Control of Oxidative Stress and Inflammation in Sickle Cell Disease with the Nrf2 Activator Dimethyl Fumarate

Abstract

Aims: Heme derived from hemolysis is pro-oxidative and proinflammatory and promotes vaso-occlusion in murine models of sickle cell disease (SCD), suggesting that enhanced detoxification of heme may be beneficial. Nuclear factor erythroid-2-related factor-2 (Nrf2) transcription pathway is the principal cellular defense system responding to pro-oxidative and proinflammatory stress. Dimethyl fumarate (DMF), a drug approved for treatment of multiple sclerosis, provides neuroprotection by activating Nrf2-responsive genes. We hypothesized that induction of Nrf2 with DMF would be beneficial in murine SCD models.

Results: DMF (30 mg/kg/day) or vehicle (0.08% methyl cellulose) was administered for 3-7 days to NY1DD and HbSS-Townes SCD mice. Vaso-occlusion, a hallmark of SCD, measured in sickle mice with dorsal skinfold chambers, was inhibited by DMF. The inhibitory effect of DMF was abrogated by the heme oxygenase-1 (HO-1) inhibitor tin protoporphyrin. DMF increased nuclear Nrf2 and cellular mRNA of Nrf2-responsive genes in livers and kidneys. DMF increased heme defenses, including HO-1, haptoglobin, hemopexin, and ferritin heavy chain, although plasma hemoglobin and heme levels were unchanged. DMF decreased markers of inflammation, including nuclear factor-kappa B phospho-p65, adhesion molecules, and toll-like receptor 4. DMF administered for 24 weeks to HbSS-Townes mice decreased hepatic necrosis, inflammatory cytokines, and irregularly shaped erythrocytes and increased hemoglobin F, but did not alter hematocrits, reticulocyte counts, lactate dehydrogenase, plasma heme, or spleen weights, indicating that the beneficial effects of DMF were not attributable to decreased hemolysis.

Innovation: These studies identify Nrf2 activation as a new therapeutic target for the treatment of SCD.

Conclusion: DMF activates Nrf2, enhances antioxidant defenses, and inhibits inflammation and vaso-occlusion in SCD mice. Antioxid. Redox Signal. 26, 748-762.

Keywords: HO-1; Nrf2; haptoglobin; hemopexin; sickle cell disease.

FIG. 1.

DMF inhibits heme-induced vaso-occlusion (stasis) in sickle mice. (A) NY1DD (n = 4/group) and (B) HbSS-Townes (n = 3/group) sickle mice with implanted dorsal DSFCs were administered DMF (30 mg/kg) or vehicle for 3 or 7 days, respectively. At baseline, on the last day of treatment, 20–30 flowing venules were selected and mapped using intravital microscopy. After venule selection and mapping, heme (3.2 μmol/kg) was infused into the tail vein. At 1 and 4 h after heme infusion, the same venules were re-examined to determine if they were flowing or not flowing (stasis). The percent stasis was calculated at each time point. In one cohort of NY1DD mice (n = 4) in (A), the HO-1 inhibitor, SnPP was administered (40 μmol/kg, i.p.) for 3 consecutive days before stasis measurement. Values are mean ± SD. *p < 0.001 for DMF versus vehicle. DMF, dimethyl fumarate; DSFC, dorsal skinfold chamber; HO-1, heme oxygenase-1; SnPP, tin protoporphyrin.

FIG. 2.

DMF increases nuclear Nrf2 expression in livers and kidneys of sickle mice. (A, B) NY1DD (n = 4/group) and (C, D) HbSS-Townes (n = 3/group) sickle mice with implanted DSFCs were administered DMF (30 mg/kg) or vehicle for 3 or 7 days, respectively. The mice were sacrificed, and the livers and kidneys were removed and frozen 4 h after the infusion of heme and the measurement of stasis. Liver and kidney nuclear extracts were isolated, run on Western blots, and immunostained for Nrf2 (98 kDa) and GAPDH (36 kDa). Nrf2, nuclear factor erythroid-2-related factor-2.

FIG. 3.

DMF increases HO-1 expression in livers and kidneys of sickle mice. (A, B) NY1DD (n = 4/group) and (C, D) HbSS-Townes (n = 3/group) sickle mice with implanted DSFCs were administered DMF (30 mg/kg) or vehicle for 3 or 7 days, respectively. The mice were sacrificed, and the livers and kidneys were removed and frozen 4 h after the infusion of heme and the measurement of stasis. Liver and kidney microsomes were isolated, run on Western blots, and immunostained for HO-1 (32 kDa) and GAPDH (36 kDa).

