressed in bone marrow-derived macrophages, the functional evolution of CD8 + T cells is impaired due to low GSH expression [135]. ...
... Since the naïve T CD8 + cells cannot produce GSH, macrophages produce GSH to help T cells develop their function. Accordingly, it has been shown that when Nrf2 has not expressed in bone marrow-derived macrophages, the functional evolution of CD8 + T cells is impaired due to low GSH expression [135]. Nrf2-null mice demonstrated higher levels of ROS and increased MDSC numbers than the wild type due to an abortive antigen recognition pathway and cellular energy metabolism in T CD8 + cells [78,[137][138][139]. ...
NF-E2-related factor 2 (Nrf2) transcription factor has contradictory roles in cancer, which can act as a tumor suppressor or a proto-oncogene in different cell conditions (depending on the cell type and the conditions of the cell environment). Nrf2 pathway regulates several cellular processes, including signaling, energy metabolism, autophagy, inflammation, redox homeostasis, and antioxidant regulation. As a result, it plays a crucial role in cell survival. Conversely, Nrf2 protects cancerous cells from apoptosis and increases proliferation, angiogenesis, and metastasis. It promotes resistance to chemotherapy and radiotherapy in various solid tumors and hematological malignancies, so we want to elucidate the role of Nrf2 in cancer and the positive point of its targeting. Also, in the past few years, many studies have shown that Nrf2 protects cancer cells, especially leukemic cells, from the effects of chemotherapeutic drugs. The present paper summarizes these studies to scrutinize whether targeting Nrf2 combined with chemotherapy would be a therapeutic approach for leukemia treatment. Also, we discussed how Nrf2 and NF-κB work together to control the cellular redox pathway. The role of these two factors in inflammation (antagonistic) and leukemia (synergistic) is also summarized.
... Previous studies have suggested that xCT-expressing APCs import cystine and supply T cells with cysteine in trans (26)(27)(28). Given that endogenous APCs in CD45.1 mice have the capacity to express xCT, it is plausible that host APCs in the CD45.1 congenic mice rescued the proliferation of transferred xCT-deficient T cells. ...
... Although T cells require GSH for proliferation in vitro and in vivo (22,23), previous reports have been conflicting as to whether T cells accumulate cysteine directly through xCT-mediated import of cystine (19)(20)(21)33) or indirectly via uptake of cysteine secreted by APCs (26)(27)(28). Our results indicate that cystine is necessary and sufficient to support the proliferation of purified T cell cultures in standard culture conditions. ...
Significance
xCT, the cystine–glutamate antiporter, has been implicated in supporting both tumor growth and T cell proliferation; thus, antitumor effects of systemic xCT inhibition may be blunted by compromised antitumor immunity. This report details the unexpected finding that xCT is dispensable for T cell proliferation in vivo and for antitumor immune responses. Consequently, tumor cell xCT loss acts synergistically with the immunotherapeutic agent anti–CTLA-4, laying the foundation for utilizing specific xCT inhibitors clinically to expand the efficacy of existing anticancer immunotherapeutics.
... Anti-cancer immune responses are typically mediated by CD8 + cytotoxic T lymphocytes (CTLs), CD4 + T h 1 helper cells, and natural killer (NK) cells (Vinay et al., 2015). NRF2 contributes to CD8 + T cell function by controlling GSH production and ROS levels (Morito et al., 2003;Sha et al., 2015). Early-phase activated T cells cannot synthesize GSH, so they depend on antigen-presenting cells, such as macrophages, to supply it. ...
... Early-phase activated T cells cannot synthesize GSH, so they depend on antigen-presenting cells, such as macrophages, to supply it. Nrf2 À/À bone marrow-derived macrophages (BMDMF) have decreased levels of cysteine and GSH, as a consequence of reduced expression of xCT and GCLM, and fail to fully activate CD8 + T cells (Sha et al., 2015). Moreover, in Keap1-kd mice, chemically induced carcinogenesis is reduced due to NRF2-dependent anti-cancer immunity (Satoh et al., 2016). ...
The transcription factor NRF2 is the master regulator of the cellular antioxidant response. Though recognized originally as a target of chemopreventive compounds that help prevent cancer and other maladies, accumulating evidence has established the NRF2 pathway as a driver of cancer progression, metastasis, and resistance to therapy. Recent studies have identified new functions for NRF2 in the regulation of metabolism and other essential cellular functions, establishing NRF2 as a truly pleiotropic transcription factor. In this review, we explore the roles of NRF2 in the hallmarks of cancer, indicating both tumor suppressive and tumor-promoting effects.
... NRF2 induces extensive transcriptional and metabolic reprogramming in myeloid cells (46). Beyond attenuating intrinsic tumoricidal functions, as our studies show, elevated NRF2 activity in macrophages shapes an immunosuppressive milieu that impairs cytotoxic T-cell functionality and fosters immune tolerance through Treg recruitment (47,48). Concurrently, NRF2 activation modulates extracellular matrix composition (49), potentially promoting tumor invasiveness. ...
Microscopic hemorrhage is a common aspect of cancers, yet its potential role as an independent factor influencing both cancer progression and therapeutic response is largely ignored. Recognizing the essential function of macrophages in red blood cell disposal, we explored a pathway that connects intratumoral hemorrhage with the formation of cancer-promoting tumor-associated macrophages (TAMs). Using spatial transcriptomics, we found that NRF2-activated myeloid cells possessing characteristics of procancerous TAMs tend to cluster in peri-necrotic hemorrhagic tumor regions. These cells resembled anti-inflammatory erythrophagocytic macrophages. We identified heme, a red blood cell metabolite, as a pivotal microenvironmental factor steering macrophages toward protumorigenic activities. Single-cell RNA-seq and functional assays of TAMs in 3D cell culture spheroids revealed how elevated intracellular heme signals via the transcription factor NRF2 to induce cancer-promoting TAMs. These TAMs stabilized epithelial-mesenchymal transition, enhancing cancer invasiveness and metastatic potential. Additionally, NRF2-activated macrophages exhibited resistance to reprogramming by IFNγ and anti-CD40 antibodies, reducing their tumoricidal capacity. Furthermore, MC38 colon adenocarcinoma-bearing mice with NRF2 constitutively activated in leukocytes were resistant to anti-CD40 immunotherapy. Overall, our findings emphasize hemorrhage-activated NRF2 in TAMs as a driver of cancer progression, suggesting that targeting this pathway could offer new strategies to enhance cancer immunity and overcome therapy resistance.
... Invariably, in Nrf2-deficient (Nrf22/2) mice challenged with LPS or tumor necrosis factor (TNF)-a, the activity of IKK was aggravated and led to raised phosphorylation and degradation of IkB [36]. Nrf2 furthermore generates an anti-inflammatory phenotype that modulates the functions of CD8+ T cells [37] as well as in macrophages and microglia [38][39][40]. This is because Nrf2 augments cysteine and GSH levels in macrophages via regulation of the cystine/glutamate transporter and the GSH-synthesizing enzyme gglutamyl cysteine ligase modulator and catalytic subunits (g-glutamyl cysteine ligase modulator subunit (GCLM) and g-glutamyl cysteine ligase catalytic subunit (GCLC)). ...
An autoimmune disease is the consequence of the immune system attacking healthy cells, tissues, and organs by mistake instead of protecting them. Inflammation and oxidative stress (OS) are well-recognized processes occurring in association with acute or chronic impairment of cell homeostasis. The transcription factor Nrf2 (nuclear factor [erythroid-derived 2]-like 2) is of major importance as the defense instrument against OS and alters anti-inflammatory activities related to different pathological states. Researchers have described Nrf2 as a significant regulator of innate immunity. Growing indications suggest that the Nrf2 signaling pathway is deregulated in numerous diseases, including autoimmune disorders. The advantageous outcome of the pharmacological activation of Nrf2 is an essential part of Nrf2-based chemoprevention and intervention in other chronic illnesses, such as neurodegeneration, cardiovascular disease, autoimmune diseases, and chronic kidney and liver disease. Nevertheless, a growing number of investigations have indicated that Nrf2 is already elevated in specific cancer and disease steps, suggesting that the pharmacological agents developed to mitigate the potentially destructive or transformative results associated with the protracted activation of Nrf2 should also be evaluated. The activators of Nrf2 have revealed an improvement in the progress of OS-associated diseases, resulting in immunoregulatory and anti-inflammatory activities; by contrast, the depletion of Nrf2 worsens disease progression. These data strengthen the growing attention to the biological properties of Nrf2 and its possible healing power on diseases. The evidence supporting a correlation between Nrf2 signaling and the most common autoimmune diseases is reviewed here. We focus on the aspects related to the possible effect of Nrf2 activation in ameliorating pathologic conditions based on the role of this regulator of antioxidant genes in the control of inflammation and OS, which are processes related to the progression of autoimmune diseases. Finally, the possibility of Nrf2 activation as a new drug development strategy to target pathogenesis is proposed.
... As a master regulator for redox homeostasis, NRF2 indirectly regulates NF-kB activity and also induces anti-inflammatory phenotype, which modulates CD8 + T cells and macrophage function. Pharmaceutical amplification of NRF2 prevents acute inflammatory liver injury in T-cell mediated model of acute inflammatory liver injury [35][36][37]. ...
Mesua ferrea Linn. (MF) is a medicinal plant whose stem bark has historically been used to treat skin disorders, gastrointestinal, and anti-inflammatory conditions. Although the MF stem bark’s contribution to inflammation was assessed, its exact mode of action remained unknown. This study aimed to investigate the pharmacological mechanisms of MF against inflammation utilizing network pharmacology, molecular docking, and molecular dynamics (MD) simulation. An integrated network pharmacology approach was used to predict the pharmacological basis and potential mechanisms by which these ingredients may treat and prevent inflammation. This approach included target identification, network construction, topological analysis, gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis, molecular docking, and MD simulation. Utilizing the pre- Absorption, Distribution, Metabolism, Elimination, and Toxicity tool, the drug-likeness of the phytoconstituents was evaluated. Protein-protein interaction network and protein interaction network was constructed. The results indicated that androgen receptor, Estrogen Receptor alpha, CYP19A1, retinoic acid receptor alpha, nuclear factor erythroid 2-related factor 2, thyrotropin receptor, nuclear factor kappa B subunit 1, and Albumin are crucial proteins engaged directly or indirectly in inflammatory pathways and illnesses. Finally, the targets are validated by molecular docking and MD simulation. MF may be effective for alleviating inflammatory conditions and the mechanism of action is characterized by multi-compound, multi-target, and multi-pathways. Thus, our study provides certain evidence for the development and utilization of medicinal plants.
Keyword: Mesua ferrea, Network pharmacology, Gene ontology, Molecular docking, MD simulation.
... pathway to suppress ferroptosis, relating to cysteine, system Xc-, Se, GSH. The β-mercaptoethanol may play a role in inhibiting ferroptosis by driving a highly efficient cystine/cysteine redox cycle (Sha et al., 2015). Cycloheximide is a potent inhibitor of ferroptosis, by increasing the concentration of GSH (Rashad et al., 2022). ...
Ferroptosis is a form of regulated cell death characterized by iron overload, overwhelming lipid peroxidation, and disruption of antioxidant systems. Emerging evidence suggests that ferroptosis is associated with pregnancy related diseases, such as spontaneous abortion, pre-eclampsia, gestational diabetes mellitus, intrahepatic cholestasis of pregnancy, and spontaneous preterm birth. According to these findings, inhibiting ferroptosis might be a potential option to treat pregnancy related diseases. This review summarizes the mechanisms and advances of ferroptosis, the pathogenic role of ferroptosis in pregnancy related diseases and the potential medicines for its treatment.
... The Nrf2-Keap1 axis also causes effects in a cluster of differentiation 8 (CD8+) antigenspecific T cells. Sha et al. [147] reported that the loss of Nrf2 in bone marrow-derived macrophages impairs antigen-driven CD8+ T cell function by limiting GSH and Cys availability. The Nrf2 signaling pathway also affects dendritic cells by suppressing their functions and facilitating the immune escape of antigens. ...
Inflammatory bowel diseases (IBDs) are related to nuclear factor erythroid 2-related factor 2 (Nrf2) dysregulation. In vitro and in vivo studies using phytocompounds as modulators of the Nrf2 signaling in IBD have already been published. However, no existing review emphasizes the whole scenario for the potential of plants and phytocompounds as regulators of Nrf2 in IBD models and colitis-associated colorectal carcinogenesis. For these reasons, this study aimed to build a review that could fill this void. The PubMed, EMBASE, COCHRANE, and Google Scholar databases were searched. The literature review showed that medicinal plants and phytochemicals regulated the Nrf2 on IBD and IBD-associated colorectal cancer by amplifying the expression of the Nrf2-mediated phase II detoxifying enzymes and diminishing NF-κB-related inflammation. These effects improve the bowel environment, mucosal barrier, colon, and crypt disruption, reduce ulceration and microbial translocation, and consequently, reduce the disease activity index (DAI). Moreover, the modulation of Nrf2 can regulate various genes involved in cellular redox, protein degradation, DNA repair, xenobiotic metabolism, and apoptosis, contributing to the prevention of colorectal cancer.
... Similarly, dysregulation of glutathione biosynthesis in hepatocytes leads to steatosis that may be accompanied by mitochondrial damage and hepatic failure [55]. Moreover, glutathione biosynthesis is critical for immune cell function and several reports have illustrated this fact, in particular in the context of the immune response and T-cell activation [56]. Interestingly, in our Go enrichment analysis, DEGs also were directly related to oxidative phosphorylation and immune response. ...
Polycystic ovary syndrome (PCOS) is a common condition in reproductive-aged women that induces reproductive and metabolic derangements. Women with PCOS seem to have disturbances in lipid metabolism in the adipose tissue. Nevertheless, gene expression in adipose tissue of PCOS women and its relation to other disturbances have been fragmentarily investigated. We utilized microarray data to identify the most important up- and down-regulated candidate genes in adipose tissue of PCOS women in contrast to healthy women using the meta-analysis technique. Microarray data produced from three independent experiments (n = 3) conducted on adipose tissue in women with PCOS were retrieved from ArrayExpress. Then, the datasets were merged using the metaSeq package in Rstudio and differentially expressed genes (DEGs) were selected in the studies. The integrative bioinformatics analyses of candidate genes were performed by gene ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, and protein–protein interaction (PPI) network construction. Of these, 12 up-regulated genes and 12 down-regulated genes were identified and assessed as the most highly up-regulated and down-regulated genes in adipose tissue of women with PCOS. These DEGs that were annotated by KEGG analysis were mainly involved in PI3K-Akt, MAPK, Rap1, and Ras signaling pathways, and pathways in cancer such as hepatocellular carcinoma and gastric cancer, as well as metabolic pathways, and brain disorder pathways such as Alzheimer’s disease and Huntington disease pathways. In the PPI networks, PRDM10, FGFR2, IGF1R, and FLT1 were the key nodes in the up-regulated networks, while the NDUFAB1 and NME2 proteins were key in the down-regulated networks. Overall, these findings provide insight into the gene expression in adipose tissue of PCOS women and its relation to other disturbances.
