Keap1-Nrf2 System: Potential Role in Prevention of Sickle Cell Disease Organs Damages and Inflammation
Authors: Keleku-Lukwete Nadine , Suzuki Mikiko , Otsuki Akihito , Tsuchida Kouhei , Katayama Saori , Hayashi Makiko , Naganuma Eriko , Moriguchi Takashi , Tanabe Osamu , Engel James Doug , Imaizumi Masue , Yamamoto Masayuki 
Published: October 2019

Abstract
Chronic hemolysis in sickle cell disease (SCD) gives rise to intermittent vessel occlusion. Recurrent ischemia-reperfusion generates high levels of reactive oxygen species (ROS) that leads to cell damage. On the other hand, lysed red blood cells (RBC) released free heme into blood stream, which contributes to generation of oxidant microenvironment. ROS burden generated by heme and ischemia-reperfusion injury contributes to endothelial cell activation that promotes inflammatory response with activation of inflammatory mediators. Sickle cell patients bearing high white blood cell (WBC) count develop severe complications of the disease.
Nrf2 is a transcription factor that mediates adaptation to oxidative stress and cell defense. Under homeostatic conditions, Nrf2 is trapped by Keap1 and degraded by proteasome pathway. Upon exposure to stress stimuli, such as ROS and electrophiles, Nrf2 is stabilized and activates transcription of cytoprotective and antioxidants genes. Therefore, we hypothesized that Nrf2 activation might be important for tissue protection in SCD.
To evaluate the therapeutic effect of Nrf2 activation on SCD, we used a SCD knock-in mouse model bearing human mutated globin loci. Since Keap1 negatively regulated Nrf2 in normal conditions, we crossed the SCD model mice with Keap1 hypomorphic knockdown (Keap1F/-) mice to generate compound mutant (SCD::Keap1F/-) mice, in which Nrf2 was constitutively activated. Histological analysis of the liver and lung revealed that congestive reaction and necrotic area observed in the SCD mice were significantly reduced in the SCD::Keap1F/- mice. Moreover, liver damage marker alanine transferase (ALT) were also decreased in SCD::Keap1F/- mice compared with SCD mice. We further examined inflammation status using human IL6 reporter mouse system and found that inflammation, which was mainly observed in lung of SCD mice, was markedly improved in the SCD::Keap1F/- mice. Expression levels of inflammatory cytokines IL6 and IL1β in the lung as well as adhesion molecules VCAM and P-selectin in the aorta of SCD::Keap1F/- mice were lower than those of the SCD mice. These results indicate that Nrf2 activation improves organ damage and inflammation in the SCD mice.
On the other hand, hemolysis of sickle cells and compensatory stress erythropoiesis did not change substantially between the SCD and the SCD::Keap1F/- mice. These results indicate that Nrf2 activation improves organ damage and inflammation independently from improvement of hemolysis. Previous reports show that free heme released from sickle cells gives rise to ROS-mediate pathological process as inflammation and organ damage in SCD. We therefore measured plasma free heme and downstream product indirect bilirubin in the SCD::Keap1F/- mice, and found that both heme and indirect bilirubin was decreased in the SCD::Keap1F/- mice. These results demonstrate that Nrf2 activation improves SCD symptoms at least in part by elimination of free heme.
To determine whether chemical compounds that serve as Nrf2 inducers have a protective potential of SCD mice organs, we treated 6-weeks aged mice with an Nrf2 inducer CDDO-Im (20 μmol/kg) 3 times per week for 3 weeks. CDDO-Im administration progressively reduced WBC numbers in the SCD::Keap1F/- mice. Also we observed decrease in the expression level of IL6 and IL1β in the lung and necrotic area in the liver in CDDO-Im-treated SCD::Keap1F/- mice. These results indicate that administration of a chemical Nrf2 inducer relieves inflammation and organ damage in the SCD mice.
Collectively, these data provide the evidence that Nrf2 activation improves ROS-mediated organ damages and inflammation. Associated in the therapy of SCD, Nrf2 inducers could be of benefit to SCD patients.

Disclosures
No relevant conflicts of interest to declare.