Spaceflight and simulated microgravity cause a significant reduction of key gene expression in early T-cell activation

Abstract

Healthy immune function depends on precise regulation of lymphocyte activation. During the National Aeronautics and Space Administration (NASA) Apollo and Shuttle eras, multiple spaceflight studies showed depressed lymphocyte activity under microgravity (μg) conditions. Scientists on the ground use two models of simulated μg (sμg): 1) the rotating wall vessel (RWV) and 2) the random positioning machine (RPM), to study the effects of altered gravity on cell function before advancing research to the true μg when spaceflight opportunities become available on the International Space Station (ISS). The objective of this study is to compare the effects of true μg and sμg on the expression of key early T-cell activation genes in mouse splenocytes from spaceflight and ground animals. For the first time, we compared all three conditions of microgravity spaceflight, RPM, and RWV during immune gene activation of Il2, Il2rα, Ifnγ, and Tagap; moreover, we confirm two new early T-cell activation genes, Iigp1 and Slamf1. Gene expression for all samples was analyzed using quantitative real-time PCR (qRT-PCR). Our results demonstrate significantly increased gene expression in activated ground samples with suppression of mouse immune function in spaceflight, RPM, and RWV samples. These findings indicate that sμg models provide an excellent test bed for scientists to develop baseline studies and augment true μg in spaceflight experiments. Ultimately, sμg and spaceflight studies in lymphocytes may provide insight into novel regulatory pathways, benefiting both future astronauts and those here on earth suffering from immune disorders.

Keywords: T-cell activation; immunosuppression; spaceflight and microgravity.

Fig. 1.

Rotating wall vessel (RWV) culture system. A: RWV attached to Rotary Cell Culture System (Synthecon) B: up-close RWV. The RWV holds 10 ml of cell culture medium and contains an internal membrane to facilitate gas exchange. Cells in the RWV are rotated synchronously such that the time averaged gravitational vector on cells is a 10⁻³ g force that approximates microgravity (μg).

Fig. 2.

Random positioning machine (RPM) and Opticell System. A: desktop RPM (Dutch Space, Leiden, The Netherlands). B: up-close OptiCell vessel. The OptiCell holds 10 ml of cell culture medium and the outer membrane is semipermeable, allowing for gas exchange. Cells on the RPM are rotated at random directions and speeds forcing the gravity vector to approach a residual force as low as 10⁻⁵ g.

Fig. 3.

Gene expression in STS-131 spaceflight and ground samples. Six key gene expressions in STS-131 spaceflight versus ground controls, indicating a significant decrease in early T-cell activation in spaceflight. Mice splenocytes were harvested from spaceflown and ground control animals and subsequently activated for 2.5 h. qRT-PCR was used to analyze targets Il2, Il2rα, Ifnγ, Tagap, Iigp1, and Slamf1 in both spaceflight and ground samples. Each data point represents the mean ± SD of four independent biological samples. Error bars represent the standard deviation. *P < 0.05; **P < 0.01; ***P < 0.001 using ANOVA with a post hoc Tukey test.

Fig. 4.

Gene expression in simulated μg samples using RWVs. Gene expression of harvested mouse splenocytes placed in RWVs simulating μg (sμg) versus 1g controls, indicating a significant decrease in early T-cell activation. Samples were prerotated in RWVs for 1 h to adjust them to the simulated μg environment and then activated for 2.5 h. qRT-PCR was used to measure expression of Il2, Il2rα, Ifnγ, Tagap, Iigp1, and Slamf1. Each data point represents the mean ± SD of four independent biological samples. Error bars represent the standard deviation. *P < 0.05; **P < 0.01; ***P < 0.001 using ANOVA with a post hoc Tukey test.

Fig. 5.

Gene expression in simulated μg samples using RPM. Gene expression of harvested mouse splenocytes placed in a RPM sμg versus 1g controls, indicating a significant decrease in early T-cell activation. Samples were prerotated in the RPM for 24 h to adjust them to the sμg environment and then activated for 2.5 h. qRT-PCR was used to measure expression of Il2, Il2rα, Ifnγ, Tagap, Iigp1, and Slamf1. Samples were prerotated in the RPM for 24 h to adjust them to the sμg environment and then activated for 2.5 h. Each data point represents the mean ± SD of five independent biological samples. Error bars represent the standard deviation. *P < 0.05; **P < 0.01; ***P < 0.001 using ANOVA with a post hoc Tukey test.

Fig. 6.

T-cell activation pathway regulating six key activation genes. Hypothesized T-cell activation pathway, curated from the literature, involving six key early activation genes (Il2, Il2rα, Ifnγ, Tagap, Iigp1, and Slamf1) (2, 5, 6, 8, 15, 23, 28, 32, 38, 42). Pathway shows the interactions between signaling pathways regulating transcription of the six key genes investigated. Red discs represent receptors. Orange, yellow, green and light blue discs represent kinases, adaptor proteins, inhibitors and transcription factors, respectively. Dark blue and purple discs represent GTPases and GTPase-activating proteins.