Nrf2-null Mice Research Articles

Targeting antioxidant factor Nrf2 by raffinose ameliorates lipid dysmetabolism-induced pyroptosis, inflammation and fibrosis in NAFLD

BackgroundNon-alcoholic fatty liver disease (NAFLD) is a persistent liver condition that affects both human health and animal productive efficiency on a global scale. A number of naturally occurring compounds activate nuclear factor erythroid 2-related factor 2 (Nrf2) as a transcription factor with important protective effects against many liver diseases, including NAFLD. Raffinose (Ra), an oligosaccharide extracted from several plants, exhibits diverse biological functions. However, the uncertainty lies in determining whether the activation of Nrf2 by Ra can provide a preventive effect on liver lipotoxicity. PurposeThe aim of this study was to shed light on the molecular pathways by which Ra possesses its protective benefits against NAFLD. MethodsExperimental protocols were established using WT and Nrf2-null (Nrf2−/−) mice. Liver samples from each group were collected for Western blot, RT-qPCR, H & E, Sirius red and Oil red O staining. Additionally, serums were processed for ELISA. ALM12 cells were gathered for Western blot and immunofluorescence. Moreover, to elucidate the molecular mechanism of Ra, molecular docking was performed. ResultsOur results indicated that Ra remarkably alleviated liver lipotoxic in vivo and in vitro. Ra treatment effectively corrected hepatic steatosis, the release of AST, ALT, TG, and TC, as well as the depletion of HDL and LDL. Meanwhile, Ra efficiently prevented inflammation by inhibiting the TLR4-MyD88-NF-κB pathway and pyroptosis. Additionally, these findings implied that Ra reduced the production of fibrosis-related proteins, which enhanced collagen deposition. Molecular docking revealed that Ra possessed the ability to bind specific regions of Nrf2, resulting in the enhancement of Nrf2 activation and nuclear translocation. Ra treatment restored serum redox factors and antioxidant enzymes to normal levels; however, these alterations were clearly reversed in Nrf2−/− mice. ConclusionThis study reveals novel information on Ra's protective benefits against liver injury caused by abnormal lipid metabolism; these effects are mostly mediated by Nrf2 activation, suggesting a potential new medicine or treatment strategy for NAFLD.

Nfe2l2/NRF2 Deletion Attenuates Tumorigenesis and Increases Bacterial Diversity in a Mouse Model of Lynch Syndrome.

Lynch syndrome (LS) is the most prevalent heritable form of colorectal cancer. Its early onset and high lifetime risk for colorectal cancer emphasize the necessity for effective chemoprevention. NFE2L2 (NRF2) is often considered a potential druggable target, and many chemopreventive compounds induce NRF2. However, although NRF2 counteracts oxidative stress, it is also overexpressed in colorectal cancer and may promote tumorigenesis. In this study, we evaluated the role of NRF2 in the prevention of LS-associated neoplasia. We found increased levels of NRF2 in intestinal epithelia of mice with intestinal epithelium-specific Msh2 deletion (MSH2ΔIEC) compared with C57BL/6 (wild-type) mice, as well as an increase in downstream NRF2 targets NAD(P)H dehydrogenase (quinone 1) and glutamate-cysteine ligase catalytic subunit. Likewise, NRF2 levels were increased in human MSH2-deficient LS tumors compared with healthy human controls. In silico analysis of a publicly accessible RNA sequencing LS dataset also found an increase in downstream NRF2 targets. Upon crossing MSH2ΔIEC with Nrf2null (MSH2ΔIECNrf2null) mice, we unexpectedly found reduced tumorigenesis in MSH2ΔIECNrf2null mice compared with MSH2ΔIEC mice after 40 weeks, which occurred despite an increase in oxidative damage in MSH2ΔIECNrf2null mice. The loss of NRF2 impaired proliferation as seen by Ki67 intestinal staining and in organoid cultures. This was accompanied by diminished WNT/β-catenin signaling, but apoptosis was unaffected. Microbial α-diversity increased over time with the loss of NRF2 based upon 16S rRNA gene amplicon sequencing of murine fecal samples. Altogether, we show that NRF2 protein levels are increased in MSH2 deficiency and associated neoplasia, but the loss of NRF2 attenuates tumorigenesis. Activation of NRF2 may not be a feasible strategy for chemoprevention in LS. Prevention Relevance: Patients with LS have an early onset and high lifetime risk for colorectal cancer. In this study, we show that NRF2 protein levels are increased in MSH2 deficiency and associated neoplasia, but the loss of NRF2 attenuates tumorigenesis. This suggests that NRF2 may not be a tumor suppressor in this specific context.

