Dietary genistein supplementation alters mRNA expression profile and alternative splicing signature in the thymus of chicks with lipopolysaccharide challenge

Abstract

Genistein is abundant in the soybean products, which exerts prominent effects on immune function. Little information is available about the effect of dietary genistein on thymic transcriptome, especially when suffering from lipopolysaccharide challenge. In this study, 180 one-day-old male broilers were randomly allocated to 3 groups: nonchallenged chicks given a basal diet (CON), and lipopolysaccharide-challenged chicks fed a basal diet (LPS), or lipopolysaccharide-challenged chicks fed a basal diet supplemented with 40 mg/kg genistein (GEN). Lipopolysaccharide injection induced thymocyte apoptosis and inflammatory reactions in the chicks. The results showed dietary genistein significantly reduced the percentage of CD3+ T lymphocytes by 10.04% and CD4+/CD8+ T lymphocyte ratio by 21.88% in the peripheral blood induced by lipopolysaccharide injection (P < 0.05). In addition, genistein significantly reduced the thymus index by 50% and apoptotic index by 12.34% induced by LPS challenge (P < 0.05). Transcriptomic analysis identified 1,926 DEGs (1,014 upregulated and 912 downregulated, P < 0.05) between GEN and LPS groups, which altered the mRNA expression profile and signaling pathways (Toll-like receptor, and NOD-like receptor signaling pathway) in the thymus. Furthermore, 5 splicing (AS) isoforms of the Drosophila Disabled-2 (DAB2) gene were detected, which were significantly upregulated in the GEN group compared with that in the LPS group. In summary, dietary genistein supplementation altered the RNA expression profile and AS signatures in the thymus, and alleviated immune response against lipopolysaccharide challenge.

Keywords: chicken; genistein; lipopolysaccharide; thymus; transcriptome.

Graphical abstract

Figure 1

Effects of genistein on the classification of peripheral blood T lymphocyte by flow cytometer analysis. (A) Square dot graph of two parameters (FL1 INT LOG/FL2 INT LOG), (B) linear gate analysis of CD3+ T cells in peripheral blood T lymphocyte (FL1 INT LOG), (C) Prism analysis of CD3+, CD4+ and CD8+ T lymphocyte percentage. Data are presented as mean value ± SD (n = 8). Values without the same mark (A, B) represent statistically significant differences (P < 0.05). Abbreviations: CON, nonchallenge control; LPS, lipopolysaccharide-challenged group; GEN, lipopolysaccharide-challenged group fed diet supplemented with 40 mg/kg genistein.

Figure 2

Effects of genistein on the classification of peripheral blood T lymphocyte by flow cytometer analysis. (A) Statistical analysis of CD3+ T lymphocyte. (B) Statistical analysis of CD4+/CD8+ T lymphocyte ratio. Data are presented as mean value ± SD (n = 8). Values without the same mark (A, B) represent statistically significant differences (P < 0.05). Abbreviations: CON, nonchallenge control; LPS, lipopolysaccharide-challenged group; GEN, lipopolysaccharide-challenged group fed diet supplemented with 40 mg/kg genistein.

Figure 3

Histological observation of chick thymus by he staining. Scale bars = 500 μm. Abbreviations: CON, nonchallenge control; LPS, lipopolysaccharide-challenged group; GEN, lipopolysaccharide-challenged group fed diet supplemented with 40 mg/kg genistein.

Figure 4

Thymus histological observation and tunel assay. (A) Tunel assay of thymus sections at 21 d of age by immunofluorescence. The blue color represents the total cells in the thymus, and the red color represents the apoptosis cells. (B) Apoptotic index of thymus at 21 d of age. AC, apoptosis cells. Data are presented as mean value ± SD (n = 8). Values without the same mark (A, B) represent statistically significant differences (P < 0.05). Scale bars = 200 μm. Abbreviations: CON, nonchallenge control; LPS, lipopolysaccharide-challenged group; GEN, Lipopolysaccharide-challenged group fed diet supplemented with 40 mg/kg genistein.

Figure 5

(A) The relative gene expression abundance from RNA-Seq data as determined by using Cufdiff software (n = 3). (B) The relative mRNA expressions of random selected genes in the chick thymus as determined by using qRT-PCR. Data are presented as mean value ± SEM (n = 8). Values without the same mark (A–C) represent statistically significant differences (P < 0.05). Abbreviations: CXCL12, motif chemokine 12; CCR2, C-C chemokine receptor type 2; COX1, cytochrome c oxidase subunit 1; COII, cytochrome c oxidase subunit 2; CON, nonchallenge control; DAB2, disabled homolog 2; GEN, lipopolysaccharide- challenged group fed diet supplemented with 40 mg/kg genistein; LPS, lipopolysaccharide- challenged group; NFKBIE, NF-kappa-B inhibitor epsilon; TNFSF8, tumor necrosis factor ligand superfamily member 8.

