Simvastatin-Mediated Nrf2 Activation Induces Fetal Hemoglobin and Antioxidant Enzyme Expression to Ameliorate the Phenotype of Sickle Cell Disease.

Sickle cell disease (SCD) is a pathophysiological condition of chronic hemolysis, oxidative stress, and elevated inflammation. The transcription factor Nrf2 is a master regulator of oxidative stress. Here, we report that the FDA-approved oral agent simvastatin, an inhibitor of hydroxymethyl-glutaryl coenzyme A reductase, significantly activates the expression of Nrf2 and antioxidant enzymes. Simvastatin also induces fetal hemoglobin expression in SCD patient primary erythroid progenitors and a transgenic mouse model. Simvastatin alleviates SCD symptoms by decreasing hemoglobin S sickling, oxidative stress, and inflammatory stress in erythroblasts. Particularly, simvastatin increases cellular levels of cystine, the precursor for the biosynthesis of the antioxidant reduced glutathione, and decreases the iron content in SCD mouse spleen and liver tissues. Mechanistic studies suggest that simvastatin suppresses the expression of the critical histone methyltransferase enhancer of zeste homolog 2 to reduce both global and gene-specific histone H3 lysine 27 trimethylation. These chromatin structural changes promote the assembly of transcription complexes to fetal γ-globin and antioxidant gene regulatory regions in an antioxidant response element-dependent manner. In summary, our findings suggest that simvastatin activates fetal hemoglobin and antioxidant protein expression, modulates iron and cystine/reduced glutathione levels to improve the phenotype of SCD, and represents a therapeutic strategy for further development.

Salubrinal induces fetal hemoglobin expression via the stress-signaling pathway in human sickle erythroid progenitors and sickle cell disease mice.

Sickle cell disease (SCD) is an inherited blood disorder caused by a mutation in the HBB gene leading to hemoglobin S production and polymerization under hypoxia conditions leading to vaso-occlusion, chronic hemolysis, and progressive organ damage. This disease affects ~100,000 people in the United States and millions worldwide. An effective therapy for SCD is fetal hemoglobin (HbF) induction by pharmacologic agents such as hydroxyurea, the only Food and Drug Administration-approved drug for this purpose. Therefore, the goal of our study was to determine whether salubrinal (SAL), a selective protein phosphatase 1 inhibitor, induces HbF expression through the stress-signaling pathway by activation of p-eIF2α and ATF4 trans-activation in the γ-globin gene promoter. Sickle erythroid progenitors treated with 24µM SAL increased F-cells levels 1.4-fold (p = 0.021) and produced an 80% decrease in reactive oxygen species. Western blot analysis showed SAL enhanced HbF protein by 1.6-fold (p = 0.0441), along with dose-dependent increases of p-eIF2α and ATF4 levels. Subsequent treatment of SCD mice by a single intraperitoneal injection of SAL (5mg/kg) produced peak plasma concentrations at 6 hours. Chronic treatments of SCD mice with SAL mediated a 2.3-fold increase in F-cells (p = 0.0013) and decreased sickle erythrocytes supporting in vivo HbF induction.

Benserazide racemate and enantiomers induce fetal globin gene expression in vivo: Studies to guide clinical development for beta thalassemia and sickle cell disease.

Increased expression of developmentally silenced fetal globin (HBG) reduces the clinical severity of ß-hemoglobinopathies. Benserazide has a relatively benign safety profile having been approved for 50 years in Europe and Canada for Parkinson's disease treatment. Benserazide was shown to activate HBG gene transcription in a high throughput screen, and subsequent studies confirmed fetal hemoglobin (HbF) induction in erythroid progenitors from hemoglobinopathy patients, transgenic mice containing the entire human ß-globin gene (ß-YAC) and anemic baboons. The goal of this study is to evaluate efficacies and plasma exposure profiles of benserazide racemate and its enantiomers to select the chemical form for clinical development. Intermittent treatment with all forms of benserazide in ß-YAC mice significantly increased proportions of red blood cells expressing HbF and HbF protein per cell with similar pharmacokinetic profiles and with no cytopenia. These data contribute to the regulatory justification for development of the benserazide racemate. Additionally, dose ranges and frequencies required for HbF induction using racemic benserazide were explored. Orally administered escalating doses of benserazide in an anemic baboon induced γ-globin mRNA up to 13-fold and establish an intermittent dose regimen for clinical studies as a therapeutic candidate for potential treatment of ß-hemoglobinopathies.

