Chronic Exposure to Dim Light at Night Disrupts Cell-Mediated Immune Response and Decreases Longevity in Aged Female mice

Abstract

Circadian rhythms are endogenous biological cycles that regulate physiology and behavior for optimal adaptive function and survival; they are synchronized to precisely 24 hours by daily light exposure. Disruption of the daily light-dark (LD) cycle by exposure to artificial light at night (ALAN) dysregulates core clock genes and biological function. Exposure to ALAN has been associated with increased health risks in humans, and elderly individuals are at elevated risk for poor outcome from disease and often experience elevated exposure to ALAN due to increased care requirements. The role of disrupted circadian rhythms in healthy, aged animals remains unspecified; thus, we hypothesized that disrupted circadian rhythms via chronic exposure to dim ALAN (dLAN) impairs immune response and survival in aged mice. 20-month-old C57BL/6 male and female mice were exposed to 24 weeks of LD conditions or dLAN (5 lux), then, cell-mediated immune response was assessed using a delayed-type hypersensitivity test. Aged female mice exposed to dLAN displayed dysregulated hypersensitivity and inflammation as a measure of cell-mediated immune response and decreased lifespan compared to females housed in dark nights. Nighttime lighting did not affect cell-mediated immune response or lifespan in males but dysregulated body mass and increased adrenal mass after immune challenge after chronic exposure to dLAN. Together, these data indicate that chronic exposure to dLAN affects lifespan in aged females and suggest that females are more susceptible to the detrimental consequences of disrupted circadian rhythms.

Introduction

Organisms have evolved endogenous rhythms that reflect recurring natural solar days comprising bright days and dark nights. Circadian rhythms have a period of ~24 hours and are synchronized to precisely 24 hours by the light cues from the external environment to optimize physiological and behavioral function, as well as survival. Circadian rhythms are generated at the cellular level by a transcriptional-translational feedback loop composed of core clock genes including Clock, Bmal1, Per, and Cry. External factors, or zeitgebers (time givers), act as cues to entrain the circadian system to match the solar day; the most common zeitgeber among vertebrates is light. In mammals, light input is transmitted from intrinsically photosensitive ganglion cells (ipRGCs) in the retina to the central biological clock, relaying environmental photic information to the suprachiasmatic nucleus (SCN) of the hypothalamus, which is responsible for synchronizing the central and peripheral biological clocks to the exogenous environment (Honma, 2018).

With the rapid adoption of artificial lighting, individual organisms and ecosystems are exposed to increasing levels of lighting that can divorce internal temporal rhythms from natural solar days (Cinzano et al., 2001). Exposure to aberrant lighting during the night disrupts circadian rhythms of core clock gene expression resulting in altered behavior and physiology. Increasing evidence suggests that low or dim levels of illuminance levels of light at night (LAN, e.g., 5 lux) that mimic the amount of light pollution in urban environments can affect several physiological parameters (Navara & Nelson, 2007; Nelson & DeVries, 2017); night-time light exposure has been associated with health consequences including disruptions to metabolism (Fonken et al., 2013) and immune function (Bedrosian et al., 2011; Cissé et al., 2017), as well as an increased risk for cancer (Walker et al., 2020). The presence, intensity, and extent of exposure to artificial light at night has significantly increased over the past century (Cinzano et al., 2001); nighttime artificial lighting increased by 2.2% per year between 2012 to 2016 alone (Kyba et al., 2017). Furthermore, light at night affects organisms at the individual, population, and ecosystem level, indicating its growing impact and importance (Navara & Nelson, 2007; Nelson & DeVries, 2017).

