Eomesodermin of atlantic salmon: an important regulator of cytolytic gene and interferon gamma expression in spleen lymphocytes

Abstract

Eomesodermin (Eomes), a T-bet homologue expressed in activated CD8+T cells was recently proposed to act as a master regulator of cytotoxic CD8+ T cell effector function and offers an exciting avenue for future exploration. Here, we have identified and characterized the full-length Atlantic salmon Eomes cDNA (2477 bp). Promoter analysis of the salmon Eomes showed the presence of important putative transcription binding sites like SP1, FOXO, Oct-1, SMAD, STAT, IRF, and Ets-1. The basal core region responsible for the promoter activity was located between base -199 and +59. Quantitative PCR analysis revealed that the Atlantic salmon Eomes was ubiquitously expressed in all the tissues studied but strongly expressed in the ovary, spleen, brain, and the head kidney. Moreover, the involvement of Eomes in Atlantic salmon immune response and its relation with the cytolytic activity was demonstrated by investigating the early time dependent expression profile of Eomes and CD8α followed by high interferon gamma (IFN-γ) and granzyme A expression during challenge with live Aeromonas salmonicida and Infectious Pancreatic Necrosis (IPN) virus. Therefore, we further analyzed the regulated expression and function of this transcription factor in spleen lymphocytes. Overexpression of Eomes induced IFN-γ, and granzyme A expression but not perforin expression, whereas small interfering RNA (siRNA) mediated suppression of Eomes expression led to significantly reduced IFN-γ production. Thus, Eomes may be critical in cytolytic gene expression and function in fish similar to mammals. Furthermore, IFN-α, and mitogens induced Eomes expression. Taken together, this is the first study on the promoter activity and regulatory role of Eomes in fish.

Figure 1. Nucleotide and deduced amino acid sequence of Atlantic salmon Eomes cDNA.

Uppercase denotes the UTR’s and lowercase denotes the coding regions. The T-box domain is underlined. Start and stop codons are shaded and marked with bold letters. The asterisk indicates the stop codon. The RNA instability motif (ATTTA) is underlined. The putative polyadenylation signal is bold and underlined.

Figure 2. Atlantic salmon Eomes promoter sequence

with consensus transcription factor binding sites. The consensus transcription binding sites were predicted by MatInspector and TRANSFAC. Putative transcription factor binding sites are underlined, while (−) sign indicates the binding sites identified on the negative strand. TATA box and transcription start site (+1) are shaded.

Figure 3. Structural and functional analysis of the 5′-upstream region of the salmon Eomes gene.

HeLa cells were transiently transfected with 0.3 µg of Eomes promoter/luciferase plasmid and 0.1 µg of Eomes promoter/SEAP plasmid (internal control). HeLa cells were stimulated with LPS and after 24 h luciferase activities were measured. Units of luciferase activity were normalized to activity of cotransfected pSEAP (relative luciferase activity). The error bars represent S.E.M values (n = 3). Asterisk (*) above the bars shows significant difference (P<0.05) compared to respective control and the different letters (a and b) on the bars denote significant difference compared to the smallest construct. The bent arrow represents the salmon Eomes transcriptional start site.

Figure 4. Tissue distribution of Eomes expression in Atlantic salmon.

Expression of salmon Eomes in different organs as detected by real-time PCR. Gene expression data were normalized to EF-1α expression using skin as a calibrator. Bar represents the mean ± S.E.M (n = 6). Asterisk (*) above the bar shows significant difference (P<0.05) compared with the organ that showed the lowest expression (skin). The value above the bars shows average real-time CT values of six fish.

Figure 5. In vivo regulation of Eomes expression in Atlantic salmon post infection.

(A) Tissue specific expression of granzyme A, IFN-γ, CD8α, and Eomes at different time-points after A. salmonicida challenge, and the overall correlation between them shown from top to bottom respectively, in the left panel. (B) Tissue specific expression of granzyme A, IFN-γ, CD8α, at different time-points after IPN virus challenge, and the overall correlation between them including Eomes shown from top to bottom respectively, in the right panel (Eomes expression not shown since there was no significant differences). Data were normalized to EF-1α expression at each time-points and presented as mean ± S.E.M (n = 6). Statistical differences (P<0.05, P<0.01, and P<0.001) between different time-points compared to control are indicated by asterisk (*, **, and ***) respectively, above the bars.

Figure 6. Regulators of Eomes expression in salmon lymphocytes.

Spleen leukocytes were stimulated for 24 h, 48 h, and 72 h with ConA+PHA+huIL-2, IFN-α (0.5 µg/ml, and 5 ng/ml), and the mRNA levels of Eomes (A), and IFN-γ (B) were determined by real-time PCR. Gene expression is normalized against EF-1α and is shown relative to the mean of the non-stimulated cells. Each bar represents the mean ± SE of triplicate samples. Different letters denote statistically significant differences between the groups.

Figure 7. Eomes mediated effects on salmon spleen lymphocytes.

Salmon leukocytes were transiently transfected with Eomes/pcDNA3.1 vector or control vector (pmaxGFP vector) (A) EomessiRNA or control siRNA (B). After 5 h, cells were stimulated with ConA+PHA+recombinant huIL-2. 24 h post stimulation, lymphocytes were sorted by FACS, collected and analyzed for the expression of Eomes, IFN-γ, granzymeA, T-box21 and EF-1α by QPCR. Eomes, IFN-γ, granzymeA, T-box21 mRNA expression in cells transfected with control siRNA were set to 100%. Statistical differences (P<0.005, and P<0.0004) between different treatment and control are indicated by asterisks (*, **) above the bars, respectively.

Figure 8. Immunofluorescence

study following in vitro modulation of Eomes in Atlantic salmon. (A) siRNA knockdown of Eomes expression (shown in pink) in spleen lymphocytes after 48 h. (B) Myc-labelled Eomes ectopic expression was detected with fluorescence labelling (pink) using alexafluor 594. Nuclear staining was performed with DAPI (blue).