NRF2 mediates γ-globin gene regulation through epigenetic modifications in a ß-YAC transgenic mouse model.

IMPACT STATEMENT: Sickle cell disease is an inherited hemoglobin disorder that affects over 100,000 people in the United States causing high morbidity and early mortality. Although new treatments were recently approved by the FDA, only one drug Hydroxyurea induces fetal hemoglobin expression to inhibit sickle hemoglobin polymerization in red blood cells. Our laboratory previously demonstrated the ability of the NRF2 activator, dimethyl fumarate to induce fetal hemoglobin in the sickle cell mouse model. In this study, we investigated molecular mechanisms of γ-globin gene activation by NRF2. We observed the ability of NRF2 to modulate chromatin structure in the human ß-like globin gene locus of ß-YAC transgenic mice during development. Furthermore, an NRF2/TET3 interaction regulates γ-globin gene DNA methylation. These findings provide potential new molecular targets for small molecule drug developed for treating sickle cell disease.

MIR-144-mediated NRF2 gene silencing inhibits fetal hemoglobin expression in sickle cell disease.

Inherited genetic modifiers and pharmacologic agents that enhance fetal hemoglobin (HbF) expression reverse the clinical severity of sickle cell disease (SCD). Recent efforts to develop novel strategies of HbF induction include discovery of molecular targets that regulate γ-globin gene transcription and translation. The purpose of this study was to perform genome-wide microRNA (miRNA) analysis to identify genes associated with HbF expression in patients with SCD. We isolated RNA from purified reticulocytes for microarray-based miRNA expression profiling. Using samples from patients with contrasting HbF levels, we observed an eightfold upregulation of miR-144-3p (miR-144) and miR-144-5p in the low-HbF group compared with those with high HbF. Additional analysis by reverse transcription quantitative polymerase chain reaction confirmed individual miR-144 expression levels of subjects in the two groups. Subsequent functional studies in normal and sickle erythroid progenitors showed NRF2 gene silencing by miR-144 and concomitant repression of γ-globin transcription; by contrast, treatment with miR-144 antagomir reversed its silencing effects in a dose-dependent manner. Because NRF2 regulates reactive oxygen species levels, additional studies investigated mechanisms of HbF regulation using a hemin-induced oxidative stress model. Treatment of KU812 cells with hemin produced an increase in NRF2 expression and HbF induction that reversed with miR-144 pretreatment. Chromatin immunoprecipitation assay confirmed NRF2 binding to the γ-globin antioxidant response element, which was inhibited by miR-144 mimic treatment. The genome-wide miRNA microarray and primary erythroid progenitor data support a miR-144/NRF2-mediated mechanism of γ-globin gene regulation in SCD.

Constitutively CD40-activated B cells regulate CD8 T cell inflammatory response by IL-10 induction.

B cells are exposed to high levels of CD40 ligand (CD40L, CD154) in chronic inflammatory diseases. In addition, B cells expressing both CD40 and CD40L have been identified in human diseases such as autoimmune diseases and lymphoma. However, how such constitutively CD40-activated B cells under inflammation may impact on T cell response remains unknown. Using a mouse model in which B cells express a CD40L transgene (CD40LTg) and receive autocrine CD40/CD40L signaling, we show that CD40LTg B cells stimulated memory-like CD4 and CD8 T cells to express IL-10. This IL-10 expression by CD8 T cells was dependent on IFN-I and programmed cell death protein 1, and was critical for CD8 T cells to counterregulate their overactivation. Furthermore, adoptive transfer of naive CD8 T cells in RAG-1(-/-) mice normally induces colitis in association with IL-17 and IFN-γ cytokine production. Using this model, we show that adoptive cotransfer of CD40LTg B cells, but not wild-type B cells, significantly reduced IL-17 response and regulated colitis in association with IL-10 induction in CD8 T cells. Thus, B cells expressing CD40L can be a therapeutic goal to regulate inflammatory CD8 T cell response by IL-10 induction.

CD4(+)CD25(+) regulatory T cells resist a novel form of CD28- and Fas-dependent p53-induced T cell apoptosis.

