Probiotics ingestion prevents HDAC11-induced DEC205+ dendritic cell dysfunction in night shift nurses

It is known that the day-night shift-rotation has a negative impact on the immune system. The underlying mechanism remains to be further investigated. Probiotics have regulatory effects on immune functions. This study aims to investigate the role of probiotic ingestion in preventing the DEC205+ dendritic cell (decDC) dysfunction in day-night shift-engaging nurses. In this study, blood samples were collected from day-night shift-rotating nurses who took or did not take yogurt (containing C. Butyricum) during the night shift (NS). decDC functions were evaluated with pertinent immunological approaches. We observed that the immune tolerogenic functions and interleukin (IL)-10 expression were impaired in decDCs of nurses after NS. HDAC11 was detected in decDCs that was markedly up regulated after NS. The HDAC11 levels were negatively correlated with the immune tolerogenic functions in decDCs. Ingestion of probiotic-containing yogurt during NS efficiently suppressed Bmal1 and HDAC11 levels as well as up regulated the immune regulatory functions in decDCs. In conclusion, NS has a negative impact on decDC immune tolerogenic functions, which can be prevented by ingesting probiotics-containing yogurt during NS.


NS impairs decDC immune tolerogenic functions.
To assess the impact of NS on immune regulatory functions, peripheral blood samples were collected from nurses before and after NS. PBMCs were isolated from blood samples and analyzed by flow cytometry. The results showed that the frequency of decDCs was not significantly altered by NS (Fig. 1A,B). We then isolated decDCs from PBMCs. The decDCs were cocultured with CD4 + CD25 − T cells in the presence of PMA/ionomycin (non-specific cell activators) for 4 days. The cells were analyzed by flow cytometry. The results showed that NS significantly suppressed the immune tolerogenic functions of decDCs; markedly less type 1 regulatory T cells (Tr1) were induced by NSp decDCs (decDCs were collected from nurses after NS) than NSb decDCs (decDCs were collected from nurses before NS. Figure 1C-E). The results indicate that although NS does not alter the frequency of decDCs, it affects the decDC immune tolerogenic capacity. decDC IL-10 expression is down regulated by NS. IL-10 is one of the major immune regulatory mediators of decDCs 15 . To test the effects of NS on IL-10 expression in decDCs, we isolated decDCs from PBMCs. The cells were analyzed by RT-qPCR and Western blotting. We observed that the IL-10 expression in decDCs was significantly reduced after NS ( Fig. 2A,B). We also identified a positive correlation between the IL-10 expression and the Tr1 induction ability in decDCs (Fig. 2C). The results suggest that NS down regulates the IL-10 expression in decDCs, thus, impairs decDC immune regulatory capacity.

NS increases HDAC11 expression in decDCs.
Published data indicate that HDAC11 can inhibit IL-10 expression 16 . Thus, we next assessed the possible association between NS and HDAC11 in decDCs. We found that the HDAC11 levels were markedly increased in decDCs after NS. The results indicate that NS can up regulate

Bmal1 increases HDAC11 expression in decDCs.
To test the role of Bmal1 in regulating the HDAC11 expression, decDCs were isolated from healthy control (HC) subjects; the cells were transfected with Bmal1-expression plasmids to overexpress Bmal1 (Fig. 5A). This markedly increased the levels of HDAC11 in the decDCs (Fig. 5B,C), and the levels of IL-10 expression were down regulated subsequently (Fig. 5D). To verify the role of HDAC11 in the Bmal1-suppressed IL-10 expression in decDCs, HDAC11-deficient decDCs were prepared ( Fig. 5E) and transfected with Bmal1-expression plasmids. The depletion of HDAC11 indeed abolished the Bmal1-suppressed IL-10 expression in decDCs (Fig. 5D). In addition, depletion of Bmal1 ( Fig. 5F) also increased the IL-10 expression (Fig. 5D). The results demonstrate that Bmal1 can suppress the IL-10 expression in decDCs through up regulating the HDAC11 expression. On the other hand, the depletion of HDAC11 did not affect the Bmal1 expression in decDCs (Fig. 5G), indicating that Bmal1 is in the upper stream of the signal transduction pathway.

