Elevated ecto-5'-nucleotidase: a missing pathogenic factor and new therapeutic target for sickle cell disease

Abstract

Although excessive plasma adenosine is detrimental in sickle cell disease (SCD), the molecular mechanism underlying elevated circulating adenosine remains unclear. Here we report that the activity of soluble CD73, an ectonucleotidase producing extracellular adenosine, was significantly elevated in a murine model of SCD and correlated with increased plasma adenosine. Mouse genetic studies demonstrated that CD73 activity contributes to excessive induction of plasma adenosine and thereby promotes sickling, hemolysis, multiorgan damage, and disease progression. Mechanistically, we showed that erythrocyte adenosine 5'-monophosphate-activated protein kinase (AMPK) was activated both in SCD patients and in the murine model of SCD. AMPK functions downstream of adenosine receptor ADORA2B signaling and contributes to sickling by regulating the production of erythrocyte 2,3-bisphosphoglycerate (2,3-BPG), a negative allosteric regulator of hemoglobin-O₂ binding affinity. Preclinically, we reported that treatment of α,β-methylene adenosine 5'-diphosphate, a potent CD73 specific inhibitor, significantly decreased sickling, hemolysis, multiorgan damage, and disease progression in the murine model of SCD. Taken together, both human and mouse studies reveal a novel molecular mechanism contributing to the pathophysiology of SCD and identify potential therapeutic strategies to treat SCD.

Graphical abstract

Figure 1.

Genetic deletion of CD73 in SCD mice decreases hemolysis and increases erythrocyte lifespan by attenuating plasma CD73 activity and plasma adenosine level. (A-B) Plasma adenosine level and plasma CD73 activity level in WT and SCD mice. (C) Correlation of plasma CD73 activity and plasma adenosine level in SCD mice. (D) Plasma CD73 activity and (E) plasma adenosine level in WT BMT mice, SCD BMT mice, and SCD/CD73^−/− BMT mice. (F) Representative image of sickled erythrocytes and the percentage of sickled erythrocytes in SCD BMT mice and SCD/CD73^−/− BMT mice (magnification ×400). (G) Lifespan of erythrocytes in SCD BMT and SCD/CD73^−/−BMT mice. (H) Plasma total hemoglobin and (I) plasma bilirubin in WT BMT mice, SCD BMT mice, and SCD/CD73^−/− BMT mice. Data are expressed as mean ± SEM. n = 5 for each group; *P < .05 vs WT, **P < .05 vs SCD, Student t test.

Figure 2.

Genetic deletion of CD73 in SCD mice decreases multiorgan damage including lung, spleen, and liver. (A) H&E staining of lungs, spleens, and livers of WT BMT, SCD BMT, and SCD/CD73^−/− BMT mice (magnification ×200). (B-D) Semiquantitative analysis of H&E-stained sections show decreased lung congestion, spleen necrosis, and liver necrosis in SCD/CD73^−/− BMT mice compared with SCD BMT mice. (E-G) MPO activity in multiple organs including lung, spleen, and liver in SCD/CD73^−/− BMT mice compared with SCD BMT mice. Data are expressed as mean ± SEM. n = 5 for each group; *P < .05 vs WT, **P < .05 vs SCD, Student t test.

Figure 3.

CD73 is important for elevation of plasma adenosine, erythrocyte p-AMPK, erythrocyte 2,3-BPG mutase activity, and erythrocyte 2,3-BPG in both SCD mice and SCD patients. (A) Erythrocyte p-AMPK, (B) erythrocyte 2,3-BPG mutase activity, (C) erythrocyte 2,3-BPG level, and (D) hemoglobin oxygen release capacity (P50) in WT BMT, SCD BMT, and SCD/CD73^−/− BMT mice. (E) Plasma CD73 activity, (F) plasma adenosine level, (G) erythrocyte p-AMPK levels quantified by ELISA, (H) erythrocyte 2,3-BPG mutase activity, and (I) 2,3-BPG level in normal individuals and sickle cell disease patients. Data are expressed as mean ± SEM. n = 5 for each group; *P < .05 vs control, **P < .05 vs SCD, Student t test.

Figure 4.

AMPK underlies the detrimental role of CD73-mediated adenosine signaling in erythrocytes in SCD patients. Plasma adenosine levels were correlated to (A) plasma hemoglobin and (B) bilirubin. Plasma CD73 activity was correlated to plasma (C) hemoglobin and (D) bilirubin. (E-H) Changes in erythrocyte-phosphorylated AMPK level, 2,3-BPG mutase activity, 2,3-BPG concentrations and percentage of sickled cells in erythrocytes isolated from SCD patients after incubation under hypoxic conditions in the absence or presence of Bay Compound (ADORA2B agonist), AICAR (AMPK agonist), Bay Compound + Compound C (AMPK antagonist), Compound C, respectively. *P < .05 vs normoxic condition; **P < .05 vs untreated samples under hypoxic condition; #P < .05 versus Bay Compound–treated samples under hypoxic condition. ##P < .05 vs untreated samples under hypoxic condition.

Figure 5.

APCP treatment decreases plasma CD73 activity, plasma adenosine level, erythrocyte phosphorylation of AMPK, erythrocyte 2,3-BPG mutase activity, and erythrocyte 2,3-BPG level. (A-B) Plasma CD73 activity and plasma adenosine level in SCD mice with or without APCP treatment. (C-F) Erythrocyte-phosphorylated AMPK level, 2,3-BPG mutase activity, 2,3-BPG concentrations, and P50 in SCD mice with or without APCP treatment. (G-I) Plasma hemoglobin, plasma total bilirubin, and erythrocyte lifespan in SCD mice with or without APCP treatment. Data are expressed as mean ± SEM. n = 5 for each group; *P < .05 vs SCD, Student t test.

Figure 6.

APCP treatment in SCD mice decreases multiorgan damage, including lung, spleen, and liver. (A) H&E staining of lungs, spleens, and livers of SCD mice with or without APCP treatment (magnification ×200). (B-D) Semiquantitative analysis of H&E-stained sections showed decreased lung congestion, spleen necrosis, and liver necrosis in SCD mice following APCP treatment. (E) BALF total cell count, (F) BALF albumin concentration, and (G) BALF IL-6 concentration in SCD mice with or without APCP treatment. (H) MPO activity in multiple organs, including lung, spleen, and liver in SCD mice with or without APCP treatment. Data are expressed as mean ± SEM. *P < .05 vs SCD, Student t test. (I) Working model: CD73 is essential for induction of plasma adenosine in SCD. Elevated plasma adenosine contributes to erythrocyte sickling, tissue injury, and dysfunction by engagement of ADORA2B on erythrocytes to induce 2,3-BPG mutase activity and 2,3-BPG production. AMPK is a key enzyme that functions downstream of ADORA2B to activate 2,3-BPG mutase and promote 2,3-BPG production and subsequent erythrocyte sickling. Preclinically, inhibition of CD73 is a promising therapeutic strategy to treat SCD or prevent hypoxia-induced tissue damage. APCP treatment decreases multiorgan damage, including lung, spleen, and liver in SCD mice.