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.

the recent launch of the Mars Curiosity rover has renewed international interest in space exploration, with crewed missions to asteroids, Moon, and Mars planned in the future. However, the impact of long-term spaceflight on human health is a considerable concern. One major challenge to crews on future expeditions is extended exposure to environments of microgravity (μg) (13, 17, 35, 36). Past studies have shown adverse effects of μg on several physiological systems, including a notable reduction of the adaptive immune response (35). To mitigate immune dysfunction in crews on future missions to Mars and beyond, it is critical to understand the mechanism of how μg impairs normal immune response; a collateral benefit is that spaceflight gives us a unique opportunity to study regulatory mechanisms of depressed immune response. As we discover novel regulatory checkpoints in early activation, some may prove useful drug targets for inflammatory diseases, such as rheumatoid arthritis, Crohn's disease, and celiac disease (16, 29, 39, 43). Because logistics and cost are major constraints to spaceflight studies of lymphocytes, it is essential to use alternate models that simulate μg to test hypotheses, design experimental parameters, and augment spaceflight experiments.

Immunosuppression during spaceflight was first observed in returning astronauts of the Apollo and Skylab missions (26). Over half of the Apollo astronauts experienced respiratory, gastrointestinal, urinary tract, or skin infections upon return to earth (19). Studies of returned Soyuz crew members also showed depressed lymphocyte activation compared with levels prespaceflight (27). Immunosuppression during spaceflight may increase the risk of opportunistic infections. Shuttle astronauts on short duration (11 day) spaceflight had significant increases in early viral gene transcription of the Epstein-Barr virus (EBV) compared with healthy controls, while astronauts onboard the ISS for long-duration (180 day) spaceflight had latent and lytic EBV gene expression that resembled activation patterns seen during infectious mononucleosis (13).

While some in vivo changes in astronaut lymphocyte function could be due to neuroendocrine stress factors, in in vitro studies of space-flown lymphocytes, our laboratory and others have demonstrated that immunosuppression occurs independent of systemic factors in μg (11, 12, 35). Studies carried out on several Spacelab flights showed a dramatic loss of human T-cell activation in flown peripheral blood leukocytes compared with ground controls. Spacelab 1 experiments saw a 97% depression in activation by concanavalin A (ConA) and subsequent spaceflights have demonstrated similar findings (12, 13). In particular, interleukin-2 (IL-2), interleukin-2 receptor α (IL-2Rα), interferon-γ (IFNγ), and tumor necrosis factor-α (TNFα) are significantly depressed in human T-cells under μg conditions (4, 7, 11). Experiments onboard the ISS comparing gene expression in activated μg and 1g onboard controls conclusively proved that μg causes immunosuppression in T-cells (7, 34).

Conducting experiments in an environment of true μg requires a roundtrip ticket into space, a feat that is both expensive and challenging. Simulated μg (sμg) models allow scientists to gather preliminary data without the cost and logistical challenges of spaceflight. Rotating wall vessels (RWVs) and three-dimensional clinostats, also called random positioning machines (RPMs), are two systems commonly used to simulate μg on the ground. The RPM was developed by Hoson et al. (21, 22) at Osaka City University and consists of two frames that rotate independently about distinct orthogonal axes while controlled by randomized software. Cells are placed on the inner frame and rotated in random directions and speeds. The gravity vector is forced into constant motion and can approach a residual force as low as 10⁻⁵g (24). RWVs were developed by the National Aeronautics and Space Administration (NASA) Johnson Space Center (JSC) Biotechnology Group as a model system for the low-shear, low-turbulence conditions predicted for cell culture in space (41). Suspended cells are rotated synchronously in the vessel such that the fluid dynamic effect on them mimics a particle allowed to free fall in a column of fluid. The time-averaged gravitational vector on individual cells is a residual 10⁻³g force that approximates μg (18, 33).

The objective of the present study was to evaluate the gene expression of several early signals known to change in spaceflight T-cell activation in mice splenocytes with those exposed to RPM- and RWV-sμg. We chose six key T-cell activation genes upregulated during activation and downregulated in μg. Four of the genes [interferon 2 (Il2), Il2rα, Ifnγ, and T-cell activation RhoGTPase activating protein (Tagap)] are known from our human studies of early T-cell activation during spaceflight (4, 7). We have discovered that two genes interferon-inducible GTPase (Iigp1) and signal lymphocytic activation molecule (Slamf1) are also early T-cell activation genes.

