Immunomodulatory Effects and Induction of Apoptosis by Different Molecular Weight Chitosan Oligosaccharides in Head Kidney Macrophages From Blunt Snout Bream (Megalobrama amblycephala)

Abstract

Prophylactic administration of immunopotentiators has been tested and practiced as one of the most promising disease prevention methods in aquaculture. Chitosan oligosaccharide (COS), as an ideal immunopotentiator, is mainly used as feed additives in aquaculture, and the antimicrobial and immune enhancement effects are highly correlated with molecular weight (MW), but little is known about the mechanisms in teleost. Here, we isolated and purified macrophages in head kidney from blunt snout bream (Megalobrama amblycephala), stimulated them with three different MW (~500 Da, ~1000 Da and 2000~3000 Da) COSs, performed RNA-sequencing, global transcriptional analyses, and verification by quantitative real-time PCR (qRT-PCR) and immunofluorescent staining methods. Differential expression gene (DEG) analysis indicated that gene expression patterns are different and the proportion of unique genes are relatively high in different treatment groups. Biological process and gene set enrichment analysis (GSEA) demonstrated that all three COSs activate resting macrophages, but the degrees are different. Weighted gene co-expression network analysis (WGCNA) reflected gene modules correlated to MW, the module hub genes and top GO terms showed the activation of macrophage was positively correlated with the MW, and larger MW COS activated cell death associated GO terms. Further use of the screening and enrichment functions of STRING and Pfam databases discovered that apoptosis-related pathways and protein families were activated, such as the P53 pathway and caspase protein family. qRT-PCR results showed that as the stimulation time extends, the innate immune-related and P53 pathways are gradually activated, and the degree of activation is positively correlated with the stimulation time. In addition, apoptosis was detected by immunofluorescent staining in three groups. Therefore, the use of COS has two sides-it can activate the immune system against pathogen invasion, but with the increase in stimulation time and MW, macrophage apoptosis is induced, which may be caused by abnormal replication of DNA and excessive inflammation. This study provides a theoretical basis for the rational use of COS as an immunopotentiator in aquaculture.

Keywords: Chitosan oligosaccharide; Megalobrama amblycephala; P53 pathway; apoptosis; macrophage; transcriptome.

Figure 1

Differentially expressed gene (DEG) analysis of different MW COS-stimulated macrophages (P < 0.001). (A) A heatmap was used to classify the DEG expression patterns in all groups, the x-axis represents the experimental conditions (4 h). (B) Venn diagrams of DEGs in COS3, COS6, and COS13-19 groups. Left panel: up-regulated genes; right panel: down-regulated genes.

Figure 2

Functional enrichment analysis identified major biological processes and pathways in COS3, COS6, and COS13-19 groups. (A–C) GO enrichment analysis of upregulated genes in COS3, COS6, and COS13-19, respectively. (D–F) Gene set enrichment analysis (GSEA) of all genes in COS3, COS6, and COS13-19.

Figure 3

Weighted gene co-expression network analysis (WGCNA) yields behaviorally relevant modules. (A) Network topology for different soft-thresholding powers. Numbers in the plots indicate the corresponding soft thresholding powers. The approximate scale-free topology can be attained at the soft-thresholding power of 14. (B) Gene dendrogram obtained by clustering the dissimilarity based on consensus topological overlap, with the corresponding module colors indicated by the color row. Each colored row represents a color-coded module which contains a group of highly connected genes. In total, 17 modules were identified. (C) Pie chart visualizing the number and percentage of genes in each module. (D) Correlation between modules and traits. The upper number in each cell refers to the correlation coefficient of each module in the trait, and the lower number is the corresponding P-value. Red represents high adjacency (positive correlation) and green represents low adjacency (negative correlation). (E–G) Scatter plots of gene significance (GS) for COS3, COS6, and COS13-19 groups vs. the module membership (MM) in the magenta, midnightblue, and blue modules, respectively. Pearson's r (“cor”) and P-value as determined by Fisher's z-transformation are indicated above each plot.

Figure 4

Gene network and enrichment analysis of selected modules. (A–C) Top hub genes of the magenta, midnightblue and blue modules are shown. Gene importance was assigned according to circle diameter, and color depth. (D–F) GO enrichment analysis of magenta, midnightblue, and blue modules genes. Top 5 GO biological process terms are shown. (G) KEGG pathway and (H) protein families and domain enrichment analysis of blue module genes screened from the STRING and Pfam databases.

Figure 5

qRT-PCR identified the key gene expressions of the MAPK and NF-κB pathways. (A–H) The key genes of the MAPK pathway. (I–K) The key genes of NF-κB pathway. The samples were analyzed at 0, 4, 8, and 16 h post-stimulation. β-actin was used as internal reference. Each experiment was executed in triplicate. Data are shown as mean ± SE (N = 3). The asterisk indicates significant difference (**P < 0.01, *P < 0.05) compared with 0 h (set as 1).

Figure 6

COSs induce apoptosis of blunt snout bream head kidney macrophages via the P53 pathway in vitro. (A–E) qRT-PCR identified the expression of key genes of the P53 pathway. The samples were analyzed at 0, 4, 8, and 16 h post-stimulation. β-actin was used as internal reference. Each experiment was executed in triplicate. Data were shown as mean ± SE (N = 3). The asterisk indicates significant difference (**P < 0.01, *P < 0.05) compared with 0 h (set as 1). (F) Cells were cultured for 16 h at 28°C after stimulation and then stained. Annexin V is the green fluorescent marker, labeling cells in early apoptosis. PI (Propidium Iodide) is the red fluorescent marker, labeling cells in late apoptosis. Bars: 25 μm.