The effects of Mycobacteria and xenobiotics on the expression of pro-inflammatory proteins in fish phagocytes

The capacity of macrophages to become fully activated is important for the elimination of intracellular pathogens such as Mycobacteria. Given the importance of certain proteins related to inflammation in mammals, this research planned to first identify novel pro-inflammatory proteins in innate resistance of teleosts to serve as biological markers of inflammation in fish. A cDNA library was constructed from striped bass, Morone saxatilis, leukocytes isolated from animals infected with Mycobacterium fortuitum, which was screened with a polyclonal antibody generated against striped bass macrophages. Phage containing striped bass leukocyte cDNA positively recognized by the striped bass macrophage antibody were excised and re-circularized into plasmids for transformation into E. coli. Once propagated, plasmids were purified, sequenced, and analyzed using the NCBI BLAST search within the GenBank(TM) database. Two cDNA products were identified as being metallothionein (MT) and protein kinase C-delta (PKC-delta). These cDNA products were cloned into affinity protein expression vectors and recombinant protein expression was induced. Purified recombinant proteins were then used to generate polyclonal antibodies.; The next objective of this research was to determine the expression profile of pro-inflammatory proteins and monitor oxidative burst activity in channel catfish and striped bass leukocytes following exposure to Mycobacteria and xenobiotic compounds. Along with antibodies generated against MT and PKC-delta recombinant proteins, antibodies specific for inducible nitric oxide synthase (iNOS), natural resistance associated macrophage protein (nRAMP), heme oxygenase (HO), and cytochrome P450 1A (CYP1A) were used to probe fish leukocytes for expression. Differences were observed between channel catfish and striped bass leukocytes in response to M. fortuitum whole cells and extracellular products (ECP), observations showing no expression of nRAMP1 in striped bass leukocytes and the induced expression of iNOS due to ECP exposure. The cellular function assays revealed striped bass leukocytes displayed increased oxidative burst activity when compared to channel catfish accounted for by interspecies differences, and oxidative burst activity was decreased due to exposure with whole cell M. fortuitum. Therefore, this project has allowed the researchers to identify additional pro-inflammatory proteins in innate resistance of teleosts, and has used them as markers of inflammation along with previously described indicators of inflammation.