Extracellular hemin crisis triggers acute chest syndrome in sickle mice

Abstract

The prevention and treatment of acute chest syndrome (ACS) is a major clinical concern in sickle cell disease (SCD). However, the mechanism underlying the pathogenesis of ACS remains elusive. We tested the hypothesis that the hemolysis byproduct hemin elicits events that induce ACS. Infusion of a low dose of hemin caused acute intravascular hemolysis and autoamplification of extracellular hemin in transgenic sickle mice, but not in sickle-trait littermates. The sickle mice developed multiple symptoms typical of ACS and succumbed rapidly. Pharmacologic inhibition of TLR4 and hemopexin replacement therapy prior to hemin infusion protected sickle mice from developing ACS. Replication of the ACS-like phenotype in nonsickle mice revealed that the mechanism of lung injury due to extracellular hemin is independent of SCD. Using genetic and bone marrow chimeric tools, we confirmed that TLR4 expressed in nonhematopoietic vascular tissues mediated this lethal type of acute lung injury. Respiratory failure was averted after the onset of ACS-like symptoms in sickle mice by treating them with recombinant hemopexin. Our results reveal a mechanism that helps to explain the pathogenesis of ACS, and we provide proof of principle for therapeutic strategies to prevent and treat this condition in mice.

Figure 1. Sudden death associated with EHC.

(A) Relatively low dose of hemin (35 μmol/kg) caused sudden death in SS mice, but not in control AS and AA mice (n = 9). (B) Lethality associated with hemin in Townes sickle mice was dose dependent (n = 6). 100% lethality of sickle mice challenged with 70 μmol/kg hemin was confirmed using the Berkeley sickle mouse model (n = 6). (C–F) Characterization of EHC associated with sudden death in SS mice. n = 9 (SS); 6 (AA and AS). (C) Plasma Hx was significantly lower in SS mice at baseline (t = 0) and declined sharply in all 3 groups after hemin challenge. (D) SS mice had markedly higher TPH than AS and AA mice at baseline. TPH initially increased to the same level in all 3 groups. Hemin clearance by AA and AS mice and amplification by SS mice explains the significantly different values at 30 minutes. (E) metHb and (F) PFH in SS and control mice at baseline and 5 and 30 minutes after hemin challenge. *P < 0.05, ***P < 0.001.

Figure 2. SS mice with EHC develop ALI.

(A) SpO₂ and (B) breath rate, as determined by pulse oximetry, in SS mice and control animals challenged with hemin (n = 6). The number of surviving animals at each time point is indicated. (C) SO₂, measured using the VitalPath blood gas analyzer, in samples obtained from the left ventricle of anesthetized SS mice at baseline and in another cohort of SS mice 20 minutes after hemin challenge (n = 6 per group). (D) Representative H&E-stained lung sections of SS mice challenged with saline or hemin. Original magnification, ×200 (alveolar wall thickening), ×100 (all other panels). (E) Semiquantitative histological scoring of lung injury in SS mice in D. (F) Assessment of lung injury, using gravimetric analysis, in SS mice at baseline (BL) and after induction of intravascular hemolysis using hemin and phenylhydrazine (PHZ). **P < 0.01, ***P < 0.001.

Figure 3. Components of EHC associated with lethal ALI.

(A–E) Baseline and acute phase (AP) hematological parameters in SS mice that survived or succumbed to hemin. Samples for baseline value measurements were collected approximately 5 days before the experiment, and acute phase values were determined in samples collected 5 minutes after i.v. challenge with 35 μmol/kg hemin. (A) metHb. (B) Plasma Hb. (C) TPH. (D) PFH. (E) Plasma Hx. (F) The higher lung wet/dry weight ratio in SS mice with EHC that died is indicative of lung edema development. *P < 0.05, ***P < 0.001.

Figure 4. Hx is a prognostic factor in hemin-induced ALI.

(A) Survival rate of 26 SS mice with different baseline plasma Hx levels challenged with 35 μmol/kg hemin. (B) Survival rate of SS mice given rhHx (n = 8), IgG (n = 3), carrier vehicle for the Hx (n = 5), and polymyxin B (n = 5) prior to challenge with 70 μmol/kg hemin. (C) SpO₂ and (D) breath rate measured at normoxia (N) and after hypoxia/reoxygenation (H/R). (E) Representative H&E-stained lung sections of SS mice at normoxia and after hypoxia/reoxygenation. Original magnification, ×100. (F) PFH and (G) plasma Hx during normoxia and after hypoxia/reoxygenation. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5. Extracellular and intracellular hemin species cause unique organ toxicities.

