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Altered gene expression patterns of innate and adaptive immunity pathways in transgenic rainbow trout harboring Cecropin P1 transgene.

Lo JH, Lin CM, Chen MJ, Chen TT - BMC Genomics (2014)

Bottom Line: From the microarray data, a total of 2480 genes in the spleen, 3022 in the kidney, and 2102 in the liver were determined as differentially expressed genes (DEGs) in the cecropin P1 transgenic rainbow trout when compared to the non-transgenics.The identified genes involved in different pathways related to immune function are valuable indicators associated with enhanced host immunity.These genes may serve as markers for selective breeding of rainbow trout or other aquaculture important fish species bearing traits of disease resistance.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT 06269, USA. Thomas.Chen@uconn.edu.

ABSTRACT

Background: We have recently developed several homozygous families of transgenic rainbow trout harbouring cecropin P1 transgene. These fish exhibit resistance characteristic to infection by Aeromonas salmonicida and infectious hematopoietic necrosis virus (IHNV). In our earlier studies we have reported that treatment of a rainbow trout macrophage cell line (RTS11) with a linear cationic α-helical antimicrobial peptide (e.g., cecropin B) resulted in elevated levels of expression of two pro-inflammatory relevant genes (e.g., IL-1β and COX-2). Therefore, we hypothesized that in addition to the direct antimicrobial activity of cecropin P1 in the disease resistant transgenic rainbow trout, this antimicrobial peptide may also affect the expression of immune relevant genes in the host. To confirm this hypothesis, we launched a study to determine the global gene expression profiles in three immune competent organs of cecropin P1 transgenic rainbow trout by using a 44k salmonid microarray.

Results: From the microarray data, a total of 2480 genes in the spleen, 3022 in the kidney, and 2102 in the liver were determined as differentially expressed genes (DEGs) in the cecropin P1 transgenic rainbow trout when compared to the non-transgenics. There were 478 DEGs in common among three tissues. Enrichment analyses conducted by two different bioinformatics tools revealed a tissue specific profile of functional pathway perturbation. Many of them were directly related to innate immune system such as phagocytosis, lysosomal processing, complement activation, antigen processing/presentation, and leukocyte migration. Perturbation of other biological functions that might contribute indirectly to host immunity was also observed.

Conclusions: The gene product of cecropin P1 transgene produced in the disease resistant transgenic rainbow trout not only can kill the pathogens directly but also exert multifaceted immunomodulatory properties to boost host immunity. The identified genes involved in different pathways related to immune function are valuable indicators associated with enhanced host immunity. These genes may serve as markers for selective breeding of rainbow trout or other aquaculture important fish species bearing traits of disease resistance.

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An overview of microarray data analysed in three immune competent organs of cecropin P1 transgenic trout versus non-transgenic trout. A, Hierarchical clustering of spleen, kidney, and liver samples derived from two families (F703 & F180) of cecropin P1 transgenic trout. Designated numbers after each tissue indicate biological and microarray technical repeats. B, Venn diagram showing numbers of differentially expressed genes (DEGs) identified in the three organs of cecropin P1 transgenic trout. C, A list of genes selected from the 478 DEGs in common among organs related to different biological functions. Color gradient denotes relative degree of expression ratio (transgenic/non-transgenic). D, Comparison of microarray and real-time RT-qPCR results (n = 54; linear regression, R2 = 0.82).
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Fig1: An overview of microarray data analysed in three immune competent organs of cecropin P1 transgenic trout versus non-transgenic trout. A, Hierarchical clustering of spleen, kidney, and liver samples derived from two families (F703 & F180) of cecropin P1 transgenic trout. Designated numbers after each tissue indicate biological and microarray technical repeats. B, Venn diagram showing numbers of differentially expressed genes (DEGs) identified in the three organs of cecropin P1 transgenic trout. C, A list of genes selected from the 478 DEGs in common among organs related to different biological functions. Color gradient denotes relative degree of expression ratio (transgenic/non-transgenic). D, Comparison of microarray and real-time RT-qPCR results (n = 54; linear regression, R2 = 0.82).