Aging, characterized through the functional decline in physiological functions, is an unmodifiable risk factor for numerous chronic diseases; several aspects of health change as individuals age, including metabolism (van Beek et al., 2016), cognition (Duncan, 2020), and response to immune challenges (Montecino-Rodriguez et al., 2013). Endogenous and exogenous environmental factors have been suggested to drive progression of the aging process, and cellular senescence, a cell-intrinsic stress response, plays a central role in aging (López-Otín et al., 2013). Within aged populations, the molecular clock and circadian system also undergo several changes including blunted amplitude and loss of robustness, leading to susceptibility to disrupted circadian rhythms that in turn can contribute to the development of neurodegenerative diseases (Abbott & Videnovic, 2016; Kondratova & Kondratov, 2012). Earlier studies examining disrupted circadian rhythms using a chronic jet lag model (chronic phase advances) reported increased mortality in mice (Davidson et al., 2006). Chronic exposure to dLAN also decreases survival in female Drosophila (McLay et al., 2017), suggesting a sex difference in survival in response to perturbed circadian rhythms.

Disrupted circadian rhythms also alter aspects of the immune system including the regulation of proinflammatory cytokines, which in turn, can provoke uncontrolled complement activation resulting in immune dysfunction and disease, as well as shortened life expectancy (Brodsky, 2015; Inokawa et al., 2020; Jasim et al., 2019; Oster et al., 2017). For example, acute exposure to ecologically relevant levels of dim light at night (dLAN; 5 lux) suppresses immune function in hamsters (Bedrosian et al., 2011). Additionally, exposure to dLAN impairs circulating monocytes and T-cells, and altered Cd68 and Ccl2 expression in peripheral tissue in rats (Okuliarova et al., 2021). The immune system has also been implicated in cellular senescence, where impaired immune surveillance accelerates aging, accumulation of senescent cells, and chronic inflammation (Ovadya et al., 2018), however, the relationship between disruptions to circadian rhythms and its effects on immune function and aging remains unspecified. Thus, we hypothesized that disrupted circadian rhythms by exposure to chronic exposure to dim light at night impairs immune response and survival in aged mice.

Methods

Sixteen-month-old C57BL/6 never mated male and female mice were obtained from the aged mouse colony maintained by the US National Institute of Aging. Upon arrival, mice were group-housed with same-sex cohorts in the vivarium and allowed to acclimate to light-dark (LD) conditions (14h light 150 lux, lights on at 0400 h; 10 h dark 0 lux, lights off at 1800 h) to maintain a long-day phenotype (Tavolaro et al., 2015) for ~4 months prior to experimental manipulation. At 20 months (±2 months), mice were randomly assigned to treatment groups and either transferred to chronic dLAN (14h light 150 lux; 10 h dark 5 lux) at night or remained in LD conditions for 24 weeks (LD males n=14, dLAN males n=15, LD and dLAN females n=15). Mice were single housed in polypropylene cages (30×18×14 cm) to avoid the formation of social dominance hierarchies that could induce differences in stress responsivity and activity among cage mates (Horii et al., 2017; Robbers et al., 2021), and to avoid the possibility of reconstituting a group of mice mid-study due to death or aggression. Mice were maintained on a static rack with bi-weekly cage changes by husbandry staff and provided ad libitum access to standard rodent chow (Envigo Teklad 2018) and reverse osmosis filtered water.

Light at night was produced by LUMA5 LED light strips (Hitlights Inc; 1.5W/ft, 5000K “cool white”, 1200 lumens) placed equidistant in front of each cage. Light measurements were determined using a light meter (Mavolux 5032C illuminance meter (Nürnberg, Germany) from the center of an empty cage with the light sensor facing the ceiling. Due to a technical issue, seven female mice in the dim light at night treatment group were briefly exposed to ~15 lux during the dark period for 6 days during week 7, however, their data were not significantly different from other females and were included in the analyses (Supplemental Figure 1). The female cohort of mice were two months older than the males at initiation of the study due to timing constraints for behavioral testing. Body mass was measured prior to lighting treatment, then measured at 10, 20, and 24 weeks of exposure to the respective lighting conditions. Mice were monitored and survival was recorded daily. One LD male, and one female in each of the LD and dLAN conditions were removed from survival analyses due to self-injury. One male in dLAN was removed from DTH analysis due to not having sufficient pinna.

