Effect of toll-like receptor activation on thymosin beta-4 production by chicken macrophages

Thymosin beta-4 (Tβ4) is an actin-binding intracellular peptide that promotes wound healing, tissue remodeling, and angiogenesis. The mechanism of Tβ4 secretion to the extracellular environment is not understood. The macrophage is a rich source of Tβ4 which also participates in wound healing process. The objective of this study was to find how Tβ4 may be externalized. Using activation of macrophage through their toll-like receptors (TLR), the changes in cellular Tβ4 was studied. A naturally transformed chicken macrophage cell line HTC was treated with different TLR agonists and the cellular Tβ4 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 (PGN) or lipopolysacharide (LPS) caused depletions in cellular Tβ4 peptide along with its detection in the cell culture supernatant at 24 h. These TLR agonists also induced the expression of interleukins-1β, -6, and nitric oxide synthase genes at 6 h but failed to modulate Tβ4 gene at that time point indicating that the Tβ4 externalization was not associated with its production. To find whether Tβ4 externalization was associated with cell death, we measured the lactate dehydrogenase (LDH) activity of the conditioned media as an indicator of cell damage. The results showed that the TLR agonists which induced depletion of intracellular Tβ4 at 24 h also increased the LDH content of the conditioned media, suggesting that the Tβ4 in the extracellular media most likely originated from dying macrophages.