We measured gene activation and expression in T-cell response using real-time quantitative reverse transcription PCR (qRT-PCR); our results show that the patterns of gene regulation are similar for spaceflight and sμg. Activation of splenocytes in 1g caused an upregulation of all six early immune genes, whereas activation in μg and sμg produced a significant downregulation of activation. Corresponding measurements of cytokine protein confirm the trend of immunosuppression seen in cytokine mRNA gene induction. To our knowledge, this is the first study to directly compare gene activity between spaceflight and both primary ground models of sμg. Our findings confirm that RWV and RPM simulations provide excellent models of μg and are an important and useful resource for testing hypotheses before progressing to the true μg environment of spaceflight.

MATERIALS AND METHODS

Mice.

The spaceflight animals and ground controls were maintained at NASA's Kennedy Space Center Space Life Sciences Lab (KSC-SLSL) or the San Francisco Department of Veterans Affairs (VA), Veterinary Medical Unit Housing in accordance with the guidelines of the Institutional Animal Care and Use Committee and recommendations in the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996). Animal use in this study was approved by the NASA Ames Research Center/JSC Animal Care Committee or by the VA Animal Studies Subcommittee. All procedures were performed under license of these committees.

C57BL/6J wild-type female mice of similar age and weight were purchased from Jackson Laboratory (Bar Harbor, MI). Mice used for the RWV and RPM experiments were housed in the VA facilities and were used in accordance with the institutional guidelines for animal care. Mice used for shuttle transport system (STS)-131 spaceflight were shipped to the KSC-SLSL from San Francisco VA facility 10 wk before launch to ensure acclimation to environment and minimize stress from transit. Preflight weights were 22.02 ± 0.85 g for spaceflight mice and 22.02 ± 0.94 g for ground control mice. Postflight weights were 20.06 ± 1.73 g for spaceflight and 21.75 ± 0.69 g for ground conrol mice. When compared with ground mice, flight mice lost a small but statistically significant amount of weight. This was likely due to increased energy expenditure from the flight mice holding onto cages during the flight or flying around the cages (seen in video from other flights). All mice were of similar age at the time of experiment.

The Animal Enclosure Modules (AEMs) were provided by NASA Ames Research Center (Moffett Field, CA) for both spaceflight and ground controls in the spaceflight experiments. AEMs are widely used for rodent spaceflight housing. They are the standard and ideal habitat for rodent housing in spaceflight because they provide a constant and steady airflow that causes feces to flow into an air filter (1, 30). One day before launch, mice were handed over to Shuttle integration or to ground control facilities to be acclimated to the AEMs.

Spaceflight mice (n = 8) and ground mice (n = 8) were housed in AEMs and given identical food bar diets and water. The spaceflight mice were flown on board the Space Shuttle Discovery for 15 days; synchronous ground control mice held on the same sleep/wake cycle and at the same temperatures. Within 2.5 h after landing at KSC, ground and spaceflight mice were sedated, euthanized, according to IACUC institutional guidelines for animals and splenocytes were isolated and activated for cell culture and isolation.

Simulated microgravity conditions.

For RWV experiments, 10-ml disposable vessels (Fig. 1A) were used with a Rotary Cell Culture System (Synthecon, Houston TX). The system was placed in a 37°C incubator for experiments. For RPM experiments, 10-ml Nunc Opticells (Thermo Fisher Scientific, Rochester, NY) were placed on a desktop RPM (Dutch Science, Leiden, The Netherlands) at 37°C (Fig. 2).

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.

Cell culture and activation.

Splenocytes harvested from the spaceflight and ground mice were collected within 2.5 h at the KSC-SLSL as follows. Spleens were harvested and dissociated, and cells were run through a 70-μm cell strainer and treated with 1.5 ml 1× Pharm Lyse Buffer (BD Biosciences, San Jose, CA) for 7 min at room temperature. Medium was added and cells were centrifuged at 1,300 rpm for 10 min and filtered through a 40-μm cell strainer. All samples were cultured in RPMI medium supplemented with 1% (vol/vol) l-glutamine, antibiotics, glucose-pyruvate, 1.2% (vol/vol) HEPES buffer, and 10% (vol/vol) FBS and maintained at 37°C and 5% CO₂. Each mouse represented an independent biological sample.

