Soy Isoflavones Inhibit Radiation-Induced Macrophage And Neutrophil Activation And Promote Ly6c+Ly6g-Suppressive Monocytes Resulting In Lung Radioprotection

Radiation-induced lung injury results from a cascade of inflammatory processes leading to clinical pneumonitis and fibrosis. Acute and chronic inflammatory events following radiation therapy could be initiated and driven by macrophage and neutrophil activation in the lung. Our lab has previously reported that treatment with soy isoflavones mitigate inflammation and fibrosis but the mechanism of radioprotection remains unclear. Recent studies demonstrated that Ly6C+Ly6G- immature monocytes have a suppressive phenotype and play a crucial role in the resolution of acute inflammation and tissue repair. We hypothesize that soy isoflavones mediate radioprotection via modulation of radiation-induced inflammatory processes involving macrophages, neutrophils, and Ly6C+Ly6G- immature monocyte suppressor cells. BALB/c mice received a single 10 Gy thoracic irradiation with soy isoflavones given orally at 1 mg/day, prior-to and continuously after radiation. Lungs were resected at different time points after radiation and processed for immunohistochemistry (IHC) or flow cytometry analysis. In situ macrophages and neutrophils were identified on lung sections by F4/80 and Gr-1 immunostaining, respectively. Macrophage and neutrophil activations were determined by IHC staining of NOS2 and myeloperoxidase (MPO), respectively. Lungs were dissociated into single cell suspensions and cells were stained with anti-CD45, anti-Ly6C, and anti-Ly6G fluorescent antibodies to analyze monocyte populations by flow cytometry. Immunohistochemical analysis for macrophages staining reveals prominent F4/80 and NOS2 staining in radiation treated lungs (++++) 4 months after radiation, in contrast to decreased levels in radiation + soy treated lungs (++). Immunohistochemical analysis for neutrophils showed an increase in Gr-1 and MPO staining at 3 and 4 months after radiation (++++) but to a lesser extent in radiation + soy (++). Flow cytometry analysis revealed that radiation treatment significantly reduced the percentage of Ly6C+Ly6G- cells in the lung at both 1 month (13±1%, SEM) and 3 months (10±2%) after radiation, compared to 16±2% in control (p<0.02). Interestingly, at 3 months post-radiation, mice that received radiation+soy had significantly increased levels of Ly6C+Ly6G- cells (16±1%) relative to irradiated mice (p<0.01). These preliminary results suggest that soy could inhibit the radiation-induced macrophage and neutrophil activations, as determined by lower levels of NOS2 and MPO activation markers, respectively. Radiation-induced decreases in Ly6C+Ly6G- monocytes in the lung are restored to normal levels by soy isoflavones. Soy modulation of the inflammatory/suppressor cell axis that occurs after thoracic irradiation may play a critical role in mediating the radioprotective effects observed with soy.