Effect of differently coated titanium dioxide nanoparticles on the lung in wild-type and Nrf2 null mice

Effect of differently coated titanium dioxide nanoparticles on the lung in wild-type and Nrf2 null mice

Protective role of Nrf2 in zinc oxide nanoparticles-induced lung inflammation in female mice and sexual dimorphism in susceptibility

BackgroundZinc oxide nanoparticles (ZnO-NPs) are currently employed in various products such as rubber, paint, and cosmetics. Our group reported recently that Nrf2 protein provides protection against pulmonary inflammation induced by ZnO-NPs in male mice. The current study investigated the effect of Nrf2 deletion on the lung inflammatory response in female mice exposed to ZnO-NPs. MethodsAn equal number of female Nrf2-/- mice and female Nrf2+/+ mice (24 each) were allocated into three equal groups, and each was exposed to ZnO-NPs at either 0, 10 or 30 µg ZnO-NPs/mouse through pharyngeal aspiration. Bronchoalveolar lavage fluid (BALF) and lungs were examined 14 days later to determine the number of inflammatory cells, the protein level, and for scoring inflammation histopathologically. The mRNA levels of Nrf2-dependent antioxidant enzymes and proinflammatory cytokine in lung tissue were also measured. ResultsExposure to ZnO-NPs increased all types of BALF cells and lung inflammation scores in both of female Nrf2-null (Nrf2-/-) and wild-type (Nrf2+/+) mice, and Nrf2 deletion enhanced ZnO-NPs-induced increase in the number of eosinophils in BALF. Exposure to ZnO-NPs dose-dependently increased the level of oxidized glutathione (GSSG), and mRNA levels of proinflammatory cytokines/chemokines; KC, MIP-2, IL-6, IL-1β and MCP-1 only in wild-type mice. Nrf2 deletion decreased total glutathione levels and basal mRNA levels of SOD1 and NQO1, and increased the basal mRNA level of above proinflammatory cytokines/chemokines. Nrf2 deletion enhanced ZnO-NPs-induced downregulation of GcLc, GR and TGF-β and upregulation of HO-1 and TNF-α. Taken together with our previous results in male mice, our results showed a lower susceptibility of females to lung tissue inflammation, relative to males, irrespective of Nrf2 deletion, and that enhancement of ZnO-NPs-induced upregulation of HO-1 and TNF-α and downregulation of GcLc, GR and TGF-β by deletion of Nrf2 is specific to female mice. ConclusionWe conclude that Nrf2 provides protection in female mice against increase in BALF eosinophils, probably through down-regulation of proinflammatory cytokines/chemokines and upregulation of oxidative stress-related genes. The study also suggests lower susceptibility to lung tissue inflammation in female mice relative to their male counterparts and the synergistic effects of Nrf2 and exposure to ZnO-NPs on mRNA expression of GcLc, GR, HO-1, TGF-β or TNF-α in female mice.

Nrf2 deficiency causes hepatocyte dedifferentiation and reduced albumin production in an experimental extrahepatic cholestasis model.

The transcription factor Nrf2 modulates the initiation and progression of a number of diseases including liver disorders. We evaluated whether Nrf2 mediates hepatic adaptive responses to cholestasis. Wild-type and Nrf2-null mice were subjected to bile duct ligation (BDL) or a sham operation. As cholestasis progressed to day 15 post-BDL, hepatocytes in the wild-type mice exhibited a tendency to dedifferentiate, indicated by the very weak expression of hepatic progenitor markers: CD133 and tumor necrosis factor-like weak induced apoptosis receptor (Fn14). During the same period, Nrf2 deficiency augmented this tendency, manifested by higher CD133 expression, earlier, stronger, and continuous induction of Fn14 expression, and markedly reduced albumin production. Remarkably, as cholestasis advanced to the late stage (40 days after BDL), hepatocytes in the wild-type mice exhibited a Fn14+ phenotype and strikingly upregulated the expression of deleted in malignant brain tumor 1 (DMBT1), a protein essential for epithelial differentiation during development. In contrast, at this stage, hepatocytes in the Nrf2-null mice entirely inhibited the upregulation of DMBT1 expression, displayed a strong CD133+/Fn14+ phenotype indicative of severe dedifferentiation, and persistently reduced albumin production. We revealed that Nrf2 maintains hepatocytes in the differentiated state potentially via the increased activity of the Nrf2/DMBT1 pathway during cholestasis.

Oltipraz ameliorates the progression of steatohepatitis in Nrf2-null mice fed a high-fat diet.

Oltipraz, a synthetic dithiolethione, has chemopreventive effect through nuclear factor erythroid 2-related factor 2 (Nrf2) activation. Nrf2 is known to be involved in the development of experimental steatohepatitis in rodents. In this study, to evaluate the effect of oltipraz on lipid and bile acid metabolism, wild-type and Nrf2-null mice were fed the standard diet (containing 4% soybean oil) with or without oltipraz. Based on these results, we examined the effect of oltipraz on the experimental steatohepatitis in high-fat diet (containing 4% soybean oil and 20% lard) fed Nrf2-null mice. Oltipraz induced hepatic mRNA expression of peroxisome proliferator-activated receptor α, carnitine palmityl transferase 1, and bile salt export pump by Nrf2 independent mechanisms. In Nrf2-null mice fed a high-fat diet for 12 weeks, moderate to severe inflammation and fibrosis were observed. Oral administration of oltipraz suppressed the degree of inflammation and fibrosis in Nrf2-null mouse liver fed a high-fat diet. These histopathological findings approximately corresponded to the data of mRNA expression of tumor necrosis factor α, monocyte chemoattractant protein-1, Timp-1, and collagen type 1α1. These results indicated that oltipraz administration ameliorated liver injury by Nrf2 independent manner in a model of steatohepatitis generated by Nrf2-null mice with high-fat diet.

Pharmacological Activation of Nrf2 Enhances Functional Liver Regeneration.