Samples from spaceflight mice and ground mice were loaded into 12-well plates. Treated cells (n = 4 per gravity condition) were activated with 25 μl/1.5 ml Dynabeads Mouse T-Activator cluster of differentiation 3 (CD3)/cluster of differentiation 28 (CD28) (CD3/CD28, Invitrogen, Carlsbad, CA); nontreated cells (n = 4) were kept under the same conditions without bead activation. Cells were pelleted then fixed with 600 μl RNAlater (Ambion, Austin, TX) after 2.5 h and stored at −80°C until further analysis.

RWV and RPM splenocytes were harvested from C57BL/6J wild-type female mice (RWV, n = 4; RPM, n = 5) as described above. RWV samples were loaded into 10-ml RWVs, and RPM samples were loaded into 10-ml Nunc Opticell culture chambers. Splenocytes were conditioned to sμg in both RWV and RPM by prerotation of 1.5 h. Samples were activated with 60 μl/10 ml Dynabeads Mouse T-Activator CD3/CD28 for 2.5 h and fixed in RNAprotect (Qiagen, Valencia, CA). Control samples of 1g, in their respective vessels, were activated and placed in a static position. Nontreated samples were kept under the same conditions as the controls without bead activation. Cells were pelleted then fixed with 600 μl RNAprotect (Qiagen) after 2.5 h of activation and stored at −80°C until further analysis.

To test whether a prerotation period is necessary in the T-cell, sμg samples were incubated at 37°C and rotated for 0 and 1.5 h in RWVs before activation (Table 1).

Table 1.

Preconditioning is not necessary for the effects of splenocyte activation in sμg

	sμg Activated
1g Activated			0 h Prerotation		1.5 h Prerotation
Gene	Average Increase in Gene Induction	SD	Average Increase in Gene Induction	SD	Average Increase in Gene Induction	SD
IL-2	179.86‡	52.49	8.52‡	3.34	11.52‡	4.22
IFNγ	61.92‡	14.88	1.50‡	0.32	2.47‡	1.62
IIGP1	10.73‡	2.45	1.42‡	0.18	2.73‡	1.61
IL-2Rα	3.71‡	1.10	0.50‡	0.07	0.60‡	0.13
TAGAP	3.65‡	0.67	0.48‡	0.22	0.65	0.17
SLAMF1	2.78‡	0.71	0.45‡	0.09	0.63‡	0.17

Average increase of gene expression in simulated microgravity (sμg)-activated C57BL/6J WT mouse splenocytes with 0 h and 1.5 h prerotation. All activated samples were stimulated with Dynabeads Mouse T-Activator CD3/CD28 for 2.5 h; sμg-activated samples were prerotated in RWVs before activation. Each data point represents the mean ± SD of four independent biological samples. See text for definitions of abbreviations.

^‡P < 0.001 comparing 1g-activated and nontreated (data not shown) and μg-activated and 1g-activated samples.

RNA isolation and real-time quantitative RT-PCR.