Figure 6

Cluster analysis of RNA-Seq data. (A) Scatter plot of DEGs (LPS vs. CON). (B) Scatter plot of DEGs (GEN vs. LPS). Red points represent upregulated genes with |Fold change| >1 and P_value < 0.05. Green points represent upregulated genes with |Fold change| <1 and P_value < 0.05. Blue points represent genes with no significant difference. (C) The hot map of hierarchical clustering analysis using genes expression values between the CON, LPS and GEN groups. Red color indicates high gene expression; blue color indicates low gene expression. (D) Venn diagram of differentially expressed genes among the CON, LPS and GEN groups. Abbreviations: CON, nonchallenge control; LPS, lipopolysaccharide-challenged group; GEN, lipopolysaccharide-challenged group fed diet supplemented with 40 mg/kg genistein.

Figure 7

Bioinformatics analysis of RNA-Seq data. (A and B) The top 10-enriched items in each main category (biologic process, cell component, and molecular function) of the GO database at all levels using upregulated and downregulated DEGs between the LPS vs. CON groups. (C and D) The top 10-enriched items in each main category (biologic process, cell component, and molecular function) of the GO database at all levels using upregulated and downregulated DEGs between the GEN vs. LPS groups. (E and F) The top enriched KEGG items using DEGs of the LPS vs. CON groups and the GEN vs. LPS groups, respectively. (G and H) Protein–protein interaction analysis using DEGs of the LPS vs. CON groups and the GEN vs. LPS groups, respectively. Circular nodes represent genes/proteins; rectangles represent KEGG pathways or GO Biologic Process terms. The pathways are colored with a gradient from yellow to blue, in which yellow indicates a smaller P value, and blue indicates a larger P value. GO biologic processes are colored red. In the fold-change analysis, genes/proteins are colored red for upregulation or green for downregulation. Abbreviations: CON, nonchallenge control; LPS, lipopolysaccharide-challenged group; GEN, lipopolysaccharide-challenged group fed diet supplemented with 40 mg/kg genistein.

Figure 7

Bioinformatics analysis of RNA-Seq data. (A and B) The top 10-enriched items in each main category (biologic process, cell component, and molecular function) of the GO database at all levels using upregulated and downregulated DEGs between the LPS vs. CON groups. (C and D) The top 10-enriched items in each main category (biologic process, cell component, and molecular function) of the GO database at all levels using upregulated and downregulated DEGs between the GEN vs. LPS groups. (E and F) The top enriched KEGG items using DEGs of the LPS vs. CON groups and the GEN vs. LPS groups, respectively. (G and H) Protein–protein interaction analysis using DEGs of the LPS vs. CON groups and the GEN vs. LPS groups, respectively. Circular nodes represent genes/proteins; rectangles represent KEGG pathways or GO Biologic Process terms. The pathways are colored with a gradient from yellow to blue, in which yellow indicates a smaller P value, and blue indicates a larger P value. GO biologic processes are colored red. In the fold-change analysis, genes/proteins are colored red for upregulation or green for downregulation. Abbreviations: CON, nonchallenge control; LPS, lipopolysaccharide-challenged group; GEN, lipopolysaccharide-challenged group fed diet supplemented with 40 mg/kg genistein.

Figure 8

The enriched PANTHER GO-Slim using genes with AS. (A) PANTHER GO-Slim biological processes using genes with AS between LPS and CON groups at all levels. (B) PANTHER GO-Slim biological processes using genes with AS between LPS and CON groups at level 1: immune system process. (C) PANTHER GO-Slim biological processes using genes with AS between GEN and LPS groups. (D) PANTHER GO-Slim molecular functions using genes with AS between HGE and CON groups at level 1: immune system process. (E) The visual demonstration of DAB2 in the style of skipped exon. The chromosome coordinates and positive and negative chain information of the three exons with the alternative splicing event are titled. Reads for cross exon alignment are represented by arcs connecting the boundary of the exon junction. The thickness of the arc is directly proportional to the number of reads compared to the junction. Meanwhile, the number on the arc indicates the number of junction reads, and the gene and inclevel value of the alternative splicing event are marked on the top right.