FIG. 4.

DMF increases FHC in livers and kidneys of sickle mice. (A, B) NY1DD (n = 4/group) and (C, D) HbSS-Townes (n = 3/group) sickle mice with implanted DSFCs were administered DMF (30 mg/kg) or vehicle for 3 or 7 days, respectively. The mice were sacrificed, and the livers and kidneys were removed and frozen 4 h after the infusion of heme and the measurement of stasis. Liver and kidney microsomes were isolated, run on Western blots, and immunostained for FHC (21 kDa) and GAPDH (36 kDa). FHC, ferritin heavy chain.

FIG. 5.

DMF increases plasma haptoglobin and hemopexin levels sickle mice. Plasma was collected from the abdominal aortas of anesthetized sickle mice without implanted DSFCs and heme infusion before collection. (A) NY1DD (n = 5/group) and (B) HbSS-Townes (n = 3/group) sickle mice were administered DMF or vehicle for 7 days. Plasma was run on Western blots and immunostained for haptoglobin (45 kDa), hemopexin (68 kDa), or IgG (155 kDa). (C) Total plasma heme levels were also measured in NY1DD and HbSS-Townes mice after 2 or 24 weeks of treatment, respectively.

FIG. 6.

DMF decreases markers of inflammation in livers of NY1DD mice. NY1DD (n = 4/group) sickle mice with implanted DSFCs were administered DMF (30 mg/kg) or vehicle for 3 days. The mice were sacrificed, and the livers were removed and frozen 4 h after the infusion of heme and the measurement of stasis. Liver subfractions were isolated, run on Western blots, and immunostained for (A) microsomal TLR4 (95 kDa) and GAPDH (36 kDa), (B) nuclear phospho- and total p65 NF-kB (65 kDa), and (C) microsomal VCAM-1 (100 kDa), ICAM-1 (99 kDa), and E-selectin (67 kDa). ICAM-1, intercellular adhesion molecule-1; NF-κB, nuclear factor-kappa B; TLR4, toll-like receptor 4; VCAM-1, vascular cell adhesion molecule-1.

FIG. 7.

Chronic DMF administration decreases hepatic necrosis in livers of sickle mice. HbSS-Townes (n = 4/group) sickle mice were treated with oral DMF (∼30 mg/kg/day) or vehicle in drinking water for 24 weeks, beginning at 4 weeks of age. Liver sections were stained with hematoxylin and eosin. (A) “Acute” infarcts were defined as eosinophilic areas of coagulative necrosis without significant reactive cellular infiltrates, reflecting relatively recent acute or subacute necrosis. (B) “Chronic” infarcts were somewhat less clearly delineated and comprised infiltrates of mononuclear cells (lymphocytes and macrophages) along with variable degrees of fibroplasia and fibrosis. (C) Areas of acute and chronic hepatic necrosis were measured, combined, and averaged for each liver. Each box plot represents the minimum box plot represent the minimum, first quartile, median, third quartile, and maximum. *p = 0.039 for DMF versus vehicle. (D) Hepatic mRNA levels of proinflammatory cytokines IL-6, IL-1β, and IL-18 are expressed relative to 18S rRNA. *p < 0.05 for DMF versus vehicle. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 8.

Proposed model of heme detoxification and clearance by DMF in sickle cell disease. DMF and its primary intestinal metabolite MMF can both react with cysteine-151 on Keap1/Nrf2 complexes in the cytoplasm (53). The most probable reaction is a Michael addition of the nucleophilic sulfhydryl on Cys151 to the electrophilic alkene bond of MMF (20). Alkylation of the Keap1 sulfhydryl allows Nrf2 to dissociate from Keap1. Nrf2 then accumulates in the nucleus where it binds other transcription factors and ARE located in the regulatory regions of a battery of cellular defense genes and thereby activates transcription of those genes. Among the proteins induced are HO-1, FHC, and other cytoprotectants that can detoxify heme and haptoglobin and hemopexin that can bind hemoglobin and heme in the plasma and transport them safely to hepatocytes and macrophages for processing and degradation. In addition, γ-globin genes are expressed in hematopoietic cells, allowing the formation of HbF in RBC. ARE, antioxidant response elements; HbF, hemoglobin F; Keap1, kelch-like ECH-associated protein 1; MMF, monomethyl fumarate; RBC, red blood cells.