FOXP3 Expression as a marker for suppressive immune cells in the gastrointestinal tract of hibernators

Abstract

Hibernating mammals rely on stored fat instead of ingested food as a fuel source during the winter. This prolonged fast affects various systems and causes them to make changes to survive the long hibernation season. One system that is greatly affected is the gastrointestinal (GI) tract, which shrinks in mass and loses some of its important barrier functions. The GI tract is home to a large number of immune cells in all mammals. The number of GI immune cells increases during hibernation, which would be a sign of inflammation in other animals. Hibernators, however, show no other signs of inflammatory disease. A possible explanation for this is that within the increased numbers of immune cells in gut of hibernators there are increased numbers of suppressive immune cells. The main type of suppressive immune cell is the regulatory T-cell (Treg). Tregs suppress other immune cells and prevent autoimmune disease. All Tregs express the protein FoxP3, which is a transcription factor responsible for the development and function of Tregs. I hypothesized that the increased numbers of immune cells in the small intestine of hibernators do not induce an inflammatory phenotype due to a larger percentage of FoxP3+ regulatory T-cells (Tregs) among them. In order to test this, I isolated intraepithelial lymphocytes and lamina propria leukocytes from small intestine of summer and hibernating thirteen-lined ground squirrels (Ictidomys tridecemlineatus) and analyzed them for FoxP3 expression using flow cytometry. I found general increases in FoxP3+ for LPL cells during hibernation compared to summer. Some variability exists in the hibernating animals based on season. CD3+FoxP3+ T-cells did not vary significantly between summer and hibernators, but a significant population of CD3loFoxP3+ or CD3-FoxP3+ cells were found in hibernators. These data suggest seasonal shifts in the intestinal immune phenotype of hibernators. qPCR results of the colon showed that FoxP3 RNA expression was not different between summer and either hibernating state, but late season hibernators had significantly more FoxP3 transcript than early season hibernators. The expression of FoxP3 RNA in the ileum did not vary significantly between summer and hibernators. These data indicate changes in leukocyte populations in the gastrointestinal tract of hibernating ground squirrels. Identification of seasonal changes in intestinal Treg populations will lead to future studies of how squirrels control immune activity during hibernation and could help identify the overactive components of the GI tract immune system in patients on total parenteral nutrition.