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. 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. 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.

Free Heme Induces a Heme Importer, Heme Catabolism and an Angiogenic Factor in Multiple Organs in Mice: Implications for Organ-Specific Response to Intravascular Hemolysis

Heme (iron protoporphyrin IX) is a major toxic component of hemoglobin released from red cell turnover especially during intravascular hemolysis such as in sickle cell disease, malaria and paroxysmal nocturnal hemoglobinuria. Heme oxygenase 1 (HO-1) is an enzyme that degrades heme, primarily generated by damaged or senescent red blood cells, releasing iron from protoporphyrin and generating equimolar amounts of biliverdin and carbon monoxide. The HO-1 heme catabolic pathway provides an important defense from oxidant and inflammatory consequences of exposure to free heme. HO-1 induction occurs as a transcriptional response to heme through the NRF2 oxidant-sensing transcription factor. Placental Growth Factor (PGF) is an angiogenic growth factor secreted by proliferating erythroblasts during normal development, which we previously have found is also activated by heme, secondarily regulating expression of the vasoconstrictor endothelin-1 that promotes pulmonary hypertension. We assessed the transcriptional response of HO-1 and PGF in different organs in C57BL/6J wild type (WT) and Nrf2 null mice following acute exposure to heme.WT and Nrf2 null mice were injected intravenously with saline or hemin (120 µmoles/kg body weight), followed by harvest of the bone marrow, spleen, liver, kidney, lung and heart 3 hours after injection. Real time PCR was used to quantify transcripts and relative changes in expression. Heme oxygenase-1 mRNA levels rose sharply in organs from WT mice that received hemin compared with saline control: liver (207-fold), heart (201-fold), kidney (101-fold), lung (69-fold), bone marrow (13-fold) and spleen (7-fold). In Nrf2 null mice, induction of HO-1 mRNA expression by heme was blunted as expected, with the greatest HO-1 mRNA induction in the kidney, followed by the heart, liver, lung, bone marrow and spleen. Our findings suggest that PGF expression is Nrf2-dependent primarily in the bone marrow, lung and kidney (4-fold, 1.65-fold and 1.89-fold, respectively, WT compared to Nrf2 null). Interestingly, in some of the organs, we observed an inverse relationship between HO-1 inducibility and PGF expression in organs from Nrf2 null mice injected with hemin, suggesting that reduced clearance of heme promotes more robust PGF induction. We observed the highest basal expression of PGF (1.23 and 2.21 DeltaCt compared to GUSB) in the heart, rising even higher with heme exposure (0.51 and 0.80 DeltaCt) in WT and Nrf2 null mice respectively. This translates to 70% and 57% respectively of the level of the “housekeeping gene” GUSB, which constitutes relatively high-level expression. In WT mice injected with hemin, the heme importer Hrg1 mRNA induction was mild but significant (heart 2.5 fold, kidney 2.1-fold, and lung 2.2-fold), which are also the organs with highest HO-1 inducibility (all p<0.05). Hrg1 mRNA induction was strongly dependent on Nrf2. Hrg1 activation in the liver increased by 1.82-fold in WT mice injected with hemin (p=0.097).The liver has been shown by others to take up free heme avidly, but these are the first data to demonstrate widely ranging differences in Nrf2-dependent heme response among other organs, implying their uptake of heme. We hypothesize that these differences in heme-induced HO-1 expression may reflect proportional differences in heme import, potentially via the Hrg1 heme importer that we find to be heme-inducible. There is likely also a role for the CD93 receptor for heme-hemopexin complexes and the FLVCR2 heme importer. This means that many organs may have some ability to take up free heme. Also, the ability of many organs to import extracellular heme might potentially play a role in diseases with robust release of heme into the circulation, such as β-thalassemia, sickle cell disease, malaria and other hemolytic anemias. PGF expression in the heart has previously shown by others to play a role in cardiac development and hypertrophy, and our new results point to cardiac PGF as a new hypothetical target of heme-induced dysregulation. Understanding the mechanism of organ-specific response to heme and PGF induction could expand the range of heme-mediated toxicity and pathophysiology of the heme-Nrf2-PGF-endothelin axis in sickle cell disease. [Display omitted] DisclosuresOfori-Acquah:NuvOx Pharma: Patents & Royalties. Kato:Global Blood Therapeutics: Consultancy; Bayer: Research Funding; MAST Therapeutics: Research Funding; Novartis: Consultancy.

Heme Regulates the Expression in Erythroblasts of Placenta Growth Factor, a Contributor to Pulmonary Hypertension in Sickle Cell Disease, through the Oxidant Response Transcription Factor NRF2