... In this context, studies have uncovered multiple roles for the GSH system in these cells. Among other things, it was shown that (1) GSH protects monocytes/macrophages from oxidized low-density lipoproteininduced injury (Wang et al, 2006); (2) GSH oxidation, triggered by metabolic stress, increases macrophage chemotactic activity (Qiao et al, 2009); (3) GSH availability regulates macrophagedependent activation of CD8 + T cells (Sha et al, 2015); and (4) glutaredoxin 1 plays an important role in immunometabolic regulation during nutrient stress (Ahn et al, 2022). Regarding the Trx system, reports have shown that the macrophage Trx system regulates inflammatory pathways by modulating the activation of the transcription factor, NF-jB, and the NLRP3 inflammasome (Heilman et al, 2011;Isakov et al, 2014;Muri et al, 2020). ...
Aims: Oxidative modifications of cysteine thiols regulate various physiological processes including inflammatory responses. The thioredoxin system plays a key role in thiol redox control. The aim of this study was to characterize the dynamic cysteine proteome of human macrophages upon activation by the prototypical pro-inflammatory agent, bacterial lipopolysaccharide (LPS), and/or perturbation of the thioredoxin system. Results: Herein, we profiled the cellular and redox proteome of human THP-1-derived macrophages during the early phase of LPS activation and/or inhibition of thioredoxin system activity by auranofin, by employing a peptide-centric, resin-assisted capture, redox proteomic workflow. Among 4200 identified cysteines, the oxidation of nearly 10% was selectively affected by LPS or auranofin treatments. Notably, the proteomic analysis uncovered a subset of ~100 thiols, mapped to proteins involved in diverse processes, whose oxidation is antagonistically regulated by LPS and thioredoxin. Compared to the redox proteome, the cellular proteome was largely unchanged, highlighting the importance of redox modifications as a mechanism that allows for rapid modulation of macrophage activities in response to a pro-inflammatory or a pro-oxidant insult. Structural-functional analyses provided mechanistic insights into redox regulation of selected proteins, including the glutathione synthesizing enzyme, glutamate cysteine ligase and the autophagy adaptor SQSTM1/p62, suggesting mechanisms by which macrophages adapt and fine-tune their responses according to a changing inflammatory and redox environment. Innovation: This study provides a rich resource for further characterization of redox mechanisms that regulate macrophage inflammatory activities. Conclusion: The dynamic thiol redox proteome allows macrophages to efficiently respond and adapt to redox and inflammatory challenges.
... Reduced glutathione (GSH) and S-nitrosoglutathione (GSNO) levels were determined by an HPLC method upon derivatization with Ellmann's reagent (5,5 ′ -dithiobis-(2-nitrobenzoic acid), DTNB) as previously described [42,44,45]. Briefly, samples were incubated with 20 mM DTNB for 5 min and stored at − 80 • C before measuring the specific DTNB-GSH adduct as well as free nitrothiobenzoate (NTB) via HPLC. ...
Nitrosation of critical thiols has been elaborated as reversible posttranslational modification with regulatory function in multiple disorders. Reversibility of S-nitrosation is generally associated with enzyme-mediated one-electron reductions, catalyzed by the thioredoxin system, or by nitrosoglutathione reductase.
In the present study, we confirm previous evidence for a non-enzymatic de-nitrosation of nitrosoglutathione (GSNO) by superoxide. The interaction leads to the release of nitric oxide that subsequently interacts with a second molecule of superoxide (O2•−) to form peroxynitrite. Despite the formation of peroxynitrite, approximately 40–70% of GSNO yielded reduced glutathione (GSH), depending on the applied analytical assay. The concept of O2•− dependent denitrosation was then applied to S-nitrosated enzymes. S-nitrosation of isocitrate dehydrogenase (ICDH; NADP⁺-dependent) was accompanied by an inhibition of the enzyme and could be reversed by dithiothreitol. Treatment of nitrosated ICDH with O2•− indicated ca. 50% recovery of enzyme activity. Remaining inhibition was largely consequence of oxidative modifications evoked either by O2•− or by peroxynitrite. Recovery of activity in S-nitrosated enzymes by O2•− appears relevant only for selected examples. In contrast, recovery of reduced glutathione from the interaction of GSNO with O2•− could represent a mechanism to regain reducing equivalents in situations of excess O2•− formation, e.g. in the reperfusion phase after ischemia.
... Nrf2 upregulates MARCO, a scavenger receptor for bacteria, which increases binding and phagocytosis, thereby enhancing bacterial clearance [68]. Nrf2 also upregulates stimulation of antigen-specific CD8+ T cells [69]. Nrf2 activation suppresses the inflammatory response of dendritic cells [70]. ...
Oxidative stress is a major component of cell damage and cell fat, and as such, it occupies a central position in the pathogenesis of metabolic disease. Nuclear factor-erythroid-derived 2-related factor 2 (Nrf2), a key transcription factor that coordinates expression of genes encoding antioxidant and detoxifying enzymes, is regulated primarily by Kelch-like ECH-associated protein 1 (Keap1). However, involvement of the Keap1–Nrf2 pathway in tissue and organism homeostasis goes far beyond protection from cellular stress. In this review, we focus on evidence for Nrf2 pathway dysfunction during development of several metabolic/inflammatory disorders, including diabetes and diabetic complications, obesity, inflammatory bowel disease, and autoimmune diseases. We also review the beneficial role of current molecular Nrf2 agonists and summarize their use in ongoing clinical trials. We conclude that Nrf2 is a promising target for regulation of numerous diseases associated with oxidative stress and inflammation. However, more studies are needed to explore the role of Nrf2 in the pathogenesis of metabolic/inflammatory diseases and to review safety implications before therapeutic use in clinical practice.
... Additionally, reduced glutathione was determined by HPLC analysis after derivatization using Ellman's reagent (5,5'-dithiobis-(2-nitrobenzoic acid), DTNB), as previously described [65,66]. The method is based on the reaction of the thiol with DTNB to form a mixed disulfide nitro-5-thiobenzoic acid (NTB). ...
While the importance of the iron-load of lipocalin-2 (Lcn-2) in promoting tumor progression is widely appreciated, underlying molecular mechanisms largely remain elusive. Considering its role as an iron-transporter, we aimed at clarifying iron-loaded, holo-Lcn-2 (hLcn-2)-dependent signaling pathways in affecting renal cancer cell viability. Applying RNA sequencing analysis in renal CAKI1 tumor cells to explore highly upregulated molecular signatures in response to hLcn-2, we identified a cluster of genes (SLC7A11, GCLM, GLS), which are implicated in regulating ferroptosis. Indeed, hLcn-2-stimulated cells are protected from erastin-induced ferroptosis. We also noticed a rapid increase in reactive oxygen species (ROS) with subsequent activation of the antioxidant Nrf2 pathway. However, knocking down Nrf2 by siRNA was not sufficient to induce erastin-dependent ferroptotic cell death in hLcn-2-stimulated tumor cells. In contrast, preventing oxidative stress through N-acetyl-l-cysteine (NAC) supplementation was still able to induce erastin-dependent ferroptotic cell death in hLcn-2-stimulated tumor cells. Besides an oxidative stress response, we noticed activation of the integrated stress response (ISR), shown by enhanced phosphorylation of eIF-2α and induction of ATF4 after hLcn-2 addition. ATF4 knockdown as well as inhibition of the ISR sensitized hLcn-2-treated renal tumor cells to ferroptosis, thus linking the ISR to pro-tumor characteristics of hLcn-2. Our study provides mechanistic details to better understand tumor pro-survival pathways initiated by iron-loaded Lcn-2.
... Other researches showed that expression of several other macrophage-specific genes, such as macrophage receptor with collagenous structure, a receptor required for bacterial phagocytosis, or CD36, a scavenger receptor for oxidized low-density lipoprotein the gene encoding the proinflammatory cytokine, IL-17D, contains AREs, and this Nrf2-T helper (Th) 17 axis seems to confer protection against tumorigenesis and viral infections could be directly regulated by Nrf2. From all of these, it was confirmed that Nrf2 acts as an antiinflammatory factor crucial in controlling the intensity and duration of inflammatory responses (Ahmed, Luo, Namani, Wang, & Tang, 2017;Banning & Brigelius-Flohé, 2005;Saddawi-Konefka et al., 2016;Sha et al., 2015).Various autoimmune disease phenotypes, such as vitiligo, multiple sclerosis (MS), and systemic lupus erythematosus (SLE), were found at the end of the Nrf2 diseasome. ...
Nuclear factor-erythroid 2-related factor 2 (Nrf2), is a transcriptional signaling pathway that plays a crucial role in numerous clinical complications. Pivotal roles of Nrf2 have been proved in cancer, autoimmune diseases, neurodegeneration, cardiovascular diseases, diabetes mellitus, renal injuries, respiratory conditions, gastrointestinal disturbances, and general disorders related to oxidative stress, inflammation, apoptosis, gelatinolysis, autophagy, and fibrogenesis processes. Green tea catechins as a rich source of phenolic compounds can deal with various clinical problems and manifestations. In this review, we attempted to focus on intervention between green tea catechins and Nrf2. Green tea catechins especially epigallocatechin gallate (EGCG) elucidated the protective role of Nrf2 and its downstream molecules in various disorders through Keap-1, HO-1, NQO-1, GPx, GCLc, GCLm, NF-kB cross-link, kinases, and apoptotic proteins. Subsequently, we compiled an updated expansions of the Nrf2 role as a gate to manage and protect different disorders and feasible indications of green tea catechins through this
signaling pathway. The present review highlighted recent evidence-based data in silico, in vitro, and in vivo studies on an outline for future clinical trials.
... There were studies showed that glutamate cysteine ligase (GCL) was the rate-limiting enzyme in the synthesis of GSH, and the cystine antiporter-xCT transported the precursors of GSH synthesis. Nrf2 may influence the modulatory subunit and catalytic subunit (GCLM, GCLC) of GCL and the cystine antiporter xCT, to affect the level of GSH [33,34]. However, there was also a report that the increased expression of GCLC and xCT was independent on Nrf2 in colorectal carcinoma cells [35]. ...
The incidence of mastitis is high during the postpartum stage, which causes severe pain and hinders breast feeding in humans and reduces milk production in dairy cows. Studies suggested that inflammation in multiple organs is associated with oxidative stress and nuclear factor E2-related factor 2 (Nrf2)-antioxidant response element pathway is one of the most important antioxidant pathways, but the effects of Nrf2 on antioxidation in the mammary gland during mastitis are still unclear. In this study, intramammary lipopolysaccharide (LPS) challenge was carried out in wild-type (WT) and Nrf2 knockout mice. Results showed that the expression of Nrf2 affected the expression of milk protein genes (Csn2 and Csn3). Importantly, LPS treatment increased the expression of Nrf2 and HO-1 and the content of glutathione in the mammary gland of WT mice, but not in Nrf2(-/-) mice. The expression levels of glutathione synthesis genes (GCLC, GCLM, and xCT) were lower in Nrf2(-/-) mice than in WT mice. Moreover, mitochondrial-dependent apoptotic and endoplasmic reticulum stress were significantly relieved in WT mice compared with that in Nrf2(-/-) mice. In summary, the expression of Nrf2 may play an important role in prevention of oxidative and organelle stresses during endotoxin-induced mastitis in mouse mammary gland.
... Unexpectedly, bone-marrow-derived Nrf2-deficient macrophages show no increased ROS production or alteration in co-stimulatory protein expression when activated. Nevertheless, antigen-driven CD8 + T cell functions are impaired [112]. Mechanistically, limited GSH and Cys availability leads to reduced T cell responses, pointing again to the antioxidant role of GSH and thiol groups. ...
The transcription factor NF-E2 p45-related factor 2 (Nrf2) is an established master regulator of the anti-oxidative and detoxifying cellular response. Thus, a role in inflammatory diseases associated with the generation of large amounts of reactive oxygen species (ROS) seems obvious. In line with this, data obtained in cell culture experiments and preclinical settings have shown that Nrf2 is important in regulating target genes that are necessary to ensure cellular redox balance. Additionally, Nrf2 is involved in the induction of phase II drug metabolizing enzymes, which are important both in degrading and converting drugs into active forms, and into putative carcinogens. Therefore, Nrf2 has also been implicated in tumorigenesis. This must be kept in mind when new therapy approaches are planned for the treatment of sepsis. Therefore, this review highlights the function of Nrf2 in sepsis with a special focus on the translation of rodent-based results into sepsis patients in the intensive care unit (ICU).
... However, Nfe2l2-null MDSCs have higher levels of intracellular ROS that suppress CTLs proliferation and induces T-cell anergy, a state of loss of CTLs antigen recognition, resulting in the increased tumor metastasis in a xenograft model of lung cancer [139,140]. Nfe2l2 deletion in the bone marrow-derived macrophages (BMDM) decreases the levels of GSH by downregulation of GCLM and xCT, which leads to limited the GSH availability to CTLs, thereby inhibiting antigen-induced CTLs proliferation and function [141]. Activated NRF2 in cancer cells can induce IL-17D expression that recruits NK cells, resulting in increased anti-tumor immunity and NK-dependent tumor regression [135]. ...
Nuclear factor erythroid 2-related factor 2 (NRF2) is a transcription factor that regulates the cellular defense against toxic and oxidative insults through the expression of genes involved in oxidative stress response and drug detoxification. NRF2 activation renders cells resistant to chemical carcinogens and inflammatory challenges. In addition to antioxidant responses, NRF2 is involved in many other cellular processes, including metabolism and inflammation, and its functions are beyond the originally envisioned. NRF2 activity is tightly regulated through a complex transcriptional and post-translational network that enables it to orchestrate the cell’s response and adaptation to various pathological stressors for the homeostasis maintenance. Elevated or decreased NRF2 activity by pharmacological and genetic manipulations of NRF2 activation is associated with many metabolism- or inflammation-related diseases. Emerging evidence shows that NRF2 lies at the center of a complex regulatory network and establishes NRF2 as a truly pleiotropic transcription factor. Here we summarize the complex regulatory network of NRF2 activity and its roles in metabolic reprogramming, unfolded protein response, proteostasis, autophagy, mitochondrial biogenesis, inflammation, and immunity.
... Direct impact of Nrf2 on the T cells possesses a complex mechanism as the process of the interference of Nrf2 gets restricted to the availability of the glutathione which leads to the process of inhibition of cell proliferation function [126,127]. ...
Background
Rheumatoid arthritis comprises the roots of 19th century and is an autoimmune and chronic inflammatory disorder leading to progressive joint destruction. This erosive joint damage is linked with infiltration of leukocytes along with inflammatory destruction and blood cell formation within the synovial membrane, deprivation of cartilage and bone that leads to incapacitative pain. The changes in synovium include its proliferation that leads to pannus formation and this ultimately leads to the invasion and erosions causing the destruction of joints. It is also defined as the destructive or chronic disease with a longer time duration that takes articular consideration as a feature.
Objective
The factors that can lead to RA includes inflammatory cascades, increased levels of (TNF-α) tumor necrosis factor α, IL-1b and IL-17 (interleukins) along with reduced levels of Nrf2 factors (nuclear factor-erythroid 2-related factor-2). Nrf2 binds effectively to antioxidant response elements (ARE) that mainly encodes majority of the phase II antioxidant enzymes as well as stress receptive proteins including glutathione S-transferase (GSH), heme oxygenase-1 (HO-1), peroxiredoxin I, all these act by cellular defense mechanism and removes the cytotoxic electrophiles along with the ROS that is reactive oxygen species. Nrf2 also responds to the inflammatory stimulus and protect the tissues from the inflammatory tissues.
