In this study we used the E. coli endotoxin lipopolysaccharide (LPS), at the relatively high doses of 12.5mg/Kg or 5mg/Kg to induce endotoxemic shock in mice. This is a severe challenge to the immune system that in many ways mimics sepsis (52
), the tenth leading cause of death in the United States (53
). We observed that chronic circadian disruption administered over 4 weeks with a weekly 6h phase advance resulted in a magnified response to the endotoxin. This change was not due to the shifters being at a different circadian phase than the controls at the time of challenge (48
), and indeed required multiple shifts, as 2 or 4 weeks of additional adjustment time to the advanced light cycle following a single shift did not produce the same result as 4 consecutive shifts.
Hypothermia in response to LPS was the prevalent response from both control and shifted mice. Cytokine signaling has been established as a mediator in the thermoregulatory response during experimental sepsis and endotoxemic shock (54
). Mild fever or hypothermia during infection can both serve as effective strategies for host defense (56
), but both can also be dangerous if uncontrolled. Control animals were significantly better at emerging from hypothermia to survive the challenge in our study. In this regard, our initial dose of LPS (12.5 mg/Kg) tested the ability of the immune system to regulate and control the inflammatory response during endotoxemic shock. Our data indicate that TNF-α levels are similar among shifters and controls at both 90m and 24h after challenge. Since this protein is activated early in the toll-like receptor 4 – mediated signaling cascade after LPS stimulation, the data suggest that the initial response to LPS is unchanged between groups. It is possible however that measurement of TNF-α at an earlier stage could reveal an altered response, since macrophages from shifted mice do show a heightened IL-6 response to LPS stimulation in vitro
at even earlier time points. At 24h after challenge we did observe higher circulating levels of other cytokines such as Interleukin-1β, GM-CSF, Interleukin-12 and Interleukin-13, which are all involved in activation of the system downstream of LPS binding to its receptor. In contrast, Interleukin-10, a potent anti-inflammatory mediator, was marginally reduced in shifters. Among other actions, IL-10 is known to inhibit IFN-γ, TNF-α, IL-12, IL-1 and IL-6 (57
), promoting control of inflammation and eventual resolution of the challenge. In fact, presence of IL-10 in the circulation has a protective role against LPS-induced endotoxemia in mice (58
). These results indicate that the exacerbated response to LPS following CJL is at least partially due to a disregulated cytokine response involving too much activation, and insufficient deactivation. Thus our results suggest the need to investigate how altered circadian rhythmicity may impinge on the mechanisms for resolution of endotoxemic shock.
Our results show that even a single experience of jet lag worsened the response to high-dose LPS challenge. This raises the possibility that outbreaks of illness during and immediately following trans-meridian travel (59
) may be at least partially due to the effects of jet lag on the immune system. Immune system changes may also underlie the increased risks of disease in shift-working populations. Certainly, altered innate immune function could upset the balance between host and intestinal microbiota (60
) and could exacerbate age-related pathologies such as heart disease, ulcers and cancers, all of which are more prevalent among shift workers (61
). Importantly, our data with a still high, but milder LPS dose (5 mg/Kg), indicate that multiple shifts are required for a maximal response, further indicating that our results may be related to the health consequences of long-term shift work.
Our results provide a potential mechanism for earlier observations that showed jet lag-related increases in non-specific death (18
) and colitis in response to an intestinal irritant (17
). Importantly, the observation that poor circadian lighting environments may exacerbate pathological immune responses suggests that intensive care units should at least minimize disruption of daily lighting conditions in order to reduce the risks associated with post-surgical or injury-related infections and sepsis.
One potential mediator of the relationship between the immune and circadian systems is the pineal hormone melatonin (63
). Melatonin has well-known effects on the immune system, and may contribute to the control of cytokine action before and during the innate immune response (68
). However, since melatonin is not produced by the C57BL/6 mouse due to a mutation in serotonin N-acetyltransferase (70
), our mouse model of CJL allows us to focus on the important mechanisms by which circadian disruption may directly alter the regulation of inflammation in the absence of melatonin.
We observed that isolated peritoneal macrophages harvested from shifted mice exhibited an enhanced response to LPS in vitro, identifying these immune cells as a specific target of CJL. A clock in these cells has been recently described which regulates gene expression (38
), phagocytosis (38
) and LPS sensitivity (43
). CJL may fundamentally alter this circadian regulation of macrophage function.
SLEEP AND STRESS
Sleep disruption can have profound effects on the immune system. Alterations of the sleep-wake cycle affect the number of circulating lymphocytes, natural killer cells, antibody titers, and levels of cytokines (19
), which translate into impaired immune function when an immune challenge is presented (27
). Shift work may disrupt sleep and thereby lead to secondary effects on health; however, in many cases it is difficult to distinguish between effects attributed to sleep loss versus effects on circadian regulation.
In this study, we could not detect evidence of sleep deprivation as a result of our jet lag paradigm. Instead of sleep loss, the data indicate that mice may gradually adjust the phase of their sleep-wake rhythm in a manner very similar to that observed in records of core body temperature (as in ). Therefore, we do not attribute the exacerbated response to LPS evident in shifted mice to a loss of sleep. If anything we instead found an increase in REM sleep during adjustment to the 4th
shift, which may account for the increase in brief arousals we observed on the same day (wake nearly always follows REM). This REM increase could be attributable to mild stress (72
) associated with the CJL schedule, although our previous (18
) and current work (Figure S2
) strongly suggest that neither hormonal nor behavioral measures of stress are increased during or after chronic jet lag. Furthermore a recent study indicates that the peak value, rhythm amplitude and waveform of corticosterone excretion are not altered during the 2 weeks following a single phase advance (73
). Altogether, these data strongly suggest that immune changes during and after CJL are due to a mechanism independent of sleep loss or stress.
