141 Lactococcus Lactis CNCM I-1631 Targets Oxidative Stress to Ameliorate Inflammation in Early-Onset Experimental Colitis

Abstract

Background&Aims:Circadian rhythms govern a wide array of intestinal epithelial functions in mammals, including nutrient absorption, microbial sensing, migration, and self-renewal. In the absence of coordinating signals generated by the central pacemaker activity of the suprachiasmatic nucleus, mammalian cells derived from peripheral tissues typically require an external cue such as a serum shock or steroid pulse to synchronize circadian rhythms in culture. Building on our recent finding of robust circadian rhythms in mouse small intestinal organoids (enteroids) derived from PERIOD2:LUCIFERASE (PER2:LUC) mice, we tested the capacity of PER2::LUC enteroids to synchronize circadian rhythms in the absence of these external cues.Methods:We generated PER2::LUC jejunal enteroids and maintained them in an incubating luminometer to measure real time oscillations of PER2 abundance. Enteroid experiments were performed under typical culture conditions in the presence or absence of MatrigelTM. Prior to bioluminescent recordings, populations of enteroids were subjected to a 2-hour 50% serum shock vs. a dexamethasone pulse to synchronize clock vs. no external synchronization. Separately, unsynchronized enteroids were incubated in the presence or absence of steroid receptor antagonist RU-486. Results: Serum-shocked and dexamethasone-treated enteroids displayed synchronized circadian rhythms of PER2 abundance immediately upon recording. The amplitude and persistence of these rhythms were increased in enteroids embedded in MatrigelTM vs. those suspended in media. In the absence of a serum shock or dexamethasone pulse, enteroids spontaneously developed synchronized circadian rhythms within 12 hours of recording. RU486 treatment prior to bioluminescent recording significantly delayed the onset of this spontaneous synchronization and dampened the amplitude of PER2 oscillations by 2to 6-fold. Conclusions: PERIOD2::LUCIFERASE enteroids autonomously synchronize circadian rhythms, providing further evidence of a tightly regulated peripheral clock intrinsic to the intestinal epithelium. Additional studies addressing the role of glucocorticoids in coordinating circadian rhythms in enteroids and in vivo should yield novel insights regarding circadian regulation of gut homeostasis and stem cell dynamics and pharmacologic effects of glucocorticoids on the intestinal epithelium.