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Transcriptomic analysis of sea cucumber (Holothuria leucospilota) coelomocytes revealed the echinoderm cytokine response during immune challenge.
Wu X
,
Chen T
,
Huo D
,
Yu Z
,
Ruan Y
,
Cheng C
,
Jiang X
,
Ren C
.
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BACKGROUND: The sea cucumber Holothuria leucospilota belongs to echinoderm, which is evolutionally the most primitive group of deuterostomes. Sea cucumber has a cavity between its digestive tract and the body wall that is filled with fluid and suspended coelomic cells similar to blood cells. The humoral immune response of the sea cucumber is based on the secretion of various immune factors from coelomocytes into the coelomic cavity. The aim of this study is to lay out a foundation for the immune mechanisms in echinoderms and their origins in chordates by using RNA-seq.
RESULTS: Sea cucumber primary coelomocytes were isolated from healthy H. leucospilota and incubated with lipopolysaccharide (LPS, 10 μg/ml), polyinosinic-polycytidylic acid [Poly (I:C), 10 μg/ml] and heat-inactived Vibrio harveyi (107 cell/ml) for 24 h, respectively. After high-throughput mRNA sequencing on an Illumina HiSeq2500, a de novo transcriptome was assembled and the Unigenes were annotated. Thirteen differentially expressed genes (DEGs) were selected randomly from our data and subsequently verified by using RT-qPCR. The results of RT-qPCR were consistent with those of the RNA-seq (R2 = 0.61). The top 10 significantly enriched signaling pathways and immune-related pathways of the common and unique DEGs were screened from the transcriptome data. Twenty-one cytokine candidate DEGs were identified, which belong to 4 cytokine families, namely, BCL/CLL, EPRF1, IL-17 and TSP/TPO. Gene expression in response to LPS dose-increased treatment (0, 10, 20 and 50 μg/ml) showed that IL-17 family cytokines were significantly upregulated after 10 μg/ml LPS challenge for 24 h.
CONCLUSION: A de novo transcriptome was sequenced and assembled to generate the gene expression profiling across the sea cucumber coelomocytes treated with LPS, Poly (I:C) and V. harveyi. The cytokine genes identified in DEGs could be classified into 4 cytokine families, in which the expression of IL-17 family cytokines was most significantly induced after 10 μg/ml LPS challenge for 24 h. Our findings have laid the foundation not only for the research of molecular mechanisms related to the immune response in echinoderms but also for their origins in chordates, particularly in higher vertebrates.
Fig. 1. Experimental design and transcriptome information. a Experimental design. Sea cucumber coelomocytes isolated from H. leucospilota were challenged with LPS (10âμg/ml), Poly (I:C) (10âμg/ml) and V.harveyi (107 cell/ml) for 24âh with three biological duplicates. b The length distribution of all-Unigene. The X-axis represents the sequence size, and the Y-axis represents the number of Mix-Unigene. The orange bar shows the number of unigene which is the representative sequences, and the blue bar shows the number of transcripts which include the rough sequences. c Venn diagram of Unigene annotation. The databases used for gene annotation include NRãKOGãKEGGãSwissProt and InterPro. d Species distribution of Unigene annotation in NR database
Fig. 2. Comparative transcriptome analysis of DEGs among different immune challenges. a Venn diagram of unique and common DEGs among the immune challenges of LPSãPoly (I:C) and V. harveyi. b the number of up- and down-regulated DEGs in each immune challenge group compared with control group (the first 3 combination bar) and the pairwise comparison of the three different immune challenge groups (the last 3 combination bar). c Volcano map of DEGs in LPS-vs-CT group: X-axis represents the fold change value after log2 conversion, and Y-axis represents the fold change after -log10 conversion. Red dots represent the up-regulated DEGs, blue dots represent the down-regulated DEGs, and gray dots represents the non-DEGs. d comparison results between RNA-Seq by RT-qPCR data in LPS-vs-CT group. X axis shows the fold changes of gene expression in RT-qPCR data, while Y axis represents the fold changes of gene expression in RNA-Seq data
Fig. 3. Co-expressed DEGs of functional classification. a GO classification of a co-expressed gene. The X-axis represents the enrichment factor value, and the Y-axis represents the path name. b Functional classification of KEGG of co-expressed genes. c Pathway enrichment distribution of co-expressed genes. The X-axis represents the enrichment factor value, and the Y-axis represents the path name. The color represents q-value, which is corrected p-value ranging from 0~1, and less q-value means greater intensiveness. The size of the point represents the number of DEGs (the larger the point, the larger the number; the smaller the point, the smaller the number). Rich Factor refers to the enrichment Factor value, which is the quotient between the foreground value (number of DEGs) of a certain pathway on the annotation and the background value (number of all genes) of a certain pathway on the annotation. The larger the data is, the more obvious the enrichment result will be. d GO classification and enrichment of differentially expressed genes under different immune challenges. The Y axis (horizontal direction) represents the number of genes, and the X axis (vertical direction) represents the specific classification under the three functional categories of GO. The red bars represent the GO classification entries annotated by the DEGs in LPS compared to the control group, and accordingly, the purple and blue represent those of Poly (I:C) and V. harveyi
Fig. 4. Changes in expression of cytokine genes upon immune challenges of LPS, Poly (I: C) and V. harveyi.
a The periphery of the circle is the X-axis which presents cytokine genes, while the radius of the circle is the Y-axis which presents the log 2 fold changes value of LPS, Poly (I:C) and V. harveyi immune challenges of corresponding cytokine genes. The closer from the edge of the circle the higher the up regulated expression of corresponding gene is, and the closer from the center of the circle the higher the down regulated expression of corresponding gene is. b two-dimensional hierarchical clustering performed on the clusters of cytokine genes FPKM under three different stimulis. The pink and blue color represents the up- and down-regulation, respectively
Fig. 5. Bioinformatics analysis of selected cytokines. a Phylogenetic analysis of the selected cytokines family with maximum-likelihood (ML) method. b The structural domains of some of the cytokines
Fig. 6. Dose-increased expression of cytokine genes. Transcriptional expression of H. leucospilota cytokines gene, with the β-actin as reference gene, in the sea cucumber coelomocytes treated respectively with LPS concentration of 10.0âμg/ml, 20.0âμg/ml, 50.0âμg/ml for 24âh. The data presented are expressed as the meanâ±âS.E. (nâ=â3). The same letter in the experimental groups represent a similar level (pâ>â0.05, ANOVA followed by Fisherâs LSD test)
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