Heme Overload Triggers Xanthine Oxidase Release and Mediates the Inflammatory Response

Heme crisis drives a number of hemolytic conditions including malaria, sepsis, blood transfusions, and cardiac bypass. Free heme, released from hemoglobin, causes endothelial damage via direct and iron (Fe)-mediated generation of reactive species, as well as activation of endothelial cells and macrophages leading to an inflammatory response. An enzyme that has been shown to have elevated activity in many hemolytic conditions is xanthine oxidase (XO). XO generates hydrogen peroxide (H2O2) as a byproduct of the oxidation of hypoxanthine and xanthine in the final steps of the purine degradation pathway. While XO activity is known to be increased in hemolytic diseases, its exact role has yet to be established. In order to study the role of XO in hemolytic disease, we developed a novel model of heme crisis in which we injected mice with two identical doses of hemin one hour apart and monitored the mice for 24 hours in order to deplete heme scavenging mechanisms before inducing heme crisis. Heme crisis induced damage was evaluated by hemopexin ELISA, plasma XO activity by HPLC, HE however, prior to hemin injection mice were pretreated with the FDA approved, XO inhibitor febuxostat (10 mg/kg/day) in drinking water. Liver, lung and kidney injury and inflammation was again evaluated with H&E staining, qRT-PCR of inflammatory cytokines, and hematological analysis. In addition, the interaction between heme and XO was explored in vitro using evaluation of hemin degradation via spectrophotometry and computational modeling. We found that mice treated with two doses of 50 μmol/kg hemin had a 92.3% decrease in hemopexin, and a 20-fold increase in plasma XO activity compared to controls. H&E staining showed severe liver hemorrhaging, increased cell infiltration in the lung, and cellular disorganization in the kidney. The pro-inflammatory cytokines, IL-6, TNFα, and IL-1β, were all significantly increased in the liver, lung, and kidney, with IL-6 having the greatest fold change in all three organs. Systemic inflammation was also suggested via significant increases in circulating monocytes and granulocytes. Additionally, hematological analysis showed decreased RBCs and platelets, indicating additional hemolysis and platelet activation. While these markers of injury and inflammation were observed with 50 μmol/kg hemin, lower doses of hemin showed no effect. Together, these results indicate that our heme crisis model mimics the pro-inflammatory state, and organ damage observed in patients during severe hemolysis. Interestingly, when mice were pre-treated with febuxostat, organ damage was observed at lower doses of hemin (25 μmol/kg) compared to untreated mice, as observed by H&E staining. Inhibition of XO also had a significant impact on the inflammatory response. While circulating monocytes were decreased in mice pre-treated with febuxostat, the pro-inflammatory cytokines IL-6, TNFα, and IL-1β, were further exacerbated in the liver, lung, and kidney. This suggests that XO may play a role in mediating the inflammatory response induced by heme crisis. To explore how XO could mediate the inflammatory response we conducted in vitro enzymatic XO experiments with hemin. We found that XO was able to degrade hemin as observed by a decrease in absorbance at 618 nm. Additionally, based on a spectral shift observed when hemin and XO were incubated together, we hypothesized that XO may have the ability to bind hemin. This was further supported by computational modeling in which a potential heme binding site was discovered in the FAD domain of XO with a kd=128 nM. This suggests that XO may have the ability to bind hemin. Thus, during substrate oxidation, H2O2 is produced in the same XO domain of the potential heme binding site, allowing for increased chance of the H2O2 induced heme splitting reaction. We further hypothesize that the uric acid produced by xanthine oxidation may serve as an Fe chelator to scavenge free Fe released by a heme splitting reaction. By creating a microenvironment that can split heme and scavenge Fe, XO may be able to mediate the inflammatory response induced by heme crisis. Disclosures Straub: Bayer Pharmaceuticals: Research Funding.