Probiotics prevent NS-induced decDC dysfunction. Previous reports indicate that probiotics improve
immune functions 17 . Thus, we tested the effects of probiotic-containing yogurt ingestion on counteracting the NS-induced decDC dysfunction. Blood samples were collected from nurses ingested 100 ml milk or 100 g yogurt (containing about 10 10 C. butyricum) during NS. decDCs were isolated from the blood samples and analyzed for the tolerogenic properties. We found that yogurt ingestion for one month in NS efficiently maintained the capacity of decDCs in the induction of Tr1 cells (Fig. 6A,B) and the production of IL-10 ( Fig. 6C), and kept the levels of HDAC11 and Bmal1 not increased by NS (Fig. 6D,E). The results demonstrate that ingestion of probiotic containing yogurt protects decDCs of NS nurses from impairing the immune tolerogenic functions.

Discussion
The present data show that NS can down regulate decDCs' immune tolerogenic capacity by inhibiting IL-10 expression. We observed that NS increased the expression of Bmal1 in decDCs; Bmal1 increased the expression of HDAC11; the latter suppressed IL-10 expression in decDCs. Ingestion of probiotic containing yogurt prevented the NS-induced decDC dysfunction.
The day-night shift rotation occurs frequently in people engaging specific occupation. NS is a regular shift in hospitals. Published data reveal that NS can alter the circadian rhythm 18 . There are several diseases occur in people engaging day-night shift rotation more often than people with other careers. For example, Night-shift work is one of the causative factors associating with poorer glycaemic control in type 2 diabetes patients 2 . Engaging day-night shift rotation for one year has risk of incident hypertension 3 . Nurses with day-night shift rotation reported higher prevalence of frequent or chronic headaches 4 . The present data provide further evidence that NS may contribute to compromising human health.
The data show that, after NS, decDC immune tolerogenic efficacy is compromised. decDCs are an important cell fraction in immune regulatory activities in the body. By releasing immune regulatory mediators, decDCs induce regulatory T cells 19 . Previous reports indicate that DCs are dysfunctional in subjects with food allergy 20 . In steroid resistant asthma patients, the myeloid DC phenotype is increased 21 . Yet, the etiology of decDC dysfunction is less understood currently. The present data show that the immune tolerogenic capacity of decDCs are impaired in subjects after NS, suggesting that NS may be one of the causative factors inducing decDC dysfunction.
We found that the IL-10 expression in decDCs was decreased after NS. IL-10 is a canonical immune regulatory mediator in decDCs 22 . IL-10 deficiency can result in immune inflammation 23 and cannot generate immune tolerance 24 . In this study, we observed that the NS-induced Bmal1 was negatively correlated with IL-10 expression in decDCs. Bmal1 is one of the circadian rhythm proteins. Expression of Bmal1 can be regulated by circadian rhythm disruptions. Sleep deprivation is one of the factors increasing Bmal1 expression 25 . Our data show that NS www.nature.com/scientificreports www.nature.com/scientificreports/ up regulated Bmal1 expression in decDCs. As both Bmal1 and IL-10 expression were altered by NS, there may be a link between these two factors in decDCs.
We also found that the levels of HDAC11 were increased in decDCs after NS. HDAC11 is a member of the HDAC family. HDAC family plays an important role in regulating gene transcription under physiological conditions 26 . Dysregulation of HDAC is associated with the pathogenesis of many diseases. Inhibition of HDAC has been employed in the treatment of many diseases such as cancer 27 , diabetes 28 and allergic diseases 29 . Our data are consistent with those previous reports. We found that HDAC11 was negatively correlated with the levels of IL-10 in decDCs. Furthermore, the data indicate that NS can increase HDAC11 levels in decDCs, suggesting that NS is one of the factors regulating the activities of HDAC11.
The present data show that NS increases HDAC11 and then impairs decDCs' tolerogenic capacity. This is a harmful event to the immune homeostasis of the body. However, there are not many remedies available to prevent impairing DC's tolerogenic property or restore the impaired DC's tolerogenic capacity. The present results contribute to this area by showing that ingestion of probiotic containing yogurt can prevent the NS-induced decDC dysfunction. Probiotics have been used as a supplement to therapeutics for immune disorders 17 . Our data are in line with previous studies by showing that ingestion of probiotics can prevent the effects of NS on compromising immune tolerant activities in the body.
In summary, the present data show that NS can compromise decDCs' tolerogenic capacity by increasing Bmal1 and HDAC11, and suppressing IL-10 expression in decDCs, which can be prevented by ingestion of probiotic containing yogurt.