Patients with sickle cell disease (SCD) have elevated plasma levels of placenta growth factor (PGF), which promotes expression of the pulmonary vasoconstrictor endothelin-1 (ET-1) contributing to pulmonary hypertension, an important age-related and life-limiting complication of SCD. In SCD patients, markers of high iron burden are associated with the highest PGF levels, leading us to the investigation of a mechanism where excessive heme induces PGF expression. Our lab's published work has demonstrated a significant heme-bound iron (hemin) stimulation of the PGF promoter in human K562 cells and in primary human erythroid cells. Our initial experiments with N-acetylcysteine and the nonspecific oxidant hydrogen peroxide, suggested to us a role of oxidant stress in PGF regulation, hypothetically through the NRF2 antioxidant response mechanism.We conducted gene expression analysis by quantitative real time PCR for PGF in human K562 erythroblastoid cells, erythroid-differentiated human and mouse primary bone marrow cells before and after exposure to hemin. We tested the effect of Nrf2 agonists and an antagonist. We analyzed expression of heme oxygenase-1 as a positive control, and expression of several Nrf2 family members, as well as baseline genes. We performed siRNA gene knockdown experiments in cultured cells, chromatin immunoprecipitation experiments and intravenous injection of hemin (120 μmol/kg) in the tail veins of wild type and Nrf2 null mice, followed by assay of plasma PGF by ELISA and qPCR of bone marrow.We find that heme regulates the expression of several members of the Nrf2 family, which in turn may appear to affect PGF expression. More specifically, the antioxidant transcription factory family members NRF2, MafF, MafG and BACH1 are transcriptionally activated in a specific temporal pattern after heme exposure. NFE2 and MafK transcripts were simultaneously repressed by heme treatment. The well-characterized non-oxidative NRF2 agonist sulforaphane activated the endogenous PGF promoter and the NRF2 inhibitor brusatol inhibited heme induction of the endogenous PGF promoter. Specific gene knockdown results using siRNA supports additive roles for NRF2 and its heterodimeric partner MafG in activating the PGF promoter in response to heme, presumably as a heterodimer. We verified direct binding of NRF2 on the PGF promoter by chromatin immunoprecipitation experiments. While PGF regulation shows quite many common aspects to HO-1 regulation (heme and oxidative stress response), we find that protoporphyrin induces HO-1 (known to require BACH-1), but not PGF.In addition to the experimental data from our human K562 cell culture models, we have found corroborating results in primary erythroid-differentiated human and murine hematopoietic cells. In vivo, intravenous injection of heme in wild type mice produces robust secretion of PGF protein into plasma within 3 hours (192 ± 16 vs. 527 ± 62 pg/mL, P <0.0001, mean ± SEM, one-way ANOVA with Sidak's multiple comparison test) and this heme induction of plasma PGF protein is blunted in the Nrf2 null mice (527 ± 62 vs. 343 ± 21 pg/mL, P=0.004).Our results support a mechanism in which accelerated heme exposure in SCD promotes robust expression of PGF in erythroblasts during erythroid differentiation, through a pathway that involves EKLF, NRF2 and MafG, culminating in expression of the vascoconstrictor endothelin-1. This mechanism helps to explain the clinical observation that adults with SCD heavily exposed to red cell transfusion are more likely to develop high endothelin levels and pulmonary hypertension, as a potential consequence of excess heme trafficking from the turnover of transfused red cells. These results might inspire greater adherence to existing approved therapies to chelate iron in SCD. [Display omitted] DisclosuresOfori-Acquah:NuvOx Pharma: Patents & Royalties. Kato:Bayer: Research Funding; Global Blood Therapeutics: Consultancy; MAST Therapeutics: Research Funding; Novartis: Consultancy.

The role of the stress-activated transcription factor Nrf2 in OVA-induced food allergy

Abstract Food allergies are rising at a striking rate, but the underlying cause is unclear. The purpose of the studies was to determine the effect of the food preservative tert-butylhydroquinone (tBHQ) in a mouse model of food allergy. Our preliminary studies show that skin-sensitization with OVA induced an elevated OVA-IgE level in mice on a tBHQ diet. Likewise, in response to oral challenge, a greater decrease in body temperature during anaphylaxis was observed in the tBHQ group. Previously published in vitro studies suggest that tBHQ promotes Th2 polarization through activation of the stress-activated transcription factor, Nrf2. Therefore, we proposed the hypothesis that tBHQ exacerbates allergic response in OVA sensitized mice through activation of Nrf2. We implemented adoptive transfers of CD4 T cells from wild-type or Nrf2-null mice into immunodeficient mice (all mice received wild-type B cells). The study revealed marked genotype differences. Notably, dietary tBHQ had little effect on food allergy in recipients of Nrf2-deficient CD4 T cells. These mice showed attenuated sensitization as compared to wild-type CD4 T cells recipients. Strikingly, mice receiving Nrf2-deficient T cells did not become anaphylactic following challenge. In contrast, tBHQ caused an increase in OVA-IgE levels and a greater drop in body temperature in mice receiving wild-type CD4 T cells, suggesting these effects are Nrf2 dependent. In addition, recipients of Nrf2-deficient CD4 T cells showed an attenuated percentage of Th2 cells and mast cell degranulation response, following challenge. Taken together, these data suggest a critical role for Nrf2 during food allergy and demonstrate that tBHQ exacerbates food allergy in a CD4 T cell-Nrf2 dependent manner.