Mentions: Tissue samples of spleen, kidney, and liver were collected from two different families of cecropin P1 transgenic or non-transgenic fish of equal body sizes and sexes (at one year of age). Experimental details of tissue RNA preparation, labelling of cRNA, and microarray hybridization were as described in Methods. As the initial step of preliminary analysis, normalized expression dataset was tested by Wilcoxon Rank-Sum test (p <0.05) to filter out probes reporting inconsistent signals. This generated 23563 reliable candidates in the liver and 28945 in kidney samples. Due to small number of spleen sample repeats (n = 4), only those bearing reliable signal (≥3 up- or down-regulation) were selected, which resulted in 33729 candidates. Of these candidates there were a total of 13027 probes in common among three different tissues, including those with designated gene names, those with only descriptive annotation, and those destined unknowns due to lack of homolog to any genes of other animal species based on the original probe annotation[11]. Hierarchical clustering of 16 sets of data containing these 13027 probes revealed a tissue-specific expression profile. There was a shorter distance between sample clusters of spleen and kidney as opposed to that of liver (Figure 1A). The clustering of samples of the same tissue suggested limited biological and technical variations, indicating data consistency. A closer expression profile between spleen and kidney versus that of liver was in line with their similar roles in terms of immune function since kidney and spleen are the primary and secondary immune organs in teleost fish[12]. To further refine the data, the candidates with expression ratio over two-fold (transgenic/non-transgenic >2; <0.5) were gated and defined as differentially expressed genes (DEGs). Elimination of redundancy (probes annotated with identical gene name) and unknowns reduced the numbers of DEG to 2480 in the spleen, 2102 in the liver, and 3022 in the kidney. There were 478 DEGs in common among three tissues (Figure 1B; Additional file1 for complete gene list). Genes selected from these DEGs implicated in different biological functions show consistent or inverse expression pattern among tissues (Figure 1C). To evaluate gene expression values obtained by microarray analysis, selected genes covering a diverse range of expression ratios were confirmed with real-time RT-qPCR analysis. Comparison of these two independent methods revealed a general agreement and a high degree of linear correlation (R2 = 0.82, Figure 1D), indicating the expression dataset was sound.Figure 1


Altered gene expression patterns of innate and adaptive immunity pathways in transgenic rainbow trout harboring Cecropin P1 transgene.

Lo JH, Lin CM, Chen MJ, Chen TT - BMC Genomics (2014)

An overview of microarray data analysed in three immune competent organs of cecropin P1 transgenic trout versus non-transgenic trout. A, Hierarchical clustering of spleen, kidney, and liver samples derived from two families (F703 & F180) of cecropin P1 transgenic trout. Designated numbers after each tissue indicate biological and microarray technical repeats. B, Venn diagram showing numbers of differentially expressed genes (DEGs) identified in the three organs of cecropin P1 transgenic trout. C, A list of genes selected from the 478 DEGs in common among organs related to different biological functions. Color gradient denotes relative degree of expression ratio (transgenic/non-transgenic). D, Comparison of microarray and real-time RT-qPCR results (n = 54; linear regression, R2 = 0.82).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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Fig1: An overview of microarray data analysed in three immune competent organs of cecropin P1 transgenic trout versus non-transgenic trout. A, Hierarchical clustering of spleen, kidney, and liver samples derived from two families (F703 & F180) of cecropin P1 transgenic trout. Designated numbers after each tissue indicate biological and microarray technical repeats. B, Venn diagram showing numbers of differentially expressed genes (DEGs) identified in the three organs of cecropin P1 transgenic trout. C, A list of genes selected from the 478 DEGs in common among organs related to different biological functions. Color gradient denotes relative degree of expression ratio (transgenic/non-transgenic). D, Comparison of microarray and real-time RT-qPCR results (n = 54; linear regression, R2 = 0.82).
Mentions: Tissue samples of spleen, kidney, and liver were collected from two different families of cecropin P1 transgenic or non-transgenic fish of equal body sizes and sexes (at one year of age). Experimental details of tissue RNA preparation, labelling of cRNA, and microarray hybridization were as described in Methods. As the initial step of preliminary analysis, normalized expression dataset was tested by Wilcoxon Rank-Sum test (p <0.05) to filter out probes reporting inconsistent signals. This generated 23563 reliable candidates in the liver and 28945 in kidney samples. Due to small number of spleen sample repeats (n = 4), only those bearing reliable signal (≥3 up- or down-regulation) were selected, which resulted in 33729 candidates. Of these candidates there were a total of 13027 probes in common among three different tissues, including those with designated gene names, those with only descriptive annotation, and those destined unknowns due to lack of homolog to any genes of other animal species based on the original probe annotation[11]. Hierarchical clustering of 16 sets of data containing these 13027 probes revealed a tissue-specific expression profile. There was a shorter distance between sample clusters of spleen and kidney as opposed to that of liver (Figure 1A). The clustering of samples of the same tissue suggested limited biological and technical variations, indicating data consistency. A closer expression profile between spleen and kidney versus that of liver was in line with their similar roles in terms of immune function since kidney and spleen are the primary and secondary immune organs in teleost fish[12]. To further refine the data, the candidates with expression ratio over two-fold (transgenic/non-transgenic >2; <0.5) were gated and defined as differentially expressed genes (DEGs). Elimination of redundancy (probes annotated with identical gene name) and unknowns reduced the numbers of DEG to 2480 in the spleen, 2102 in the liver, and 3022 in the kidney. There were 478 DEGs in common among three tissues (Figure 1B; Additional file1 for complete gene list). Genes selected from these DEGs implicated in different biological functions show consistent or inverse expression pattern among tissues (Figure 1C). To evaluate gene expression values obtained by microarray analysis, selected genes covering a diverse range of expression ratios were confirmed with real-time RT-qPCR analysis. Comparison of these two independent methods revealed a general agreement and a high degree of linear correlation (R2 = 0.82, Figure 1D), indicating the expression dataset was sound.Figure 1

