Endocrinology. 2015 Nov;156(11):4302-11. doi: 10.1210/en.2015-1490.
Glucocorticoids Reset the Nasal Circadian Clock in Mice.
Aya Honma1, 2, Yoshiko Yamada2, Yuji Nakamaru1, Satoshi Fukuda1, Ken-ichi Honma2, Sato Honma2
1 Department of Otolaryngology-Head and Neck Surgery, Hokkaido University Graduate School of Medicine, Sapporo, Japan
2 Department of Chronomedicine, Hokkaido University Graduate School of Medicine, Sapporo, Japan
The symptoms of allergic rhinitis show marked day-night changes that are likely to be under the control of the circadian clock, but the mechanism of this control is poorly understood. Because most peripheral tissues have endogenous circadian clocks, we examined the circadian rhythm of the clock gene product PERIOD2 (PER2) in the nasal mucosa of male mice using a luciferase reporter and demonstrated for the first time the phase-dependent effects of dexamethasone (DEX) on nasal PER2 rhythm in vivo and ex vivo. The phase shifts in PER2 rhythm caused by DEX were observed around the peak phase of serum glucocorticoids, suggesting that the circadian rhythm of endogenous glucocorticoids regulates the peripheral clock of the mouse nasal mucosa. From the viewpoint of circadian physiology, the best time to administer intranasal steroid treatment for allergic rhinitis would be when no phase shift is caused by DEX: in the early evening in diurnal humans.
The mammalian circadian clock comprises the master clock in the hypothalamic suprachiasmatic nucleus (SCN) and peripheral clocks in a variety of tissues throughout the body (1). Symptoms of allergic rhinitis (AR) exhibit prominent circadian variation, worsening in the early morning (2). Circadian rhythms in neural and humoral systems, and in immune reactivity to antigens are considered as possible triggers of these symptoms. However, little is known as to the circadian rhythms in the nasal tissues. In addition, because several peripheral clocks are synchronized by glucocorticoids (3), an intranasal steroid, a topical treatment for AR, may effect on the nasal circadian clock.
In this study, using mice carrying a luciferase reporter, we sought a peripheral clock generating autonomous circadian rhythms in the nasal mucosa. In this mice (PER2::LUC knock-in mice), a gene coding firefly luciferase is inserted immediately downstream to the gene coding clock gene Period (Per) 2. Thus, in response to intrinsic as well as extrinsic signals activating Per2 expression, PER2 and luciferase fused proteins are synthesized in cells (4). And when we culture the tissue of these PER2::LUC mice in the medium containing luciferin, the substrate of the luciferase with high membrane permeability, luciferase reacts with luciferin to emit bioluminescence, the level of which is proportional to the amount of luciferase synthesized in the cell. Thus, in PER2::LUC mice, the level of bioluminescence reflects that of PER2 in the cell.
We measured bioluminescence of cultured nasal mucosa to demonstrate the presence of an autonomous circadian clock. We also examined the effects of dexamethasone (DEX) on the nasal clock, since glucocorticoids are widely used as a treatment for AR, while it could be resetting time cues since the blood glucocorticoids exhibit robust circadian rhythm.
All mice were reared under controlled environmental conditions: a 12-hour light, 12-hour dark cycle. We cultured nasal mucosa of male PER2::LUC knock-in mice and measured bioluminescence levels by using a photomultiplier for more than 6 consecutive days. DEX was applied either in culture media at 4 different circadian phases (ex vivo experiment), or injected intraperitoneally at one of 4 phases on the day of tissue sampling (in vivo experiment) and effects on the circadian phase of bioluminescence rhythms were analyzed.
Mouse nasal mucosa exhibited a robust circadian rhythm in PER2 levels with the peak in the evening, consistent with PER2 immunohistochemistry (Fig. 1) and mRNA levels as measured by RT-PCR. PER2 signals were especially strong in the epithelial cells.
Fig.1: Immunostaining of the respiratory epithelia. Tissues were collected either in the morning at 6 a.m. (left) or evening at 6 p.m. (right), and immunostained with anti-PER2 antibody. PER2 (dark staining of 3,3’ -diaminobenzidine) was highly expressed in the evening especially in the epithelial cells, vascular endothelial cells (arrow), and nerve termini (arrowhead). Scale bars: 100 µm.