RHYTHM ALTERATION BY CJL
To determine the extent to which circadian rhythms in the brain and peripheral tissues, including the immune system, were affected by chronic jet lag, we compared the rhythms of mPer2-luciferase in cultured explants taken from naïve and CJL-exposed mice. We observed tissue-specific effects of CJL on circadian parameters suggestive of dysfunction in systemic circadian organization. Phase was fully adjusted for SCN, liver, spleen and thymus on Day 6 after the 4th
shift, and was therefore similar among shifted and control mice. However, amplitude of the mPer2:LUC rhythm was increased in the SCN, but suppressed in the thymus (and perhaps liver) by 4 weeks of CJL. Peripheral rhythm suppression during CJL observed here and in other studies (15
) may indicate impaired communication with the SCN, an altered pattern of food intake, or an impairment in the local generation of coherent rhythmicity (perhaps via a loss of synchrony among hepatocytes and thymocytes). It is tempting to speculate that changes in rhythmicity due to CJL might impinge upon proper organ function during endotoxemic challenge, as altered liver function resulted from a liver-specific Bmal1 deletion (74
). Since the liver contains the largest pool of macrophages in the body, it is responsible for clearance of endotoxin and is a major source of inflammatory mediators during the early stages of inflammation (75
). Thus, the liver may be an important target for jet lag-related morbidity. Our data further indicate that peritoneal macrophages also exhibit significant changes in at least one important circadian clock gene: Bmal1. Relative abundance of Bmal1 is rhythmic in control mice (see also (38
)), but constitutively low in shifted mice. While mPer2 appears to still be rhythmic in shifted mice, the data indicate that the phase of the peak may be delayed by the CJL history. Selective loss of clock gene rhythms is not an unprecedented observation. Tissue-specific ablation of the endogenous clock in the liver by rev-erbα overexpression resulted in the loss of Bmal1 rhythmicity, but the retention of Per 2 rhythmicity, potentially due to the influence of afferent signals to the liver arising from rhythmic environmental signals or clocks in other loci (76
). The specific loss of this gene leads to compromised immune function among other pathologies (41
). Our environmental manipulation has resulted in at least a partial loss of circadian regulation in this cell population, and this may underlie the dramatic change in the cellular response to LPS that we have observed.
The circadian period of SCN explants was lengthened by CJL exposure, but liver period was dramatically shortened. Aftereffects of short and long T-cycles (experimental light-dark cycles different from a period of 24h) on behavioral and physiological rhythms are well-established (77
), and our CJL paradigm may share features with a short (22.83h) T-cycle, since mice are required to phase advance by 6h every 7 days in order to achieve entrainment before the next shift. Our data are consistent with intriguing but still unexplained data indicating that the isolated SCN expresses a T-cycle aftereffect that is opposite to that seen in behavior (79
The opposite direction of the period and amplitude effects of CJL on central versus peripheral targets may indicate a fundamental change in whole-animal circadian organization, the consequences of which may be an increased risk of pathology (41
). The shorter period for liver would be expected to change the response of the liver clock to SCN or environmental (e.g. food) signals, thereby altering its ability to entrain to those signals.
We and others (83
) have observed a suppression of rhythmicity during sepsis. Our observations of altered rhythmic organization prior to the challenge in CJL-exposed mice may make the system more reactive, thereby exacerbating the pathological response. Importantly, it has been suggested that circadian organization during
sepsis is functionally important to recovery (85
). If so, one might predict that restoration of rhythmicity during the challenge will enhance recovery from systemic inflammatory states.
We propose that circadian desynchrony alters components of the innate immune system to affect their response to a challenge. However, we cannot rule out that the effect we observe could result from the chronic alteration of the lighting environment acting directly upon the immune system without the involvement of the circadian clock. Future studies will be needed to further clarify the role of the circadian system in the exacerbated response to LPS that we have described. For example, models in which clock function is altered genetically could be challenged with LPS with and without light manipulation. It seems likely that the clock system is involved, as earlier studies suggest that schedule parameters such as shift direction impinge upon the effects of CJL on health (18
), and that generalized stress is likely not involved
Our data identify one important physiological target for chronic jet lag: the innate immune system and inflammatory processes. We specifically have identified peritoneal macrophages as a cellular target of this manipulation. However, these data do not rule out other systems that may also be negatively impacted by circadian disruption. Furthermore, our model of CJL in mice simulates some but not all aspects of human shift work. Dramatic changes in the inflammatory response in our model were evident without the contribution of sleep loss, melatonin suppression, and psychosocial stress in our model, all of which are known to alter immunity in humans. However, we suggest that our results point toward a common mechanism in which lack of adequate circadian regulation results in long-lasting and potentially fatal consequences during an immune challenge. Loss or alteration of clock components has been implicated recently in dozens of diseases in both animal models and human populations, but to our knowledge, this is the first report in which a purely environmental manipulation causes disregulation of inflammatory responses potentially mediated by loss or alteration of clock function. Further investigation into the health effects of circadian disruption is clearly warranted.