CD14 in the TLRs signaling pathway is associated with the resistance to E. coli F18 in Chinese domestic weaned piglets.

Zhengchang Wu,Shenglong Wu,Wenhua Dong,Wenbin Bao,Guo-qiang Zhu,Ying Liu

doi:10.1038/srep24611

Abstract

Escherichia coli F18 (E. coli F18) is mainly responsible for post-weaning diarrhea (PWD) in piglets. The genetic basis and regulatory mechanism of E. coli F18 resistance in Chinese domestic weaned piglets remain unclear. Meishan piglets were used as model animals to test their susceptibility to E. coli F18. By performing a comparative transcriptome study on duodenum tissues of sensitive and resistant pigs, we identified 198 differentially expressed genes (DEGs; 125 upregulated and 73 downregulated) in the resistant pigs. DEGs were predominately involved in immune system pathways, including the Toll-like receptor (TLR) signaling pathway. qPCR and western blot showed CD14, IFN-α, TLR4 and IL-1β, etc. in the TLR signaling pathway had significantly higher expression levels in lipopolysaccharide (LPS)-induced small intestinal epithelial cell lines (IPEC-J2) than those in normal IPEC-J2 cells. Immunohistochemical analysis showed the increased expression of CD14 gene in the E. coli F18-resistant individuals. After CD14 knockdown, the levels of cytokines IL-6 and IL-12 were significantly reduced in IPEC-J2 cell supernatants. The adhesion ability of F18ab strain with IPEC-J2 cells was significantly increased (p < 0.01). This study revealed the TLR signaling pathway, and especially CD14, probably plays an important role in resistance to E. coli F18 infection in Chinese domestic piglets.

Highlights

We collected feces from piglets in the same litter weaned after 30 days in the same feeding environment, which were used to detect common pathogens, including general pathogenic Escherichia coli (E. coli F18, E. coli K88) and viruses (transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), rotavirus (RV)) (PCR primers are shown in Supplementary Table S1)
In all piglets detected by the binding assay, we strictly identified piglets displaying no adherence with F18-expressing fimbriae of the standard Enterotoxigenic Escherichia coli (ETEC) strain as E. coli F18resistant individuals (Fig. 1A)
We identified an interesting pathway in the “Immune System” pathways, namely TLR4-mediated Toll-like receptor signaling pathway, where CD14, OPN, CXCL11 genes were significantly enriched

Summary

Introduction

It is difficult to analyze the molecular mechanism of E. coli F18 resistance using RNA-Seq technology in native Chinese pig breeds because of the lack of extreme phenotype individuals for E. coli F18 infection. One of main purposes of this study was to obtain E. coli F18-resistant and -sensitive individuals in native Chinese pig breeds. We obtained many differentially expressed genes (DEGs) between the resistant group and sensitive group, which were analyzed by Gene Ontology (GO) and pathway enrichment analysis to identify important pathway or functional genes related to E. coli F18 infection. This study determined the molecular regulation mechanisms and the critical genes involved in regulating piglets’ responses to E. coli F18 infection, which will pave the way for further genetic engineering to improve resistance to E. coli F18 in Chinese native pig breeds

Methods

Results

Conclusion