Activation of Human CD8+ T Cells with Nitroso Dapsone-Modified HLA-B*13:01-Binding Peptides.

Previous studies have shown that cysteine-reactive drug metabolites bind covalently with protein to activate patient T cells. However, the nature of the antigenic determinants that interact with HLA and whether T cell stimulatory peptides contain the bound drug metabolite has not been defined. Because susceptibility to dapsone hypersensitivity is associated with the expression of HLA-B*13:01, we have designed and synthesized nitroso dapsone-modified, HLA-B*13:01 binding peptides and explored their immunogenicity using T cells from hypersensitive human patients. Cysteine-containing 9-mer peptides with high binding affinity to HLA-B*13:01 were designed (AQDCEAAAL [Pep1], AQDACEAAL [Pep2], and AQDAEACAL [Pep3]), and the cysteine residue was modified with nitroso dapsone. CD8+ T cell clones were generated and characterized in terms of phenotype, function, and cross-reactivity. Autologous APCs and C1R cells expressing HLA-B*13:01 were used to determine HLA restriction. Mass spectrometry confirmed that nitroso dapsone-peptides were modified at the appropriate site and were free of soluble dapsone and nitroso dapsone. APC HLA-B*13:01-restricted nitroso dapsone-modified Pep1- (n = 124) and Pep3-responsive (n = 48) CD8+ clones were generated. Clones proliferated and secreted effector molecules with graded concentrations of nitroso dapsone-modified Pep1 or Pep3. They also displayed reactivity against soluble nitroso dapsone, which forms adducts in situ, but not with the unmodified peptide or dapsone. Cross-reactivity was observed between nitroso dapsone-modified peptides with cysteine residues in different positions in the peptide sequence. These data characterize a drug metabolite hapten CD8+ T cell response in an HLA risk allele-restricted form of drug hypersensitivity and provide a framework for structural analysis of hapten HLA binding interactions.

Multi-Excitation Raman Spectroscopy for Label-Free, Strain-Level Characterization of Bacterial Pathogens in Artificial Sputum Media.

The current methods for diagnosis of acute and chronic infections are complex and skill-intensive. For complex clinical biofilm infections, it can take days from collecting and processing a patient's sample to achieving a result. These aspects place a significant burden on healthcare providers, delay treatment, and can lead to adverse patient outcomes. We report the development and application of a novel multi-excitation Raman spectroscopy-based methodology for the label-free and non-invasive detection of microbial pathogens that can be used with unprocessed clinical samples directly and provide rapid data to inform diagnosis by a medical professional. The method relies on the differential excitation of non-resonant and resonant molecular components in bacterial cells to enhance the molecular finger-printing capability to obtain strain-level distinction in bacterial species. Here, we use this strategy to detect and characterize the respiratory pathogens Pseudomonas aeruginosa and Staphylococcus aureus as typical infectious agents associated with cystic fibrosis. Planktonic specimens were analyzed both in isolation and in artificial sputum media. The resonance Raman components, excited at different wavelengths, were characterized as carotenoids and porphyrins. By combining the more informative multi-excitation Raman spectra with multivariate analysis (support vector machine) the accuracy was found to be 99.75% for both species (across all strains), including 100% accuracy for drug-sensitive and drug-resistant S. aureus. The results demonstrate that our methodology based on multi-excitation Raman spectroscopy can underpin the development of a powerful platform for the rapid and reagentless detection of clinical pathogens to support diagnosis by a medical expert, in this case relevant to cystic fibrosis. Such a platform could provide translatable diagnostic solutions in a variety of disease areas and also be utilized for the rapid detection of anti-microbial resistance.

Pharmacological Activation of Nrf2 Enhances Functional Liver Regeneration.

