Study of calcitriol anti-aging effects on human natural killer cells in vitro

ABSTRACT Vitamin D is widely considered to have a regulatory effect on the immune system. Some clinical investigations have shown that the demand for vitamin D increases with age. Calcitriol is the biologically active form of vitamin D. However, its effect on human natural killer (NK) cells remains unclear. Therefore, in this study, we investigated the anti-aging and immunomodulatory effects of calcitriol on NK cells using a series of immunological methods to explore its important role in innate immunity. We found that calcitriol reversed the expression of aging-related biomarkers in NK cells and inhibited their expansion by maintaining these cells in the G1 phase, without any apoptosis and exhaustion. Calcitriol repressed the release of inflammation-related cytokines, such as interleukin-5 (IL-5), interleukin-13 (IL-13), interferon-gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α). The degranulation of NK cells was downregulated by calcitriol when these cells were co-cultured with K562 tumor cells. We also found that calcitriol upregulated the aging-related sirtuin 1- protein/kinase R-like endoplasmic reticulum kinase (SIRT1/pERK) pathway and SIRT1-deltaExon8 (SIRT1-∆Exon8) expression by activating the vitamin D receptor (VDR). Moreover, calcitriol could be a potential negative regulator of NK cell apoptosis and mitochondrial inactivation which caused by oxidative stress. Thus, calcitriol exhibits anti-aging effects on human NK cells in vitro by activating the SIRT1-PERK axis and resisting oxidative senescence.


Introduction
Many studies have found that aging exerts a direct influence on several diseases, such as infectious diseases, cardiovascular diseases, neurodegenerative diseases, autoimmune disorders, and cancer. Organism aging is accompanied by immunosenescence. The common features of aging mainly include inflammageing with chronic low inflammation [1], genome instability, protein expression imbalance, mitochondrial inactivation, cellular senescence, and changes in intercellular communication [2].
As the main component of innate immunity, NK cells play an important role in the antiinfection and anti-tumor effects as well as the regulation of the immune system [3]. NK cells can eliminate the target cells directly or via antibody-dependent cellular cytotoxicity (ADCC), instead of exhibiting any antigen-or antibodyspecific responses [4]. NK cells are also associated with hypersensitivity reactions and autoimmune diseases [5]. When the immune system getting aged, the expression of surface activation receptors such as the natural killer group 2 member D (NKG2D) [6], killer immunoglobulin-like receptor (KIR), surface marker cluster of differentiation 57 (CD57) [7], cluster of differentiation 16 (CD16) [8] increased. These molecule could activate the killing function of NK cells, which may leads to cellular imbalance and inflammation in elder. Meanwhile, the inhibitory molecules of NK cells such as natural killer group 2 member A (NKG2A), natural killer group 2 member C (NKG2C), and natural cytotoxicity receptors (NCRs) [6] decreased. Other aging-related markers of NK cells are T cell immunoglobulin and mucin domain-containing protein 3 (TIM3) and programmed cell death-1 (PD-1) increased. The upregulation of PD-1 and TIM3 during immunosenescence may inhibit the functions of NK cells and leads to NK cell depletion [9].
Another factor leading to immunosenescence is immune-inflammation [10]. High levels of inflammatory cytokines released by NK cells have been reported to cause DNA damage, oxidative stress, and cell senescence. Moreover, the cellular imbalance of NK cells and inflammation status may result in autoimmune disease [11]. The levels of interleukin-1 (IL-1), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-10 (IL-10), and tumor necrosis factor-alpha (TNF-α) are higher in elderly people [12]. Although the number of total immune cells decreased with aging, the NK cell population remarkably increased, which may be caused by the infiltration of inflammatory cells [13].
Vitamin D exists in two active forms in the human body as vitamin D2 (ergocalciferol) and vitamin D3 (calcitriol). The main function of vitamin D is to promote the absorption of calcium and phosphorus. Vitamin D also regulates the immune functions [14]. Although many reports have focused on its anti-infection and anti-tumor functions [15], calcitriol exhibits antiinflammatory effects as well [16]. The vitamin D receptor (VDR) is expressed on various immune cells [17]. Decrease in the production of inflammatory cytokines contributes to the reduction in inflammatory reactions. Calcitriol is known to suppress the production of TNF-α, interleukin-1 (IL-1), interleukin-2 (IL-2), interferon-gamma (IFN-γ), and other inflammatory cytokines in B and T cells. It can also release antiinflammatory cytokines like IL-4 and IL-10 [18] and regulate autophagy against inflammatory cell infiltration [19].
The antioxidant properties of calcitriol have been reported in depression, fatty liver disease [20], cardiovascular disease, and other inflammation-related diseases. Free radicals and reactive oxygen species (ROS) also accumulate in cells and tissues during aging. Published data have demonstrated that oxidative stress affects a variety of signaling pathways in cells [21], including the SIRT1, mitogen-activated protein kinase (MAPK), and nuclear factor-κB (NF-κB) pathways. The SIRT1 gene is considered as a longevity gene and the suppression of this gene results in disorders related to protein expression, cell cycle, and metabolism [22]. SIRT1-ΔExon8 is a novel isoform of SIRT1, which existed in mammals by alternatively splicing. SIRT1-ΔExon8 was also considered as a co-regulatory factor of fulllength SIRT1 [23].
So far, very few studies focus on the effects of aging and oxidative senescence on NK cells. The effects of calcitriol on SIRT1-ΔExon8 have not been reported as well. Therefore, this study aimed at investigating the effects of calcitriol on the aging and oxidative senescence of NK cells, as these cells occupy a central position in the human innate immune system.