Ethic statement. The experimental procedures were approved by the Human Ethics Committee at Shanxi
Medical University. All methods were performed in accordance with the relevant guidelines and regulations. A written informed consent was obtained from each human subject.
Human subjects. Nurses engaging the day-night rotation shift for 1-4 years were randomly recruited into this study. The demographic data are presented in Table 1. Subjects with any of the following conditions were excluded from this study, including allergic diseases, autoimmune diseases, severe organ diseases, cancer, under treatment with corticosteroids or other immune tolerogenic agents for any reasons. www.nature.com/scientificreports www.nature.com/scientificreports/

Preparation of peripheral blood mononuclear cells (PBMC).
Blood samples were collected from nurses through ulnar vein puncture before and after each NS shift (the NS shift time was 12 h) at 20 ml per case. To avoid the blood drawing stress, nurses were explained the study purposes before the experiments. PBMCs were isolated from blood samples by Percoll gradient density centrifugation.
Cell culture. Cells were cultured in RPMI1640 medium. The medium was supplemented with fetal calf serum (10%), glutamine (2 mM), penicillin (100 U/ml) and streptomycin (0.1 mg/ml). The medium was changed in 2-3 days. Cell viability was greater than 99% as assessed by Trypan blue exclusion assay.
Immune cell isolation. DCs, B cells and CD4 + T cells were isolated from PBMCs by flow cytometry cell sorting. The cells were labeled by incubating with fluorescence-conjugated antibodies and sorted with a flow cytometer. If the purity of isolated cells did not reach 95%, the cell sorting was repeated.
Flow cytometry. Cells were collected from relevant experiments. In the surface staining, cells were stained with fluorescence labeled antibodies of interest or isotype IgG for 30 min at 4 °C. In the case of intracellular staining, cells were fixated with 1% paraformaldehyde (containing 0.1% Triton X-100 to increase cell membrane permeability) for 1 h. Then, cells were stained with fluorescence labeled antibodies of interest or isotype IgG for 30 min at 4 °C. After washing with PBS, cells were analyzed with a flow cytometer (FACSCanto II; BD Bioscience). Data were analyzed using a software package, FlowJo (TreeStar Inc., Ashland, OR) Data obtained from isotype IgG staining were used as a gating reference.
Assessment of decDC tolerogenic functions. Naive CD4 + CD25 − T cells were isolated from blood samples collected from healthy subjects; decDCs were isolated from blood samples collected from nurses before and after NS. CD4 + CD25 − T cells were cultured with decDCs at a ratio of 5:1 in the presence of PMA (50 ng/ml) www.nature.com/scientificreports www.nature.com/scientificreports/ and ionomycin (100 ng/ml) for 4 days. Then, the cells were analyzed by flow cytometry. The frequency of IL-10 + Foxp3 + iTr1 cells was determined and used as an indicator of decDC immune tolerogenic functions.

Real-time quantitative RT-PCR (RT-qPCR).
Total RNA was extracted from cells collected from relevant experiments and converted to cDNA with a reverse transcription kit following the manufacturer's instructions. The samples were amplified in a qPCR device with the SYBR Green Master Mix in the presence of relevant primers ( Table 2). The results were processed with the 2 −∆∆Ct method and presented as fold change against controls.
Western blotting. Total proteins were extracted from cells collected from relevant experiments, fractioned by SDS-PAGA and transferred onto a PVDF membrane. After blocking with 5% skim milk for 30 min, the membrane was incubated with the primary antibodies (diluted 1:200-300) of interest overnight at 4 °C, washed with TBST (Tris-buffered saline containing 0.1% Tween 20) 3 times, incubated with peroxidase-labeled secondary antibodies for 2 h and washed with TBST 3 times. Immunoblots on the membrane were developed by the enhanced chemiluminescence and photographed in an imaging device (UVP imaging; Cambridge, UK).   Table 2. Primers used in the study.