BACKGROUND AND AIMS: The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) regulates an array of cytoprotective genes, yet studies in transgenic mice have led to conflicting reports on its role in liver regeneration. We aimed to test the hypothesis that pharmacological activation of Nrf2 would enhance liver regeneration. APPROACH AND RESULTS: Wild-type and Nrf2 null mice were administered bardoxolone methyl (CDDO-Me), a potent activator of Nrf2 that has entered clinical development, and then subjected to two-thirds partial hepatectomy. Using translational noninvasive imaging techniques, CDDO-Me was shown to enhance the rate of restoration of liver volume (MRI) and improve liver function (multispectral optoacoustic imaging of indocyanine green clearance) in wild-type, but not Nrf2 null, mice following partial hepatectomy. Using immunofluorescence imaging and whole transcriptome analysis, these effects were found to be associated with an increase in hepatocyte hypertrophy and proliferation, the suppression of immune and inflammatory signals, and metabolic adaptation in the remnant liver tissue. Similar processes were modulated following exposure of primary human hepatocytes to CDDO-Me, highlighting the potential relevance of our findings to patients. CONCLUSIONS: Our results indicate that pharmacological activation of Nrf2 is a promising strategy for enhancing functional liver regeneration. Such an approach could therefore aid the recovery of patients undergoing liver surgery and support the treatment of acute and chronic liver disease.

Characterization of Clozapine-Responsive Human T Cells.

Use of the atypical antipsychotic clozapine is associated with life-threatening agranulocytosis. The delayed onset and the association with HLA variants are characteristic of an immunological mechanism. The objective of this study was to generate clozapine-specific T cell clones (TCC) and characterize pathways of T cell activation and cross-reactivity with clozapine metabolites and olanzapine. TCC were established and characterized by culturing PBMCs from healthy donors and patients with a history of clozapine-induced agranulocytosis. Modeling was used to explore the drug-HLA binding interaction. Global TCC protein changes were profiled by mass spectrometry. Six well-growing clozapine-responsive CD4+ and CD8+ TCC were used for experiments; activation of TCC required APC, with clozapine interacting directly at therapeutic concentrations with several HLA-DR molecules. TCC were also activated with N-desmethylclozapine and olanzapine at supratherapeutic concentrations. Marked changes in TCC protein expression profiles were observed when clozapine treatment was compared with olanzapine and the medium control. Docking of the compounds into the HLA-DRB1*15:01 and HLA-DRB1*04:01 binding clefts revealed that clozapine and olanzapine bind in a similar conformation to the P4-P6 peptide binding pockets, whereas clozapine N-oxide, which did not activate the TCC, bound in a different conformation. TCC secreted Th1, Th2, and Th22 cytokines and effector molecules and expressed TCR Vß 5.1, 16, 20, and 22 as well as chemokine receptors CXCR3, CCR6, CCR4, and CCR9. Collectively, these data show that clozapine interacts at therapeutic concentrations with HLA-DR molecules and activates human CD4+ T cells. Olanzapine only activates TCC at supratherapeutic concentrations.

Integrated transcriptomic and proteomic analyses uncover regulatory roles of Nrf2 in the kidney.

The transcription factor Nrf2 exerts protective effects in numerous experimental models of acute kidney injury, and is a promising therapeutic target in chronic kidney disease. To provide a detailed insight into the regulatory roles of Nrf2 in the kidney, we performed integrated transcriptomic and proteomic analyses of kidney tissue from wild-type and Nrf2 knockout mice treated with the Nrf2 inducer methyl-2-cyano-3,12-dioxooleano-1,9-dien-28-oate (CDDO-Me, also known as bardoxolone methyl). After 24 h, analyses identified 2561 transcripts and 240 proteins that were differentially expressed in the kidneys of Nrf2 knockout mice, compared with those of wild-type counterparts, and 3122 transcripts and 68 proteins that were differentially expressed in wild-type mice treated with CDDO-Me, compared with those of vehicle control. In the light of their sensitivity to genetic and pharmacological modulation of renal Nrf2 activity, genes/proteins that regulate xenobiotic disposition, redox balance, the intra/extracellular transport of small molecules, and the supply of NADPH and other cellular fuels were found to be positively regulated by Nrf2 in the kidney. This was verified by qPCR, immunoblotting, pathway analysis, and immunohistochemistry. In addition, the levels of NADPH and glutathione were found to be significantly decreased in the kidneys of Nrf2 knockout mice. Thus, Nrf2 regulates genes that coordinate homeostatic processes in the kidney, highlighting its potential as a novel therapeutic target.

Inhibition of metabotropic glutamate receptor 5 induces cellular stress through pertussis toxin-sensitive Gi-proteins in murine BV-2 microglia cells.

