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 calcitriol's anti-aging and immunomodulatory effects 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 is 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.

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1. 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 inflame aging 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 anti-infection 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 antibody-specific 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], a cluster of differentiation 16 (CD16) [8] increased. This molecule could activate the killing function of NK cells, which may lead to cellular imbalance and inflammation in the 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 primary function of vitamin D is to promote the absorption of calcium and phosphorus. Vitamin D also regulates immune functions [14]. Although many reports have focused on its anti-infection and anti-tumor functions [15], calcitriol exhibits anti-inflammatory effects as well [16]. The vitamin D receptor (VDR) is expressed on various immune cells [17]. A 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 anti-inflammatory 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 full-length 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.

2. Materials and methods 

2.1 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. 

2.2 Flow cytometric analysis

After treatment with calcitriol for 72 h, the NK cells were stained with anti-human FITC-CD3, PerCp-CD56, APC-CD16, PE-NKG2A, PE-TIM3, PE-PD1, and PE-KIR (BioLegend, California, USA). All the FACS assays were performed using DxFLEX Flow Cytometer (Beckman, California, USA).

2.3 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].

2.4 NK cell-killing assay

The human erythroleukemic cells, K562 cells (ATCC, Virginia, USA), were incubated with  10 uM 3,3 -dioctadecyl-oxacarbocyanine (DIOBeyotime, Shanghai, China) for 15 min and then added to the calcitriol-treated NK cells at different ratios (E/T' = 1:1, 5:1 and 10:1) for 4 h, respec-tively (27].

2.5 Oxidative senescence induction and X-gal staining analysis 

The in vitro NK cell aging model using hydrogen peroxide (H2O2). In order to determine the anti-oxidation 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.

2.6 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. 

2.7 Western blotting 

The cells were lysed using the radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China) with phosphatase inhibitor PhosSTOP (Roche, Basel, Switzerland) and 1 μM phenylmethylsulfonyl fluoride (PMSF) (Beyotime, Shanghai, China). Total protein was quantified using the Bradford Protein Assay Kit (TAKARA, Tokyo, Japan). The protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to 0.22 μm polyvinylidene fluoride (PVDF) membranes (Immobilon-P membrane; Millipore, Massachusetts, USA). After blocking, the membranes were probed with primary antibodies and incubated with the secondary antibodies [30]. Membranes were detected using the Odyssey system (LI-COR, Nebraska, USA).

2.8 Statistical analysis

The data was analyzed by one-way analysis of variance (ANOVA) using GraphPad Prism and presented as mean ± standard deviation (SD). 6846 W. LI ET AL.Differences were considered statistically significant at P < 0.05. The results were also analyzed using the GraphPad software.

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