Sickle Cell Hemoglobin Impairs Lysosomal Processing in Macrophages Leading to Inflammasome Activation and Inflammation

BACKGROUND: Sickle cell anemia (SCA) is a chronic hemolytic disease that is characterized by chronic presence of low-level plasma hemoglobin (Hb, 3-10 µM). Plasma Hb can reduce NO availability, induce endothelium damage and activate coagulation cascade and inflammation leading to development of vasculopathies. The Hb scavenging proteins, haptoglobin and hemopexin, are depleted in SCA resulting in increased levels of circulating Hb. Hb is recognized by macrophages/monocytes scavenger receptors and acquired by endocytosis resulting in degradation of Hb in lysosomes. Single nucleotide mutation in the β- globin gene (HbS) promotes its polymerization at low pH which is characteristic for lysosomes.

HYPOTHESIS: We hypothesize that HbS forms polymers inside macrophage lysosome impairing its processing by lysosomal enzymes and causing lysosome injury and inflammation.

METHODS: The study was approved by Howard University review board (IRB) and all subjects provided written inform consent prior the sample collection. Whole blood samples were obtained from five SCA patients and five normal control subjects. Human THP-1 promonocytic cells were differentiated into macrophages with PMA and treated with either purified HbS and HbA (Sigma-Aldrich) or whole red blood cell lysate for 3 hrs. After washing, the cells were further cultured for 24-72 hrs. Conditioned media was collected at different time points and cytokine levels were measured by BioPlex Kit (BioRad). Trypan blue assays were used to measure cell proliferation. Lysosome dye (LysoTraker red DND99, Molecular Probe), plasma membrane dye (CellMask deep red plasma membrain stain, Thermo-Fisher Scientific), DAPI (Sigma-Aldrich) and phalloidin-FITC (Sigma-Aldrich) were used for visualization of cell structures. LPS treatment (1µg/mL) was used for generation of M1 macrophages; IL-4 treatment (5µg/mL) was used for generation of M2 macrophages.

RESULTS: Treatment of THP1- derived macrophages with lysates of red blood cells obtained from SCA patients induced accumulation of lysosomes, induced apoptosis and stimulated cell proliferation. No significant accumulation of lysosomes, apoptosis and proliferation were observed after treatment of macrophages with lysates of red blood cells obtained from control subjects with normal hemoglobin. Treatment of macrophages with purified HbS but not HbA also induced lysosome accumulation and macrophages proliferation. No activation of inflammation was observed at 24 hrs after treatment with either HbS or HbA proteins. HbS treatment induced a delayed, 72 hrs post treatment, activation of inflammasome evidenced by IL-18 and IL-1β production and secretion of pro-inflammatory cytokines (IL-2, IL-6 and TNF-α). HbS treatment also induced secretion of M-CSF and GM-CSF growth factors.

CONCLUSION: Lysosomal processing of HbS was delayed in human macrophages, leading to lysosome accumulation, inflammasome activation, macrophage proliferation and inflammation. This observation can explain long term chronic inflammation observed in patients with SCA.

ACKNOWLEDGMENTS: This work was supported by NIH Research Grants 1P50HL118006, 1R01HL125005 and 5G12MD007597. The content is solely the responsibility of the authors and does not necessarily represent the official view of NHLBI, NIMHD or NIH.