Summary                         

Red blood cells (RBCs) are highly susceptible to various changes in the internal environment, such as oxidative stress, osmotic shock, energy depletion. These changes can lead to morphological and functional alterations of RBCs, eventually accelerating the clearance of them. Macrophages remove senescent or damaged RBCs from the circulation by the process called erythrophagocytosis, which is essential for iron/heme metabolism and homeostasis. It is generally believed that erythrophagocytosis is executed by hepatic and splenic macrophages. However, which kind of macrophages have a key function in RBCs clearance remains elusive. Some studies demonstrated that they are mainly cleared by splenic red pulp macrophages, while other researchers consider hepatic macrophages. The aim of this study was to assess stressed RBCs erythrophagocytosis and organ distribution in vivo with the application of ¹⁸F-FDG PET/CT. RBCs were induced under high temperature (48°C) to prepare stressed RBCs. Fluorescence-activated cell sorting (FACS) was used to analyse reactive oxygen species (ROS) generation, intracellular Ca²⁺ concentration and membrane phosphatidylserine externalization of RBCs. ¹⁸F-FDG was used to label RBCs and assess the erythrophagocytosis. Finally, ¹⁸F-FDG PET/CT was applied to reveal and measure the organ distribution of stressed RBCs in mice. ¹⁸F-FDG PET/CT imaging showed that stressed RBCs were mainly trapped in spleen, while untreated RBCs remained in circulation system.

Results from nanoScan PET/CT

After isolating RBCs from the blood of BALB/c mice, cells were incubated for 30min at 48°C to induce stressed RBCs, while untreated RBCs were not. RBCs were incubated at 37°C for 1h to reduce the intracellular glucose concentration. RBCs were mixed with osmotic pressure-adjusted ¹⁸F-FDG solution and incubated at 37°C for 30min. Then, RBCs were washed with PBS to eliminate extracellular ¹⁸F-FDG. ¹⁸F-FDG-labelled RBCs were injected via the lateral tail vein of mice. Mice were anesthetized and maintained in anaesthesia state by isoflurane throughout imaging procedure. Imaging was performed using nanoScan PET/CT system. Image acquisition was started after intravenous injection and lasted for 2h. The reconstructed images were analysed by Nucline NanoScan 3.00.018.0000. In the images of BALB/c mice, volume of interest (VOI) was placed in each organ, and the radioactivity was expressed as the percentage of injected dose per gram (%ID/g). Time-activity curves of cardiac, liver, spleen, lung, and kidney were obtained.

Results show

• Utreated RBCs mainly accumulated in the heart as they remained in cardiovascular system

• Stressed RBCs were trapped in the spleen

Current study proved that ¹⁸F-FDG labelling of RBCs is suitable to assess erythrophagocytosis and in vivo organ distribution of stressed RBCs and untreated RBCs. Data demonstrated increased erythrophagocytosis in stressed RBCs. Additionally, PET/CT imaging showed that splenic radioactivity increased rapidly in mice injected with stressed RBCs.

https://www.nature.com/articles/s41598-021-82100-y