The temporal expression pattern of classical MHC class I in sleep-restricted mice: Generalizations and broader implications

Abstract

The intricate relationship between sleep and leukocyte trafficking has garnered intense attention, particularly their homing dynamics to secondary lymphoid organs under normal and restricted sleep (SR). Considering the scarcity of information regarding circadian rhythms in major histocompatibility class I (MHC-I) expression in SR, we designed a study that assessed the temporal expression of MHC-I in murine lymph nodes and spleen and the subsequent effects of sleep recovery. Male C57BL/6, housed in 12:12 light/dark cycle, were grouped into control (C) and SR. SR was carried for one week before lymphoid tissues were sampled at selected time points and assessed for leukocyte number and MHC-I expression. SR resulted in 21% decrease in granulocyte and 24% increase in agranulocyte numbers. In C, MHC-I expression pattern in lymph nodes was bimodal and relatively higher than splenocytes during the animal's active phase (110.2 ± 1.8 vs 81.9 ± 3.8, respectively; p = 0.002). Splenocytes; however, showed a bimodal pattern upon SR, with higher protein levels during the rest than the activity period (154.6 + 36.2 vs 99.5 + 15.9, respectively; p = 0.002), suggesting preparedness for a potential infection. Furthermore, SR caused a significant drop in MHC-I expression at the onset of rest with 57% and 30% reduction in lymph nodes and splenocytes, respectively. However, the overall protein expression collectively taken from both lymphoid tissues remained stable, emphasizing its indispensable role in immunological homeostasis. This stability coincided with the restoration of protein levels to baseline after a short sleep recovery period, resembling a reset for MHC-I antigen presentation following a week of SR. Understanding the interplay between MHC-I expression and contextual factors could enhance treatment protocols, refining the efficacy and time precision of glucocorticoid-based therapies in immune modulation.

Keywords: C57BL/6 mice; Circadian rhythms; Leukocytes; Major histocompatibility complex class I; Sleep restriction.

Fig. 1

Schematic representation of the time points for lymphoid tissue collection in mice maintained on a 12:12 light/dark cycle or under sleep restriction (SR). Dots represent different time points of tissue collection; black bars, dark period (ZT12); open bars, light period (ZT0); arrows point at the beginning and end of the SR time period.

Fig. 2

Identifying different subsets of leukocytes and their viability from isolated mouse splenocytes. A) Three pools of leukocytes, represented by 3 distinct peaks, were distinguished based on cell size or FSC signal at ZT5. In terms of increasing size, lymphocytes have the smallest FSC signal, followed by monocytes and granulocytes. B) Cell viability was monitored by tracking PI signal by flow cytometry. Almost all cells showed negative PI fluorescence as depicted by low FL2 signal. This demonstrates a minute death of cells upon isolation and homogenization.

Fig. 3

A representative scatter plot showing granulocyte number in isolated lymphoid tissue of control and sleep restricted mice. The total cell count, as well as the relative proportions of agranulocytes, were determined based on SSC and FSC signals at ZT5. Compared to control, SR animals had lower granulocyte number (16.7 % ± 1.99 vs 44.2 % ± 1.9, respectively; p=.004); higher lymphocyte and monocyte counts (79.7% ± 3.19 vs 53.6% ± 1.13, respectively; p= .009). FSC, forward scatter; SR, sleep restriction; SSC, side scatter.

Fig. 4

A representative MHC expression profile in lymph node isolates collected at ZT5 from C57BL/6 mice. The relative MFI counts of MHC I immunoreactive cells were plotted in C (solid line) and SR (dashed line) animals. The number of immunolabelled cells in SR groups showed a slight increase in MHC class I expression. C, control; SR, sleep restriction.

Fig. 5

Graphical representation of the diurnal MFI MHC-I signals in the lymph node (A) and spleen (B) of control and sleep restricted mice. A) Unlike splenocytes, lymph node cells displayed a bimodal pattern of protein expression, with relatively high levels at ZT0, ZT10, and ZT18, under C conditions. This pattern was altered following SR, where MHC-I levels increased at ZT5 and reached maximal levels at ZT13. B) In splenocytes, MHC-I levels were relatively low throughout most of the selected time points (except at ZT0 and ZT18), whereas SR caused a different pattern of protein expression during the day, with 2 peaks at ZT5 and ZT13. MFI, mean fluorescence intensity; C, control group; SR, sleep restricted group; bottom open bar, light period (lights on at 0700 h or ZT0); bottom black bar, dark period; top open bar, rest period of SR (onset at ZT3); top gray bar, activity period of SR (18 h duration). * p-value <0.05 indicates significant difference between C and SR groups. Values represent mean ± standard deviation.

Fig. 6

Graphical comparison of MHC-I expression in the lymph nodes and spleen of control and experimental group before and following recovery sleep. There was no significant difference in protein expression among all groups at the selected time points in both lymphoid tissues. Values are expressed as mean ± standard deviation.