... The tumor microenvironment harbors both tumor-promoting and tumor-antagonizing immune cells with antitumor immunity being mediated by CD8 + cytotoxic T lymphocytes, CD4 + T helper (Th) 1 cells and natural killer cells (65). NRF2 activation in macrophages can enhance cytotoxic T lymphocyte function by providing them with GSH and cysteine to support their activation and proliferation (95). Conversely, Nfe2l2 gene ablation or pharmacological inhibition of γ-GCS-dependent GSH synthesis as well as knockdown of SLC7A11 in bone marrow-derived macrophages attenuates cytotoxic T lymphocyte activation (64). ...
Nuclear factor erythroid 2-related factor 2 (NRF2) is a master transcriptional regulator of genes whose products defend our cells for toxic and oxidative insults. Although NRF2 activation may reduce cancer risk by suppressing oxidative stress and tumor promoting inflammation, many cancers exhibit elevated NRF2 activity either due to mutations that disrupt the negative control of NRF2 activity or other factors. Importantly, NRF2 activation is associated with poor prognosis and NRF2 has turned out to be a key activator of cancer supportive anabolic metabolism. In this review, we summarize the diverse roles played by NRF2 in cancer focusing on metabolic reprogramming and tumor-promoting inflammation.
... Other researches showed that expression of several other macrophage-specific genes, such as macrophage receptor with collagenous structure, a receptor required for bacterial phagocytosis, or CD36, a scavenger receptor for oxidized low-density lipoprotein the gene encoding the proinflammatory cytokine, IL-17D, contains AREs, and this Nrf2-T helper (Th) 17 axis seems to confer protection against tumorigenesis and viral infections could be directly regulated by Nrf2. From all of these, it was confirmed that Nrf2 acts as an antiinflammatory factor crucial in controlling the intensity and duration of inflammatory responses (Ahmed, Luo, Namani, Wang, & Tang, 2017;Banning & Brigelius-Flohé, 2005;Saddawi-Konefka et al., 2016;Sha et al., 2015).Various autoimmune disease phenotypes, such as vitiligo, multiple sclerosis (MS), and systemic lupus erythematosus (SLE), were found at the end of the Nrf2 diseasome. ...
Circular RNAs (cirRNAs) are long, noncoding endogenous RNA molecules and covalently closed continuous loop without 5′–3′ polarity and polyadenylated tail which are largely concentrated in the nucleus. CirRNA regulates gene expression by modulating microRNAs and functions as potential biomarker. CirRNAs can translate in vivo to link between their expression and disease. They are resistant to RNA exonuclease and can convert to the linear RNA by microRNA which can then act as competitor to endogenous RNA. This chapter summarizes the evolutionary conservation and expression of cirRNAs, their identification, highlighting various computational approaches on cirRNA, and translation with a focus on the breakthroughs and the challenges in this new field.
... NRF2 also induces an anti-inflammatory phenotype that modulates the functions of CD8 + T cells (Sha et al., 2015) as well as in macrophages and microglia ( Rojo et al., 2010( Rojo et al., , 2014aBrune et al., 2013). This is because NRF2 increases cysteine and GSH levels in macrophages through regulation of the cystine/glutamate transporter and the GSH-synthesizing enzyme g-glutamyl cysteine ligase modulator and catalytic subunits [g-glutamyl cysteine ligase modulator subunit (GCLM) and g-glutamyl cysteine ligase catalytic subunit (GCLC)]. ...
Systems medicine has a mechanism-based rather than a symptom- or organ-basedapproach to disease and identifies therapeutic targets in a nonhypothesisdrivenmanner. In this work, we apply this to transcription factor nuclear factor (erythroid-derived 2)-like 2 (NRF2) by cross-validating its position in a protein-protein interaction network (the NRF2 interactome) functionally linked to cytoprotection in low-grade stress, chronic inflammation, metabolic alterations, and reactive oxygen species formation. Multiscale network analysis of these molecularprofiles suggests alterations ofNRF2 expression and activity as a common mechanism in a subnetwork of diseases (the NRF2 diseasome). This network joins apparently heterogeneous phenotypes such as autoimmune, respiratory, digestive, cardiovascular, metabolic, and neurodegenerative diseases, along with cancer. Importantly, this approach matches and confirms in silico several applications for NRF2-modulating drugs validated in vivo at different phases of clinical development. Pharmacologically, their profile is as diverse as electrophilic dimethyl fumarate, synthetic triterpenoids like bardoxolone methyl and sulforaphane, protein-protein or DNA-protein interaction inhibitors, and even registered drugs such as metformin and statins, which activate NRF2 and may be repurposed for indications within the NRF2 cluster of disease phenotypes. Thus, NRF2 represents one of the first targets fully embraced by classic and systems medicine approaches to facilitate both drug development and drug repurposing by focusing on a set of disease phenotypes that appear to be mechanistically linked. The resulting NRF2 drugome may therefore rapidly advance several surprising clinical options for this subset of chronic diseases.
... Because CD8 + T cells do not express these Nrf2-target gene enzymes, which are required for efficient CD8 + T cell activity, the T cells need to take up the thiols from the medium. Consequently, cytotoxicity of wild type CD8 + T cells towards antigens presented by Nrf2-deficient macrophages was significantly reduced [46]. ...
Tight regulation of inflammation is very important to guarantee a balanced immune response without developing chronic inflammation. One of the major mediators of the resolution of inflammation is the transcription factor: the nuclear factor erythroid 2-like 2 (Nrf2). Stabilized following oxidative stress, Nrf2 induces the expression of antioxidants as well as cytoprotective genes, which provoke an anti-inflammatory expression profile, and is crucial for the initiation of healing. In view of this fundamental modulatory role, it is clear that both hyper- or hypoactivation of Nrf2 contribute to the onset of chronic diseases. Understanding the tight regulation of Nrf2 expression/activation and its interaction with signaling pathways, known to affect inflammatory processes, will facilitate development of therapeutic approaches to prevent Nrf2 dysregulation and ameliorate chronic inflammatory diseases. We discuss in this review the principle mechanisms of Nrf2 regulation with a focus on inflammation and autophagy, extending the role of dysregulated Nrf2 to chronic diseases and tumor development.
... 46,47 Antitumor immunity is largely dependent on CD8 + T cells and macrophages, 48 and a study has shown that NRF2 activation in macrophages is necessary for CD8 + T cell function. 49 Regardless of the cause of tumor initiation (environmental exposure, replication-associated mutations, inheritance of susceptibility genes), inhibition of NRF2 seems to be a very promising strategy to slow down tumor growth or to sensitize tumors to chemo or radiation therapies. NRF2 is very frequently upregulated in cancer and NRF2 target genes are involved in processes related to the hallmarks of cancer 50 (sustained proliferation, insensitivity to apoptosis, metabolic reprogramming, angiogenesis, etc), as well as chemoresistance. ...
Targeting the transcription factor NRF2 has been recognized as a feasible strategy for cancer prevention and treatment, but many of the mechanistic details underlying its role in cancer development and progression are lacking. Therefore, careful mechanistic studies of the NRF2 pathway in cancer initiation and progression are needed to identify which therapeutic avenue—activation or inhibition—is appropriate in a given context. Moreover, while numerous reports confirm the protective effect of NRF2 activation against chemical carcinogenesis little is known of its role in cancer arising from spontaneous mutations. Here, we tested the effects of NRF2 modulation (activation by sulforaphane or inhibition by brusatol) in lung carcinogenesis using a chemical (vinyl carbamate) model in A/J mice and a genetic (conditional KrasG12D oncogene expression, to simulate spontaneous oncogene mutation) model in C57BL/6J mice. Mice were treated with NRF2 modulators before carcinogen exposure or KrasG12D expression to test the role of NRF2 in cancer initiation, or treated after tumor development to test the role of NRF2 in cancer progression. Lung tissues were analyzed to determine tumor burden, as well as status of NRF2 and KRAS pathways. Additionally, proliferation, apoptosis, and oxidative DNA damage were assessed. Overall, NRF2 activation prevents initiation of chemically induced cancer, but promotes progression of pre-existing tumors regardless of chemical or genetic etiology. Once tumors are initiated, NRF2 inhibition is effective against the progression of chemically and spontaneously induced tumors. These results have important implications for NRF2-targeted cancer prevention and intervention strategies. This article is protected by copyright. All rights reserved
... In this regard, an unbalance in macrophage type populations and an active role of monocytes in angiotensin IImediated vascular dysfunction and fibrogenesis have been reported [54,55]. The homeostasis of GSH is critical for immune cell f unction and several reports have illustrated this fact, in particular in the context of atherogenesis and T-cell activation [56][57][58] In our endothelial dysfunction model, due to the nature of the endothelial reactivity experiments it is reasonable to assume a limited contribution of the immune response to the endothelial relaxation impairment. Nevertheless, in the case of renal injury associated to the UUO model, macrophage activation of the proinflammatory M1 population and recruitment of immune (mainly T) cells play an active role. ...
Glutathione (GSH) biosynthesis is essential for cellular redox homeostasis and antioxidant defense. The rate-limiting step requires glutamate-cysteine ligase (GCL), which is composed of the catalytic (GCLc) and the modulatory (GCLm) subunits. To evaluate the contribution of GCLc to endothelial function we generated an endothelial-specific Gclc haplo-insufficient mouse model (Gclc e/+ mice). In murine lung endothelial cells (MLEC) derived from these mice we observed a 50% reduction in GCLc levels compared to lung fibroblasts from the same mice. MLEC obtained from haplo-insufficient mice showed significant reduction in GSH levels as well as increased basal and stimulated ROS levels, reduced phosphorylation of eNOS (Ser 1177) and increased eNOS S-glutathionylation, compared to MLEC from wild type (WT) mice. Studies in mesenteric arteries demonstrated impaired endothelium-dependent vasodilation in Gclc(e/+) male mice, which was corrected by pre-incubation with GSH-ethyl-ester and BH4. To study the contribution of endothelial GSH synthesis to renal fibrosis we employed the unilateral ureteral obstruction model in WT and Gclc(e/+) mice. We observed that obstructed kidneys from Gclc(e/+) mice exhibited increased deposition of fibrotic markers and reduced Nrf2 levels. We conclude that the preservation of endothelial GSH biosynthesis is not only critical for endothelial function but also in anti-fibrotic responses.
... cDNA synthesis from 1 μg RNA was run according to the Maxima First Strand cDNA Synthesis Kit for RT-qPCR (Thermo Scientific). qPCR was performed as described previously [16]; actin was used as an internal control. Primer sequences used to detect cytokine mRNAs are listed in S2 Table. ...
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic, Th17-derived cytokine thought to critically contribute to the pathogenesis of diverse autoimmune diseases, including rheumatoid arthritis and psoriasis. Treatment with monoclonal antibodies that block GM-CSF activity is associated with favorable therapeutic effects in patients with rheumatoid arthritis. We evaluated the role of GM-CSF as a potential target for therapeutic interference in psoriasis using a combined pharmacologic and genetic approach and the mouse model of imiquimod-induced psoriasiform dermatitis (IMQPD). Neutralization of murine GM-CSF by an anti-GM-CSF antibody ameliorated IMQPD. In contrast, genetic deficiency in GM-CSF did not alter the course of IMQPD, suggesting the existence of mechanisms compensating for chronic, but not acute, absence of GM-CSF. Further investigation uncovered an alternative pathogenic pathway for IMQPD in the absence of GM-CSF characterized by an expanded plasmacytoid dendritic cell population and release of IFNα and IL-22. This pathway was not activated in wild-type mice during short-term anti-GM-CSF treatment. Our investigations support the potential value of GM-CSF as a therapeutic target in psoriatic disease. The discovery of an alternative pathogenic pathway for psoriasiform dermatitis in the permanent absence of GM-CSF, however, suggests the need for monitoring during therapeutic use of long-term GM-CSF blockade.
... In this regard, an unbalance in macrophage type populations and an active role of monocytes in angiotensin IImediated vascular dysfunction and fibrogenesis have been reported [54,55]. The homeostasis of GSH is critical for immune cell f unction and several reports have illustrated this fact, in particular in the context of atherogenesis and T-cell activation [56][57][58] In our endothelial dysfunction model, due to the nature of the endothelial reactivity experiments it is reasonable to assume a limited contribution of the immune response to the endothelial relaxation impairment. Nevertheless, in the case of renal injury associated to the UUO model, macrophage activation of the proinflammatory M1 population and recruitment of immune (mainly T) cells play an active role. ...
Glutathione (GSH) biosynthesis is essential for cellular redox homeostasis and antioxidant defence. The rate-limiting step requires glutathione cysteine ligase (GCL), which is composed of the catalytic (GCLc) and modulatory (GCLm) subunits. To evaluate the contribution of GCLc to endothelial function we generated endothelial-specific GCLc haploinsufficient (GCLc e/+ mice). In murine lung endothelial cells (MLEC) derived from these mice we corroborated a 50% reduction in GCLc levels compared to fibroblasts from the same mice. MLEC obtained from haploinsufficient mice showed significant reduction in GSH levels as well as increased basal and stimulated ROS levels, reduced phosphorylation of eNOS (Ser 1176) and increased eNOS S-glutathionylation, compared to MLEC from wild type (WT) mice. Studies in mesenteric arteries demonstrated impaired endothelium-dependent vasodilation in GCLc e/+ male mice, which was corrected by pre-incubation with ethyl-ester GSH and BH4. To study the contribution of endothelial GSH synthesis to renal fibrosis we employed the unilateral ureteral obstruction model in WT and GCLc e/+ mice. We observed that obstructed kidneys from GCLc e/+ mice exhibited increased deposition of fibrotic markers and reduced Nrf2 levels. We conclude that the preservation of endothelial GSH biosynthesis is critical for endothelial function and anti-fibrotic response.
... It has been shown that the immunosuppressive activity by MDSCs is primarily regulated by the intracellular ROS level (37) and that the Nrf2-mediated antioxidant system appears to play a crucial role for the reduction of immunosuppressive activity in MDSCs (24,38). Recently, ex vivo experiment of bone marrow-derived macrophage using Nrf2-deficient mice showed that Nrf2 contributes to CD8 þ T-cell function by regulating g-GCS and xCT (39). We recently reported that the immune microenvironment of Keap1-kd mice leads to resistance against metastasis of lung cancer cells and that activation of Nrf2 by chemical inducers reduces ROS levels in MDSCs, which in turn strengthens host immunity against metastatic cancer cells (38). ...
Nrf2 activation promotes resistance to chemical carcinogenesis in animal models, but activating mutations in Nrf2 also confer malignant characters to human cells by activating anti-oxidative/detoxifying enzymes and metabolic reprogramming. In this study, we examined how these contradictory activities of Nrf2 - cancer chemoprevention and cancer cell growth enhancement - can be reconciled in an established mouse model of urethane-induced lung carcinogenesis. Using Keap1-knockdown (kd) mice which express high levels of Nrf2, we found that urethane was rapidly excreted into the urine, consistent with an upregulation in the expression of urethane detoxification genes. Consequently, urethane-induced tumors were significantly smaller and less frequent in Keap1-kd mice than in wild-type mice. In contrast, tumor cells derived from Keap1-kd mice and transplanted into nude mice exhibited higher tumorigenicity compared with cells derived from wild-type mice. To identify the factors contributing to the tumor growth phenotype in the transplantation model, we performed a microarray analysis and found that many antioxidative stress genes were upregulated in the Keap1-kd-derived tumors. Therefore, we suggest that Nrf2 activation in cancer cells enhances their tumorigenicity, but global Nrf2 activation, as in Keap1-kd mice, simultaneously enhances anticancer immunity, thereby suppressing the growth potential of Keap1-kd tumors. Our findings provide relevant insight into the dual role of Nrf2 in cancer and warrant further studies of Nrf2 function during different stages of carcinogenesis.
