Chronic shift‐lag promotes NK cell ageing and impairs immunosurveillance in mice by decreasing the expression of CD122

Abstract Long‐term subjection to shift work increases the risk of cancer. The purpose of the present study was to explore the mechanism by which chronic circadian disruption impairs natural killer (NK) cell immunosurveillance. Mice were subjected to light‐dark reverse every 4 days for 12 weeks to disrupt normal circadian rhythm. NK cell development and function were evaluated by flow cytometry. The mRNA and protein levels of period 1 (per1) and per2 were suppressed, while circadian locomotor output cycle kaput (CLOCK) was increased in the shifted mice, indicating successful generation of the circadian rhythm disruption mouse model. Chronic shift‐lag promoted NK cell ageing, which is likely due to the reduction in Ly49 family receptor expression in shifted NK. We further studied the effects of circadian rhythm disruption on NK cell function. Chronic shift‐lag inhibited NK cell secretion of granular CD107a and interferon gamma. Moreover, chronic shift‐lag attenuated the clearance of MHC‐I–deficient tumour cells by NK cells in vivo and promoted lung metastasis of B16F10 melanomas. Furthermore, chronic shift‐lag reduced NK cell killing function, which may be due to the suppression of Eomes transcription factor expression, which inhibiting the transcription of CD122. In conclusion, our findings suggest that chronic circadian disruption attenuates NK cell cytolytic activity by decreasing the expression of CD122.

Eomes is highly expressed in NK and memory CD8 + T cells, functioning to promote CD122 expression and maintain IL-15 response. Eomes promotes NK cell development and maturation, and T-bet is necessary for NK cell terminal maturation. 7 Circadian rhythm (CR) is an endogenous autonomous rhythmic clock controlling the physiological activity of an organism to form a 24-hour day-night cycle. The major pacemaker of CR lies in the suprachiasmatic nucleus (SCN), which plays an important role in maintaining systemic CR by releasing endogenous regulatory factors. 8 The molecular mechanism for maintaining circadian rhythm in the SCN and organs is regulated by feedback from the core circadian clock genes, including periods (Per1-3), Cryptochrome (Cry1 and Cry2) and CLOCK. 9 Although the importance of circadian rhythm in the immune system has been recognized, little is known regarding CR in the regulation of NK cell function. NKs are innate immune cells important for combating cell deterioration and tumour metastasis.
Organismal circadian disorders are likely to alter NK cell function.
Some studies have reported that NK cell functions, including the release of cytokines and cytolytic factors, are strictly controlled by circadian rhythm. 10 However, there is little evidence to prove the relationship between the disruption of circadian rhythm and NK cell development and function. Hence, the purpose of the present study was to investigate whether circadian rhythm disruption impairs NK cell function and explore the underlying mechanism. Under the shift-lag schedule, circadian rhythm was examined by monitoring circadian genes. NK cell maintenance and development were evaluated under both resting and stimulating conditions. Finally, it was explored whether NK cell immunosurveillance impairment due to chronic shift-lag induces circadian rhythm disruption; an RMA-S tumour cell clearance assay and B16 melanoma lung metastasis assay were used to examine NK cell anti-tumour function. We assumed that chronic shift-lag would suppress NK cell cytolytic activity towards tumour cells by promoting NK cell ageing and down-regulation of CD122.

| Animals, experimental design and animal ethics statement
The animal research protocols used in the present study were

| Real-time PCR
Total RNA was extracted from splenic NK cells obtained by flow sorting, from which cDNA was generated using a reverse transcription system (TAKARA). Quantitative real-time polymerase chain reaction (RT-qPCR) was performed on a BioRad CFX96 qPCR system using the SYBR ExScript PCR Kit (TAKARA). The primer sequences of the selected genes used in the present study are shown in Table 1. The relative expression levels were calculated using the 2 −∆∆CT method as previously described. 11 The results were normalized to the mRNA expression level of GAPDH.

| Small interfering RNA transfection
NK cells sorted from control or chronic shift-lag mice were seeded onto 12-well plates at a density of 2 × 10 5 /well in DMEM containing 10% foetal calf serum and 1000 U/mL IL-2. The following day, cells were transfected with 25 nmol/L Per1 (s71484, Thermo Fisher) and Per2 (s71485, Thermo Fisher) small interfering RNA (siRNA) or 50 nmol/L negative control siRNA (4390844, Thermo Fisher) using

| Flow cytometry
Flow cytometry analysis was carried out on a BD CANTO (BD Biosciences), and cell sorting was performed on a BD Aria II (BD Biosciences

| Detection of CD107a expression and intracellular staining of IFN-γ
Each mouse was injected intraperitoneally with 200 µg poly I:C to activate NK cells. After 18 hours, poly I:C-activated spleen cells After 14 days, the mice were culled; the lungs were weighed, and the number of tumour nodules was counted. To clarify the role of NK cells, antibodies were used to eliminate NK cells in mice. Following B16 melanoma injection, each mouse was intraperitoneally injected with 1 mg PK136 antibody three times a week.

