Ghrelin Modulates Differential Expression of Genes Relevant to Immune Activities and Antimicrobial Peptides in Primary Head Kidney Cells of Rainbow Trout (Oncorhynchus mykiss).

Ghrelin is a peptide hormone/cytokine that regulates metabolic processes and plays essential roles in the immune system. To evaluate the immunomodulatory actions of ghrelin isoforms in rainbow trout (RT), an in vitro model was utilized with primary cells isolated from fish head kidney (HKD). These RT-HKD cells were treated with synthetic rainbow trout ghrelin and its truncated isoform, desVRQ-ghrelin, over time (0, 2, 4, and 24 h). Reverse transcriptase-coupled qPCR was used to measure the differential expression patterns of genes relevant to various immune processes and genes of antimicrobial peptides. Ghrelin isoform treatments resulted in functional perturbations that displayed overlapping and divergent patterns of gene expression. The differing actions between the two ghrelin isoforms on various assessed genes, and at differing time points, suggested that the two analogs may activate unique pathways, thereby eliciting distinct responses in fish immunity.

Cecropin P1 antimicrobial peptide modulates differential expression of immune relevant genes in rainbow trout (Oncorhynchus mykiss) gill cell line, RTgill-W1.

Accumulated evidence indicates that antimicrobial peptides modulate immune activities in fish. In this study, we profiled the differential expression patterns of representative immune relevant genes in an epithelial-like cell line of rainbow trout gill, RTgill-W1, in response to exposure of cecropin P1 antimicrobial peptide. RTgill-W1 cells were treated with synthetic cecropin P1 over time (0, 2, 4 and 24 h) with or without the present of lipopolysaccharide (LPS) or polyinosinic polycytidylic acid (PolyI:C). The relative abundances of each mRNA were measured by real-time quantitative PCR. The dose-response study revealed significant perturbations of mRNA levels of genes related to pro-inflammation, acute phase, surface proteins and transcription factors at 30 µM of cecropin P1. In addition, cecropin P1 altered the differential expression patterns that were induced by LPS or PolyI:C, at different time points in RTgill-W1. Overall, our results indicate that cecropin P1 exhibits pro-inflammation activity, modulate cell-cell interaction and cytokine signal transduction in rainbow trout gill cell, and may suggest a potential application of this peptide as an immune adjuvant for disease control in aquaculture.

Molecular Analysis and Sex-specific Response of the Hepcidin Gene in Yellow Perch (Perca Flavescens) Following Lipopolysaccharide Challenge.

Hepcidin antimicrobial peptide (hamp) is active in teleosts against invading pathogens and plays important roles in the stress and immune responses of finfish. The response of hamp gene was studied in yellow perch (yp) (Perca flavescens) challenged with lipopolysaccharides to understand if this immunity response is sex-specifically different. The cloned hamp gene consists of an open-reading frame of 273 bp and encodes a deduced protein of 90 amino acids (a.a.), which includes a signal peptide of 24 a.a., a pro-domain of 40 a.a. and a mature peptide of 26 a.a. Yp hamp involves 8 cysteine residues with 4 disulfide bonds, and a protein with an internal alpha helix flanked with C- and N-terminal random coils was modeling predicted. RT-qPCR was used to analyze the relative abundances (RAs) of hamp mRNA in the livers of juvenile female and male yellow perch challenged with lipopolysaccharide. The expression levels of hamp were significantly elevated by 3 h (RA = 7.3) and then peaked by 6 h (RA = 29.4) post-treatment in females but the peak was delayed to 12 h (RA = 65.4) post-treatment in males. The peak mRNA level of challenged males was shown 7.6-fold higher than females. The post-treatment responses in both genders decreased to their lowest levels by 24 h and 48 h. Overall, female perch had an earlier but less-sensitive response to the lipopolysaccharide challenge than male.

Structure and regulation of the NK-lysin (1-4) and NK-lysin like (a and b) antimicrobial genes in rainbow trout (Oncorhynchus mykiss).