Cell culture and reagents
Three human peripheral blood samples were acquired from donors who did not have any active autoimmune disease, acute or chronic inflammatory disease, cancer, or other immune-related diseases. The age of the donors ranged from 48-65 years. Peripheral blood mononuclear cells (PBMCs) were isolated using Lymphoprep Ficoll. The NK cells were expanded and maintained using an NK Cell Culture Kit (MoreCell, Shenzhen, China) at 37°C/5% CO2.

Cell expansion measurement, cytokine-releasing and cell cycle assay
Cell expansion was determined using a Cell Counting Kit-8 [24] (CCK8; Beyotime, Shanghai, China). NK cells were treated with calcitriol for 48 h and the supernatant was collected for the Cytokine-releasing assay. The cytokine levels were examined using the LEGEND Plex Human Inflammation Panel (BioLegend, California, USA). First, the cytokine capture beads were incubated with the standards or samples and then further incubated with biotinylated detection antibodies. The biotinylated detection antibody-binding solution, streptavidin (SA)-PE, was subsequently added to provide the fluorescent signal [25]. These signals were then analyzed using the FACS assay.
Cell cycle was measured using a Cell Cycle Detection Kit (Beyotime, Shanghai, China). NK cells were treated with calcitriol for 72 h. These cells were fixed in 70% ice-cold ethanol and then dyed with ribonuclease A (RNase A) and propidium iodide (PI) (Beyotime, Shanghai, China) [26].

Oxidative senescence induction and X-gal staining analysis
The in vitro NK cell aging model using hydrogen peroxide (H2O2). In order to determine the antioxidation effect of calcitriol, NK cells were first treated with calcitriol and then exposed to 100 μM H2O2 for 24 h.
The activity of β-galactosidase was measured using 5-bromo-4-chloro-3-indolyl-β -D-galactopyranoside (X-gal) staining. The treated cells were fixed in 4% paraformaldehyde and stained with β-galactosidase staining solution (Beyotime, Shanghai, China) overnight at 37°C [28]. The stained cells were then suspended in PBS and images of these cells were acquired using a Leica fluorescence inversion microscope system. Three microscopic images were randomly collected from different positions at 40× magnification for each group. The percentage of X-gal-stained cells was calculated and used to assess the aging of cells using the ImageJ software V.1.45S.

