Apoptosis and differential gene expression of Mycobacterium bovis-infected bovine macrophages

Mycobacteria are one of the most successful bacterial parasites because they can survive in the hostile environment of the macrophage. In the present study, an extensive search for bovine macrophage genes differentially expressed at 0, 12, and 24h after Mycobacterium bovis infection was conducted. Differential expression of 7 genes was successfully confirmed. Sequence homology identified probe 18 as thymosin beta-10, probe 26 as a cysteine protease, probe 27 as the signaling lymphocytic activation molecule, probe 30 as the alveolar macrophage-derived chemotactic factor II, probe 35 as ferritin heavy chain, probe 36 as osteopontin and probe 47 as the serum amyloid A protein.; M. bovis infection induced macrophage apoptosis. Macrophages infected with a MOI of 25:1 developed chromatin condensation and DNA fragmentation at 4h and 8h respectively, whereas, changes in chromatin condensation induced by MOIs of 10:1 and 1:1 required a longer time and had a reduced number of apoptotic cells.; M. bovis infection induced both upregulation of thymosin beta-10 mRNA and apoptosis in bovine monocyte-derived macrophages suggesting that macrophage apoptosis may be linked to the overexpression of thymosin beta-10 which is known to prevent actin polymerization and accelerate apoptosis. In an attempt to link the upregulation of thymosin beta-10 in macrophages with the induction of apoptosis, overexpression and antisense analysis of a mouse macrophage cell line transfected with a construct carrying the thymosin beta-10 full length cDNA in the sense orientation and with a second construct containing a shorter version in the opposite orientation were performed. All the clones overexpressing the transgene were found to undergo apoptosis at higher rates than parental cells, with statistical differences detected in two of six clones. The expression of thymosin beta-10 in the antisense orientation did not ameliorate the rate of apoptosis. Although results from this study are not conclusive, our evidence links the upregulation of thymosin beta-10 in M. bovis infected macrophages with the increase in cell death through apoptosis.