Nk-lysin (Nkl), an antimicrobial peptide (AMP) product of natural killer cells and cytotoxic T cells in mammals, has recently been characterized in a number of finfish species. In this study, we identified six genes with sequence homology to Nkl and characterized their patterns of mRNA expression and abundances in rainbow trout (Oncorhynchus mykiss). The cDNA sequences for the six Nkls encoded precursor peptides of 128-133 aa in length, and mature peptides of 109-111 aa in length. Genomic DNA of the nkl1-4 genes consisted of five exons and four introns, whereas the nkl-like a & b genes consisted of four exons and three introns. Chromosomal locations of these peptides show that nkl1 was located on chromosome arm 25q, whereas the other five nkl genes were clustered on chromosome arm 19q. Phylogenetic analysis revealed a conserved structure of Nkls among the teleosts and further protein sequence analyses suggests that all six nkl genes fall within the Nkl sub-family of the Saposin family of proteins. Patterns of tissue-specific mRNA expression were asymmetric among the six trout Nkl homologues, with nkl1, nkl3, and nkl-like a & b occurring in immune competent organs such as spleen, gill, intestine and kidney, as well as pineal gland, brain and oocytes. However, nkl2 and nkl4, showed primary abundances in brain, pineal gland and oocyte tissues. Using mRNA sequencing, in whole-body pools of juvenile trout fry (1 g bw) exposed to Flavobacterium psychrophilum infection, we observed modest up-regulation (2-3 fold) of five (nkl 2-4 and nkl-like a & b) of the six nkl mRNAs over the five-day post-challenge time-course. However, no upregulation could be recorded in spleen tissue measured by qPCR in juvenile trout (270 g bw). Using mRNA sequencing again, mRNA abundances were determined in gill of juvenile trout (~57.7 g bw) exposed to various aquaculture stressors. The results indicated that all six nkls (nkl1-4 and nkl-like a and nkl-like b) were downregulated when exposed to high temperature, and that nkl1 was significantly downregulated following salinity challenge. Overall, these newly characterized AMPs may contribute to host innate immunity as they are modulated following pathogen challenge and by physiological stressors.

BioTarget: A Computational Framework Identifying Cancer Type Specific Transcriptional Targets of Immune Response Pathways.

Transcriptome data can provide information on signaling pathways active in cancers, but new computational tools are needed to more accurately quantify pathway activity and identify tissue-specific pathway features. We developed a computational method called "BioTarget" that incorporates ChIP-seq data into cellular pathway analysis. This tool relates the expression of transcription factor TF target genes (based on ChIP-seq data) with the status of upstream signaling components for an accurate quantification of pathway activity. This analysis also reveals TF targets expressed in specific contexts/tissues. We applied BioTarget to assess the activity of TBX21 and GATA3 pathways in cancers. TBX21 and GATA3 are TF regulators that control the differentiation of T cells into Th1 and Th2 helper cells that mediate cell-based and humoral immune responses, respectively. Since tumor immune responses can impact cancer progression, the significance of our pathway scores should be revealed by effective patient stratification. We found that low Th1/Th2 activity ratios were associated with a significantly poorer survival of stomach and breast cancer patients, whereas an unbalanced Th1/Th2 response was correlated with poorer survival of colon cancer patients. Lung adenocarcinoma and lung squamous cell carcinoma patients had the lowest survival rates when both Th1 and Th2 responses were high. Our method also identified context-specific target genes for TBX21 and GATA3. Applying the BioTarget tool to BCL6, a TF associated with germinal center lymphocytes, we observed that patients with an active BCL6 pathway had significantly improved survival for breast, colon, and stomach cancer. Our findings support the effectiveness of the BioTarget tool for transcriptome analysis and point to interesting associations between some immune-response pathways and cancer progression.

A pathway-focused RT-qPCR array study on immune relevant genes in rainbow trout (Oncorhynchus mykiss) harboring cecropin P1 transgene.