(A–D) SS mice were given 70 and 25 μmol/kg hemin to induce EHC and NCEH, respectively (n = 6–11). (A) SpO₂ measured 20 minutes after induction. (B) Prognosis of SS mice. Death occurred within 2 hours in 100% of EHC mice and 12–72 hours later in 45% of NCEH mice. (C and D) Plasma concentration of (C) ALT and (D) AST, determined at baseline and either 20 minutes (EHC) or 24 hours (NCEH) after hemin challenge. (E) Representative H&E-stained sections of postmortem lung and liver in SS mice with EHC and NCEH and Perl’s Prussian blue (PPB) staining of the liver sections. Note the region of tissue damage (TD) likely caused by apoptosis and necrosis in the NCEH liver. Distribution of iron in Kupffer cells (arrow) and hepatocytes (arrowhead) was unique, with diffuse cytoplasmic staining in the hepatocytes. Original magnification, ×100 (H&E); ×200 (Perls); ×400 (insets). **P < 0.01, ***P < 0.001.

Figure 6. TAK-242 blocks ALI development in SS mice with EHC.

(A) PFH in SS mice previously given i.v. injection of TAK-242 (5 mg/kg) or vehicle (intralipid; 50 μl) followed by hemin challenge. (B) Lung function in 9 hemin-challenged SS mice pretreated with TAK-242 (n = 5) or vehicle (n = 4). The number of surviving animals at each time point is indicated. (C) Survival of hemin-challenged SS mice pretreated with TAK-242 or vehicle. (D) Representative H&E-stained lung sections of hemin-challenged SS mice pretreated with vehicle or 5 mg/kg TAK-242. Original magnification, ×100. (E) Cumulative lung injury score in each group in D. Data are mean ± SEM (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 7. TLR4 is required for hemin-induced ALI.

(A) Nonanemic B6TLR4^–/– and B6TLR4^+/+ mice (n = 5–6) were injected with 210 μmol/kg hemin to raise PFH to levels comparable to those of SS mice with EHC. (B) Lung function, determined by SpO₂. (C) Survival rate showing no lethality in hemin-challenged B6TLR4^–/– mice. (D) Representative H&E-stained lung sections of B6TLR4^–/– and B6TLR4^+/+ mice challenged with saline or hemin. Original magnification, ×100. (E) Cumulative lung injury score. Data are mean ± SEM (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 8. TLR4 mediates ALI in chimeric SS mice with EHC.

(A and B) Lung function, determined by (A) SpO₂ and (B) breath rate, in SS^TLR4+/+ and SS^{NH/TLR4–/–} chimeras at baseline and 20 minutes after EHC induction by 70 μmol/kg hemin. (C) Survival in SS^TLR4+/+ and SS^{NH/TLR4–/–} chimeras after hemin-induced EHC. (D) Representative H&E-stained lung sections. Original magnification, ×100. (E) Cumulative lung injury score. Data are mean ± SEM (n = 3). (F) Lung wet/dry weight ratios showed lung edema in SS^TLR4+/+, but not SS^{NH/TLR4–/–}, chimeras (n = 3–4). *P < 0.05, **P < 0.01.

Figure 9. Hx and TAK-242 treatment prevents respiratory failure in SS mice.

(A) Survival of SS mice treated as indicated at the onset of hemolytic crisis induced with 35 μmol/kg hemin. (B) SpO₂, determined 20 minutes after initiation of therapy. Note the lower mean SpO₂ in the nontreated (NT) group and 3 of the mice given 7.5 mg/kg TAK-242. TAK-242^S, survived; TAK-242^DS, did not survive. (C) TPH 30 minutes after treatment. Values for rhHx-treated mice were significantly reduced compared with nontreated and TAK-242 groups. (D) Representative H&E-stained lung sections of organs harvested after the end of the experiment (rhHx and TAK-242^S) or immediately after death (nontreated and TAK-242^DS). Original magnification, ×100. (E) Cumulative lung injury score. Data are mean ± SEM (n = 3). (F) Lung wet/dry weight ratio (n = 3–7). (G) SpO₂ and (H) breath rate of SS mice treated several minutes after the onset of hemolytic crisis and after showing evidence of respiratory distress. The number of surviving animals at each time point is indicated. *P < 0.05, **P < 0.01, ***P < 0.001.