Delayed-Type Hypersensitivity Test (DTH)

To assess cell-mediated immune response, mice were sensitized to a chemical antigenic challenge as previously described (Bilbo and Nelson, 2003). Briefly, under light anesthesia 25 μl of 2–4 dinitro-1-fluorobenzene (DNFB; Sigma, St. Louis, MO; 0.5% volume in a 4:1 acetone/olive oil vehicle) was applied to a 2 × 3 cm shaved region of the dorsum for 2 consecutive days starting at week 22. Prior to manipulation, mice acclimated to the experimental room for 30 min and the thickness of the right and left pinna was measured using a constant-loaded thickness gauge (Mitutoyo #7309, Kawasaki, Kamagawa, Japan) at the same time point each day (zeitgeber time; ZT 8–10, 1200 h to 1400 h). Mice were lightly anesthetized with isoflurane vapors during DNFB challenge and pinnae thickness measurements. Seven days post sensitization, mice were re-challenged with 20 μl DNFB (0.2% volume DNFB in vehicle) to the external right pinna, the left pinna received the vehicle solution. Pinnae thickness was measured for the following 6 days. All experiments were approved by West Virginia University Institutional Animal Care and Use Committee and animals were maintained in accordance with NIH Animal Welfare guidelines.

Statistical Analyses

All data were analyzed independently for each sex. Survival was assessed through the Kaplan-Meier method. Survival curves comparing lighting conditions were analyzed using the Mantel-Cox test. Body mass was assessed as a percent change from baseline using a repeated measures two-way ANOVA analysis with lighting condition and time as independent variables. Mice that died during the experimental timeline and study were removed from body mass analysis. A repeated measures two-way ANOVA was used to analyze the delayed-type sensitivity test with lighting condition and time post sensitization as variables. Adrenal and spleen masses were reported as a percentage of body mass and were analyzed comparing lighting conditions using an unpaired 2-tailed t test. Post-hoc analyses were performed using Fisher’s LSD test. An outlier test was performed prior to analysis and a maximum of one outlier per group was removed a priori; an outlier was defined as having a within-group Z score >2. Data were tested for normality using Shapiro-Wilks test. Statistical analyses were performed using GraphPad Prism 9.0 software, and data are presented as the mean ± standard error of the mean (SEM). Mean differences were considered statistically significant if p≤0.05. A table summarizing statistical tests results and significance has been included in Supplementary Table 2.

Results

Dim Light at Night Reduces Lifespan in Female Aged Mice

After 24 weeks of chronic exposure to light at night, female mice exposed to dim light at night displayed reduced lifespan compared to females housed in dark nights assessed through the Kaplan-Meier Mantel-Cox test (p<0.05) (Fig 1A). There were no significant differences in survival between males in either lighting condition ( p>0.05) (Fig 1A).

Figure 1.

Survival and body mass after 24 weeks of exposure to dLAN. (a) dLAN decreased lifespan in females compared to LD counterparts (p=0.05), (LD female and dLAN female n=14). No observed effect of lighting condition on survival in males (p>0.05), (LD male n=13, dLAN male n=15). (b) There was a significant interaction of lighting condition on body mass in males (p<0.05). Males exposed to dLAN initially lost a smaller percentage of their body mass at week 10, then lost a significantly larger percentage of their body mass at 24 weeks compared to LD males. There were no differences in body mass when comparing lighting conditions in females (p>0.05). Mean ± S.E.M.; *p≤0.05 (&; denotes an interaction effect).

Dim Light at Night Altered Weight Loss in dLAN Males

Our analysis revealed a main effect of time (F_2,48=22.92; p<0.05) on body mass in males and an interaction effect between lighting and time on male body mass reported as a percentage compared to baseline (F_2,48 =4.629; p<0.05) (Fig 1B). When comparing male body mass at 10, 20, and 24 weeks, dLAN males lost proportionally less body mass compared to males exposed to dark nights at 10 weeks, while males housed under dLAN lost significantly more body mass at 24 weeks compared to 10 weeks, however, post-hoc comparisons were not statistically different when comparing lighting conditions across each time point (Male LD n=13, dLAN n=13). There was no effect of lighting condition on body mass in females (p>0.05), however, mice that died during the experimental timeline across both sexes were excluded from body mass analysis due to statistical requirements.