The role of the stress‐activated transcription factor Nrf2 in OVA‐induced food allergy

Immune-mediated adverse reactions to food allergens are rising at a striking rate, but the underlying cause is unknown. The purpose of the present studies was to determine the effect of the food preservative tert-butylhydroquinone (tBHQ) on anaphylaxis in a mouse model of food allergy. Our preliminary studies demonstrate that skin-sensitization with OVA induced a markedly higher OVA-specific IgE response in mice on a tBHQ diet. Likewise, in response to OVA challenge by the oral route, a greater decrease in body temperature during anaphylaxis was observed in the tBHQ-treated group. Previously published in vitro studies suggest that tBHQ promotes Th2 polarization, a key step in the development of allergy, through activation of the stress-activated transcription factor, Nrf2. Therefore, we proposed the hypothesis that tBHQ exacerbates allergic response in ovalbumin (OVA) sensitized mice through activation of Nrf2. We implemented adoptive transfers of CD4 T cells from wild-type or Nrf2-null mice into immunodeficient SCID mice (all mice also received wild-type B cells). The study revealed marked genotype differences. Notably, dietary tBHQ had little effect on food allergy in recipients of Nrf2-deficient CD4 T cells. These mice showed markedly attenuated sensitization as compared to mice who received wild-type CD4 T cells. Strikingly, mice receiving Nrf2-deficient T cells did not become anaphylactic following OVA challenge. In contrast, tBHQ caused an increase in OVA-IgE plasma levels and a greater drop in body temperature in mice receiving wild-type CD4 T cells, suggesting these effects are Nrf2 dependent. In addition, recipients of Nrf2-deficient CD4 T cells showed a greatly attenuated percentage of Th2 cells and mucosal mast cell degranulation response, following OVA challenge. Taken together, these data suggest a critical role for Nrf2 during food allergen-induced sensitization and anaphylaxis and demonstrate that tBHQ exacerbates food allergy in a CD4 T cell- Nrf2 dependent manner in mice.

Dimethyl Fumarate Reduces Oxidative Stress and Pronociceptive Immune Responses in a Murine Model of Complex Regional Pain Syndrome.

Complex regional pain syndrome (CRPS) is a highly disabling cause of pain often precipitated by surgery or trauma to a limb. Both innate and adaptive immunological changes contribute to this syndrome. Dimethyl fumarate (DMF) works through the nuclear factor erythroid 2-related factor 2 (Nrf2) transcription factor and other targets to activate antioxidant systems and to suppress immune system activation. We hypothesized that DMF would reduce nociceptive, functional, and immunological changes measured in a model of CRPS. Male C57BL/6 mice were used in the well-characterized tibial fracture model of CRPS. Some groups of mice received DMF 25 mg/kg/d orally, per os for 3 weeks after fracture versus vehicle alone. Homozygous Nrf2 null mutant mice were used as test subjects to address the need for this transcription factor for DMF activity. Allodynia was assessed using von Frey filaments and hindlimb weight-bearing data were collected. The markers of oxidative stress malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) were quantified in the skin of the fractured mice using immunoassays along with the innate immune system cytokines IL-1β and IL-6. The accumulation of IgM in the fractured limbs and lymph node hypertrophy were used as indexes of adaptive immune system activation, and the passive transfer of serum from wildtype fractured mice to B cell-deficient fractured muMT mice (mice lacking B cells and immunoglobulin) helped to assess the pronociceptive activity of humoral factors. We observed that oral DMF administration strongly prevented nociceptive sensitization and reduced uneven hindlimb weight bearing after fracture. DMF was also very effective in reducing the accumulation of markers of oxidative stress, activation of innate immune mediator production, lymph node hypertrophy, and the accumulation of IgM in fractured limbs. The sera of fractured vehicle-treated but not DMF-treated mice conferred pronociceptive activity to recipient mice. Unexpectedly, the effects of DMF were largely unchanged in the Nrf2 null mutant mice. Oxidative stress and immune system activation are robust after hindlimb fracture in mice. DMF strongly reduces activation of those systems, and the Nrf2 transcription factor is not required. DMF or drugs working through similar mechanisms might provide effective therapy for CRPS or other conditions where oxidative stress causes immune system activation.

Withaferin A alleviates fulminant hepatitis by targeting macrophage and NLRP3

Fulminant hepatitis (FH) is an incurable clinical syndrome where novel therapeutics are warranted. Withaferin A (WA), isolated from herb Withania Somnifera, is a hepatoprotective agent. Whether and how WA improves D-galactosamine (GalN)/lipopolysaccharide (LPS)-induced FH is unknown. This study was to evaluate the hepatoprotective role and mechanism of WA in GalN/LPS-induced FH. To determine the preventive and therapeutic effects of WA, wild-type mice were dosed with WA 0.5 h before or 2 h after GalN treatment, followed by LPS 30 min later, and then killed 6 h after LPS treatment. To explore the mechanism of the protective effect, the macrophage scavenger clodronate, autophagy inhibitor 3-methyladenine, or gene knockout mouse lines NLR family pyrin domain containing 3 (Nlrp3)-null, nuclear factor-erythroid 2-related factor 2 (Nrf2)-null, liver-specific AMP-activated protein kinase (Ampk)a1 knockout (Ampka1ΔHep) and liver-specific inhibitor of KB kinase β (Ikkb) knockout (IkkbΔHep) mice were subjected to GalN/LPS-induced FH. In wild-type mice, WA potently prevented GalN/LPS-induced FH and inhibited hepatic NLRP3 inflammasome activation, and upregulated NRF2 and autophagy signaling. Studies with Nrf2-null, Ampka1ΔHep, and IkkbΔHep mice demonstrated that the hepatoprotective effect was independent of NRF2, hepatic AMPKα1, and IκκB. Similarly, 3-methyladenine cotreatment failed to abolish the hepatoprotective effect of WA. The hepatoprotective effect of WA against GalN/LPS-induced FH was abolished after macrophage depletion, and partially reduced in Nlrp3-null mice. Consistently, WA alleviated LPS-induced inflammation partially dependent on the presence of NLRP3 in primary macrophage in vitro. Notably, WA potently and therapeutically attenuated GalN/LPS-induced hepatotoxicity. In conclusion, WA improves GalN/LPS-induced hepatotoxicity by targeting macrophage partially dependent on NLRP3 antagonism, while largely independent of NRF2 signaling, autophagy induction, and hepatic AMPKα1 and IκκB. These results support the concept of treating FH by pharmacologically targeting macrophage and suggest that WA has the potential to be repurposed for clinically treating FH as an immunoregulator.

