Research Article: A reductionist, in vitro model of environmental circadian disruption demonstrates SCN-independent and tissue-specific dysregulation of inflammatory responses

Date Published: May 28, 2019

Publisher: Public Library of Science

Author(s): Adam Stowie, Ivory Ellis, Kandis Adams, Oscar Castanon-Cervantes, Alec J. Davidson, Shin Yamazaki.


Environmental circadian disruption (ECD), characterized by repeated or long-term disruption in environmental timing cues which require the internal circadian clock to change its phase to resynchronize with the environment, is associated with numerous serious health issues in humans. While animal and isolated cell models exist to study the effects of destabilizing the relationship between the circadian system and the environment, neither approach provides an ideal solution. Here, we developed an in vitro model which incorporates both elements of a reductionist cellular model and disruption of the clock/environment relationship using temperature as an environmental cue, as occurs in vivo. Using this approach, we have demonstrated that some effects of in vivo ECD can be reproduced using only isolated peripheral oscillators. Specifically, we report exaggerated inflammatory responses to endotoxin following repeated environmental circadian disruption in explanted spleens. This effect requires a functional circadian clock but not the master brain clock, the suprachiasmatic nucleus (SCN). Further, we report that this is a result of cumulative, rather than acute, circadian disruption as has been previously observed in vivo. Finally, such effects appear to be tissue specific as it does not occur in lung, which is less sensitive to the temperature cycles employed to induce ECD. Taken together, the present study suggests that this model could be a valuable tool for dissecting the causes and effects of circadian disruption both in isolated components of physiological systems as well as the aggregated interactions of these systems that occur in vivo.

Partial Text

The circadian system is a highly conserved biological timing system in organisms from single cell bacteria to humans. In mammals, cell-autonomous clocks are present in most cells but are synchronized by a central master clock, the suprachiasmatic nucleus (SCN) of the anterior hypothalamus. Most physiological and behavioral processes are subject to circadian regulation which temporally optimizes the internal environment and allows anticipation of recurrent events in the external environment. Disruption of the circadian system in humans, as occurs during chronic shift work or multiple time-zone travel, has been linked to increased incidence and severity of several cancers [1–3], autoimmune disorders [4], obesity [5], diabetes [6], and stroke [7]. Animal models of shiftwork have also been employed to demonstrate the deleterious effects of circadian disruption [8, 9]. Though the mechanism(s) mediating the development of so called “shift work diseases” is not presently known, one potential explanation is that immune function is directly impacted by misalignment of circadian clocks with the external environment.

Organs were removed and micro-dissected into roughly equally sized pieces then sealed individually in 35mm dishes with 2ml of DMEM supplemented with 10mM HEPES, 2% B27, 2.5mL pen/strep, and 42μg D-(+) Glucose [8, 28, 29]. For luminometry experiments, tissue was cultured in media containing 1mM beetle luciferin (Molecular Imaging Products). Because of the phase-resetting effects of media changes no media changes were performed in while the explants were in a temperature cycle. Explants were assigned to experimental conditions at random to control for potential differences in explant size or cellular composition of individual explants.

In the present study we have demonstrated that at least some of the in vivo effects of circadian disruption on immune function can be recapitulated using a reductionist model of environmental circadian disruption. Given the importance of understanding how circadian disruption negatively impacts physiology, this model, particularly used in conjunction with in vivo studies, could be an important tool to identify mechanisms of circadian disruption at the cellular and organismal level. Importantly, because the organotypic culture approach in the present work did not allow for the identification of cell types or putative mechanisms involved in mediating these effects, future work should take this model even further and address these questions in a cell-type specific manner.




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