... Tert-butyl hydroquinone, an NRF2 inducer, increases cystine uptake by 2-fold in mouse embryonic fibroblasts (MEFs) [25], and system x c À activity is lost in Nrf2 À / À MEFs [4]. In macrophages [26], endothelial cells infected with Kaposi's Sarcoma-Associated Herpesvirus [27], and rat microglial cells [28], xCT upregulation is dependent on NRF2. However, in SH-SY5Y cells, NRF2 does not directly mediate the upregulation of xCT [29]. ...
Cancer cells adapt to high levels of oxidative stress in order to survive and proliferate by activating key transcription factors. One such master regulator, the redox sensitive transcription factor NF E2 Related Factor 2 (NRF2), controls the expression of cellular defense genes including those encoding intracellular redox-balancing proteins involved in glutathione (GSH) synthesis. Under basal conditions, Kelch-like ECH-associated protein 1 (KEAP1) targets NRF2 for ubiquitination. In response to oxidative stress, NRF2 dissociates from KEAP1, entering the nucleus and binding to the antioxidant response element (ARE) in the promoter of its target genes. Elevated reactive oxygen species (ROS) production may deplete GSH levels within cancer cells. System xc(-), an antiporter that exports glutamate while importing cystine to be converted into cysteine for GSH synthesis, is upregulated in cancer cells in response to oxidative stress. Here, we provided evidence that the expression of xCT, the light chain subunit of system xc(-), is regulated by NRF2 in representative human breast cancer cells. Hydrogen peroxide (H2O2) treatment increased nuclear translocation of NRF2, also increasing levels of xCT mRNA and protein and extracellular glutamate release. Overexpression of NRF2 up-regulated the activity of the xCT promoter, which contains a proximal ARE. In contrast, overexpression of KEAP1 repressed promoter activity and decreased xCT protein levels, while siRNA knockdown of KEAP1 up-regulated xCT protein levels and transporter activity. These results demonstrate the importance of the KEAP1/NRF2 pathway in balancing oxidative stress in breast cancer cells through system xc(-). We have previously shown that xCT is upregulated in various cancer cell lines under oxidative stress. In the current investigation, we focused on MCF-7 cells as a model for mechanistic studies.
Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.
Sarcoma is a malignant tumor that originates from mesenchymal tissue. The common treatment for sarcoma is surgery supplemented with radiotherapy and chemotherapy. However, patients have a 5-year survival rate of only approximately 60%, and sarcoma cells are highly resistant to chemotherapy. Ferroptosis is an iron-dependent nonapoptotic type of regulated programmed cell death that is closely related to the pathophysiological processes underlying tumorigenesis, neurological diseases and other conditions. Moreover, ferroptosis is mediated via multiple regulatory pathways that may be targets for disease therapy. Recent studies have shown that the induction of ferroptosis is an effective way to kill sarcoma cells and reduce their resistance to chemotherapeutic drugs. Moreover, ferroptosis-related genes are related to the immune system, and their expression can be used to predict sarcoma prognosis. In this review, we describe the molecular mechanism underlying ferroptosis in detail, systematically summarize recent research progress with respect to ferroptosis application as a sarcoma treatment in various contexts, and point out gaps in the theoretical research on ferroptosis, challenges to its clinical application, potential resolutions of these challenges to promote ferroptosis as an efficient, reliable and novel method of clinical sarcoma treatment.
Alzheimer's disease (AD) is the most common form of dementia. Unfortunately, despite numerous studies, an effective treatment for AD has not yet been established. There is remarkable evidence indicating that the innate immune mechanism and adaptive immune response play significant roles in the pathogenesis of AD. Several studies have reported changes in CD8⁺ and CD4⁺ T cells in AD patients.
This mini-review article discusses the potential contribution of CD4⁺ and CD8⁺ T cells reactivity to amyloid β (Aβ) protein in individuals with AD. Moreover, this mini-review examines the potential associations between T cells, heme oxygenase (HO), and impaired mitochondria in the context of AD. While current mathematical models of AD have not extensively addressed the inclusion of CD4⁺ and CD8⁺ T cells, there exist models that can be extended to consider AD as an autoimmune disease involving these T cell types. Additionally, the mini-review covers recent research that has investigated the utilization of machine learning models, considering the impact of CD4⁺ and CD8⁺ T cells.
Nucleus pulposus, annulus fibrosus, and cartilage endplate constitute an avascular intervertebral disc (IVD), which is crucial for spinal and intervertebral joint mobility. As one of the most widespread health issues worldwide, intervertebral disc degeneration (IVDD) is recognized as a key contributor to back and neck discomfort. A number of degenerative disorders have a strong correlation with ferroptosis, a recently identified novel regulated cell death (RCD) characterized by an iron-dependent mechanism and a buildup of lipid reactive oxygen species (ROS). There is growing interest in the part ferroptosis plays in IVDD pathophysiology. Inhibiting ferroptosis has been shown to control IVDD development. Several studies have demonstrated that in TBHP-induced oxidative stress models, changes in ferroptosis marker protein levels and increased lipid peroxidation lead to the degeneration of intervertebral disc cells, which subsequently aggravates IVDD. Similarly, IVDD is significantly relieved with the use of ferroptosis inhibitors. The purpose of this review was threefold: 1) to discuss the occurrence of ferroptosis in IVDD; 2) to understand the mechanism of ferroptosis and its role in IVDD pathophysiology; and 3) to investigate the feasibility and prospect of ferroptosis in IVDD treatment.
Both genomic instability and the presence of chronic inflammation are involved in carcinogenesis and tumor progression. These alterations predispose the cancer cells to undergo metabolic reprogramming as well as the epithelial-mesenchymal transition (EMT). These pathways allow cancer cells to avoid apoptosis and stimulate tumor progression. EMT is an important early event in tumor cell invasion, which can be regulated through inflammatory signaling pathways. Cancer cells undergoing EMT are vulnerable to cell death by the process of ferroptosis. Ferroptosis is a form of regulated cell death involving iron-dependent lipid peroxidation, designed to maintain cellular homeostasis. Several reports have linked ferroptosis, inflammation, and cancer. Ferroptosis inhibitors and EMT inducers have been used to understand the anti-inflammatory and anticancer effects in experimental models. A better understanding of the crosstalk between ferroptosis and EMT, and the involvment of inflammatory mediators may accelerate the discovery of therapeutic strategies to eradicate cancer cells and overcome drug-resistance.
(250words)
Background
Glutathione (γ-L-glutamyl-L-cysteinyl-glycine, GSH) is a tripeptide that is part of the antioxidant defense system and contributes to numerous redox-regulatory processes. In vivo, reduced GSH and oxidized glutathione disulfide (GSSG) are present in redox equilibrium and their ratio provides important information on the cellular redox state. Here, we compared three different methods for in vivo quantification of glutathione in tissues of hypertensive rats, an accepted animal model of oxidative stress.
Methods and Results
In the present study we used hypertensive rats (infusion of 1 mg/kg/d angiotensin-II for 7 days) to determine the levels of reduced GSH and/or GSH/GSSG ratios in different tissue samples. We used an HPLC-based method with direct electrochemical detection (HPLC/ECD) and compared it with Ellman’s reagent (DTNB) dependent derivatization of reduced GSH to the GS-NTB adduct and free NTB (UV/Vis HPLC) as well as with a commercial GSH/GSSG assay (Oxiselect). Whereas all three methods indicated overall a decreased redox state in hypertensive rats, the assays based onHPLC/ECD and DTNB derivatization provided the most significant differences.
Conclusions
We applieda direct, fast and sensitive method for electrochemical GSH detection in tissues from hypertensive animals, and confirmed its reliability for in vivo measurements by head-to-head comparison with two other established assays. The HPLC/ECD but not DTNB and Oxiselect assays yielded quantitative GSH data but all 3 assays reflected nicely the qualitative redox changes and functional impairment in hypertensive rats. However, especially our GSH/GSSG values are lower than reported by others pointing to problems in the work-up protocol.
Human CD4⁺CD25hiFOXP3⁺ regulatory T (Treg) cells are key players in the control of immunological self-tolerance and homeostasis. Here, we report that signals of pseudo-starvation reversed human Treg cell in vitro anergy through an integrated transcriptional response, pertaining to proliferation, metabolism, and transmembrane solute carrier transport. At the molecular level, the Treg cell proliferative response was dependent on the induction of the cystine/glutamate antiporter solute carrier (SLC)7A11, whose expression was controlled by the nuclear factor erythroid 2-related factor 2 (NRF2). SLC7A11 induction in Treg cells was impaired in subjects with relapsing-remitting multiple sclerosis (RRMS), an autoimmune disorder associated with reduced Treg cell proliferative capacity. Treatment of RRMS subjects with dimethyl fumarate (DMF) rescued SLC7A11 induction and fully recovered Treg cell expansion. These results suggest a previously unrecognized mechanism that may account for the progressive loss of Treg cells in autoimmunity and unveil SLC7A11 as major target for the rescue of Treg cell proliferation.
Ferroptosis is an iron-dependent cell death, which is different from apoptosis, necrosis, autophagy, and other forms of cell death. The process of ferroptotic cell death is defined by the accumulation of lethal lipid species derived from the peroxidation of lipids, which can be prevented by iron chelators (e.g., deferiprone, deferoxamine) and small lipophilic antioxidants (e.g., ferrostatin, liproxstatin). This review summarizes current knowledge about the regulatory mechanism of ferroptosis and its association with several pathways, including iron, lipid, and cysteine metabolism. We have further discussed the contribution of ferroptosis to the pathogenesis of several diseases such as cancer, ischemia/reperfusion, and various neurodegenerative diseases (e.g., Alzheimer’s disease and Parkinson’s disease), and evaluated the therapeutic applications of ferroptosis inhibitors in clinics.
The basic leucine zipper transcription factor Nrf2 is the primary regulator of cellular oxidative stress. Activation of Nrf2 is regarded as a potential preventive and therapeutic strategy. However, aberrant hyperactivation of Nrf2 is found in a variety of cancers and promotes cancer progression and metastasis. Moreover, constitutive activation of Nrf2 confers cancer cells resistance to chemo- and radiotherapy. Thus, inhibiting Nrf2 could be a new therapeutic strategy for cancer. With the aim of accelerating the discovery and development of novel Nrf2 inhibitors, we summarize the biological and pathological functions of Nrf2 in cancer. Furthermore, the recent studies of small molecular Nrf2 inhibitors and potential Nrf2 inhibitory mechanisms are also summarized in this review.
Oxidative stress is an important cause of neurodegenerative diseases. Antioxidant is an potential important method to treat such diseases. The aim of this study is to discover new and effective antioxidants and their mechanism. The neuroprotective effect of six curcumin pyrozole compounds were first evaluated on sodium nitroprusside (SNP) - induced PC12 cell injury by testing cell viability and LDH release. The results showed that four compounds (C1-C4) have more significant protective effects compared to curcumin and edaravone. Furthermore, compounds C1-C4 can attenuate the intracellular ROS, and compound C3 is the most effective one which can preservate the mitochondria function by inhibiting the mitochondrial membrane potential loss and enhance nuclear translocation of Nrf2 in PC12 cell. These results indicated that C3 may be a potential candidate drug for treating neurodegenerative diseases.
The red fluorescent hybrid nanoparticles (NPG@PFBD) have been designed and synthesized successfully. The NPG@PFBD nanoparticles are composed of fluorescent semiconducting polymer (PFBD) and n-propyl gallate (NPG). Because of the coordination between the gallate and Cu2+, Cu2+ could attach to the hybrid nanoparticles and quenched their fluorescence through photoinduced electron transfer (PET). In addition, in the presence of biothiols, such as glutathione (GSH), Cu2+ would bind to the thiol. The PET between hybrid nanoparticles and Cu2+ was weakened, and the fluorescence signal of the nanoparticles was recovered. A facile detection method for Cu2+ and biothiols was therefore developed. The detection limit for Cu2+ and GSH were 40 nM and 90 nM, respectively. The proposed fluorescent hybrid nanoparticles have great potential application for Cu2+ and GSH detection in related biological field.
Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a master regulator of cellular protective processes. Rheumatic diseases are chronic conditions characterized by inflammation, pain, tissue damage and limitations in function. Main examples are rheumatoid arthritis, systemic lupus erythematosus, osteoarthritis and osteoporosis. Their high prevalence constitutes a major health problem with an important social and economic impact. A wide range of evidence indicates that Nrf2 may control different mechanisms involved in the physiopathology of rheumatic conditions. Therefore, the appropriate expression and balance of Nrf2 is necessary for regulation of oxidative stress, inflammation, immune responses, and cartilage and bone metabolism. Numerous studies have demonstrated that Nrf2 deficiency aggravates the disease in experimental models while Nrf2 activation results in immunoregulatory and anti-inflammatory effects. These reports reinforce the increasing interest in the pharmacologic regulation of Nrf2 and its potential applications. Nevertheless, a majority of Nrf2 inducers are electrophilic molecules which may present off-target effects. In recent years, novel strategies have been sought to modulate the Nrf2 pathway which has emerged as a therapeutic target in rheumatic conditions.
Adriamycin (ADR) induces the over-expression of P-glycoprotein (P-gp) and multiple drug resistance in breast cancer cells. However, the biochemical process and underlying mechanisms are not clear. Our previous study revealed that ADR increased reactive oxygen species (ROS) generation and decreased glutathione (GSH) biosynthesis, while N-acetylcysteine, the ROS scavenger, reversed the over-expression of P-gp. The present study showed that ADR inhibited the influx of cystine (the source material of GSH) and the activity of the SLC7A11 transporter (in charge of cystine uptake) in MCF-7 cells. For the first time, we showed that the down-regulation/silence of SLC7A11, or cystine deprivation, or enhanced ROS exposure significantly increased P-gp expression in MCF-7 cells. The down-regulation of SLC7A11 markedly enhanced ROS induced P-gp over-expression and drug resistance in MCF-7 cells; a combination of either an inhibited/silenced SLC7A11 or cystine deprivation and increased ROS dramatically promoted P-gp expression, which could be reversed by N-acetylcysteine. In contrast, the over-expression of SLC7A11, or supplementation with sufficiently cystine, or treatment with N-acetylcysteine significantly decreased P-gp expression and activity. It was suggested that ROS and SLC7A11/cystine were the two relevant factors responsible for the expression and function of P-gp, and that SLC7A11 might be a potential target modulating ADR resistance.