RNA was isolated using the miRNeasy Mini Kit (Qiagen) according to the manufacturer's protocol. After isolation, samples were stored at −80°C until further analysis. For the RT reaction, RNA (0.3 μg) was added to 30 μl RT reaction buffer containing 5 mM MgCl₂, 10 mM Tris·HCl (pH 8.3), 50 mM KCl, 1 mM dNTP, 2.5 μM oligo d(T) primer, 2.5 U/μl murine leukemia virus reverse transcriptase, and 1 U/μl RNase inhibitor. The reaction was incubated at 25°C for 10 min, 37°C for 2 h, inactivated at 85°C for 5 min, and held at 4°C. cDNA (2 μl) from the RT reaction was added to 20 μl qRT-PCR containing 10 μl 2X-SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) and 12 pmol oligonucleotide primers. PCRs were carried out in a Bio-Rad CFX96 Touch Real-Time PCR Detection System or a Bio-Rad MyIQ Single Color Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA). The thermal profile was 50°C for 2 min, 95°C for 10 min to activate the Taq polymerase, followed by 40 amplification cycles, consisting of denaturation at 95°C for 1 min 40 s, annealing at 63°C for 1 min 10 s, and elongation at 72°C for 1 min 40 s. Fluorescence of the qRT-PCR reaction was measured using Bio-Rad CFX Manager software and used to quantify mRNA expression. At the end of the amplification period, melting curve analysis was performed to confirm the specificity of the amplicon. RNA samples were normalized to cyclophilin (Cphi) internal standard. Relative fold increase of gene expression was calculated by using the 2^ΔΔCt equation, where C_t represents the comparative threshold. Relative gene absolute abundance calculations were made by using the equation 1/(2^{Ct gene T−Ct CPHI T}) as previously described (25). The resulting values were then multiplied by 100,000 for better graphical presentation. All data derived using qRT-PCR was from multiple experiments with four or more independent biological samples. Data are representative of multiple experiments using mouse splenocytes: Human T cell activation also had similar immunosuppression in spaceflight, RPM, and RWV. Statistical analysis was performed using ANOVA with a post hoc Tukey test. Primers used were designed using OLIGO Primer Analysis Software (Molecular Biology Insights, Cascade, CO) in the Hughes-Fulford laboratory and manufactured by Eurofins Genomics (Huntsville, Al). In some cases, the sequences for primers were developed directly from Primer Bank (40).

Cytokines.

Splenocytes were isolated and activated in STS-131 flown (n = 8) and ground control (n = 8) mice as described above. After 22 h of activation, cells were pelleted and supernatants were collected. Concentrations of IL2 and IFNγ in supernatants were measured with the Mouse Cytokine 20-Plex Panel kit (Invitrogen) using the Bio-Plex 200 System (Bio-Rad). Because IL2 and IFNγ concentrations were high, supernatants from activated samples were diluted 10-fold to obtain accurate measurements.

IL2Rα ELISA.

Supernatants were collected as described above. Mouse IL2Rα was measured using a mouse IL2Rα ELISA kit (Corning Life Sciences, Corning, NY) according to the manufacturer's protocol. The optical density value was measured using a Spectra MR (Dynex, Chantilly, VA). Because IL2Rα concentrations were high, supernatants from activated samples were diluted 10-fold to obtain accurate measurements.

RESULTS

qRT-PCR was used to compare the expression of six specific early T-cell activation markers between spaceflight samples under true μg and under sμg in RWV and RPM samples (Figs. 3–5). True μg and sμg caused a blunted T-cell activation response in the mouse splenocyte gene expression patterns.

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. 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.

Gene expression in activated murine splenocytes in STS-131 spaceflight and ground controls.

Splenocytes were purified from mice that had been flown on the STS-131 mission in AEMs. Four or more independent biological samples were used in these experiments. To control for experimental variables, splenocytes were simultaneously purified from animals that had been kept in AEMs on the ground for the duration of the spaceflight. We selectively performed qRT-PCR on several genes that have been identified in early T-cell activation pathways. All splenocyte RNA samples were tested for quantitative expression of Il2, Il2rα, Ifnγ, Tagap, Iigp1, and Slamf1 (Fig. 3). Relative decrease of gene induction ranged from 28.15% up to 66.62% of ground-activated splenocytes. Expression of the housekeeping genes cyclophilin (Cphi) was unchanged between spaceflight and ground or 1g and sμg samples (data not shown). Activated samples (μg and ground) showed significant increases in expression over naïve controls (Fig. 3). Expression of all genes except Tagap decreased significantly in μg-activated cells stimulated with Dynabeads.

Gene expression in nontreated, 1g-activated and sμg-activated murine splenocytes.