Effects of ablation and activation of Nrf2 on bile acid homeostasis in male mice

The role of nuclear factor erythroid 2-related factor 2 (Nrf2) in bile acid (BA) homeostasis remains controversial. In this study, activation of Nrf2 was achieved either pharmacologically by CDDO-imidazolide (CDDO-Im) or genetically through a “gene dose-response” model consisting of Nrf2-null, wild-type (WT), Keap1-knockdown (Keap1-KD), and Keap1-hepatocyte knockout (Keap1-HKO) mice. In WT mice, CDDO-Im increased bile flow and decreased hepatic BAs, which was associated with a down-regulation of the canalicular BA efflux transporter Bsep and an increase in biliary BA excretion. In contrast, hepatic Bsep and biliary BA excretion were not altered in Keap1-KD or Keap1-HKO mice, suggesting that Nrf2 is not important for regulating Bsep or BA-dependent bile flow. In contrast, hepatic Mrp2 and Mrp3 were up-regulated by both pharmacological and genetic activations of Nrf2. Furthermore, ileal BA transporters (Asbt and Ostβ) and cholesterol transporters (Abcg5 and Abcg8) were down-regulated by both pharmacological and genetic activations of Nrf2, suggesting a role of Nrf2 in intestinal absorption of BAs and cholesterol. In Nrf2-null mice, CDDO-Im down-regulated hepatic BA uptake transporters (Ntcp, Oatp1a1, and Oatp1b2), leading to a 39-fold increase of serum BAs. To conclude, the present study demonstrates that activation of Nrf2 in mice up-regulates Mrp2 and Mrp3 in the liver and down-regulates BA and cholesterol transporters in the intestine.

Tissue distribution, hormonal regulation, ontogeny, diurnal expression, and induction of mouse cystine transporters Slc3a1 and Slc7a9

Slc7a11 (xCT) and Slc3a1 (rBAT) are cystine uptake transporters that maintain intracellular concentrations of cysteine, the rate-limiting amino acid in glutathione synthesis. This study was conducted to first determine the tissue distribution of the two transporters in male and female mice. Because Slc3a1 was the primary cystine transporter in liver, its sex-divergent expression, ontogeny, diurnal rhythm and whether its mRNA expression is altered by transcription factors (AhR, CAR, PXR, PPARα, and Nrf2) was also investigated. Slc7a11 was expressed highest in brain and gonads. Slc3a1 was expressed highest in kidney and intestine, followed by liver. Duodenal and hepatic Slc3a1 was higher in females than males. Hepatic Slc3a1 was high during darkness and low during daytime. Hepatic Scl3a1 was lowest pre-birth, increased to near maximal levels at birth, decreased back to pre-birth levels between Days 3–10, and then returned to peak levels by Day 45. Except for CAR, activation of transcription factors did not increase hepatic mRNA expression of Slc3a1. Chemical activation of CAR significantly induced Slc3a1 1.4-fold in wild-type but not CAR-null mice. Slc3a1 mRNA was higher in livers of AhR- and Nrf2-null mice compared to wild-type mice. High doses of diquat but not acetaminophen induced Slc3a1, suggesting Slc3a1 may respond to oxidative stress but not necessarily to GSH depletion. Overall, Slc7a11 is mainly expressed in brain and gonads, whereas Slc3a1 is mainly expressed in kidney, small intestine and liver, and its hepatic expression is regulated by diurnal rhythm and certain xenobiotic treatments.

Dendrobium nobile Lindl. alkaloids-mediated protection against CCl4-induced liver mitochondrial oxidative damage is dependent on the activation of Nrf2 signaling pathway

The activation of nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated signaling pathway has been involved in the mechanisms of a variety of protective agents against cellular oxidative stress. We recently demonstrated that Dendrobium nobile Lindl. alkaloids (DNLA), the active ingredients of Dendrobium, protects mice from CCl4-induced liver injury, dependent on the Nrf2 signaling pathway. The present study was aimed to determine whether the protection against mitochondrial oxidative damage plays a role in the mode of action of DNLA on CCl4-induced liver injury, and to further investigate whether the DNLA-conferred mitochondrial beneficial effects is dependent on the activation of Nrf2 signaling. The CCl4-induced acute liver injury model was employed in both wild-type (WT) and Nrf2-knockout (Nrf2−/−) mice. The results showed that in WT mice DNLA reduced CCl4-induced liver injury, accompanied by a significant reduction in CCl4-induced mitochondrial oxidative stress as evidenced by a decrease in mitochondrial H2O2 content and MDA production, and a marked increase in GSH level and Mn-SOD activity. However, these protective effects were significantly attenuated in Nrf2−/− mice. Furthermore, the administration of DNLA improved mitochondrial oxygen consumption, elevated ATP production, and decreased CCl4-induced apoptosis in the WT mice, whereas the DNLA-mediated protections on mitochondrial function were diminished in the Nrf2 null mice. These results demonstrate that the improvement of mitochondrial oxidative stress and mitochondrial dysfunction is implicated in the mechanism of DNLA-mediated protection on CCl4-induced liver injury, and this DNLA-modulated mode of action is dependent on the activation of Nrf2 signaling pathway.