BACKGROUND: Activation of metabotropic glutamate receptor 5 (mGluR5) by (RS)-2-chloro-5-hydroxyphenylglycine (CHPG) was shown to suppress microglia activation and decrease the release of associated pro-inflammatory mediators. In contrast, the consequences of mGluR5 inhibition are less well understood. Here, we used BV-2 cells, retaining key characteristics of primary mouse microglia, to examine whether mGluR5 inhibition by 2-methyl-6-(phenylethynyl)-pyridine (MPEP) enhances cellular stress and production of inflammatory mediators. METHODS: BV-2 cells were treated with MPEP, followed by determination of cellular stress using fluorescent dyes and high-content imaging. The expression of inflammatory mediators, endoplasmic reticulum (ER)-stress markers and phosphorylated AMPKα was analyzed by quantitative PCR, ELISA and Western blotting. Additionally, phospholipase C (PLC) activity, cellular ATP content and changes in intracellular free Ca(2+) ([Ca(2+)]i) were measured using luminescence and fluorescence assays. RESULTS: Treatment of BV-2 microglia with 100 µM MPEP increased intracellular reactive oxygen species (ROS), mitochondrial superoxide, mitochondrial mass as well as inducible nitric oxide synthase (iNOS) and IL-6 expression. Furthermore, MPEP reduced cellular ATP and induced AMPKα phosphorylation and the expression of the ER-stress markers CHOP, GRP78 and GRP96. The MPEP-dependent effects were preceded by a rapid concentration-dependent elevation of [Ca(2+)]i, following Ca(2+) release from the ER, mainly via inositol triphosphate-induced receptors (IP3R). The MPEP-induced ER-stress could be blocked by pretreatment with the chemical chaperone 4-phenylbutyrate and the Ca(2+) chelator BAPTA-AM. Pretreatment with the AMPK agonist AICAR partially abolished, whilst the inhibitor compound C potentiated, the MPEP-dependent ER-stress. Importantly, the PLC inhibitor U-73122 and the Gi-protein inhibitor pertussis toxin (PTX) blocked the MPEP-induced increase in [Ca(2+)]i. Moreover, pretreatment of microglia with AICAR, BAPTA-AM, U-73122 and PTX prevented the MPEP-induced generation of oxidative stress and inflammatory mediators, further supporting a role for Gi-protein-mediated activation of PLC. CONCLUSIONS: The results emphasize the potential pathophysiological role of mGluR5 antagonism in mediating oxidative stress, ER-stress and inflammation through a Ca(2+)-dependent pathway in microglia. The induction of cellular stress and inflammatory mediators involves PTX-sensitive Gi-proteins and subsequent activation of PLC, IP3R and Ca(2+) release from the ER.

A blinded in vitro analysis of the intrinsic immunogenicity of hepatotoxic drugs: implications for preclinical risk assessment.

In vitro preclinical drug-induced liver injury (DILI) risk assessment relies largely on the use of hepatocytes to measure drug-specific changes in cell function or viability. Unfortunately, this does not provide indications toward the immunogenicity of drugs and/or the likelihood of idiosyncratic reactions in the clinic. This is because the molecular initiating event in immune DILI is an interaction of the drug-derived antigen with MHC proteins and the T-cell receptor. This study utilized immune cells from drug-naïve donors, recently established immune cell coculture systems and blinded compounds with and without DILI liabilities to determine whether these new methods offer an improvement over established assessment methods for the prediction of immune-mediated DILI. Ten blinded test compounds (6 with known DILI liabilities; 4 with lower DILI liabilities) and 5 training compounds, with known T-cell-mediated immune reactions in patients, were investigated. Naïve T-cells were activated with 4/5 of the training compounds (nitroso sulfamethoxazole, vancomycin, Bandrowski's base, and carbamazepine) and clones derived from the priming assays were activated with drug in a dose-dependent manner. The test compounds with DILI liabilities did not stimulate T-cell proliferative responses during dendritic cell-T-cell coculture; however, CD4+ clones displaying reactivity were detected toward 2 compounds (ciprofloxacin and erythromycin) with known liabilities. Drug-responsive T-cells were not detected with the compounds with lower DILI liabilities. This study provides compelling evidence that assessment of intrinsic drug immunogenicity, although complex, can provide valuable information regarding immune liabilities of some compounds prior to clinical studies or when immune reactions are observed in patients.