Dim Light at Night Dysregulates Cell-Mediated Immune Response in Female Mice

Re-exposure to DNFB induced swelling in the challenged pinna compared to baseline for both treatment groups (p<0.05; data not shown). There was an interaction effect of lighting condition and time in females (Fig. 2A; F_5,65 = 2.39, p<0.05); dLAN females initially had comparable swelling to LD female mice but displayed decreased right pinna swelling beginning after day 3 post sensitization; post-hoc comparisons were not statistically different when comparing lighting conditions across each day (p>0.05). No significant difference between lighting conditions was observed among males (F_1,24 = 1.16, p>0.05.

Figure 2.

(a) dLAN significantly impaired female inflammatory response during the later days of the delayed-type hypersensitivity response, depicted by decreased cell-mediated pinna swelling reported as a percent change from baseline (p<0.05). There was no significant difference between LD and dLAN males (p>0.05). (b) After correction for differences in body mass, males exposed to chronic dLAN displayed increased adrenal mass as a percentage of body mass compared to LD males (p<0.05) and no difference in females in both lighting treatments (p>0.05) (LD females n=10, dLAN females n=6, LD males n=11, dLAN males n=13). Mean ± S.E.M.; *p≤0.05. (# denotes a main effect of sex, @ denotes a main effect of lighting condition, and & denotes an interaction effect).

Dim Light at Night Increases Adrenal Mass in Aged Male Mice

Males housed in dLAN and after immune challenge had significantly greater adrenal masses reported as a percentage of body weight compared to dark night conditions (Fig. 2B; p<0.05, unpaired, 2-tailed t test), whereas there were no differences in adrenal masses among females (Fig. 2B; p>0.05, unpaired, 2-tailed t test). There were no effects of lighting condition on spleen masses among males (Fig. 2C; p>0.05, unpaired, 2-tailed t test) or females (Fig. 2C; p>0.05, unpaired, 2-tailed t test).

Discussion

Here, we report the presence of a sex difference in the effects of chronic exposure to dim light at night on lifespan in aged mice. These findings are consistent with previous reports in other species, such as Drosophila, where females housed in nighttime lighting had reduced survival, and is the first report, to our knowledge, that chronic exposure to dim light at night late in lifespan affects survival in otherwise healthy, aged mice. Our results suggest that dLAN accelerates aging in a sex specific manner, denoted through a shortened lifespan compared to LD females. Additionally, females exposed to dLAN displayed dysregulated T-cell mediated hypersensitivity and inflammation through DTH reaction, as a measure of cell-mediated immune response. These results are consistent with previous reports that 4 weeks of dim light at night is sufficient to suppress immune responses in young Siberian hamsters (Bedrosian et al., 2011). Other studies evaluating models of disruption of circadian rhythms or genetic clock gene manipulations have also identified that even in the absence of immune challenge, disruption of circadian rhythms can produce pro-inflammatory cytokines and increase immune activation (Bedrosian et al., 2013) and does so in as few as 3 nights of exposure to dLAN in otherwise healthy mice (Walker et al., 2020). We observed no differences in cell-mediated immune challenge or lifespan in males; however, male mice housed in chronic dLAN conditions displayed dysregulated body mass and increased adrenal mass compared to mice housed in dark nights, suggesting that dLAN may differentially affect pathways and mechanisms between sexes in aged mice.