Nrf2 deficiency in mice attenuates erythropoietic stress-related macrophage hypercellularity

Erythropoiesis in the bone marrow and spleen depends on intricate interactions between the resident macrophages and erythroblasts. Our study focuses on identifying the role of nuclear factor erythroid 2-related factor 2 (Nrf2) during recovery from stress erythropoiesis. To that end, we induced stress erythropoiesis in Nrf2+/+ and Nrf2-null mice and evaluated macrophage subsets known to support erythropoiesis and erythroid cell populations. Our results confirm macrophage and erythroid hypercellularity after acute blood loss. Importantly, Nrf2 depletion results in a marked numerical reduction of F4/80+/CD169+/CD11b+ macrophages, which is more prominent under the induction of stress erythropoiesis. The observed macrophage deficiency is concomitant to a significantly impaired erythroid response to acute stress erythropoiesis in both murine bone marrow and murine spleen. Additionally, peripheral blood reticulocyte count as a response to acute blood loss is delayed in Nrf2-deficient mice compared with age-matched controls (11.0 ± 0.6% vs. 14.8 ± 0.6%, p ≤ 0.001). Interestingly, we observe macrophage hypercellularity in conjunction with erythroid hyperplasia in the bone marrow during stress erythropoiesis in Nrf2+/+ controls, with both impaired in Nrf2-/- mice. We further confirm the finding of macrophage hypercellularity in another model of erythroid hyperplasia, the transgenic sickle cell mouse, characterized by hemolytic anemia and chronic stress erythropoiesis. Our results revealed the role of Nrf2 in stress erythropoiesis in the bone marrow and that macrophage hypercellularity occurs concurrently with erythroid expansion during stress erythropoiesis. Macrophage hypercellularity is a previously underappreciated feature of stress erythropoiesis in sickle cell disease and recovery from blood loss.

A low glycemic diet protects disease-prone Nrf2-deficient mice against age-related macular degeneration

Age-related macular degeneration (AMD) is a major blinding disease, affecting over 14% of the elderly. Risk for AMD is related to age, diet, environment, and genetics. Dietary modulation of AMD risk is a promising treatment modality, but requires appropriate animal models to demonstrate advantages of diet. Mice lacking the antioxidant transcription factor Nrf2 (Nfe2l2) develop age-related retinopathy relevant to human AMD. Here we evaluated the effect of consuming high glycemic (HG) or low glycemic (LG) diets until 18-months of age on development of features relevant to AMD in Nrf2-null mice. Nrf2-null mice that consumed HG diets developed atrophic AMD, characterized by photoreceptor degeneration, retinal pigment epithelium (RPE) atrophy and pigmentary abnormalities, basal laminar deposits, and loss of the choriocapillaris. In contrast, Nrf2-null-mice that consumed LG diets did not develop retinal disease phenotypes. Consumption of HG diets was associated with accumulation of advanced glycation end-products in the RPE and systemically, whereas consumption of the LG diet was associated with increased levels of anti-glycative and anti-oxidative detoxification machinery. Together our data indicate that the Nrf2-null HG mouse is a good model for atrophic AMD studies and that the LG diet can activate protective pathways to prevent AMD, even in a genetically predisposed animal.

Rutaecarpine inhibits KEAP1-NRF2 interaction to activate NRF2 and ameliorate dextran sulfate sodium-induced colitis

Inflammatory bowel disease (IBD) represents a group of chronic relapsing intestinal disorders. Rutaecarpine (RUT), isolated from the Traditional Chinese Medicine (TCM) of Evodia rutaecarpa, was reported to suppress IBD. However, the mechanism by which RUT ameliorates dextran sulfate sodium (DSS)-induced IBD is largely unknown. By use of nuclear factor-erythroid 2–related factor 2 (NRF2) knockout mice, cell-based studies, surface plasmon resonance (SPR), western blotting analysis, and molecular docking studies, the mechanism by which RUT affects DSS-induced colitis was explored. In DSS-treated wild-type mice but not in Nrf2-null mice, RUT significantly improved colitis as revealed by rescued body weight loss, improved histology and inflammation, and induced expression of NRF2 target genes in colon and ileum. Cell-based studies showed that RUT significantly increased the LD50 for hydrogen peroxide (H2O2)-induced cell damage, activated NRF2 nuclear translocation, and suppressed the production of reactive oxygen species in H2O2-treated HCT116 cells, activated NRF2 luciferase reporter activities in HCT116 cells and HepG2 cells, and induced expression of NRF2 target genes in primary intestinal epithelial cells. Molecular docking in silico and SPR assays indicated that RUT interacted with kelch-like ECH-associated protein 1 (KEAP1), and extracellular incubation studies revealed that RUT bound to the KEAP1 kelch domain with a calculated equilibrium dissociation constant Kd of 19.6 μM. In conclusion, these results demonstrate that RUT ameliorates DSS-induced colitis, dependent on NRF2, and could be a potential therapeutic option for IBD patients. Mechanistically, RUT potentiates NRF2 nuclear translocation to upregulate NRF2-mediated antioxidant response by directly inhibiting KEAP1-NRF2 interaction.

