Abstract
Purpose :
Hematopoietic stem cell transplantation is a curative procedure for many blood disorders, but graft versus host disease (GVHD) remains its major complication. Irradiation to ablate recipient’s diseased bone marrow prior to transplantation is a major trigger for GVHD. Due to its surface location, the eye is especially susceptible to irradiation damage. Macrophages are sentinel, phagocytic cells involved in clearance of cellular debris and dead cells after tissue injury. The present study is designed to investigate whether irradiation could trigger macrophage phagocytosis, activation and polarization.
Methods :
Bone marrow-derived macrophages were cultured from the femur and tibia marrow of BALB/c mice in the presence of macrophage colony stimulating factor. Cultured macrophages were exposed to 7Gy irradiation using an x-ray irradiator. Effect of irradiation on macrophage phagocytosis and apoptosis was quantified using Alexa Fluor-594 conjugated zymosan A particles and Annexin V-APC Conjugated apoptosis kit using confocal microscopy and flow cytometry, respectively. Real-time PCR was used to quantify the cytokines and markers of classical and alternative macrophage activation status.
Results :
Our data demonstrates that exposing macrophages to irradiation caused a >2-fold increase in the uptake of zymosan particles compared to control macrophages not exposed to irradiation. Irradiation did not affect macrophage apoptosis or necrosis (control 85.6± 4.5% viable cells and 14.1±4.5% apoptotic cells vs irradiated 85.7±3% viable cells and 19.8±5.8% apoptotic cells). Further, irradiation caused 2-fold ± 0.29 increase in IL-1b, 4.2-fold ± 1.21 increase in TNF-a, 2-fold ±0.99 increase in IL-6 and 2.4-fold ± 0.77 increase in CCL2 expression. While irradiation did not cause any upregulation of M1 macrophage markers i.e., CD80 and iNOS, it did cause a robust increase in gene expression of M2 phenotype markers (2.7-fold ± 1.5 increase in CD206, 16.4-fold ± 4.1 increase in arginase-1, 4-fold ± 0.4 increase in IL-10).
Conclusions :
Our data demonstrates that irradiation causes a remarkable activation of macrophages, as shown by enhanced phagocytosis and the increase in gene expression of macrophage-derived cytokines and chemokines. These radiation-exposed macrophages show a gene signature of predominantly M2 spectrum phenotype.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.