| In mammals, it is well documented that forkhead box protein 3 (Foxp3) is a crucial transcription regulator in the generation and function of CD4+CD25+ regulatory T cells (Tregs), which are essential mediators of peripheral immunological tolerance by active suppression. It is ever thought that Foxp3 is specific in CD4+CD25+Tregs. However, several findings have overturned the paradigm and indicate that Foxp3 is also expressed in non-hematopoietic cells, including epithelial cells of breast, lung and prostate, tumor cells of different tissue origins. Collectively, these data imply that Foxp3 might have broad biological roles outside of Tregs in mammals.The teleost immune system possesses most elements of the adaptive immune system present in mammals. In addition, like in mammals, T-cell receptor repertoire is generated through complex re-combinatorial and mutational processes, during which potentially harmful self-reactive immune receptors can be generated. In theory, teleosts can also produce auto-reactive lymphocytes, but it is still unclear whether teleosts possess functional immunity regulation cell or molecule like Foxp3 in mammals. In recent years, the ortholog of the mammalian Foxp3 has been identified in zebrafish (Danio rerio), rainbow trout(Oncorhynchus mykiss), pufferfish (Tetraodon nigroviridis) Atlantic salmon(Salmo salar), grass carp (Ctenopharyngodon idellus) and Nile tilapia (Oreochromis niloticus). However, most study were confined to mRNA levels, the expression of Foxp3 in protein and cellular levels remains to be clarified in fish. In our previous study, the tilapia (tilapia referred to Nile tilapia (Oreochromis niloticus)) Foxp3 cDNA was isolated and identified, moreover, the N-terminus of full-length Foxp3 (110 aa) (s-ntFoxp3) was expressed in E.coli. In the present study, the recombinant protein of s-ntFoxp3 was purified and used to produce a polyclonal antibody (Ab) specific to tilapia Foxp3, namely aFoxp3.The specificity of aFoxp3 was detected by SDS-PAGE and Western blot. The expression of Foxp3 was further studied by immunohistochemistry (IHC) and flow cytometry. On the other hand, the experimental autoimmune encephalomyelitis (EAE) model was constructed by immunizing tilapia with the automous brain proteins. In order to investigate the biological acitivity of Foxp3 in EAE, its expression changes in different tissues were detected during the process of EAE. The results were as follows.1) The results by SDS-PAGE showed that the recombinant protein s-ntFoxp3 has the same molecular weight with the predicted size. By Western blotting analysis, one strong band with ～44 kD of molecular weight which is compatible with the predicted size of F-Foxp3 was detected in the crude protein extracts of the thymus, spleen, head kidney, intestine, stomach, kidney, and gills from the Nile tilapia by aFoxp3, whereas no band was detected in the crude protein extracts of the muscle. foxp3 expression at the mRNA level was investigated by RT-PCR, and the results were essentially consistent with those of Western blotting. Taken together, our results indicate that aFoxp3 has high specificity and can provide a valuable research tool for further study.2) IHC analysis indicates that Foxp3 was expressed in peripheral blood mononuclear cells (PBMC) and certain packed lymphocytes in particular, Moreover, flow cytometry analysis indicates that the percentage of Foxp3+ cells among PBMC was approximately 5.7±2.0%(n=5) in healthy adults and could be significantly up-regulated after phytohemagglutinin (50 μg/ml) stimulation in vitro at 6,12 and 24 h, up to 21.90%,19.1%,7.86% after PHA stimulation comparing 7.38% 6.47%,2.7% in the controls.3) The results of IHC analysis suggest that the cellular expression patterns of Foxp3 might be conserved and predominantly limited to lymphocyte lineage in thymus, spleen, head kidney, etc; Foxp3 at mRNA and protein levels was detected in the non-lymphoid tissue stomach and in the non-hematopoietic cells.4) After immunization tilapia by intraperitoneal injection with it’s brain tissue homogenate emulsified in FIA, the behavior and body weight of tilapias were observed at different times, moreover, morphological changes of the brain, kidney, intestine and liver sections were detected by HE staining. Comparing with the control, our results showed that the automous brain protein-immunized tilapias appeared some deviant changes, such as ambulation slow, response brady, food intake reduced and body weight gain decreased; the brain tissue sections in experimental group appeared typical morphological changes similar to those of mammalian EAE after 12 days immunization, such as inflammation infiltration, meningeal thickening, perivascular cuffing, and no obvious morphological changes were observed after 1,3,6, 7,9 days immunization. In addition, some pathological changes, such as hyperemia and inflammation infiltration, were observed in the immune-related tissues. The results show that the tilapia could develop autoimmune diseases such as EAE after immunization by automous brain tissue homogenate emulsified in FIA.5) During 1-12 days after immunization, the percentage of Foxp3+ cells among PBMC was significantly up-regulated, with 13.5% and 7.98% in the control increasing to 63.6% and 16.2% at 1,12 d, respectively. From 15 d onset, the percentage of Foxp3+ cells among PBMC decreased, and was even lower than the control group, with 5.37%, 5.93% in the control decreasing to 3.34% and 2.78% at 15 and 18 d, respectively. Moreover, the expression of Foxp3 at mRNA and protein levels in spleen, head kidney and kidney were detected by real-time PCR and IHC. Our results indicated that the expression of Foxp3 were significantly up-regulated in the above-mentioned tissues during 1-12 days after immunization and down-regulated from 15 donwards, which was consistent with the results by the flow cytometry analysis. Therefore, it is reasonable to propose that Foxp3 might be involved with the onset and development of the autoimmune diseases such as EAE in lower vertebrates like tilapia.In the present study, an Ab specific to ntFoxp3 was produced and characterized. To our best knowledge, it is the first time to demonstrate that Foxp3 protein expression occur not only in hematopoietic cells of lymphoid organ systems but also non-hematopoietic cells of non-lymphoid organ in fish. Conservative cellular expression of Foxp3 in teleost and mammalian suggest that Foxp3 may have conservative biological function. On the other hand, EAE in tilapia was constructed successfully, and the expression of Foxp3 detected by flow cytometry analysis, IHC and real-time PCR suggest that Foxp3 might play an important role in autoimmune diseases such as EAE in lower vertebrates like fish tilapia. |
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