Elsevier

Brain, Behavior, and Immunity

Volume 88, August 2020, Pages 220-229
Brain, Behavior, and Immunity

Circadian desynchronization alters metabolic and immune responses following lipopolysaccharide inoculation in male mice

https://doi.org/10.1016/j.bbi.2020.05.033Get rights and content

Highlights

  • Circadian desynchronization alters the metabolic and behavioral response to LPS.

  • Sickness behaviors are prolonged in circadian desynchronized mice.

  • Changes in brain and peripheral cytokines are associated with these effects.

Abstract

Metabolism and inflammation are linked at many levels. Sickness behaviors are elicited by the immune system’s response to antigenic stimuli, and include changes in feeding and metabolism. The immune system is also regulated by the circadian (daily) clock, which generates endogenous rhythms, and synchronizes these rhythms to the light-dark cycle. Modern society has resulted in chronic misalignment or desynchronization of the circadian clock and the external environment. We have demonstrated that circadian desynchronization (CD) in mice alters metabolic function, and also affects both peripheral and central immune responses following a low-dose lipopolysaccharide (LPS) challenge. However, it is unclear how this altered immune response impacts sickness behaviors and metabolism following challenge. To test this, we housed male mice in circadian desynchronized (10-hours light:10-hours dark) or control (12-hours light:12-hours dark) conditions for 5–6 weeks. We then challenged mice with LPS (i.p., 0.4 mg/kg) or PBS and measured changes in body mass, feeding, drinking and locomotion using a comprehensive phenotyping system. Plasma, liver, and brain were collected 36 h post-inoculation (hpi) and inflammatory messengers were measured via multiplex cytokine/chemokine array and qPCR. We find that recovery of locomotion and body mass is prolonged in CD mice following LPS challenge. Additionally, at 36 hpi the expression of several proinflammatory cytokines differ depending on pre-inoculation lighting conditions. Our findings add to the growing literature which documents how desynchronization of circadian rhythms can lead to disrupted immune responses and changes in metabolic function.

Introduction

Virtually all organisms on Earth exhibit oscillations in physiology and behavior over an approximate 24-hour day (Man et al., 2016). These circadian rhythms in physiology and behavior enable organisms to anticipate and respond to the external environment driven by the rotation of the Earth about its axis (Man et al., 2016). Circadian rhythms are generated by molecular clocks in nearly every mammalian cell and tissue (Scheiermann et al., 2013). Under normal physiological conditions, these clocks are synchronized to each other by multiple pathways, including the autonomic nervous system and the hypothalamic-pituitary-adrenal (HPA) axis, which are directly regulated by the suprachiasmatic nucleus (SCN) of the hypothalamus (Kalsbeek et al., 2006).

Physiological and behavioral processes that are regulated by the circadian system include the sleep-wake cycle, locomotor activity, cardiovascular system, hormone secretion, body temperature, metabolism, and immunity, to name a few (Scheiermann et al., 2018). Considering the wide variety of processes that are regulated by the circadian system, it is not surprising that the desynchronization between the SCN and peripheral clocks is associated with various human pathologies including obesity, diabetes, cancer, and cardiovascular disease (Early and Curtis, 2016). With more than 15% of the United States’ working population regularly working night shifts, the development of these human pathologies in night shift workers is a considerable concern (Early and Curtis, 2016). Furthermore, circadian misalignment has become commonplace as more people travel across time zones and engage in activities at times when the circadian system promotes resting (Early and Curtis, 2016). Therefore, human pathologies associated with circadian desynchronization (CD) are not only a concern for night shift workers but also for the greater population. Our previous work has demonstrated that we can induce CD by housing mice in a 20 h long light-dark cycle of 10 h light, and 10 h darkness (LD10:10). Relatively short-term housing in this environment (4-6wks) leads to metabolic dysregulation, including weight gain, as well as increased plasma leptin, insulin, and triglycerides (Karatsoreos et al., 2011). Remarkably, these effects are not associated with sleep deprivation, but instead changes in sleep timing and sleep quality (Phillips et al., 2015).

The immune system is regulated by the circadian clock. Some of the first experimental evidence that the immune system was regulated by the circadian clock was reported 60 years ago by Halberg et al. (1960), that revealed challenging the immune system of mice with bacterial lipopolysaccharide (LPS) at the end of the resting phase resulted in higher mortality rates than at other times of day. More recently, this phenomenon has been observed in non-pathogen induced inflammatory disease. For instance, there is a predictable time of symptom onset for immune conditions such as rheumatoid arthritis and asthma (Scheiermann et al., 2018). We now know that numerous immune cells contain intrinsic circadian clocks, including monocytes, macrophages, mast cells, neutrophils, eosinophils, natural killer cells, CD4+ T lymphocytes, and B lymphocytes (Scheiermann et al., 2018).

Emerging evidence supports the idea that the circadian clock prepares the mammalian immune system for defense against pathogens during the time that mammals are most likely to encounter pathogens, i.e., their active phase (Tognini et al., 2017). However, this increased resistance against pathogens comes with an increased susceptibility to these same pathogens at the beginning of the resting phase (Tognini et al., 2017).

The present studies demonstrate that CD prolongs recovery of locomotor activity and body mass, as measured by a comprehensive behavioral phenotyping system, in mice challenged with LPS. It also results in changes in both circulating cytokines and immune mediators, and expression of immune factors within the brain.

Section snippets

Animals

Adult male C57/BL6Nhsd mice (5–6 weeks, n = 64; Envigo) were used in two independent experiments. Upon arrival, all mice were single housed in standard shoebox cages with food and water available ad libitum. The light cycles were maintained at 12-h light and 12-h dark (LD12:12) for at least 6 days to allow for acclimatization to the new environment. Following the acclimatization period, half of the mice (n = 16 from each experiment) were randomly assigned to CD (10-h light, 10-h dark, LD10:10),

Baseline behavioral measures

We have previously shown that CD leads to increased body mass gain and this weight gain is associated with elevated plasma triglyceride, leptin and insulin levels (Karatsoreos et al., 2011). Using the Promethion metabolic and behavioral phenotyping system, we assessed whether there were changes in overall total locomotion, feeding and drinking activities. We did not observe a statistically significant difference in total average daily locomotion (Mann-Whitney U test: p = 0.22), food consumption

Discussion

Our previous results (Phillips et al., 2015) demonstrated that our CD paradigm alters the central and peripheral immune response to a low-dose LPS challenge 3 hpi. However, it was not known how this altered inflammatory response to LPS impacted physiological and behavioral responses. We hypothesized that this altered inflammatory response to LPS induced by CD would impact changes in body mass and sickness behavior following LPS challenge. Using survival analysis, we demonstrated that CD mice

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by grants from the National Institutes of Health (DK119811) and the National Science Foundation (CAREER 1553067) to INK. We would also like to acknowledge the skilled animal care staff at Washington State University, as well as Naomi Wallace who assisted with tissue collections, and Andy He for his general help in the laboratory.

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