Lupus nephritis (LN) is the major clinical manifestation of systemic lupus erythematosus. LN is promoted by T helper 17 (Th17) cells, which are the major pro-inflammatory T cell subset contributing to autoimmunity regulation. Nuclear factor erythroid 2-related factor 2 (NRF2) is critical for suppressing reactive oxygen species (ROS) and relieving oxidant stress by regulating antioxidant gene expression. Previous studies have demonstrated that Nrf2 deficiency promotes drug-induced or spontaneous LN. However, whether NRF2 regulates Th17 function during LN development is still unclear. In this study, we introduced Nrf2 deficiency into a well-known LN model, the B6/lpr mouse strain, and found that it promoted early-stage LN with altered Th17 activation. Th17 cells and their relevant cytokines were dramatically increased in these double-mutant mice. We also demonstrated that naïve T cells from the double-mutant mice showed significantly increased differentiation into Th17 cells in vitro, with decreased expression of the Th17 differentiation suppressor Socs3 and increased phosphorylation of STAT3. Our results demonstrated that Nrf2 deficiency promoted Th17 differentiation and function during LN development. Moreover, our results suggested that the regulation of Th17 differentiation via NRF2 could be a therapeutic target for the treatment of subclinical LN patients.
Glutathione (GSH) is an abundant intracellular tripeptide that has been implicated as an important regulator of T cell proliferation. The effect of pharmacological regulators of GSH and other thiols on murine T cell signaling, proliferation, and intracellular thiol levels was examined. L-Buthionine-SR-sulfoximine (BSO), an inhibitor of GSH synthesis, markedly reduced GSH levels and blocked T cell proliferation without significant effect on cell viability. N-acetylcysteine markedly enhanced T cell proliferation without affecting GSH levels. Cotreatment of T cells with N-acetyleysteine and BSO failed to restore GSH levels, but completely restored the proliferative response. Both 2-ME and L-cysteine also reversed the BSO inhibition of T cell proliferation. Intracellular L-cysteine levels were reduced with BSO treatment and restored with entreatment with NAC or L-cysteine. However, 2-ME completely reversed the BSO inhibition of proliferation without increasing intracellular cysteine levels. Therefore, neither GSH nor cysteine is singularly critical in limiting T cell proliferation. Reducing equivalents from free thiols were required because oxidation of the thiol moiety completely abolished the effect. Furthermore, BSO did not change the expression of surface activation markers, but effectively blocked IL-2 and IL-6 secretion. Importantly, exogenous IL-2 completely overcame BSO-induced block or T cell proliferation. These results demonstrate that T cell proliferation is regulated by thiol-sensitive pathway involving IL-2.
Activated macrophages express high levels of Nrf2, a transcription factor that positively regulates the gene expression of
antioxidant and detoxication enzymes. In this study, we examined how Nrf2 contributes to the anti-inflammatory process. As
a model system of acute inflammation, we administered carrageenan to induce pleurisy and found that in Nrf2-deficient mice,
tissue invasion by neutrophils persisted during inflammation and the recruitment of macrophages was delayed. Using an antibody
against 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), it was observed that macrophages from pleural lavage accumulate 15d-PGJ2. We show that in mouse peritoneal macrophages 15d-PGJ2 can activate Nrf2 by forming adducts with Keap1, resulting in an Nrf2-dependent induction of heme oxygenase 1 and peroxiredoxin
I (PrxI) gene expression. Administration of the cyclooxygenase 2 inhibitor NS-398 to mice with carrageenan-induced pleurisy
caused persistence of neutrophil recruitment and, in macrophages, attenuated the 15d-PGJ2 accumulation and PrxI expression. Administration of 15d-PGJ2 into the pleural space of NS-398-treated wild-type mice largely counteracted both the decrease in PrxI and persistence of
neutrophil recruitment. In contrast, these changes did not occur in the Nrf2-deficient mice. These results demonstrate that
Nrf2 regulates the inflammation process downstream of 15d-PGJ2 by orchestrating the recruitment of inflammatory cells and regulating the gene expression within those cells.
Abstract The antiporter system x(c)(-) imports the amino acid cystine, the oxidized form of cysteine, into cells with a 1:1 counter-transport of glutamate. It is composed of a light chain, xCT, and a heavy chain, 4F2 heavy chain (4F2hc), and, thus, belongs to the family of heterodimeric amino acid transporters. Cysteine is the rate-limiting substrate for the important antioxidant glutathione (GSH) and, along with cystine, it also forms a key redox couple on its own. Glutamate is a major neurotransmitter in the central nervous system (CNS). By phylogenetic analysis, we show that system x(c)(-) is a rather evolutionarily new amino acid transport system. In addition, we summarize the current knowledge regarding the molecular mechanisms that regulate system x(c)(-), including the transcriptional regulation of the xCT light chain, posttranscriptional mechanisms, and pharmacological inhibitors of system x(c)(-). Moreover, the roles of system x(c)(-) in regulating GSH levels, the redox state of the extracellular cystine/cysteine redox couple, and extracellular glutamate levels are discussed. In vitro, glutamate-mediated system x(c)(-) inhibition leads to neuronal cell death, a paradigm called oxidative glutamate toxicity, which has successfully been used to identify neuroprotective compounds. In vivo, xCT has a rather restricted expression pattern with the highest levels in the CNS and parts of the immune system. System x(c)(-) is also present in the eye. Moreover, an elevated expression of xCT has been reported in cancer. We highlight the diverse roles of system x(c)(-) in the regulation of the immune response, in various aspects of cancer and in the eye and the CNS. Antioxid. Redox Signal. 00, 000-000.
Because naïve T cells are unable to import cystine due to the absence of cystine transporters, it has been suggested that T cell activation is dependent on cysteine generated by antigen presenting cells. The aim of this study was to determine at which phases during T cell activation exogenous cystine/cysteine is required and how T cells meet this requirement. We found that early activation of T cells is independent of exogenous cystine/cysteine, whereas T cell proliferation is strictly dependent of uptake of exogenous cystine/cysteine. Naïve T cells express no or very low levels of both cystine and cysteine transporters. However, we found that these transporters become strongly up-regulated during T cell activation and provide activated T cells with the required amount of cystine/cysteine needed for T cell proliferation. Thus, T cells are equipped with mechanisms that allow T cell activation and proliferation independently of cysteine generated by antigen presenting cells.
Interleukin-2 (IL-2) signals influence various lymphocyte subsets during differentiation, immune responses and homeostasis. As discussed in this Review, stimulation with IL-2 is crucial for the maintenance of regulatory T (T(Reg)) cells and for the differentiation of CD4(+) T cells into defined effector T cell subsets following antigen-mediated activation. For CD8(+) T cells, IL-2 signals optimize both effector T cell generation and differentiation into memory cells. IL-2 is presented in soluble form or bound to dendritic cells and the extracellular matrix. Use of IL-2 - either alone or in complex with particular neutralizing IL-2-specific antibodies - can amplify CD8(+) T cell responses or induce the expansion of the T(Reg) cell population, thus favouring either immune stimulation or suppression.
Dendritic cells (DCs) are critical mediators of immunity and immune tolerance by orchestrating multiple aspects of T cell activation and function. Immature DCs (iDCs) expressing low levels of co-stimulatory receptors are highly efficient at antigen capture but are poor activators of T cells. Maturation of DCs is associated with increased expression of co-stimulatory molecules. Co-stimulatory receptor gene expression is regulated by intracellular redox, NF-κB, and MAPK pathways and by histone deacetylase (HDAC) activity. The transcription factor, Nrf2, is important for maintaining intracellular glutathione (GSH) levels and redox homeostasis and has been implicated in modulating DC co-stimulatory receptor expression. It is unclear whether Nrf2 mediates this effect by GSH-dependent mechanisms and whether it influences DC signaling pathways. Using bone marrow-derived iDCs from Nrf2(+/+) and Nrf2(-/-) mice, we demonstrate that Nrf2(-/-) iDCs have lower basal GSH levels, enhanced co-stimulatory receptor expression, impaired phagocytic functions, and increased antigen-specific CD8 T cell stimulation capacity. Interestingly, lowering GSH levels in Nrf2(+/+) iDCs did not recapitulate the Nrf2(-/-) iDC phenotype. Loss of Nrf2 resulted in elevated basal levels of reactive oxygen species but did not affect basal NF-κB activity or p38 MAPK phosphorylation. Using pharmacological inhibitors, we demonstrate that enhanced co-stimulatory receptor phenotype of Nrf2(-/-) iDC does not require ERK activity but is dependent on HDAC activity, indicating a potential interaction between Nrf2 function and HDAC. These results suggest that Nrf2 activity is required to counter rises in intracellular reactive oxygen species and to regulate pathways that control DC co-stimulatory receptor expression.
Myeloid-derived suppressor cells (MDSC) are present in most cancer patients and are potent inhibitors of T-cell-mediated antitumor immunity. Their inhibitory activity is attributed to production of arginase, reactive oxygen species, inducible nitric oxide synthase, and interleukin-10. Here we show that MDSCs also block T-cell activation by sequestering cystine and limiting the availability of cysteine. Cysteine is an essential amino acid for T-cell activation because T cells lack cystathionase, which converts methionine to cysteine, and because they do not have an intact xc- transporter and therefore cannot import cystine and reduce it intracellularly to cysteine. T cells depend on antigen-presenting cells (APC), such as macrophages and dendritic cells, to export cysteine, which is imported by T cells via their ASC neutral amino acid transporter. MDSCs express the xc- transporter and import cystine; however, they do not express the ASC transporter and do not export cysteine. MDSCs compete with APC for extracellular cystine, and in the presence of MDSCs, APC release of cysteine is reduced, thereby limiting the extracellular pool of cysteine. In summary, MDSCs consume cystine and do not return cysteine to their microenvironment, thereby depriving T cells of the cysteine they require for activation and function.
We demonstrate that the mechanism of redox remodeling during mouse T-cell activation involves secretion of glutathione by dendritic cells and its subsequent cleavage to cysteine. Extracellular cysteine accumulation results in a lower redox potential, which is conducive to proliferation, and changes the net redox status of exofacial protein domains. Regulatory T cells inhibit this redox metabolite signaling pathway, which represents a previously unrecognized mechanism for immunosuppression of effector T cells.
One of the functions of the immune system is to recognize and destroy abnormal or infected cells to maintain homeostasis. This is accomplished by cytotoxic lymphocytes. Cytotoxicity is a highly organized multifactor process. Here, we reviewed the apoptosis pathways induced by the two main cytotoxic lymphocyte subsets, natural killer (NK) cells and CD8+ T cells. In base to recent experimental evidence, we reviewed NK receptors involved in recognition of target-cell, as well as lytic molecules such as perforin, granzymes-A and -B, and granulysin. In addition, we reviewed the Fas-FasL intercellular linkage mediated pathway, and briefly the cross-linking of tumor necrosis factor (TNF) and TNF receptor pathway. We discussed three models of possible molecular interaction between lytic molecules from effector cytotoxic cells and target-cell membrane to induction of apoptosis.
Buthionine sulfoximine (S-n-butyl homocysteine sulfoximine), the most potent of a series of analogs of methionine sulfoximine thus far studied (Griffith, O.W., Anderson, M.E., and Meister, A. (1979) J. Biol. Chem. 254, 1205-1210), inhibited gamma-glutamylcysteine synthetase about 20 times more effectively than did prothionine sulfoximine and at least 100 times more effectively than methionine sulfoximine. The findings support the conclusion that the S-alkyl moiety of the sulfoximine binds at the enzyme site that normally binds the acceptor amino acid. Thus, the affinity of the enzyme for the S-ethyl, S-n-propyl, and S-n-butyl sulfoximines increases in a manner which is parallel to those of the corresponding isosteric acceptor amino acid substrates, i.e. glycine, alanine, and alpha-aminobutyrate. Buthionine sulfoximine did not inhibit glutamine synthetase detectably, nor did it produce convulsions when injected into mice. Injection of buthionine sulfoximine into mice decreased the level of glutathione in the kidney to a greater extent (less than 20% of the control level) than found previously after giving prothionine sulfoximine. alpha-Methyl buthionine sulfoximine was also prepared and found to be almost as effective as buthionine sulfoximine; this compound would not be expected to undergo substantial degradative metabolism. Buthionine sulfoximine and alpha-methyl buthionine sulfoximine may be useful agents for inhibition of glutathione synthesis in various experimental systems.
Regulation of proliferation of normal human T lymphocytes (T cells) by glutathione (GSH) was explored with T-cell activation models that do not require accessory cell signals. L-Buthionine-(S,R)-sulfoximine (BSO), which inactivates gamma-glutamylcysteine synthetase and therefore inhibits GSH synthesis, inhibited proliferation elicited by monoclonal antibodies directed at cluster designation 2 (CD2) and CD3 antigens, or by sn-1,2-dioctanoylglycerol and ionomycin. L-Buthionine-(R)-sulfoximine, which does not inactivate gamma-glutamylcysteine synthetase, did not affect proliferation. BSO-induced inhibition of accessory cell-independent T-cell proliferation was not reversed by recombinant human interleukin 2, despite activation-dependent expression of interleukin 2 receptor alpha by T cells treated with BSO. However, BSO-associated inhibition of T-cell proliferation was reversed by GSH or GSH ester. These studies, which show that GSH can directly modulate proliferation of highly purified T cells, suggest that GSH is essential for steps close to or at DNA synthesis. The availability of methods for decreasing and for increasing GSH levels suggest therapies to produce (i) immunosuppression (of value in organ transplantation), and (ii) immunopotentiation (of potential value in treatment of immunodeficiency states such as AIDS).
Even a moderate increase in the cellular cysteine supply elevates the intracellular glutathione (GSH) and glutathione disulfide (GSSG) levels and potentiates immunological functions of lymphocytes in vitro. At low GSSG levels, T cells cannot optimally activate the immunologically important transcription factor NF kappa B, whereas high GSSG levels inhibit the DNA binding activity of NF kappa B. The effects of GSSG are antagonized by reduced thioredoxin (TRX). As the protein tyrosine kinase activities p56lck and p59fyn are activated in intact cells by hydrogen peroxide, they are likely targets for GSSG action. These redox-regulated enzymes trigger signal cascades for NF kappa B activation and transduce signals from the T cell antigen receptor, from CD4 and CD8 molecules, and from the IL-2 receptor beta-chain. The effector phase of cytotoxic T cell responses and IL-2-dependent functions are inhibited even by a partial depletion of the intracellular GSH pool. As signal transduction is facilitated by prooxidant conditions, we propose that the well-known immunological consequences of GSH depletion ultimately may be results of the accompanying GSSG deficiency. As HIV-infected patients and SIV-infected rhesus macaques have, on the average, significantly decreased plasma cyst(e)ine and intracellular GSH levels, we also hypothesize that AIDS may be the consequence of a GSSG deficiency as well.
Nitric oxide (NO) has been invoked as an important pathogenic factor in a wide range of immunologically mediated diseases. The present study demonstrates that macrophage-derived NO may conversely function to fine tune T cell-mediated inflammation via reversible dephosphorylation of intracellular signaling molecules, which are involved in the control of T cell proliferation. Thus, T cells activated in the presence of alveolar macrophages are unable to proliferate despite expression of IL-2R and secretion of IL-2. This process is reproduced by the NO generator S-nitroso-N-acetylpenicillamine and is inhibitable by the NO synthase inhibitor N(G)-methyl-L-arginine. Analysis of T cell lysates by immunoprecipitation with specific Abs and subsequent immunoblotting indicated marked reduction of tyrosine phosphorylation of Jak3 and STAT5 mediated by NO. Further studies indicated that NO-mediated T cell suppression was reversible by the guanylate cyclase inhibitors methylene blue and LY-83583 and was reproduced by a cell-permeable analogue of cyclic GMP, implicating guanylate cyclase activation as a key step in the inhibition of T cell activation by NO.
