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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online October 6, 2005 as doi:10.1096/fj.05-3778fje. |
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,||,1
* Lab of Cell Growth, Veteran’s Affairs Medical Center, San Francisco, California, USA;
Swiss Federal Institute of Technology, Zurich, Switzerland;
Lab of Cell Growth, Northern California Institute for Research and Education, San Francisco, California, USA;
Department of Physiological, Biochemical, and Cellular Sciences, University of Sassari, Sassari, Italy; and
|| Lab of Cell Growth, University of California San Francisco, San Francisco, California, USA
1Correspondence: Veteran’s Affairs Medical Center, 4150 Clement St., MC151F, San Francisco, CA 94121, USA. E-mail: millie.hughes-fulford{at}med.va.gov
SPECIFIC AIMS
We performed statistical microarray analysis of early activation of primary human T cells to identify genes induced in normal gravity and evaluate the differential transcriptional response in simulated freefall on the Random Positioning Machine, an environment much like microgravity experienced during space travel. We used cluster analyses and immunoblots against components of the PKA, PKC, and PI3-K pathways to find major gravity-sensitive signaling pathways regulating gene expression that could account for the increased susceptibility to infection documented in astronauts dating back to Apollo and Skylab.
PRINCIPAL FINDINGS
1. Gravity influences the gene expression distribution in T cells during activation
Microarray analysis of differential gene expression revealed that T cells treated with mitogen and CD28 costimulation in a vectorless gravity (vg) environment generated by a random positioning machine (RPM) failed to achieve the expression distribution characteristic of activation in normal gravity. The primary T cells studied exhibited significant changes in gene expression only after activation in normal gravity, while gene induction was suppressed in cells treated in vg. 99 genes were significantly up-regulated > 2-fold after activation at 1 g (Fig. 1
A). Only 9 of the 99 genes passed our criteria for statistically significant up-regulation in a vg environment. Principal component analysis of the sample profiles revealed that T cells under different treatment and gravity conditions clustered into separate quadrants reflecting their distinct gene expression signatures (Fig. 1B
). Activated T cells cultured in normal gravity clustered into a separate class in the second quadrant. Untreated and treated T cells cultured in vg clustered together in the third quadrant.
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2. Early T cell activation is dependent on key signaling pathways that are impaired in vectorless gravity
Cluster analysis, gene ontologies, and the literature on biological and molecular function of the significant genes were used to search for common regulatory elements and function. 28% of the significantly induced genes were either a component of the NF-
B signaling pathway, contained computed NF-B binding sites in the promoter, or had experimental evidence for transcriptional regulation by NF-B. Every gene in this group except OAS3 was suppressed in vg. The NF-B signaling pathway appeared to exert transcriptional control over the greatest number of genes that were significantly induced in normal gravity and suppressed in vg.
3. CREB transcription factor activation occurs in 1 g, but not in vg
We previously showed that PKA and PKC are key early regulators in T cell activation. Since the majority of the genes were regulated by NF-B, CREB, and AP-1, we studied the pathways that regulated these transcription factors. We found that the PKA pathway was down-regulated in vg. To validate the gene expression analysis, we performed immunoblots against phosphorylated CREB. T cells activated with Con A and anti-CD28 antibody for 30 min at 1 g showed significant up-regulation of CREB phosphorylation compared with 1 g controls (Fig. 2
A). In contrast, activation of CREB by phosphorylation failed to occur in T cells treated with mitogen in vg. The protein abundance of activated CREB was significantly different between the treated 1 g and vg samples. Since NF-B, AP-1, and CREB are all regulated by PKA and are transcription factors predicted by microarray analysis to be involved in the altered gene expression in vg, the data suggest that PKA is a key player in the loss of T cell activation in altered gravity.
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4. LAT adaptor protein is comparably activated in both 1 g and vg
PI3-K, PKC, and its upstream regulator pLAT were not significantly down-regulated by vg. To investigate PKC signaling pathway involvement, we performed immunoblots against phosphorylated linker of activation in T cells (LAT), an upstream adaptor protein that activates the PKC pathway. LAT phosphorylation occurred at significant levels after T cells were incubated with Con A and anti-CD28 antibody for 30 min in both 1 g and vg compared with unactivated controls (Fig. 2B
). Phosphorylation of PI3-K was not down-regulated in vg (Fig. 2C
).
