The necrotic areas were decreased by almost half upon EC-specific Nrf2 activation (Figure 3E). protection in SCD. However, little is known regarding the mechanisms by which Nrf2 ameliorates SCD pathology or how some cells respond to Nrf2 stimuli to alleviate SCD pathology. Here, we asked whether monocytes/granulocytes and/or endothelial cells are particularly critical in alleviating the pathology of SCD. By targeting these cells with a Cre recombinase system, we generated SCD::Keap1F/F::LysM-Cre and Tie1-Cre mice with constitutive Nrf2 activation in monocytes/granulocytes and endothelial cells, respectively. Analyses of SCD::Keap1F/F::LysM-Cre and SCD::Keap1F/F::Tie1-Cre mice revealed significantly reduced inflammation, along with decreased white EG00229 blood cell counts and lower gene in SCD mice to activate Nrf2 specifically in myeloid lineage cells and ECs. This study revealed that Nrf2 activation in myeloid lineage cells attenuates inflammation and protects the liver against avascular necrosis. In addition to promoting heme clearance from the circulation, Nrf2 activation in myeloid lineage cells prevents the tissue accumulation of toxic heme and iron and promotes heme degradation and iron elimination in organs. Nrf2 activation in ECs protects tissues and cells from heme extravasation, reinforces the integrity of the vascular endothelium, and upregulates the expression of genes encoding scavenging proteins and antioxidant enzymes. These results unequivocally demonstrate that to protect tissues from SCD pathology, Nrf2 activation is required in both myeloid lineage cells and ECs in a distinct but overlapping manner. Materials and methods Mice The Animal Care and Use Committee of Tohoku University approved all animal experiments. We used both male and female homozygous SCD model (h/h, S/S) mice generated by Townes and colleagues9 and allele in myeloid cells or ECs was achieved by crossing Keap1F/F mice with mice harboring recombinase under the regulation of the lysozyme M (test was used to calculate statistical significance ( .05 or ** .01. Results Nrf2 Rabbit polyclonal to ABCD2 activation in monocytes/granulocytes ameliorates organ damage in SCD mice To determine the beneficial effect of Nrf2 activation in particular cells, we conditionally induced Nrf2 in monocytes/granulocytes by deleting the gene, a negative regulator of Nrf2, in SCD mice9,24 by breeding 2 distinct mouse genotypes. Nonphenotypic floxed-Keap1 (referred to as Keap1F/F) mice, EG00229 which were previously described,21 were inbred with LysM-Cre mice to generate myeloid cellCspecific Keap1-deficient mice25 (Keap1F/F::LysM-Cre). We confirmed the activation of Nrf2 based on the upregulated expression of reduced nicotinamide adenine dinucleotide phosphate:quinone oxidoreductase (and heme oxygenase 1 (mRNA expression was significantly higher in the livers, lungs, kidneys, and aortas of SCD::Keap1F/F::LysM-Cre mice than in those of SCD::Keap1F/F mice, showing increases of approximately twofold, fivefold, more EG00229 than fourfold, and threefold, respectively (supplemental Figure 1B). Similarly, mRNA expression was much higher in the livers and kidneys of SCD::Keap1F/F::LysM-Cre mice than in those of SCD::Keap1F/F mice (greater than twofold and greater than sixfold higher, respectively), whereas the expression of mRNA was threefold higher in the lungs of SCD::Keap1F/F::LysM-Cre mice than in those of SCD::Keap1F/F mice (supplemental Figure 1C). Our results confirmed the activation of Nrf2 in SCD::Keap1F/F::LysM-Cre mice. We also confirmed the recombination of Keap1 in the lungs, liver, spleen, kidney, and peritoneal macrophages of SCD::Keap1F/F::LysM-Cre mice based on the presence of the 288-bp amplicon from the knockout allele by polymerase chain reaction (supplemental Figure 2A-B). Except for the deletion of Keap1 in SCD::Keap1F/F::LysM-Cre mice, no other significant phenotypic changes were observed. Body weight and organ weight were within the same range in both EG00229 genotypes (supplemental Figure 2C). To examine whether Nrf2 activation in myeloid cells affects the RBC phenotype of SCD, we analyzed RBC indices, reticulocyte counts, and RBC lifespan. We found that RBC numbers and hemoglobin levels were moderately but significantly lower in SCD::Keap1F/F::LysM-Cre mice than in SCD::Keap1F/F mice, indicating that anemia was not relieved by Nrf2 activation (supplemental Figure 2D). Reticulocyte counts were comparable between SCD::Keap1F/F and SCD::Keap1F/F::LysM-Cre mice (supplemental Figure 3A). In addition, the lifespan of RBCs was not altered between SCD::Keap1F/F and SCD::Keap1F/F::LysM-Cre mice (supplemental Figure 3B-C). These results indicate that hemolysis is not rescued by Nrf2 activation in myeloid cells. Genetic alteration of can upregulate Nrf2 in SCD mice and improve lung and liver inflammation.12 To assess the effects of Nrf2 in the lungs, we examined the histology of the lungs. Congestion and edema.