To investigate the effects of sμg on early activation genes, we determined whether expression of immediate early genes was also inhibited in RWV and RPM sμg. Expression of Il2, Il2rα, Ifnγ, Tagap, Iigp1, and Slamf1 were measured by qRT-PCR (Figs. 4 and 5). For cells in both sμg environments, expression of all genes in 1g-activated samples increased significantly compared with nontreated control. Immunosuppression was almost complete in the μg models, with Ifny, Tagap, and Iigp1 failing to achieve significant activation over naïve controls in RWV-sμg (Fig. 4); in addition, Il2, Il2rα, Ifnγ, and Iigp1 were significantly suppressed in RPM-sμg (Fig. 5). In the RWV, immunosuppression ranged from 74.56% to as high as 95.30% of controls, showing a significant effect of sμg on immune activation. Suppression in the RPM was also significant, ranging from a 49.88% to 86.20% depression.

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.

Prerotation is not required for μg to inhibit expression.

In some cell types and pathways, a prerotation period is required for a downregulation of gene expression (20). To test the need to acclimate splenocytes to a sμg environment in RWV, gene expression was compared between naïve cells, bead activated (BA) 1g and μg samples that were either prerotated in sμg before activation or left at 1g for the preincubation period, and subsequently rotated in sμg after activation (Table 1). All genes for 1g-activated samples were significantly upregulated relative to the nontreated controls. All genes were significantly downregulated in both the 0 and 1.5-h prerotated BA sμg relative to the BA 1g samples, and there was no significant difference in expression between the two sμg conditions.

Chemokine and cytokine production are suppressed in μg.

Protein levels were measured for IL2, IL2Rα, and IFNγ in spaceflight and ground samples to confirm that immunosuppression in mRNA levels reflected changes in protein production. Gene expression of IL2, IL2Rα, and IFNγ were measured in cells from flown and ground animals that were fixed 22 h following activation with Dynabeads. Synthesis of all three proteins was significantly decreased in flight cell supernatants compared with ground controls and corresponded to the changes seen in mRNA levels (Table 2).

Table 2.

Immunosuppression of mRNA corresponds to suppression of protein synthesis

	1g	μg	% Inhibition
RNA fold increase over naïve spleneocytes
IL-2	1268.33‡	423.33†	66.62‡
IL-2Rα	16.36‡	9.86‡	39.73‡
IFNγ	20.05‡	9.07‡	54.76‡
Protein fold increase over naïve spleneocytes
IL-2	19.37‡	5.59	71.13‡
IL-2Rα	27.67‡	16.04‡	42.03‡
IFNγ	2174.64‡	527.18*	75.75‡

Fold change of mRNA and protein levels of IL2, IL2Rα, and IFNγ in STS-131 spaceflight (n = 8) versus ground controls (n = 8) showing a significant decrease in T-cell activation in both following spaceflight. Mice splenocytes were harvested from space-flown and ground control animals and subsequently activated for 2.5 h (mRNA) or 22 h (protein) with Dynabeads Mouse T-Activator CD3/CD28. qRT-PCR was used to analyze targets Il2, Il2rα, and Ifnγ in both spaceflight and ground samples. Protein levels in spaceflight and ground samples were measured using the Mouse Cytokine 20-Plex Panel kit (IL2, IFNγ) or an ELISA kit (IL2Rα).

^*P < 0.05;

^†P < 0.01;

^‡P < 0.001 using ANOVA with a post hoc Tukey test.

DISCUSSION

The immune system evolved under earth's gravity to defend against potentially harmful infectious agents. It is well established that spaceflight impairs normal immune function, with μg indicted as a primary culprit (7, 35, 36). Maintaining a balance of pathogen clearance and avoidance of self-attack requires strict regulation of activation pathways in the immune system. Impaired activation can cause dysfunctional immune responses and the inability to clear routine pathogens, such as in X-linked lymphoproliferative syndrome (XLP), which is characterized by extreme sensitivity to the EBV (15). Uncontrolled activation can lead to autoimmune disorders such as Sjögren's syndrome and systemic lupus erythematosus (39) and inflammatory diseases (16, 43). Immunosuppression during spaceflight has been recognized since the Apollo missions, and recent studies have highlighted the risk of opportunistic infection in astronauts, such as the reemergence of latent EBV in both short- and long-duration spaceflight (13).