The two-signal model states that activation of naive T cells requires a signal 1 stimulus through the TCR and a co-stimulatory signal 2. By contrast, signal 1 alone is sufficient for pre-activated T cells. Recently, however, it has been shown that under certain conditions T cells can bypass the requirement for co-stimulation. For example, CD28-deficient mice, when immunized with lymphocytic choriomeningitis virus, mount a vigorous cytotoxic T lymphocyte response and clear the virus. As a continuous effort to unravel the mechanisms of T cell activation, we previously reported activation of hybridoma T cells by recombinant single-chain MHC molecules in the absence of antigen-presenting cells. In such reconstitution experiments, since the signals delivered to the T cells are well controlled, the contribution of any known or unknown signals can be ruled out. In the present study, we analyzed the requirements for activation of naive T cells by using splenocytes from TCR transgenic mice as a source of responding cells. We observed that naive CD8+ T cells are fully activated by signal 1 alone, but that co-stimulation lowers their activation threshold. Previously activated T cells are fully responsive, even when the first stimulation was performed in the absence of co-stimulation. They display a low activation threshold and are insensitive to co-stimulation. The physiological relevance of this finding and its consequences for immunotherapy as well as for our understanding of self-tolerance are discussed.
Transport system xc- found in plasma membrane of cultured mammalian cells is an exchange agency for anionic amino acids with high specificity for anionic form of cystine and glutamate. We have isolated cDNA encoding the transporter for system xc- from mouse activated macrophages by expression in Xenopus oocytes. The expression of system xc- activity in oocytes required two cDNA transcripts, and the sequence analysis revealed that one is identical with the heavy chain of 4F2 cell surface antigen (4F2hc) and the other is a novel protein of 502 amino acids with 12 putative transmembrane domains. The latter protein, named xCT, showed a significant homology with those recently reported to mediate cationic or zwitterionic amino acid transport when co-expressed with 4F2hc. Thus xCT is a new member of a family of amino acid transporters that form heteromultimeric complex with 4F2hc, with a striking difference in substrate specificity. The expression of system xc- was highly regulated, and Northern blot analysis demonstrated that the expression of both 4F2hc and xCT was enhanced in macrophages stimulated by lipopolysaccharide or an electrophilic agent. However, the expression of xCT was more directly correlated with the system xc- activity.
Most current models of T cell activation postulate a requirement for two distinct signals. One signal is delivered through the TCR by engagement with peptide/MHC complexes, and the second is delivered by interaction between costimulatory molecules such as CD28 and its ligands CD80 and CD86. Soluble peptide/MHC tetramers provide an opportunity to test whether naive CD8+ T cells can be activated via the signal generated through the TCR-alphabeta in the absence of any potential costimulatory molecules. Using T cells from two different TCR transgenic mice in vitro, we find that TCR engagement by peptide/MHC tetramers is sufficient for the activation of naive CD8+ T cells. Furthermore, these T cells proliferate, produce cytokines, and differentiate into cytolytic effectors. Under the conditions where anti-CD28 is able to enhance proliferation of normal B6 CD4+, CD8+, and TCR transgenic CD8+ T cells with anti-CD3, we see no effect of anti-CD28 on proliferation induced by tetramers. The results of this experiment argue that given a strong signal delivered through the TCR by an authentic ligand, no costimulation is required.
T lymphocytes are defective in cystine uptake and thus require exogenous thiols for activation and function. Here we show that monocyte-derived human dendritic cells (DCs) release cysteine in the extracellular space. Cysteine generation is increased by lipopolysaccharide and tumor necrosis factor alpha, and by contact with T cells specifically recognizing soluble or alloantigens. These stimuli also induce thioredoxin (TRX) accumulation in DCs. However, only the contact with antigen-specific T cells triggers TRX secretion by the antigen-presenting cells. Fewer extracellular thiols are recovered after DC-T cell interactions when cystine uptake or TRX activity are inhibited. In addition, glutamate (Glu) and anti-TRX-inactivating antibodies inhibit antigen-dependent T lymphocyte proliferation. These findings indicate that, during antigen presentation, DCs uptake cystine and release cysteine and TRX, thus providing a reducing microenvironment that facilitates immune response.
In mammalian cultured cells, the cystine/glutamate exchange transport mediated by system x(c)- is important to maintain intracellular GSH levels. System x(c)- consists of two protein components, xCT and the heavy chain of 4F2 antigen. The activity of system x(c)- is induced by various stimuli, including electrophilic agents like diethyl maleate. In the present study, we have investigated the mechanism of the transcriptional regulation of xCT mRNA by diethyl maleate. The xCT gene consisted of twelve exons and sequence analysis identified four electrophile response element (EpRE)-like sequences between -230 and -1 in the 5'-flanking region, designated EpRE-1 to EpRE-4. To identify sequences mediating the constitutive and induced expression of xCT, a series of 5'-deletion mutants created from the 5'-flanking region were cloned into a luciferase reproter vector and transfected into BHK21 cells. The 5'-deletion analysis revealed that the sequence between -116 and -82 is essential for the basal expression and the sequence between -226 and -116 containing EpRE-1 is essential in response to diethyl maleate. Mutational analysis demonstrated that EpRE-1 is critically involved in the response to diethyl maleate. Other stress agents like arsenite, cadmium, and hydroquinone seemed to induce system x(c)- activity via the same sequence. Furthermore, the experiments using the mouse embryonic fibroblasts derived from the Nrf2-deficient mice revealed that the induction of xCT gene by electrophilic agents is mediated by Nrf2. EpRE occurs in a broad spectrum of genes for the proteins that are involved in the defense against xenobiotics and regulates their expression. The present results have demonstrated that xCT is a novel member of this protein family.
Interferon-gamma (IFNgamma) is important in regulating the adaptive immune response, and most current evidence suggests that it exerts a negative (proapoptotic) effect on CD8(+) T cell responses. We have developed a novel technique of dual adoptive transfer, which allowed us to precisely compare, in normal mice, the in vivo antiviral responses of two T cell populations that differ only in their expression of the IFNgamma receptor. We use this technique to show that, contrary to expectations, IFNgamma strongly stimulates the development of CD8(+) T cell responses during an acute viral infection. The stimulatory effect is abrogated in T cells lacking the IFNgamma receptor, indicating that the cytokine acts directly upon CD8(+) T cells to increase their abundance during acute viral infection.
Thioredoxin is a redox-regulating protein, the expression of which is induced by various forms of oxidative stress. Thioredoxin controls the interactions of various transcription factors through redox regulation. In K562 cells, we have previously reported that hemin induces activation of the thioredoxin gene by regulating NF-E2-related factor (Nrf2) through the antioxidant responsive element (ARE). We showed here that tert-butylhydroquinone (tBHQ), an electrophile stressor, activates the thioredoxin gene through the ARE. In an electrophoretic mobility shift assay, a specific Nrf2/small Maf binding complex was induced by tBHQ and bound to the ARE. Overexpression of Nrf2 increased the tBHQ-induced thioredoxin gene activation through the ARE, whereas that of Jun and Fos suppressed the activation. The tBHQ-induced ARE binding activity was completely abrogated by an oxidizing agent, diamide, whereas 2-mercaptoethanol (2-ME) reversibly recovered the inhibitory effects of diamide, suggesting that ARE binding activity is redox-dependent. Moreover, overexpression of thioredoxin enhanced the ARE-mediated thioredoxin gene activation by tBHQ. Therefore, ARE-mediated induction of thioredoxin expression is a mechanism of enhancing signal transduction through the ARE in electrophile-induced stress responses.
Glutamate cysteine ligase (GCL), which synthesizes gamma-glutamyl-cysteine (gamma-GC), is the rate-limiting enzyme in GSH biosynthesis. gamma-GC may be produced by the catalytic subunit GCLC or by the holoenzyme (GCLholo), which comprises GCLC and the modifier subunit GCLM. The Gclm(-/-) knock-out mouse shows tissue levels of GSH that are between 9 and 40% of the Gclm(+/+) wild-type mouse. In the present study, we used recombinant GCLC and GCLM and Gclm(-/-) mice to examine the role of GCLM on gamma-GC synthesis by GCLholo. GCLM decreased the Km for ATP by approximately 6-fold and, similar to other species, decreased the Km for glutamate and increased the Ki for feedback inhibition by GSH. Furthermore, GCLM increased by 4.4-fold the Kcat for gamma-GC synthesis; this difference in catalytic efficiency of GCLholo versus GCLC allowed us to derive a mathematical relationship for gamma-GC production and to determine the relative levels of GCLholo and GCLC; in homogenates of brain, liver, and lung, the ratio of GCLC to GCLholo was 7.0, 2.0, and 3.5, respectively. In kidney, however, the relationship between GCLC and GCLholo was complicated. Kidney contains GCLholo, free GCLC, and free GCLM, and free GCLC in kidney cannot interact with GCLM. Taken together, we conclude that, in most tissues, GCLM is limiting, suggesting that an increase in GCLM alone would increase gamma-GC synthesis. On the other hand, our results from kidney suggest that gamma-GC synthesis may be controlled post-translationally.
The generation of T cell immunity requires the acquisition and presentation of Ag on bone marrow-derived APCs. Dendritic cells (DC) are believed to be the most potent bone marrow-derived APCs, and the only ones that can stimulate naive T cells to productively respond to Ags. Because macrophages (Mphi) are bone marrow-derived APCs that are also found in tissues and lymphoid organs, can acquire and present Ag, and can express costimulatory molecules, we have investigated their potential to stimulate primary T cell responses in vivo. We find that both injected Mphi and DCs can migrate from peripheral tissues or blood into lymphoid organs. Moreover, injection of peptide-pulsed Mphi or DCs into mice stimulates CD8 T cells to proliferate, express effector functions including cytokine production and cytolysis, and differentiate into long-lived memory cells. Mphi and DCs stimulate T cells directly without requiring cross-presentation of Ag on host APCs. Therefore, more than one type of bone marrow-derived APC has the potential to prime T cell immunity. In contrast, another bone marrow-derived cell, the T lymphocyte, although capable of presenting Ag and homing to the T cell areas of lymphoid organs, is unable to stimulate primary responses. Because Mphi can be very abundant cells, especially at sites of infection and inflammation, they have the potential to play an important role in immune surveillance and the initiation of T cell immunity.
CD8+ T lymphocytes are excellent sources of IFN-gamma; however, the molecular mechanisms that dictate IFN-gamma expression upon TCR stimulation in these cells are not completely understood. In this study, we evaluated the involvement of NFAT1 in the regulation of IFN-gamma gene expression in murine CD8+ T cells and its relevance during Th differentiation. We show that CD8+, but not CD4+, T cells, represent the very first source of IFN-gamma upon primary T cell activation, and also that the IFN-gamma produced by naive CD8+ T cells may enhance CD4+ Th1 differentiation in vitro. TCR stimulation rapidly induced IFN-gamma expression in CD8+ T lymphocytes in a cyclosporin A-sensitive manner. Evaluation of CD8+ T cells showed that calcium influx alone was sufficient to activate NFAT1 protein, transactivate IFN-gamma gene promoter, and induce IFN-gamma production. In fact, NFAT1-deficient mice demonstrated highly impaired IFN-gamma production by naive CD8+ T lymphocytes, which were totally rescued after retroviral transduction with NFAT1-encoding vectors. Moreover, NFAT1-dependent IFN-gamma production by the CD8+ T cell compartment was crucial to control a Th2-related response in vivo, such as allergic inflammation. Consistently, CD8alpha- as well as IFN-gamma-deficient mice did not mount a Th1 immune response and also developed in vivo allergic inflammation. Our results clearly indicate that IFN-gamma production by CD8+ T cells is dependent of NFAT1 transcription factor and may be an essential regulator of Th immune responses in vivo.
Glutathione (GSH) is an abundant intracellular tripeptide that has been implicated as an important regulator of T cell proliferation. The effect of pharmacological regulators of GSH and other thiols on murine T cell signaling, proliferation, and intracellular thiol levels was examined. l-Buthionine-S,R-sulfoximine (BSO), an inhibitor of GSH synthesis, markedly reduced GSH levels and blocked T cell proliferation without significant effect on cell viability. N-acetylcysteine markedly enhanced T cell proliferation without affecting GSH levels. Cotreatment of T cells with N-acetylcysteine and BSO failed to restore GSH levels, but completely restored the proliferative response. Both 2-ME and l-cysteine also reversed the BSO inhibition of T cell proliferation. Intracellular l-cysteine levels were reduced with BSO treatment and restored with cotreatment with NAC or l-cysteine. However, 2-ME completely reversed the BSO inhibition of proliferation without increasing intracellular cysteine levels. Therefore, neither GSH nor cysteine is singularly critical in limiting T cell proliferation. Reducing equivalents from free thiols were required because oxidation of the thiol moiety completely abolished the effect. Furthermore, BSO did not change the expression of surface activation markers, but effectively blocked IL-2 and IL-6 secretion. Importantly, exogenous IL-2 completely overcame BSO-induced block of T cell proliferation. These results demonstrate that T cell proliferation is regulated by thiol-sensitive pathway involving IL-2.
CD8(+) T lymphocytes are excellent sources of IFN-gamma; however, the molecular mechanisms that dictate IFN-gamma expression upon TCR stimulation in these cells are not completely understood. In this study, we evaluated the involvement of NFAT1 in the regulation of IFN-gamma gene expression in murine CD8(+) T cells and its relevance during Th differentiation. We show that CD8(+), but not CD4(+), T cells, represent the very first source of IFN-gamma upon primary T cell activation, and also that the IFN-gamma produced by naive CD8(+) T cells may enhance CD4(+) Th1 differentiation in vitro. TCR stimulation rapidly induced IFN-gamma expression in CD8(+) T lymphocytes in a cyclosporin A-sensitive manner. Evaluation of CD8(+) T cells showed that calcium influx alone was sufficient to activate NFAT1 protein, transactivate IFN-gamma gene promoter, and induce IFN-gamma production. In fact, NFAT1-deficient mice demonstrated highly impaired IFN-gamma production by naive CD8(+) T lymphocytes, which were totally rescued,after retroviral transduction with NFAT1-encoding vectors. Moreover, NFAT1-dependent IFN-gamma production by the CD8(+) T cell compartment was crucial to control a Th2-related response in vivo, such as allergic inflammation. Consistently, CD8 alpha- as well as IFN-gamma-deficient mice did not mount a Th1 immune response and also developed in vivo allergic inflammation. Our results clearly indicate that IFN-gamma production by CD8(+) T cells is dependent of NFAT1 transcription factor and maybe an essential regulator of Th immune responses in vivo.
Macrophages are mononuclear phagocytes that are widely distributed throughout the body. These cells can contribute to development and homeostasis and participate in innate and adaptive immune responses. The physiology of macrophages can vary tremendously depending on the environment in which they reside and the local stimuli to which they are exposed. Macrophages are prodigious secretory cells, and in that role can promote and regulate immune responses and contribute to autoimmune pathologies. Macrophages are highly phagocytic, and in this capacity have long been considered to be essential immune effector cells. The important roles of macrophages in maintaining homeostasis and in contributing to tissue remodeling and wound healing is sometimes overlooked because of their vital role in host defense. © 2015 by John Wiley & Sons, Inc.
