Dysregulation of inflammatory responses by chronic circadian disruption

Abstract

Circadian rhythms modulate nearly every mammalian physiological process. Chronic disruption of circadian timing in shift work or during chronic jet lag in animal models leads to a higher risk of several pathologies. Many of these conditions in both shift workers and experimental models share the common risk factor of inflammation. In this study, we show that experimentally induced circadian disruption altered innate immune responses. Endotoxemic shock induced by LPS was magnified, leading to hypothermia and death after four consecutive weekly 6-h phase advances of the light/dark schedule, with 89% mortality compared with 21% in unshifted control mice. This may be due to a heightened release of proinflammatory cytokines in response to LPS treatment in shifted animals. Isolated peritoneal macrophages harvested from shifted mice exhibited a similarly heightened response to LPS in vitro, indicating that these cells are a target for jet lag. Sleep deprivation and stress are known to alter immune function and are potential mediators of the effects we describe. However, polysomnographic recording in mice exposed to the shifting schedule revealed no sleep loss, and stress measures were not altered in shifted mice. In contrast, we observed altered or abolished rhythms in the expression of clock genes in the central clock, liver, thymus, and peritoneal macrophages in mice after chronic jet lag. We conclude that circadian disruption, but not sleep loss or stress, are associated with jet lag-related dysregulation of the innate immune system. Such immune changes might be a common mechanism for the myriad negative health effects of shift work.

Figure 1. Resynchronization after 1 or 4 weekly 6h phase-advances

A. Mean group core Tb from mice resynchronizing to a 4^th 6h advance shift of the light-cycle, and control unshifted mice. The current light-dark cycle for both groups is shown at the bottom of Day 1. Note the initial 6h phase difference on Day 1, and the gradual leftward adjustment made by the shifters, until the two records overlay each other by the last day, indicating synchronization. B. A map of 7 phase markers in shifted and control mice. Shifted mice were analyzed for Day 6 after the first or the 4^th advance shift. The LPS challenge occurred on Day 7. All measures except for locomotor activity (LMA) onset are the time of the rhythm peak.

Figure 2. The effects of LPS challenge after chronic jet lag

A. Mean core body temperature (±SEM) after LPS challenge in control (black) and 4-week CJL-exposed (red) mice. Chamber temperature is shown in blue to indicate the increase in environmental temperature during the light phase of each day. The nighttime is indicated by shading. B. Average temperature in 6h time bins was calculated for each mouse for the 2 days after LPS injection, and then group means (±SEM) were compared. * p<0.05. C. Survival curve for 4-week shifters and controls after LPS challenge. D. 6h temperature comparison after LPS challenge for mice exposed to 1 phase shift, compared against control mice. Same conventions as B. E. Survival curve for LPS challenge after 1 shift. Same conventions as C.

Figure 3. Disregulated response of the innate immune system during LPS challenge due to chronic jet lag

Mice exposed to 4 weekly 6h advances of the light-cycle and unshifted control mice were injected with LPS, then bled at 90min or 24h. Cytokines were quantified in serum using a Milliplex MAP assay kit. IL-1β: Interleukin 1-beta; GM-CSF: Granulocyte-macrophage colony-stimulating factor; IL-12: Interleukin 12; IL-12: Interleukin 13; IL-10: Interleukin 10; TNF-α: Tumor necrosis factor alpha.

Figure 4. Ontogeny and cellular targets of immune changes following CJL

A. To determine whether 4 shifts are actually necessary to produce changes in the immune response, mice were shifted once, then allowed to recover for 1 week, 2 weeks or 4 weeks before being injected with 5mg/kg LPS. Control unshifted mice and 4 week-shifted mice are shown for comparison. A single shift produced no significant change in the serum responses of IL-6, IL-18, Macrophage inflammatory protein-2 (MIP-2) or leukemia inhibitory factor (LIF) following this smaller dose of LPS, after any recovery period. The 4-week shifted group exhibited increased levels of all of these factors after challenge relative to controls. B. Peritoneal macrophage quantity did not differ statistically (p > 0.05) among 4-week shifters versus controls (inset), but stimulation of these cultures with LPS induced higher IL-6 release in vitro from cells harvested from shifted mice. HPF: high-powered field (for cell counting).

Figure 5. Chronic jet lag does not cause sleep deprivation

A. Vigilance states were quantified for each day shown: baseline recording, the first and 6^th day of Shift 1, and the first and 6^th day of Shift 4. * p<0.05; Tukey post-hoc comparison. B. Fragmentation measures for each day. Same conventions as A. C. Mean (±SEM) waveform per group for total sleep for the baseline recording, and the first and 6^th day of the 4^th shift. Nighttime is indicated by shading

Figure 6. Altered molecular rhythms in central and peripheral tissues following CJL

Mean (±SEM) circadian amplitude (A) and period (B) of mPer2:LUC rhythms for 4 tissues in vitro, harvested from 4-week shifted versus control mice. * p<0.05; ** p<0.01, Students t-test. C. Relative mRNA abundance of Period2 and Bmal1 in peritoneal macrophages harvested from shifted and control mice at 8 daily time points. ANOVA: Per2 Control p<0.01, Shifted: p<0.02; Bmal1 Control p=0.06, Shifted p=0.64; n=3–4 per time point for all groups.