Using the absence of gravity and its resultant immunosuppression also provides new opportunities to discover novel pathways controlling the immune system. Spaceflight experiments are by definition the ideal environment for studying cell function in μg. However, because of the expense and rare launch opportunities of sending studies into space, ground models of simulated μg are a necessary step in testing hypotheses and collecting preliminary data before advancing studies to spaceflight. The ability to activate lymphocytes in μg has been known for some time. Cogoli-Greuter and others have previously shown that cell-cell contact and aggregate formation are not impaired when cells are activated in both true and sμg (9, 10, 31). Levels of suppression in all six genes are more apparent in immediate exposure to sμg, as seen in RPM (49.88–86.20%) and RWV (74.56%-95.30%) sμg. The effects of μg persisted on return to normal gravity. Spaceflight animals were not in μg from deorbit until cell activation (∼12 h) and showed highly significant suppression (28.15%-66.62%) of immune activation for Il2, Il2rα, Ifnγ, Iigp1, and Slamf1.

This is the first time that the gene expression of activated mouse splenocytes and naïve splenocytes has been compared side-by-side with these three types of μg. We compared the profiles of six genes (Il2, Il2rα, Ifnγ, Iigp1, Slamf1, and Tagap) with increased expression in early T-cell activation. Il2, Il2rα, and Ifnγ were picked because of their well-known and key roles in lymphocyte activation and because they have been identified in several past studies as being downregulated in human T-cells during spaceflight (4, 7, 11). Tagap was selected due to our previous findings from human spaceflight experiments (4, 7). We recently discovered that Slamf1 and Iigp1 were downregulated 2.5 h after activation as shown by microarray analysis of splenocytes from mice flown on STS-131 (unpublished observations; M. Hughes-Fulford). Confirmation of the downregulation of these two genes in μg using qRT-PCR is reported here for the first time.

The transcription factor, c-Rel, plays a central role in early immune activation (7) and in the regulation of several of our six early activation genes studied here (Fig. 6). Iigp1 is an interferon-inducible GTPase in mice that belongs to a family of 47-kDa family of GTPases expressed early in the activation response and has been identified only in the mouse (3). Our data shows it is upregulated as early as 2.5 h after activation in mouse splenocytes. Iigp1 upregulation is reliant upon the presence of Ifnγ but is also strongly activated by T-cell receptor (TCR)/CD28-mediated activation (42). This is in agreement with our present study, where we found TCR/CD28-mediated activation upregulated Iigp1 expression in 1-g ground conditions. Further studies in murine T-cells have indicated that Iigp1 may be a potential target of c-Rel (5). This is consistent with the downregulation of Iigp1 seen in all three microgravity conditions we tested, since c-Rel itself experiences a significant reduction in gene expression in activated T-cells in spaceflight (7).

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.

Tagap is a T-cell activation-specific GTPase-activating protein. In humans, Tagap expression is strongest at 1.5 h and diminishes at later time points (unpublished observation; M. Hughes-Fulford). In spaceflight samples from mouse splenocytes, the immunosuppression by microgravity is not seen, perhaps due to the time delay from deorbit until activation. Two and a half hours after activation in the RWV there is significant immunosuppression of Tagap expression. In the RPM, there is suppressed activation of TAGAP compared with 1-g controls.

Slamf1 regulation and signaling has been characterized by several past studies and reviews (6, 8, 15, 23, 28). Slamf1 belongs to the signaling lymphocyte-activation molecule family that provides a costimulatory activation signal and is induced in naïve T-cells following activation. It has been implicated as an activation gene in macrophages and is expressed ∼3 h after IFNγ/TCR costimulation, with maximum expression 6 to 8 h after stimulation (8). Here we show that Slamf1 is upregulated as early as 2.5 h in mice splenocytes. After TCR stimulation, engagement of the SLAM receptor results in a costimulatory signal that later helps to fine-tune TCR signals (6, 8). Our 1g activation data confirm the upregulation of Slamf1 in early activation, and present novel evidence of its downregulation across all three models of μg.

Dysregulation of SLAM signaling has severe consequences. XLP is characterized by a mutation in the gene encoding SAP (SLAM-associated protein), which binds to activated SLAM. Patients with XLP are characterized by dysfunctional response to EBV infection. In normal lymphocytes, increased levels of SAP following TCR activation leads to decreased IFNγ production. In the absence of SAP, such as in XLP patients, IFNγ-producing cells hyperproliferate and T helper 2 (T_H2) cells develop abnormally (15). EBV is reactivated in astronauts during spaceflight, though the specific mechanism of reactivation has not yet been identified (13, 37). It is possible that the dysregulation of Slamf1 expression in μg contributes to the reemergence of latent EBV in short- and long-duration spaceflight.