Exposure to dLAN has been previously reported to disrupt molecular rhythms and core clock genes during disruption of circadian rhythms (Fonken et al, 2013). Indeed, with the widespread adoption of ALAN, the delineation between light days and dark nights is blurred, in turn resulting in disruption of clock gene function. Low levels of nighttime lighting, (i.e., 5 lux), are consistent with the exposure of populations residing in light polluted urban and suburban settings (Gaston et al. 2012). Furthermore, (5 lux) ALAN is ecologically relevant due to its behavioral and biological effects (Nelson and Navara, 2007; Gaston et al. 2013). Although we did not directly measure clock gene expression, several studies have demonstrated that there are intact circadian rhythms in this strain (Panagiotou & Deboer, 2020) and that constant levels of “daytime lighting” dysregulates clock gene expression (Hong et al., 2020). Furthermore, constant lighting in younger, 2-month-old CBA mice disrupted estrous function, increased spontaneous tumors, and reduced lifespan (Anisimov et al, 2004).

The relationship between circadian rhythms and aging is complex; the bi-directional relationship can affect aspects of biological function including aging. Introductions of mutations in core clock genes bmal1 and period in Drosophila and mice accelerates age-related impairments including tissue decline, cognitive impairments, and shortened lifespan (Kondratov et al, 2006; Krishnan et al, 2009). Previous studies have also suggested the existence of a sex difference in circadian rhythm misalignment. For example, sex differences in hypothalamic-pituitary-adrenal (HPA) function have been observed in which stress-response activation is altered in women compared to men (Kudielka & Kirschbaum, 2005). There are also notable sex differences of circadian modulation of the HPA axis attributed to differences in gonadal steroid hormones; estrogens generally enhance, whereas androgens inhibit, HPA function (Yan et al., 2016). Further, females also display significantly more disturbances in energy homeostasis, primarily via increased energy expenditure and lipid oxidation rates, compared to temporally misaligned males (Qian et al., 2019). These results are consistent with previous studies of other forms of chronic circadian rhythm disruption, including a repeated jet-lag model, in which aged mice appear susceptible to shortened lifespans compared to younger mice (Davidson et al., 2006). Whereas dLAN may seem fairly innocuous compared to constant lighting or a chronic jetlag model, dLAN disrupts molecular biological rhythms in multiple species (Bedrosian et al., 2013; Fonken et al., 2013). It is possible that the effects we report here on cell-mediated immune response and longevity in female mice are caused by circadian misalignment dysregulating physiological function.

In our study, mice were unmated and remained gonadally intact throughout the duration of their lifespan; these factors have previously been demonstrated to affect lifespan and longevity (Russell, 1996). In female mice, reproductive changes and decline are detectable by 13–14 months of age (Nelson et al., 1995), and accelerates such that 80% of female mice are acyclic or exhibit irregular cycles at 17 months of age, and 100% by 25 months (Frick et al., 2000). Thus, because the mice in our study were assigned to experimental lighting groups at approximately 20 months of age, it is likely that the majority experienced reproductive decline prior to the experimental lighting exposure. We did not gonadectomize aged mice as we addressed the experimental question of how exposure to chronic lighting conditions would affect otherwise typically aging mice. Whether gonadectomy would alter the outcome of our study, implicating a role for gonadal steroids across the lifespan, is an interesting question that should be answered in future studies.

Age and survivor effects are another consideration for interpreting this study. We enrolled mice at 20±2 months of age, at which point 75–90% of the population is still typically alive (NIA colony statistics). Whether dLAN has similar effects on survival in younger and older mice will need to be determined in the future studies. For the present study, males and females were obtained from the same National Institute of Aging (NIA) cohort, but the mean ages of females and males at exposure to dLAN were different by 2 months because we needed to adjust the experimental start timelines due to time constraints for behavioral/immune testing. Thus, data from females and males in this study were analyzed separately. However, it is important to emphasize that the greatest effects of dLAN on survival in females occurred in the first 80 days of exposure to dLAN, when the female mice were ~22–24.5 months of age. Male mice were approximately this age between week 8 and 12 of dLAN exposure and yet there was no similar effect of dLAN on survival among males during this period, however, a greater sample size of males may be necessary to identify differences between survival (Yuan et al. 2009) and future studies will need to be conducted.