| Blockade of CD122 with an anti-CD122 monoclonal antibody
The control mice were kept under a normal LD cycle, and the chronic shift-lag mice were subjected to a reverse LD cycle every 4 days for 12 weeks. During the final 2 weeks, the mice received 200 μg anti-CD122 monoclonal antibody (TMβ1, Bio X Cell) intraperitoneally twice a week. The expression level of CD122 in NK cells was examined by FACS.

| Chronic shift-lag interferes with circadian gene expression and inhibits the expression of CD107a and IFN-γ on NK cells
Circadian rhythm is regulated by a transcriptional-translational feedback loop that involves a family of clock genes. The clock gene family includes aryl hydrocarbon receptor nuclear translocator-like 1 (Bmal1), CLOCK, per1, per2, and cryptochrome (cry1 and cry2). In addition, the orphan nuclear receptor REV-ERB and ROR families are also reported to be feedback regulatory targets of CLOCK-Bmal1. 14 It is possible to change the circadian clock in mammals by changing the light period or food pattern; therefore, changing these signals can cause circadian clock disruption. To determine whether chronic shift-lag disrupts circadian clock genes in splenic NK cells, we detected the mRNA levels of several circadian genes, such as per1, per2, per3, Bmal1, and CLOCK, in NK cells after chronic shift-lag. In comparison with the control, chronic shift-lag inhibited the mRNA expression of per1 and per2 and enhanced the mRNA expression of CLOCK ( Figure 1A-E). These results were consistent with the corresponding protein expression levels ( Figure 1F). To verify the relationship between these gene changes and NK cell function, splenic NK cells were sorted from control and chronic shift-lag mice and transfected with specific per1 and per2 siRNA. It was found that the expression levels of per1 and per2 in NK cells were significantly decreased following knockdown ( Figure 1G). Further, the mRNA expression levels of CD107a and INF-γ in NK cells were evaluated, and it was found that they were significantly decreased in NK cells from chronic shift-lag mice ( Figure 1H,I). We speculated that this may be F I G U R E 1 Chronic shift-lag disrupts circadian rhythm and inhibits the expression of CD107a and IFN-γ in NK cells. Splenic NK cells from control or chronic shift-lag mice were sorted by flow cytometry. RT-qPCR was used to analyse the mRNA levels of circadian rhythm genes such asper1 (A), per2 (B), per3 (C), Bmal1 (D) and CLOCK (E) in NK cells. The protein levels of per1, per2 and CLOCK were examined by Western blotting (F). Sorted NK cells were transfected with 25 nmol/L per1 and per2 siRNA or negative control siRNA and harvested after 48 h for protein analysis by Western blotting (G). The mRNA levels of CD107a and INF-γ were detected by RT-qPCR with or without Per1 and Per2 knockdown (H, I). Data are shown as the means ± SD. Unpaired Student'st tests (two-tailed) were performed using the Prism software.P-value of <.05 was considered significant. *P < .05, **P < .01 the result of disruption of the circadian rhythm in NK cells by chronic shift-lag. Therefore, we further evaluated the mRNA expression levels of CD107a and INF-γ in NK cells following knockdown of per1 and per2, and it was found that they were decreased, with no significant difference between the control and chronic shift-lag groups ( Figure 1H,I). These results indicate that chronic shift-lag disrupts the expression of NK cell circadian genes and reduces the mRNA levels of the NK cell function-related genes CD107a and IFN-γ.

| Chronic shift-lag slightly decreases the proportion and number of NK cells in the spleen and lungs
Maintaining an appropriate number of NK cells in the resident organs is extremely important for immune monitoring 15 ; hence, we wondered whether circadian rhythm disruption affects the physiology of NK cells. The proportion and absolute number of NK cells in different organs and tissues from mice subjected to chronic shift-lag were detected by FACS. It was found that the proportion and number of NK cells in the spleen and lungs from chronic shift-lag mice were decreased slightly but those in the bone marrow, lymph nodes and liver were unchanged. The proportion of NK cells in the spleen and lungs was decreased by 75% and 60%, respectively (Figure 2A,B), and the absolute number was decreased by 73% and 50%, respectively, after chronic shift-lag ( Figure 2C). These results suggest that circadian rhythm disruption may reduce the number of resident NK cells in the spleen and lungs, impairing NK cell function.

