H. Ahn, E. J. Chung, A. White, S. Kwon, Y. Jung, and D. E. Citrin; Radiation Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD
Purpose/Objective(s): Prior studies have demonstrated sex-based differences in sensitivity to radiation-induced lung injury (RILI) but the mechanisms of this effect are unclear. We compared RILI across three mouse strains and both sexes using in vivo and in vitro models. Materials/
Methods: Male and female mice (8-10 weeks old) of each strain (C57/L, C57BL6/NCr, C3H/HeN) were exposed to 15Gy thoracic irradiation (IR, n=20/group) to assess survival. Additional mice (n=10/group) were treated identically for tissue collection at the inflection point on the survival curve (20 wks for C57BL6/NCr). Lung injury was assessed with Masson-Trichrome and Aniline blue staining with Ashcroft scoring. Type II pneumocytes (AECII) and macrophages (M?) in lung were examined by immunohistochemistry for pro-SPC (AECII), F4/80 (total M?), and Arginase-1 (M2 M?). Primary AECII senescence (in vitro) was assessed by ß-galactosidase activity assays after IR. Primary M? prepared from bone marrow derived monocytes of each strain and sex were polarized with Il-13, Il-4, or LPS in vitro and polarization assessed by quantitative PCR. Results: No significant difference in survival between males and females was observed after 15Gy thoracic IR in C57L and C3H/HeN mice. In contrast, irradiated C57BL6/NCr males exhibited a longer median survival than females (males: 30wks, female: 24wks, p<0.0001) and were further studied. Female C57BL6/NCr mice developed greater lung injury after IR as assessed by Ashcroft score compared to C57BL6/NCr males (no IR female: 1.8±0.29, 15Gy female: 4.8±0.17, no IR male: 2.25±0.20, 15Gy male: 3.5±0.17, p<0.001 for female 15Gy vs. male 15 Gy). In contrast, collagen accumulation as measured by Aniline blue staining area (% of total lung) was similar in response to IR between males and females (no IR female: 16.1%±0.80, 15Gy female: 29.5±1.95, no IR male: 18.4±0.65, 15Gy male: 26.6±1.24, p=0.3733 for female 15Gy vs. male 15 Gy). On Masson Trichrome staining, female mice were appreciated to have increase cellularity consistent with inflammation in profibrotic regions compared to males after IR. Immunohistochemical assessment revealed a greater accumulation of total M? in the perifibrotic regions and in the whole lung of C57BL6/NCr females after IR compared to males, most of which stained positive for the M2 marker Arginase-1 (no IR female: 2.88/high power field±0.38, 15Gy female: 10.46±1.78, no IR male: 1.08±0.23, 15Gy male: 4.58±0.85, p<0.001 for female 15Gy vs. male 15 Gy). No difference in the rate of AECII senescence was observed between C57BL6/NCr males and females, however after IR female mice had significantly reduced total AECII compared to males by 20 wks after IR. Primary M? derived from the three strains responded in a distinct fashion to polarizing stimuli. Conclusion: This comparison study across three different mouse strains reveals distinct differences in sensitivity to RILI between male and female C57BL6/NCr mice that is accompanied by differences in AECII loss and macrophage accumulation.