Identification of flucloxacillin-modified hepatocellular proteins: implications in flucloxacillin-induced liver injury.

Flucloxacillin is a ß-lactam antibiotic associated with a high incidence of drug-induced liver injury. Although expression of HLA-B*57:01 is associated with increased susceptibility, little is known of the pathological mechanisms involved in the induction of the clinical phenotype. Irreversible protein modification is suspected to drive the reaction through the provision of flucloxacillin-modified peptides that are presented to T-cells by the protein encoded by the risk allele. In this study, we have shown that flucloxacillin binds to multiple proteins within human primary hepatocytes, including major hepatocellular proteins (hemoglobin and albumin) and mitochondrial proteins. Inhibition of membrane transporters multidrug resistance-associated protein 2 (MRP2) and P-glycoprotein (P-gp) appeared to reduce the levels of covalent binding. A diverse range of proteins with different functions was found to be targeted by flucloxacillin, including adaptor proteins (14-3-3), proteins with catalytic activities (liver carboxylesterase 1, tRNA-splicing endonuclease subunit Sen2, All-trans-retinol dehydrogenase ADH1B, Glutamate dehydrogenase 1 mitochondrial, Carbamoyl-phosphate synthase [ammonia] mitochondrial), and transporters (hemoglobin, albumin, and UTP-glucose-1-phosphate uridylyltransferase). These flucloxacillin-modified intracellular proteins could provide a potential source of neoantigens for HLA-B*57:01 presentation by hepatocytes. More importantly, covalent binding to critical cellular proteins could be the molecular initiating events that lead to flucloxacillin-induced cholestasis Data are available via ProteomeXchange with identifier PXD038581.

Physical and functional interaction of sequestosome 1 with Keap1 regulates the Keap1-Nrf2 cell defense pathway.

Nrf2 regulates the expression of numerous cytoprotective genes in mammalian cells. The activity of Nrf2 is regulated by the Cul3 adaptor Keap1, yet little is known regarding mechanisms of regulation of Keap1 itself. Here, we have used immunopurification of Keap1 and mass spectrometry, in addition to immunoblotting, to identify sequestosome 1 (SQSTM1) as a cellular binding partner of Keap1. SQSTM1 serves as a scaffold in various signaling pathways and shuttles polyubiquitinated proteins to the proteasomal and lysosomal degradation machineries. Ectopic expression of SQSTM1 led to a decrease in the basal protein level of Keap1 in a panel of cells. Furthermore, RNA interference (RNAi) depletion of SQSTM1 resulted in an increase in the protein level of Keap1 and a concomitant decrease in the protein level of Nrf2 in the absence of changes in Keap1 or Nrf2 mRNA levels. The increased protein level of Keap1 in cells depleted of SQSTM1 by RNAi was linked to a decrease in its rate of degradation; the half-life of Keap1 was almost doubled by RNAi depletion of SQSTM1. The decreased level of Nrf2 in cells depleted of SQSTM1 by RNAi was associated with decreases in the mRNA levels, protein levels, and function of several Nrf2-regulated cell defense genes. SQSTM1 was dispensable for the induction of the Keap1-Nrf2 pathway, as Nrf2 activation by tert-butylhydroquinone or iodoacetamide was not affected by RNAi depletion of SQSTM1. These findings demonstrate a physical and functional interaction between Keap1 and SQSTM1 and reveal an additional layer of regulation in the Keap1-Nrf2 pathway.

Nrf2 is overexpressed in pancreatic cancer: implications for cell proliferation and therapy.