Nrf2 Null Mice Are Deficient in CD169+ Macrophages, Associated with Impaired Erythroid Response and Delayed Recovery from Acute Blood Loss

Erythropoiesis occurs in specialized niches in the bone marrow consisting of a central macrophage, surrounded by differentiating erythroblasts. This central macrophage has been identified by several markers including, CD169 (Sialoadhesin or Siglec-1), F4/80, CD11b, VCAM-1, ER-HR3 and Ly-6G. These CD169+ macrophages support erythropoiesis both at steady state and during stress. Nuclear factor erythroid 2-related factor 2 (Nrf2) is the master regulator of the cellular oxidative defense system. It modulates hematopoietic stem cells but its loss produces no visible phenotype in steady state hematological parameters. However, the importance of Nrf2 and macrophage subsets has not been fully characterized during recovery from stress erythropoiesis. We examined specific subsets of CD169+ macrophage populations in Nrf2 knockout (Nrf2−/−) mice as well as the role of Nrf2 in recovery from stress erythropoiesis in vivo. We quantified the expression of CD169, F4/80 and CD11b, markers of central macrophages, in the BM and spleen of Nrf2+/+ and Nrf2-/- mice at steady state. Surprisingly, Nrf2-/- mice showed a phenotype characterized by lower percentages of cells expressing known macrophage markers. We observed a significant decrease of 47% (p≤0.01), 24% (p≤0.01) and 50% (p≤0.01) in BM macrophage subpopulations expressing F4/80hiCD169hi, F4/80hiCD11bhi and CD169hiCD11bhi respectively, in age-matched Nrf2-/- mice compared to Nrf2+/+ control mice (Fig. 1a). In the spleen, we also observed a similar significant deficiency in BM macrophages (p≤0.01). Further validating this phenotype, immunofluorescence staining of isolated spleen tissue showed that expression of CD169+ macrophages was dramatically lower in spleen sections of Nrf2-/- mice than in Nrf2+/+ control mice. We hypothesized that our macrophage-deficient mice would display a defect in recovery from blood loss. Five to seven days after acute blood loss, immature erythroid progenitors (CD71hiTer119hi) increased in marrow by about 5-fold in Nrf2+/+ mice (p≤0.001, Fig. 1b), and mature erythroid progenitors (CD71loTer119hi) increased in marrow by 12-fold (p≤0.05) but the erythroid marrow response was impaired significantly in the macrophage deficient Nrf2-/- mice. To extend our observations regarding macrophage deficiency and impaired erythroid response, we chose a more functional outcome of recovery from anemia after high-grade blood loss produced by daily phlebotomies over 3 consecutive days to induce stress erythropoiesis in Nrf2+/+ and Nrf2-/- mice. We found significantly lower packed cell volume values specifically on Days 2, 4 and 10, implying delayed erythroid recovery (p≤0.05, two-way ANOVA). The Nrf2-/- mice also showed a significant decline in total hemoglobin than the Nrf2+/+ mice (p≤0.05). Additionally, peripheral blood reticulocyte response to blood loss is delayed in Nrf2 deficient mice compared to age-matched controls (11.0 ± 0.6% vs. 14.8 ± 0.6%, p≤0.001). We analyzed expression of heme-oxygenase 1 (HO-1), a well-known Nrf2-regulated gene. HO-1 mRNA expression increased 3-fold and 23-fold in Nrf2+/+ mice animals subjected to phlebotomy and hemin treatment compared to 2-fold and 12-fold expression in Nrf2-deficient mice (p≤0.05). We demonstrate for the first time that Nrf2-deficient mice have a deficiency of macrophages that includes subsets considered erythroblastic island (EI) macrophages, and that this deficiency is associated with impaired erythroid response to induced stress. Secondly, our multiple phlebotomies data in aggregate demonstrate that Nrf2-/- mice deficient in BM macrophages have significant delay in functional erythroid response and recovery from experimentally-induced anemia. Thirdly, impaired inducibility of HO-1 is a known feature of Nrf2-/- mice, which we confirmed in our results, could be contributing to the impairment in erythroid response. However, it is not likely that restricted iron trafficking to erythroid progenitors occurs in Nrf2-/- mice, since there is no characteristic alteration of mean corpuscular volume and mean corpuscular hemoglobin in peripheral blood. This is an area worthy of additional investigation. We conclude that the Nrf2 gene plays a previously unappreciated role in erythroid biology that appears to be mediated through macrophage function. Disclosures Ofori-Acquah: Shire Human Genetic Therapies Inc: Other: Financial Relationship. Kato:Bayer: Research Funding; Novartis, Global Blood Therapeutics: Consultancy, Research Funding.