DEX shifted the phase of the circadian clock in the nasal mucosa and the response depended on the phase of DEX application. PER2 rhythm in the nasal mucosa phase-delayed when DEX was applied in the evening and phase-advanced when it was applied in the midnight. The nasal clock is phase-shifted by glucocorticoids at the time when blood glucose is high in nocturnal mice. On the other hand, DEX did not shift the rhythm when administered in the morning.
It was reported that immune reactivity to antigens and activation in inflammatory mediators had circadian rhythms and played an important role in biophylaxis (5,6). Since the nasal mucosa is located in the very front of the upper airway, preventing our body from physical, chemical and bacteriological stimuli, the circadian clock in the nasal mucosa may be beneficial defense system forecasting the cyclic changes in environments.
Our results suggest that intranasal steroid spray shifts the nasal clock depending on the time of administration, thus treatment at wrong time of day may result in desynchronization of nasal clock from the master clock in the SCN and other peripheral clocks. In addition, once the nasal clock is phase-shifted, it would be even more difficult to find the right time for the following treatment. Such repeated internal desynchronization, due to consecutive phase shifting, has been reported to increase the risk of a number of medical problems (7,8). From the viewpoint of circadian physiology, the best time of intranasal steroids treatment would be when no phase-shift in the nasal clock occurs (Fig. 2).
The importance of the study: Our data suggest that the nasal mucosa has a stable circadian clock, and endogenous glucocorticoids are potent synchronizers of the nasal clock. We recommend applying steroid spray without inducing a phase shift to wrong direction. And the subjective morning is the recommended time in mice for intranasal steroid treatment without resetting the nasal clock, which corresponds to the early evening in diurnal humans.
Fig.2: Scheme showing the circadian system and the time-dependent glucocorticoids effects on the nasal clock.
The master clock in the SCN is entrained to a day–night cycle by photic signals and coordinates the peripheral clocks. In humans, intranasal steroids treatment in the evening wouldn’t shift the nasal clock. On the other hand, treatment in the morning may reset the nasal clock to cause desynchrony from the master and other peripheral clocks.
Acknowledgements: This study was supported in part by Creation of Innovation Centers for Advanced Interdisciplinary Research Areas Program, Ministry of Education, Culture, Sports, Science and Technology, Japan and JSPS KAKENHI (No. 24390055).
- Reppert SM, Weaver DR. 2002 Coordination of circadian timing in mammals. Nature 418: 935–941.
- Reinberg A, Gervais P, Levi F, Smolensky M, Del Cerro L, Ugolini C. 1998 Circadian and circannual rhythms of allergic rhinitis: an epidemiologic study involving chronobiologic methods. J Allergy Clin Immunol 81: 51-62.
- Balsalobre A, Brown SA, Marcacci L, Tronche F, Kellendonk C, Reichardt HM, Schütz G, Schibler U. 2000 Resetting of circadian time in peripheral tissues by glucocorticoid signaling. Science 289: 2344-2347.
- Yoo SH, Yamazaki S, Lowrey PL, Shimomura K, Ko CH, Buhr ED, Siepka SM, Hong HK, Oh WJ, Yoo OJ, Menaker M, Takahashi JS. 2004 PERIOD2::LUCIFERASE real-time reporting of circadian dynamics reveals persistent circadian oscillations in mouse peripheral tissues. Proc Natl Acad Sci USA 101: 5339-46.
- Silver AC, Arjona A, Walker WE, Fikrig E. 2012 The circadian clock controls toll-like receptor 9-mediated innate and adaptive immunity. Immunity 36: 251-61.
- Nakao A, Nakamura Y, Shibata S. 2015 The circadian clock functions as a potent regulator of allergic reaction. Allergy 70: 467-73.
- Costa G. 2003 Shift work and occupational medicine: an overview. Occup Med 53: 83-8.
- Davidson AJ, Sellix MT, Daniel J, Yamazaki S, Menaker M, Block GD. 2006 Chronic jet-lag increases mortality in aged mice. Curr Biol 16: R914-16.