Transcriptomic Analyses Reveal the Protective Immune Regulation of Conjugated Linoleic Acids in Sheep Ruminal Epithelial Cells

Figures

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  FIGURE 1. Characterization of ruminal epithelial cells from sheep (RECs). (A) Morphology of isolated primary RECs. The cell aggregates were well-attached in 24 h of culture. (B) Immunofluorescence staining of primary RECs. Cytokeratin 19: fluorescent image showing stained cytokeratin 19; DAPI: negative control when only secondary antibody was used; nuclei were stained with DAPI. (C) Morphology of successfully immortalized REC induced by SV40T after puromycin selection. (D) Morphology of immortal RECs after 30 passages of culture without showing any signs of senescence.

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  FIGURE 2. Verification of the pro-inflammatory effect of LPS and anti-inflammatory effect of CLA. (A) Relative gene expression of IL-6, IL-8 and NF-κB in RECs after stimulating with 0.1, 1, and 10 μg/ml LPS for 3 h respectively. (B) Relative gene expression of TNF-α, IL-6 and IL-8 in RECs with the 100 μM CLA pretreatment for 24 h during 0.1 μg/ml LPS stimulation. *indicates significant difference compared to the CON group (*P < 0.05, **P < 0.01), # indicates significant difference compared to the LPS group (#P < 0.05, ##P < 0.01).

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  FIGURE 3. Overview of the differentially expressed genes (DEGs) after different treatments. (A) Venn diagrams of known DEGs based on comparisons of different groups. Cut-off for the differential expression criteria were Padj < 0.01 and FC > 1.5. (B) Hierarchical clustering of the overlapping DEGs among different groups. The color code indicates highly expressed genes (red) and genes expressed at low levels (blue).

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  FIGURE 4. Functional annotation of the DEG genes according to the GO analyses. (A) GO enrichment analysis of the overlapping DEGs that were downregulated in the CLA + LPS group and upregulated in the LPS group. (B) GO enrichment analysis of the DEGs that were upregulated in the CLA + LPS group compared with gene expression in the LPS group. Only the top 20 GO terms are shown (Padj < 0.01).

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  FIGURE 5. Functional annotation of the DEG genes according to the KEGG analyses. (A) KEGG enrichment analysis of the overlapping DEGs that were downregulated in the CLA + LPS group and upregulated in the LPS group. (B) KEGG enrichment analysis of the DEGs that were upregulated in the CLA + LPS group compared with the gene expression in the LPS group. Circles represent the numbers of enriched genes, and colors indicate the Padj values.

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  FIGURE 6. Protein-protein interaction (PPI) network of DEGs. (A) PPI network of the overlapping DEGs that were downregulated in the CLA + LPS group and upregulated in the LPS group. (B) PPI network of the DEGs that were upregulated in the CLA + LPS group compared with the expression level in the LPS group. The node color is determined by the clustering coefficient (low values are red, and high values are blue), and the node size is proportional to the number of degrees. Each edge represents the interaction (a thicker edge indicates lower betweenness).

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  FIGURE 7. Gene expression analysis of the DEGs involved in the immune response, cell proliferation and lipid metabolism among different treatments. Gene names are shown on the x-axis, and the fold change value is shown on the y-axis. * indicates significant difference compared to the CON group (*P < 0.05, **P < 0.01), #indicates significant difference compared to the LPS group (#P < 0.05, ##P < 0.01).

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  FIGURE 8. Schematic diagram summarizing the mechanisms underlying the protective effect of CLA against LPS-induced inflammation in RECs. CLA alleviated ruminal epithelial cell inflammation by inhibiting the proinflammatory process and by regulating cell proliferation and lipid metabolism, which are potentially related to injury repair and the immune response.