Recently, our laboratory had produced five families of transgenic rainbow trout harboring cecropin P1 transgene, and via repeated challenge studies these fish exhibited a significant elevation of resistance to infection by microbial pathogens. By cDNA microarray and mRNA deep sequencing (mRNA-seq) analyses on two of the five families of cecropin P1 transgenic fish, differentially expressed genes (DEGs) relevant to the innate and adaptive immune pathways in three different immune-related tissues, (i.e. spleen, kidney and liver) were profiled. These results supported our hypothesis that in addition to its direct microbicidal activity, the transgene product of cecropin P1 induces immunomodulatory activity in the transgenic host. Here, we have adapted the technique of quantitative reverse transcription real time PCR (RT-qPCR) array to analyze the expression of genes relevant to the innate and adaptive immune pathways in the rest three families. A RT-qPCR array was constructed with oligonucleotide primers of fifty-two innate/adaptive immune relevant DEGs shown to be the most perturbed by cecropin P1 transgene product in previous studies. Messenger RNA isolated from the spleen, kidney and liver of transgenic fish and non-transgenic fish control were studied on this array. Results of RT-qPCR array revealed that statistically significant perturbations of gene expression were detected in pathways of cytokine/chemokine signaling, Toll-like receptor signaling, complement cascade, antigen processing/presentation, lysosomal phagocytosis and leukocyte trans-endothelial migration in the transgenic spleen; extracellular matrix (ECM) organization and leukocyte trans-endothelial migration pathways in the transgenic kidney; lysosomal activity pathway in the transgenic liver. Furthermore, genes related to the pathways of the peroxisome proliferator-activated receptors (PPAR) signaling, lipid metabolism process and arachidonic acid metabolism were also impacted in the transgenic liver. Findings of the current study are in good agreement with those discoveries in previous two transgenic families by cDNA microarray and mRNA-seq analyses.

RNA-Seq analysis of differentially expressed genes relevant to innate and adaptive immunity in cecropin P1 transgenic rainbow trout (Oncorhynchus mykiss).

BACKGROUND: In the past years, our laboratory successfully generated transgenic rainbow trout bearing cecropin P1 transgene. These fish exhibited resistant characteristic to infection by Aeromonas salmonicida, Infectious Hematopoietic Necrosis Virus (IHNV) and Ceratomyxa shasta (a parasitic pathogen). Previously, treating rainbow trout macrophage cells (RTS-11) with cecropin B, pleurocidin and CF17, respectively, resulted in elevated expression of two pro-inflammatory genes, e.g. cyclooxygenase-2 (cox-2) and interleukin-1ß (il-1ß). In addition, a profiling of global gene expression by 44 k salmonid microarray analysis was conducted, and the results showed that immune relevant processes have been perturbed in cecopin P1 transgenic rainbow trout. Therefore, we hypothesized that cecropin P1 may not only eliminate pathogens directly, but also modulate the host immune systems, leading to increased resistance against pathogen infections. To confirm this hypothesis, we performed de novo mRNA deep sequencing (RNA-Seq) to analyze the transcriptomic expression profiles in three immune competent tissues of cecropin P1 transgenic rainbow trout. RESULTS: De novo sequencing of mRNA of the rainbow trout spleen, liver and kidney tissues were conducted by second-generation Illumina system, followed by Trinity assembly. Tissue specific unigenes were obtained, and annotated according to the Gene Ontology (GO) and the Nucleotide Basic Local Alignment Search Tool (BLAST). Over 2000 differentially expressed genes (DEGs) were determined by normalized ratio of Reads Per Kilobase of transcript per million mapped reads (RPKM) among the transgenic and non-transgenic fish in a tissue specific manner, and there were 82 DEGs in common among the three tissues. In addition, the enrichment analysis according to Gene Ontology Biological Process (GO:BP), and Kyoto Encyclopedia of Genes and Genomes (KEGG) based pathway analysis associated with innate/adaptive immunity of fish were also performed to illustrate the altered immune-related functions in each tissue. CONCLUSIONS: According to the RNA-Seq data, the correlations between alteration of gene expression profiles and the functional perturbations of the host immune processes were revealed. In comparison with the results of cDNA microarray analysis conducted by Lo et al., the overall results supported our hypothesis that the gene product of cecropin P1 transgene may not only directly eliminate pathogens, but also modulate the host immune system. Results of this study present valuable genetic information for Oncorhynchus mykiss, and will benefit future studies on the immunology of this fish species.