BACKGROUND: Nrf2 is a key transcriptional regulator of a battery of genes that facilitate phase II/III drug metabolism and defence against oxidative stress. Nrf2 is largely regulated by Keap1, which directs Nrf2 for proteasomal degradation. The Nrf2/Keap1 system is dysregulated in lung, head and neck, and breast cancers and this affects cellular proliferation and response to therapy. Here, we have investigated the integrity of the Nrf2/Keap1 system in pancreatic cancer. RESULTS: Keap1, Nrf2 and the Nrf2 target genes AKR1c1 and GCLC were detected in a panel of five pancreatic cancer cell lines. Mutation analysis of NRF2 exon 2 and KEAP1 exons 2-6 in these cell lines identified no mutations in NRF2 and only synonomous mutations in KEAP1. RNAi depletion of Nrf2 caused a decrease in the proliferation of Suit-2, MiaPaca-2 and FAMPAC cells and enhanced sensitivity to gemcitabine (Suit-2), 5-flurouracil (FAMPAC), cisplatin (Suit-2 and FAMPAC) and gamma radiation (Suit-2). The expression of Nrf2 and Keap1 was also analysed in pancreatic ductal adenocarcinomas (n = 66 and 57, respectively) and matching normal benign epithelium (n = 21 cases). Whilst no significant correlation was seen between the expression levels of Keap1 and Nrf2 in the tumors, interestingly, Nrf2 staining was significantly greater in the cytoplasm of tumors compared to benign ducts (P < 0.001). CONCLUSIONS: Expression of Nrf2 is up-regulated in pancreatic cancer cell lines and ductal adenocarcinomas. This may reflect a greater intrinsic capacity of these cells to respond to stress signals and resist chemotherapeutic interventions. Nrf2 also appears to support proliferation in certain pancreatic adenocarinomas. Therefore, strategies to pharmacologically manipulate the levels and/or activity of Nrf2 may have the potential to reduce pancreatic tumor growth, and increase sensitivity to therapeutics.

Deciphering Adverse Drug Reactions: In Vitro Priming and Characterization of Vancomycin-Specific T Cells From Healthy Donors Expressing HLA-A*32:01.

Drug rash with eosinophilia with systemic symptoms (DRESS) is a serious adverse event associated with use of the glycopeptide antibiotic vancomycin. Vancomycin-induced drug rash with eosinophilia with systemic symptoms is associated with the expression of human leukocyte antigen (HLA)-A*32:01, suggesting that the drug interacts with this HLA to activate CD8+ T cells. The purpose of this study was to utilize peripheral blood mononuclear cell from healthy donors to: (1) investigate whether expression of HLA-A*32:01 is critical for the priming naïve of T cells with vancomycin and (2) generate T-cell clones (TCC) to determine whether vancomycin exclusively activates CD8+ T cells and to define cellular phenotype, pathways of drug presentation and cross-reactivity. Dendritic cells were cultured with naïve T cells and vancomycin for 2 weeks. On day 14, cells were restimulated with vancomycin and T-cell proliferation was assessed by [3H]-thymidine incorporation. Vancomycin-specific TCC were generated by serial dilution and repetitive mitogen stimulation. Naïve T cells from HLA-A*02:01 positive and negative donors were activated with vancomycin; however the strength of the induced response was significantly stronger in donors expressing HLA-A*32:01. Vancomycin-responsive CD4+ and CD8+ TCC from HLA-A*32:01+ donors expressed high levels of CXCR3 and CCR4, and secreted IFN-γ, IL-13, and cytolytic molecules. Activation of CD8+ TCC was HLA class I-restricted and dependent on a direct vancomycin HLA binding interaction with no requirement for processing. Several TCC displayed cross-reactivity with teicoplanin and daptomycin. To conclude, this study provides evidence that vancomycin primes naïve T cells from healthy donors expressing HLA-A*32:01 through a direct pharmacological binding interaction. Cross-reactivity of CD8+ TCC with teicoplanin provides an explanation for the teicoplanin reactions observed in vancomycin hypersensitive patients.

HLA Class-II‒Restricted CD8+ T Cells Contribute to the Promiscuous Immune Response in Dapsone-Hypersensitive Patients.

