Title: Effect of toll-like receptor activation on thymosin beta-4 production by chicken macrophages Authors
|Kannan, Lakshmi -|
|Liyanage, Rohana -|
|Lay, Jackson -|
Submitted to: Molecular and Cellular Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 21, 2010
Publication Date: July 8, 2010
Citation: Kannan, L., Rath, N.C., Liyanage, R., Lay, Jr., J.O. 2010. Effect of toll-like receptor activation on thymosin beta 4 production by chicken macrophages. Molecular and Cellular Biochemistry. 344(1-2):55-63. Interpretive Summary: Thymosin beta 4 (Tb4) is a small peptide present in all cells. It is known to help in wound healing. However, it is not clear how this peptide is released from the cell to help in healing the process. Using a type of chicken immune cells grown in culture, we used chemicals to cause these cells to release Tb4. This research provides a mechanism on how local wound healing may be possible.
Technical Abstract: Thymosin beta 4 (Tb4) is an actin binding intracellular peptide that promotes wound healing, tissue remodeling, and angiogenesis. The regulation of Tb4 secretion to the extracellular environment is not understood. The macrophage is a rich source of Tb4 which also participates in wound healing process. Hence, the objective of this study was to find how Tb4 may be externalized. Using activation of macrophage through their toll-like receptors (TLR), the changes in cellular Tb4 were studied. Chicken HTC cells were treated with different TLR agonists and the cellular Tb4 changes was determined at 6 and 24 h after stimulations using stable isotope labelling of amino acids in cell culture (SILAC) and mass spectrometry. Real time PCR was used to determine changes in gene expression. The results showed that TLR agonists such as peptidoglycan or LPS caused depletions in cellular Tb4 peptide along with its detection in the cell culture supernatant at 24 h. These TLR agonists also induced the expression of interleukins-1b,-6, and nitric oxide synthase genes at 6 h but failed to modulate Tb4 gene expression indicating that the Tb4 externalization was not associated with its production. To find how Tb4 could have externalized, we determined lactate dehydrogenase (LDH) activity of the conditioned media as a measure of cell death. The results showed that the TLR agonists which induced depletion of intracellular Tb4 at 24 h also caused increases in LDH content of the conditioned media, suggesting that the Tb4 in the extracellular media originated from dying macrophages.