The two-signal model states that activation of naive T cells requires a signal 1 stimulus through the TCR and a co-stimulatory signal 2. By contrast, signal 1 alone is sufficient for pre-activated T cells. Recently, however, it has been shown that under certain conditions T cells can bypass the requirement for co-stimulation. For example, CD28-deficient mice, when immunized with lymphocytic choriomeningitis virus, mount a vigorous cytotoxic T lymphocyte response and clear the virus. As a continuous effort to unravel the mechanisms of T cell activation, we previously reported activation of hybridoma T cells by recombinant single-chain MHC molecules in the absence of antigen-presenting cells. In such reconstitution experiments, since the signals delivered to the T cells are well controlled, the contribution of any known or unknown signals can be ruled out. In the present study, we analyzed the requirements for activation of naive T cells by using splenocytes from TCR transgenic mice as a source of responding cells. We observed that naive CD8 1 T cells are fully activated by signal 1 alone, but that co-stimulation lowers their activation threshold. Previously activated T cells are fully responsive, even when the first stimulation was performed in the absence of co-stimulation. They display a low activation threshold and are insensitive to co-stimulation. The physiological relevance of this finding and its consequences for immunotherapy as well as for our understanding of self-tolerance are discussed.
Aims:
During sepsis, macrophages are alternatively activated toward an M2-like phenotype on contact with apoptotic cells (ACs) or their secretion products. Simultaneously, NADPH oxidase-dependent reactive oxygen species (ROS) formation is attenuated, thus contributing to immune paralysis. However, the exact mechanism remains elusive. Here, we provide mechanistic insights into diminished mRNA stability of the NADPH oxidase Nox2 on macrophage M2 polarization and therefore reduced ROS formation in sepsis.
Results:
Murine J774A.1 macrophages were stimulated with conditioned medium (CM) of apoptotic T cells, which reduced Nox2 mRNA and protein expression, consequently decreasing ROS production. An mRNA pulldown approach coupled to mass spectrometry analysis identified the RNA-binding protein SYNCRIP attached to the Nox2 mRNA 3' untranslated region (3'UTR). The binding of SYNCRIP to the 3'UTR of Nox2 mRNA is attenuated after treatment with CM of apoptotic T cells, followed by Nox2 mRNA destabilization. In in vivo models of polymicrobial sepsis such as cecal ligation and puncture, SYNCRIP was strongly downregulated, which was associated with a decreased Nox2 expression in peritoneal macrophages.
Innovation:
Downregulation of SYNCRIP in macrophages after contact to material of ACs destabilized Nox2 mRNA and impaired ROS formation, thereby contributing to an M2 phenotype shift of macrophages in sepsis.
Conclusion:
M2 polarization of macrophages in sepsis results in an attenuated SYNCRIP binding to the 3'UTR of Nox2 mRNA, destabilizing Nox2 mRNA abundance and expression. Consequently, ROS formation needed to fight against recurrent infections is impaired. In conclusion, SYNCRIP-regulated Nox2 mRNA degradation mediates the hypoinflammatory phase of sepsis.
Recently, we demonstrated that gene ablation of mitochondrial manganese superoxide dismutase and aldehyde dehydrogenase-2 markedly contributed to age-related vascular dysfunction and mitochondrial oxidative stress. The present study has sought to investigate the extent of vascular dysfunction and oxidant formation in glutathione peroxidase-1-deficient (GPx-1(-/-)) mice during the aging process with special emphasis on dysregulation (uncoupling) of the endothelial NO synthase. GPx-1(-/-) mice on a C57 black 6 (C57BL/6) background at 2, 6, and 12 months of age were used. Vascular function was significantly impaired in 12-month-old GPx-1(-/-) -mice as compared with age-matched controls. Oxidant formation, detected by 3-nitrotyrosine staining and dihydroethidine-based fluorescence microtopography, was increased in the aged GPx-1(-/-) mice. Aging per se caused a substantial protein kinase C- and protein tyrosine kinase-dependent phosphorylation as well as S-glutathionylation of endothelial NO synthase associated with uncoupling, a phenomenon that was more pronounced in aged GPx-1(-/-) mice. GPx-1 ablation increased adhesion of leukocytes to cultured endothelial cells and CD68 and F4/80 staining in cardiac tissue. Aged GPx-1(-/-) mice displayed increased oxidant formation as compared with their wild-type littermates, triggering redox-signaling pathways associated with endothelial NO synthase dysfunction and uncoupling. Thus, our data demonstrate that aging leads to decreased NO bioavailability because of endothelial NO synthase dysfunction and uncoupling of the enzyme leading to endothelial dysfunction, vascular remodeling, and promotion of adhesion and infiltration of leukocytes into cardiovascular tissue, all of which was more prominent in aged GPx-1(-/-) mice.
Thioredoxin is a redox-regulating protein, the expression of which is induced by various forms of oxidative stress. Thioredoxin controls the interactions of various transcrip-tion factors through redox regulation. In K562 cells, we have previously reported that hemin induces activation of the thioredoxin gene by regulating NF-E2-related factor (Nrf2) through the antioxidant responsive element (ARE). We showed here that tert-butylhydroquinone (tBHQ), an electrophile stressor, activates the thioredoxin gene through the ARE. In an electrophoretic mobility shift assay, a specific Nrf2/small Maf binding complex was induced by tBHQ and bound to the ARE. Over-expression of Nrf2 increased the tBHQ-induced thior-edoxin gene activation through the ARE, whereas that of Jun and Fos suppressed the activation. The tBHQ-induced ARE binding activity was completely abrogated by an oxidizing agent, diamide, whereas 2-mercaptoetha-nol (2-ME) reversibly recovered the inhibitory effects of diamide, suggesting that ARE binding activity is redox-dependent. Moreover, overexpression of thioredoxin enhanced the ARE-mediated thioredoxin gene activation by tBHQ. Therefore, ARE-mediated induction of thior-edoxin expression is a mechanism of enhancing signal transduction through the ARE in electrophile-induced stress responses. Oncogene (2003) 22, 1860–1865. doi:10.1038/sj.onc.1206369
The present report provides the first extensive characterization of the OT-I TCR transgenic line, which produces MHC class I-restricted, ovalbumin-specific, CD8+ T cells (OT-I cells). These cells are shown to be positively selected in vivo in H-2b C57BL/6 mice and in bm5 mice, which express the Kbm5 mutant molecule. In contrast, OT-I cells were not selected by mutant Kb molecules in bm1, bm3, bm8, bm10, bm11 or bm23 mice. Interestingly, however, when positive selection was examined in vitro in foetal thymic organ culture (FTOC), bm1 and bm8 were still poorly selective, but the bm3 haplotype now selected as efficiently as B6. The ability to select in vitro correlated with the capacity to present the ovalbumin (OVA) peptide to OT-I cells, as measured by induction of an OVA-specific proliferative response. These results suggest that a lower affinity TCR:MHC interaction may be necessary for positive selection in FTOC compared with selection in situ.Keywords: CD8+ T lymphocyte, cytotoxic T lymphocyte, ovalbumin, thymus, transgenic T cell receptor
The induction of phase II detoxifying enzymes is an important defense mechanism against intake of xenobiotics. While this group of enzymes is believed to be under the transcriptional control of antioxidant response elements (AREs), this contention is experimentally unconfirmed. Since the ARE resembles the binding sequence of erythroid transcription factor NF-E2, we investigated the possibility that the phase II enzyme genes might be regulated by transcription factors that also bind to the NF-E2 sequence. The expression profiles of a number of transcription factors suggest that an Nrf2/small Maf heterodimer is the most likely candidate to fulfill this rolein vivo.To directly test these questions, we disrupted the murinenrf2 genein vivo.While the expression of phase II enzymes (e.g., glutathione S-transferase and NAD(P)H: quinone oxidoreductase) was markedly induced by a phenolic antioxidantin vivoin both wild type and heterozygous mutant mice, the induction was largely eliminated in the liver and intestine of homozygousnrf2-mutant mice. Nrf2 was found to bind to the ARE with high affinity only as a heterodimer with a small Maf protein, suggesting that Nrf2/small Maf activates gene expression directly through the ARE. These results demonstrate that Nrf2 is essential for the transcriptional induction of phase II enzymes and the presence of a coordinate transcriptional regulatory mechanism for phase II enzyme genes. Thenrf2-deficient mice may prove to be a very useful model for thein vivoanalysis of chemical carcinogenesis and resistance to anti-cancer drugs.
This study is to investigate the role of Nrf2 in suppressing LPS-mediated inflammation in ex vivo macrophages by polyunsaturated fatty acids (PUFA) docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Primary peritoneal macrophages from Nrf2 wild-type (+/+; WT) and Nrf2 knockout (-/-; KO) mice were treated with lipopolysaccharides (LPS) in the presence or absence of DHA or EPA. Quantitative real-time PCR (qPCR) analyses showed that LPS potently induced cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) in the macrophages collected from Nrf2 (+/+) wild-type mice. DHA and EPA inhibited LPS-induced COX-2, iNOS, IL-1β, IL-6, or TNF-α, but increased hemeoxygenase (HO-1) expression. DHA was found to be more potent than EPA in inhibiting COX-2, iNOS, IL-1β, IL-6, and TNF-α mRNA expression. DHA and EPA were also found to induce HO-1 and Nrf2 mRNA with a different dose-response. LPS induced COX-2, iNOS, IL-1β, IL-6, and TNF-α in the macrophages collected from Nrf2 (-/-) mice as well, however, DHA and EPA suppression of COX-2, iNOS, IL-1β, IL-6, and TNF-α was attenuated as compared to that in Nrf2 (+/+) macrophages. Taken together, using Western blotting, ELISA and qPCR approaches coupled with Nrf2 (-/-) mice, our study clearly shows for the first time that DHA/EPA would induce Nrf2 signaling pathway and that Nrf2 plays a role in DHA/EPA suppression of LPS-induced inflammation.
Glutathione (GSH) is essential for survival of eukaryotic but not in prokaryotic cells. Its functions in nucleated cells are far from being known. In fact GSH plays an important role in cell proliferation. The purpose of the present review is to summarize the relationship between glutathione and the important events that take place in the nucleus during the cell cycle. Most GSH co-localizes with nuclear DNA when cells are proliferating. However, when cells were confluent no differences between nucleus and cytoplasm could be seen. A number of relevant nuclear proteins are strictly dependent on nuclear redox status. For instance, we found that telomerase is regulated by shifts in glutathione redox potential within values similar to those found in vivo, and alterations in telomerase activity are coordinated with changes in critical cell cycle proteins, particularly Id2 and E2F4. More studies are required to establish the role of nuclear glutathione in the epigenetic control of histone function. The information provided in the present review suggests an important role of nuclear glutathione as a key regulator of epigenetic events that may be critical in the regulation of cell proliferation.
Macrophages are mononuclear phagocytes that are widely distributed throughout the body. These cells can contribute to development and homeostasis and participate in innate and adaptive immune responses. The physiology of macrophages can vary tremendously depending on the environment in which they reside and the local stimuli to which they are exposed. Macrophages are prodigious secretory cells, and in that role can promote and regulate immune responses and contribute to autoimmune pathologies. Macrophages are highly phagocytic, and in this capacity have long been considered to be essential immune effector cells. The important roles of macrophages in maintaining homeostasis and in contributing to tissue remodeling and wound healing is sometimes overlooked because of their vital role in host defense.
Glutathione (GSH) is a tripeptide composed of glutamate, cysteine, and glycine. The first and rate-limiting step in GSH synthesis is catalyzed by glutamate cysteine ligase (GCL, previously known as gamma-glutamylcysteine synthetase). GCL is a heterodimeric protein composed of catalytic (GCLC) and modifier (GCLM) subunits that are expressed from different genes. GCLC catalyzes a unique gamma-carboxyl linkage from glutamate to cysteine and requires ATP and Mg(++) as cofactors in this reaction. GCLM increases the V(max) and K(cat) of GCLC, decreases the K(m) for glutamate and ATP, and increases the K(i) for GSH-mediated feedback inhibition of GCL. While post-translational modifications of GCLC (e.g. phosphorylation, myristoylation, caspase-mediated cleavage) have modest effects on GCL activity, oxidative stress dramatically affects GCL holoenzyme formation and activity. Pyridine nucleotides can also modulate GCL activity in some species. Variability in GCL expression is associated with several disease phenotypes and transgenic mouse and rat models promise to be highly useful for investigating the relationships between GCL activity, GSH synthesis, and disease in humans.
Oxidative stress is important in dendritic cell (DC) activation. Environmental particulate matter (PM) directs pro-oxidant activities that may alter DC function. Nuclear erythroid 2 p45-related factor 2 (Nrf2) is a redox-sensitive transcription factor that regulates expression of antioxidant and detoxification genes. Oxidative stress and defective antioxidant responses may contribute to the exacerbations of asthma. We hypothesized that PM would impart differential responses by Nrf2 wild-type DCs as compared with Nrf2(-/-) DCs. We found that the deletion of Nrf2 affected important constitutive functions of both bone marrow-derived and highly purified myeloid lung DCs such as the secretion of inflammatory cytokines and their ability to take up exogenous Ag. Stimulation of Nrf2(-/-) DCs with PM augmented oxidative stress and cytokine production as compared with resting or Nrf2(+/+) DCs. This was associated with the enhanced induction of Nrf2-regulated antioxidant genes. In contrast to Nrf2(+/+) DCs, coincubation of Nrf2(-/-) DCs with PM and the antioxidant N-acetyl cysteine attenuated PM-induced up-regulation of CD80 and CD86. Our studies indicate a previously underappreciated role of Nrf2 in innate immunity and suggest that deficiency in Nrf2-dependent pathways may be involved in susceptibility to the adverse health effects of air pollution in part by promoting Th2 cytokine responses in the absence of functional Nrf2. Moreover, our studies have uncovered a hierarchal response to oxidative stress in terms of costimulatory molecule expression and cytokine secretion in DCs and suggest an important role of heightened oxidative stress in proallergic Th2-mediated immune responses orchestrated by DCs.
Glutathione (GSH) is known to play an important role in various lymphocyte functions. We now report that different T cell subsets express different requirements for intracellular GSH. Depletion of intracellular GSH by buthionine sulfoximine (BSO), a specific inhibitor of GSH biosynthesis, decreases the proportion of CD8+ cells (i.e., increases the CD4+/CD8+ ratio), and inhibits particularly the generation of large blast-like CD8+ cells and cytotoxic T lymphocyte (CTL) activity. CTL activity is restored by administration of exogenous GSH. Differential effects of GSH depletion were also seen at the level of individual T cell clones. The CD4+ helper T cell clone D10.G4.1.HD was found to express a high rate of interleukin 2 (IL-2) dependent DNA synthesis even after severe depletion of intracellular GSH, whereas other T cell clones including the clone 29 were severely inhibited by BSO. The results of these studies suggest that the decreased intracellular GSH levels of HIV-1 seropositive persons are probably not (directly) responsible for the selective depletion of the CD4+ T cell subset but may be responsible for a cellular dysfunction of the CD8+ subset and for the ultimate failure of the CTL to control the viral infection in these patients.