HLA-B∗13:01 is associated with dapsone (DDS)-induced hypersensitivity, and it has been shown that CD4+ and CD8+ T cells are activated by DDS and its nitroso metabolite (nitroso dapsone [DDS-NO]). However, there is a need to define the importance of the HLA association in the disease pathogenesis. Thus, DDS- and DDS-NO‒specific CD8+ T-cell clones (TCCs) were generated from hypersensitive patients expressing HLA-B∗13:01 and were assessed for phenotype and function, HLA allele restriction, and killing of target cells. CD8+ TCCs were stimulated to proliferate and secrete effector molecules when exposed to DDS and/or DDS-NO. DDS-responsive and several DDS-NO‒responsive TCCs expressing a variety of TCR sequences displayed HLA class-I restriction, with the drug (metabolite) interacting with multiple HLA-B alleles. However, activation of certain DDS-NO‒responsive CD8+ TCCs was inhibited with HLA class-II block, with DDS-NO binding to HLA-DQB1∗05:01. These TCCs were of different origin but expressed TCRs displaying the same amino acid sequences. They were activated through a hapten pathway; displayed CD45RO, CD28, PD-1, and CTLA-4 surface molecules; secreted the same panel of effector molecules as HLA class-I‒restricted TCCs; but displayed a lower capacity to lyse target cells. To conclude, DDS and DDS-NO interact with a number of HLA molecules to activate CD8+ TCCs, with HLA class-II‒restricted CD8+ TCCs that display hybrid CD4‒CD8 features also contributing to the promiscuous immune response that develops in patients.

CDDO-imidazolide Targets Multiple Amino Acid Residues on the Nrf2 Adaptor, Keap1.

Synthetic triterpenoids including CDDO, its methyl ester (CDDO-Me, bardoxolone methyl), and its imidazolide (CDDO-Im) enhance Nrf2-mediated antioxidant and anti-inflammatory activity in many diseases by reacting with thiols on the adaptor protein, Keap1. Unlike monofunctional CDDO-Me, the bifunctional analog, CDDO-Im, has a second reactive site (imidazolide) and can covalently bind to amino acids other than cysteine on target proteins such as glutathione S-transferase pi (GSTP), serum albumin, or Keap1. Here we show for the first time that bifunctional CDDO-Im (in contrast to CDDO-Me), as low as 50 nM, can covalently transacylate arginine and serine residues in GSTP and cross-link them to adjacent cysteine residues. Moreover, we show that CDDO-Im binds covalently to Keap1 by forming permanent Michael adducts with eight different cysteines, and acyl adducts with lysine and several tyrosine residues. Modeling studies suggest that the Tyr 85 adduct stabilizes the Keap1-Cul3 complex, thereby enhancing the potency of CDDO-Im.

Development of an Improved T-cell Assay to Assess the Intrinsic Immunogenicity of Haptenic Compounds.

The prediction of drug hypersensitivity is difficult due to the lack of appropriate models and known risk factors. In vitro naïve T-cell priming assays that assess immunogenicity have been developed. However, their application is limited due requirements for 2 batches of autologous dendritic cells (DC) and inconsistent results; a consequence of single well readouts when exploring reactions where compound-specific T-cell frequency is undefined. Hence, we aimed to develop an improved, but simplified assay, termed the T-cell multiple well assay (T-MWA), that permits assessment of drug-specific activation of naïve T cells, alongside analysis of the strength of the induced response and the number of cultures that respond. DC naïve T-cell coculture, depleted of regulatory T cells (Tregs), was conducted in up to 48 wells for 2 weeks with model haptens (nitroso sulfamethoxazole [SMX-NO], Bandrowski's base [BB], or piperacillin [PIP]). Cultures were rechallenged with hapten and T-cell proliferation was measured using [3H]-thymidine incorporation. Priming of naïve T cells was observed with SMX-NO, with no requirement for DC during restimulation. Greater than 65% of cultures were activated with SMX-NO; with 8.0%, 30.8%, and 27.2% characterized as weak (stimulation index [SI] =1.5-1.9), moderate (SI = 2-3.9), and strong responses (SI > 4), respectively. The number of responding cultures and strength of the response was reproducible when separate blood donations were compared. Coinhibitory checkpoint blockade increased the strength of the proliferative response, but not the number of responding cultures. Moderate to strong priming responses were detected with BB, whereas PIP stimulated only a small number of cultures to proliferate weakly. In drug-responsive cultures inducible CD4+CD25+FoxP3+CD127low Tregs were also identified. To conclude, the T-MWA offers improvements over existing assays and with development it could be used to study multiple HLA-typed donors in a single plate format.

NRF2 regulates the glutamine transporter Slc38a3 (SNAT3) in kidney in response to metabolic acidosis.