Staining and analysis of the mitochondrial membrane potential
Chloromethyl-X-Rosamine (CMXRos) and Hoechst (Beyotime, Shanghai, China) was used to assess the mitochondrial membrane potential. The cell nuclei (blue) were located by Hoechst staining, while the mitochondrial membrane potential was determined by the mitochondrial specificitydenoting fluorescent dye, CMXRos. The Hoechst + CMXRos-cells (marked by white arrows) showed low activity. The relative fluorescence level was calculated by dividing the number of Hoechst positive cells by CMXRos-positive cells [29]. Cells were incubated with 200 nM CMXRos and Hoechst (30 min, 37°C) and detected using a Leica inverted microscope at excitation wavelengths of 579 nm and 350 nm. The percentage of Hoechst + CMXRos + cells was calculated as described in Section 2.5.

Statistical analysis
The data was analyzed by one-way analysis of variance (ANOVA) using GraphPad Prism and presented as mean ± standard deviation (SD).
Differences were considered statistically significant at P < 0.05. The results were also analyzed using the GraphPad software.

Results
This study focused on the anti-aging effects of calcitriol on NK cells. The results showed that Calcitriol decreased the expression of aging-related biomarkers including CD16, TIM3, PD-1, KIR and increased the expression of NKG2A of NK cells. it also inhibited NK cell proliferation and arrest them in G1 phase. Calcitriol decreased the releasing of inflammatory factors such as IL-5, IL-13, IFN-γ, TNF-α and had a certain antioxidation effects, which could contribute to its anti-aging effects.

Calcitriol reversed the expression of the surface aging markers, slowed down the NK cell amplification, and maintained NK cells in the G1 phase.
Senescent NK cells showed high expression of agingrelated biomarkers, such as CD16, PD-1, TIM3, and KIR, and low expression of NKG2A. We found that the expression levels of the aging-related biomarkers NKG2A We expanded cells from the human peripheral blood samples in vitro. Then we isolated the NK cells (CD3-CD56+) by magnetic bead sorting (figure 1f). The sorted NK cells were then cultured with different concentrations of calcitriol or rapamycin for 72 h. CCK8 staining results showed that the growth rate of the calcitriol-treated NK cells of high concentration (10 − [7] M) was slower than that of the negative control group (NC) (*P < 0.05) (Figure 1g). In addition, calcitriol regulated the NK cell cycle. After treatment with calcitriol for 48 h, NK cells showed a high calcitriol concentrationdependent shift toward the G1 phase (*P < 0.05), while the proportion of cells in the G2 phase (#P < 0.05) decreased. The proportion of calcitrioltreated NK cells in the S phase showed no change, regardless of the concentration of calcitriol ( Figure 1h, Supplementary Figure 2). We then tested whether this calcitriol-derived G1 phase arrest induced NK cell apoptosis and found that the NK cell apoptotic biomarker, T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif (ITIM) domain (TIGIT), showed no significant change (P > 0.05, NS) (Figure 1i). These results implied that addition of calcitriol reduced the growth rate of NK cells, decreased their proliferation, and induced G1 phase arrest of the NK cells instead of causing apoptosis.

Calcitriol decreased the release of inflammatory cytokines in NK cells and inhibited the cytotoxicity of NK cells
Inflammageing is a chronic low-grade inflammation. It accelerates the aging of NK cells, as NK cells participate in this chronic inflammation. Figure 2a demonstrates that the levels of IL-5, IL-13, IFN-γ, and TNF-α significantly decreased in all groups than in the negative control (*P < 0.05). IFN-γ and TNF-α are well-known pro-inflammatory cytokines but some studies have also shown the proinflammatory properties of IL-5. IL-13 has been reported to contribute to different types of mucosal inflammation, such as allergic asthma, ulcerative colitis, eosinophilic esophagitis, and several diseases related to fibrosis.
However, the cytotoxicity of NK cells mainly involves the release of inflammatory cytokines and degranulation of cells. We labeled K562 cells with DIO and co-cultured the DIO-K562 cells with calcitriol-treated NK cells for 4 h. The percentage of DIO + PI + cells were lower in all the effector (E)/target (T) ratio groups than in the negative control (*P < 0.05) (Figure 2b,c). The deduced expression of cluster of differentiation 107 (CD107) indicated lower degranulation (*P < 0.05) (Figure 2d). These results show the inhibition of the NK-killing function by calcitriol.

