Role of Luteal Cell-derived Exosomes in Communication with Immune Cells

Open Access
Author:
Branham, Katie L
Graduate Program:
Animal Science
Degree:
Master of Science
Document Type:
Master Thesis
Date of Defense:
April 03, 2015
Committee Members:
  • Joy Lee Pate, Thesis Advisor
  • Troy Ott, Thesis Advisor
  • Francisco Javier Diaz, Thesis Advisor
Keywords:
  • bovine
  • corpus luteum
  • exosomes
  • macrophages
  • T cells
  • immune cells
Abstract:
The corpus luteum (CL) is a vital organ for the production of progesterone (P4) which maintains pregnancy in ruminants. Immune cells infiltrate the CL and increase in number during luteal regression. These immune cells communicate with luteal cells to either sustain the CL for maintenance of pregnancy or promote regression of the CL, resulting in progression to the next estrous cycle. Luteal cell communication with T lymphocytes (T cells) can program the T cell responses, and luteal cells from fully functional CL can activate T cells through paracrine mediators. The mediators of this communication are not well understood. To investigate these interactions, we hypothesized that luteal cells secrete extracellular vesicles, such as exosomes, and that these exosomes serve as a means of communication from luteal steroidogenic cells to resident immune cells. Exosomes are 15-100 nm vesicles released from cells that contain protein, miRNA, and mRNA, which can later function of target cells. Currently, there is no research on the role of exosomes in the CL or the interaction between luteal cell-derived exosomes and the immune system. Thus, the objective of the first study was to determine an optimal method for the isolation of exosomes from luteal cell-conditioned media. Luteal cells were isolated and cultured for 48 hours. Media was collected, concentrated and exosomes were isolated by three methods: ultracentrifugation and two commercial exosome isolation reagents. Analysis of exosomes using electron microscopy revealed the expected cup-shaped morphology. Concentration of exosomes isolated from regressing luteal cell-conditioned media was less than from midcycle luteal cell-conditioned media. An experiment was conducted to determine if luteal cell-derived exosomes affected T cell function by interacting with surface markers on T cells or internalization. To determine if T cells can internalize luteal cell-derived exosomes, luteal cell-derived exosomes were co-cultured with T cells and analyzed using FlowSight imaging cytometry and confocal microscopy. This experiment revealed that the majority of T cells internalized exosomes. Next, a study was conducted to determine the effect of exosomes on T cell function. The maximum T cell proliferation achieved was 23%. T cells were also collected and their phenotype was determined by quantitative polymerase chain reaction (qPCR) for expression of TH1 (T helper cell 1) and TH2 (T helper cell 2) mRNA expression. Midcycle luteal cell-derived exosomes increased TNF and decreased IL10 mRNA expression while regressing luteal cell-derived exosomes decreased TNF, IFNG, IL4 and IL10 mRNA expression. Finally, an experiment was conducted to determine if luteal-derived exosomes affect monocyte differentiation and if so, determine the characteristics of the macrophages that arise. Midcycle or regressing luteal cell-derived exosomes were added to PBMCs along with GMCSF or MCSF and mRNA expression for macrophage cell type-specific genes was determined. Midcycle luteal cell-derived exosomes drove monocyte differentiation into macrophages that were characterized by highly significant expression of TNF, IL1B, NOS2, CD36, IL10 and TGFB while regressing luteal cell-derived exosomes induced greater expression of TNF, NOS2, CCL24 and IL10. In summary, bovine luteal cells from both physiological states released exosomes into culture media with luteal cells from regressing CL releasing fewer exosomes than midcycle luteal cells. Exosomes were internalized into T cells, and midcycle luteal cell-derived exosomes induced T cell proliferation. Also, midcycle luteal cell-derived exosomes were able induce a TH1 phenotype while regressing exosomes down regulated all T cell markers measured. Finally, midcycle and regressing luteal cell-derived exosomes drove monocyte differentiation that was dependent on both prior priming toward M1 or M2 and on source of the exosomes. In conclusion, bovine luteal cells release exosomes that may facilitate communication with immune cells and provide an alternative method to direct cell-cell contact by which luteal cells communicate with immune cells to control the luteal microenvironment.