As seen in Fig. 6, after binding SLAM, SAP recruits and activates the src kinase Fyn (14). The signaling pathway is also thought to include SHIP1, which associates with Fyn, and the adaptor molecules Dok1 and Dok2. Tyrosine phosphorylated Dok2 was found to associate with RasGAP. How this association affects downstream signals is unknown, but it is of interest as Ras is a well-known element of the TCR/CD28-mediated signaling pathway (28). In leprosy patients, activation of SLAM triggered a signaling cascade that induced activation of the NF-κB complex, resulting in increased IFNγ production (32). SLAM may also signal independently of SAP: studies in SAP-deficient mice showed that cross-linking SLAM resulted in increased IFNγ production and activation of the Ser/Thr kinase Akt independent of SAP (Fig. 6) (23). Collectively, these data demonstrate the complexity of the SLAM-mediated costimulatory pathway in T-cell activation. The downstream impact of Slamf1 dysregulation in spaceflight is an interesting question for further study, given the risk of opportunistic reemergence of EBV in astronauts (13).

Our qRT-PCR data confirms that gene expression of key genes in activated mice splenocytes placed in the RWV and RPM is very similar to the expression profile of the same genes analyzed in humans and mice flown on ISS. Although the fold change of gene expression with activation is higher in spaceflight samples than in ground sμg, the expression of key genes Il2, Il2rα, Ifnγ, Iigp1, Slamf1, and Tagap follow the same trend of immunosuppression in both ground sμg (RWV and RPM) and in spaceflight. Suppression of the six early activation genes is clearly demonstrated across all three models of μg.

Protein levels were not detectable at the 2.5-h time point for both ground- and spaceflight-activated samples. This is not surprising, since the induced gene expression is at an early time point and the splenocytes do not have the time required for transcription, translation, and accumulation of detectable levels of protein. We have been able to detect protein synthesis by multiplex methods 22 h after activation in splenocytes from spaceflight and ground mice; there was strong and significant suppression of the gene product, which correspond well with mRNA data (Table 2). IL2, IL2Rα, and IFNγ protein levels were significantly inhibited (42.03%-75.75%) and closely mirrored the trend and magnitude of inhibition of gene expression at 2.5 h (39.73%-66.62%). These data show that the effects of μg persist for some time following return to normal gravity. Variation in incubation chamber shape could account for the differences in the absolute values of activation seen across the models, as the shape of the container may affect aspects of T-cell/Dynabead interaction. However, the importance of these differences are minor in light of the strong overall trend of significant immunosuppression seen in gene induction in all three models of reduced gravity, and the overall performance the sμg models was excellent compared with true spaceflight.

Perspectives and Significance

Our results show that simulated models of microgravity induce immunosuppression in splenocytes similar to that seen in splenocytes from mice exposed to true microgravity in spaceflight. These findings demonstrate the crucial role of sμg models in illuminating novel mechanisms that regulate T-cell function and in augmenting spaceflight studies. Ultimately, insights into lymphocyte regulatory pathways gained from sμg and spaceflight studies will benefit not only future astronauts, but also those on earth who suffer from immune disorders.

GRANTS

This work was supported by National Aeronautics and Space Administration Grant NNX09AH21G and National Institute on Aging Grant 5UH3AG037628.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: E.M.M., M.C.Y., T.T.C., and M.H.-F. performed experiments; E.M.M., M.C.Y., T.T.C., and M.H.-F. analyzed data; E.M.M., M.C.Y., T.T.C., and M.H.-F. interpreted results of experiments; E.M.M., M.C.Y., T.T.C., and M.H.-F. prepared figures; E.M.M., M.C.Y., T.T.C., and M.H.-F. drafted manuscript; E.M.M., M.C.Y., T.T.C., and M.H.-F. edited and revised manuscript; E.M.M., M.C.Y., T.T.C., and M.H.-F. approved final version of manuscript; M.H.-F. conception and design of research.