T1749 Radiation-Induced Colonic Epithelial Cell Apoptosis and Crypt Killing Is Promoted By Intestinal Microbes and MyD88

Background.Injury to the intestinal mucosa during cancer radiotherapy is associated with significant morbidity, and may limit the radiation dose.Epithelial stem cell apoptosis resulting in subsequent crypt death is a central mechanism of radiation-induced intestinal injury.Prior work in germ-free mice revealed that absence of intestinal microbiota protected against the radiation-induced intestinal injury.We explored cellular and molecular factors underlying the pathogenic role of intestinal microbes in the causation of the radiation-induced intestinal injury.Methods.To lower intestinal microbial colonization, established protocols using neomycin, metronidazole, ampicillin and vancomycin in drinking water were applied to C57 BL/6 mice.In other experiments, Myd88 knockout and control mice were utilized.Mice were irradiated using 137 Cs source.Crypt killing was quantified using the microcolony assay applied 4.5 days post irradiation.Crypt cell apoptosis was quantified 4.5 hours after irradiation by analysis of H&E-stained sections, and TUNEL.Cell proliferation was assessed by PCNA staining.Results are expressed as mean ± SD.Results.Radiation doses from 4 to 20 Gy resulted in progressive colonic crypt death in normal mice.In contrast, antibiotictreated mice were markedly protected against crypt death.At 14 Gy radiation, the mean 4.5 day colonic crypt survival was 5.4% in control mice, compared to 73.3% in antibiotictreated mice (p<0.01).At this time-point, control mice had fewer PCNA positive cells than antibiotic-treated mice, 1.6±0.1 cells per crypt section versus 6.1±0.5 respectively (p<0.01), consistent with a protective effect of antibiotics.Antibiotic-treated mice also had better intestinal function compared to control mice: at 14 Gy, mean daily water intake was 2.6 g versus 1.4 g (p<0.05), and daily food intake was 3.2 g versus 1.4 g (p<0.05) in antibiotic and control groups, respectively.4.5 hours after 14 Gy, the mean number of apoptotic epithelial cells per crypt section was 5.8±0.5 in control mice compared to 0.6± 0.38 in antibiotic-treated mice (p<0.01).Myd88 knockouts, compared to normal control mice suffered less radiation-induced apoptosis: at 14 Gy, 1.7±0.6 apoptotic cells per crypt section versus 5.0±0.8respectively, and had greater crypt survival of 53.1±9.3%versus 5.9 ±4.1%.Conclusions.These results suggest that microbial signaling via Myd-88 promotes radiationinduced colonic epithelial cell apoptosis and subsequent crypt death.This indicates that intestinal microbes regulate DNA damage responses in the colon and reveal previously unrecognized regulatory mechanisms of intestinal radiation.