Effects of simulated microgravity on primary human NK cells

Abstract

The deleterious effects of microgravity on lymphocytes have been demonstrated in previous studies. However, research on the effects of microgravity on human natural killer (NK) cells remains exceedingly limited. In this study, we demonstrated that NK cell cytotoxicity was significantly decreased under simulated microgravity (SMG) conditions (p<0.05). Several processes, including apoptosis, receptor expression, and cytokine secretion, were investigated in human NK cells under SMG. We observed decreased cytotoxicity, concurrent with increased apoptosis and necrosis, in NK cells after exposure to SMG (p<0.05). Additionally, interferon (IFN)-γ and perforin expression decreased significantly, and the expression of granzyme-B was only slightly reduced. Meanwhile, SMG selectively inhibited the expression of certain surface receptors on NK cells. Specifically, the expression of NKG2A and NKG2D were significantly downregulated under SMG, but the expression of NKp30 and NKp44 was not affected. We also found that interleukin (IL)-15 alone or in combination with IL-12 could counteract the inhibition of NK cell cytotoxicity under SMG. Our findings indicate that human NK cells were sensitive to SMG, as reflected by their decreased cytotoxicity. Factors such as increased early apoptosis and late apoptosis/necrosis and the decreased expression of INF-γ, cytolytic proteins, and cell surface receptors may be responsible for the loss of cytotoxicity in human NK cells under SMG. A combination of IL-12 and IL-15 may be useful as a therapeutic strategy for overcoming the effects of microgravity on human NK cells during long space missions.

FIG. 1.

The 2-D RWV SMG system. The clinostat is equipped with computerized temperature and motor controls. Cells were initially seeded into the chambers. The chambers were filled with complete medium, and care was taken to exclude air bubbles. These chambers were rotated at 30 rpm around the horizontal or vertical axis. The 1GC controls were static control cultures kept in the same room as the clinostat. Color images available online at www.liebertonline.com/ast

FIG. 2.

Flow cytometry assay of primary human NK cells. Flow cytometry assay of primary human NK cells from one donor; 91.74% of the cells were NK cells. PBMCs were co-cultured with stimulating cells and harvested after 21 days of ex vivo expansion. All pellets were stained with CD56+16-PE and CD3-FITC mAbs and analyzed by flow cytometry. The percentage of NK cells (CD56⁺16⁺CD3⁻) in the PBMC population was tested.

FIG. 3.

Cytotoxicity of NK cells after exposure to SMG conditions for different durations. SMG-treated NK cells were mixed with K562 cells in an E:T ratio of 5:1. After co-culture for 4 h, CCK-8 was used to detect the remaining vital cells, and the killing rate (%) was calculated using the following equation: [1 − (OD_e+t − OD_e)/OD_t]×100% (OD_e, average OD₄₅₀ of triplicate wells for NK cell control; OD_t, average OD₄₅₀ of triplicate wells for K562 cell control; OD_e+t, average OD₄₅₀ of triplicate wells for NK cells plus K562 cells). The data represent the mean±SD of six independent experiments. Multivariate ANOVA and LSD test, *p<0.05 compared with the RC group; ^#p<0.05 compared with the 1GC group.

FIG. 4.

Apoptosis and necrosis of NK cells after 48 h of exposure to SMG. NK cells were stained with FITC-conjugated Annexin V and PI, followed by flow cytometry analysis. (A) A representative flow cytometry assay of NK cells from one donor. The percentages of viable (Annexin V−/PI−), early apoptotic (Annexin V+/PI−), and late apoptotic/necrotic (Annexin V+/PI+) populations of NK cells are shown. (B) The percentages of apoptotic cells are summarized in the bar graph; each column represents the mean±SD of four independent experiments. One-way ANOVA and LSD test, *p<0.05 compared with the RC group; ^#p<0.05 compared with the 1GC group.

FIG. 5.

IFN-γ and perforin secretion levels of NK cells after 48 h of exposure to SMG. NK cells were stimulated with K562 cells for 4 h, supernatants were collected, and the concentrations of IFN-γ and perforin were detected using the appropriate ELISA kits. Each sample was tested twice. The data represent the mean±SD of four independent experiments. One-way ANOVA and LSD test, *p<0.05 compared with the RC group, ^#p<0.05 compared with the 1GC group.

FIG. 6.

Protein expression profiles of NKG2A, NKG2D, NKp30, and NKp44 receptors on NK cells after 48 h of exposure to SMG. (A) Representative flow cytometry analysis of PBMC from one donor. NK cells were stained with PE-conjugated mAbs followed by flow cytometric analysis. (B) Data are for positive percentages and represent the mean±SD of four independent experiments. One-way ANOVA and LSD test, *p<0.05 compared with the RC group, ^#p<0.05 compared with the 1GC group.

FIG. 7.

Recovery of cytotoxicity in NK cells following exposure to SMG treatment. After 48 h of exposure to SMG, NK cells were removed and cultured under normal gravity conditions (1g), and cells were sampled from each group at 0, 3, 5, and 7 days to determine the cytotoxicity of the NK cells. The data represent the mean±SD of four independent experiments. Multivariate ANOVA and LSD test. *p<0.05 compared with the RC group, ^#p<0.05 compared with the 1GC group.

FIG. 8.

The effects of interleukins on the cytotoxicity of NK cells under SMG. Interleukins were supplied to each experimental group. The final concentration of each interleukin was IL-2, 500 U/mL; IL-12, 10 ng/mL; and IL-15, 10 ng/mL. The cytotoxicity of NK cells in each group was assayed as previously described. The data represent the mean±SD of six independent experiments. Multivariate ANOVA and LSD test, *p<0.05 compared with blank control.