Ag receptor stimulation of preactivated T cells causes rapid cell death in an IL-2- and Fas-dependent manner. This phenomenon, known as activation-induced cell death (AICD), plays a pivotal role in the removal of Ag-reactive T cells after initial expansion. In this study, we report a novel form of T cell apoptosis that is distinct from classic AICD. When peripheral T cells were activated with anti-CD3 and anti-CD28 Abs precoated onto plastic plates, CD4(+)CD25(-) and CD8 T cells initially expanded but underwent massive apoptosis after 4 d. Unlike classic AICD, this type of T cell apoptosis pathway requires engagement of CD28 and expression of p53, a tumor-suppressor gene. The most striking feature of this form of apoptosis was regulatory T cell resistance. Under the same stimulating conditions, CD4(+)CD25(+) T cells grew continuously beyond 4 d. Consequently, when the entire CD4 population was cultured with plate-bound anti-CD3 plus anti-CD28 Ab, CD4(+)CD25(+)FoxP3(+) regulatory T cells outgrew nonregulatory T cells and expanded >7000-fold after 11 d. The data presented herein demonstrate a novel process of Ag-induced T cell death by sustained TCR and CD28 engagement and represent a simple and efficient procedure for the expansion of regulatory T cells in vitro.

Role of CD28 in fatal autoimmune disorder in scurfy mice.

Scurfy mice develop CD4 T-cell-mediated lymphoproliferative disease leading to death within 4 weeks of age. The scurfy mutation causes loss of function of the foxp3 gene (foxp3(sf)), which is essential for development and maintenance of naturally occurring regulatory CD4 T cells (nTregs). In humans, mutations of the foxp3 gene cause immune dysregulation, polyendocrinopathy, enteropathy, and X-linked syndrome (IPEX). In most patients with IPEX and also in scurfy mice, T cells show hyperreactivity and levels of Th1- and Th2-associated cytokines are substantially elevated. We report that removal of CD28 expression rescued scurfy mice from early death. Longer-term surviving CD28-deficient scurfy mice still had lymphoproliferative disorder, but their CD4 T cells showed decreased interferon-gamma and no sign of interleukin-4 or interleukin-10 hyperproduction. Furthermore, injection of CTLA4-Ig to block CD28-B7 interactions substantially improved the survival of scurfy mice by blocking effector T-cell differentiation. These data support the hypothesis that CD28-B7 interactions play a critical role in the etiology of lethal autoimmune disease in scurfy mice by stimulating the differentiation of antigen-activated naive T cells into effector T cells.

Enrichment of regulatory CD4(+)CD25(+) T cells by inhibition of phospholipase D signaling.

Antigen stimulation of lymphocytes induces upregulation of phospholipase D (PLD) activity, but the biological significance of PLD-mediated signaling in T cells has not been well established. Here we demonstrate that PLD signaling is essential for proliferation of mouse CD8(+) T cells and CD4(+)CD25(-) T cells, but is not required for proliferation of CD4(+)CD25(+) regulatory T cells. We exploited this observation to develop an efficient method to enrich for regulatory T cells starting from preparations of total CD4(+) T lymphocytes. Inhibition of PLD signaling blocked effector T-cell proliferation after T cell-antigen receptor (TCR) engagement, but had no significant effect on the proliferation of CD4(+)CD25(+) T cells with regulatory functions. Consequently, cells expanded in vitro for one week by antigen receptor stimulation with PLD signal inhibition were markedly enriched for regulatory T cells.

Coupling between B cell receptor and phospholipase C-gamma2 is essential for mature B cell development.

Two signaling pathways known to be essential for progression from immature to mature B cells are BAFF receptor (BAFF-R) and the B cell receptor (BCR). Here, we first show that phospholipase C (PLC)-gamma2 is required for a BAFF-R-mediated survival signal. Then, we have examined the question of whether the reduced number of mature B cells in PLC-gamma2-/- mice is caused by a defect in either BCR or BAFF-R signaling. We find that a PLC-gamma2 SH2 mutant, which inhibits coupling between BCR and PLC-gamma2, fails to restore B cell maturation, despite supporting BAFF-dependent survival. Therefore, our data suggest that the BAFF-R-mediated survival signal, provided by PLC-gamma2, is not sufficient to promote B cell maturation, and that, in addition, activation of PLC-gamma2 by BCR is required for B cell development.