Gene expression alterations in activated human T-cells induced by modeled microgravity.

ABSTRACT

Studies conducted in real Space and in ground-based microgravity analog systems (MAS) have demonstrated changes in numerous lymphocyte functions. In this investigation we explored whether the observed functional changes in lymphocytes in MAS are associated with changes in gene expression. NASA-developed Rotating Wall Vessel (RWV) bioreactor was utilized as a MAS. Activated T lymphocytes were obtained by adding 100 ng/ml of anti-CD3 and 100 U/ml of IL-2 in RPMI medium to blood donor mononuclear cells for 4 days. After that the cells were washed and additionally cultured for up to 2 weeks with media (RPMI, 10% FBS and 100 U/ml IL-2) replacement every 3-4 days. Flow cytometry analysis had proven that activated T lymphocytes were the only cells remaining in culture by that time. They were split into two portions, cultured for additional 24 h in either static or simulated microgravity conditions, and used for RNA extraction. The gene expression was assessed by Affymetrix GeneChip Human U133A array allowing screening for expression of 18,400 genes. About 4-8% of tested genes responded to MG by more than a 1.5-fold change in expression; however, reproducible changes were observed only in 89 genes. Ten of these genes were upregulated and 79 were downregulated. These genes were categorized by associated pathways and viewed graphically through histogram analysis. Separate histograms of each pathway were then constructed representing individual gene expression fold changes. Possible functional consequences of the identified reproducible gene expression changes are discussed.