In a study designed to test the role of extracellular heme in SCD pathogenesis , Ghosh et al. made a serendipitous observation that provides insight into how the concentration of heme is elevated in SCD plasma: heme is amplified. The investigators infused groups of SS, AS, and AA mice with a bolus of purified heme, and as expected this raised the TPH in all the animals to a similar concentration immediately after the challenge. Paradoxically, the TPH in the SS mice continued to increase surpassing the bolus by ~50 % within 30 min. Meanwhile, both control animals (AS and AA) rapidly cleared the exogenous heme from their circulation. This finding showed that there is auto-amplification of extracellular heme in SCD plasma . More importantly, it uncovered for the first time a phenomenon of extracellular heme crisis; an acute uncontrolled release of heme into the circulation that was associated with: (a) enhanced oxidation of intracellular HbS, (b) impaired reduction of intracellular metHbS, (c) enhanced heme-induced hemolysis, and (d) rapid depletion of hemopexin. This phenomenon promoted the development of a lethal ALI reminiscent of ACS in the SS mice [54]. Extracellular heme crisis may be inevitable specifically within occluded vascular segments with impaired blood flow in patients with severe VOC who develop acute hemolysis.

NETs are released into the plasma by activated neutrophils. They consist of a mesh of extracellular DNA, nucleosomes, and histones decorated with granular enzymes, typically neutrophil elastase. It is thought that NETs trap pathogens with their high concentrations of enzymes . They were first implicated in SCD pathogenesis in a prospective study including 70 patients with VOC [84]. During VOC, levels of both nucleosomes and elastase-α1-antitrypsin complexes increased significantly as compared to steady-state values. Levels of nucleosomes correlated significantly with elastase-α1-antitrypsin complex levels during painful crisis, and the levels were highest in patients with ACS [84]. Extracellular heme stimulates the release of NETs by increasing intracellular neutrophil ROS formation (Fig. 3.1). Plasma from both humans and mice with SCD promotes NET release in vitro, and infusions of hemopexin reduced the number of pulmonary NETs, and the concentration of plasma nucleosomes in the Berkeley sickle mouse. The production of NETs by neutrophils during TNF-α exposure led to an ACS-like condition and sudden death in these animals following cremasteric surgical preparation [78]. DNase I treatment prolonged survival however hemopexin infusions did not, probably, because of the relatively low dose that was used in this study (0.15 mg hemopexin per mouse) [78]. In SCD patients, infections, which increase systemic TNF-α, are important contributing factors to ACS development , thus enhancing the idea that NETs may play an important role in the pathobiology of this lung condition.

Complete saturation of hemopexin yielding at least ~1.5 μM of extracellular heme that is seemingly free from other plasma binding partners triggers a lethal ALI in mice [54]. Given the low plasma hemopexin reserve in SCD, this ALI is inducible in ~70 % of sickle mice (Townes and Berkeley) with a relatively low dose of purified heme (i.v. 35 μmol/kg). This preclinical ACS model shares several similarities with the condition in humans. It is characterized by acute onset, acute anemia, pulmonary infiltration, hypoxemia, hypercapnia, acidosis, pulmonary edema, and sudden death (Table 3.1). This model suggests that continuous supply of extracellular heme fuels ACS, and that targeting this supply can avert respiratory failure even when the initial insult has caused some lung injury. Indeed, this may be one mechanism through which blood transfusion halts ACS, by reducing the concentration of extracellular heme in the intravascular space below the threshold required to cause endothelial barrier disruption.

Directly targeting heme with recombinant hemopexin infusions (1 mg per mouse) produces almost immediate improvement in oxygen saturation, followed steadily by restoration of breath rate in SS mice with hemolytic crisis and respiratory distress [54]. Hemopexin has as unique ability to rapidly squelch the redox activities of heme [85]. This suggests that reactive oxygen species (ROS) signaling contributes to the acute injury in the murine ACS lung, as has previously been suggested for the human ACS lung [68]. In addition, the lethal lung injury in the murine model requires a functional TLR4, which is redox-sensitive. The TLR4 antagonist TAK-242 is effective in preventing lung injury in the murine ACS model [54].

TLR4 mutant mice are protected from respiratory disease when challenged with a high dose of heme that causes a lethal ALI in congenic control mice. This means that the basic mechanism of the ALI, and perhaps other injuries caused by extracellular heme in SCD is mediated by TLR4. Using bone marrow chimeric mice, TLR4 expressed by nonhematopoietic vascular tissues (most likely the endothelium), has been found to mediate heme-induced ALI/ACS [54], and stasis in sickle mice. The receptor had been a major therapeutic target in sepsis resulting in the development of TAK-242 [86, 87] and Eritoran [88]. Both drugs have failed phase III sepsis trials nonetheless they may be ideal candidates for repurposing in SCD [89–91]. In preclinical studies, TAK-242 was effective in preventing respiratory failure in sickle mice only when it was given before or immediately after the induction of hemolytic crisis [54]. Since a majority of ACS patients present initially with well-defined antecedent acute events that are interceded by hemolysis, acute prophylaxis using TLR4 blockade may be an effective strategy to alter the clinical course of this condition.