| Chronic shift-lag inhibits NK cell proliferation, promotes apoptosis and decreases activation in the spleen
Next, we explored why circadian rhythm disruption reduces NK cell number in mice. We focused on the two major pathways that affect NK cell homeostasis: cell proliferation and apoptosis. Apoptotic NK cells were labelled with Annexin V and detected by FACs, and it was found that apoptosis of total NK cells in the spleen was significantly increased following chronic shift-lag. Apoptosis of effective mature NK cells (CD27 + CD11b + ) was also enhanced after chronic shift-lag.
On the other hand, apoptosis of ageing NK cells (CD27 − CD11b + ) was inhibited following chronic shift-lag ( Figure 3A Figure 4A,B). The number of effective mNK cells was reduced and that of ageing NK cells was increased in the spleen ( Figure 4C).

NK cells recognize tumour cells by activating and inhibiting recep-
tors on the cell surface. 16 Ly49 family receptors can interact with

MHC-I molecules to activate or inhibit NK cells. Normal cells express
MHC-I, which interacts with Ly49 family receptors to transmit inhibitory signals to NK cells, while aberrant cells expressing a low level of MHC-I transmit an activation signal for killing. 17

| Chronic shift-lag impairs NK cell-mediated immunosurveillance
Up  For the B16 metastasis assay, the indicated mice pretreated with or without an anti-NK1.1 antibody were intravenously injected with 2 × 10 5 B16 cells. After 14 d, the number of tumour nodules was counted (G, H). Each symbol represents an individual mouse. All the data represent at least three independent experiments. Data are shown as the means ± SD. Unpaired Student'st tests (two-tailed) were performed using the Prism software. P-value of <.05 was considered significant. *P < .05, **P < .01 and ***P < .001

| Chronic shift-lag disturbs the expression of T-bet, Eomes and CD122 on NK cells
E4BP4 are important transcription factors expressed during NK cell maturation; thus, we evaluated their protein expression levels by flow cytometry. The results show that the expression of T-bet in NK cells from chronic shift-lag mice was significantly increased, while that of Eomes was decreased ( Figure 7A-D). These results are consistent with those of previous studies that found T-bet and Eomes to be antagonistic in NK cells. Next, we further tested the key transcription factor E4BP4, which determines the survival of NK cells, and it was found that the expression of E4BP4 in NK cells from chronic shift-lag mice was normal ( Figure 7E,F). Eomes plays an important role in NK cell maturation by regulating CD122 expression; therefore, we evaluated the level of CD122 expression inNK cells from chronic shift-lag mice.
The results show that the level of CD122 in NK cells from chronic shift-lag mice was consistent with that of Eomes, both of which were significantly reduced ( Figure 7G-H). Therefore, chronic shift-lag disrupts the balance between T-bet and Eomes in NK cells, reducing CD122 expression, which weakens the response of NK cells to IL-15.

| Blockade of CD122 decreases the secretion of CD107a and INF-γ from NK cells
To verify that chronic shift-lag damages NK cell function by inhibiting CD122, we used an anti-CD122 monoclonal antibody to block CD122 during the final 2 weeks of chronic shift-lag mouse model establishment. Following completion of the model, CD122 expression on NK cells was examined, and it was found that anti-CD122 antibody treatment significantly reduced the expression of CD122 on NK cells ( Figure 8A,B). We further explored the effect of CD122 blockade on the function of NK cells by evaluating their ability to secrete CD107a and IFN-γ following stimulation with tumour cells.
It was found that after blocking CD122, the ability of NK cells to secrete CD107a and IFN-γ was significantly reduced; however, interestingly, there was no significant difference between the control and chronic shift-lag groups ( Figure 8C-F). These results indicate that blockade of CD122 inhibits NK cell function and further suggests that chronic shift-lag impairs NK cell function by inhibiting CD122 expression.

| D ISCUSS I ON
Circadian rhythm regulates many physiological processes including immune cell development and function. 19 The SCN is the centra lcontroller of the circadian rhythm and regulates the peripheral clock through the action of hormones or other signals. 20 For example, immune tissue receives rhythmic input, thereby regulating clock gene expression in the immune system including NK cells. 10

Since the number of functional NK cells is decreased in chronic
shift-lag mice, we wondered whether chronic shift-lag impairs the function of NK cells. We found that secretion of CD107a by NK cells with circadian rhythm disorder is significantly reduced irre-

| CON CLUS IONS
In conclusion, the present study demonstrates that circadian