Expression of the glutamine transporter SNAT3 increases in kidney during metabolic acidosis, suggesting a role during ammoniagenesis. Microarray analysis of Nrf2 knock-out (KO) mouse kidney identified Snat3 as the most significantly down-regulated transcript compared to wild-type (WT). We hypothesized that in the absence of NRF2 the kidney would be unable to induce SNAT3 under conditions of metabolic acidosis and therefore reduce the availability of glutamine for ammoniagenesis. Metabolic acidosis was induced for 7 days in WT and Nrf2 KO mice. Nrf2 KO mice failed to induce Snat3 mRNA and protein expression during metabolic acidosis. However, there were no differences in blood pH, bicarbonate, pCO2, chloride and calcium or urinary pH, ammonium and phosphate levels. Normal induction of ammoniagenic enzymes was observed whereas several amino acid transporters showed differential regulation. Moreover, Nrf2 KO mice during acidosis showed increased expression of renal markers of oxidative stress and injury and NRF2 activity was increased during metabolic acidosis in WT kidney. We conclude that NRF2 is required to adapt the levels of SNAT3 in response to metabolic acidosis. In the absence of NRF2 and SNAT3, the kidney does not have any major acid handling defect; however, increased oxidative stress and renal injury may occur.

Hydrogen sulfide attenuates calcification of vascular smooth muscle cells via KEAP1/NRF2/NQO1 activation.

BACKGROUND AND AIMS: Vascular calcification is a common health problem related to oxidative stress, inflammation, and circulating calciprotein particles (CPP). Hydrogen sulfide is an endogenous signaling molecule with antioxidant properties and potential for drug development targeting redox signaling. Yet, its molecular mechanisms of action in vascular smooth muscle cell (VSMC) calcification have not been delineated. We therefore sought to identify key pathways involved in the calcification-inhibitory properties of sulfide employing our recently developed CPP-induced VSMC calcification model. METHODS: Using next-generation sequencing, we investigated the transcriptomic changes of sodium hydrosulfide-treated versus non-treated calcifying VSMCs. The potential role of candidate genes and/or regulatory pathways in prevention of calcification was investigated by small interfering RNA (siRNA). RESULTS: CPP led to a pronounced accumulation of cell-associated calcium, which was decreased by sulfide in a concentration-dependent manner. Both, CPP-induced hydrogen peroxide production and enhanced pro-inflammatory/oxidative stress-related gene expression signatures were attenuated by sulfide-treatment. Gene ontology enrichment and in silico pathway analysis of our transcriptome data suggested NAD(P)H dehydrogenase [quinone] 1 (NQO1) as potential mediator. Corroborating these findings, silencing of Kelch-like ECH-associated protein 1 (KEAP1), an inhibitor of nuclear factor (erythroid-derived 2)-like 2 (NRF2) nuclear activity, enhanced NQO1 expression, whereas NRF2 silencing reduced the expression of NQO1 and abrogated the calcification-suppressing activity of sulfide. Moreover, immunofluorescence microscopy and Western blot analysis confirmed nuclear translocation of NRF2 by sulfide in VSMC. CONCLUSIONS: Sulfide attenuates CPP-induced VSMC calcification in vitro via the KEAP1-NRF2 redox sensing/stress response system by enhancing NQO1 expression.

Dibutyltin promotes oxidative stress and increases inflammatory mediators in BV-2 microglia cells.

The organotin dibutyltin (DBT) is used as biocide and as stabilizer in the manufacture of silicones, polyvinyl chloride plastics, polyurethanes and polyester systems. Although the immuno- and neurotoxicity of DBT has been recognized, the underlying mechanisms remained unclear and the impact of DBT on microglia cells has not yet been established. We now used cultured mouse BV-2 cells as a model of activated microglia to investigate the impact of DBT on oxidative stress and pro-inflammatory cytokines. DBT, at subcytotoxic concentrations, increased intracellular reactive oxygen species (ROS), mitochondrial mass, mitochondrial ROS, and the mRNA expression of inducible nitric oxide synthase (iNOS) and NADPH-dependent oxidase-2 (NOX-2). ATP levels were decreased by DBT, followed by activation of AMP-activated protein kinase (AMPK). Moreover, DBT potentiated the expression of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Inhibition of NOX-2 diminished both ROS production and induction of IL-6 expression. The DBT-mediated increase in NF-κB activity and subsequent up regulation of IL-6 was abolished by co-treatment with a NF-κB inhibitor. Treatment of cells with pharmacological inhibitors indicated a role for mitogen-activated protein kinases (MAPKs), PI3K/Akt, protein kinase C (PKC) and phospholipase C (PLC) in the DBT-induced toxicity. Finally, the calcium chelator BAPTA-AM diminished oxidative stress and induction of IL-6 expression, indicating the involvement of increased intracellular calcium in the enhanced microglia activity upon exposure to DBT. Together, the results suggest that a potentiation of oxidative stress and pro-inflammatory cytokine expression in microglia cells contribute to the toxicity of DBT in the CNS.