Calcitriol activated the expression of SIRT1-∆Exon8 and anti-aging-related SIRT1/pERK pathway
To investigate the anti-aging effects of calcitriol on NK cells, we detected the aging-related signaling pathways by immunoblotting. Western blotting analysis was performed to assess the expression levels of VDR, SIRT1-∆Exon8, SIRT1, and PERK in the calcitrioltreated NK cells. Statistical analysis showed that calcitriol activated the VDR/SIRT1/pERK axis at high concentrations. Meanwhile, calcitriol activated the expression of SIRT1-∆Exon8 (*P < 0.05). (Figure 3a).

Calcitriol resisted the aging of NK cells induced by oxidative stress in vitro
It is widely accepted that aging is caused by oxidative damage accumulation due to the large body of evidence found over the years [31]. We cultured the NK cells with different concentrations of H 2 O 2 for 24 h and found that 100 μM was the nearest concentration to the median lethal dose (LD 50 ) (Figure 3b). The expression of TIM3 in the NK cell population increased by the addition of H 2 O 2 , while the expression decreased after treatment with calcitriol (*P < 0.05) (Figure 3c). In addition, aging NK cells were stained blue in the presence of β-galactosidase. This blue color of the cells is a specific hallmark of senescent cells. Statistical analysis demonstrated that untreated NK cells have less cellular β-galactosidase activity, while the NK cells exposed to H 2 O 2 displayed elevated βgalactosidase activity. Calcitriol reduced the βgalactosidase-positive population, indicating the presence of fewer aging cells (*P < 0.05) (Figure 3d).
Decreased mitochondrial membrane potential has also been observed in a variety of aging cells from several mammalian species. We evaluated the mitochondrial membrane potential of the NK cells in this study. The statistical results showed that H 2 O 2 significantly decreased the cellular activity of NK cells compared to that of the untreated cells, while calcitriol increased the damage caused by H 2 O 2 (*P < 0.05) (Figure 3e).

Calcitriol resisted the apoptosis of NK cells induced by oxidative stress in vitro
Next, we measured the number of apoptotic cells. The flow cytometry results showed that treatment with 100 μM H 2 O 2 for 24 h led to significant apoptosis of the NK cells. However, treatment  with calcitriol decreased the apoptotic ratio (*P < 0.05) (Figure 4a).
All the above results demonstrated the resistance of calcitriol against oxidative stress caused by H 2 O 2 . However, we detected apoptotic proteins in NK cells under oxidative stress. Before treatment with H 2 O 2 , 10 − [7] M calcitriol was added to the NK cells for 24 h. The levels of apoptosis-related proteins, caspase-3 p17 and caspase-3 p20, increased significantly after treatment with H 2 O 2 . Calcitriol decreased H 2 O 2 -derived apoptosis and downregulated the expression of caspase-3, p17, and caspase-3 p20 (*P < 0.05) (Figure 4b). These results indicated that calcitriol resisted apoptosis induced by H 2 O 2 .