Aging is associated with changes in body mass composition, and body mass loss late in life in aged populations is correlated with adverse health consequences in rodents and humans (Ray et al., 2010; Nagy and Pappas, 2019). In the current study, a significant interaction between time and lighting condition was observed among males; during the first 10 weeks of exposure, dLAN males initially lost less mass proportionally than LD males, but by 20 weeks of exposure this pattern reversed and dLAN males lost more mass proportionally than LD males. In contrast, there were no significant differences in body mass among females at any of the time points. However, these analyses included only mice that survived to the end of the experiment, and because fewer dLAN females survived than LD females, it is possible that the differential survival skewed the body mass data if the mice that died had the greatest body mass loss.

Females housed in dLAN also exhibited changes to cell-mediated immune response that could contribute to accelerated aging and reduced lifespan that we did not observe in males. We observed increased adrenal mass in males housed in chronic dLAN compared to dark night conditions, which suggests that chronic dLAN may dysregulate stress response in males, but not females. Chronic stress is often accompanied by increased adrenal masses (Ulrich-Lai et al., 2006). Previous studies evaluating other models of disrupted circadian rhythms, including light pulses during the night (Ishida et al., 2005) or jet lag paradigms/constant illumination, report elevated glucorticoid concentrations (Dunnet al., 1972; Sakellaris et al., 1975). No studies to date have reported altered stress responses in aged males evoked by exposure to dim light at night; however, elderly mice may have increased risk and susceptibility to the consequences of disrupted circadian rhythms due to aging. Future studies should further characterize increased adrenal masses and stress responses to delineate whether this has functional significance. Together, these data support the proposition that chronic dLAN differentially affects aspects of physiology in aged populations.

Optimal immune response is dependent on a functioning circadian system (Okuliarova et al., 2021). Thus, we chose to use the DTH test, which is a measure of cell-mediated immunity that can be used as an indirect measure of immune response mediated by memory T-cells, antigen presenting cells, effector T cells, cytokine production, and inflammation at the exposure site. DTH is also used as an assay for increased risk of mortality and poor health in aged human populations (Wayne et al., 1990), and natural aging has been associated with reduced B and T cell production, and reduced lymphocyte function (Montecino-Rodriguez et al., 2013). Previous studies indicate that disrupted circadian rhythms alters circulating immune cells. For example, 5 weeks of dLAN reduces white blood cell populations, including monocytes and T-cells (Okuliarova et al., 2021). Furthermore, chronic disruption of circadian rhythms accelerated aging in young 10-week-old mice, likely via low-grade inflammation (Inokawa et al., 2020). The altered DTH response we observed after exposure to chronic dLAN suggests that altered immune function may also be a contributing factor to accelerated aging in this study.

In summary, our results suggest that chronic exposure to dim light at night dysregulates cell-mediated hypersensitivity reactions as a measure of cell-mediated immune response and accelerates aging, demonstrated through reduced lifespan in female mice. Further, we report that males housed in dLAN display dysregulated body mass after chronic exposure to dim light at night and increased adrenal mass which is often associated with elevated stress exposure and response. This may be a concern given that aged human populations have a particularly high incidence of chronic exposure to artificial light at night (Scheuermaier et al., 2010), in part due to the increased care requirements of elderly populations. Dysregulation of immune response can result in increased hospital visits, further exposing patients to night-time artificial lighting from recovery units (Durrington et al., 2017). Future studies should be aimed towards identifying underlying mechanisms for sex differences in response to circadian misalignment and examine how restoration of naturalistic lighting in environments to normalize circadian rhythms could improve immune function and prolong longevity.

Data Availability Statement:

The data generated and analyzed during this study is available from Mendeley Data. Liu, Jennifer (2022), “Chronobiology International - Data Set”, Mendeley Data, V1, doi: 10.17632/85dyc9v5xk.1