5. Real-time RT-PCR confirms the transcriptional suppression of 10 genes integral to effective T cell-mediated immune response in vectorless gravity
Quantitative real-time PCR validated our microarray findings of early gene induction in 1 g and suppression in vg for IFNG, XCL2, IL2RA, IL2, CSF2, STAT1, LTA, TNFA, MIF, and NFKB1. All 10 genes examined experienced statistically significant up-regulation after activation in 1 g. Only CSF2, IL2RA, and XCL2 passed our criteria for statistically significant up-regulation in vg, and, even then, transcriptional induction of CSF2 was reduced by 53% and induction of IL2RA and XCL2 were both reduced by 95%. The real-time PCR data confirmed that cytokines, chemokines, and their receptors experienced the greatest degree of up-regulation after activation in 1 g and the steepest decline in relative induction in vg.
CONCLUSIONS AND SIGNIFICANCE
Immune suppression in the absence of gravity poses a novel barrier to the early immune response. Using microarray analysis, we found that 99 genes were significantly up-regulated during early T cell activation in normal gravity. However, when we removed the gravity vector, the majority of those genes showed no significant mitogen induced gene expression. The data presented here suggests that gravity is a key regulator of the cell-mediated immune response and that its absence either slows, impedes, or fully blocks signaling pathways essential for early T cell activation. We found that expression of downstream targets of signal transduction cascades, including genes involved in proliferation, apoptosis, biosynthesis, and secretion, are repressed in the absence of gravity (Fig. 3
).
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The transcription factors exerting the greatest regulatory control over gene expression during early T cell activation were NF-B, CREB, ELK, AP-1, and STAT, accounting for 59% of induced genes. As seen in Fig. 3
, activation of NF-B, CREB, ELK, and AP-1 can share subsets of the same upstream kinases to cross-talk with one another and achieve gene induction within the nucleus. Mechanical stimuli activate these same pathways that are inhibited in microgravity. We found that phosphorylation of both PKC and its upstream regulator pLAT were not down-regulated in vg.
NF-B and CREB were the top two transcription factors predicted by microarray analysis to be involved in gravity-mediated gene expression. Since the PKA signaling pathway regulates these transcription factors, we thought that PKA signaling could be affected by vg. We report here that the activation of CREB by phosphorylation within 30 min was significantly blocked by a vg environment (Fig. 2A
). The loss of CREB phosphorylation implicates a failure of the PKA signal transduction pathway during T cell exposure to mitogen in vg. PKA has been shown to be intricately involved in activating NF-B in addition to CREB. PKA may have a central role interconnecting CREB and NF-B needed during early induction of gene expression in normal gravity. We looked at the activation of PKC and PI3-K signaling pathways as well as LAT, an upstream activator of PLC
. pLAT is phosphorylated by 30 min after mitogen exposure; LAT phosphorylation was not affected by vg (Fig. 2B
). We found that PKC phosphorylation is intact in vg (data not shown). We detected no significant changes in PI3-K phosphorylation at either 1 g or vg by 30 min (Fig. 2C
).
Our immune system protects us from infection and disease, yet T cells lose their adaptability in spaceflight. This study builds and extends upon earlier discoveries that immune suppression is a phenomenon of manned space travel. The physical mechanisms by which the T cell responds to gravity remain an intriguing enigma and the Space Shuttle and International Space Station offer abundant opportunities for both in vitro and in vivo studies. The genes identified and classified in this study can serve as a springboard for further investigations into immune function in normal and altered gravity. Such studies will advance our understanding of the human immune system in health and disease and give us insight into and an appreciation for the fundamental biological laws governing Earth’s gravity-based life.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-3778fje;
Supported By Grants: VA Merit, NCC-2-1361 and GCRC M01RR0083.
This article has been cited by other articles:
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  H. L. Nichols, N. Zhang, and X. Wen Proteomics and genomics of microgravity Physiol Genomics, September 14, 2006; 26(3): 163 - 171. [Abstract] [Full Text] [PDF]
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