PI-10: DIETARY IRON RESTRICTION PROTECTS AGAINST VASO-OCCLUSION AND ORGAN DAMAGE IN SICKLE CELL DISEASE

Abstract

Purpose: Sickle cell disease (SCD) is the most commonly inherited monogenic hemoglobinopathy and results from a mutation in the β-globin gene (HbS), leading to red blood cell (RBC) sickling, vaso-occlusive episodes (VOE), and organ damage. Chronic hemolysis, inflammation, and repeated red blood cell transfusions in SCD can disrupt iron homeostasis. Patients who receive multiple blood transfusions develop iron-overload, and another subpopulation of SCD patients manifest iron deficiency. SCD mice exhibit increased iron absorption via upregulation of the intestinal transcription factor hypoxia inducible factor-2a(HIF-2a) leading to the development of iron overload. Disruption of intestinal HIF-2a or limiting iron intake by dietary iron restriction in SCD mice, significantly reduced systemic iron overload, lowered cellular sickle hemoglobin as reflected by reduced RBC mean corpuscular hemoglobin concentration (MCHC), and consequently improved hemolysis and ameliorated anemia. Understanding iron homeostasis is therefore critical to SCD pathogenesis and holds the promise for identifying novel approaches to treat SCD. Materials and methods: SCD mice were generated by transplanting bone marrow cells from Berkeley SCD (Tg[Hu-miniLCRα1GγAγδβS]Hba−/− Hbb−/−) mice into 8-week old, lethally-irradiated C57BL/6J mice. Immediately after transplantation, SCD mice were fed either a regular-iron diet (Ctrl) containing 185 parts per million (ppm) iron or an otherwise identical iron-restricted diet (IRD) containing 3 ppm iron. VOE and organ damage analysis were done 3-4 months after treatments. Results: IRD resulted in a reduced organ iron concentration (Fig-1A) in SCD compared to Ctrl group, confirming that dietary iron restriction ameliorates organ iron deposition in SCD mice. SCD mice treated with IRD exhibited reduced MCHC (Fig-1B) and serum indirect bilirubin(IDBILC) (Fig-1C) indicating a decrease in cellular sickle hemoglobin and hemolysis. Furthermore, IRD reduced the fraction of aged neutrophils by >50% in SCD mice (Fig-1D), which corresponded with improved VOE parameters. Specifically, intravital microscopy (IVM) demonstrated elevated mean centerline RBC velocity and blood flow rate and decreased leukocyte adhesion in IRD compared to Ctrl mice (Fig-1E-G). Importantly, IRD led to prolonged survival after TNF-a challenge in SCD mice (Fig-1H). IRD resulted in a dramatic reduction in lung leukocyte infiltration, indicating significantly reduced lung inflammation in SCD mice (Fig-2A). Compared to Ctrl liver samples, IRD resulted in significantly less liver necrosis, leukocyte infiltration and fibrosis (Fig-2B,C), decreased serum liver enzymes (ALT, AST; Fig-2D), and reduced levels of total and direct bilirubin (TBILC, DBILC; Fig-2D).We further hypothesized that, in addition to reducing iron concentration in organs, IRD ameliorates organ damage by preventing translocation of microbial compounds.IRD inhibits translocation of FITC-Dextran by 5-fold compared to Ctrl suggesting functionally improved gut permeability (Fig-2E). We detected significantly decreased TLR2 ligands in serum of IRD mice (Fig-2F) suggesting ligands from gram-positive bacteria (TLR2 agonists) may play a role in triggering SCD complications. Conclusion: Our study demonstrates for the first time that dietary iron restriction reduces VOE severity and alleviates chronic organ damage in SCD mice. Novel approaches that target iron availability/absorption in the gut, the gut microbiome and aged neutrophil crosstalk and gut barrier integrity, provide an important area of further investigation with the potential of improving hemolysis, VOE, and organ damage in SCD patients.(A)The bone-marrow/spleen/liver Non-heme iron content. (B)MCHC, (C)IDBILC,(D)aged neutrophils percentage in Ctrl/IRD SCD mice. IVM parameters (E) (Vrbc) (F) blood flow rate (J) the number of adherent leukocytes and (J) survival rate after TNF-a injection(A)Leukocyte infiltration in lung and (B)liver tissue. (C)Liver necrosis, leukocyte infiltration, and fibrosis. (D)Liver ALT and AST, TBILC, and DBILC from sera. (E)serum FITC-Dextran and (F) TLR2 ligands activity in Ctrl/IRD SCD mice The authors do not declare any conflict of interest