Discussion
The long-term interaction between inflammation and oxidative stress is an important factor in aging. In addition, inflammation and oxidative stress are synergistic [32]. Excess ROS generated by oxidative stress attack the body cells. During this process, inflammatory factors are released by the activated immune system to kill the abnormal cells, which leads to further ROS generation. Lack of calcitriol can cause autoimmune diseases, chronic metabolic diseases, and tumors. However, little is known about the anti-aging effects of calcitriol on the immune system, especially on NK cells.
In this study, we investigated the anti-aging effects of calcitriol on NK cells. We found that calcitriol increased the expression of NKG2A, while decreasing the expression of KIR in vitro. The CD56 − CD16 + subpopulation of NK cells, which is known to be related to chronic inflammation, is significantly increased in the elderly [33]. Therefore, the reduction in CD16 expression induced by calcitriol might be attributed to its anti-inflammatory effect. TIM-3 and PD-1 have been reported to be the inhibitory and depletion receptors of NK cells. Our study showed that calcitriol downregulated the expression levels of TIM-3 and PD-1 and prevented apoptosis of NK cells.
In addition, calcitriol inhibited the expansion of NK cells and maintained the NK cell population in G1 phase in vitro. However, the cells in the S-phase were not affected. TIGIT, which is known as a checkpoint factor of NK cells, remained unchanged. These results indicated that calcitriol did not lead to the death of NK cells, but it only slowed down the rate of NK cell expansion.
It's well known that inflammation leads to immunosenescence [34,35]. Furthermore, we found that calcitriol reduced the levels of IL-5, IL-13, IFN-γ, and TNF-α in NK cells. The cytotoxicity of NK cells is partly mediated by the release of inflammatory factors [36]. Moreover, there was a decrease in the mortality of K562 tumor cells and degranulation of NK cells. These results were in line with our expectations. Rapamycin is a well-known drug that reduces the metabolic rate to prevent aging [37] which used as the positive control in this study. It results in immunosuppression and increases the potential risk of tumor and tumor burden. This burden has been confirmed in mouse models of breast cancer [38]. Whether calcitriol exhibits similar effects to rapamycin remains unknown. More animal and clinical studies should be performed to verify their effects in the human body.
Finally, we studied the effects of calcitriol on antioxidant senescence. Resveratrol, a well-known anti-aging drug, can downregulate the NF-κB pathway by activating the SIRT1/PERK axis. This downregulation has also been shown to be beneficial in neurodegenerative disorders and cardiovascular diseases [39]. SIRT1 deacetylation is associated with metabolic control and mitochondrial biogenesis. In this type of regulation, elevated SIRT1 levels can promote the accumulation of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) in the nucleus, which results in the transcription of genes that are necessary for mitochondrial function [40]. Besides, SIRT1 suppresses the mammalian target of rapamycin (mTOR) signaling pathway, which can protect the nerves and resist brain aging in mice [41]. In this study, we identified a similar regulatory pathway for calcitriol. Calcitriol activated SIRT1 via VDR and raised the SIRT1/pERK axis. In addition, we found calcitriol upregulated the expression of SIRT1-∆Exon8. Stresses are known as an up-regulatory factor of SIRT1-∆Exon8. Although SIRT1-∆Exon8 itself displays reduced p53 deacetylation activity, it exerts an additive deacetylation effect on p53 when expressed together with full-length SIRT1 [42]. This effect indicated the anti-apoptotic function of calcitriol on NK cells. However, the role of SIRT1-∆Exon8 in age-related pathways remains to be better understood.
Since the decline of SIRT1 might be due to oxidative damage [43], we established an acutely aging NK cell model in vitro using H 2 O 2 . The oxidative aging induced by H 2 O 2 increased the expression of TIM3 as well as the activity of β-galactosidase in NK cells. We found that calcitriol resisted the oxidative damage and maintained the mitochondrial activity and cell viability of NK cells, while preventing cell apoptosis. However, calcitriol has not yet been identified as an anti-aging drug in clinical medicine as more evidence is needed regarding the anti-aging effects of this drug in vivo. In summary, we provided preliminary evidence to implicate the anti-aging effects of calcitriol on NK cells in vitro. We believe that the effects of this drug should be further explored in future animal and clinical studies.

Conclusion
In this study, we demonstrated the anti-aging effects of calcitriol on NK cells. Calcitriol reversed the expression of aging biomarkers on NK cells. It slowed down NK amplification and kept them in G1 phase. In addition, calcitriol inhibited the release of inflammatory cytokines and downregulated degranulation of NK cells. Activation of SIRT1-∆Exon8 and VDR/ SIRT1/pERK axis by calcitriol might be the main mechanism of calcitriol anti-aging effect based on all above. Finally, calcitriol resisted the oxidative senescence of NK cells in vitro. Further studies should focus on animal and clinical research.

Highlights
(1) Calcitriol decreased the expression of ageing-related biomarkers including CD16, TIM3, PD-1, KIR and increased the expression of NKG2A of NK cells. (2) Calcitriol was able to inhibit NK cell proliferation and arrest them in G1 phase. (3) Calcitriol decreased the releasing of major inflammatory factors such as IL-5, IL-13, IFN-γ and TNF-α of NK cells.
(4) The anti-ageing effect of calcitriol on NK cells can be achieved through the Sirt1-pERK signal pathway.