Study Of Calcitriol Anti-aging Effects On Human Natural Killer Cells in Vitro Ⅱ

3. 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 release of inflammatory factors such as IL-5, IL-13, IFN-γ, and TNF-α and had certain antioxidation effects, which could contribute to its anti-aging effects.

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3.1 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 aging-related 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 (Figure 1a) (*P < 0.05) increased, while the expression of CD16 (Figure 1b), TIM3 (Figure 1c), PD-1 (Figure 1d), and KIR (Figure 1e) (*P < 0.05) were decreased after treatment with calcitriol for 72 h. The reversal effects were positively correlated with the concentration of calcitriol (Supplementary Figure 1). 

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 concentration-dependent shift toward the G1 phase (*P < 0.05), while the proportion of cells in the G2 phase (#P < 0.05) decreased. The proportion of calcitriol-treated 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 the 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.

3.2 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 pro-inflammatory 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.

3.3 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).

Figure 1. Aging-related phenotype, cell expansion, and cell cycle analysis of NK cells. (a-e) The human NK cells treated with calcitriol or rapamycin for 72 h showed the following cell biomarkers: natural killer group 2 member A (NKG2A), cluster of differentiation 16 (CD16), T cell immunoglobulin and mucin domain-containing protein 3 (TIM3) programmed cell death-1 (PD-1), and killer immunoglobulin-like receptor (KIR), which were related to aging. 50 nM rapamycin (RAPA) was used as the positive control. n = 3 for each group. *P < 0.05 and **P < 0.01 were compared with the negative control (NC) group. (f) NK cells (right) isolated from human peripheral blood mononuclear cells (PBMCs, left) by Magnetic bead sorting. (g) Cell proliferation analysis measured by cell counting kit 8 (CCK8). (h) Effect of calcitriol or rapamycin on the cel cyce in sorted nk cels after treatment for 72 h, wp  0.0,  0.05, and fp < 0.05 indicated the significant differences in the G1, S, and G2 phases, respectively. All the groups were compared with NC, respectively. n = 3. ) The percentage of T cellimmunoa globulin and immunoreceptor tyrosine-based inhibitory motif(M) domain (TGI)-positive NK cels after treatment with calcitriol or rapamycin for 72 h.*p < 0.05 and **p < 0.01 were compared with NC.n = 3.

3.4 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 H2O2 for 24 h and found that 100 μM was the nearest concentration to the median lethal dose (LD50) (Figure 3b). The expression of TIM3 in the NK cell population increased by the addition of H2O2, 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 H2O2 displayed elevated β- galactosidase activity. Calcitriol reduced the β- galactosidase-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 H2 O2 significantly decreased the cellular activity of NK cells compared to that of the untreated cells, while calcitriol increased the damage caused by H2 O2 (*P < 0.05) (Figure 3e).

Figure 2. Effects of calcitriol on the functions of NK cells. (a) Analysis of the cytokine released by the calcitriol-treated NK cells in  48 h. b) Fluorescence-activated cel sorting (FACS) results showed dead K562 cells labeled by 3,3 -octadecyl-oxacarbocyanineDlO) and propidium iodide (P).K562 cells were co-cultured with different ratios of calcitriol-treated Nk cells for 4 h. (C) FACSinalysis of dead K562 cels in killing assays. (d) The degranulation effects of treated Nk cells in killing assays. n = 3 for eaciyroup. *p < 0.05 and **p < 0.01 were compared with NC

3.5 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 H2O2 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 H2O2. However, we detected apoptotic proteins in NK cells under oxidative stress. Before treatment with H2O2, 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 H2O2. Calcitriol decreased H2O2-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 H2O2.

Figure 3. The anti-aging effects of calcitriol on NK cells. (a) The expression of the vitamin D receptor (VDR), sirtuin 1 (SIRT1), SIRT1- ∆Exon8, and protein kinase R-like endoplasmic reticulum kinase (pERK) in NK cells was detected by western blotting analysis after treatment with calcitriol for 48 h. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the loading control. Bar diagram illustrating the western blotting results (n = 3). *P < 0.05 and **P < 0.01 were compared with NC (n = 3). (b) Dosage mortality curve of NK cells after treatment with hydrogen peroxide (H2O2) for 24 h. (c) Percentage of TIM3 cells exhibiting high expression after treatment with 100 μM H2O2 for 24 h. (d) The senescence-associated β-galactosidase staining and analysis results. The senescent cells were stained in blue and indicated by red arrows. Scale bar, 50 μm. (e) NK cells were stained with Hoechst and Chloromethyl-X-Rosamine (CMXRos) to determine the cell location and mitochondrial membrane potential. The aging and apoptotic cells were Hoechst+ CMXRos− and indicated by white arrows. Scale bar, 50 μm. Statistical analysis showed relative fluorescence intensity. *P < 0.05 and **P < 0.01 were compared with H2O2 group in figures C, D, and E (n = 3).

Figure 4. The anti-apoptotic effects of calcitriol on NK cells. (a) Anti-apoptotic effects of calcitriol in H2O2-induced injury. FACS analysis showed the percentage of apoptotic NK cells after treatment with 100 μM H2O2 for 24 h. (b) The expression levels of caspase-3 p20 and caspase-3 p17 in NK cells were detected by western blotting analysis after treatment with 100 μM H2O2 for 24 h. GAPDH was used as the loading control. Bar diagram illustrating the western blotting results (n = 3). *P < 0.05 was compared with the H2O2 group.

4. 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 Figure 4. The anti-apoptotic effects of calcitriol on NK cells. (a) Anti-apoptotic effects of calcitriol in H2O2-induced injury. FACS analysis showed the percentage of apoptotic NK cells after treatment with 100 μM H2O2 for 24 h. (b) The expression levels of caspase-3 p20 and caspase-3 p17 in NK cells were detected by western blotting analysis after treatment with 100 μM H2O2 for 24 h. GAPDH was used as the loading control. Bar diagram illustrating the western blotting results (n = 3). *P < 0.05 was compared with the H2O2 group. BIOENGINEERED 6851calcitriol 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 is 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 the 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 H2O2. The oxidative aging induced by H2O2 increased the expression of TIM3 as well as the activity of β-galactosidase in NK cells. We found that calcitriol resisted 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.

5. 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 down-regulated 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 release of major inflammatory factors such as IL-5, IL-13, IFN-γ and TNF-α of NK cells.(4) The anti-aging effect of calcitriol on NK cells can be achieved through the Sirt1- pERK signal pathway. 

Acknowledgments We would like to thank all the staff members in our group for their support and the Shenzhen Science and Technology Innovation Committee for their funding support for this study.

Funding This work was financially supported by the Shenzhen Science and Technology Innovation Committee (JCYJ20170412155231633), Shenzhen Key Medical Discipline Construction Fund (No. SZXK062), Chengdu Wecistanche Biotech, and the Sanming Project of Medicine in Shenzhen (No. SZSM202011010); Shenzhen Science and Technology Innovation Committee [JCYJ20170412155231633]; Shenzhen Key Medical Discipline Construction Fund [No. SZXK062]; Sanming Project of Medicine in Shenzhen [No. SZSM202011010].

Ethics approval All procedures were approved by the Ethics Committee of Shenzhen Luohu People’s Hospital (ZLNK 12/2019, Shenzhen, China). Informed consent was obtained for experimentation with human samples. 

Disclosure statement All authors approved the submission of this manuscript. The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper.

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