L-Buthionine-(S,R)-sulfoximine (BSO) specifically depletes GSH synthesis by inactivating gamma-glutamylcysteine synthetase, whereas 2-ME augments intracellular GSH concentration. These reagents were used to examine GSH regulation of the proliferation and function of human PBL in response to IL-2 or OKT-3 mAb directed at the CD3 T cell Ag. 2-ME enhanced both IL-2-induced proliferation of PBL and CD3- large granular lymphocytes (LGL) and OKT-3 mAb-induced proliferation of CD3+ T cells. BSO partially suppressed activation-induced proliferation in CD3- LGL and CD3+ T cells and totally inhibited the positive co-proliferative regulation by 2-ME in these cells. By contrast, neither BSO nor 2-ME appeared to affect the activation-dependent differentiation of cytotoxic lymphocytes. The absence of effect of 2-ME or BSO on activation-induced PBL NK activity and T cell cytotoxic potential was supported by their negligible effect on the induction of two different markers of activated cytotoxic lymphocytes, namely pore-forming protein gene expression and benzoyloxycarbonyl-1-L-lysine thiobenzylester-esterase activity. BSO inhibition of CD3- LGL proliferation accounted for the inhibitory effects of BSO on both IFN-gamma production in IL-2-stimulated PBL cultures and IL-2-induced PBL lymphokine activated killer activity. The modulatory effects of 2-ME and BSO on lymphocyte proliferation regardless of phenotype (LGL vs T cell) or stimulation (IL-2, via CD3, lectin, etc.) and the functional differentiation of cytotoxic lymphocytes independent of proliferation suggests that these cells share a common site of GSH regulation close to or at the level of DNA synthesis.
Macrophages consume cystine and generate approximately equivalent amounts of acid-soluble thiol. Stimulation of macrophages with bacterial lipopolysaccharide (LPS) or tumor necrosis factor (TNF) strongly augments the amount of thiol released into the culture supernatant. Cysteine constitutes most of the acid-soluble thiol. The intracellular glutathione level and the DNA synthesis activity in mitogenically stimulated lymphocytes are strongly increased by either exogenously added cysteine, or (syngeneic) macrophages. This cysteine dependency is observed even in the presence of relatively high extracellular cystine concentration as they occur in the blood plasma. The extracellular cysteine concentration also has a strong influence on the intracellular glutathione concentration, viability, and DNA synthesis of cycling T cell clones. Moreover, the cysteine concentration in the culture medium on Day 3 and Day 4 of a 5-day allogeneic mixed lymphocyte culture (i.e., in the late phase of incubation) has a strong influence on the generation of cytotoxic T cell activity, indicating that regulatory effects of cysteine are not restricted to the early phase of the blastogenic response. The inhibitory effect of cysteine starvation on the DNA synthesis of the T cell clones and on the activation of cytotoxic T lymphocytes can be explained essentially by the depletion of intracellular glutathione, since similar effects are observed after treatment with buthionine sulfoximine (BSO), a specific inhibitor of the glutathione biosynthesis. BSO has practically no influence, however, on the N alpha-benzyloxycarbonyl Ne-t-butyloxycarbonyl-L-lysine-thiobenzyl-ester (BLT)-esterase activity and hemolytic activity of the cell lysates from cytotoxic T cells against sheep red blood cells (perforin activity). Taken together, our experiments indicate that cysteine has a regulatory role in the immune system analogous to the hormone-like lymphokines and cytokines. It is released by macrophages at a variable and regulated rate and regulates immunologically relevant functions of lymphocytes in the vicinity.
The glutathione (GSH) synthesis inhibitor, buthionine sulfoximine (BSO) was tested for cytotoxicity and thiol depletion in murine and human tumor cells in vitro, and for its antitumor activity and toxicity in vivo. The cell lines used in these studies included murine L-1210 leukemia, human RPMI 8226 myeloma, MCF-7 breast cancer and WiDr colon carcinoma. Soft agar colony forming assays showed that BSO was most effective at reducing tumor colony formation when exposed continuously to cells in vitro. Drug concentrations which inhibited colony formation to 50% of control levels ranged from 2.0-6.2 mM (for 1 hour exposures), 2-100 mM for 24 hour exposures and 0.4-1.40 microM (for continuous BSO exposures). Human myeloma cells proved most sensitive to BSO. In vitro cytotoxicity correlated with depletion of intracellular nonprotein sulfhydryls to less than or equal to 10% of control values in both L-1210 and 8226 cells. This was routinely achieved with prolonged exposures to mM BSO concentrations for greater than 24 hours. Normal mice tolerated high BSO doses (up to 5.0 g/kg) without evidence of acute toxicity. BSO was not active against L-1210 leukemia-bearing DBA/2 mice. When tested in vivo against MOPC-315 plasmacytoma-bearing BALB/c mice, BSO was not active at doses up to 4.0 g/kg. In contrast, the bifunctional alkylating agent melphalan (L-PAM) was active against MOPC-315 and this activity was enhanced by a 24 hour pretreatment of mice with 50 mg/kg of L-BSO.(ABSTRACT TRUNCATED AT 250 WORDS)
"Professional" antigen-presenting cells (APC) such as macrophages or dendritic cells display not only antigens to specific T cell receptors but deliver in addition certain "costimulatory signals." Macrophages also release substantial amounts of cysteine and raise thereby the intracellular glutathione (GSH) levels of activated T cells in their vicinity. We therefore studied the hypothesis that stimulation with "nonprofessional" APC that fail to deliver cysteine may induce a type of immunological unresponsiveness that is caused by GSH deficiency and prevented by administration of exogenous GSH. Our experiments showed that cytotoxic T cell (CTL) responses are indeed strongly suppressed in vivo and in vitro by different types of nonprofessional APC and reconstituted by administration of GSH at the time of immunization if these APC are administered at relatively high doses. At lower doses, however, the same cell types may stimulate CTL responses without exogenous GSH, and the responses may be even inhibited in these cases by GSH. CTL responses were not suppressed by high numbers of professional APC, and GSH had no substantial effect on CTL responses after in vivo immunization with professional APC or a cysteine-releasing tumor variant. However, exogenous GSH reconstituted the otherwise weak CTL response in vitro against splenic adherent cells from athymic (nude) mice. Experiments with cloned T cells (D10.G4.1) finally showed that stimulation with graded concentrations of mitogen causes a dose-dependent decrease of intracellular GSH levels. These effects are expected to play a role in "high zone tolerance," in CTL responses against certain types of tumors, and in pathological conditions with a GSH deficiency.
The expression of activation antigens, namely CD25, CD69, CD71, and HLA-DR on T cells from 15 healthy individuals stimulated with different mitogens and specific antigens was evaluated by immunofluorescence assay and flow cytometric analysis and compared with cell proliferation as a function of [3H]thymidine incorporation. CD69 was the earliest expressed antigen on stimulated cells, while HLA-DR was the latest. Regardless of the stimulus used, lymphocytes expressing CD25 and CD71 were always more numerous than cells expressing CD69 and HLA-DR. Variations in the proportion of CD4+ and CD8+ T cells expressing each activation marker were observed with different antigenic stimuli. The expression of each activation marker showed overall agreement with the [3H]thymidine incorporation assay in discriminating between positive and negative immune response. However, no correlation was observed between the percentage of CD25-, CD69-, CD71-, and HLA-DR-positive T cells and the amount of [3H]thymidine incorporation. Moreover, low doses of mitogens and antigens as well as short time of stimulation were sufficient to induce T cells to express activation antigens but not to proliferate. Our data show that results obtained by flow cytometry and [3H]thymidine incorporation may differ qualitatively, at least under certain conditions; this suggests that the 2 assays are complementary, and when combined, may gives a clearer understanding of events leading to efficient cell-mediated immune response.
Prostaglandins (PG) are synthesized by two isoforms of the enzyme PG G/H synthase [cyclooxygenase (COX)]. To examine selectivity of tolerated doses of an inhibitor of the inducible COX-2 in humans, we examined the effects of celecoxib on indices of COX-1-dependent platelet thromboxane (Tx) A2 and on systemic biosynthesis of prostacyclin in vivo. Volunteers received doses of 100, 400, or 800 mg of celecoxib or 800 mg of a nonselective inhibitor, ibuprofen. Ibuprofen, but not celecoxib, significantly inhibited TxA2-dependent aggregation, induced ex vivo by arachidonic acid (83 +/- 11% vs. 11. 9 +/- 2.2%; P < 0.005) and by collagen. Neither agent altered aggregation induced by thromboxane mimetic, U46619. Ibuprofen reduced serum TxB2 (-95 +/- 2% vs. -6.9 +/- 4.2%; P < 0.001) and urinary excretion of the major Tx metabolite, 11-dehydro TxB2 (-70 +/- 9.9% vs. -20.3 +/- 5.3%; P < 0.05) when compared with placebo. Despite a failure to suppress TxA2-dependant platelet aggregation, celecoxib had a modest but significant inhibitory effect on serum TxB2 4 hr after dosing. By contrast, both ibuprofen and celecoxib suppressed a biochemical index of COX-2 activity (endotoxin induced PGE2 in whole blood ex vivo) to a comparable degree (-93.3 +/- 2% vs. -83 +/- 6.1%). There was no significant difference between the doses of celecoxib on COX-2 inhibition. Celecoxib and ibuprofen suppressed urinary excretion of the prostacyclin metabolite 2,3 dinor 6-keto PGF1alpha. These data suggest that (i) platelet COX-1-dependent aggregation is not inhibited by up to 800 mg of celecoxib; (ii) comparable COX-2 inhibition is attained by celecoxib (100-800 mg) and ibuprofen (800 mg) after acute dosing; and (iii) COX-2 is a major source of systemic prostacyclin biosynthesis in healthy humans.
Electrophiles and reactive oxygen species have been implicated in the pathogenesis of many diseases. Transcription factor
Nrf2 was recently identified as a general regulator of one defense mechanism against such havoc. Nrf2 regulates the inducible
expression of a group of detoxication enzymes, such as glutathione S-transferase and NAD(P)H:quinone oxidoreductase, via antioxidant response elements. Using peritoneal macrophages from Nrf2-deficient
mice, we show here that Nrf2 also controls the expression of a group of electrophile- and oxidative stress-inducible proteins
and activities, which includes heme oxygenase-1, A170, peroxiredoxin MSP23, and cystine membrane transport (system xc
−) activity. The response to electrophilic and reactive oxygen species-producing agents was profoundly impaired in Nrf2-deficient
cells. The lack of induction of system xc
− activity resulted in the minimum level of intracellular glutathione, and Nrf2-deficient cells were more sensitive to toxic
electrophiles. Several stress agents induced the DNA binding activity of Nrf2 in the nucleus without increasing its mRNA level.
Thus Nrf2 regulates a wide-ranging metabolic response to oxidative stress.
Transcriptional activation of genes that play a role in detoxification of xenobiotics and defense against oxidative stress is mediated in part by the antioxidant response element (ARE). For example, it has been shown that the promoters for both the heavy and light chain gamma-glutamylcysteine synthetase (GCS(H) and GCS(L)) genes require the ARE. CNC-bZIP factors, together with small Maf proteins, have been shown to bind as heterodimers to the NF-E2/AP-1 element, which is similar to the consensus sequence for the ARE. Nrf1 and Nrf2, two widely expressed CNC-bZIP factors, have been implicated in the regulation of genes involved in oxidative stress response. In this study, we examined the effect of nrf2 mutation on the expression of genes involved in glutathione synthesis. We observed that transcripts for gcs(H) and gcs(L) genes were decreased in nrf2(-/-) fibroblasts and livers. Correspondingly, glutathione levels were decreased in Nrf2 deficient livers and fibroblasts. By transient transfection studies in nrf2(-/-) fibroblasts, we show that transcriptional activation of reporter constructs bearing the human GCS(L) promoter, as well as the functional ARE of GCS(H) promoter, required the activator protein Nrf2. By electrophoretic mobility shift assay, recombinant Nrf2 binds the ARE of the GCS(L) and GCS(H) promoters. Overexpression of Nrf2 cDNA restored glutathione (GSH) levels in nrf2(-/-) fibroblasts, which correlated with increased steady state levels of gcs(H) and gcs(L) transcripts. These results establish a link between Nrf2 transcription factor and GSH biosynthesis.
All cells depend on nutrients acquired from the extracellular environment to support the biochemical processes required for cell growth. Although unicellular organisms such as bacteria and yeast have developed strategies to survive nutrient limitation, the growth of these organisms is constrained by nutrient availability. In contrast, mammalian cells exist in a nutrient-rich environment where cell growth is not limited by substrate availability, but rather by the levels of extrinsic growth factors (1, 2). Because of the limits imposed by growth factor control, cellular proliferation is restricted even under nutrient conditions that are highly favorable for unlimited cell growth. However, it is becoming apparent that nutrient availability also plays an important role in controlling mammalian cell growth. An example of this form of growth control is found in the work of Angelini et al. (3) reported in this issue that describes how dendritic cells control the proliferation of T cells by regulating the availability of the amino acid cysteine.
Activated macrophages express high levels of Nrf2, a transcription factor that positively regulates the gene expression of antioxidant and detoxication enzymes. In this study, we examined how Nrf2 contributes to the anti-inflammatory process. As a model system of acute inflammation, we administered carrageenan to induce pleurisy and found that in Nrf2-deficient mice, tissue invasion by neutrophils persisted during inflammation and the recruitment of macrophages was delayed. Using an antibody against 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), it was observed that macrophages from pleural lavage accumulate 15d-PGJ(2). We show that in mouse peritoneal macrophages 15d-PGJ(2) can activate Nrf2 by forming adducts with Keap1, resulting in an Nrf2-dependent induction of heme oxygenase 1 and peroxiredoxin I (PrxI) gene expression. Administration of the cyclooxygenase 2 inhibitor NS-398 to mice with carrageenan-induced pleurisy caused persistence of neutrophil recruitment and, in macrophages, attenuated the 15d-PGJ(2) accumulation and PrxI expression. Administration of 15d-PGJ(2) into the pleural space of NS-398-treated wild-type mice largely counteracted both the decrease in PrxI and persistence of neutrophil recruitment. In contrast, these changes did not occur in the Nrf2-deficient mice. These results demonstrate that Nrf2 regulates the inflammation process downstream of 15d-PGJ(2) by orchestrating the recruitment of inflammatory cells and regulating the gene expression within those cells.
Co-signalling molecules are cell-surface glycoproteins that can direct, modulate and fine-tune T-cell receptor (TCR) signals. On the basis of their functional outcome, co-signalling molecules can be divided into co-stimulators and co-inhibitors, which promote or suppress T-cell activation, respectively. By expression at the appropriate time and location, co-signalling molecules positively and negatively control the priming, growth, differentiation and functional maturation of a T-cell response. We are now beginning to understand the power of co-inhibitors in the context of lymphocyte homeostasis and the pathogenesis of human diseases. In this article, I focus on several newly described co-inhibitory pathways in the B7–CD28 family.
The transcription factor Nrf2 regulates the basal and inducible expression of numerous detoxifying and antioxidant genes. The cytoplasmic protein Keap1 interacts with Nrf2 and represses its function. Analysis of keap1-knockout mice provides solid evidence that Keap1 acts as a negative regulator of Nrf2 and as a sensor of xenobiotic and oxidative stresses. The simultaneous ablation of the keap1 and nrf2 genes reversed all apparent phenotypes of the Keap1-deficient mice, suggesting that Nrf2 is a primary target of Keap1. The Nrf2-Keap1 system is now recognized as one of the major cellular defence mechanisms against oxidative and xenobiotic stresses. Furthermore, extensive studies have suggested that the Nrf2-Keap1 system contributes to protection against various pathologies, including carcinogenesis, liver toxicity, respiratory distress and inflammation.