Transcription Factor T-Bet in Atlantic Salmon: Characterization and Gene Expression in Mucosal Tissues during Aeromonas Salmonicida Infection

Abstract

The T-box transcription factor T-bet is expressed in a number of hematopoietic cell types in mammals and plays an essential role in the lineage determination of Th1 T-helper cells and is considered as an essential feature for both innate and adaptive immune responses in higher vertebrates. In the present study, we have identified and characterized the full-length Atlantic salmon T-bet cDNA (3502 bp). The putative primary structure of the polypeptide deduced from the cDNA sequence contained 612 aa, which possessed a T-box DNA binding domain. Phylogenetic study and gene synteny revealed it is as a homolog to mammalian T-bet. Quantitative PCR analysis of different tissues in healthy fish showed that salmon T-bet gene was highly expressed in spleen, followed by head kidney, and was expressed in intestine, skin, and liver at lower levels. Moreover, the time-dependent expression profile of T-bet, interferon gamma (IFNγ), interleukin-22 (IL-22), and natural killer enhancement factor in mucosal tissues during water-borne infection with live Aeromonas salmonicida, indicated the involvement of T-bet in mucosal immune response in Atlantic salmon.

Studies on the effects of LPS, ß‐glucan and metabolic inhibitors on the respiratory burst and gene expression in Atlantic salmon macrophages

“…The procedure was the same as described by Kumari et al. (). In short, the reaction mixtures were incubated at 95°C (10 min), thereafter, 40 cycles of 95°C (15 s), 60°C (1 min) and 95°C for 15 s. Amplifications were specific in all cases, and amplification was not observed in any of the negative controls (non‐template control).…”

“…Invitrogen, USA). To synthesize first-strand cDNA, a SuperScript III RNase reverse transcriptase (Invitrogen) was used, as described by Kumari et al (2015).…”

“…The procedure was the same as described by Kumari et al (2015). In short, the reaction mixtures were incubated at 95°C (10 min), thereafter, 40 cycles of 95°C (15 s), 60°C (1 min) and 95°C for 15 s.…”

Overexpression of T-bet, GATA-3 and TGF-ß Induces IFN-γ, IL-4/13A, and IL-17A Expression in Atlantic Salmon

“…For the construction of plasmid-encoding TGF-ß, T-bet and GATA-3 genes with the reporter gene (RFP)-Tag, the open reading frames (ORFs) of salmon TGF-ß gene (BT059581), T-bet (GU979861) and GATA-3 (EU418015) were all retrieved from the GenBank and cloned, as described by Kumari et al (2009Kumari et al ( , 2015 [30,31]. From the spleen cDNA library, the genes were sub-cloned into a pTagRFP-N vector (Evrogen) by PCR, with the aid of restriction enzymes SacII and Xho I.…”

“…RNA isolation and cDNA synthesis were performed according to Kumari et al (2015) [31]. The synthesized cDNA was diluted 10-fold with MilliQ water, and 5 μL of this dilution was used as a template in a 20 μL reaction volume.…”