Document Type

Thesis - University Access Only

Award Date

2012

Degree Name

Master of Science (MS)

Department / School

Biology and Microbiology

First Advisor

Radney S. Kaushik

Abstract

The innate immune responses play an important role in body's defense with its preformed and specific receptors localized on the cell surface and endosomes of all the immune cells and non-immune cells of the body. Enteric mucosal cells are in constant contact with microflora including commensals and pathogens. These cells are the first line of defense in the enteric infections. Porcine intestinal epithelial cell culture models had been used for studying the host-pathogen interactions of various bacterial and viral diseases. This study was conducted to understand the transcriptional changes in innate immunity-related genes when porcine intestinal epithelial (I PEC-J2 and I PEC-1) cells were stimulated with bacteria-associated molecular patterns, porcine enterotoxigenic Escherichia coli (ETEC), and heat-labile toxins. The bacterial ligands used for the study were lipopolysaccharide (LPS), peptidoglycan (PGN), flagellin (FLA), CpG and GpC oligodeoxynucleotide (ODN). Cells without any treatment acted as a negative control and GpC acted as a control for CpG ODN .The changes in the gene expression of toll-like receptors (TLRs 1-10), MD-2, nucleotide-binding oligomerization domain (NOD) -like receptors (NLRs; NOD-1,-2), RIG-1-like receptors (RLRs; RIG-1,and MDA-5), cytokines ( IL-1a, IL-1 J3, TNF-a, IL-6, IL-6, - 7, IL-12p35 and IL-12p40, IL-15, -18, IFN-a, and IFN-J3), chemokines (IL-8, MIF, MCP-1, CCL-20, and Osteopontin), antimicrobial peptides (AMPs) such as J3 Defensin -1, and -2 (BD-1, and -2) relative to porcine Cyclophilin-A were quantified using quantitative real time RT-PCR. The significant modulatory changes were observed in both cell lines in response to bacterial ligands at 3 and 24h time points. There was no significant modulation in the TLRs-2, -4,-5 and -9 gene expression that specifically detect the PGN, LPS, FLA and CpG respectively. However, a cross regulation of certain TLRs by various bacterial ligands was observed in both IPEC-J2 and IPEC-1 cells. IPEC-J2 and IPEC-1 cells were significantly different in the expression of TLRs, NLRs and RLRs at both time points. Various cytokines, chemokines and AMPs gene expressions also significantly varied with different treatments. These findings clearly indicated that both IPEC-J2 and IPEC-1 cells sufficiently respond upon stimulation wth various bacterial ligands. Previous studies showed that porcine intestinal epithelial cell lines have a better binding to porcine ETEC over human intestinal epithelial cell lines. So these cell lines can be used for studying the patheogenesis of porcine ETEC, which is of great economic importance in swine industry. For the ETEC study, we used two wild type porcine K88ac ETEC strains 3030-2 and 2534-86, a LT toxin deficient K88ac E. coli strain (1836-2) and G58-1 a non-pathogenic, non-fimbriated, wild-type E.coli strain at multiplicity of infection 10: 1. Heat labile toxins LT and L Tb were also used in this study. G58-1 strain stimulated cells and cells with media alone served as negative controls. The time points used for the ETEC study were 3 and 6h. We found that wild type fimbriated strains (3030-2 and 2534-86) significantly decreased the expression of TLR-7 gene. However, both wild type ETEC strains and fimbriated 1836-2 strain increased the expression of pro-inflammatory cytokines IL-1 a, IL-1 J3, TNF-a, and CCL-20. The toxins LT and L Tb did not induce significant differential expression of pro-inflammatory cytokines. The significant findings obtained from the two studies helped us to understand the transcriptional changes occurring in a controlled in vitro environment. Further investigations will help in understanding more about enteric mucosa! innate immune responses to ETEC and other associated enteric bacterial infections, and may provide new insights to the prevention and treatment of these diseases.

Library of Congress Subject Headings

Intestines -- Infections
Natural immunity
Escherichia coli infections in swine
Epithelial cells

Publisher

South Dakota State University

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Rights Statement

In Copyright