Chemical tuning enhances both potency toward nrf2 and in vitro therapeutic index of triterpenoids.

The transcription factor Nrf2 protects against a number of experimental pathologies, and is a promising therapeutic target. The clinical investigation of a potent Nrf2-inducing agent, the triterpenoid (TP) bardoxolone methyl (BARD), was recently halted due to adverse cardiovascular events in chronic kidney disease patients, although the underlying mechanisms are yet to be resolved. The majority of small molecule Nrf2 inducers are electrophilic and trigger Nrf2 accumulation via the chemical modification of its redox-sensitive repressor Keap1. Therefore, it is pertinent to question whether the therapeutic targeting of Nrf2 could be hindered in many cases by the inherent reactivity of a small molecule inducer toward unintended cellular targets, a key mechanism of drug toxicity. Using H4IIE-ARE8L hepatoma cells, we have examined the relationship between (a) Nrf2 induction potency, (b) toxicity and (c) in vitro therapeutic index (ratio of b:a) for BARD and a number of other small molecule activators of Nrf2. We show that BARD exhibits the highest potency toward Nrf2 and the largest in vitro therapeutic index among compounds that have been investigated clinically (namely BARD, sulforaphane and dimethylfumarate). Through further examination of structurally related TPs, we demonstrate that an increase in potency toward Nrf2 is associated with a relatively smaller increase in toxicity, indicating that medicinal chemistry can be used to enhance the specificity of a compound as an inducer of Nrf2 signaling whilst simultaneously increasing its therapeutic index. These findings will inform the continuing design and development of drugs targeting Nrf2.

Suppression of the Nrf2-dependent antioxidant response by glucocorticoids and 11ß-HSD1-mediated glucocorticoid activation in hepatic cells.

BACKGROUND: Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a key transcription factor regulating a plethora of detoxifying enzymes and antioxidant genes involved in drug metabolism and defence against oxidative stress. The glucocorticoid receptor (GR) is a ligand-induced transcription factor involved in the regulation of energy supply for metabolic needs to cope with various stressors. GR activity is controlled by glucocorticoids, which are synthesized in the adrenal glands and regenerated mainly in the liver from inactive cortisone by 11ß-hydroxysteroid dehydrogenase-1 (11ß-HSD1). METHODS AND PRINCIPAL FINDINGS: Using transfected HEK-293 cells and hepatic H4IIE cells we show that glucocorticoids, activated by 11ß-HSD1 and acting through GR, suppress the Nrf2-dependent antioxidant response. The expression of the marker genes NQO1, HMOX1 and GST2A was suppressed upon treatment of 11ß-HSD1 expressing cells with cortisone, an effect that was reversed by 11ß-HSD1 inhibitors. Furthermore, our results demonstrate that elevated glucocorticoids lowered the ability of cells to detoxify H(2)O(2). Moreover, a comparison of gene expression in male and female rats revealed an opposite sexual dimorphism with an inverse relationship between 11ß-HSD1 and Nrf2 target gene expression. CONCLUSIONS: The results demonstrate a suppression of the cellular antioxidant defence capacity by glucocorticoids and suggest that elevated 11ß-HSD1 activity may lead to impaired Nrf2-dependent antioxidant response. The gender-specific differences in hepatic expression levels of 11ß-HSD1 and Nrf2 target genes and the impact of pharmacological inhibition of 11ß-HSD1 on improving cellular capacity to cope with oxidative stress warrants further studies in vivo.