Effects on the intestinal morphology, inflammatory response and microflora in piglets challenged with enterotoxigenic Escherichia coli K88.

Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrhea in piglets, which leads to great economic losses. In this study, the ternary crossbred weaned piglets were orally administered with 1.5 × 1011 CFU ETEC K88 for three days. The results showed the ratio of villus length to crypt depth decreased in the duodenum and ileum after ETEC K88 infection. The expression of tight junction proteins ZO-1 in the jejunum and ileum, occludin in the jejunum and colon, and claudin-1 in the colon were down-regulated. The expression of IL-8 in the duodenum and jejunum, IL-13 in the colon, and TNF-α in the jejunum and colon were up-regulated. The expression of pBD1 in the colon, pBD2 in the jejunum, and pBD3 in the duodenum increased after infection. Meanwhile, the expression of TLR4, p38 MAPK and NF-κB p65 increased in all intestinal segments. Moreover, the expression of IL-8 in superficial cervical lymph nodes (SCLN), TNF-α in mesenteric lymph nodes (MLN), and IL-13 in inguinal lymph nodes (ILN) and MLN were up-regulated. The expression of pBD1 and pBD2 in SCLN and MLN, and pBD3 in SCLN were up-regulated. Acidobacteria and Proteobacteria were the most abundant phyla in both groups by analysis of intestinal microflora using 16 s rRNA sequencing, and the relative abundances of bacteria were found to be changed by Metastats software and LEfSe analysis. Our results indicated that cytokines and pBDs had different roles in different intestinal segments or different lymph nodes against ETEC K88, and gut microbiota was influenced after infection.

Transcriptome Analysis Reveals the Multiple Functions of pBD2 in IPEC-J2 Cells against E. coli.

Defensins play an important role in fighting bacteria, and are a good candidate for bactericidal agents. However, the function and mechanism of defensins in regulating host responses against bacteria is unclear. In this study, transcriptome analysis was used to study the comprehensive functions of pBD2 in IPEC-J2 cells against E. coli. In total, 230 differentially expressed genes (DEGs) were identified in IPEC-J2 cells between the control and E. coli groups, and were found by KEGG analysis to be involved in many signaling pathways related to immunity. Furthermore, 812 DEGs were observed between E. coli and E. coli +pBD2 groups, involved in the ribosome, oxidative phosphorylation, and certain disease pathways. Among these, 94 overlapping DEGs were in the two DEG groups, and 85 DEGs were reverse expression, which is involved in microRNA in cancer, while PTEN and CDC6 were key genes according to PPI net analysis. The results of qRT-PCR verified those of RNA-seq. The results indicated that pBD2 plays an important role against E. coli by acting on the genes related to immune response, cell cycle, ribosomes, oxidative phosphorylation, etc. The results provide new insights into the potential function and mechanism of pBD2 against E. coli. Meanwhile, this study provides a certain theoretical basis for research and the development of novel peptide drugs.

Recombinant porcine beta defensin 2 alleviates inflammatory responses induced by Escherichia coli in IPEC-J2 cells.

pBD2 is one of the porcine beta defensins with broad antimicrobial activity, and plays an important role in immune regulation. However, the activities and mechanisms of pBD2 regulating host resistance to Escherichia coli infection are unclear. In this study, the immunomodulatory activity and mechanisms of recombinant pBD2 against Escherichia coli infection were explored in IPEC-J2 cells. Recombinant pBD2 had no obvious effect on the growth of cells below 80 µg/mL, however, it reduced the number of E. coli adhering to cells. Furthermore, pBD2 restored the abnormal expression of ZO-1 and occludin in cells challenged with E. coli. pBD2 treatment also reduced cell apoptosis and decreased the expression of the apoptosis-related genes Cox-2 and Caspase-3, and decreased the expression of the pro-inflammatory IL-6, IL-8, IL-1α and TNF-α, and Cxcl2 and Ccl20. pBD2 also reduced the expression of TAK1, and inhibited the phosphorylation of NF-κB p65 following E. coli infection. In addition, pBD2 was localized in the cytoplasm. Collectively, pBD2 appeared to penetrate cells and alleviate inflammatory responses via the TAK1-NF-κB signaling pathway. Our results revealed the immunomodulatory activity of recombinant pBD2 against E. coli and provided insights into the molecular mechanisms that protected cells from E. coli infection.