C57BL/6 Mouse Peritoneal Macrophages (Fresh Cells)

Ionizing Radiation Induces Extracellular Trap Release from Macrophages

Macrophages are key innate immune cells in the host defense against pathogens. Ionizing radiation can impair macrophage functions such as phagocytosis and activate them, potentially exacerbating tissue injury. Macrophage extracellular traps (METs) are formed upon stimulation of macrophages with PAMPs (pathogen-associated molecular pattern) or DAMPs (damage-associated molecular pattern). We hypothesized that macrophages exposed to ionizing radiation can release extracellular traps.

Peritoneal macrophages were collected from C57BL/6 mice and subjected to 5 Gy radiation. We performed assays to detect METs, including the immunofluorescence of citrullination of histone H3 (CitH3) and cell-free DNA measurement in cell culture medium as well as cell death. The results demonstrated MET formation in irradiated macrophages compared to the control (Fig. 1A). The proportion of CitH3 over the total number of nuclei in the microscopic fields was significantly elevated upon the exposure of 5 Gy radiation from 3.2 to 13.1% (Fig. 1B). The measurement of the absolute number of CitH3-positive DNA segments also showed a 3.5-fold increase compared to that of non-irradiated control cells. Total MET area with 5 Gy radiation was increased 2.4-fold compared to the control cell culture (Fig. 1C). We also measured the concentration of cell-free DNA, which is free-floating DNA in the cell culture media. The concentration of cell-free DNA was increased to 170% by the irradiation (Fig. 1D). These data show that radiation increases CitH3 and MPO, which are frequently used as MET markers.