Bottom Line: From the microarray data, a total of 2480 genes in the spleen, 3022 in the kidney, and 2102 in the liver were determined as differentially expressed genes (DEGs) in the cecropin P1 transgenic rainbow trout when compared to the non-transgenics.The identified genes involved in different pathways related to immune function are valuable indicators associated with enhanced host immunity.These genes may serve as markers for selective breeding of rainbow trout or other aquaculture important fish species bearing traits of disease resistance.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT 06269, USA. Thomas.Chen@uconn.edu.

ABSTRACT

Background: We have recently developed several homozygous families of transgenic rainbow trout harbouring cecropin P1 transgene. These fish exhibit resistance characteristic to infection by Aeromonas salmonicida and infectious hematopoietic necrosis virus (IHNV). In our earlier studies we have reported that treatment of a rainbow trout macrophage cell line (RTS11) with a linear cationic α-helical antimicrobial peptide (e.g., cecropin B) resulted in elevated levels of expression of two pro-inflammatory relevant genes (e.g., IL-1β and COX-2). Therefore, we hypothesized that in addition to the direct antimicrobial activity of cecropin P1 in the disease resistant transgenic rainbow trout, this antimicrobial peptide may also affect the expression of immune relevant genes in the host. To confirm this hypothesis, we launched a study to determine the global gene expression profiles in three immune competent organs of cecropin P1 transgenic rainbow trout by using a 44k salmonid microarray.

Results: From the microarray data, a total of 2480 genes in the spleen, 3022 in the kidney, and 2102 in the liver were determined as differentially expressed genes (DEGs) in the cecropin P1 transgenic rainbow trout when compared to the non-transgenics. There were 478 DEGs in common among three tissues. Enrichment analyses conducted by two different bioinformatics tools revealed a tissue specific profile of functional pathway perturbation. Many of them were directly related to innate immune system such as phagocytosis, lysosomal processing, complement activation, antigen processing/presentation, and leukocyte migration. Perturbation of other biological functions that might contribute indirectly to host immunity was also observed.

Conclusions: The gene product of cecropin P1 transgene produced in the disease resistant transgenic rainbow trout not only can kill the pathogens directly but also exert multifaceted immunomodulatory properties to boost host immunity. The identified genes involved in different pathways related to immune function are valuable indicators associated with enhanced host immunity. These genes may serve as markers for selective breeding of rainbow trout or other aquaculture important fish species bearing traits of disease resistance.

Show MeSH
Related in: MedlinePlus