Targeted Therapy of Acute Chest Syndrome with Recombinant Heme Oxygenase-1 Revealed By the Resistance of Young Transgenic Sickle Cell Mice to Heme-Induced Acute Lung Injury
Authors: Hazra Rimi , Ghosh Samit , Flage Bethany , Crouthamel Matthew , Pan Clark , Sun Xiuxia , Zhang Bohong , Sypek Joseph , Ofori-Acquah Solomon Fiifi 
Published: February 2020

Acute chest syndrome (ACS) is a major pulmonary complication of sickle cell disease (SCD) with currently no mechanistic-based therapies. Although ACS is more frequent in childhood, survival among children 1-9 years is ~10-fold higher compared to adults 20 years and older. Hitherto, mechanisms responsible for the high ACS survival in children have not been defined. We previously reported that acute elevation of circulating heme causes a TLR4-dependant lethal acute lung injury (ALI) in transgenic mice with homozygous SCD (SS) reminiscent of severe ACS (Ghosh et al., J Clin Invest, 2013). In the current study we discovered that young (4-6 weeks) and adult (12 weeks) SS mice suffer comparable severity of hemolytic crises when challenged with intravenous hemin (35 μmoles/kg bw). However, virtually none of the young mice develop ALI while nearly all the adults succumb with respiratory failure. We discovered that young SS mice rapidly clear excess heme from the circulation suggesting this was the reason for their resistance to intravenous hemin. Interestingly, young SS mice have significantly lower plasma hemopexin levels than adult SS mice, which excludes the classical heme scavenging pathway as the reason for their heme resistance. Previous studies have linked genetically high heme oxygenase-1 (HO-1) expression with low-risk of ACS, and raised steady-state levels of plasma heme with high-risk ACS in children. To explore these associations further, we studied a large cohort of patients and discovered for the first time that the concentration of plasma HO-1 in SCD children 1-9 yrs is 2-fold higher than adults 20 yrs and older (23.6±1.1, n=191 versus mean 10.7±0.6, n=67); these findings were phenocopied in transgenic SS mice. Plasma fractionization experiments revealed that HO-1 co-localizes with the enzymatic and co-factor machinery required for heme degradation indicating that heme can be degraded in the blood circulation. To test this idea ~3 weeks old SS mice were treated with the HO-1 inhibitor tin protoporphyrin (SnPP) or vehicle, and challenged with intravenous hemin five days later to induce ALI/ACS. Plasma HO activity declined by ~76% in SnPP-treated mice and was unchanged in the vehicle treated animals (n=12-14, p&lt;0.001). A significant majority (10/12; 83%) of the vehicle-treated mice survived, while a majority of the SnPP-treated mice (10/14; 71%) developed lethal ALI/ACS. In another series of experiments young SS mice were treated for three months with Nrf2 activator to block age-dependent HO-1 decline, and then treated with vehicle or SnPP prior to ALI/ACS induction. The SnPP-treated group had 58% reduction in plasma HO-1 (p&lt;0.05), failed to clear heme and developed lethal ALI/ACS, while a majority (63%) of the vehicle treated SS mice with persistently raised HO-1 activity in adulthood survived. Finally, adult SS mice were treated acutely with novel truncated human HO-1 recombinant proteins or a carrier vehicle immediately prior to induction of ALI/ACS. Recombinant variant 4 (V4) possessing an Fc component that enhances protein half-life provided 100% protection while 70% of vehicle treated mice succumbed with ALI/ACS (Mantel-Cox log-rank p&lt;0.001). While the arterial oxygen saturation level (% SpO2) in vehicle treated SS mice declined sharply reflecting severe respiratory distress, V4-treated mice maintained normal SpO2 (p&lt;0.001). In conclusion, we have determined that HO-1 activity in blood plasma declines with aging in SCD patients and identify this phenomenon as the basis of poor survival among adult SS mice with induced ALI/ACS. Furthermore, we provide proof-of-principle in mice that a novel human recombinant HO-1 protein can attenuate lung injury and improve survival from ALI/ACS in SCD.

Disclosures

Ofori-Acquah: Shire Human Genetic Therapies Inc: Other: Financial Relationship.