Evaluating the effect of TLR4-overexpressing on the transcriptome profile in ovine peripheral blood mononuclear cells

Xiaofei Guo,Yihai Li,Jing Yang,Chunxiao Dong,Yao Li,Xiaosheng Zhang,Guoshi Liu,Zhengxing Lian,Jinlong Zhang

doi:10.1186/s40709-020-00124-3

Abstract

BackgroundToll-like receptor 4 (TLR4) plays an important role in the elimination of Gram-negative bacteria infections and the initiation of antiinflammatory response. Using the technology of pronuclear microinjection, genetically modified (GM) sheep with TLR4 overexpression were generated. Previous studies have shown that these GM sheep exhibited a higher inflammatory response to Gram-negative bacteria infection than wild type (WT) sheep. In order to evaluate the gene expression of GM sheep and study the co-expressed and downstream genes for TLR4, peripheral blood mononuclear cells (PBMC) from TLR4-overexpressing (Tg) and wild type (WT) sheep were selected to discover the transcriptomic differences using RNA-Seq.ResultAn average of 18,754 and 19,530 known genes were identified in the Tg and WT libraries, respectively. A total of 338 known genes and 85 novel transcripts were found to be differentially expressed in the two libraries (p < 0.01). A differentially expressed genes (DEGs) enrichment analysis showed that the GO terms of inflammatory response, cell recognition, etc. were significantly (FDR < 0.05) enriched. Furthermore, the above DEGs were significantly (FDR < 0.05) enriched in the sole KEGG pathway of the Phagosome. Real-time PCR showed the OLR1, TLR4 and CD14 genes to be differentially expressed in the two groups, which validated the DEGs data.ConclusionsThe RNA-Seq results revealed that the overexpressed TLR4 in our experiment strengthened the ovine innate immune response by increasing the phagocytosis in PBMC.

Highlights

Toll-like receptor 4 (TLR4) plays an important role in the elimination of Gram-negative bacteria infections and the initiation of antiinflammatory response
The RNA-Seq results revealed that the overexpressed TLR4 in our experiment strengthened the ovine innate immune response by increasing the phagocytosis in peripheral blood mononuclear cells (PBMC)
These results indicated that TLR4-overexpressing sheep were successfully generated

Summary

Introduction

Toll-like receptor 4 (TLR4) plays an important role in the elimination of Gram-negative bacteria infections and the initiation of antiinflammatory response. Previous studies have shown that these GM sheep exhibited a higher inflammatory response to Gram-negative bacteria infection than wild type (WT) sheep. In order to evaluate the gene expression of GM sheep and study the co-expressed and downstream genes for TLR4, peripheral blood mononuclear cells (PBMC) from TLR4-overexpressing (Tg) and wild type (WT) sheep were selected to discover the transcriptomic differences using RNA-Seq. Most Gram-negative bacteria are considered to be harmful to their hosts, many of them (such as Brucella melitensis, Salmonella enterica and Escherichia coli) are pathogenic to both animals and humans. Our further findings for Salmonella enterica infection indicated that the overexpression of TLR4 enhances phagocytosis through PI3K signalling, as well as the subsequent activation of actin polymerization and scavenger receptors in sheep monocytes/macrophages [10]

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Discussion

Conclusion